發(fā)布時間：2018-06-09 05:06

  本文選題：生物學(xué)功能 + 啟動子　； 參考：《長江大學(xué)》2017年碩士論文

【摘要】：研究表明植物轉(zhuǎn)脂蛋白(lipid transfer proteins,LTPs)參與多種生物學(xué)過程,但在非逆境脅迫下的作用鮮有報道。本研究將玉米中一個轉(zhuǎn)脂蛋白成員——ZmLTP3基因的正義鏈和反義鏈分別連接上表達(dá)載體再利用花粉管通道法轉(zhuǎn)入到玉米品種2416中,驗證其生物學(xué)功能。同時克隆ZmLTP3基因的上游啟動子序列(1302bp),并連接表達(dá)載體轉(zhuǎn)化擬南芥,通過GUS染色驗證其是否具有啟動子活性。明確其上游調(diào)控機(jī)制。研究結(jié)果表明:1.干旱脅迫下過量表達(dá)ZmLTP3基因的玉米株系生理生化指標(biāo)都優(yōu)于對照,隨著干旱處理時間的延長,轉(zhuǎn)基因株系株高、莖粗、葉面積、側(cè)根數(shù)、根長、鮮重、干重、三個保護(hù)酶(SOD、POD、CAT)活性、葉綠素含量都顯著高于對照。MDA的含量和外滲電導(dǎo)率顯著低于對照,復(fù)水后仍有差異;高鹽脅迫下過量表達(dá)ZmLTP3基因的玉米株系生理生化反應(yīng)也均優(yōu)于對照,隨著高鹽處理時間的延長,轉(zhuǎn)基因株系株高、莖粗、葉面積、側(cè)根數(shù)、根長、鮮重、干重、三個保護(hù)酶(SOD、POD、CAT)活性、葉綠素含量都顯著高于對照,MDA的含量和外滲電導(dǎo)率顯著低于對照。反義表達(dá)在干旱和高鹽脅迫下轉(zhuǎn)基因株系與對照差異均不顯著。2.用PCR儀克隆啟動子序列,連接T載體測序驗證,測序結(jié)果表明,從設(shè)計的上游引物到ATG處只有1302 bp,而不是NCBI數(shù)據(jù)庫中的2492 bp,測序結(jié)果用Plantcare在線分析發(fā)現(xiàn)在克隆序列中含有CAAT-box、TATA-box等啟動子必需作用元件,除此之外還含有眾多響應(yīng)生物脅迫、非生物脅迫的元件。3.將啟動子序列與GUS報告基因相連構(gòu)建植物表達(dá)載體,轉(zhuǎn)化擬南芥。轉(zhuǎn)基因擬南芥GUS組織化學(xué)染色結(jié)果表明,此序列具備啟動子活性。
[Abstract]:Studies have shown that the plant translipoprotein lipid transfer proteinsm (LTPs) is involved in many biological processes, but the role of LTPsunder non-stress is rarely reported. In this study, a translipoprotein member of maize, the sense chain and antisense chain of ZmLTP3 gene, were connected to the expression vector respectively and then transferred into maize variety 2416 by pollen tube pathway to verify its biological function. At the same time, the upstream promoter sequence of ZmLTP3 gene was cloned and transformed into Arabidopsis thaliana by ligating the expression vector, and the promoter activity was verified by Gus staining. Clarify its upstream regulation mechanism. The results of the study show that 1: 1. Under drought stress, the physiological and biochemical indexes of maize lines over-expressing ZmLTP3 gene were superior to those of the control. With the extension of drought treatment, the transgenic lines had higher plant height, stem diameter, leaf area, lateral root number, root length, fresh weight and dry weight. The activity of three protective enzymes, chlorophyll content were significantly higher than that of the control. MDA content and the conductivity of exosmosis were significantly lower than those of the control, and there were still differences after rehydration, and the physiological and biochemical responses of the maize lines overexpressing ZmLTP3 gene under high salt stress were also better than those of the control. With the prolongation of the time of high salt treatment, the plant height, stem diameter, leaf area, lateral root number, root length, fresh weight, dry weight, three protective enzymes (SOD, POD, CAT) activity of transgenic lines were increased. The content of chlorophyll was significantly higher than that of the control, and the content of MDA and the conductivity of the exosmosis were significantly lower than that of the control. There was no significant difference in antisense expression between transgenic lines and control under drought and high salt stress. The promoter sequence was cloned by PCR, and the ligated T vector was sequenced. From the designed upstream primer to ATG, there were only 1302 BP, instead of 2492 BP in the NCBI database. Plantcare online analysis showed that the cloned sequence contained the essential elements of promoter such as CAAT-box TATA-box, in addition to many responsive biological stress. Abiotic stress element. Plant expression vector was constructed by linking promoter sequence with Gus reporter gene and transformed into Arabidopsis thaliana. Gus histochemical staining of transgenic Arabidopsis thaliana showed that this sequence had promoter activity.
【學(xué)位授予單位】：長江大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S513

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本文編號：1999055