本文選題：GhSR13 + 陸地棉�。� 參考：《河南大學(xué)》2016年碩士論文

【摘要】：隨著土地環(huán)境不斷惡化,鹽堿地面積不斷擴大,如何有效利用這些鹽堿地,成為擺在我們面前的現(xiàn)實問題。棉花具有很高的耐鹽性和耐旱性,可是在實際的生產(chǎn)過程中,可利用的耐鹽耐旱品種并不多見,這就需要通過基因工程手段或者分子生物學(xué)手段,尋找出一些耐鹽基因,然后運用基因工程技術(shù),培育出優(yōu)良的棉花品種。前期利用基因芯片測序技術(shù),得到一條EST序列信息,通過NCBI數(shù)據(jù)庫比對分析,陸地棉數(shù)據(jù)庫公布的基因信息,成功克隆了該基因的CDS全長序列,并命名為GhSR13。該基因CDS全長1476 bp,編碼492個氨基酸。隨后進行有關(guān)GhSR13序列生物信息學(xué)的分析。首先,對GhSR13的蛋白家族進行分析發(fā)現(xiàn),該基因?qū)儆贒DE_Tnp_1_6家族。隨后結(jié)合NCBI數(shù)據(jù)庫,以及最近公布的二倍體和四倍體棉花4數(shù)據(jù)庫,做了棉花同源種屬的同源性分析和不同種屬的同源性分析,并對該EST序列的開放閱讀框以及保守結(jié)構(gòu)域進行了預(yù)測分析。通過同源性比對,在番茄以及擬南芥中都發(fā)現(xiàn)了與GhSR13同源的基因,同源性達到了50%以上,在番茄中該同源基因已經(jīng)被證明參與鹽脅迫反應(yīng)途徑,而在擬南芥中,該同源基因也被證明響應(yīng)鹽脅迫途徑。在得到了該基因的CDS序列后,開展了以下實驗。我們首先對該基因是否響應(yīng)鹽脅迫作了定量分析,在用200 mM NaCl處理后,在0h,1 h,4 h,6 h,12 h,24 h,分別提取棉花的真葉和根的RNA反轉(zhuǎn)錄為cDNA后進行定量分析。結(jié)果表明,在根中,GhSR13的表達量發(fā)生了明顯的變化,我們推測測其可能參與了鹽脅迫反應(yīng)途徑。隨后,構(gòu)建了GhSR13-GFP和GhSR13-GUS等一系列的載體,進一步研究該基因是否響應(yīng)鹽脅迫。GFP熒光亞細胞定位結(jié)果表明,GhSR13蛋白定位于細胞核。GUS染色結(jié)果表明,該基因主要在根成熟區(qū)表達。通過構(gòu)建表達有GhSR13-PYES2的酵母菌株并進行鹽處理發(fā)現(xiàn),在200 mM Na Cl,10 m M Li Cl處理條件下,含有目的基因的酵母菌落生長狀況均不如空白對照,進一步表明該基因是參與了鹽脅迫反應(yīng)途徑。在隨后的VIGS實驗中發(fā)現(xiàn),600 mM NaCl處理后的棉花葉片發(fā)生明顯的萎蔫,而且被干涉掉的基因棉花植株萎蔫程度要比對照高,表明在基因干涉掉后,加劇植物對鹽脅迫途徑的調(diào)控反應(yīng),造成植株對鹽的耐受性降低。通過將棉花基因轉(zhuǎn)入擬南芥材料獲得轉(zhuǎn)基因植株,選取了3個株系(OE-2,OE-3,OE-4)進行鹽處理的表型實驗,結(jié)果表明,(OE-2,OE-3,OE-4)這三個轉(zhuǎn)基因種子在萌發(fā)上對鹽的敏感性明顯要比野生型高,根伸長也表現(xiàn)出對鹽更高的敏感性。以上結(jié)果均表明GhSR13基因參與棉花鹽脅迫響應(yīng)調(diào)節(jié)。
[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.
【學(xué)位授予單位】：河南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：S562;Q943.2

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1 熊金濤;棉花鹽脅迫應(yīng)答基因GhSR13的克隆和功能分析[D];河南大學(xué);2016年

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本文編號：2091869