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甜菜磷脂酰肌醇转运蛋白基因SbSEC14的克隆及低温胁迫下的表达分析
Phosphatidylinositol Transporters Gene SbSEC14 C in Sugarbeet: Cloning and Expression Analysis Under Low Temperature Stress
磷脂酰肌醇转运蛋白(PITPs)广泛存在于真核生物细胞中,能够在体外膜脂质双层之间调控磷脂酰肌醇(PtdIns)或者磷脂酰胆碱(PtdCho)单体的独立转运。参与磷酸肌醇代谢、膜运输、极性生长、信号转导、逆境胁迫、胞浆运动和细胞周期调节等多种重要的生命过程,在植物的逆境响应以及发育调节中具有重要的作用。为了研究甜菜磷脂酰肌醇转运蛋白基因及其在低温胁迫下的表达情况。本研究以甜菜基因组数据库中一条预测的磷脂酰肌醇转运蛋白基因CRS1为模板,用基因克隆的方法得到一条全长765 bp,开放阅读框596 bp,编码198个氨基酸的甜菜SEC14基因,命名为SbSEC14。理化性质分析表明,该蛋白质为不稳定亲水蛋白;蛋白质二、三级结构分析表明,该蛋白质α-螺旋所占的比例最高,为47.47%,β-转角所占比例最低,为5.56%;蛋白质保守结构分析表明,该蛋白质有典型的SEC14结构域;蛋白质系统进化树分析表明,甜菜SbSEC14基因与菠菜、藜麦的磷脂酰肌醇运转蛋白基因亲缘关系最近;实时荧光定量结果表明,SbSEC14基因在甜菜中组成型表达,在甜菜叶中的表达量最高,在甜菜根中的表达量最低,在叶中表达量约为根中的2.5倍。甜菜幼苗4℃处理0、2、6、12、24 h,该基因在植株处理0~2 h时表达量呈上升趋势,在植株处理2~6 h时表达量呈下降趋势,在植株处理6~24 h时表达量趋于平稳状态。因此,预测该基因与甜菜抗低温胁迫相关。
Phosphatidylinositol transporters (PITPs) are widely present in eukaryotic cells and can regulate the independent transport of phosphatidylinositol (PtdIns) or phosphatidylcholine (PtdCho) monomers between lipid bilayers in vitro. They are involved in many important life processes such as phosphoinositide metabolism, membrane transport, polar growth, signal transduction, stress, cytoplasmic movement, and cell cycle regulation. They play an important role in plant stress response and development regulation. In order to study the sugarbeet phosphatidylinositol transporter gene and its expression under low temperature stress, in this study, using a predicted phosphatidylinositol transporter gene CRS1 from sugarbeet genome database as a template, a beet SEC14 gene with a length of 765 bp, an open reading frame of 596 bp, and a coding of 198 amino acids was obtained by gene cloning. It was named SbSEC14. The analysis of the physicochemical properties showed that the protein was an unstable hydrophilic protein. The secondary and tertiary structure analysis of the protein showed that the protein's α-helix accounted for the highest proportion, 47.47%, and the β-turn angle accounted for the lowest, 5.56%. The conserved structural analysis of the protein showed that the protein had a typical SEC14 domain. The phylogenetic tree analysis of the protein showed that the beet SbSEC14 gene was most closely related to spinach and quinoa's. Real-time fluorescence quantitative results showed that the constitutive expression of SbSEC14 gene appeared in sugarbeet, the highest expression was in sugarbeet leaves, the lowest expression was in sugarbeet roots, and the expression in leaves was about 2.5 times that of roots. Sugarbeet seedlings were treated at 4℃ for 0, 2, 6, 12, and 24 h, the expression level of this gene showed an upward trend when the plants were treated for 0-2 h, the expression level was decreased when the plants were treated for 2-6 h and the expression level tended to be steady for 6-24 h. Therefore, it is predicted that this gene is related to the resistance of sugarbeet to low temperature stress.
甜菜 / 磷脂酰肌醇运转蛋白 / SEC14 / 低温 / 非生物胁迫 {{custom_keyword}} /
sugarbeet / phosphatidylinositol transporter / SEC14 / low temperature / abiotic stress {{custom_keyword}} /
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There has recently been rapid progress in understanding receptors that generate intracellular signals from inositol lipids. One of these lipids, phosphatidylinositol 4,5-bisphosphate, is hydrolysed to diacylglycerol and inositol trisphosphate as part of a signal transduction mechanism for controlling a variety of cellular processes including secretion, metabolism, phototransduction and cell proliferation. Diacylglycerol operates within the plane of the membrane to activate protein kinase C, whereas inositol trisphosphate is released into the cytoplasm to function as a second messenger for mobilizing intracellular calcium.
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Protein kinase C has a crucial role in signal transduction for a variety of biologically active substances which activate cellular functions and proliferation. When cells are stimulated, protein kinase C is transiently activated by diacylglycerol which is produced in the membrane during the signal-induced turnover of inositol phospholipids. Tumour-promoting phorbol esters, when intercalated into the cell membrane, may substitute for diacylglycerol and permanently activate protein kinase C. The enzyme probably serves as a receptor for the tumour promoters. Further exploration of the roles of this enzyme may provide clues for understanding the mechanism of cell growth and differentiation.
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Diphosphorylated inositol polyphosphates, also referred to as inositol pyrophosphates, are important signaling molecules that regulate critical cellular activities in many eukaryotic organisms, such as membrane trafficking, telomere maintenance, ribosome biogenesis, and apoptosis. In mammals and fungi, two distinct classes of inositol phosphate kinases mediate biosynthesis of inositol pyrophosphates: Kcs1/IP6K- and Vip1/PPIP5K-like proteins. Here, we report that PPIP5K homologs are widely distributed in plants and that Arabidopsis thaliana VIH1 and VIH2 are functional PPIP5K enzymes. We show a specific induction of inositol pyrophosphate InsP8 by jasmonate and demonstrate that steady state and jasmonate-induced pools of InsP8 in Arabidopsis seedlings depend on VIH2. We identify a role of VIH2 in regulating jasmonate perception and plant defenses against herbivorous insects and necrotrophic fungi. In silico docking experiments and radioligand binding-based reconstitution assays show high-affinity binding of inositol pyrophosphates to the F-box protein COI1-JAZ jasmonate coreceptor complex and suggest that coincidence detection of jasmonate and InsP8 by COI1-JAZ is a critical component in jasmonate-regulated defenses.
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We report the crystal structure of the catalytic domain of human ADAR2, an RNA editing enzyme, at 1.7 angstrom resolution. The structure reveals a zinc ion in the active site and suggests how the substrate adenosine is recognized. Unexpectedly, inositol hexakisphosphate (IP6) is buried within the enzyme core, contributing to the protein fold. Although there are no reports that adenosine deaminases that act on RNA (ADARs) require a cofactor, we show that IP6 is required for activity. Amino acids that coordinate IP6 in the crystal structure are conserved in some adenosine deaminases that act on transfer RNA (tRNA) (ADATs), related enzymes that edit tRNA. Indeed, IP6 is also essential for in vivo and in vitro deamination of adenosine 37 of tRNAala by ADAT1.
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Phosphoinositides (PIs) make up only a small fraction of cellular phospholipids, yet they control almost all aspects of a cell's life and death. These lipids gained tremendous research interest as plasma membrane signaling molecules when discovered in the 1970s and 1980s. Research in the last 15 years has added a wide range of biological processes regulated by PIs, turning these lipids into one of the most universal signaling entities in eukaryotic cells. PIs control organelle biology by regulating vesicular trafficking, but they also modulate lipid distribution and metabolism via their close relationship with lipid transfer proteins. PIs regulate ion channels, pumps, and transporters and control both endocytic and exocytic processes. The nuclear phosphoinositides have grown from being an epiphenomenon to a research area of its own. As expected from such pleiotropic regulators, derangements of phosphoinositide metabolism are responsible for a number of human diseases ranging from rare genetic disorders to the most common ones such as cancer, obesity, and diabetes. Moreover, it is increasingly evident that a number of infectious agents hijack the PI regulatory systems of host cells for their intracellular movements, replication, and assembly. As a result, PI converting enzymes began to be noticed by pharmaceutical companies as potential therapeutic targets. This review is an attempt to give an overview of this enormous research field focusing on major developments in diverse areas of basic science linked to cellular physiology and disease.
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Phosphatidylinositol transfer proteins (PITPs) are highly conserved polypeptides that bind phosphatidylinositol or phosphatidylcholine monomers, facilitating their transfer from one membrane compartment to another . Although PITPs have been implicated in a variety of cellular functions, including lipid-mediated signaling and membrane trafficking, the precise biological roles of most PITPs remain to be elucidated . Here we show for the first time that a class I PITP is involved in cytokinesis. We found that giotto (gio), a Drosophila gene that encodes a class I PITP, serves an essential function required for both mitotic and meiotic cytokinesis. Neuroblasts and spermatocytes from gio mutants both assemble regular actomyosin rings. However, these rings fail to constrict to completion, leading to cytokinesis failures. Moreover, gio mutations cause an abnormal accumulation of Golgi-derived vesicles at the equator of spermatocyte telophases, suggesting that Gio is implicated in membrane-vesicle fusion. Consistent with these results, we found that Gio is enriched at the cleavage furrow, the ER, and the spindle envelope. We propose that Gio mediates transfer of lipid monomers from the ER to the equatorial membrane, causing a specific local enrichment in phosphatidylinositol. This change in membrane composition would ultimately facilitate vesicle fusion, allowing membrane addition to the furrow and/or targeted delivery of proteins required for cytokinesis.
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Root hairs are a major site for the uptake of water and nutrients into plants, and they form an increasingly important model system for the study of development in higher plants. We now report on the molecular genetic analysis of the srh1 mutant in Arabidopsis thaliana impaired in root hair tip growth. We show that srh1 is a new allele of cow1 (can of worms1) and we identified the COW1 gene using a positional cloning strategy. The N-terminus of the COW1 protein is 32% identical to an essential phosphatidylinositol transfer protein (PITP), the yeast Sec14 protein (sec14p) while the C-terminus is 34.5% identical to a late nodulin of Lotus japonicus, Nlj16. We show that expression of the COW1 lipid-binding domain complements the growth defect associated with Sec14p dysfunction in yeast. In addition, we show that GFP fused to the COW1 protein specifically accumulates at the site of root hair outgrowth. We conclude that the COW1 protein is a PITP, essential for proper root hair growth.
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苏世超, 唐益苗, 徐磊, 等. 普通小麦TaSEC14p-5基因的克隆及表达分析[J]. 农业生物技术学报, 2016,24(8):1129-1137.
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KEY MESSAGE: A Sec14-like protein, ZmSEC14p , from maize was structurally analyzed and functionally tested. Overexpression of ZmSEC14p in transgenic Arabidopsis conferred tolerance to cold stress. Sec14-like proteins are involved in essential biological processes, such as phospholipid metabolism, signal transduction, membrane trafficking, and stress response. Here, we reported a phosphatidylinositol transfer-associated protein, ZmSEC14p (accession no. KT932998), isolated from a cold-tolerant maize inbred line using the cDNA-AFLP approach and RACE-PCR method. Full-length cDNA that consisted of a single open reading frame (ORF) encoded a putative polypeptide of 295 amino acids. The ZmSEC14p protein was mainly localized in the nucleus, and its transcript was induced by cold, salt stresses, and abscisic acid (ABA) treatment in maize leaves and roots. Overexpression of ZmSEC14p in transgenic Arabidopsis conferred tolerance to cold stress. This tolerance was primarily displayed by the increased germination rate, root length, plant survival rate, accumulation of proline, activities of antioxidant enzymes, and the reduction of oxidative damage by reactive oxygen species (ROS). ZmSEC14p overexpression regulated the expression of phosphoinositide-specific phospholipase C, which cleaves phosphatidylinositol 4,5-bisphosphate (PIP2) and generates second messengers (inositol 1,4,5-trisphosphate and 1,2-diacylglycerol) in the phosphoinositide signal transduction pathways. Moreover, up-regulation of some stress-responsive genes such as CBF3, COR6.6, and RD29B in transgenic plants under cold stress could be a possible mechanism for enhancing cold tolerance. Taken together, this study strongly suggests that ZmSEC14p plays an important role in plant tolerance to cold stress.
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bHLH transcription factors play important roles in the abiotic stress response in plants, but their characteristics and functions in Tartary buckwheat (Fagopyrum tataricum), a traditional coarse cereal with a strong stress tolerance, haven't been sufficiently studied. Here, we found that the expression of a bHLH gene, FtbHLH2, was induced significantly by cold treatments in Tartary buckwheat seedlings. Subcellular localization indicated that FtbHLH2 localized in nucleus. Its overexpression in Arabidopsis increased tolerance to cold. The Arabidopsis plants overexpressing FtbHLH2 displayed higher root length and photosynthetic efficiency, and had lower malondialdehyde (MDA) and reactive oxygen species (ROS) after cold treatment compared to wild type (WT) plants. Meanwhile, the expression levels of some stress-related genes in transgenic plants were remarkably higher than that in wild type under normal and/or stress conditions. Furthermore, transgenic Arabidopsis lines with the FtbHLH2 promoter had higher GUS activity after cold stress. On the whole, the results suggest that FtbHLH2 may play a positive regulatory in cold stress of Tartary buckwheat.
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To elucidate the molecular mechanisms of plant immune responses, we isolated genes whose expression was regulated by inoculation with Ralstonia solanacearum. Here, we report the characterization of Nicotiana benthamiana belonging to the SEC14-gene superfamily designated as Nicotiana benthamiana SEC14 (NbSEC14). NbSEC14 rescued growth defects and impaired invertase secretion associated with the yeast sec14p temperature-sensitive mutant, while recombinant NbSec14 protein had phospholipids transfer activity. NbSEC14 expression was up-regulated in N. benthamiana leaves after inoculation with virulent or avirulent R. solanacearum. Expression of NbSEC14 was induced by treatment with chitin, flg22, and by Agrobacterium-mediated transient expression of INF1 elicitin, AvrA from R. solanacearum, and co-expression of the capsid protein from Tobacco mild green mosaic virus with its cognate resistance L1 protein. NbSEC14-silenced plants showed accelerated growth of both the virulent and avirulent R. solanacearum as well as acceleration of disease development. This study may provide useful information for the further analysis of the function of plant Sec14 protein homologs in the regulation of plant immune responses.
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Rice is cultivated in water-logged paddy lands. Thus, rice root hairs on the epidermal layers are exposed to a different redox status of nitrogen species, organic acids, and metal ions than root hairs growing in drained soil. To identify genes that play an important role in root hair growth, a forward genetics approach was used to screen for short-root-hair mutants. A short-root-hair mutant was identified and isolated by using map-based cloning and sequencing. The mutation arose from a single amino acid substitution of OsSNDP1 (Oryza sativa Sec14-nodulin domain protein), which shows high sequence homology with Arabidopsis COW1/AtSFH1 and encodes a phosphatidylinositol transfer protein (PITP). By performing complementation assays with Atsfh1 mutants, we demonstrated that OsSNDP1 is involved in growth of root hairs. Cryo-scanning electron microscopy was utilized to further characterize the effect of the Ossndp1 mutation on root hair morphology. Aberrant morphogenesis was detected in root hair elongation and maturation zones. Many root hairs were branched and showed irregular shapes due to bulged nodes. Many epidermal cells also produced dome-shaped root hairs, which indicated that root hair elongation ceased at an early stage. These studies showed that PITP-mediated phospholipid signaling and metabolism is critical for root hair elongation in rice.
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A full-length patellin1 (PATL1) cDNA was cloned and characterized from zucchini (Cucurbita pepo). PATL1, originally discovered in the higher plant Arabidopsis thaliana, is a plant Sec14-related protein that localizes to the cell plate during the late stages of cytokinesis. PATL1 is related in sequence to other eukaryotic proteins involved in membrane trafficking and is thought to participate in vesicle trafficking events associated with cell plate maturation. The zucchini PATL1 (CpPATL1) cDNA predicts a 605 amino acid protein which consists of an acidic N-terminal domain (pI=4.2) followed by a Sec14 lipid-binding domain and a C-terminal Golgi dynamics domain (GOLD). The predicted CpPATL1 protein sequence shows a high degree of similarity to Arabidopsis PATL1, especially in the Sec14 (84%) and GOLD domains (87%). A phylogenetic analysis of all available full-length PATL sequences revealed that the PATLs belong to four distinct clades; CpPATL1 is a member of the PATL1/2 clade. RT-PCR analysis showed that the CpPATL1 gene is highly expressed throughout the plant. The domain structure, as well as biochemical fractionation studies, which demonstrated that CpPATL1 is a peripheral membrane protein, support a role in membrane trafficking events.
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Phosphatidylinositol transfer proteins (PITPs) modulate signal transduction pathways and membrane-trafficking functions in eukaryotes. Here, we describe the characterization of a gene family from Lotus japonicus that encodes a novel class of plant PITP-like proteins (LjPLPs) and that is regulated in an unusual nodule-specific manner. Members of this gene family were identified based on their nucleotide sequence homology with a previously described cDNA, LjNOD16, which encodes the L. japonicus late nodulin Nlj16. Nlj16 or highly related amino acid sequences are shown to constitute C-terminal domains of LjPLPs and are suggested to function as specific plasma membrane targeting modules. The expression patterns of one member of this gene family (LjPLP-IV) revealed that LjNOD16 mRNA synthesis in nodules is the result of the transcriptional activity of a nodule-specific promoter located in an intron of the LjPLP-IV gene. This intron-borne bidirectional promoter also generates nodule-specific antisense transcripts derived from the N-terminal PITP domain coding region of the LjPLP-IV gene. We propose that Nlj16 protein synthesis and LjPLP-IV antisense transcript generation are components of an elaborate mechanism designed to control LjPLP synthesis and/or functioning in nodules.
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Protein-lipid interactions are important for protein targeting, signal transduction, lipid transport, lipid biosynthesis, lipid metabolism, and the maintenance of cellular compartments and membranes. Specific lipid-binding protein domains, such as PH, FYVE, PX, PHD, C2 and SEC14 homology domains, mediate interactions between proteins and specific phospholipids. Here we review the published literature, plus some of our most recent unpublished findings, regarding the biology of the SEC14 domain, also known as CRAL_TRIO domain.
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The Rho guanine nucleotide exchange factor protoDbl is involved in different biochemical pathways affecting cell proliferation and migration. The N-terminal sequence of protoDbl contains negative regulatory elements that restrict the catalytic activity of the DH-PH module. Here, we report the identification of a new mouse protoDbl splice variant lacking exon 3. We found that the splice variant mRNA is expressed in the spleen and bone marrow lymphocytes, adrenal gland, gonads and brain. The protoDbl variant protein was detectable in the brain. The newly identified variant displays the disruption of the SEC14 domain, positioned on exons 2 and 3 in the protoDbl N-terminal region. We show here that an altered SEC14 sequence leads to enhanced Dbl translocation to the plasma membrane and to augmented transforming and exchange activity.
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SEC14 lipid transfer proteins are important regulators of phospholipid metabolism. Structural, genetic and cell biological studies in yeast suggest that they help phosphatidylinositol (PtdIns)/phosphoinositide (PIP) kinases to overcome their intrinsic inefficiency to recognize membrane-embedded substrate, thereby playing a key role in PIP homeostasis. Genomes of higher plants encode a high number and diversity of SEC14 proteins, often in combination with other domains. The Arabidopsis SEC14-Nlj16 protein AtSFH1, an important regulator of root hair development, plays an important role in the establishment of PIP microdomains. Key to this mechanism is a highly specific interaction of the Nlj16 domain with PtdIns(4,5)P2 and an interaction-triggered oligomerization of the protein. Nlj16/PtdIns(4,5)P2 interaction depends on a polybasic motif similar to those identified in other regulatory proteins.
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图1 2010—2018年全国土地出让金收入及其在地方公共预算中占比图2 2014—2018年杭州市土地财政情况图3 2009—2018年中国M2和GDP情况/
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