本文關鍵詞： 翅堿蓬 啟動子 FPNI-PCR 順式作用元件　出處：《沈陽農業(yè)大學》2017年碩士論文　論文類型：學位論文

【摘要】：本研究以盤錦紅海灘的翅堿蓬為試驗材料,首先進行實時熒光定量PCR技術分析脫水素基因(DHN)在鹽脅迫條件下基因的表達量,驗證啟動子的類型,然后采用FPNI-PCR方法克隆SsDHN基因的啟動子,運用生物信息學軟件進行預測分析,然后根據啟動子區(qū)域的作用元件進行缺失,構建缺失片段表達載體,再將缺失片段重組表達載體質粒通過農桿菌介導法轉化煙草葉片,進行瞬時表達分析。結果如下:1.NaCl脅迫處理下翅堿蓬SsDHN基因表達分析將40 d的翅堿蓬幼苗在300 mM NaCl脅迫下處理不同的時間,qRT-PCR分析結果表明SsDHN基因在12h基因的表達量達到最高,基因在葉中表達量高于根,表達量表現(xiàn)為先上升后下降,研究結果表明SsDHN啟動子是鹽脅迫誘導型啟動子。2.SsDH 基因全長、啟動子序列的克隆和序列分析根據本課題組獲得的SsDH cDNA序列,克隆得到了SsDH 基因全長,為1253bp,序列中包含一個兩端的邊界為GT-AG的560bp的內含子。采用FPNI-PCR法克隆到了872 bp啟動子,進行PlantCARE分析表明,序列中含有一些保守序列TATA-box、CAAT-box 等,還包含一些如:ABRE、ARE、MRE、CCGTCC-box、CGTCA-motif、GC-motif、TCA-motif、TGACG-motif和MBSI與逆境脅迫相關的順式作用元件等。3.構建缺失表達載體將獲得的5'端缺失片段連接到TA載體上命名為:pMD18T-F1、pMD18T-F2、pMD18T-F3、pMD18T-F4,雙酶切獲得粘性末端的缺失片段與pCAMBIA1303質粒大骨架連接,構建了 4個重組表達載體分別命名為:SsDHNp1-GUS、SsDHNp2-GUS、SsDHN沖3-GUS、SsDHNp4-GUS,經PCR法、雙酶切和序列比對驗證重組表達載體構建成功。4.啟動子的分析采用農桿菌介導法將缺失片段重組表達載體質粒轉化煙草葉圓片,進行GUS瞬時表達分析,試驗表明該啟動子可能存在響應NaCl脅迫、MeJA、ABA、SA的順式作用元件。
[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.
【學位授予單位】：沈陽農業(yè)大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：Q943.2
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本文編號：1512570