丹参C2-SRC2-like基因的克隆及其表达分析
发布时间：2019-01-08 07:58
【摘要】：丹参(Salvia miltiorrhiza Bunge)是唇形科鼠尾草属的一种常见药用植物,入药部分为其干燥的根和茎,水溶性的酚酸类次生代谢物是其重要的药用成分。植物中的钙离子作为胞内第二信使参与植物生长发育、代谢和胁迫应答等的信号转导过程。本研究使用的材料为悬浮培养的丹参细胞,从丹参中克隆获得一条含有C2-SRC2特异结合位点的、编码能够结合钙离子的C2-SRC2-like蛋白的基因,将其命名为丹参C2-SRC2-like蛋白(C2-SRC2-like基因),并利用生物信息学在线软件分析其核酸和氨基酸序列。分别将丹参培养细胞进行水杨酸、氯化钙和缺钙处理,利用实时定量荧光PCR(qRT-PCR)分析丹参C2-SRC2-like基因表达情况。进一步构建C2-SRC2-like的过表达和反义表达载体,并用叶盘法侵染丹参叶片得到过表达和反义表达植株,探究C2-SRC2-like蛋白在丹参酚酸类次生代谢物生物合成过程中的作用。取得了以下主要研究结果:1、克隆出丹参C2-SRC2-like基因,得到了cDNA全长。丹参C2-SRC2-like基因包括一个109 bp的5’-非翻译区、1134 bp的开放阅读框和273 bp的3’-非翻译区,全长共1516 bp,能够编码377个氨基酸。2、生物信息学分析预测C2-SRC2-like蛋白分子量为38.9 kDa,等电点为6.9,是不具信号肽、含有一个跨膜结构的亲水性蛋白。该蛋白可能位于细胞核,具有C2-SRC2特异结合位点,属于C2超家族。与芝麻SRC2-like蛋白的氨基酸序列相似性最高,为72%。3、丹参悬浮培养细胞进行激素(SA)、信号分子(Ca2+)和缺钙处理发现,水杨酸、过量钙能使C2-SRC2-like基因在m RNA水平相对表达量显著提高,且分别在50 min和40 min达到峰值,为对照组的1.9倍和2.3倍;缺钙处理后C2-SRC2-like基因在m RNA水平显著并持续降低。4、成功构建了C2-SRC2-like基因的过表达和反义表达载体,为后期研究C2-SRC2-like蛋白在丹参酚酸类次生代谢物生物合成过程中的作用和其生物学功能奠定基础。5、成功将C2-SRC2-like基因的过表达和反义表达载体转入农杆菌GV3101,并用叶盘法侵染丹参叶片得到过表达和反义表达植株,在基因水平上检测出过表达和反义表达载体均已成功转入丹参无菌苗,为研究C2-SRC2-like蛋白在丹参酚酸类次生代谢物生物合成过程中的作用研究提供材料。
[Abstract]:Salvia miltiorrhiza (Salvia miltiorrhiza Bunge) (Salvia miltiorrhiza) is a common medicinal plant in the genus Salvia. Calcium ions in plants are involved in the signal transduction of plant growth and development, metabolism and stress response as the second messenger in the cell. In this study, a C2-SRC2-like protein encoding calcium binding site was cloned from Salvia miltiorrhiza cells cultured in suspension culture, and a specific binding site of C2-SRC2-like was obtained from Salvia miltiorrhiza (Salvia miltiorrhiza). It was named Salvia miltiorrhiza C2-SRC2-like protein (C2-SRC2-like gene) and its nucleic acid and amino acid sequences were analyzed by bioinformatics online software. Salicylic acid, calcium chloride and calcium deficiency were treated with salicylic acid, calcium chloride and calcium deficiency in Salvia miltiorrhiza cultured cells. The expression of C2-SRC2-like gene in Salvia miltiorrhiza was analyzed by real-time quantitative PCR (qRT-PCR). The overexpression and antisense expression vectors of C2-SRC2-like were further constructed, and the overexpression and antisense expression of C2-SRC2-like protein were obtained by leaf disk method. The role of C2-SRC2-like protein in the biosynthesis of Salvianolic acid secondary metabolites was investigated. The main results are as follows: 1. The C2-SRC2-like gene of Salvia miltiorrhiza was cloned and the full length of cDNA was obtained. The C2-SRC2-like gene of Salvia miltiorrhiza contains an untranslated region of 10 9 bp, an open reading frame of 1134 bp and a 3 '-untranslated region of 273 bp. The total length of 1516 bp, can encode 377 amino acids. Bioinformatics analysis predicted that the molecular weight of C2-SRC2-like protein was 38.9 kDa, isoelectric point 6.9, which was not a signal peptide and contained a transmembrane hydrophilic protein. This protein may be located in the nucleus, with C2-SRC2 specific binding site, belonging to the C 2 superfamily. The amino acid sequence of Sesame SRC2-like protein was similar to that of Sesame seed protein (722.3. Salvia miltiorrhiza suspension culture cells were treated with hormone (SA), signaling molecule (Ca2) and calcium deficiency), salicylic acid, salicylic acid, Excess calcium significantly increased the relative expression of C2-SRC2-like gene at m RNA level, and reached the peak at 50 min and 40 min, which was 1.9-fold and 2.3-fold higher than that in the control group. After calcium deficiency treatment, C2-SRC2-like gene was significantly decreased at m RNA level. 4. The overexpression and antisense expression vector of C2-SRC2-like gene were successfully constructed. In order to study the role and biological function of C2-SRC2-like protein in the biosynthesis of Salvianolic acid secondary metabolites. 5. The overexpression and antisense expression vector of C2-SRC2-like gene were successfully transferred into Agrobacterium tumefaciens GV3101,. Overexpression and antisense expression plants were obtained from the leaves of Salvia miltiorrhiza infected with leaf disk method. The overexpression and antisense expression vectors were detected at the gene level and were successfully transferred into the sterile seedlings of Salvia miltiorrhiza. To study the role of C2-SRC2-like protein in Salvianolic acid secondary metabolites biosynthesis.
【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
，
本文编号：2404322
[Abstract]:Salvia miltiorrhiza (Salvia miltiorrhiza Bunge) (Salvia miltiorrhiza) is a common medicinal plant in the genus Salvia. Calcium ions in plants are involved in the signal transduction of plant growth and development, metabolism and stress response as the second messenger in the cell. In this study, a C2-SRC2-like protein encoding calcium binding site was cloned from Salvia miltiorrhiza cells cultured in suspension culture, and a specific binding site of C2-SRC2-like was obtained from Salvia miltiorrhiza (Salvia miltiorrhiza). It was named Salvia miltiorrhiza C2-SRC2-like protein (C2-SRC2-like gene) and its nucleic acid and amino acid sequences were analyzed by bioinformatics online software. Salicylic acid, calcium chloride and calcium deficiency were treated with salicylic acid, calcium chloride and calcium deficiency in Salvia miltiorrhiza cultured cells. The expression of C2-SRC2-like gene in Salvia miltiorrhiza was analyzed by real-time quantitative PCR (qRT-PCR). The overexpression and antisense expression vectors of C2-SRC2-like were further constructed, and the overexpression and antisense expression of C2-SRC2-like protein were obtained by leaf disk method. The role of C2-SRC2-like protein in the biosynthesis of Salvianolic acid secondary metabolites was investigated. The main results are as follows: 1. The C2-SRC2-like gene of Salvia miltiorrhiza was cloned and the full length of cDNA was obtained. The C2-SRC2-like gene of Salvia miltiorrhiza contains an untranslated region of 10 9 bp, an open reading frame of 1134 bp and a 3 '-untranslated region of 273 bp. The total length of 1516 bp, can encode 377 amino acids. Bioinformatics analysis predicted that the molecular weight of C2-SRC2-like protein was 38.9 kDa, isoelectric point 6.9, which was not a signal peptide and contained a transmembrane hydrophilic protein. This protein may be located in the nucleus, with C2-SRC2 specific binding site, belonging to the C 2 superfamily. The amino acid sequence of Sesame SRC2-like protein was similar to that of Sesame seed protein (722.3. Salvia miltiorrhiza suspension culture cells were treated with hormone (SA), signaling molecule (Ca2) and calcium deficiency), salicylic acid, salicylic acid, Excess calcium significantly increased the relative expression of C2-SRC2-like gene at m RNA level, and reached the peak at 50 min and 40 min, which was 1.9-fold and 2.3-fold higher than that in the control group. After calcium deficiency treatment, C2-SRC2-like gene was significantly decreased at m RNA level. 4. The overexpression and antisense expression vector of C2-SRC2-like gene were successfully constructed. In order to study the role and biological function of C2-SRC2-like protein in the biosynthesis of Salvianolic acid secondary metabolites. 5. The overexpression and antisense expression vector of C2-SRC2-like gene were successfully transferred into Agrobacterium tumefaciens GV3101,. Overexpression and antisense expression plants were obtained from the leaves of Salvia miltiorrhiza infected with leaf disk method. The overexpression and antisense expression vectors were detected at the gene level and were successfully transferred into the sterile seedlings of Salvia miltiorrhiza. To study the role of C2-SRC2-like protein in Salvianolic acid secondary metabolites biosynthesis.
【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
，
本文编号：2404322
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