Current Issue http://journals.caass.org.cn/zgnykx EN-US http://journals.caass.org.cn/zgnykx/EN/current.shtml http://journals.caass.org.cn/zgnykx 5 【Objective】 Rice is an important food crop, providing staple food for more than half of the world’s population. Panicle traits are the main factors affecting rice yield. Discover the elite haplotype of the panicle regulation gene, and provide important germplasm and gene resources for pyramiding breeding. 【Method】 In this study, recombinant inbred lines (RILs) derived from a cross between SN265 and R99 were re-sequenced through high-throughput sequencing. QTL analysis and candidate gene identification were conducted on the grain number on the primary branch, the grain number on the secondary branch, and the grain shape. The sequences of candidate genes were compared using the long-read sequence assemblies of SN265 and R99. The combination of candidate genes that can maximize grain yield was selected among RILs. Finally, the super rice variety SN265 was improved using CRISPR/Cas9 gene editing technology. 【Result】 The R99 had significantly more grain number per panicle and grain number on the secondary branch, whereas SN265 had significantly more grain number on the primary branch. The grain of R99 is slender, and the grain of SN265 is short and round. The RILs were sequenced with approximately 6.25-fold depth. For parent lines, 30.0-fold depth and 32.0-fold depth data were generated for R99 and SN265, respectively. Subsequently, a bin map was constructed by 1456445 high-quality SNPs. The genetic map containing 3 569 recombinant blocks, with an average length of 58.17 kb. The QTL analysis detected a QTL on Chr.9 for grain number per panicle and grain number on both primary and secondary branch, a QTL on Chr.1 for grain number per panicle and grain number on the secondary branch, a QTL on Chr.5 for grain shape. The candidate gene prediction and sequence comparison showed that DEP1 regulated the grain number on both primary and secondary branches of rice, Gn1a mainly regulated the grain number on secondary branches of rice, and qSW5 mainly regulated the grain shape. The yield of the combination of Gn1a^R99/DEP1^SN265/qSW5^SN265 alleles showed an advantage in yield performance among the RILs. We further conducted a molecular design breeding to SN265 by knocking out the Gn1a locus using CRISPR/Ca9 gene editing technology, and the grain number per panicle of the transgenic plants increased significantly compared to that of SN265. 【Conclusion】 This study used RILs derived from a XI/GJ cross and high-throughput sequencing technology to conduct QTL analysis of rice panicle traits, revealed the effects of DEP1, Gn1a, and qSW5 on grain number per panicle and grain shape, and clarified that Gn1a^SN265/ DEP1^R99/qSW5^R99 was the best gene combination in RILs. The yield per plant was further improved by knocking out the Gn1a locus of SN265. This study provided important germplasm and gene resources for pyramiding breeding with elite alleles.

]]> 【Background】 Food safety is key for ensuring national security. Rice is the staple food crop upon which people life depend. It is an important breeding target to improve its yield. Rice yield is composed of panicle number per plant, grain number per panicle and grain weight, among which grain weight relates closely to grain size and filling degree. However, these traits are controlled by multiple genes, and their genetic basis are complex. Chromosome segment substitution lines (CSSLs) can accurately dissect QTL for complex trait into a single Mendel’s factor, which is closely linked with the breeding work, so they are ideal materials for genetic research and breeding. 【Objective】 In the early stage, we fine-mapped a seed shattering gene SH6 using a rice chromosome segment substitution line Z481 carrying four substitution segments, However, there are still some significant differences in the panicle traits between Z481 and its recipient parent Nipponbare. It is important to understand how to distribute for these QTLs controlling panicle traits on 4 substitution segments of Z481 and then to dissect them into single segment substitution lines (SSSLs) for map-cloning of target QTL in theory meaning and for rice breeding by design in application value.【Method】 Here, the secondary F₂ population constructed by crossing Nipponbare with Z481 was used to map QTL for these traits by mixed linear model (MLM) method in SAS9.3 statictic shoftware (P<0.05), and then by MAS method to develop SSSLs and dual-segment substitution lines (DSSLs) in F₃ derived from 42 F₂ indiviuals according to their genotypes and phynotypes. Finally, the additive effect and epistasis effect of QTL were analyzed using these SSSLs and DSSLs by ONE-WAY ANOVA,TWO-WAY ANOVA, LSD and Duncan’s multiple comparasion (P<0.05) in IBM SPSS Statistics 25.0.【Result】 12 QTLs controlling rice panicle traits are mapped from the secondary F₂ population constructed by Nipponbare/Z481, and 11 single segment substitution lines (S1-S11) and 3 dual-segment substitution lines (D1-D3) with each corresponding single substitution segment are developed. Among them, 8 QTLs (qGL1, qGL3, qGL6, qG-W1, qGW3, qRLW1, qRLW3, qRLW6) can be verified by 11 SSSLs, indicating that these QTLs are genetically stable. In addition, 33 QTLs such as qGL1-2, qGL1-3, qGL3-2 etc. are only detected by 11 single segment substitution lines. Among them, 15 QTLs such as qNSB1-1 etc. might be novel QTLs identified in the study. Furthermore, the epistasis effect between non-allelic QTLs was analyzed by three DSSLs and corresponding SSSLs, the results showed that pyramid of different QTL produce various epistasis effect. For example, the pyramid of qGL3 (a=1.26) and qGL6-2(a=0.86) yield epistasis effect of -0.77, according to the genetic model of DSSL, D2 with the genetic effect of 1.35 produce longer grain length than any of two SSSLs with qGL3 or qGL6-2; the pyramid of qGWT3-2 (a=3.18) and qGWT6-2 (a=3.39) produce epistasis effect of -5.46, making the 1000-grain weight of D2 significantly smaller than that of the corresponding SSSLs due to its genetic effect of 1.11.【Conclusion】 In total 45 QTLs for rice panicle traits are deteted on the 4 substitution segments of Z481 and then further dissected into 11 secondary SSSLs. SSSL have higher efficiency for QTL detection than the F₂ population. The additive effect and epistasis effect of these QTLs detected by SSSL and DSSL are necessary for breeders to predict the phenotype of the designed genotype according to these genetic informations and then to screen favorable SSSLs to breed by design.

]]> 【Objective】 Spikelet is a unique floral organ of the grass family. In rice, the spikelet, as the basic unit and unique structure of inflorescence, has an important impact on the yield and quality. Therefore, studying the development of rice spikelets and floral organs can provide the foundation for the formation of rice yield and quality. 【Method】 Two rice allelic mutants, polarity defect of lateral organs 2-1 and polarity defect of lateral organs 2-2 (pdl2-1 and pdl2-2) with similar mutant phenotypes were obtained using Ethyl Methane Sulfonate (EMS) mutagenesis in the indica rice maintenance line Xinong 1B. Because of their phenotypic similarity, pdl2-1 (named pdl2) was selected as the material for further analysis. Microscopic observation and paraffin sectioning techniques were used to analyze their spikelet mutant phenotypes; agronomic trait examination was used to analyze the effect of lemma on rice yield; map-based cloning were used to verify the function of PDL2; in situ hybridization and real-time fluorescence quantitative PCR (RT-qPCR) were used to analyze the expression pattern of PDL2. 【Result】 The results of the phenotypic analysis showed that the lemma of pdl2 mutant was significantly narrower and could not be closely hooked to the palea compared with that of wild type, resulting in spikelet dehiscence and partially exposed inner whorl floral organs. However, the morphology and number of stamens, pistils, and lodicules were normal. Further paraffin section results showed that the reduced volume and number of silicified and vesicular cells in the mutant lemma, as well as the reduced spacing of vascular bundles, were responsible for the significant narrowing of the width of the lemma in pdl2. Agronomic traits examined showed that the mutation in the pdl2 lemma eventually caused the seeds to be teardrop-shaped and resulted in a significant decrease in yield traits such as seed setting rate and 1000-grain weight in the pdl2 mutant. Genetic analysis and map-based cloning showed that PDL2 is a single recessive nuclear gene, which is OsDCL4 gene localized on chromosome 4. PDL2 encodes a Dicer-like protein that plays an essential role in the rice ta-siRNA synthesis pathway, and the pdl2 mutant is a novel weak allelic mutation of OsDCL4 gene. The expression pattern analysis showed that the PDL2 gene was constitutively expressed in all whorls of floral organs. Mutation of PDL2 affected the ta-siRNA synthesis and the expression of genes related to the establishment of adaxial-abaxial polarity, thus leading to the disorder of adaxial-abaxial polarity establishment in the lemma of pdl2. And the characteristics of floral organs were normal. 【Conclusion】 Mutation of lemma degeneration gene PDL2 disrupts the establishment of lemma adaxial-abaxial polarity and affects the development of lemma and the formation of yield traits.

]]> 【Objective】 Rice yield is composed of effective panicle number per unit area, grains per panicle and grain weight, in which grain weight is mainly determined by grain morphology. Screening and identification of new grain type mutation materials and genes can lay a foundation for molecular design breeding of yield traits. 【Method】 A short and wide grain mutant short and widen grain1 (swg1) was identified in the mutant population of indica rice maintainer line Xida1B(XD1B) induced by ethyl methane sulfonate (EMS). The grain morphology and other agronomic characters were analyzed, and the glume was observed and analyzed by histocytology. Gene mapping was carried out by BSA method, and candidate genes were identified by genetic complementarity experiment. qRT-PCR was used to analyze the expression pattern of the gene and the expression level of other genes related to grain shape and cell development.【Result】 The analysis of agronomic characters showed that the grain length of swg1 mutant was significantly lower and the grain width was significantly higher than that of wild type, showing the phenotype of short and wide grains, and further histological and cytological analysis showed that the shortening of longitudinal cells of glume was the main reason for the shortening of grain length, while the increase of grain width was due to the increase of the number and size of transverse cells of glume at the same time. The results of genetic analysis showed that the mutation was controlled by a recessive single gene, and the candidate gene for SWG1 was determined to be LOC_Os07g42410 by map-based cloning and genetic complementary verification, which encoded a plant-specific transcription factor. qRT-PCR analysis showed that the expression of this gene had no obvious tissue specificity, and its expression is strong in stem, leaf and young panicle. According to the analysis of the expression of known genes related to grain shape, cell cycle and cell expansion, it was found that GS5 and GW8, which positively regulate the number and/or size of glume transverse cells to determine grain width, were significantly up-regulated in the mutants, while GW7/GL7 genes, which positively regulated the number and size of longitudinal cells and negatively regulated the number and size of transverse cells, were significantly down-regulated in the mutants. Some genes related to cell cycle and cell expansion also showed significant differences between mutants and wild types. 【Conclusion】 SWG1 encodes a plant-specific transcription factor, which affects glume cell proliferation and cell expansion by regulating grain shape genes GS5, GW8 and GW7/GL7, thus determining rice grain length and width.

]]> 【Objective】 Starch degradation is involved in lodicule absorbing abundant water and swelling during rice floret opening, but the amylase genes associated with this process have not been identified yet. 【Method】 To identify the swelling of tissues during floret opening, the in vitro rice panicles absorbed diluted Fuchsin basic and the dye remains were observed after florets were closing again. The starch grain distribution in rice florets before and during anthesis from stage 11 to stage 14 (according to 14 stages of rice anther development) was detected using iodide staining. The spatial-temporal expression patterns of 10 α-amylase genes were detected by RT-PCR, RT-qPCR and GUS staining. 【Result】 Before floret opening, the stamens, pistils and lodicules are enclosed by the lemma and palea through marginal tissues of palea (mtp). Rapid swelling of the lodicules causes floret opening by separating the lemma from the palea. After the in vitro panicles absorbed diluted Fuchsin basic during floret opening, the dye remains were observed located in the joint between mtp and lodicules and filaments. Iodide staining showed that the starch grains were mainly located in the stamens and mtp and a small amount of starch grains in the lodicules at stage 12 (before floret opening), whereas the starch grains in the mtp and lodicules were almost completely degraded at stage 13-14 (during floret opening). RT-PCR showed that OsRAmy2A and OsRAmy3D began to express from stage 12 and were expressed with high levels at stage 13-14. The expression levels of the two genes decreased at DAP1 (1 day after pollination). OsRAmy3E and OsRAmy3F kept expressed during this process. The expression level of OsRAmy3E was higher than that of OsRAmy3F. The RT-qPCR analysis showed that the expression level of OsRAmy2A increased most dramatically at stage 13-14, followed by OsRAMy3A and OsRAMy3E. Further, the transgenic plants expressing the GUS reporter gene driven by the OsRAmy2A promoter were generated. The GUS signaling was located only in the lemma, palea and mtp at stage 12 and the expression of the GUS gene driven by the RAmy2A promoter was induced in the mtp, lodicules and filaments at stage 13-14. 【Conclusion】 These data indicated that starch grain degradation in the mtp and lodicules at stage 13-14 might be related with high expression levels of some α-amylase genes such as OsRAmy2A and OsRAmy3D, probably involved in controlling lodicules swelling and floret opening in rice.

]]> 【Objective】 In order to provide the theoretical basis for the development of high-yield and high-efficiency maize production technology in this area, the characteristics of dry matter accumulation and distribution and the response of grain yield to the return of green manure to the field combined with nitrogen reduction were analyzed in the oasis irrigation area. 【Method】 The field experiment was carried out in Hexi Oasis Irrigation Area, Gansu Province from 2020 to 2021. The combination of green manure returning to the field and different nitrogen reduction ratios (green manure combined with nitrogen reduction 0%, N100; green manure combined with nitrogen reduction 10%, N90; green manure combined with nitrogen reduction 20%, N80; green manure combined with nitrogen reduction 30%, N70; green manure combined with nitrogen reduction 40%, N60) on the distribution of dry matter accumulation in maize and impact on production. 【Result】 After the jointing stage, the aboveground dry matter accumulation under N80 and N90 treatments was significantly higher than that of N70 and N60 treatments. At the mature stage, the aboveground dry matter accumulation under N80 increased by 13.3%-23.2% compared with N70 and N60 treatments, and N90 was higher than N70 and N60 treatments. Compared with the N70 and N60 treatments, the maximum growth rate and average growth rate of the aboveground dry matter under N80 were significantly increased by 9.5%-21.2% and 13.0%-23.2%, respectively; N90 significantly increased by 10.2%-21.8% and 13.9%-23.7% compared with N70 and N60 treatments, both of which effectively delayed the decrease of aboveground dry matter accumulation rate of maize from silking stage to grain filling stage. Compared with N70 and N60, the maximum growth rate of dry matter under N80 treatment was 2.44 d and 2.77 d earlier than that under N70 and N60, respectively, and N90 was 1.92 d and 2.3 d earlier than the N70 and N60 treatments, respectively. N80 and N90 promoted the distribution of dry matter in the ears of maize, and effectively increased the contribution rate of dry matter transport before flowering to grain dry matter accumulation. At the same time, the post-flowering dry matter accumulation under N80 increased by 12.2% and 20.4% compared with N70 and N60 treatments, respectively. Compared with the N70 and N60 treatments, the post-flowering dry matter accumulation under N90 was increased by 12.4% and 20.5%, respectively, and the difference was significant. There was no significant difference in maize grain yield among N100, N90, and N80 treatments, but the maize grain yield under N80 increased by 16.8% and 27.4%, respectively. Compared with N70 and N60 treatments, the yield under N90 treatment increased by 17.4% and 27.9%, respectively, with significant differences. 【Conclusion】 The return of leguminous green manure to the field combined with 10% and 20% nitrogen reduction treatments increased the aboveground dry matter accumulation and accumulation rate of maize, promoted the distribution of dry matter in the ears at maturity, and improved the pre-flowering dry matter transport on grain dry matter. The cumulative contribution rate could be used as the recommended nitrogen application method for high maize yield in oasis irrigated areas.

]]> 【Objective】 The purpose of this study was to investigate the effects of exogenous tryptophan on senescence of sorghum (Sorghum bicolor L.) seedling leaves under low nitrogen stress, and to explore the relationship between C/N balance and senescence of sorghum seedlings leaves, so as to provide effective regulation means for sorghum resistance to low nitrogen stress. 【Method】 In a hydroponic culture experiment, the low nitrogen tolerance sorghum line (398B) and the low nitrogen sensitive sorghum line (CS3541) were selected as the experimental materials. Two nitrogen levels were set at normal nitrogen (5 mmol·L^-1) and low nitrogen (0.5 mmol·L^-1), and 50 mg·L^-1 exogenous tryptophan was applicated by spraying. After 10 days application, leaf morphology, tissue structure, photosynthetic activity, chlorophyll fluorescence parameters, content of carbon and nitrogen metabolism-related substances and enzyme activities, C/N and senescence related gene expression levels were determined, and the correlation between C/N and senescence genes in sorghum seedlings under low nitrogen stress was analyzed.【Result】 (1) Compared with the normal nitrogen treatment, low nitrogen stress significantly reduced the leaf area of 398B and CS3541, while exogenous tryptophan significantly increased the leaf area of 398B and CS3541 by 36.72% and 52.06%. Meanwhile, leaf dry weight and leaf fresh weight of 398B and CS3541 were significantly increased by exogenous tryptophan under low nitrogen stress. (2) Compared with the normal nitrogen treatment, the rosette structure of 398B was relatively complete under low nitrogen stress, while exogenous tryptophan kept the leaf cells orderly and the rosette structure clear. In addition, exogenous tryptophan significantly increased the chlorophyll content of 398B leaves (36.85%), but did not significantly increase the pigment content of CS3541 leaves under low nitrogen stress. (3) Under low nitrogen stress, the exogenous tryptophan treatment resulted in higher PSII maximum photochemical efficiency (F_v/F_m) and non-photochemical quenching (NPQ) capacity, increased leaf photosynthetic rate, and maintained stronger photosynthetic capacity than that without tryptophan. (4) The treatment with exogenous tryptophan reduced the accumulation of sugar (soluble sugar, sucrose and starch) in leaves, but significantly increased the nitrogen content in leaves, correspondingly increased the carbon and nitrogen metabolism enzymes activities, and decreased the C/N in leaves. (5) Exogenous tryptophan positively regulated the expressions of senescence related genes SbLHCB and SBSGR-2, and negatively regulated the expressions of SbNAC6, SbPaO3, SbPPDK-2 and SbSAG12-2 under low nitrogen conditions. In addition, C/N was positively correlated with the expression of SbLHCB and SbSGR-2, and negatively correlated with the expression of SbNAC6, SbPaO3, SbPPDK-2 and SbSAG12-2.【Conclusion】 Under low nitrogen stress, exogenous tryptophan affected leaf morphology and photosynthetic characteristics by reducing C/N and senescence gene expression, and delayed leaf senescence by regulating leaf carbon and nitrogen metabolism, thus enhancing the tolerance of sorghum seedlings under low nitrogen stress. Tryptophan application would be a strategy to weaken low nitrogen stress in the future sustainable agricultural production.

]]> 【Objective】 To explore the recognition mechanisms of BminOBP6 and its ligands, a C terminus-truncated BminOBP6 (TOBP6) was constructed in this study. Subsequently, the binding affinities of TOBP6 and the wild type protein BminOBP6 to 1-undecanol and (+)-limonene under different pH conditions were determined.【Method】 3D structure of BminOBP6 was built by homology modeling through the online software SWISS MODEL. Based on the established BminOBP6 3D model, the C terminus sequence after sixth α-helix of BminOBP6 was determined. This C terminus sequence is the sequence that will be removed from BminOBP6 in the subsequent study. Specific primers were designed to amplify the encoding sequence of BminOBP6 C terminus- truncated mutant TOBP6. Then the recombinant expression vector pET-32a/TOBP6 was constructed. Recombinant expression vectors pET-32a/TOBP6 and pET32a/BminOBP6 that has been made before in our lab were transformed into Escherichia coli cells BL21 (DE3) to express the TOBP6 and BminOBP6 recombinant proteins, respectively. Fluorescence competitive binding assays were conducted to determine the binding affinities of TOBP6 and BminOBP6 to 1-undecanol and (+)-limonene under different pH (7.4 and 5.0) conditions.【Result】 Amino acid sequence alignments revealed that BminOBP6 shared 62.6% identity with Culex quinquefasciatus CquiOBP1, and thus the 3D structure of CquiOBP1 was used as the template in the homology modeling of BminOBP6. The 3D structure of BminOBP6 indicated that the C terminus sequence after α6 of BminOBP6 is a sequence of 8 amino acid residues (D117-P124). Based on this result, the encoding sequence of TOBP6 was cloned and its recombinant expression vector pET-32a/TOBP6 was built. Recombinant expression vectors pET-32a/TOBP6 and pET32a/BminOBP6 were then transformed into E. coli cells BL21 (DE3) to express the TOBP6 and BminOBP6 recombinant proteins, respectively. The fluorescence competitive binding results showed that the binding affinity of BminOBP6 to 1-undecanol and (+)-limonene was quite strong at pH 7.4, the K_i values were 6.89 and 9.50 μmol·L^-1, respectively. However, when the pH decreased to 5.0, BminOBP6 completely lost its binding capacity to 1-undecanol and exhibited an obvious decrease in binding to (+)-limonene (the K_i value increased from 9.50 μmol·L^-1 to 31.26 μmol·L^-1). The binding affinity of TOBP6 to 1-undecanol and (+)-limonene was completely abolished regardless of pH conditions.【Conclusion】 Changes in pH have a significant effect on the ligand binding and release of BminOBP6, and the C terminus of BminOBP6 is crucial for BminOBP6 to bind its ligands.

]]> 【Objective】 The objective of this study is to clarify the differentially expressed cytochrome P450 genes and phylogenetic relationships in response to quercetin in Hyphantria cunea, and to explore the dosage effect of cytochrome P450 genes in response to quercetin, and the role of cytochrome P450 genes in detoxification adaptation to quercetin in H. cunea.【Method】 Based on the transcriptome data of quercetin-induced midgut of H. cunea, the significantly differentially expressed cytochrome P450 genes were screened. MEGA X software was used to analyze the phylogenetic relationships of the target genes with cytochrome P450 genes of other related species. RT-qPCR was used to detect the expression of the five cytochrome P450 genes in different quercetin concentrations (0.5%, 1.0%, 2.0% and 4.0%). The full-length cDNA sequence of CYP6ZB2 was cloned, and the effect of CYP6ZB2 gene-silenced on the body weight and survival rate of H. cunea was investigated by RNA interference (RNAi) technique.【Result】 Five cytochrome P450 genes were identified from the differentially expressed genes in the transcriptome, and were named CYP4G296, CYP4G297, CYP333A30, CYP304F33 and CYP6ZB2 by Standardized Cytochrome P450 Nomenclature Committee (GenBank accession numbers: ON032363-ON032367). Phylogenetic tree analysis showed that these five genes were divided into four clades (CYP2, CYP3, CYP4 and the mitochondrial CYP clade). RT-qPCR results showed that cytochrome P450 genes had a significant dosage effect in response to quercetin, and the expressions of CYP4G297, CYP304F33, CYP333A30 and CYP4G296 were inhibited by different concentrations of quercetin. The expression of CYP6ZB2 was significantly increased at 0.5%-2.0% concentration of quercetin, but it was significantly inhibited at 4.0% quercetin. The full-length cDNA sequence of CYP6ZB2 was cloned. CYP6ZB2 had a complete open reading frame (ORF) encoding 500 amino acids. RNAi results showed that the expression level of CYP6ZB2 was significantly inhibited by injection of two doses (500 and 1 000 ng) dsCYP6ZB2, and the maximum reduction in CYP6ZB2 expression level was 96.9% after injection of 1 000 ng dsCYP6ZB2 for 48 h. The weight increase of CYP6ZB2 gene-silenced H. cunea larvae fed on 0.5% quercetin was lower than that of the control group. Furthermore, the survival rate (53.33%) was 61.5% of that of the untreated group and 72.7% of that of the ddH₂O group (P<0.05).【Conclusion】 Five cytochrome P450 genes were identified in the transcriptome data of quercetin-induced midgut of H. cunea, and quercetin had a significant dosage effect on the five genes, indicating that the response patterns of different cytochrome P450 genes to quercetin were different. RNAi can significantly inhibit the expression level of CYP6ZB2, and quercetin has a significant negative impact on the growth of H. cunea, suggesting that CYP6ZB2 plays an important role in quercetin detoxification in H. cunea.

]]> 【Objective】 The effects of long-term fertilization on the distribution of organic carbon (OC) and total nitrogen (TN_OIC) of organo-mineral complexes in red paddy soil were studied, so as to provide a basis for long-term management and cultivation of soil fertility in red paddy soil. 【Method】 The red paddy soils in long-term fertilization experiment were studied (Since 1984), which included four treatments: no fertilization (CK), inorganic P and K fertilizers (PK), inorganic N, P and K fertilizers (NPK), and NPK plus manure (NPKM, 70%NPK plus 30% manure). Soil samples of 0-20 cm soil layer were collected, and the distribution of organo-mineral complexes at each particle level (<2 μm, 2-10 μm, 10-20 μm, 20-50 μm, and 50-250 μm) was analyzed. The effects of fertilization on the content, storage of organic carbon and total nitrogen as well as the carbon-nitrogen ratio (C/N) were discussed. The effects of OC and TN_OIC contents in organo-mineral complex on contribution rate of SOC and TN contents in red paddy soil were investigated too.【Result】 Compared with the CK treatment, the fertilization treatment significantly increased the particle size ratio of 20-50 μm and decreased the particle size ratio of <10 μm. Compared with other fertilization treatments, NPKM treatment increased the proportion of 50-250 μm grain size complex more significantly. Different fertilization treatments had different effects on the content of OC and TN_OIC in grain size. Compared with PK, the content of OC and TN_OIC in grain size from 50 μm to 250 μm under NPK treatment increased 36.3% and 80.6%, respectively. Compared with NPK, the content of OC and TN_OIC in 50-250 μm granular complex increased by 35.4% and 19.5% under NPKM treatment, respectively. The OC and TN_OIC storage of the organic and inorganic complexes were mainly distributed at the 10-20 μm particle level. And fertilization significantly reduced the storage of <2 μm particle-level complex OC and TN_OIC, but increased the storage of OC and TN_OIC at 20-50 μm particle-level. Compared with PK treatment, the OC storages of <2 μm and 50-250 μm particle-level complexes treated by NPK increased by 18.5% and 31.2%, respectively, and the storages of TN_OIC increased by 18.8% and 73.7%, respectively. Compared with NPK, the NPKM treatment reduced the OC and TN_OIC storages of the <2 μm particle-level complex by 25.6% and 27.4%, respectively, while OC and TN_OIC storages of 50-250 μm particle-level complex increased by 56.3% and 38.6%, respectively. Fertilization significantly influenced the C/N ratio of 50-250 μm particles. Compared with PK, the C/N ratio of 50-250 μm fraction treated with NPK decreased by 24.6%; compared with NPK, the C/N ratio of 50-250 μm fraction treated with NPKM was increased by 13.4%. Fertilization significantly affected the contribution rate of organo-mineral complexes of each particle size to SOC and TN content. Compared with PK, the contribution rate of NPK treatment 50-250 μm particle-level complex to SOC and TN increased by 17.4% and 47.4%, respectively. Compared with NPK, the contribution rate of NPKM treatment 50-250 μm particle to SOC and TN were reduced by 39.5% and 32.8%, respectively.【Conclusion】 In red paddy soil, the long-term fertilization promoted the formation of large-grain organo-mineral complexes. The nitrogen input in the chemical fertilizers significantly increased the grain-level organic carbon, total nitrogen content and storage of the granular grade of 50-250 μm. The organo-mineral combined application was conducive to increasing the soil organic carbon content and the proportion of large-grain organo-mineral complexes, which was helping to preserve the inorganic nitrogen fertilizer application. Therefore, organic and inorganic compound application was an effective measure for long-term fertilization management of red paddy soils.

]]> 【Objective】 The aim of this study was to ascertain the effects of straw returning quantity on the soil physical characteristics and to establish a recycling model for planting by-products, so as to provide a scientific basis for the utilization of straw resources in the central hilly area of Sichuan basin.【Method】 Herein, based on long-term field trials (2006-present) using a combination of in situ monitoring and computed tomography microscanning (CT), the effects of different amounts of straw returned to the field (0 straw returned (RMW₀), 30% straw returned (RMW₃₀), 50% straw returned (RMW₅₀), and 100% straw returned (RMW₁₀₀)) on the physical characteristics at the cultivated soil layer of the winter wheat-summer maize rotation system were examined.【Result】 (1) Straw returned to the field could significantly improve soil permeability, water holding capacity and hydraulic conductivity, and the improving effect increased significantly with the amount of straw returned to the field. Compared with RMW₀, soil bulk density under RMW₃₀, RMW₅₀, and RMW₁₀₀ reduced significantly by 15.2%, 11.7%, and 17.9%, respectively; whereas, soil porosity under these treatments were significantly increased by 18.4%, 13.7%, and 21.3%, respectively. In addition, the saturated hydraulic conductivity of RMW₁₀₀ treatment was as high as 1.62 mm·min^-1, and the soil hydraulic conductivity was superior to other treatments. (2) Straw returning promoted the development of existing pores into larger ones and significantly improved pore uniformity and connectivity. The RMW₁₀₀ and RMW₅₀ treatments improved the macropore composition of the soil better than that under the RMW₃₀ and RMW₀ treatments. The average pore diameter of the RMW₁₀₀ treatment tended to be larger and inter-pore connectivity was optimal. The homogeneity of the pores under the RMW₅₀ treatment was significantly improved and the pore size distribution was more appropriate than that under other treatments. (3) Compared with RMW₀ treatment, the number of >2 mm agglomerates increased significantly and the number of 0.25-2 mm agglomerates decreased significantly after straw returned to the field, which was beneficial to the formation of large soil water-stable agglomerates and promoted the transformation of medium to large agglomerates. Both RMW₅₀ and RMW₁₀₀ treatments improved significantly better than that under RMW30 treatment. (4) Principal components analysis showed soil bulk density, water-stable aggregate with diameter larger than 0.25 mm and large pore were the main indicators of the physical characteristics of cultivated soils in calcareous purple soils. The first and second principal components explained 57.8% and 23.6% of the physical properties of the soil, respectively. The physical characteristics under RMW₅₀ and RMW₁₀₀ treatments were close to each other, and showed significant divergence from the RMW₀ and RMW₃₀ treatments on the PC1 and PC2 axes. 【Conclusion】 On the basis of no significant difference of crop yield in the central hilly area of Sichuan basin, there were differences in the effects of different straw returning quantities on the physical properties of cultivated soil layer, with no significant differences between 50% and 100% straw returning effects, but significantly better than that of 30% and 0 of straw incorporation. The specific straw application rate should be selected according to the local conditions.

]]> 【Objective】 Grape seeds have high bioactivity because they are rich in many metabolites. The objective of this study was to comprehensively identify the metabolite components in grape seeds, to compare and to analyze the differences of metabolites among different varieties, and to explore the relationship between metabolites in grape seeds and skin color and variety origin, so as to provide a reference basis for further development and utilization of grape seeds. 【Method】 The mature seeds of purple-skinned Kyoho (V. labruscana: V. labrusca × V. vinifera, JFS), pink-skinned Wink (V. vinifera, WKS), and yellow-skinned Italia (V. vinifera, YDS) were used for widely targeted metabolomics analysis by UPLC-MS/MS. The metabolites were identified and compared by multivariate statistical methods. 【Result】 The quality of metabolomics data was good, and the data of samples within groups was repetitive and the differences in the data of samples among groups were existed. A total of 514 metabolites were identified in the seeds of three grape varieties, including 6 primary metabolites, such as amino acids and lipids, and 20 secondary metabolites, such as proanthocyanidins and resveratrol. Among different varieties, the metabolite components were similar but the metabolite contents were significantly different. The relative contents of most metabolites were high in the dark variety Kyoho, followed by the light variety Wink, but low in the colorless variety Italy, indicating that the metabolite contents in grape seeds might be positively correlated with the skin color. The relative contents of metabolites in the seeds of Wink and Italia were similar, while they were greatly different from those of Kyoho, indicating that the metabolite contents in grape seeds might be related to the variety origin. The differential metabolites among different varieties were mainly involved in phenylpropane biosynthesis, anthocyanin synthesis, lipid metabolism, etc. pathway. There were many phenolic compounds in the differential metabolites and the metabolites with the large difference were mainly flavonoids. Grape seeds were rich in phenols and lipids. In addition to monomeric flavane-3-ols and their polymers, the relative contents of other phenolic compounds such as flavones and flavonols were also high. There was no significant difference in the relative contents of resveratrol among the three varieties. The relative contents of glyceryl phosphatide such as lysophosphatidylcholine were high, while those of linolenic acid were low. There was little difference in the relative contents of lipids among different varieties. 【Conclusion】 The metabolite components in grape seeds of different varieties were similar, while the metabolite contents were related to the skin color and the variety origin. Phenols and lipids were important components of metabolites in grape seeds and could be used as good sources for food and other processing industries.

]]> 【Objective】 Here, two ecotypes of P. betulaefolia from Huaguo Mountain, Lianyungang (the salt-tolerant ecotype, D) and Purple Mountain, Nanjing (the common ecotype, U) were collected for this research. The purpose of this study was to analyze the role of transcription factor genes in the roots of two ecotypes of P. betulaefolia differing in terms of salt stress. Transcription factors involving in the regulation of the salt tolerance of different P. betulaefolia ecotypes were identified on the grounds of differential expression under salt stress and the relationship between the methylation status and the relative expression level of relevant tolerance genes after exposure to salt stress was investigated. 【Method】 The 90-day-old P. betulaefolia seedlings were grown hydroponically in Hoagland’s nutrient solution supplemented with 200 mmol∙L^-1 NaCl, with seedlings grown in Hoagland’s nutrient solution as the control. The sodium ion content in the tissues was determined by flame graphite furnace atomic absorption spectrometry. Whole-genome DNA methylation analysis and transcriptome sequencing were performed on three replicates for the following four root samples: ecotype D and ecotype U, each grown in the presence or absence of salt stress. Bioinformatics analysis of transcription factor gene expression under salt stress at the levels of transcriptional regulation and epigenetic methylation were carried out using transcriptome sequencing data and whole-genome DNA methylation results, respectively. Then, McrBC-PCR and real-time fluorescence quantitative PCR (qPCR) were used to confirm the levels of methylation and transcription of differential transcription factor genes. 【Result】 After exogenous NaCl treatment for 24 h, the concentration of sodium ions in P. betulaefolia roots increased significantly, with the increase in sodium ion concentration in the salt-tolerant ecotype being significantly less than that in the common ecotype. In the whole seedling, the final salt concentration of tolerant ecotype was only 73.1% of that of the common ecotype. Whereas, in the roots, the sodium content of the salt-tolerant ecotype was 1.1 times of that in the common ecotype. These results indicated that the salt-tolerant ecotype could store more sodium ions in roots and limit their upward transport after salt stress. A total of 2 682 transcription factor (TF) genes from 69 gene families were detected in roots. Among them, 243 TF genes displayed differential expression in response to salt stress, including 37 AP2/ERF, 19 bHLH, 7 bZIP, 10 HD-Zip, 30 MYB, 18 NAC, 8 WRKY, and 23 ZFP family genes. The global methylation level of transcription factor genes in the genome of the salt-tolerant rootstock ecotype decreased, whereas the overall methylation level of these genes in the common ecotype increased after exposure to 200 mmol∙L^-1 NaCl. The differentially methylated regions in both ecotypes were mainly in the position of gene promoters, with the type of differentially methylated sequences being mostly mCHH, constituting more than 93% of the sum of all three types of methylated sequences. The expression levels of twenty-three transcription factor genes, which belonged to the AP2/ERF, bHLH, DREB, GRAS, GT factor, HB Zip, MYB, NAC, Trihelix, and zinc-finger ZFP gene families, were upregulated, and their methylation levels were downregulated in both two ecotypes in response to salt stress. These genes may be involved in the regulation of sodium uptake and accumulation in roots under salt stress. The expression patterns and promoter methylation of representative candidate genes identified by bioinformatics analysis were confirmed by qPCR and McrBC-qPCR.【Conclusion】 The differentially expressed genes in roots of P. betulaefolia under salt stress included 243 transcription factor genes in both ecotypes. The methylation changes in DNA sequences in eight transcription factor genes (PbERF2, PbGT3, PbZAT10.1, PbSCL33, PbDREB1, PbZAT10.2, PbERF53, and PbNAC72) were correlated with their transcriptional activity. Our results provided preliminary experimental evidence for supporting a relationship between promoter DNA methylation and expression of TF genes in P. betulaefolia in response to salt stress as part of the molecular role of TFs involved in the regulation of salt tolerance among different P. betulaefolia ecotypes, which would increase our understanding of the role of epigenetics in the response of woody trees to abiotic stress.

]]> 【Objective】 The objective of this study was to study the effects of ambient temperature on feeding and intestinal development of poultry, and to supplement the absent temperature parameters for laying hens rearing, so as to provide a certain scientific basis for the correct feeding of laying hens.【Method】 A total of 360 Issa brown laying hens aged 11 weeks were selected and divided into 5 treatment groups with 6 replicates per group and 12 hens per replicate. The experimental laying hens were transferred to 5 intelligent environmental control chicken houses for 1 week of pre-trial and 8 weeks of formal experiment, with 3 chickens per cage. The relative humidity in the chicken house was kept at 60%, and the light was kept for 8 h (9:00-17:00) every day during the prelay period. The temperature of the control group was kept unchanged at 22℃, and the four treatment groups were carried out in a manner of daily intermittent, including 24℃, 26℃, 28℃, and 30℃ at 10:00-18:00 every day, respectively, and changed to the base temperature 22℃ for the rest of the time, the heating and cooling time were within 1 h. The experiment lasted for 8 weeks. The experimental laying hens were free to eat and drink, the feed intake was counted weekly, and the samples were collected once every two weeks. Twelve hens in each group were randomly selected and weighed, and then killed by neck cutting. The weight of glandular stomach, the weight and length of duodenum, jejunum and ileum were weighed. Hypothalamus, glandular stomach and duodenum samples were frozen in liquid nitrogen and stored at -80℃. Feed intake was calculated in heat treatment period and non-heat treatment period for 3 days before the end of experiment.【Result】 Compared with T22 group, the feed intake in T30 group was significantly decreased at 13-16 week (P<0.05); the feed intake in T24 group was significantly higher than that in T28 and T30 groups at 17-20 week (P<0.05). During the heat treatment period, the feed intake of T30 group was significantly lower than that in T22 and T24 groups (P<0.05). The feed intake of T30 group was significantly lower than that in T22, T24 and T26 groups during the non-heat treatment period (P<0.05). The feed intake in the heat treatment period was significantly lower than that in the non-heat treatment period (P<0.05), and the highest feed intake was maintained in T24 group and the lowest in T30 group. Compared with T22 group, the glandular gastric index was significantly increased at 16 week (P<0.01). At 18 week, compared with T22 group, the jejunum index in T30 group was significantly lower than that in other groups (P<0.05). At 20 week, compared with T22 group, the glandular stomach index, jejunum index and ileum index in T24 group were significantly increased (P<0.05), and the jejunum index was significantly higher than that in T30 group (P<0.05). Compared with T22 group, the expression of NPY (Neuropeptide Y) in hypothalamus of all treatment groups was significantly increased at 14 week (P<0.05). The expression of AgRP (Agouti-related protein) in hypothalamus of T30 group was significantly decreased (P<0.05), and the expression of CCK (Cholecystokinin) in duodenum of T30 group was significantly increased (P<0.05). At 20 week, compared with T22 group, the expression of CART (amphetamine-regulated transcript) in hypothalamus of T24 group was significantly decreased (P<0.05), and the expression of ghrelin in glandular stomach of T24 group was significantly decreased (P<0.05).【Conclusion】 These results indicated that ambient temperature at 24℃ during the growing period could promote the development of gastrointestinal tract, increase the expression of NPY in hypothalamus, and inhibit the expression of CART and ghrelin in hypothalamus, which was beneficial to the growth and development of laying hens. However, the high temperature treatment at 30℃ damaged the intestinal tract of laying hens, inhibited the expression of hypothalamus feeding promoting factor AgRP, and promoted the expression of duodenal feeding inhibiting factor CCK, thus inhibiting feeding intake and reducing feed intake of laying hens.

]]> 【Background】 African swine fever (ASF) is an acute, highly contagious, and deadly infectious disease of pigs caused by ASF virus (ASFV), which is currently considered the biggest killer in global swine industry. To date, there is no effective vaccines or antiviral drugs for the prevention or treatment of ASF. As obligate intracellular parasites, the viruses are fully reliant on the host translation machinery to produce the polypeptides that are essential for viral replication. A central mechanism regulating translation initiation involves phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2α), which directs host translational control and adaptation to cellular stress. The regulation of eIF2α phosphorylation has been regarded as a critical step for viral infection, with important effects on virulence, tissue tropism, pathogenicity, and immunoevasion. However, the molecular mechanisms by which most of the ASFV-encoded proteins affecting eIF2α phosphorylation have not been well studied. 【Objective】 The aim of this study was to explore the mechanism of ASFV MGF110-5L-6L protein on the host cell translation block and promote the formation of stress particles, so as to provide a scientific basis for further revealing the pathogenic mechanism of African swine fever virus.【Method】 The preliminary screening by luciferase reporter assays identified that ectopic expression of ASFV MGF110-5L-6L, a previously uncharacterized member of the multigene family 110, significantly increased eIF2α phosphorylation levels. To confirm and clarify the potential role of MGF110-5L-6L expression in mediating eIF2α phosphorylation and downstream of translation control, two continuous porcine cell lines, including 3D4/21 (porcine alveolar macrophage) and PK-15 (porcine kidney), were used for the plasmid transfection and/or drug treatment and subjected to immunoblotting or confocal immunofluorescence analysis. To investigate how the ectopic expression of MGF110-5L-6L triggers cellular stress, the structure, subcellular localization and function of the MGF110-5L-6L protein were further characterized by a combination of bioinformatic prediction, immunofluorescence and immunoblotting analysis.【Result】 Here, it was confirmed that ectopic expression of MGF110-5L-6L remarkably promoted eIF2α phosphorylation and the expression of ATF4, indicating that it functions in the integrated stress response. The subsequent analyses revealed that MGF110-5L-6L expression could trigger the ER stress and activate the unfolded protein response, and the phosphorylation of eIF2α was mediated via PERK and PKR, resulting in the suppression of host translation and stress granule formation. It was further observed that MGF110-5L-6L protein had two highly conserved central cysteine-rich domains and was mainly retained in the endoplasmic reticulum (ER), and also caused a significant reorganization of the subcellular distribution and morphological characteristics of the Golgi and peroxisome, suggesting that it might interfere with ER redox homeostasis, secretory pathway, and other membrane-bound organelles to trigger cellular stress.【Conclusion】 Together, these results demonstrated a previously uncharacterized role of ASFV MGF110-5L-6L and further defined several molecular interfaces by which ASFV MGF110-5L-6L hijacks the host cell translation, which expanded the view of ASFV in determining the fate of host-pathogen interactions.

]]> 【Background】 Meridian theory is the cornerstone of Traditional Chinese Medicine (TCM), and acupoints are the key sites on the meridian, which are the implementation location of the acupuncture. However, there are different opinions on the structural basis and morphological composition of acupoints, which cannot be scientifically clarified. As a newly found type of interstitial cells, Telocytes (TCs) were suggested to be the potential essence cells of the meridian by morphological study recently, but the characteristics and distribution of TCs at acupoints need to be further elucidated. 【Objective】 This study aimed to analyze the structural differences between acupoints and non-acupoints, and to explore the morphological characteristics of TCs at acupoints. The structural relations between TCs and its surrounding components were also analyzed, so as to provide theoretical support for the study of the cellular mechanism of TCM acupuncture treatment. 【Method】 The skin tissues of Baihui (Du₂₀), Quchi (LI₁₁), Sanyinjiao (Sp₆), Danzhong (Ren₁₇), Chengjiang (Ren₂₄), Erjian (EP₄) and non-acupoints on the back and abdomen were collected from five adult healthy Hu sheep. TCs and Tps (telopodes) were stained by specific markers CD34 and Vimentin, the mast cells were labeled by TPS, and nerves were identified by PGP9.5. Extracellular vesicles were marked by TSG101. The structure and fine composition of skin acupoints and non-acupoints were analyzed by H.E and immunohistochemical techniques (IHC), and the morphological quantitative analysis of the data was carried out by using ImageJ and Image-Pro Plus statistical software. The distribution differences of TCs and its related structures at acupoints and non-acupoints were analyzed. On this basis, the morphological characteristics and stereoscopic structure of TCs were observed by transmission electron microscope (TEM), scanning electron microscope (SEM) and immunofluorescence (IF) double labeling technique, and the morphological relationship between TCs and these structures was further analyzed, thus determining the ultramorphology and material basis of acupoints. 【Result】 There were such structures as hair follicles, sebaceous glands, sweat glands and arrector pili muscle, as well as nerves, blood vessels, mast cells, collagen fiber bundles at the acupoints and non-acupoints. However, the number of nerves, blood vessels and mast cells distribution at acupoints was significantly more than that at non-acupoints (P<0.05). More importantly, TCs with slender tubular processes (telopodes, Tps) were distributed in the skins, and the distribution of TCs at the acupoints was significantly different from that of non-acupoints (P<0.05). TCs could be used as the integrator of acupoints stroma. There were extensive relationships between TCs themselves or TCs and surrounding morphological structures (including gap junctions and extracellular vesicles, etc.), which could develop a structural network system. At the ultramicro level, it was observed that the Tps was a typical beaded appearance, which was composed of alternating arrangement of the inflated part (podom, Pd) and the slender stenotic part (podomer, P). The well-developed mitochondria in the cytoplasm of podom, the cellular connection between Tps and Tps, and a large number of extracellular vesicles on or around TCs (significantly more than non-acupoints (P<0.05)) ensured the core role of TCs in the structure of acupoints. Moreover, the structural connection between TCs and epidermal derivatives also verified the relationship between acupoints and epidermal derivatives in the classic book Huangdi Neijing (The Yellow Emperor’s Canon of Internal Medicine) at the cellular level. 【Conclusion】 Structural compositions at the acupoints and non-acupoints were basically the same, while the number of TCs and Tps, nerves, blood vessels, mast cells, extracellular vesicles at acupoints was significantly more than that at non-acupoints; TCs and Tps had the functional structures of connecting and integrating various morphological components, which might be mediators or integrators of different systems at the acupoints. The cell connections among TCs, developed mitochondria and extracellular vesicles had the structural basis for cell communication and energy generation, which corresponds to the “Qi-Xue” in TCM.

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