棉花盐胁迫应答基因GhSR13的克隆和功能分析

发布时间：2018-07-03 01:58

本文选题：GhSR13 + 陆地棉　；参考：《河南大学》2016年硕士论文

【摘要】：随着土地环境不断恶化,盐碱地面积不断扩大,如何有效利用这些盐碱地,成为摆在我们面前的现实问题。棉花具有很高的耐盐性和耐旱性,可是在实际的生产过程中,可利用的耐盐耐旱品种并不多见,这就需要通过基因工程手段或者分子生物学手段,寻找出一些耐盐基因,然后运用基因工程技术,培育出优良的棉花品种。前期利用基因芯片测序技术,得到一条EST序列信息,通过NCBI数据库比对分析,陆地棉数据库公布的基因信息,成功克隆了该基因的CDS全长序列,并命名为GhSR13。该基因CDS全长1476 bp,编码492个氨基酸。随后进行有关GhSR13序列生物信息学的分析。首先,对GhSR13的蛋白家族进行分析发现,该基因属于DDE_Tnp_1_6家族。随后结合NCBI数据库,以及最近公布的二倍体和四倍体棉花4数据库,做了棉花同源种属的同源性分析和不同种属的同源性分析,并对该EST序列的开放阅读框以及保守结构域进行了预测分析。通过同源性比对,在番茄以及拟南芥中都发现了与GhSR13同源的基因,同源性达到了50%以上,在番茄中该同源基因已经被证明参与盐胁迫反应途径,而在拟南芥中,该同源基因也被证明响应盐胁迫途径。在得到了该基因的CDS序列后,开展了以下实验。我们首先对该基因是否响应盐胁迫作了定量分析,在用200 mM NaCl处理后,在0h,1 h,4 h,6 h,12 h,24 h,分别提取棉花的真叶和根的RNA反转录为cDNA后进行定量分析。结果表明,在根中,GhSR13的表达量发生了明显的变化,我们推测测其可能参与了盐胁迫反应途径。随后,构建了GhSR13-GFP和GhSR13-GUS等一系列的载体,进一步研究该基因是否响应盐胁迫。GFP荧光亚细胞定位结果表明,GhSR13蛋白定位于细胞核。GUS染色结果表明,该基因主要在根成熟区表达。通过构建表达有GhSR13-PYES2的酵母菌株并进行盐处理发现,在200 mM Na Cl,10 m M Li Cl处理条件下,含有目的基因的酵母菌落生长状况均不如空白对照,进一步表明该基因是参与了盐胁迫反应途径。在随后的VIGS实验中发现,600 mM NaCl处理后的棉花叶片发生明显的萎蔫,而且被干涉掉的基因棉花植株萎蔫程度要比对照高,表明在基因干涉掉后,加剧植物对盐胁迫途径的调控反应,造成植株对盐的耐受性降低。通过将棉花基因转入拟南芥材料获得转基因植株,选取了3个株系(OE-2,OE-3,OE-4)进行盐处理的表型实验,结果表明,(OE-2,OE-3,OE-4)这三个转基因种子在萌发上对盐的敏感性明显要比野生型高,根伸长也表现出对盐更高的敏感性。以上结果均表明GhSR13基因参与棉花盐胁迫响应调节。
[Abstract]:With the continuous deterioration of the land environment and the expansion of the saline-alkali land area, how to make effective use of the saline-alkali land has become a realistic problem in front of us. Cotton has high salt tolerance and drought tolerance, but in the actual production process, the available salt and drought tolerance varieties are rare, so we need to find some salt tolerance genes through genetic engineering or molecular biological means. Then genetic engineering technology is used to cultivate excellent cotton varieties. An EST sequence information was obtained by using gene chip sequencing technique. The full-length CDS sequence of the gene was cloned by comparison analysis of NCBI database and published gene information in upland cotton database and named GhSR13. The gene is 1476 BP in length and encodes 492 amino acids. Then the bioinformatics of GhSR13 sequence was analyzed. Firstly, the protein family of GhSR13 was analyzed. Combined with the NCBI database and the recently published diploid and tetraploid cotton databases, the autologous analysis of cotton autotaxic genera and the homology analysis of different species were made. The open reading frame and conserved domain of the EST sequence are predicted and analyzed. Through homology comparison, GhSR13 homologous gene was found in tomato and Arabidopsis thaliana, and the homology was more than 50%. In tomato, the homologous gene has been proved to be involved in salt stress response pathway, but in Arabidopsis thaliana, the homologous gene has been found in Arabidopsis thaliana. The homologous gene has also been demonstrated to respond to salt stress pathways. After obtaining the CDS sequence of the gene, the following experiments were carried out. At first, the response of the gene to salt stress was quantitatively analyzed. After treated with 200mm NaCl, the RNA of the true leaves and roots of cotton were extracted and transformed into cDNA for 24 h at 0 h, 1 h, 4 h, 6 h, and 24 h, respectively. The results showed that the expression of GhSR13 in root changed obviously, and we speculated that GhSR13 might be involved in salt stress response pathway. Subsequently, a series of vectors, GhSR13-GFP and GhSR13-GUS, were constructed to further study whether the gene was responsive to salt stress. The results of fluorescence subcellular localization showed that GhSR13 protein was located in the nucleus. Gus staining showed that the gene was mainly expressed in the mature root region. The yeast strain expressing GhSR13-PYES2 was constructed and treated with salt. It was found that the colony growth status of yeast containing target gene was lower than that of blank control under 200 mm Na Cl-10 m Li Cl treatment. It is further suggested that the gene is involved in the salt stress response pathway. In the subsequent VIGS experiment, it was found that the leaves of cotton treated with 600 mm NaCl had obvious wilting, and the wilting degree of the transgenic cotton plants was higher than that of the control, indicating that after gene interference, the wilting degree of the transgenic cotton plants was higher than that of the control. The response of plants to salt stress pathway was increased, and the tolerance of plants to salt was decreased. Transgenic plants of Arabidopsis thaliana were obtained by transferring cotton gene into Arabidopsis thaliana. Three lines (OE-2OE-3OE-4) were selected for salt treatment. The results showed that the three transgenic seeds were more sensitive to salt than wild type in germination. Root elongation also showed a higher sensitivity to salt. These results indicated that GhSR13 gene was involved in the regulation of cotton salt stress response.
【学位授予单位】：河南大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S562;Q943.2

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