茶树转录因子基因CsWRKY57和CsWRKY40的克隆及功能初探

发布时间：2018-11-13 17:06

【摘要】：茶树[Camellia sinensis(L.)O.Kuntze]是一种多年生木本植物,在我国南方广泛种植。其叶片加工而成的茶饮品,因其含有丰富的茶多酚、咖啡碱、脂多糖等,具有防癌、抗癌的作用,因此,受到全世界人民的喜爱。然而,在茶树生长发育过程中,会遭受各种生物(如病原菌、真菌等)和非生物(如干旱、高温等)因素的胁迫,严重影响茶叶的产量和品质,造成严重的经济损失。陕西气候干燥,土壤盐碱化较为严重,因此,为研究茶树在逆境条件下的调控机制,以及抗逆基因的功能分析,培育抗逆性较强的茶树新品种,本文以茶树品种“陕茶1号”为试验材料,以叶片cDNA为模板,从中克隆出茶树WRKY57和WRKY40两个基因,并对这两个基因编码的氨基酸序列进行生物信息学分析,在聚乙二醇6000(PEG 6000)、氯化钠(NaCl)、脱落酸(ABA)处理下,基因表达模式分析以及转录激活活性验证。主要研究结果如下:1.克隆了两个与茶树盐胁迫、干旱胁迫、ABA信号调控途径相关的WRKY转录因子基因CsWRKY57和CsWRKY40。生物信息学分析表明,CsWRKY57开放阅读框(ORF)为912 bp,编码303个氨基酸,预测分子量为33.5 kD,理论等电点为5.49,蛋白比对显示,CsWRKY57含有一个WRKY核心序列和一个Cx4Cx23HxH型锌指结构,属于WRKYIIc家族;Cs WRKY40开放阅读框(ORF)为963bp,编码320个氨基酸,预测分子量为35.06 kD,理论等电点为8.80,氨基酸序列比对显示,CsWRKY40含有一个约60个氨基酸的WRKY保守结构域,锌指结构为Cx5Cx23HxH型,属于WRKYIIa家族。2.利用实时荧光定量PCR(Real-time RT-PCR)分析CsWRKY57和CsWRKY40基因在100μM ABA、20%PEG6000、300 mM Na Cl胁迫处理下的表达模式,发现在这些胁迫处理下,CsWRKY57和CsWRKY40均被诱导表达,并且表达量在短时间内迅速增加,之后逐渐下降,并且不同基因在不同胁迫处理下,表达模式存在差异。3.将CsWRKY57和CsWRKY40基因分别与酵母表达载体pGBKT7连接,以空载体pGBKT7作为阴性对照,pCL1载体作为阳性对照,分别观察目的蛋白CsWRKY57和CsWRKY40的转录激活活性。结果表明:CsWRKY57和CsWRKY40蛋白均无转录激活活性。
[Abstract]:Tea tree [Camellia sinensis (L.) O.Kuntze] is a perennial woody plant widely cultivated in southern China. The tea beverage processed from its leaves is loved by people all over the world because of its rich tea polyphenols, caffeine, lipopolysaccharide and so on. However, during the growth and development of tea plant, it will be subjected to various biological (such as pathogens, fungi, etc.) and abiotic (such as drought, high temperature) stress, seriously affect the yield and quality of tea, resulting in serious economic losses. In order to study the regulation mechanism of tea plants under stress and the function analysis of stress resistance genes, new varieties of tea plants with strong resistance to stress were bred in order to study the dry climate and the serious salinization of soil in Shaanxi Province, so as to study the regulation mechanism of tea plants under stress, and to analyze the function of stress resistance genes. In this paper, two genes of tea plant WRKY57 and WRKY40 were cloned by using Shaancha 1 as experimental material and leaf cDNA as template. The amino acid sequences of these two genes were analyzed by bioinformatics. Under the treatment of polyethylene glycol 6000 (PEG 6000) and sodium chloride (NaCl), abscisic acid (ABA), gene expression patterns and transcriptional activation activity were analyzed. The main results are as follows: 1. Two WRKY transcription factor genes CsWRKY57 and CsWRKY40. related to salt stress, drought stress and ABA signaling pathway were cloned. Bioinformatics analysis showed that the CsWRKY57 open reading frame (ORF) encodes 303 amino acids with a predicted molecular weight of 33.5 kD, and a theoretical isoelectric point of 5.49. CsWRKY57 contains a WRKY core sequence and a Cx4Cx23HxH zinc-finger structure, belonging to the WRKYIIc family. The Cs WRKY40 open reading frame (ORF) is 963bpand encodes 320 amino acids, and its predicted molecular weight is 35.06 kD, with a theoretical isoelectric point of 8.80.The amino acid sequence alignment shows that CsWRKY40 contains a conserved WRKY domain of about 60 amino acids. The structure of zinc finger is Cx5Cx23HxH type, belonging to WRKYIIa family. 2. The expression patterns of CsWRKY57 and CsWRKY40 genes under 100 渭 M ABA,20%PEG6000300 mM Na Cl stress were analyzed by real-time fluorescence quantitative PCR (Real-time RT-PCR). It was found that both CsWRKY57 and CsWRKY40 were induced to express under these stress treatments. The expression level increased rapidly in a short period of time, then decreased gradually, and the expression patterns of different genes were different under different stress treatments. CsWRKY57 and CsWRKY40 genes were ligated with yeast expression vector pGBKT7 respectively, empty vector pGBKT7 was used as negative control and pCL1 vector as positive control. The transcriptional activation activities of the target protein CsWRKY57 and CsWRKY40 were observed respectively. The results showed that neither CsWRKY57 nor CsWRKY40 had transcriptional activation activity.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S571.1

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