發(fā)布時(shí)間：2018-04-13 16:57

  本文選題：Sep15-like + 抗逆��； 參考：《山東大學(xué)》2017年碩士論文

【摘要】：作物在生長過程中受到非生物逆境的脅迫通常會(huì)導(dǎo)致細(xì)胞內(nèi)氧化還原系統(tǒng)的失衡和內(nèi)質(zhì)網(wǎng)壓力增大,從而影響了作物的生長和發(fā)育。因此鑒定抗逆相關(guān)基因及解析其作用機(jī)制并應(yīng)用于育種工作,是提高作物產(chǎn)量的一個(gè)重要途徑。在動(dòng)物中,Sep15參與多種氧化還原反應(yīng)、蛋白質(zhì)折疊的質(zhì)量控制等,但在植物中其同源基因的功能未見報(bào)道。通過測(cè)序公共數(shù)據(jù)庫搜索得到了擬南芥、短柄草和小麥的Sep15-like的cDNA和gDNA序列,設(shè)計(jì)了特異性引物,克隆得到了全長Sep15-like基因序列。序列分析發(fā)現(xiàn)Sep15-like在不同植物中比較保守且具有高度相似的兩個(gè)結(jié)構(gòu)域:富含半胱氨酸的結(jié)構(gòu)域和硫氧還蛋白樣折疊結(jié)構(gòu)域;亞細(xì)胞定位實(shí)驗(yàn)顯示植物Sep15-like蛋白定位于內(nèi)質(zhì)網(wǎng)中;組織表達(dá)模式分析顯示,植物Sep15-like在根和葉中的表達(dá)量相對(duì)較高;鹽、旱脅迫下的誘導(dǎo)表達(dá)模式分析表明,植物Sep15-like在鹽、旱脅迫下其表達(dá)水平明顯上調(diào)。將AtSep15-like、BdSep15-like和TaSep15-like轉(zhuǎn)入擬南芥中,并獲得過表達(dá)株系。在萌發(fā)階段,正常條件下,除突變體萌發(fā)略差外,野生型和過表達(dá)系的萌發(fā)情況基本一致,脅迫處理?xiàng)l件下,突變體萌發(fā)情況最差,過表達(dá)系好于野生型;在幼苗階段,正常條件下,突變體生長受到抑制,而野生型和過表達(dá)系的生長狀況基本一致,脅迫處理?xiàng)l件下,突變體的根長明顯短于野生型,而過表達(dá)系相比于野生型根長增加顯著,地上部分差異也比較顯著。同樣地,BdSep15-like和TaSep15-like轉(zhuǎn)擬南芥過表達(dá)系也展現(xiàn)了類似的表型。通過對(duì)內(nèi)質(zhì)網(wǎng)壓力相關(guān)Marker基因的測(cè)定和蛋白質(zhì)羰基、巰基含量的測(cè)定,初步證明AtSep15-like基因在脅迫條件下可以有效緩解內(nèi)質(zhì)網(wǎng)壓力。通過對(duì)ROS含量和ROS信號(hào)途徑相關(guān)基因的分析,表明AtSep15-like對(duì)于ROS平衡具有顯著的調(diào)控作用。遺傳互補(bǔ)實(shí)驗(yàn)表明AtSep15-like通過AtUGGT發(fā)揮了抗逆作用。本論文的研究證明了植物Sep15-like在抗逆方面的生理功能及其與內(nèi)質(zhì)網(wǎng)壓力和活性氧之間的關(guān)系,并可能通過UGGT發(fā)揮作用。
[Abstract]:The stress of abiotic stress during the growth of crops usually leads to the imbalance of intracellular redox system and the increase of endoplasmic reticulum pressure, which affects the growth and development of crops.Therefore, it is an important way to improve crop yield to identify the genes related to stress resistance, to analyze the mechanism of their action and to apply them to breeding.Sep15 is involved in many redox reactions and quality control of protein folding in animals, but the function of its homologous genes has not been reported in plants.The cDNA and gDNA sequences of Sep15-like of Arabidopsis thaliana, Stipa sativus and wheat were obtained by sequencing the common database. Specific primers were designed and the full-length Sep15-like gene sequence was cloned.Sequence analysis showed that Sep15-like was conserved and highly similar in different plants: cysteine-rich domain and thioredoxin like folding domain, subcellular localization experiments showed that plant Sep15-like protein was located in endoplasmic reticulum (ER).Tissue expression pattern analysis showed that the expression of plant Sep15-like was relatively high in roots and leaves, and the induced expression pattern under salt and drought stress showed that the expression level of plant Sep15-like was significantly up-regulated under salt and drought stress.AtSep15-like Bd Sep15-like and TaSep15-like were transferred into Arabidopsis thaliana and the expressed lines were obtained.In the germination stage, the germination of wild type and overexpression line was basically the same except for the slightly worse germination of mutant, and under stress, the germination of mutant was the worst, the over-expression line was better than wild type, and in seedling stage, the germination of wild type and overexpression line were better than that of wild type.Under normal conditions, the growth of mutant was inhibited, but the growth status of wild type and overexpression line was basically the same. Under stress treatment, the root length of mutant was significantly shorter than that of wild type, and the root length of overexpression line increased significantly compared with that of wild type.There was also significant difference in the above-ground part.Similarly, Bd Sep15-like and TaSep15-like-transformed Arabidopsis overexpression lines showed similar phenotypes.Through the determination of endoplasmic reticulum pressure-related Marker gene and protein carbonyl and sulfhydryl content, it was preliminarily proved that AtSep15-like gene could relieve endoplasmic reticulum pressure effectively under stress.The analysis of ROS content and ROS signaling pathway showed that AtSep15-like had a significant role in regulating ROS balance.Genetic complementation experiment showed that AtSep15-like played a role of stress resistance through AtUGGT.In this paper, the physiological function of plant Sep15-like in stress resistance and its relationship with endoplasmic reticulum pressure and reactive oxygen species were proved, which may be played by UGGT.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：Q943.2

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本文編號(hào)：1745388