The Genetic Stability Research Of Some Genetic Engineering Strains Of Beauveria Bassiana

Genetic engineering is an effective approach for solving the problems of slow speed of insectkilling by entomopathogenic fungi. However, it remains problematic whether the inserted genecan be lost and whether variation and degeneration can occur in the transgenic strains duringsubculturing as those happen frequently to the wild-type strains. In the present paper, sixtransgenic strain and two wild-type strains of Beauveria bassiana during subculturing wasstudied,such as Bb13T-10-3、Bb202T-7、Bb13TPr1A-1、Bb13TPr1A-2、Bb13-C、Bb13-A-C、Bb13and Bb202. First,check out the inserted gene by PCR, then test sectorization frequencies,sporulation, germination rate of spores, the growth rate and the pathogenicity of different strainsfor subculturing up to30generations, repeat them every5generations.At last evaluate the geneticstability by comparing with their mother strainIn order to investigate the genetic stability in serial subcultivation of genetically engineeredbacteria, the superior strain was obtained through using the technology of single spore isolationused to isolate monospore in this research.We found that Bb13(initial strain) and Bb13-S (engineered strain) had the same variationtendency of sectorization rate and sporulation, measuring during the serial subcultivation. Thesectorization rate had increased with the cultivate generation at previous stage and decreased then.The sporulation had cutted down gradually during the whole course. The colony of eight strainshad changed obviously. And the change is different each other. The sectorization rate andsporulation of Bb13was higher than Bb13-S, which meaned that Bb13mutates more easily thanBb13-S. The sectorization rate of transgenic strains, Bb13T-10-3, was between83%and100%,which was higher than other strains and had kept in the serial subcultivation. But the sporulationof Bb13T-10-3was low from beginning to end,9×10~6per mL in first generation and3×10~6permL in thirty generation, which reduced66.67%and was different significantly from others(P<0.01). The Bb13T-10-3-S was more stable, whose colony had changed from the seventhgeneration and sectorization rate peaked at17%merely from10to12generation. It was fluctuantun-obviously. But it produced3.2×10~8spores per mL, which was35.4times than the firstgeneration of multi-spore strain. The sporulation was2.8×10~8per mL in30generation and lowed13.79%compared with the first generation, though the yield decreased in the serial subcultivation.It was different from Bb13-S and other six strains significantly（P＜0.01）. So Bb13T-10-3-S wasa stable and high yield transgenic strains.The sectorization rate of Bb13TPr1A-1、Bb13TPr1A-2、Bb13-C and Bb13-A-C was lower than Bb13. But their sporulation was higher than Bb13. Thedifference of sectorization rate and sporulation between the transgenic strains Bb202T-7and its initial strain Bb202is indistinctive.We determined the growth rate of transgenic strains and initial strain in the serialsubcultivation, and found that the average growth rate of1st generation transgenic strains(Bb13T-10-3) was0.56cm/d, which is significant difference from othersBb13-A-C(0.38cm/d),Bb13-C(0.38cm/d),Bb13TPr1A-1(0.38cm/d)andBb13TPr1A-2(0.38cm/d)).The average growth rate of thirty generation (Bb13T-10-3) was0.51cm/d, which was differentfrom others Bb13-A-C(0.32cm/d), Bb13-C(0.32cm/d), Bb13TPr1A-1(0.32cm/d) andBb13TPr1A-2(0.32cm/d) significantly.The biological test showed that the toxicity of the2nd and the30th of6engineering strainsand2original strains decline with the passage increased. Punctatus larvae of bioassay resultsindicate that the Project2Bb13T-10-3strain of the median lethal time increased more than24%of the original strain Bb13.The median lethal time of30th engineering strain Bb13T-10-3increased18%according to original strain Bb13.Though the sporulation condition of Bb13T-10-3,we could infer that the degradation of variation was clear. The bioassay results of2nd strainsshowed that the median lethal time of the single-spore and multi-spore isolates of Bb13T-C,Bb13T-AC, Bb13TPr1A-1and Bb13TPr1A-2were lower than Bb13; the median lethal time of thesingle-spore and multi-spore isolates of30th were lower than Bb13except Bb13TPr1A-1andBb13TPr1A-2. It indicate that the degradation was not obvious though the situation of littlechanging in sporulation.The bioassay of Monochamus alternatus show that the median lethal timeof2nd and30th of single spore and multi-spore strains were lower than original strain, Themulti-spore isolates Bb202T-7’s median lethal time was unchanged in2nd and30th generation,while the single-spore isolates Bb202T-7-S increased only0.1d.The result showed that the sectorization occurred in engineering strains as well as itswild-type strain, while the inserted gene was not lost. As compared to the sectorization rate ofBb13and one of its single spore isolate Bb13-S, that of Bb13T-10-3remained high, whilesporulation and virulence obviously declined on the pine caterpillar, Dendrolimus punctatus.However, Bb13T-10-3-S, one of the single spore isolates from Bb13T-10-3was relatively stable,with sporulation and virulence against pine caterpillars being much higher than those of Bb13andBb13-S. It was the “super strain” we demanded.