黃金茶CsDAM2基因克隆及其功能分析
發(fā)布時間：2019-07-03 11:33
【摘要】：休眠是植物芽停止生長以適應(yīng)外界不良環(huán)境的一種機制,也是植物體內(nèi)相應(yīng)基因表達調(diào)控的結(jié)果。茶樹(Camellia sinensis (L.) O.Kuntez.)作為一種多年生葉用經(jīng)濟作物,每年冬季都會遇到低溫等不適環(huán)境的脅迫,茶芽進入休眠狀態(tài),直至春季氣溫的回升和光照時間增加,打破休眠,恢復(fù)生長。因此,茶樹芽休眠及解除的早晚與茶樹高產(chǎn)優(yōu)質(zhì)及經(jīng)濟效益密切相關(guān)。目前有關(guān)茶樹芽休眠及解除的研究主要集中在生理生化變化、基因篩選與表達、抗寒或低溫脅迫轉(zhuǎn)錄因子克隆與功能驗證等方面,取得了顯著進展,但其分子機制尚有待進一步探索。黃金茶是一個原產(chǎn)于湖南湘西州保靖縣的優(yōu)異茶樹品種資源,較當?shù)仄渌铇淦贩N發(fā)芽早10-15天是其優(yōu)異特性之一,但機理不詳。本研究前期通過低溫誘導(dǎo)與差異性表達分離到多個可能與黃金茶發(fā)芽早相關(guān)的候選基因,其中之一就是轉(zhuǎn)錄因子Dormancy Associated MADS-box(CsDAM2),為此,本研究通過克隆該基因全長片段并且分析其功能,為進一步豐富茶樹芽休眠及解除的分子機制提供科學(xué)依據(jù),以及為調(diào)控茶樹春芽萌發(fā)提供新的靶基因。主要研究結(jié)果如下：1.通過設(shè)計特異性引物DMAOF\DMAOR,進行PCR反應(yīng)得到了CsDAM2目標基因的特異性產(chǎn)物。將目標基因與載體質(zhì)粒pCXSN重組,通過菌落驗證、抗性篩選和測序驗證等驗證性試驗,成功構(gòu)建了茶樹CsDAM2基因的過表達載體pCXSN-CsDAM2,并將其轉(zhuǎn)入到農(nóng)桿菌GV3101中。2.在農(nóng)桿菌GV3101的介導(dǎo)下,成功將過表達載體pCXSN-CsDAM2轉(zhuǎn)入煙草(Nicotiana tabacum L)愈傷組織中。在抗性培養(yǎng)基中經(jīng)過誘導(dǎo)分化、誘導(dǎo)生根、移栽成活,成功獲得了陽性煙草植株。提取煙草植株DNA,進行PCR檢測,驗證結(jié)果表明過表達載體pCXSN-CsDAM2已導(dǎo)入轉(zhuǎn)基因煙草。3.將轉(zhuǎn)基因煙草和野生型煙草分別在25℃、4℃、0℃培養(yǎng)24h,其電導(dǎo)率與葉綠素熒光值結(jié)果表明,在相同條件下,隨著溫度的下降轉(zhuǎn)基因煙草比野生型煙草電導(dǎo)率增加幅度較小,而葉綠素熒光值降低率較低,說明轉(zhuǎn)基因煙草抗寒性優(yōu)于野生型煙草,由此一定程度上提示了CsDAM2基因具有提高茶樹抗寒性的功能。
[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，
本文編號：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，
本文編號：2509350
本文鏈接：http://sikaile.net/kejilunwen/jiyingongcheng/2509350.html
最近更新
教材專著
