【作者】 张舒；

【导师】 郝东云；

【作者基本信息】 东北师范大学 ， 遗传学， 2017， 硕士

【摘要】 植物基因功能研究和转基因植物新品种研发,通常需要将多个基因构建在一个表达载体上转入植物体,获得聚合多个目的基因的复合功能转基因材料。但是,每个功能基因通常要各自拥有独立的调控元件系统,导致T-DNA区过长,增加了载体构建的复杂性和遗传转化难度。所以,在实际应用中,如何构架最小的植物表达载体,而装载最多的功能基因是转基因植物研究中的一个重要技术环节。通常情况下,拟转化的目标功能基因长度是不可改变的,为保持尽可能小的T-DNA区,可以改变的只是多克隆位点内的调控元件部分。这样,选用更少的调控元件是植物表达载体设计的重要考量之一。双向启动子存在于自然生物中,是一种在其启动子序列的两个方向上都具备驱动功能基因表达的调控序列,它的发现为利用更少的调控元件完成多基因聚合提供了新的研究理念和原始素材。目前,关于双向启动子的研究仅停留在对其两侧驱动报告基因的定性检测,双向启动子在玉米中的异源驱动功能和启动效率的研究尚未见报道。本研究以从拟南芥基因组中克隆的双向启动子K9I9为研究对象,以gus为报告基因,构建植物表达载体,转化玉米,gus检测转基因玉米幼胚。初步结果显示,K9I9在玉米中可以驱动报告基因转录。进一步,将K9I9与抗鳞翅目昆虫基因cryNGc和抗除草剂基因epsps共同构建双向启动表达载体cryNGC::K9I9::epsps,载体中筛选标记基因为2×35S::bar,遗传转化共获得T0世代5个转化事件25株转基因玉米新材料,以此为基础,评估K9I9在转基因玉米中的双向启动功能。RT-PCR结果显示,T0和T1代转基因玉米中,K9I9两侧的外源基因均可以正常表达;Realtime-PCR和生物信息学分析结果显示,K9I9启动子的启动效率是2×35S::bar的13.1%-70.0%,K9I9对两侧功能基因的启动效率分别为cryNGC 3.25×106 copies/μl和epsps 17.4×106 copies/μl;生物信息学分析显示,K9I9序列正反两个方向上存在常规调控元件数量和种类的差异。综上,双向启动子K9I9可以在玉米中发挥双向启动功能,但影响双向启动效率的原因有待深入研究,目前的实验结果认为拟南芥K9I9启动子可以在玉米中应用。更多还原

【Abstract】 In plant gene function research and transgenic plant breeding,there requires constructing more than one gene into a single vector that each functional gene usually has its own independent set of regulatory elements.This increases the length of the T-DNA region leading to either complexity in vector construction or the difficulty in genetic transformation.In practice,thus,construction of a smallest plant expression vector that harbors multiple genes has become an important technical aspect in transgenic plant research and application.In fact,the length of a given functional gene is immutable,what can be changed is the regulatory element section within the multiple cloning sites.Bidirectional promoter is a regulatory sequence present in many natural organisms.It promotes the expression of a given gene in both orientations of its sequence.Discovery of bidirectional promotors provide new insight in efficiently vector construction of functional gene.In literature,many efforts have been made in the functionality validation of bidirectional promoters for their both sequence orientations.However,there has hardly been report describing the promoting efficiency of bidirectional promotors.In this study,a bidirectional promoter K9I9 from Arabidopsis was adopted to construct plant expression vectors with gus as reporter gene,so as to determine the functionality and the promoting efficiency of K9I9 in transgenic maize.Pilot experiment showed that gus transcripts may be detected in the immature embryo of transgenic maize,suggesting that K9I9 functions well in transgenic maize.Further,a Lepidoptera-resistance gene cryNGc and a glyphosate-resistance gene epsps were co-constructed at different orientations of K9I9,with2×35S::bar as screening marker gene,forming a bidirectional expression vector cryNGC::K9I9::epsps.Genetic transformation of this vector gave rise to at T0 generation 25 transgenic maize of 5 events,with which the bidirectional functionality of K9I9 in transgenic maize was evaluated.RT-PCR results showed that the exogenous genes on both sides of K9I9 expressed in T0 and T1 transgenic maize;real time-PCR showed that the promoter efficiency of K9I9 was ca.13.1%-70.0% of 2x35S::bar and,K9I9 exhibited a promoting efficiency to the functional genes at both sides as cryNGC 3.25×106 copies/μl and epsps 17.4×106 copies/μl respectively.Bioinformatics analysis indicated that differences in quantity and nature of conventional regulatory elements such as TATA-box etc.are present in the forward and reverse orientations of K9I9.In summary,Arabidopsis bidirectional promoter K9I9 functions in both orientations with divergent efficiency in the transgenic maize.It is at moment unclear the reason affecting the efficiency of the bidirectional promoter.The current results suggested that the Arabidopsis K9I9 promoter may be used in maize breeding.更多还原