抑制稗草生长的木霉菌REMI转化子构建与功能初探

Preliminary Discussion on the Construction of REMI Transformant of Trichoderma for Inhibiting the Growth of Barnyard Grass and its Function

创制新的木霉菌突变株,为筛选新的抑草类化合物奠定基础。通过REMI插入突变技术,以Trichoderma atrovirideT23为出发菌,进行了REMI转化子构建。对得到的转化子进行了PCR检测,分别利用抗潮霉素和氨苄青霉素基因为引物进行特异性扩增,并对发酵产物细分为分生孢子、超声波PBS法破碎孢子和发酵液,分别对稗草生长的生物活性进行测试。获得了18个转化子,通过继代培养和PCR扩增,证明线性pV2质粒DNA已成功插入木霉菌基因组。从REMI创制的木霉菌突变株库中,筛选出8株有抑草活性转化子(TaA2、TaB3T、aB7、TaE2、TaE8T、aH8、TaJ6和TaJ9),其发酵液具有抑制稗草的活性,并对水稻生长安全。经测试发酵产物对稗草生长的生物活性,获得了具有抑草活性及稳定性均理想的转化子TaB3。REMI方法所创造的转化子可较高效地筛选出具有抑草功能的突变体。

The aim of the research was to create new mutant strains of Trichoderma and lay the foundation for screening new weed-suppressive compounds. Trichoderma atroviride T23 was used as the original strain to eonstruct REMI transformant by REMI insertional mutagenesis technology. Obtained transformants were detected by PCR to amplify specially by using the genes resistant to hygromyein and ampieillin as premiers. And the fermented products were sub-divided into conidiophore, crashed spores by ultrasonic PBS method and fermented liquid to test the biology activities of barnyard grass growth respectively. 18 transformants were obtained in this test. It was proved that linear pV2 plasmid DNA was inserted into Trichoderma genome successfully by subculture and PCR amplification. 8 transformants of weed-suppressive activity, including TaA2, TAB3, TaB7,TaE2, TaE8, TaH8, TaJ6 and TaJ9, were screened from mutant strains library of Trchoderma created by REMI and the fermented liquid of weed-suppressive activity was safe to rice production. Transformant TaB3 with ideal wecd-suppressive activity and stability was obtained by determining the biology activity of fermented products on the growth of barnyard grass.The transformant created by REMI method could screen the mutants with weed-suppressive function efficiently.