【摘要】：茶樹[Camellia sinensis(L.)O.Kuntze]是一種多年生木本植物,在我國南方廣泛種植。其葉片加工而成的茶飲品,因其含有豐富的茶多酚、咖啡堿、脂多糖等,具有防癌、抗癌的作用,因此,受到全世界人民的喜愛。然而,在茶樹生長發(fā)育過程中,會遭受各種生物(如病原菌、真菌等)和非生物(如干旱、高溫等)因素的脅迫,嚴重影響茶葉的產(chǎn)量和品質,造成嚴重的經(jīng)濟損失。陜西氣候干燥,土壤鹽堿化較為嚴重,因此,為研究茶樹在逆境條件下的調控機制,以及抗逆基因的功能分析,培育抗逆性較強的茶樹新品種,本文以茶樹品種“陜茶1號”為試驗材料,以葉片cDNA為模板,從中克隆出茶樹WRKY57和WRKY40兩個基因,并對這兩個基因編碼的氨基酸序列進行生物信息學分析,在聚乙二醇6000(PEG 6000)、氯化鈉(NaCl)、脫落酸(ABA)處理下,基因表達模式分析以及轉錄激活活性驗證。主要研究結果如下:1.克隆了兩個與茶樹鹽脅迫、干旱脅迫、ABA信號調控途徑相關的WRKY轉錄因子基因CsWRKY57和CsWRKY40。生物信息學分析表明,CsWRKY57開放閱讀框(ORF)為912 bp,編碼303個氨基酸,預測分子量為33.5 kD,理論等電點為5.49,蛋白比對顯示,CsWRKY57含有一個WRKY核心序列和一個Cx4Cx23HxH型鋅指結構,屬于WRKYIIc家族;Cs WRKY40開放閱讀框(ORF)為963bp,編碼320個氨基酸,預測分子量為35.06 kD,理論等電點為8.80,氨基酸序列比對顯示,CsWRKY40含有一個約60個氨基酸的WRKY保守結構域,鋅指結構為Cx5Cx23HxH型,屬于WRKYIIa家族。2.利用實時熒光定量PCR(Real-time RT-PCR)分析CsWRKY57和CsWRKY40基因在100μM ABA、20%PEG6000、300 mM Na Cl脅迫處理下的表達模式,發(fā)現(xiàn)在這些脅迫處理下,CsWRKY57和CsWRKY40均被誘導表達,并且表達量在短時間內迅速增加,之后逐漸下降,并且不同基因在不同脅迫處理下,表達模式存在差異。3.將CsWRKY57和CsWRKY40基因分別與酵母表達載體pGBKT7連接,以空載體pGBKT7作為陰性對照,pCL1載體作為陽性對照,分別觀察目的蛋白CsWRKY57和CsWRKY40的轉錄激活活性。結果表明:CsWRKY57和CsWRKY40蛋白均無轉錄激活活性。
[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.
【學位授予單位】：西北農林科技大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S571.1

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