黄金茶CsDAM2基因克隆及其功能分析
发布时间：2019-07-03 11:33
【摘要】：休眠是植物芽停止生长以适应外界不良环境的一种机制,也是植物体内相应基因表达调控的结果。茶树(Camellia sinensis (L.) O.Kuntez.)作为一种多年生叶用经济作物,每年冬季都会遇到低温等不适环境的胁迫,茶芽进入休眠状态,直至春季气温的回升和光照时间增加,打破休眠,恢复生长。因此,茶树芽休眠及解除的早晚与茶树高产优质及经济效益密切相关。目前有关茶树芽休眠及解除的研究主要集中在生理生化变化、基因筛选与表达、抗寒或低温胁迫转录因子克隆与功能验证等方面,取得了显著进展,但其分子机制尚有待进一步探索。黄金茶是一个原产于湖南湘西州保靖县的优异茶树品种资源,较当地其它茶树品种发芽早10-15天是其优异特性之一,但机理不详。本研究前期通过低温诱导与差异性表达分离到多个可能与黄金茶发芽早相关的候选基因,其中之一就是转录因子Dormancy Associated MADS-box(CsDAM2),为此,本研究通过克隆该基因全长片段并且分析其功能,为进一步丰富茶树芽休眠及解除的分子机制提供科学依据,以及为调控茶树春芽萌发提供新的靶基因。主要研究结果如下：1.通过设计特异性引物DMAOF\DMAOR,进行PCR反应得到了CsDAM2目标基因的特异性产物。将目标基因与载体质粒pCXSN重组,通过菌落验证、抗性筛选和测序验证等验证性试验,成功构建了茶树CsDAM2基因的过表达载体pCXSN-CsDAM2,并将其转入到农杆菌GV3101中。2.在农杆菌GV3101的介导下,成功将过表达载体pCXSN-CsDAM2转入烟草(Nicotiana tabacum L)愈伤组织中。在抗性培养基中经过诱导分化、诱导生根、移栽成活,成功获得了阳性烟草植株。提取烟草植株DNA,进行PCR检测,验证结果表明过表达载体pCXSN-CsDAM2已导入转基因烟草。3.将转基因烟草和野生型烟草分别在25℃、4℃、0℃培养24h,其电导率与叶绿素荧光值结果表明,在相同条件下,随着温度的下降转基因烟草比野生型烟草电导率增加幅度较小,而叶绿素荧光值降低率较低,说明转基因烟草抗寒性优于野生型烟草,由此一定程度上提示了CsDAM2基因具有提高茶树抗寒性的功能。
[Abstract]:Dormancy is a mechanism by which plant buds stop growing to adapt to the external bad environment, and it is also the result of the regulation of corresponding gene expression in plants. Tea (Camellia sinensis (L.) O.Kuntez.) As a kind of perennial leaf cash crop, tea buds enter dormancy under the stress of low temperature and other uncomfortable environment every winter, until the recovery of temperature and the increase of light time in spring, break dormancy and resume growth. Therefore, the dormancy and release of tea bud are closely related to the high yield, good quality and economic benefit of tea. At present, the research on dormancy and release of tea bud mainly focuses on physiological and biochemical changes, gene screening and expression, cloning and functional verification of transcription factors resistant to cold or low temperature stress, but its molecular mechanism needs to be further explored. Golden tea is an excellent tea variety resource originated in Baojing County, Xiangxi County, Hunan Province. compared with other local tea varieties, the germination time of gold tea is 10 days earlier than that of other local tea varieties, which is one of its excellent characteristics, but the mechanism is unknown. In this study, a number of candidate genes, one of which is transcription factor Dormancy Associated MADS-box (CsDAM2), were isolated by low temperature induction and differential expression. Therefore, by cloning the full-length fragment of the gene and analyzing its function, this study provides scientific basis for further enriching the molecular mechanism of dormancy and release of tea bud, and provides a new target gene for regulating spring bud germination of tea tree. The main results are as follows: 1. The specific product of CsDAM2 target gene was obtained by PCR reaction with specific primers DMAOF\ DMAOR,. The overexpression vector pCXSN-CsDAM2, of tea plant CsDAM2 gene was successfully constructed and transferred into Agrobacterium tumefaciens GV3101 by recombination of the target gene and vector plasmid pCXSN. Through colony verification, resistance screening and sequencing verification, the overexpression vector pCXSN-CsDAM2, of tea plant DNA gene was successfully constructed and transferred into Agrobacterium tumefaciens GV3101. 2. Under the mediation of Agrobacterium tumefaciens GV3101, the overexpression vector pCXSN-CsDAM2 was successfully transferred into tobacco (Nicotiana tabacum L) calli. The positive tobacco plants were successfully obtained by inducing differentiation, inducing root and transplanting in resistant medium. The DNA, of tobacco plants was extracted and detected by PCR. The results showed that the overexpression vector pCXSN-CsDAM2 had been introduced into transgenic tobacco. The conductivity and chlorophyll fluorescence value of transgenic tobacco and wild type tobacco were cultured at 25 鈩，
本文编号：2509350
[Abstract]:Dormancy is a mechanism by which plant buds stop growing to adapt to the external bad environment, and it is also the result of the regulation of corresponding gene expression in plants. Tea (Camellia sinensis (L.) O.Kuntez.) As a kind of perennial leaf cash crop, tea buds enter dormancy under the stress of low temperature and other uncomfortable environment every winter, until the recovery of temperature and the increase of light time in spring, break dormancy and resume growth. Therefore, the dormancy and release of tea bud are closely related to the high yield, good quality and economic benefit of tea. At present, the research on dormancy and release of tea bud mainly focuses on physiological and biochemical changes, gene screening and expression, cloning and functional verification of transcription factors resistant to cold or low temperature stress, but its molecular mechanism needs to be further explored. Golden tea is an excellent tea variety resource originated in Baojing County, Xiangxi County, Hunan Province. compared with other local tea varieties, the germination time of gold tea is 10 days earlier than that of other local tea varieties, which is one of its excellent characteristics, but the mechanism is unknown. In this study, a number of candidate genes, one of which is transcription factor Dormancy Associated MADS-box (CsDAM2), were isolated by low temperature induction and differential expression. Therefore, by cloning the full-length fragment of the gene and analyzing its function, this study provides scientific basis for further enriching the molecular mechanism of dormancy and release of tea bud, and provides a new target gene for regulating spring bud germination of tea tree. The main results are as follows: 1. The specific product of CsDAM2 target gene was obtained by PCR reaction with specific primers DMAOF\ DMAOR,. The overexpression vector pCXSN-CsDAM2, of tea plant CsDAM2 gene was successfully constructed and transferred into Agrobacterium tumefaciens GV3101 by recombination of the target gene and vector plasmid pCXSN. Through colony verification, resistance screening and sequencing verification, the overexpression vector pCXSN-CsDAM2, of tea plant DNA gene was successfully constructed and transferred into Agrobacterium tumefaciens GV3101. 2. Under the mediation of Agrobacterium tumefaciens GV3101, the overexpression vector pCXSN-CsDAM2 was successfully transferred into tobacco (Nicotiana tabacum L) calli. The positive tobacco plants were successfully obtained by inducing differentiation, inducing root and transplanting in resistant medium. The DNA, of tobacco plants was extracted and detected by PCR. The results showed that the overexpression vector pCXSN-CsDAM2 had been introduced into transgenic tobacco. The conductivity and chlorophyll fluorescence value of transgenic tobacco and wild type tobacco were cultured at 25 鈩，
本文编号：2509350
本文链接：https://www.wllwen.com/kejilunwen/jiyingongcheng/2509350.html
最近更新
教材专著
