翅碱蓬DHN启动子的克隆及分析

发布时间：2018-02-15 05:47

  本文关键词： 翅碱蓬 启动子 FPNI-PCR 顺式作用元件　出处：《沈阳农业大学》2017年硕士论文　论文类型：学位论文

【摘要】：本研究以盘锦红海滩的翅碱蓬为试验材料,首先进行实时荧光定量PCR技术分析脱水素基因(DHN)在盐胁迫条件下基因的表达量,验证启动子的类型,然后采用FPNI-PCR方法克隆SsDHN基因的启动子,运用生物信息学软件进行预测分析,然后根据启动子区域的作用元件进行缺失,构建缺失片段表达载体,再将缺失片段重组表达载体质粒通过农杆菌介导法转化烟草叶片,进行瞬时表达分析。结果如下:1.NaCl胁迫处理下翅碱蓬SsDHN基因表达分析将40 d的翅碱蓬幼苗在300 mM NaCl胁迫下处理不同的时间,qRT-PCR分析结果表明SsDHN基因在12h基因的表达量达到最高,基因在叶中表达量高于根,表达量表现为先上升后下降,研究结果表明SsDHN启动子是盐胁迫诱导型启动子。2.SsDH 基因全长、启动子序列的克隆和序列分析根据本课题组获得的SsDH cDNA序列,克隆得到了SsDH 基因全长,为1253bp,序列中包含一个两端的边界为GT-AG的560bp的内含子。采用FPNI-PCR法克隆到了872 bp启动子,进行PlantCARE分析表明,序列中含有一些保守序列TATA-box、CAAT-box 等,还包含一些如:ABRE、ARE、MRE、CCGTCC-box、CGTCA-motif、GC-motif、TCA-motif、TGACG-motif和MBSI与逆境胁迫相关的顺式作用元件等。3.构建缺失表达载体将获得的5'端缺失片段连接到TA载体上命名为:pMD18T-F1、pMD18T-F2、pMD18T-F3、pMD18T-F4,双酶切获得粘性末端的缺失片段与pCAMBIA1303质粒大骨架连接,构建了 4个重组表达载体分别命名为:SsDHNp1-GUS、SsDHNp2-GUS、SsDHN冲3-GUS、SsDHNp4-GUS,经PCR法、双酶切和序列比对验证重组表达载体构建成功。4.启动子的分析采用农杆菌介导法将缺失片段重组表达载体质粒转化烟草叶圆片,进行GUS瞬时表达分析,试验表明该启动子可能存在响应NaCl胁迫、MeJA、ABA、SA的顺式作用元件。
[Abstract]:In this study, the expression of dehydrin gene (DHN) under salt stress was analyzed by real-time fluorescence quantitative PCR technique, and the type of promoter was verified. Then, the promoter of SsDHN gene was cloned by FPNI-PCR method, and predicted by bioinformatics software, then the expression vector was constructed according to the deletion of the acting elements in the promoter region. The recombinant expression vector plasmid was transformed into tobacco leaves by Agrobacterium tumefaciens. The results of transient expression analysis were as follows: 1. The expression of SsDHN gene of Suaeda salsa was analyzed under NaCl stress. The results of qRT-PCR analysis showed that the expression of SsDHN gene reached the highest level at 12h after treatment with 300mm NaCl for 40 days. The results showed that SsDHN promoter was salt-stress inducible promoter. 2. SsDH gene was full length. Cloning and sequence Analysis of Promoter sequence according to the SsDH cDNA sequence obtained by our research group, the full-length SsDH gene was cloned, which contains a 560bp intron with the boundary of GT-AG at both ends. 872bp promoter was cloned by FPNI-PCR method. PlantCARE analysis showed that the sequence contained some conservative sequences, such as TATA-box, CAAT-box and so on. It also contains some cis-acting elements such as CGTCA-motifen GC-motifen TGACG-motif and MBSI associated with stress stress, etc. The 5'terminal deletion fragment obtained by constructing the deletion expression vector will be linked to TA vector and named as''pMD18T-F1' pMD18T-F2 pMD18T-F3pMD18T-F4. the deletion expression vector will be linked to the TA vector under the name of'w 'pMD18T-F2 pMD18T-F3pMD18T-F4. Plasmids linked to large skeletons, Four recombinant expression vectors were constructed and named as SsDHNp1-GUSN SsDHNp2-GUSS SsDHHNp4-GUSS by PCR method. The recombinant expression vector was constructed successfully by double enzyme digestion and sequence alignment. The recombinant expression vector plasmid was transformed into tobacco leaf disk by Agrobacterium tumefaciens mediated by Agrobacterium tumefaciens, and the transient expression of GUS was analyzed. The results showed that the promoter might have a cis-acting element in response to NaCl stress.
【学位授予单位】：沈阳农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q943.2
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本文编号：1512570