马铃薯MAPKK基因鉴定及其抗旱相关功能基因筛选

发布时间：2018-04-02 02:20

本文选题：马铃薯　切入点：MAPKK基因　出处：《甘肃农业大学》2017年硕士论文

【摘要】：MAPKKs(即丝裂原活化蛋白激酶激酶)是主要存在于动植物细胞中,是一种信号传递者,在细胞中具有重要作用,而且也是MAPK级联反应中的关键组成部分。MAPK信号通路在动植物细胞中具有明显的级联现象,是信号转导途径主要途径之一,并在植物体内起到关键作用,它能被激素、细胞因子等不同的细胞外物质刺激,从而使细胞在生长、发育等多种重要的生理过程发生一列反应来响应外界刺激胁迫。MAPK信号通路在所有的动物、植物、微生物等真核生物中广泛存在。迄今为止,MAPK级联反应只在有限的植物,如拟南芥、水稻和杨树等物种中有过相关的系统研究,而MAPKK家族基因在马铃薯中的系统研究还没有相关的文献报导。本研究通过生物信息学分析了马铃薯MAPKK基因,鉴定了5个MAPKK家族基因。通过对马铃薯模拟各种生物胁迫和非生物胁迫,筛选出了与干旱相关的MAPKK基因。构建了MAPKK1基因的过表达载体和Gateway-MAPKK1的干扰表达载体,转化了马铃薯“大西洋”和“紫花白”两个品种获得了转基因马铃薯植株。用qRT-PCR分析了转基因植株中MAPKK1基因的表达量变化。取得的主要研究成果如下:1.通过生物信息学分析,鉴定出5个马铃薯St MAPKK基因。可分为A、B、C、D四组,其中马铃薯StMAPKK基因全长cDNA的平均长度为1001 bp,从1547bp至1574bp不等。马铃薯StMAPKKs编码的氨基酸序列长度从333个氨基酸至515个氨基酸之间,StMAPKKs的预测蛋白质的分子量变化从37.26 kDa至57.45 kDa。推导的多肽的等电点(PI)是从5.47至8.87。而染色体则主要集中于12号染色体和3号染色体。2.通过qRT-PCR分析了马铃薯在生物胁迫和非生物胁迫处理下(4℃、45℃、20%PEG、200 mM NaCl、10 mM H2O2、100μM MeJA、100μM SA、100μM ABA)马铃薯StMAPKK家族基因表达量的变化。以及干旱胁迫下马铃薯StMAPKK基因的表达模式,结果表明在干旱胁迫下MAPKK中多数基因均上调表达,其中StMAPKK1、StMAPKK5上调程度最高。3.选取干旱胁迫下表达量上调最明显的基因MAPKK1用于构建过表达载体。成功构建与抗旱相关的MAPKK1过表达载体,经农杆菌转化后,分别进行PCR鉴定并成功获得相应的转基因StMAPKK1转化植株。经qRT-PCR检测,其转基因马铃薯“大西洋”品种中,StMAPKK1表达量是对照的3.2倍;而转基因马铃薯“紫花白”品种中,StMAPKK1表达量最高为对照“紫花白”品种的7.2倍。4.用Gateway技术构建MAPKK1基因干扰表达载体。经农杆菌遗传转化后,分别进行PCR鉴定并成功获得相应的转基因转化植株。经qRT-PCR检测,其转基因马铃薯“大西洋”品种中,StMAPKK1表达量最低是对照“大西洋”品种的0.09倍;而转基因马铃薯“紫花白”品种中,StMAPKK1表达量最低为对照“紫花白”品种的0.4倍。StMAPKK1基因表达量均得到明显的抑制作用。
[Abstract]:MAPKKs (mitogen-activated protein kinase kinase) is mainly found in animal and plant cells, is a signal transporter, and plays an important role in cells.It is also a key component of MAPK cascade reaction. MAPK signaling pathway has obvious cascade phenomenon in animal and plant cells, is one of the main pathways of signal transduction, and plays a key role in plants, it can be hormone,Different extracellular substances such as cytokines stimulate cells in many important physiological processes, such as growth and development, to respond to external stimuli. MAPK signaling pathways are present in all animals and plants.Eukaryotes such as microbes are widespread.Up to now, the cascade reaction of mitogen-activated kinase (MAPK) has been systematically studied in a limited number of plants, such as Arabidopsis thaliana, rice and poplar, but the systematic study of MAPKK family genes in potato has not been reported.In this study, the MAPKK gene of potato was analyzed by bioinformatics, and five MAPKK family genes were identified.The MAPKK genes related to drought were screened by simulating various biotic and abiotic stresses on potato.The overexpression vector of MAPKK1 gene and the interference expression vector of Gateway-MAPKK1 were constructed and transformed into potato "Atlantic" and "purple flower white" to obtain transgenic potato plants.The changes of MAPKK1 gene expression in transgenic plants were analyzed by qRT-PCR.The main research results are as follows: 1.Five St MAPKK genes were identified by bioinformatics analysis.The average length of full-length cDNA of potato StMAPKK gene was 1001 BP, ranging from 1547bp to 1574bp.The predicted molecular weight of the predicted protein of potato StMAPKKs encoded amino acid sequence ranged from 333 amino acids to 515 amino acids from 37.26 kDa to 57.45 kDa.The isoelectric point (Pi) of the deduced polypeptide is from 5.47 to 8.87.The chromosomes were mainly concentrated on chromosome 12 and chromosome 2. 2.The changes of StMAPKK gene expression in potato StMAPKK family under biological stress and abiotic stress were analyzed by qRT-PCR.And the expression pattern of StMAPKK gene in potato under drought stress. The results showed that most of the genes were up-regulated in MAPKK under drought stress, among which StMAPK1 + StMAPKK5 was the highest.The gene MAPKK1, which was the most obvious up-regulation gene under drought stress, was used to construct overexpression vector.The MAPKK1 overexpression vector related to drought resistance was successfully constructed. After transformed by Agrobacterium tumefaciens, the transformed plants were identified by PCR and the corresponding transgenic StMAPKK1 plants were obtained successfully.QRT-PCR analysis showed that the expression of StMAPKK1 in the transgenic potato "Atlantic" was 3.2 times higher than that in the control, while the highest expression of StMAPKK1 in the transgenic potato "purple flower white" was 7.2 times as high as that in the control.The interference expression vector of MAPKK1 gene was constructed by Gateway technique.After genetic transformation of Agrobacterium tumefaciens, the transgenic plants were identified by PCR and the corresponding transgenic plants were obtained successfully.QRT-PCR analysis showed that the expression of StMAPKK1 in transgenic potato "Atlantic" was 0.09 times of that of the control.However, the expression of StMAPKK1 in transgenic potato "Zihuabai" was the lowest, which was 0.4 times as much as that of "Zihuabai", and the expression of StMAPKK1 was significantly inhibited.
【学位授予单位】：甘肃农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q943.2;S532

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