【摘要】：【目的】克隆GmPAP4啟動子(PAP4-pro),并分析其表達特性,為進一步研究其作用機制奠定基礎(chǔ)�！痉椒ā恳罁�(jù)GmPAP4 c DNA序列(Gen Bank No.HQ162477),通過比對大豆參考基因組,設(shè)計特異引物,克隆GmPAP4啟動子序列,通過PLACE與Plant CARE在線生物信息學(xué)數(shù)據(jù)庫預(yù)測該啟動子相關(guān)調(diào)控元件。構(gòu)建GmPAP4啟動子驅(qū)動GUS表達載體(PAP4-pro-GUS)并轉(zhuǎn)化根癌農(nóng)桿菌GV3101;通過Floral dip法將PAP4-pro-GUS轉(zhuǎn)化擬南芥,利用卡那霉素(Kan)抗性篩選和特異引物的PCR鑒定,最終獲得T3轉(zhuǎn)基因擬南芥。通過對T_3轉(zhuǎn)基因擬南芥不同組織GUS染色,分析啟動子的組織表達特性,將T3轉(zhuǎn)基因擬南芥通過適磷和植酸磷處理,20 d后,取其根部進行GUS活性和表達分析,研究啟動子對不同磷環(huán)境的響應(yīng)。【結(jié)果】克隆了GmPAP4上游啟動子序列,通過PLACE與Plant CARE在線生物信息學(xué)數(shù)據(jù)庫預(yù)測顯示,GmPAP4啟動子除含有啟動子核心的調(diào)控元件外,還含有(1)組織特異調(diào)控元件:as1(根系特異表達調(diào)控元件)和Skn-1_motif(胚乳特異表達調(diào)控元件);(2)應(yīng)答元件:TC-rich repeats(逆境脅迫反應(yīng)調(diào)控元件)和Box-W3(真菌應(yīng)答相關(guān)調(diào)控元件);(3)結(jié)合位點:MBS(MYB轉(zhuǎn)錄因子的結(jié)合位點)等。不同組織GUS染色結(jié)果顯示,轉(zhuǎn)基因擬南芥整個根系GUS染色較深,莖、葉中僅微管組織有較明顯GUS染色,花瓣微管組織中也能觀察到微弱GUS染色。定量PCR結(jié)果顯示,植酸磷處理條件下轉(zhuǎn)基因擬南芥根系GUS表達比適磷處理提高了1.3倍(P0.05);同時GUS活性測定顯示,與適磷處理相比,植酸磷處理條件下轉(zhuǎn)基因擬南芥根系GUS活性提高了1.9倍(P0.05)�！窘Y(jié)論】獲得大豆GmPAP4啟動子,通過不同組織GUS染色和不同磷環(huán)境GUS表達分析顯示該啟動子主要在根部且受低磷信號誘導(dǎo)表達,為誘導(dǎo)型啟動子。
[Abstract]:[objective] to clone GmPAP4 promoter (PAP4-pro) and analyze its expression characteristics, so as to lay a foundation for further study of its mechanism. [methods] according to the GmPAP4 c DNA sequence (Gen Bank No.HQ162477), a specific primer was designed by comparing the reference genome of soybean. GmPAP4 promoter sequence was cloned and predicted by PLACE and Plant CARE online bioinformatics database. GmPAP4 promoter driven GUS expression vector (PAP4-pro-GUS) was constructed and transformed into Agrobacterium tumefaciens GV3101; by Floral dip method to transform PAP4-pro-GUS into Arabidopsis thaliana. Finally, T3 transgenic Arabidopsis thaliana was obtained by screening kanamycin (Kan) resistance and PCR identification of specific primers. The tissue expression characteristics of T 3 transgenic Arabidopsis thaliana were analyzed by GUS staining. The GUS activity and expression of T 3 transgenic Arabidopsis thaliana were analyzed by GUS activity and expression analysis in the roots of T 3 transgenic Arabidopsis thaliana treated with phosphorus and phytate for 20 days. [results] the upstream promoter sequence of GmPAP4 was cloned and predicted by PLACE and Plant CARE online bioinformatics database. It also contains (1) the binding sites of the tissue specific regulatory element: as1 (root specific expression regulator) and Skn-1_motif (endosperm specific expression regulatory element); (2) response element: TC-rich repeats (stress regulatory element) and Box-W3 (fungal response-related regulatory element); (3). Points: MBS (MYB transcription factor binding sites) and so on. The results of GUS staining in different tissues showed that the GUS staining of the whole root system of transgenic Arabidopsis thaliana was deep, only microtubule tissue was stained by GUS in stem and leaf, and weak GUS staining was also observed in petal microtubule tissue. The results of quantitative PCR showed that the expression of GUS in transgenic Arabidopsis thaliana root system was 1.3-fold higher than that of the control (P0.05), and the GUS activity of transgenic Arabidopsis thaliana was higher than that of the control (P0.05). The GUS activity of transgenic Arabidopsis thaliana roots was increased by 1.9 times (P0.05). [conclusion] Soybean GmPAP4 promoter was obtained. The results of GUS staining in different tissues and GUS expression in different phosphorus environments showed that the promoter was mainly expressed in the root and was induced by low phosphorus signal.
【作者單位】： 河北農(nóng)業(yè)大學(xué)農(nóng)學(xué)院/教育部華北作物種質(zhì)資源研究與利用重點實驗室;
【基金】：轉(zhuǎn)基因生物新品種培育科技重大專項(2014ZX0800404B) 河北省自然科學(xué)基金(C2014204035)
【分類號】：S565.1;Q943.2

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