长枝木霉菌非核糖体肽合成酶基因启动子NP249的克隆及功能验证
Cloning and Functional Verification of Non-ribosomal Peptide Synthetase Gene Promoter NP249 from Trichoderma longibrachiatum
Peptaibols是由真菌非核糖体肽合成酶(NRPS)合成的多肽抗菌素,能够抑制多种病原菌,促进植物生长和诱导细胞凋亡。为了深入探究木霉菌非核糖体肽类抗菌素合成的调控机制,为通过基因工程来提高Peptaibols产量提供帮助。通过设计引物,克隆长枝木霉菌非核糖体肽合成酶基因NP249 的上游启动子区域,然后构建到一个具有绿色荧光蛋白基因(GFP)融合表达载体,验证基因NP249的启动子。以长枝木霉菌总DNA为模板,使用引物249-1B和249-4X,通过PCR技术扩增克隆到了NRPS的启动区域,全长1 204 bp。通过启动子NP249替代载体上GFP启动子,分别用BamH Ⅰ和XmaⅠ酶切pCX-62载体和启动子片段,T4连接酶连接,构建GFP融合载体pCX-62-NP249-GFP。通过限制性内切酶介导的转化技术转化(REMI)木霉菌原生质体,得到的转化子转到含潮霉素的培养基上进行筛选,得到潮霉素抗性转化子Z249,进一步通过荧光显微镜检测转化子,转化子Z249能检测到GFP荧光。克隆到的启动子能启动GFP表达,具备启动子功能。
Peptaibols are polypeptide antibacterials synthesized by fungal non-ribosomal peptide synthetases(NRPS),which can inhibit a variety of pathogens,promote plant growth and induce cell apoptosis.In order to further explore the regulatory mechanism of non-ribosomal peptide antimicrobial synthesis in Trichoderma spp.,and to help improve the output of peptaibols through genetic engineering.By designing primers,the upstream promoter region of T.longibrachiatum non-ribosomal peptide synthetase gene NP249 was cloned,and then a green fluorescent protein gene(GFP)fusion expression vector was constructed to verify the promoter of gene NP249.A 1 204 bp activating region of NRPS was amplified by PCR using the primers 249-1B and 249-4X from total DNA of T.longibrachiatum as a template.Replaced the GFP promoter on the vector by promoter NP249.Digestion of pCX-62 vector and promoter fragment with BamH Ⅰ and Xma Ⅰ,respectively,and ligation with T4 ligase,The GFP fusion vector pCX-62-NP249-GFP was constructed,and the protoplasts of Trichoderma spp..The hydromycin resistant transformer Z249 was screened.The transformer Z249 could detect GFP fluorescence by fluorescence microscopy.The cloned promoter could initiate GFP expression and function as a promoter.
长枝木霉菌 / 非核糖体肽合成酶 / 启动子 {{custom_keyword}} /
Trichoderma longibrachiatum / Non-ribosomal peptide synthase / Promoter {{custom_keyword}} /
图2 测序后 NP249 基因片段PCR扩增电泳结果M.DL2000 DNA Marker;1,2.NP249基因片段。Fig.2 PCR amplification and electrophoresis result of NP249 gene fragment after sequence M.DL2000 DNA Marker;1,2.NP249 gene fragment. |
图4 GFP融合载体菌液PCR电泳结果M.DL2000 DNA Marker;1.目的片段;2.GFP融合载体菌液PCR。Fig.4 PCR electrophoresis result of GFP fusion vector bacterial solution M.DL2000 DNA Marker;1.Target fragment;2.GFP fusion vector bacterial solution PCR. |
图6 PCR检测转化子中潮霉素抗性基因M.DL2000 DNA Marker;1.野生型长枝木霉SMF2 DNA;2,3.转化子DNA。Fig.6 Detection of hph gene in transformants by PCR amplification M.DL2000 DNA Marker;1.DNA of Trichoderma longibrachiatum SMF2;2,3.DNA of transformants. |
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图1 启动子预测图2 测序后 NP249 基因片段PCR扩增电泳结果图3 重组质粒电泳结果图4 GFP融合载体菌液PCR电泳结果图5 GFP融合载体双酶切电泳结果图6 PCR检测转化子中潮霉素抗性基因图7 转化子菌丝荧光检测/
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