The Studies Of Constructing Antagonistical, Insecticidal And Endophytic Multifunctional-Engineering Strains

The insecticidal gene, vip3A, of WB7 strain (Bacillus thuringiensis) and the antagonistical and endophytic characters of BS-2 strain (Bacillus subtilis) were tried to integrate into a fusant by protoplast fusion. In the end, a multifunctional engineering-strain which had the insecticidal, antagonistical and endophytic characters was constructed. The study included four portions:1) The BS-2 and WB7 were tested for their induced resistance and antagonism to fungus. 2) The best situations of protoplast formation and protoplast regeneratetion of the parent strains were found through experiments. 3)The protoplasts of the two strains were fused at PEG6000 and Ca+2, and the geneticly stable fusants were screened.4) The bioassay of the fusants were validated by different experiments.The experiments showed that the induced strains still had stable resistance to the Kanamycin and Rifampin. The antagonism of induced BS-2 as the same as that of BS-2. Both of them were very strong angaist the Fusarium oxysporum f. sp. cucumerinwn. The WB7 had feeble antagonism angaist the fungus, but the induced WB7 had ndtany antagonism against it. It also showed that the antagonisms of the parent strains, induced BS-2 and induced WB7, were quite different against F. oxysporum f. sp. cucumerinum.The best conditions of protoplast formation and potoplast regeneration were very different in different srains. The experiments showed the percentage of protoplast formation was increased with the increasing of enzyme treating solution and time, but the protoplast regeneration was decreased. The best enzyme treating solution of the BS-2rif was 1.15 mg/ml, but the WB7kan was 0.76mg/ml. At this enzyme treating solution the percentages of protoplast formation and protoplast regeneration of the BS-2nf were 95.5% and 11.3% respectively. But the WB7kan were 96.77% and 10.5%.97 fusants were isolated as BS-2rif and WB7kan were fused at the PEG 6000 and Ca+2, but only 11 stable fusants were gotten after subcultureing forten times at the double resistances selecting medium. Then, the antagonism test prooved that only 6 among the 11 fusants had strong antagonism against F. oxysporum f. sp. cucumerinum.After the insecticidal test to Plutella xylostella and PCR for vip3A gene, only 3 among 6 fusnts had toxicity to the insect and vipBA gene. The three fusants were named as YB1, YB2 and YB3. Their toxicity to the P. xylostella reached 62%, 65% and 68.3% respectively. The endophytic test showed that YB2 had very strong endophytic character. Then, the YB2, which had the insecticidal, antagonistical and endophytic characters, was selected.