珙桐DiMYB1基因的克
发布时间：2018-07-24 12:08
【摘要】：珙桐(Davidia involucrata Baill.)为珙桐科珙桐属落叶乔木,第三纪古热带植物孑遗种,我国特有的珍稀植物,国家I级重点保护濒危物种,具有极高的观赏价值和学术研究价值。本研究从珙桐果实及种子转录组数据库中筛选到一个编码调控原花青素生物合成的MYB转录因子的基因DiMYB1,对其进行了克隆及功能分析。主要研究结果如下:1.珙桐DiMYB1基因的克隆及生物信息学分析。根据珙桐果实及种子转录组数据库中该基因的ORF序列设计特异引物,以珙桐败育种子的cDNA为模板进行RCR扩增得到全长序列,并在GenBank上登录,登录号为KR996175。利用在线软件对该基因序列进行了生物信息学分析。2.DiYB1基因的表达模式。①DiMYB1基因在珙桐芽、叶片、苞片、果肉、种子中的表达量qPCR分析。结果表明,DiMYB1基因在紫色幼叶中的表达量最高,其次是雄蕊,在芽、茎、成熟叶片、苞片、果肉和种子中微量表达。②DiMYB1基因在正常和败育种子中的表达模式。qPCR分析结果表明,D/MYB1基因在败育种子中的表达量显著高于正常种子,且在中期败育种子中的表达量达到最高。③DiMYB/基因在不同颜色叶片中的表达。DiMYB1基因在紫色叶片中的相对表达量最高,其次是中间色(介于紫色和淡黄绿色之间)叶片,在绿色叶片中表达量较低。3.珙桐DiMYB1基因的原核表达。构建原核表达载体pET28a(+)-DiMYB1,高效表达蛋白质,采用His标签纯化蛋白,DiMYB1蛋白以包涵体的形式存在。4.珙桐DiMYB1基因功能在拟南芥中的初步验证。构建植物超量表达载体pBI-121-DiMYB1,重组转化农杆菌菌株GV3101,采用花序浸染法转化拟南芥花序,收获种子后,通过抗性培养筛选阳性植株,自交繁殖2代后得到T2代转基因植株。与野生型拟南芥相比,DiMYB1超量表达植株的主要表型差异表现在:大部分幼苗叶片颜色变紫;多数幼苗叶片的形态和数目发生了明显变化,有的叶片出现了白化现象;少数成熟豆荚颜色变紫;有的豆荚不饱满,种子有皱缩现象;植株矮小,茎秆细长,提前进入生殖生长。后续将筛选纯合子转基因植株,验证珙桐DiMB1基因的功能。
[Abstract]:Davidia involucrata (Davidia involucrata Baill.) It is a deciduous tree of the genus Davidia involucrata, a relict species of Tertiary paleotropical plants, a rare and rare plant endemic to China, and a national class I protected endangered species, which has very high ornamental value and academic research value. In this study, a gene DiMYB1 encoding MYB transcription factor regulating procyanidins biosynthesis was screened from the database of fruit and seed transcriptome of Davidia involucrata. The gene was cloned and its function was analyzed. The main results are as follows: 1. Cloning and bioinformatics analysis of DiMYB1 gene from Davidia involucrata. According to the ORF sequence of Davidia involucrata fruit and seed transcriptome database, a specific primer was designed. The full-length RCR sequence was amplified by using the cDNA of Davidia involucrata breeders as template, and the full-length sequence was registered on GenBank. The accession number was KR996175. The expression pattern of DiYB1 gene in bud, leaf, bract, pulp and seed of Davidia involucrata was analyzed with online software. 2. The expression level of DiYB1 gene in bud, leaf, bract, pulp and seed was analyzed by qPCR. The results showed that DiMYB1 gene expression was the highest in young purple leaves, followed by stamens, buds, stems, mature leaves and bracts. The expression pattern of DiMYB1 gene in both normal and abortive breeders was analyzed by qPCR. The results showed that the expression of D _ (r) MYB1 gene in abortive breeders was significantly higher than that in normal seeds. The highest expression level of .3DiMYB/ gene was found in different color leaves, and the relative expression of DiMYB1 gene was the highest in purple leaves, followed by intermediate color leaves (between purple and yellowish green). The expression level in green leaves was lower. 3. Prokaryotic expression of DiMYB1 gene in Davidia involucrata. The prokaryotic expression vector pET28a () -DiMYB1 was constructed and the protein was highly expressed. The protein was purified by His tag and existed in the form of inclusion body. The function of Davidia involucrata DiMYB1 gene in Arabidopsis thaliana was preliminarily verified. The plant overexpression vector pBI-121-DiMYB1 was constructed and transformed into Agrobacterium tumefaciens strain GV3101. The inflorescence was transformed into Arabidopsis thaliana by inflorescence soaking. After harvesting the seeds, the positive plants were screened by resistance culture, and the transgenic plants of T2 generation were obtained after 2 generations of self-breeding. Compared with wild type Arabidopsis thaliana, the main phenotypic differences of DiMYB1 overexpression plants were as follows: the color of most seedling leaves became purple, the morphology and number of most seedling leaves changed obviously, and some leaves appeared albinism. A few mature pods became purple in color; some of them were not full and the seeds were crumpled; the plants were short and the stems were slender and entered into reproductive growth ahead of time. The homozygous transgenic plants will be screened to verify the function of DiMB1 gene in Davidia involucrata.
【学位授予单位】：中南林业科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S792.99;Q943.2
，
本文编号：2141333
[Abstract]:Davidia involucrata (Davidia involucrata Baill.) It is a deciduous tree of the genus Davidia involucrata, a relict species of Tertiary paleotropical plants, a rare and rare plant endemic to China, and a national class I protected endangered species, which has very high ornamental value and academic research value. In this study, a gene DiMYB1 encoding MYB transcription factor regulating procyanidins biosynthesis was screened from the database of fruit and seed transcriptome of Davidia involucrata. The gene was cloned and its function was analyzed. The main results are as follows: 1. Cloning and bioinformatics analysis of DiMYB1 gene from Davidia involucrata. According to the ORF sequence of Davidia involucrata fruit and seed transcriptome database, a specific primer was designed. The full-length RCR sequence was amplified by using the cDNA of Davidia involucrata breeders as template, and the full-length sequence was registered on GenBank. The accession number was KR996175. The expression pattern of DiYB1 gene in bud, leaf, bract, pulp and seed of Davidia involucrata was analyzed with online software. 2. The expression level of DiYB1 gene in bud, leaf, bract, pulp and seed was analyzed by qPCR. The results showed that DiMYB1 gene expression was the highest in young purple leaves, followed by stamens, buds, stems, mature leaves and bracts. The expression pattern of DiMYB1 gene in both normal and abortive breeders was analyzed by qPCR. The results showed that the expression of D _ (r) MYB1 gene in abortive breeders was significantly higher than that in normal seeds. The highest expression level of .3DiMYB/ gene was found in different color leaves, and the relative expression of DiMYB1 gene was the highest in purple leaves, followed by intermediate color leaves (between purple and yellowish green). The expression level in green leaves was lower. 3. Prokaryotic expression of DiMYB1 gene in Davidia involucrata. The prokaryotic expression vector pET28a () -DiMYB1 was constructed and the protein was highly expressed. The protein was purified by His tag and existed in the form of inclusion body. The function of Davidia involucrata DiMYB1 gene in Arabidopsis thaliana was preliminarily verified. The plant overexpression vector pBI-121-DiMYB1 was constructed and transformed into Agrobacterium tumefaciens strain GV3101. The inflorescence was transformed into Arabidopsis thaliana by inflorescence soaking. After harvesting the seeds, the positive plants were screened by resistance culture, and the transgenic plants of T2 generation were obtained after 2 generations of self-breeding. Compared with wild type Arabidopsis thaliana, the main phenotypic differences of DiMYB1 overexpression plants were as follows: the color of most seedling leaves became purple, the morphology and number of most seedling leaves changed obviously, and some leaves appeared albinism. A few mature pods became purple in color; some of them were not full and the seeds were crumpled; the plants were short and the stems were slender and entered into reproductive growth ahead of time. The homozygous transgenic plants will be screened to verify the function of DiMB1 gene in Davidia involucrata.
【学位授予单位】：中南林业科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S792.99;Q943.2
，
本文编号：2141333
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