半胱氨酸蛋白酶抑制剂相关基因SRAC1的功能分析

发布时间：2018-05-04 14:12

本文选题：拟南芥 + 非生物胁迫　；参考：《山东农业大学》2016年硕士论文

【摘要】：近年来,随着环境恶化,各种非生物胁迫使作物的生长发育受到严重影响,导致籽粒的产量明显下降,比如高盐可引起植物细胞膜系统破坏,细胞渗透压改变,细胞内外离子浓度改变,导致各种水解酶和蛋白酶失去活性;干旱可引起植物遭受生理干旱,细胞中的多种蛋白生理活性降低,导致作物发育畸形,从而影响产量。半胱氨酸蛋白酶抑制剂具有非常保守的结构域,包括位于多肽链中心部位的Gln-X-Val-X-Gly基序、羧基端保守的Pro/Leu-Trp基序和氨基端保守的Gly基序。半胱氨酸蛋白酶抑制剂形成一个具有槽型的三维结构,该结构与半胱氨酸蛋白酶发生互作,进而抑制半胱氨酸蛋白酶活性。在植物抵抗低温、高温、高盐和干旱等非生物胁迫以及抵抗真菌和昆虫等生物胁迫中有重要作用。已有研究发现,拟南芥半胱氨酸蛋白酶抑制剂对植物生长发育及胁迫抗性有重要影响,但半胱氨酸蛋白酶抑制剂相关基因及其编码蛋白的功能研究较少。拟南芥半胱氨酸蛋白酶抑制剂超家族相关基因是否也参与非生物胁迫响应亟待深入探索。本研究对半胱氨酸蛋白酶抑制剂超家族相关基因的基因结构进行了全面分析,并对SRAC1进行了分子克隆和功能分析:(1)生物信息学分析表明:基因的结构包括前导区、尾部区、调控区和编码区。23个半胱氨酸蛋白酶抑制剂超家族相关基因的基因长度,87%的在1500bp以内,外显子数目在1-4个之间,与已知的半胱氨酸蛋白酶抑制剂的基因长度基本一致,外显子的长度在600bp以内,与已知的半胱氨酸蛋白酶抑制剂外显子的长度基本一致。它们结构的相似性预示着它们可能有相似的功能。(2)23个半胱氨酸蛋白酶抑制剂超家族相关基因的染色体定位分析表明:它们在1-5号染色体上均有分布,并与已知的半胱氨酸蛋白酶抑制剂基因聚集成簇,预示着它们可能有相似的功能。(3)23个半胱氨酸蛋白酶抑制剂超家族相关基因的启动子分析表明:启动子不仅含有响应赤霉素、乙烯和光的元件,也含有响应高温、低温、干旱、脱落酸和茉莉酸等非生物胁迫的元件。(4)同源分析表明:半胱氨酸蛋白酶抑制剂超家族相关基因分为两类,12个相关基因与已知的半胱氨酸蛋白酶抑制在同一类别中,其中的5个基因已被发现具有抑制半胱氨酸蛋白酶的活性,预示着其他相关基因可能也能够抑制半胱氨酸蛋白酶的活性。(5)蛋白保守性分析表明:它们在N端含有相对保守的G和L-E-F-V-V-Y-R-A基序,在中部含有Y-Q-A-K-V基序,它们可能会形成与半胱氨酸蛋白酶抑制剂相似的三维结构,从而发挥同样的功能。(6)对该家族相关基因进行基因芯片分析,发现ABA处理后,At5g17090(SRAC1)的表达量明显上调,过量表达SRAC1提高了转基因株系的对Na Cl的抗性,减弱了对ABA的敏感性。(7)酶活检测结果表明SARC1可抑制木瓜蛋白酶的活性,与已知的半胱氨酸蛋白酶抑制剂的效果相当,表明SARC1蛋白具有半胱氨酸蛋白酶抑制剂活性。
[Abstract]:In recent years, with the deterioration of the environment, the growth and development of crops have been seriously affected by various abiotic stresses, resulting in a significant decrease in the grain yield. For example, high salt can cause damage to the membrane system of plant cells, change of cell osmotic pressure, change of cell concentration inside and outside, lead to the loss of all kinds of hydrolase and protease, and drought can cause plants to be damaged. Due to physiological drought, the physiological activity of various proteins in the cells is reduced, resulting in malformation of crops and the effect of production. Cysteine protease inhibitors have a very conservative domain, including the Gln-X-Val-X-Gly motif located in the central part of the polypeptide chain, the conservative Pro/ Leu-Trp motif of carboxyl terminus and the conservative Gly motif in the amino terminal. Cysteine Acid protease inhibitors form a three-dimensional structure with a trough type, which interacts with cysteine protease and inhibits cysteine protease activity. It plays an important role in plant resistance to abiotic stress, such as low temperature, high temperature, high salt and drought, and resistance to biological stress, such as fungi and insects. Cysteine protease inhibitors have important effects on plant growth and stress resistance, but the function of cysteine protease inhibitor related genes and their encoded proteins is less. The gene structure of cystine protease inhibitor superfamily related genes was analyzed comprehensively, and molecular cloning and functional analysis of SRAC1 were carried out. (1) bioinformatics analysis showed that the structure of the gene includes the gene length of the superfamily related genes in the leading region, the tail region, the regulatory area and the coding region,.23, cystine protease inhibitor. Within 87% of 1500bp, the number of exons is between 1-4, and the length of the known cysteine protease inhibitors is in the same length. The length of exons is within 600bp, which is basically consistent with the length of the known exons of cysteine protease inhibitors. Their structural resemblance indicates that they may have similar functions. (2) 23 Chromosomal location analysis of superfamily related genes of cysteine protease inhibitors showed that they were distributed on chromosome 1-5 and clustered with known cysteine protease inhibitor genes, indicating that they might have similar functions. (3) the initiation of superfamily related genes of cystine proteinase inhibitor 23.5 Subanalysis shows that the promoter not only contains elements that respond to Gibberellin, ethylene and light, but also contains elements that respond to non biological stresses such as high temperature, low temperature, drought, abscisic acid and jasmonic acid. (4) homologous analysis shows that cysteine protease inhibitor superfamily related genes are divided into two groups, 12 related genes and known cysteine proteases. In the same category, 5 of these genes have been found to inhibit cysteine protease activity, indicating that other related genes may also inhibit the activity of cysteine protease. (5) conserved analysis of protein shows that they contain relatively conservative G and L-E-F-V-V-Y-R-A motif at the N end, and contain Y-Q-A-K-V motif in the middle. They may form a three dimensional structure similar to cysteine protease inhibitors. (6) gene chip analysis of the related genes of the family found that after ABA treatment, the expression of At5g17090 (SRAC1) was obviously up-regulated, and overexpression of SRAC1 increased the resistance to Na Cl in the transgenic lines and weakened the sensitivity to ABA. (7) the results of enzyme activity detection showed that SARC1 could inhibit the activity of papain, which was equivalent to the known cysteine protease inhibitor, indicating that SARC1 protein had the activity of cysteine protease inhibitor.

【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q945.78;Q943.2

【参考文献】