【Objective】 Rice blast is one of the most devastating diseases of rice production. A broad-spectrum disease resistance gene Pigm-1 was identified but its functional pathway and interactors are unknown. The screening and identification of key proteins in the Pigm-1 signaling pathway will provide an important theoretical basis for rice disease resistance breeding. 【Method】 In this study, the decoy protein pGBKT7-Pigm-1-CC1-576 vector was constructed to detect the decoy protein self-activation, and the toxicity of the decoy protein was detected by separately transforming the plasmid pGBKT7 and pGBKT7-Pigm-1-CC1-576 into Y2H Gold yeast. The rice disease resistance R protein Pigm-1 was screened by cDNA expression yeast library induced by rice blast fungus. The sequencing results were compared and annotated by Rice Information GateWay (RIGW). The interaction of OsbHLH148 protein was verified by Luc, Co-IP and yeast two-hybrid assays, and the tissue expression of the corresponding gene of the interaction protein OsbHLH148 was analyzed by qRT-PCR. 【Result】 The self-activation test showed that the decoy protein pGBKT7-Pigm-1-CC1-576 did not self-activate when cotransformed with the AD plasmid, and the toxicity analysis showed that the decoy protein had little or no toxicity to yeast cells. A total of 124 proteins that may interact with Pigm-1 were obtained by screening the yeast library, and among these proteins, there are ethylene synthesis related, gibberellin synthesis related, active oxygen species clearly related, enzyme metabolism related, and some function unknown. The interaction between Pigm-1-CC1-576 and OsbHLH148 was verified by Luc, Co-IP and yeast two-hybrid methods. Further analysis showed that OsbHLH148 can be induced by blast fungus infection, and the tissue expression analysis showed that OsbHLH148 expression level was the highest in rice leaves at 6 weeks. 【Conclusion】 In this study, 124 proteins that may interact with Pigm-1 were obtained. One of these proteins, OsbHLH148, was selected and verified to interact with Pigm-1-CC1-576. Suggesting that OsbHLH148 may be involved in Pigm-1 mediated resistance of rice blast.
【Objective】 Rice (Oryza sativa L.) is a staple cereal crop for about half of the global population, with protein being the second-most significant nutritional component in rice grains. The storage proteins in rice grains mostly consist of glutelin, prolamin, globulin, and albumin, among which the content of easy-to-digest glutelin is the highest. Consequently, common rice increases the burden of kidney and accelerates the progression of renal disorders. The method of generating low-glutelin rice germplasm will provide novel genetic material for the cultivation of functional rice cultivars suitable for individuals with kidney diseases. 【Method】 We utilized Suxiu 867 (SX867), an elite japonica rice cultivar appropriate for cultivation in Jiangsu province, as a transgenic recipient to delete a fragment of approximately 3 500 bp between the B subfamily glutelin-coding genes GluB4 and GluB5 using CRISPR/Cas9-mediated gene editing technology. The large fragment deletion was identified by PCR using the primers corresponding to the flanking sequence of gene editing target sites, while sequence-specific primers for Cas9 and hygromycin resistance gene cassettes were used to identify the low-glutelin rice mutant absent of transgenic elements. The protein component contents of homozygous low-glutelin mutants were analyzed qualitatively and quantitatively, and the expression levels of glutelin-coding genes in rice grains were detected by quantitative PCR. The agronomic traits and quality traits of homozygous low-glutelin mutants and recipient cultivar cultivated under the same cultivation conditions were measured. 【Result】 Homozygous mutants with a 3 448 bp deletion between GluB4 and GluB5 genes were generated successfully. In the mutants, the relative proportion of glutelin decreased significantly, while that of prolamin and globulin increased significantly. The glutelin content of homozygous mutants decreased to 45.54%-49.75% compared to recipient cultivar, and the reduction level is comparable to LGC-1, a low-glutelin rice germplasm commonly used as a donor of low-glutelin trait in commercialized rice cultivars. The expression levels of B subfamily glutelin-coding genes in homozygous mutant were decreased significantly, and the changing trends was consistent with that of LGC-1 derived rice cultivar. Except that plant height decreased and grain length increased significantly, other measured agronomic and quality traits of homozygous mutants were not changed significantly compared to recipient cultivar. 【Conclusion】 Using CRISPR/Cas9-mediated gene editing technology, rice mutants with significant lower glutelin content free from transgenic elements were obtained successfully providing a convenient and quick method to generate low-glutelin germplasm.
【Objective】 Common wheat, as an important food crop, plays a crucial role in global food security. Identifying the gene regulatory networks involved in wheat grain protein synthesis and determining key candidate genes will provide theoretical support for quality breeding and improvement of wheat. 【Method】 The study used wheat grains at six developmental stages (5, 10, 15, 20, 25, and 30 days post-anthesis) as research materials to summarize the pattern of protein accumulation in wheat grains. Transcriptome data and grain protein content phenotypic data were analyzed using the WGCNA (Weighted Gene Co-expression Network Analysis) method to construct weighted gene co-expression networks and identify key hub transcription factor (TFs) genes.【Result】 The accumulation of protein content in wheat grains showed a trend of initial decline followed by an increase, reaching its lowest value (12.16%) at 25 days post-anthesis, with significant differences in protein content between adjacent developmental stages. A total of 25 427 differentially expressed genes (DEGs) were identified between adjacent developmental stages. Cluster analysis divided these DEGs into five groups (A-E), with group B containing the highest number of DEGs (10 906). A total of 1 022 transcription factors (TFs) from 49 families were identified, with the NAC family containing the most TFs (107). WGCNA analysis identified five co-expression modules significantly associated with protein content. The turquoise module showed the highest positive correlation with protein content (r=0.80, P=1×10-⁴). By integrating differentially expressed genes and weighted gene co-expression networks, six positively regulated hub TFs from the MIKC-MADS, TCP, TALE, and CPP families were identified in two modules (turquoise and blue). Further correlation analysis between the protein content phenotype of Huaimai 48 and gene expression levels at different time points revealed that the expression levels of five hub TFs were significantly positively correlated with the protein content phenotype. Specifically, TraesCS5B03G0740100 and TraesCS7D03G0590500 showed specific high expression in spike and grain tissues.【Conclusion】 The study identified important modules (turquoise and blue) related to wheat protein content accumulation, screened six hub TFs, and identified that the expression levels of two hub TFs are significantly positively correlated with protein content and are specifically highly expressed in spike and grain tissues. These genes can serve as candidate genes for regulating protein accumulation in wheat grains.
【Objective】 Under the background of global warming, this paper explored the physiological mechanism of anthocyanin content in colored wheat in response to high temperature stress in the middle of grain filling, so as to lay a theoretical basis for further coping with the high-quality cultivation of functional colored wheat varieties under climate warming. 【Method】 The experiment was conducted in Hefei High-tech Agricultural Park in the 2022-2023 and 2023-2024 growing seasons. Six colored wheat varieties with different colors were selected and subjected to high temperature stress treatment (T) for 5 days at the middle stage of filling, with the same materials grown under ambient temperature as the controls (CK). 【Result】 Under high temperature stress after anthesis, the net photosynthetic rate, stomatal conductance, transpiration rate, relative chlorophyll content (SPAD), dry matter partition, soluble sugar content, sucrose synthase activity, anthocyanin content, anthocyanidin synthase activity, chalcone synthase activity and phenylalanine ammonia-lyase activity of colored wheat were significantly reduced, and the yield of six varieties of colored wheat decreased by 9.10% to 16.94%, 1000-grain weight decreased by 7.84% to 16.94%, and anthocyanin content decreased by 7.18% to 14.17%. The yield, photosynthetic intensity, SPAD value, dry matter partition, soluble sugar content, sucrose synthase activity, anthocyanin content, anthocyanidin synthase activity, chalcone synthase activity, and phenylalanine ammonia-lyase activity of different varieties of colored wheat were: Qinbai 1>Qinlü 3>Qinzi 1>Xinchun 36>Qinhe 2>Qinlan 1, and the anthocyanin content was: Qinhei 2>Xinchun 36>Qinzi 1>Qinlü 3>Qinlan 1>Qinbai 1. The yield of heat-resistant wheat varieties of Qinbai 1, Qinlü 3 and Qinzi 1 decreased significantly less than that of heat-sensitive wheat varieties Qinhei 2, Xinchun 36 and Qinlan 1. The decreases in photosynthetic intensity, SPAD value, dry matter fraction, soluble sugar content, sucrose synthase activity, anthocyanin content, anthocyanidin synthase activity, chalcone synthase activity and phenylalanine ammonia-lyase activity of color wheat varieties Qinhei 2, Xinchun 36 and Qinzi 1 with high anthocyanin content were smaller than those of Qinlü 3, Qinlan 1 and Qinbai 1 with low anthocyanin content. Correlation analysis showed that the yield of each color wheat variety was significantly positively correlated with 1000-grain weight, sucrose content, sucrose synthase activity, flag leaf net photosynthetic rate, stomatal conductance, transpiration rate and SPAD value, anthocyanin content was significantly positively correlated with soluble sugar content, and yield was negatively correlated with anthocyanins, but the correlation was not significant. After high temperature stress after anthesis, the decomposition of sucrose bound to free anthocyanins decreased, and the decomposition of anthocyanins in grains increased, which supplemented the growth and development of wheat.【Conclusion】 The antioxidant activity of anthocyanins helped crops resist external stress, and the decline of various indexes of color wheat varieties with higher anthocyanin content was comparable to that of color wheat varieties with lower anthocyanin content under high temperature stress after anthesis, the anthocyanin content was significantly positively correlated with soluble sugar content. In conclusion, the accumulation of anthocyanin content could respond to high temperature stress, reduce the decrease of soluble sugar content, and increase the heat resistance of colored wheat.
