中间锦鸡儿4个R2R3-MYB基因的克隆表达分析

发布时间：2018-01-20 13:47

本文关键词： 中间锦鸡儿 MYB 表达分析启动子克隆　出处：《内蒙古农业大学》2017年硕士论文　论文类型：学位论文

【摘要】：干旱、盐碱、高温、低温和氧化等非生物胁迫是世界范围内作物减产的主要原因。应用转录因子改善植物抗逆性已经成为当今的研究热点。MYB类转录因子是植物中最大的转录因子家族之一,而R2R3-MYB类是植物MYB基因编码蛋白中最丰富的类型。R2R3-MYB在初生与次生代谢、生长发育及对生物与非生物胁迫的应答等各个方面具有重要功能。中间锦鸡儿(Csragana intermedia)作为重要的保水、防风、固沙类灌木,在高寒沙地被作为主要造林灌木树种之一。本研究以中间锦鸡儿为实验材料克隆到4个R2R3-MYB类转录因子,分别命名为CiMYB102、CiMYB31、CiMYB60和CiMYB185,并对其表达模式及启动子顺式作用元件进行了分析。主要结果如下:1.从干旱转录组数据库中筛选到4个R2R3-MYB并对其cDNA和gDNA进行了克隆。CiMYB102、CiMYB31、CiMY60与 CiMYB185 的 ORF 长度分别为 1107 bp、969 bp、1038 bp与906 bp,分别编码369、323、346和302个氨基酸。CiMYB102 CiMYB31、CiMYYB60与CiMYB185基因gDNA序列长度分别为1396 bp、1724 bp、1485 bp和1592 bp,其中CiMYB102、CiYB31和CiMYB60均包含2个内含子和3个外显子,而CiM7B185包含1个内含子和2个外显子。2.利用实时荧光定量PCR技术分析了 4个R2R3-MYB基因不同胁迫下的表达模式。发现CiMYB31和CiMYB185受到低温的诱导,CiMYB102受脱水、NaCl、ABA和千旱的诱导,在脱水、NaCl、UV-B处理下CiMYB60的表达量均降低。表明四个R2R3-MYB可能在中间锦鸡儿对非生物肋迫的响应过程中起作用。3.利用染色体步移技术克隆了 4个基因的启动子序列。CiMYB102、CiMYB31、CiMYB60与CiMYB185的ATG上游序列长度分别为1359 bp、902 bp、1848 bp与908 bp,分析显示启动子序列中包含一些与光反应、组织特异性表达、激素和非生物胁迫相关的响应元件。4.CiMYB102、CiMYTB60及CiMYB31在不同组织部位表达存在差异,在根中的表达量均为最低。CiMYB102在茎中表达量最高,CiMYB31在叶中表达量最高,CiMYB60在叶与茎中表达量均相对较高。5.构建了过表达载体p35s::CiMYB102-GFP、p35s::CiMYB31-GFP和p35s::CiMYB60-GFP,转化野生型拟南芥并筛选得到纯合体株系。
[Abstract]:Drought, salt, high temperature. Abiotic stress, such as low temperature and oxidation, is the main cause of crop reduction in the world. The application of transcription factors to improve plant stress resistance has become a hot topic. MYB transcription factors are the largest transcriptional factors in plants. One of the children. R2R3-MYB is the most abundant type of plant MYB gene encoding protein. R2R3-MYB is primary and secondary metabolism. Growth and development as well as response to biological and abiotic stress have important functions. Csragana intermedia is an important water conservation and windbreak. Sand-fixing shrub was used as one of the main afforestation shrub species in alpine sandy land. In this study, four transcription factors of R2R3-MYB were cloned from Caragana intermedia. They were named CiMYB102, CiMYB31, CiMYB60 and CiMYB185, respectively. The expression pattern and promoter cis-acting elements were analyzed. The main results were as follows:. 1. Four R2R3-MYB were screened from the database of drought transcriptome and their cDNA and gDNA were cloned. CiMYB102. The ORF length of CiMYB31C CiMY60 and CiMYB185 were 1038bp and 906bp, respectively. It encodes 369,323,346 and 302 amino acids. CiMYB102 CiMYB31, respectively. The length of gDNA sequence of CiMYYB60 and CiMYB185 gene was 1396 BP, 1724 BP, 1485 BP and 1592 BP, respectively. Both CiMYB102 and CiYB31 and CiMYB60 contained two introns and three exons. However, CiM7B185 contains one intron and two exons. It is analyzed by real-time fluorescence quantitative PCR. Four R2R3-MYB gene expression patterns under different stress. It was found that CiMYB31 and CiMYB185 were induced by low temperature. CiMYB102 was induced by dehydration and drought. The expression of CiMYB60 decreased under UV-B treatment, which suggested that four R2R3-MYB might play a role in the response of Caragana intermedia to abiotic costal forces. 3. Chromosome step technique was used to detect the effect of R2R3-MYB on the response of Caragana intermedia to abiotic costal forces. Up. The promoter sequence of four genes. CiMYB102. The upstream ATG length of CiMYB31C CiMYB60 and CiMYB185 was 1359bpmc902 BP 1848 BP and 908bp respectively. Analysis showed that the promoter sequence contained some response elements related to light response, tissue specific expression, hormone and abiotic stress. 4. CiMYB102. The expression of CiMYTB60 and CiMYB31 were different in different tissues, and the lowest expression was in root. CiMYB102 was the highest in stem. The expression of CiMYB60 in leaves and stems was higher than that in leaves. 5. the overexpression vector p35s:: CiMYB102-GFP was constructed. P35: s: CiMYB31-GFP and p35s: CiMYB60-GFP.The wild type Arabidopsis thaliana was transformed and homozygous lines were screened.
【学位授予单位】：内蒙古农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q943.2

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