【摘要】：本文利用Site Finding-PCR方法克隆了白樺BpGT14基因起始密碼子ATG上游2 169 bp序列,并通過PLACE啟動(dòng)子預(yù)測(cè)工具對(duì)其進(jìn)行元件分析。結(jié)果表明,該啟動(dòng)子片段含有啟動(dòng)子核心元件及多種逆境及激素響應(yīng)元件,同時(shí)具有植物苯丙烷及木質(zhì)素生物合成的MYB類轉(zhuǎn)錄因子的重要結(jié)合基序。研究選取了其中含有啟動(dòng)子核心元件的1 156 bp片段構(gòu)建了pBpGT14∷GUS植物表達(dá)載體,利用農(nóng)桿菌侵染的方法將pBpGT14∷GUS報(bào)告基因瞬時(shí)轉(zhuǎn)化煙草植株,鑒定該啟動(dòng)子在煙草中的表達(dá)活性及對(duì)非生物脅迫和激素的響應(yīng)模式。對(duì)轉(zhuǎn)基因煙草植株進(jìn)行GUS染色,結(jié)果表明該啟動(dòng)子具有啟動(dòng)活性,且在莖段處活性較高;進(jìn)一步分析非生物脅迫對(duì)煙草中GUS酶活性的影響,表明該啟動(dòng)子對(duì)ABA、NaCl、PEG及高溫處理均有明顯響應(yīng),且對(duì)于NaCl及PEG處理響應(yīng)迅速。為了更好的鑒定白樺BpGT14基因啟動(dòng)子在白樺細(xì)胞中的啟動(dòng)活性及響應(yīng)模式,本文構(gòu)建了pBpGT14∷GFP載體并瞬時(shí)轉(zhuǎn)化白樺莖段懸浮細(xì)胞,進(jìn)行研究。GFP轉(zhuǎn)錄水平分析結(jié)果與GUS酶活性結(jié)果基本一致,但其中部分時(shí)間點(diǎn)仍存在差異。選取PEG處理3、6、12及24 h的轉(zhuǎn)GFP基因白樺莖段懸浮細(xì)胞,在顯微鏡下觀察其綠色熒光蛋白,以此揭示該啟動(dòng)子對(duì)干旱的響應(yīng)模式。結(jié)果表明,該啟動(dòng)子在白樺莖段懸浮細(xì)胞中啟動(dòng)了GFP的表達(dá),在處理初期(3 h),熒光效果明顯;隨著處理時(shí)間的增加,細(xì)胞脫水明顯,且在細(xì)胞壁表現(xiàn)高亮度熒光。
[Abstract]:In this paper, the upstream 2 169 bp sequence of Bai Hua BpGT14 gene initiation codon (ATG) was cloned by Site Finding-PCR method and analyzed by PLACE promoter prediction tool. The results showed that the promoter fragment contained promoter core elements, various stress and hormone response elements, as well as important binding motifs of MYB transcription factors synthesized by plant phenylpropane and lignin biosynthesis. PBpGT14: GUS plant expression vector was constructed with 1 156 bp fragment containing promoter core element. The pBpGT14: GUS reporter gene was transiently transformed into tobacco plant by Agrobacterium tumefaciens infection. The expression activity of the promoter in tobacco and its response to abiotic stress and hormone were identified. The results of GUS staining on transgenic tobacco plants showed that the promoter had high activity at stem segments, and the effects of abiotic stress on the activity of GUS in tobacco were further analyzed. The results showed that the promoter had obvious response to ABA,NaCl,PEG and high temperature treatment, and rapid response to NaCl and PEG treatment. In order to better identify the promoter activity and response pattern of Bai Hua BpGT14 gene promoter in Bai Hua cells, pBpGT14: GFP vector was constructed and the suspension cells were transformed into the suspensions of Bai Hua stem segment. The results of transcriptional level analysis of .GFP were consistent with the results of GUS activity, but there were still some differences at some time points. In order to reveal the response pattern of the promoter to drought, the suspension cells of GFP gene Bai Hua were treated with PEG for 12 and 24 hours, and the green fluorescent protein was observed under microscope. The results showed that the promoter initiated the expression of GFP in the suspension cells of Bai Hua stem segment, and the effect of 3 h), fluorescence was obvious at the initial stage of treatment, and with the increase of treatment time, the cells were dehydrated obviously and showed high brightness fluorescence in the cell wall.
【作者單位】： 東北林業(yè)大學(xué)生命科學(xué)學(xué)院 林木遺傳育種國家重點(diǎn)實(shí)驗(yàn)室;
【基金】：中央高�；究蒲袠I(yè)務(wù)費(fèi)專項(xiàng)(2572014DA04) 國家自然科學(xué)基金項(xiàng)目(31200463,J1210053)
【分類號(hào)】：S792.153

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