發(fā)布時(shí)間：2018-02-15 05:47

  本文關(guān)鍵詞： 翅堿蓬 啟動(dòng)子 FPNI-PCR 順式作用元件　出處：《沈陽(yáng)農(nóng)業(yè)大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：本研究以盤(pán)錦紅海灘的翅堿蓬為試驗(yàn)材料,首先進(jìn)行實(shí)時(shí)熒光定量PCR技術(shù)分析脫水素基因(DHN)在鹽脅迫條件下基因的表達(dá)量,驗(yàn)證啟動(dòng)子的類型,然后采用FPNI-PCR方法克隆SsDHN基因的啟動(dòng)子,運(yùn)用生物信息學(xué)軟件進(jìn)行預(yù)測(cè)分析,然后根據(jù)啟動(dòng)子區(qū)域的作用元件進(jìn)行缺失,構(gòu)建缺失片段表達(dá)載體,再將缺失片段重組表達(dá)載體質(zhì)粒通過(guò)農(nóng)桿菌介導(dǎo)法轉(zhuǎn)化煙草葉片,進(jìn)行瞬時(shí)表達(dá)分析。結(jié)果如下:1.NaCl脅迫處理下翅堿蓬SsDHN基因表達(dá)分析將40 d的翅堿蓬幼苗在300 mM NaCl脅迫下處理不同的時(shí)間,qRT-PCR分析結(jié)果表明SsDHN基因在12h基因的表達(dá)量達(dá)到最高,基因在葉中表達(dá)量高于根,表達(dá)量表現(xiàn)為先上升后下降,研究結(jié)果表明SsDHN啟動(dòng)子是鹽脅迫誘導(dǎo)型啟動(dòng)子。2.SsDH 基因全長(zhǎng)、啟動(dòng)子序列的克隆和序列分析根據(jù)本課題組獲得的SsDH cDNA序列,克隆得到了SsDH 基因全長(zhǎng),為1253bp,序列中包含一個(gè)兩端的邊界為GT-AG的560bp的內(nèi)含子。采用FPNI-PCR法克隆到了872 bp啟動(dòng)子,進(jìn)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(guān)的順式作用元件等。3.構(gòu)建缺失表達(dá)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(zhì)粒大骨架連接,構(gòu)建了 4個(gè)重組表達(dá)載體分別命名為:SsDHNp1-GUS、SsDHNp2-GUS、SsDHN沖3-GUS、SsDHNp4-GUS,經(jīng)PCR法、雙酶切和序列比對(duì)驗(yàn)證重組表達(dá)載體構(gòu)建成功。4.啟動(dòng)子的分析采用農(nóng)桿菌介導(dǎo)法將缺失片段重組表達(dá)載體質(zhì)粒轉(zhuǎn)化煙草葉圓片,進(jìn)行GUS瞬時(shí)表達(dá)分析,試驗(yàn)表明該啟動(dòng)子可能存在響應(yīng)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(xué)位授予單位】：沈陽(yáng)農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：Q943.2
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本文編號(hào)：1512570