木霉双价基因表达载体的构建

Construction of the Bivalent-expression Vector for Trichoderma spp.

利用几丁质酶基因和β-1,4-葡聚糖酶基因构建双价基因表达载体,为木霉转化奠定基础。利用限制性内切酶酶切得到目的基因片段,通过T4 DNAligase将基因插入表达框中,并采用PCR、酶切和核苷酸序列分析验证双价基因表达载体的正确性。利用启动子CaMV35S和终止子PolyA构建几丁质酶基因chi42的表达框,获得表达载体pC1302-C42;利用启动子CaMV35S和终止子Nos构建β-1,4-葡聚糖酶基因glu14的表达框,获得表达载体pC1300-2-G14;在单基因表达载体的基础上,通过串联构建双价基因表达载体pC1300-2-G14-C42,该表达载体含有潮霉素B抗性基因。经检测证实表达元件35S-chi42-PolyA和35S-glu14-Nos连接成功。得到的双价基因表达载体pC1300-2-G14-C42可直接用于木霉等丝状真菌的遗传转化,为构建木霉广谱高效工程菌株奠定基础。

Bivalent-expression vector of chitianse gene and β-1,4-glucanase gene lays the foundation for the transformation of Trichoderma spp.. The gene fragment was obtained by digesting of restriction endonuclease, and then lagased into expression cassette by T4 DNA ligase to form the bivalent-expression vector. The vector was verified by using PCR, digestion and nucleotide sequencing. The recombinant pC1302- C42 was constructed by cloned the chitinase gene chi42 into expression cassette with promoter CaMV35S and terminator PolyA, and the recombinant pC1300-2-G14 was constructed by cloning the β-1,4-glucanase gene glu14 into expression cassette with promoter CaMV35S and terminator Nos, and then the bivalent-expression vector pC1300- 2- G14- C42 with the hygromycin B resistance gene was constructed through successful connection of 35S-chi42-PolyA and 35S-glu14-Nos. The bivalent-expression vector pC1300-2-G14-C42 can be directly applied to filamentous fungi genetic transformation, lays the foundation for screening broad spectrum engineering strain of Trichoderma spp..