本文選題：擬南芥 + 非生物脅迫　； 參考：《山東農(nóng)業(yè)大學(xué)》2016年碩士論文

【摘要】：近年來,隨著環(huán)境惡化,各種非生物脅迫使作物的生長發(fā)育受到嚴(yán)重影響,導(dǎo)致籽粒的產(chǎn)量明顯下降,比如高鹽可引起植物細(xì)胞膜系統(tǒng)破壞,細(xì)胞滲透壓改變,細(xì)胞內(nèi)外離子濃度改變,導(dǎo)致各種水解酶和蛋白酶失去活性;干旱可引起植物遭受生理干旱,細(xì)胞中的多種蛋白生理活性降低,導(dǎo)致作物發(fā)育畸形,從而影響產(chǎn)量。半胱氨酸蛋白酶抑制劑具有非常保守的結(jié)構(gòu)域,包括位于多肽鏈中心部位的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劑形成一個(gè)具有槽型的三維結(jié)構(gòu),該結(jié)構(gòu)與半胱氨酸蛋白酶發(fā)生互作,進(jìn)而抑制半胱氨酸蛋白酶活性。在植物抵抗低溫、高溫、高鹽和干旱等非生物脅迫以及抵抗真菌和昆蟲等生物脅迫中有重要作用。已有研究發(fā)現(xiàn),擬南芥半胱氨酸蛋白酶抑制劑對植物生長發(fā)育及脅迫抗性有重要影響,但半胱氨酸蛋白酶抑制劑相關(guān)基因及其編碼蛋白的功能研究較少。擬南芥半胱氨酸蛋白酶抑制劑超家族相關(guān)基因是否也參與非生物脅迫響應(yīng)亟待深入探索。本研究對半胱氨酸蛋白酶抑制劑超家族相關(guān)基因的基因結(jié)構(gòu)進(jìn)行了全面分析,并對SRAC1進(jìn)行了分子克隆和功能分析:(1)生物信息學(xué)分析表明:基因的結(jié)構(gòu)包括前導(dǎo)區(qū)、尾部區(qū)、調(diào)控區(qū)和編碼區(qū)。23個(gè)半胱氨酸蛋白酶抑制劑超家族相關(guān)基因的基因長度,87%的在1500bp以內(nèi),外顯子數(shù)目在1-4個(gè)之間,與已知的半胱氨酸蛋白酶抑制劑的基因長度基本一致,外顯子的長度在600bp以內(nèi),與已知的半胱氨酸蛋白酶抑制劑外顯子的長度基本一致。它們結(jié)構(gòu)的相似性預(yù)示著它們可能有相似的功能。(2)23個(gè)半胱氨酸蛋白酶抑制劑超家族相關(guān)基因的染色體定位分析表明:它們在1-5號染色體上均有分布,并與已知的半胱氨酸蛋白酶抑制劑基因聚集成簇,預(yù)示著它們可能有相似的功能。(3)23個(gè)半胱氨酸蛋白酶抑制劑超家族相關(guān)基因的啟動(dòng)子分析表明:啟動(dòng)子不僅含有響應(yīng)赤霉素、乙烯和光的元件,也含有響應(yīng)高溫、低溫、干旱、脫落酸和茉莉酸等非生物脅迫的元件。(4)同源分析表明:半胱氨酸蛋白酶抑制劑超家族相關(guān)基因分為兩類,12個(gè)相關(guān)基因與已知的半胱氨酸蛋白酶抑制在同一類別中,其中的5個(gè)基因已被發(fā)現(xiàn)具有抑制半胱氨酸蛋白酶的活性,預(yù)示著其他相關(guān)基因可能也能夠抑制半胱氨酸蛋白酶的活性。(5)蛋白保守性分析表明:它們在N端含有相對保守的G和L-E-F-V-V-Y-R-A基序,在中部含有Y-Q-A-K-V基序,它們可能會(huì)形成與半胱氨酸蛋白酶抑制劑相似的三維結(jié)構(gòu),從而發(fā)揮同樣的功能。(6)對該家族相關(guān)基因進(jìn)行基因芯片分析,發(fā)現(xiàn)ABA處理后,At5g17090(SRAC1)的表達(dá)量明顯上調(diào),過量表達(dá)SRAC1提高了轉(zhuǎn)基因株系的對Na Cl的抗性,減弱了對ABA的敏感性。(7)酶活檢測結(jié)果表明SARC1可抑制木瓜蛋白酶的活性,與已知的半胱氨酸蛋白酶抑制劑的效果相當(dāng),表明SARC1蛋白具有半胱氨酸蛋白酶抑制劑活性。
[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.

【學(xué)位授予單位】：山東農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：Q945.78;Q943.2

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