【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啟動子預測工具對其進行元件分析。結果表明,該啟動子片段含有啟動子核心元件及多種逆境及激素響應元件,同時具有植物苯丙烷及木質素生物合成的MYB類轉錄因子的重要結合基序。研究選取了其中含有啟動子核心元件的1 156 bp片段構建了pBpGT14∷GUS植物表達載體,利用農桿菌侵染的方法將pBpGT14∷GUS報告基因瞬時轉化煙草植株,鑒定該啟動子在煙草中的表達活性及對非生物脅迫和激素的響應模式。對轉基因煙草植株進行GUS染色,結果表明該啟動子具有啟動活性,且在莖段處活性較高;進一步分析非生物脅迫對煙草中GUS酶活性的影響,表明該啟動子對ABA、NaCl、PEG及高溫處理均有明顯響應,且對于NaCl及PEG處理響應迅速。為了更好的鑒定白樺BpGT14基因啟動子在白樺細胞中的啟動活性及響應模式,本文構建了pBpGT14∷GFP載體并瞬時轉化白樺莖段懸浮細胞,進行研究。GFP轉錄水平分析結果與GUS酶活性結果基本一致,但其中部分時間點仍存在差異。選取PEG處理3、6、12及24 h的轉GFP基因白樺莖段懸浮細胞,在顯微鏡下觀察其綠色熒光蛋白,以此揭示該啟動子對干旱的響應模式。結果表明,該啟動子在白樺莖段懸浮細胞中啟動了GFP的表達,在處理初期(3 h),熒光效果明顯;隨著處理時間的增加,細胞脫水明顯,且在細胞壁表現(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)大學生命科學學院 林木遺傳育種國家重點實驗室;
【基金】：中央高�；究蒲袠I(yè)務費專項(2572014DA04) 國家自然科學基金項目(31200463,J1210053)
【分類號】：S792.153

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