玉米ZmLTP3基因的生物学功能鉴定及启动子克隆分析

发布时间：2018-06-09 05:06

本文选题：生物学功能 + 启动子　；参考：《长江大学》2017年硕士论文

【摘要】：研究表明植物转脂蛋白(lipid transfer proteins,LTPs)参与多种生物学过程,但在非逆境胁迫下的作用鲜有报道。本研究将玉米中一个转脂蛋白成员——ZmLTP3基因的正义链和反义链分别连接上表达载体再利用花粉管通道法转入到玉米品种2416中,验证其生物学功能。同时克隆ZmLTP3基因的上游启动子序列(1302bp),并连接表达载体转化拟南芥,通过GUS染色验证其是否具有启动子活性。明确其上游调控机制。研究结果表明:1.干旱胁迫下过量表达ZmLTP3基因的玉米株系生理生化指标都优于对照,随着干旱处理时间的延长,转基因株系株高、茎粗、叶面积、侧根数、根长、鲜重、干重、三个保护酶(SOD、POD、CAT)活性、叶绿素含量都显著高于对照。MDA的含量和外渗电导率显著低于对照,复水后仍有差异;高盐胁迫下过量表达ZmLTP3基因的玉米株系生理生化反应也均优于对照,随着高盐处理时间的延长,转基因株系株高、茎粗、叶面积、侧根数、根长、鲜重、干重、三个保护酶(SOD、POD、CAT)活性、叶绿素含量都显著高于对照,MDA的含量和外渗电导率显著低于对照。反义表达在干旱和高盐胁迫下转基因株系与对照差异均不显著。2.用PCR仪克隆启动子序列,连接T载体测序验证,测序结果表明,从设计的上游引物到ATG处只有1302 bp,而不是NCBI数据库中的2492 bp,测序结果用Plantcare在线分析发现在克隆序列中含有CAAT-box、TATA-box等启动子必需作用元件,除此之外还含有众多响应生物胁迫、非生物胁迫的元件。3.将启动子序列与GUS报告基因相连构建植物表达载体,转化拟南芥。转基因拟南芥GUS组织化学染色结果表明,此序列具备启动子活性。
[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.
【学位授予单位】：长江大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S513

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