發(fā)布時間：2018-07-11 12:54

  本文選題：NsylCBL2 + 克隆��； 參考：《中國農(nóng)業(yè)科學(xué)院》2016年碩士論文

【摘要】：類鈣調(diào)神經(jīng)素B亞基蛋白(CBL)是植物中普遍存在的一種鈣感受器家族。很多研究表明CBL在植物響應(yīng)生物脅迫和非生物脅迫中起著至關(guān)重要的作用。因此,它被認(rèn)為是基因抗逆育種工作中很重要的候選基因。本研究旨在從林煙草中分離CBL基因并對其進(jìn)行功能驗證。主要工作內(nèi)容如下:1.利用同源克隆的策略,從林煙草的葉片中獲得一個基因。進(jìn)化樹分析表明該基因與其它物種中的CBL2基因在同一個分支上。因此將其命名為林煙草CBL2基因。多序列比對分析表明林煙草CBL2蛋白與擬南芥CBL2蛋白的序列相似度高達(dá)89.38%,與楊樹、水稻和玉米CBL2蛋白的序列相似度分別為86.22%、84.00%、48.21%。2.NsylCBL2的CDS序列全長為675 bp,編碼一個由223個氨基酸組成的蛋白質(zhì),其基因組包括7個內(nèi)含子和8個外顯子。NsylCBL2的4個EF手型結(jié)構(gòu)之間的距離分別為23、25和32個氨基酸。生物信息學(xué)分析表明NsylCBL2的結(jié)構(gòu)和理化性質(zhì)與其它物種中CBL2相似。3.通過浸花法將NsylCBL2轉(zhuǎn)入擬南芥中,對T1代植株進(jìn)行RCR陽性鑒定和RT-PCR表達(dá)量檢測,對T2和T3代植株進(jìn)行抗性篩選,T3代純合株系用來進(jìn)行NsylCBL2的功能驗證,擬南芥cbl2突變體從TAIR上訂購并進(jìn)行了T-DNA插入驗證。4.已有研究表明CBL2基因參與鹽和鎂的信號轉(zhuǎn)導(dǎo),因此本試驗也進(jìn)行了高鹽和高鎂處理,來檢測NsylCBL2是否也具有這樣的功能。但是萌發(fā)、表型觀察和生理數(shù)據(jù)表明過表達(dá)NsylCBL2擬南芥植株、突變體與野生型在這些處理下沒有明顯差異。因此,NsylCBL2可能不參與鹽與鎂的信號轉(zhuǎn)導(dǎo)。5.另一方面,pCHF3-NsylCBL2這個質(zhì)粒通過葉盤轉(zhuǎn)化法侵染到煙草里面。過表達(dá)轉(zhuǎn)基因煙草就已經(jīng)獲得,并且表達(dá)量水平高于普通煙草。從轉(zhuǎn)基因煙草收到種子后做特定的抗性篩選檢測測試,獲得符合3:1比例的T1代,然后種到溫室獲得T2代,供以后的實驗做準(zhǔn)備。6.獲得NsylCBL2的啟動子序列并對其進(jìn)行了預(yù)測分析,發(fā)現(xiàn)其含有多種逆境和激素相關(guān)轉(zhuǎn)錄因子。構(gòu)建了NsylCBL2啟動子與GUS基因融合的表達(dá)載體pBI101-CBL2pro。本研究是對NsylCBL2基因的首次報道。因此,本研究期望能給NsylCBL2今后的研究提供一定的指導(dǎo)。
[Abstract]:Calmodulin B subunit protein (CBL) is a common calcium receptor family in plants. Many studies have shown that CBL plays an important role in plant response to biotic and abiotic stresses. Therefore, it is considered to be an important candidate gene in gene resistance breeding. The purpose of this study was to isolate CBL gene from forest tobacco and verify its function. The main work is as follows: 1. Using the strategy of homologous cloning, a gene was obtained from the leaves of forest tobacco. Phylogenetic tree analysis showed that the gene was in the same branch as CBL2 gene in other species. Therefore, it was named CBL2 gene of forest tobacco. The sequence similarity between CBL2 protein and CBL2 protein in Arabidopsis thaliana was 89.38 and 89.38 respectively. The sequence similarity of CBL2 protein in rice and maize is 86.22 and 84.00, respectively. The CDS sequence of NsylCBL2 is 675 BP, which encodes a 223 amino acid protein. The distance between the four EF structures of its genome including 7 introns and 8 exons. NsylCBL2 was 23 25 and 32 amino acids respectively. Bioinformatics analysis showed that the structure and physicochemical properties of NsylCBL2 were similar to those of other species. NsylCBL2 was transferred into Arabidopsis thaliana by flower soaking method. RCR positive identification and RT-PCR expression of T1 generation plants were detected, and resistance screening of T _ 2 and T _ 3 generation plants were carried out to verify the function of NsylCBL2 in Arabidopsis thaliana. Arabidopsis cbl2 mutants were ordered from TAIR and verified by T-DNA insertion. It has been shown that CBL2 gene is involved in the signal transduction of salt and magnesium. However, germinating, phenotypic observation and physiological data showed that NsylCBL2 was overexpressed in Arabidopsis thaliana plants, and there was no significant difference between mutant and wild type under these treatments. Therefore, NsylCBL2 may not be involved in the signal transduction of salt and magnesium. On the other hand, the plasmid pCHF 3-asylum CBL 2 infects tobacco by leaf disc transformation. Overexpression transgenic tobacco has been obtained, and the level of expression is higher than that of ordinary tobacco. After receiving the seeds from transgenic tobacco, a specific screening test of resistance was performed to obtain T1 generation with a ratio of 3:1, and then to plant in greenhouse to obtain T2 generation, which could be used to prepare for the later experiment. The promoter sequence of asylum CBL2 was obtained and predicted. It was found that it contained many stress and hormone related transcription factors. The expression vector pBI101-CBL2pro. was constructed. This study is the first report of NsylCBL 2 gene. Therefore, this study is expected to provide some guidance for the future study of asylum CBL 2.
【學(xué)位授予單位】：中國農(nóng)業(yè)科學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：S572

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