本文選題：玉米大斑病菌　切入點：STK-EGFP融合基因　出處：《生物工程學(xué)報》2017年06期

【摘要】：STK1基因是玉米大斑病菌調(diào)控分生孢子發(fā)育、滲透脅迫調(diào)節(jié)和致病性的重要MAPK基因。本文首先構(gòu)建了含有增強型綠色熒光蛋白基因(EGFP)的畢赤酵母GSS115(Pichia pastoris GS115)表達載體p PIC3.5K-EGFP,再以玉米大班病菌模式菌株01-23的菌絲c DNA為模板,PCR擴增STK1基因,克隆到p PIC3.5K-EGFP,構(gòu)建了STK1-EGFP融合基因的GS115表達載體p PIC3.5K-STK1-EGFP。利用電擊轉(zhuǎn)化法將該融合基因表達載體轉(zhuǎn)化到GS115感受態(tài)細(xì)胞內(nèi),利用MD培養(yǎng)基篩選、PCR鑒定,獲得了STK1-EGFP融合基因的畢赤酵母轉(zhuǎn)化子。通過RT-PCR和熒光觀察,發(fā)現(xiàn)STK1基因和EGFP基因均可以高效穩(wěn)定地表達。另外,在試驗中我們還發(fā)現(xiàn),在STK1基因起始密碼子前加入Kozak序列可以使STK1-EGFP融合基因的表達強度增強4.8倍。以上研究結(jié)果為STK1基因表達蛋白的亞細(xì)胞功能定位和抗體制備奠定了基礎(chǔ)。
[Abstract]:STK1 gene is the regulation of conidial development by Mycobacterium macrophylla. The important MAPK gene of osmotic stress regulation and pathogenicity. Firstly, the expression vector of Pichia pastoris GSS115(Pichia pastoris GS115 containing enhanced green fluorescent protein gene (GSS115(Pichia pastoris GS115) was constructed, and then the mycelia c DNA of strain 01-23 was constructed. STK1 gene was amplified by PCR. The GS115 expression vector pPIC3.5K-STK1-EGFPof STK1-EGFP fusion gene was cloned into pPIC3.5K-EGFP.The expression vector pPIC3.5K-STK1-EGFPwas transformed into GS115 receptive cells by electroporation. Pichia pastoris transformant of STK1-EGFP fusion gene was obtained. By RT-PCR and fluorescence observation, it was found that both STK1 gene and EGFP gene could be expressed efficiently and stably. The expression intensity of STK1-EGFP fusion gene could be increased by 4.8-fold by adding Kozak sequence before the initiation codon of STK1 gene. These results laid a foundation for subcellular functional localization and antibody preparation of STK1 gene expressed protein.
【作者單位】： 唐山師范學(xué)院生命科學(xué)系;
【基金】：河北省高等學(xué)校科學(xué)技術(shù)研究項目(No.QN2017415) 河北省自然科學(xué)基金(No.C2014105067) 河北省留學(xué)人員科技活動擇優(yōu)資助項目(No.C2015005009) 唐山師范學(xué)院科學(xué)研究基金(Nos.2016C05,2014E04)資助~~
【分類號】：S435.131.4
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本文編號：1676332