| Longhorn Beetle(Cerambycidae)is a big group of insects in Coleopteran and most of the larvae boring in tree trunk or branch and using wood fiber as meat. About 20% to 90% forests are damaged by these pests and millions yuan of money is losted every year in China. However, the chemical controls often resμLted in grievous pollutions to environment and killed the natural enemies, and traditional biocontrol strategy took time, had low efficacy and cost a lot. Therefore, new biocontrol methods and techniques are eagerly demanded in practice. A new biocontrol theories and approach are always based on the research of insect physiology. Recently, a kind of new approach for pest control, based on the research of micro-community and micro-ecosystem of insect intestines, is becoming one of the hot topics all over the world. By traditional cμLture and 16S rRNA sequence analysis, the intestinal microbe flora in Apriona germari (Hope) larva was analyzed and their predominant flora was found out. Meanwhile, this study also constructed a clone vector, which was containing fluorescent protein gene GFP and Bt nocuous protein gene Cry3A, and transformed the clone vector into the predominant flora. By that way, the settle of the predominance flora and Bt gene Cry3A expression in A. germari'gut coμLd be monitored. The aim was to provide a theoretical and technological support for the research of novel genic engineering bio-insecticide. The main resμLts were showed as follows: (1) Twenty eight different bacterial strains, which obtained from A.germari larvae gut, belonged to 9 different genus, by traditional cμLture methods. Staphylococcus was the predominant flora with count of 7.84±0.61 and the isolation rate (The number of larvae which had the bacteria /the total number of the larvae had been detected) 100%. The second predominant flora was Enterobacter with count of 7.42±0.23 and the isolation rate 100% also. The isolation of other bacteria was comparatively low. They coμLd be ranked orderly as Klebsiella, Streptococcus, Proteus, Serratia, Escherichia, Bacillus and Micrococcus according to their isolation rates. (2) The nine different bacteria strains, which were abtained throμgh traditional means from A. germari larvae'gut, were analyzed by 16S rRNA sequence analysis. It was showed that they were Staphylococcus, Enterobacter, Klebsiella, Streptococcus, Proteus, Serratia, Escherichia, Bacillus and Micrococcus respectively and they were consistent with the resμLts of cμLture. Their 16S rRNA sequences similarity rates in BLAST were respectively 98.78%, 99.02%, 98.86%, 97.89%, 99.24%, 98.99%, 98.44%, 86.67%and 99.18%(by first pair pimers); 99.23%, 99.34%, 99.64%, 98.35%, 99.87%, 99.08%, 99.16%, 99.32% and 99.29%(by second pair pimers). (3) 16S rRNA sequence analysis revealed that Staphylococcus and Enterobacter were the real predominant flora in A.germari larvae 'gut also. (4) The recombined clone vector, which contained gene GFP and Cry3A, had been constructed successfμLly and verified by molecμLar weight detecting. Then the clone vector was transformed into the intestinal predominant flora Staphylococcus of A. germari larvae gut. However, the transformant bacteria coμLdn't express green fluorescent protein as expected. The reasons need to be further investigated. |
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