丹参NOX基因鉴定及SmRbohE基因的克隆

发布时间：2018-03-05 22:24

  本文选题：丹参　切入点：SmRboh　出处：《西北农林科技大学》2017年硕士论文　论文类型：学位论文

【摘要】：NADPH 氧化酶(NADPH oxidase,NOX)是植物产生活性氧(reactive oxygen species,ROS)的来源之一,NOX家族成员众多,功能多样,在植物生长发育过程中和生物和非生物胁迫应答的信号转导的过程中发现NADPH氧化酶有着重要的作用,而且在药用植物中发现NADPH氧化酶与植物次生代谢有紧密联系。本研究以丹参为实验材料,利用RACE扩增技术,然后从丹参中克隆得到一条完整SmRbohE基因的cDNA序列,并对其转录组数据库中NOX基因家族的cDNA和其所翻译表达的氨基酸序列进行生物信息学分析。通过实时定量PCR(qRT-PCR)分析用水杨酸、茉莉酸甲酯、过氧化氢处理丹参悬浮细胞后其丹参NOX家族基因的相对表达量以及在不同组织部位的表达特异性。构建了SmRbohE过表达载体和RNAi沉默(RNA interference,RNAi)载体,把表达载体转入农杆菌感受态细胞中,并用农杆菌侵染丹参无菌苗叶片得到转基因丹参,从而研究在次生代谢中丹酚酸B合成过程中SmRbohE的作用。实验取得了下列主要研究成果:1.依据NOX蛋白特有功能域,在现有转录组数据库基础上对丹参NOX同源基因进行检索,鉴定到3个丹参NOX同源基因;采用cDNA末端快速扩增技术的方式,补充得到SmRbohE基因。综上,共鉴定到3个丹参MOX同源基因。2.生物信息学预测丹参SmRbohA、SmRbohC、SmRbohE的蛋白结构域,发现3个蛋白都有NADPH氧化酶结构域,而且都有一个跨膜结构域因此都是跨膜蛋白,丹参SmRbohA蛋白有938个氨基酸,等电点为9.23,分子量为107303.32,平均疏水性-0.251。丹参SmRbohC蛋白有935个氨基酸,等电点为9.06,分子量为105232.13,平均疏水性-0.340。丹参SmRbohE蛋白有908个氨基酸,等电点为8.98,分子量为101983.07,平均疏水性-0.124,丹参 SmRbohA、SmRbohC、SmRbohE 均无信号肽,不是分泌蛋白。构建丹参中NOX家族基因系统发育树,SmRbohA与SiRbohA相似度最高为90%,SmRbohA与AtRbohF相似度为78%,SmRbohE与SiRbohE相似度最高为85%,SmRbohE与AtRbohE相似度为64%。SmRbohC与SiRbohC相似度最高为90%,SmRbohC 与 AtRbohC 相似度为 68%。3.克隆出SmRbohE的基因,得到了其cDNA全长,命名为SmRbohE。SmRbohE全长 cDNA 全长为 3020 bp,包含 147bp 的 5'-URT,146 bp 的 3'-URT 和一个 2727 bp的开放阅读框(ORF)。4.利用qRT-PCR技术检测NOX基因家族在丹参植株根、茎、花、叶和愈伤组织中的表达量,SmRbohA在叶和愈伤组织中表达量最高,在花中也有很高的表达,SmRbohC在叶中表达量最高,SmRbohE在叶和愈伤组织中表达量最高。用不同激素、信号分子处理丹参悬浮培养细胞,结果表明,水杨酸、茉莉酸甲酯、过氧化氢均能使SmRbohA、SmRbohC、SmRbohE在mRNA水平显著提高,其中SA、H202的处理效果最显著。5.构建了SmRboh 基因的过表达和RNAi沉默表达载体,并转化过表达和RNAi沉默表达植株,为后期研究SmRbohE在丹酚酸B合成积累中的作用提供基础。
[Abstract]:NADPH oxidase NADPH-oxidase NOX is one of the sources of reactive oxygen species-ROSs produced by plants. NADPH oxidase was found to play an important role in the growth and development of plants and in the signal transduction of biological and abiotic stress response. NADPH oxidase was found to be closely related to plant secondary metabolism in medicinal plants. In this study, a complete cDNA sequence of SmRbohE gene was cloned from Salvia miltiorrhiza by RACE amplification. The cDNA of NOX gene family and the amino acid sequence translated and expressed in its transcriptional database were analyzed by bioinformatics. The real-time quantitative analysis was performed with salicylic acid and methyl jasmonate. After being treated with hydrogen peroxide, the relative expression amount of NOX family genes and the expression specificity in different tissues of Salvia miltiorrhiza suspension cells were obtained. SmRbohE overexpression vector and RNAi silencing RNA interference RNAi vector were constructed. Transgenic salvia miltiorrhiza (Salvia miltiorrhiza) was obtained by infusing Agrobacterium tumefaciens into the competent cells of Agrobacterium tumefaciens. Thus, the role of SmRbohE in the synthesis of Salvianolic acid B in secondary metabolism was studied. The following main research results were obtained: 1. According to the specific functional domain of NOX protein, the homologous gene of Salviae miltiorrhiza NOX was searched on the basis of existing transcriptional database. Three homologous genes of Salvia miltiorrhiza NOX were identified and the SmRbohE gene was supplemented by cDNA terminal rapid amplification technique. In summary, three homologous genes of MOX were identified. Bioinformatics was used to predict the protein domain of SmRbohCnSmRbohCnrbohE of Salvia miltiorrhiza. It was found that all three proteins had NADPH oxidase domain, and all of them had a transmembrane domain. The SmRbohA protein of Salvia miltiorrhiza had 938 amino acids, the isoelectric point was 9.23, the molecular weight was 107303.32, and the average hydrophobicity was -0.251. The SmRbohC protein of Salvia miltiorrhiza had 935 amino acids. The isoelectric point was 9.06, the molecular weight was 105232.13, and the average hydrophobicity was -0.340. The SmRbohE protein had 908 amino acids, the isoelectric point was 8.98, the molecular weight was 101983.07, and the average hydrophobicity was -0.124. The highest similarity between SmRbohA and AtRbohF is 78Sm RbohA and the highest similarity between SmRbohA and SiRbohE is 850.SmRbohE and AtRbohE are 64.SmRbohC and SiRbohC have the highest similarity between SmRbohC and AtRbohC. Clone the SmRbohE gene for 68. 3. The full length of its cDNA, named SmRbohE.SmRbohE, is 3020bp. it contains 147bp cDNA with 147bp 5- URT146bp and an open reading frame of 2727bp. QRT-PCR technique is used to detect the NOX gene family in root, stem and flower of Salvia miltiorrhiza (Salvia miltiorrhiza). The expression of SmRbohA in leaf and callus was the highest, and that of SmRbohC in flower was the highest. SmRbohE was the highest in leaf and callus. The results showed that salicylic acid, methyl jasmonate and hydrogen peroxide could significantly increase the level of SmRbohCon SmRbohE in the suspension culture cells of Salvia miltiorrhiza. 5. The overexpression of SmRboh gene and RNAi silencing expression vector were constructed, and the overexpression and RNAi silencing expression plants were transformed to provide the basis for studying the role of SmRbohE in the synthesis and accumulation of Salvianolic acid B.
【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】：S567.53
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本文编号：1572176