【摘要】：利用農(nóng)桿菌介導(dǎo)的基因轉(zhuǎn)化(Agrobacterium-mediated transformation,ATMT)技術(shù)構(gòu)建玉米彎孢葉斑病菌突變體庫,并從中篩選到了一個(gè)毒素合成相關(guān)基因clt-1。對(duì)clt-1基因編碼的蛋白質(zhì)進(jìn)行生物信息學(xué)分析,結(jié)果表明CLT-1的分子質(zhì)量為81.984 8k Da,等電點(diǎn)p I為8.62,不穩(wěn)定指數(shù)為55.08;CLT-1是親水性蛋白,包含1個(gè)膜外區(qū)域。在亞細(xì)胞水平上,CLT-1定位于細(xì)胞核內(nèi);在氨基酸序列方面,CLT-1含有多個(gè)蘇氨酸、絲氨酸和酪氨酸激酶磷酸化位點(diǎn);在功能方面,CLT-1包含BTB特征結(jié)構(gòu)域,可能參與一些功能蛋白質(zhì)活性的調(diào)控。利用GFP(green fluorescent protein)基因作為報(bào)告基因,構(gòu)建了用于鑒定clt-1基因啟動(dòng)子活性的p C1300th-Pclt1-GFP真菌表達(dá)載體,采用ATMT方法轉(zhuǎn)化彎孢菌萌發(fā)的分生孢子,通過PCR及GFP熒光檢測(cè)clt-1基因啟動(dòng)子在彎孢菌中調(diào)控GFP基因表達(dá)的活性。結(jié)果表明,在共聚焦激光掃描顯微鏡下觀察到菌絲和孢子發(fā)綠色熒光,說明彎孢菌clt-1基因啟動(dòng)子具有較強(qiáng)驅(qū)動(dòng)外源gfp基因表達(dá)的活性。
[Abstract]:A mutant library of Curvularia corniculata was constructed by Agrobacterium-mediated transformation technique, from which a toxin synthesis-associated gene clt-1 was screened. The bioinformatics analysis of the protein encoded by clt-1 gene showed that the molecular weight of CLT-1 was 81.984 kDa, the isoelectric point I was 8.62, and the instability index was 55.08% CLT-1, which was a hydrophilic protein containing an extracellular region. At the subcellular level, CLT-1 is located in the nucleus; in amino acid sequence, the CLT-1 contains several threonine, serine and tyrosine kinase phosphorylation sites; and in the functional aspect, CLT-1 contains the characteristic domain of BTB. It may be involved in the regulation of the activity of some functional proteins. Using GFP (green fluorescent protein) gene as reporter gene, the expression vector of p C1300th-Pclt1-GFP fungi was constructed to identify the promoter activity of clt-1 gene. The conidia of Curvularia sprouting were transformed by ATMT method. PCR and GFP fluorescence were used to detect the activity of clt-1 promoter regulating GFP gene expression in Curvularia. The results showed that the green fluorescence of mycelium and spores was observed under confocal laser scanning microscope, which indicated that the promoter of clt-1 gene of Curvularia had strong activity to drive the expression of exogenous gfp gene.
【作者單位】： 上海交通大學(xué)農(nóng)業(yè)與生物學(xué)院農(nóng)業(yè)部都市農(nóng)業(yè)(南方)重點(diǎn)開放實(shí)驗(yàn)室;上海交通大學(xué)國家微生物代謝重點(diǎn)實(shí)驗(yàn)室;
【基金】：國家自然科學(xué)基金(31471734) 國家玉米產(chǎn)業(yè)技術(shù)體系(CARS-02)資助項(xiàng)目
【分類號(hào)】：S435.13

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