粉红粘帚霉67-1转几丁质酶基因转化子生物学特性及高效工程菌株的筛选

Biological characteristics of chitinase gene transformants of Gliocladium roseum 67-1 and screening of efficient biocontrol strains

通过原生质体转化法,制备粉红粘帚霉67-1转几丁质酶基因的转化子,通过潮霉素抗性平板初筛及PCR复筛,共获得530个转化子。对其生物学性状测定表明,3株转化子产孢量明显增加,3株转化子生长量显著提高,比色法测定发现40%的转化子几丁质酶活高于野生型菌株,对峙试验发现25%的转化子抑菌效果明显提高。初步筛选出生长快速、产孢量多、几丁质酶高、对菌核抑制效果明显的转化菌株TC-8、TC-64、TC-298、TC-406、TC-514,对其进行抗大豆菌核病防效试验测定,结果表明TC-8、TC-64、TC-298转化子防病效果明显优于67-1原始菌株和化学农药,通过实时荧光定量PCR验证,发现此3株工程菌株几丁质酶表达量明显增加。

Using protoplast conversion method to prepar the converter of Gliocladium roseum 67-1 Chitanase Transgenic, The study got 530 of the transformants through the hyg-resistant plates prescreening and PCR affination. The biometrics showed that 3 sporulation of the transformants and the amount of growth 3 transformants were markedly increased, colorimetry determination had found 40%of the chitinase activity was higher than wild strain, The confrontation experiment showed that 25% of the transformants, the bacteriostatie efficacy had increased. These results preliminary screen out that TC-8, TC-64, TC-298, TC-406, TC-514 were fast growth, quantity sporulation, higher chitinase activity and good inhibit effect of the Selerotia. The determination control effect of soybean Sclerotinia test showed that TC-8, TC-64, TC-298 had better prevent disease efficacy than 67-1original strain and chemical pesticides. The real time PCR indicated that 3 engineering strain chitinase activity expression of quantity had markedly increased.