| Longhorn Beetle (Coleopteran: Cerambycidae) is a big group of insects distribuding worldwide and most of the larvae boring and feeding in tree trunks or branches. About 20% to 90% forests are damaged by these pests and millions yuan of money losted every year in China. Forest trees, fruit trees, mulbery trees, tea trees, cotton, wood material, evern furnitures and houses can be damaged. The pest control is very different because of the covert and long time life of the beetle's larva.Now the control methods for the pests mainly included some traditional strategys such as hooking and kill larvae, smashing eggs, pluging up breath hole and the chemical spray. However, most of traditional means have limitations of low efficancy, high cost and and time taking. And the chemical control often results in grievous environmental pollutions, killing natural enemies or even hard to reach the body of pest. Therefore, new biocontrol strategys and techniques are eagerly demanded in practice. A new pest control theories and approach is always based on the research of insect physiology and biochemique. 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.In this present, the intestinal microbe flora in Apriona germari (Hope) larvae was analyzed and identified by traditional culture and 16S rDNA sequence analysis. Whith this understanding, a study on the transforming Bt specific insecticidal protein gene cry3A into predominant bacteria and resident bacteria was carried for the first time to construct the recombinant pesticidal engineering bacteria which could settle down and reproduce in intestine of the larva, and express insecticidal crystal protein. The study result would exploit a new approach for covert pest biocontrol and provide a theoretical and technological support for the research of novel genic engineering bio-insecticide development.The main results were showed as follows:(1) Eighteen different bacterial strains were isolated and identified from A.germari larvae gut by traditional culture and identification methods. They were Klebsilla Oxytoca, Pseudomonas fluorescen, Pseudomonas putida, Shiqella flexneri, Staphylococcus haemolyticu, Staphylococcus homis, Shiqella boydii, Micrococcus luteus, Micrococcus kristinae, Breneria quercina, Serratia ficaria, Brevibacillus brevis, Bacillus thuringiensis, Escherichia coli, Naxibacter haematophilus, Enterobacter aerogens, Enterobacter cloacae, Enterobacter absburiae. Among them, 4 species bacterial Staphylococcus haemolyticu, Staphylococcus homis, Brevibacillus brevis and Bacillus thuringiensis can be isolated from Apriona germari (Hope) larvae intestine all the year, but other microorganisms just can be isolated from March to November ( the active period). So these 4 species bacteria were presumed resident endogenetic bacteria of Apriona germari larvae intestine, whereas other bacteria may be transient flora which get in intestine byfeeding or contact from enviorment. The data statistic of the bacterial clones showed that S. haemolyticu and S. homis were confirmed the endogenetic predominant flora with count of 7.74±0.61 and 7.66±0.25, and the isolation rate (The number of larvae which had the bacteria /the total number of the larvae had been detected) were 100% and 98.09%. The other isolations were comparatively low. They could be ranked orderly by the measure as Enterobacter aerogens, Escherichia coli, Micrococcus luteus, Brevibacillus brevis, Bacillus thuringiensis,Enterobacter cloacae, Micrococcus kristinae, Shiqella boydii, Shiqella flexneri, Klebsilla Oxytoca, Enterobacter absburiae, Serratia ficaria, Pseudomonas putida, Pseudomonas fluorescens, Breneria quercina, Naxibacter haematophilus according to their isolation rates.(2) The 18 different bacteria strains, which isolated by traditional cultural means from A. germari larvae'gut, were analyzed by 16S rDNA sequence analysis. The result consists with the results of normal classification by morphology, biochemical methods. The homologous rates between isolations 16S rDNA sequences and the recording 16SrDNA sequences in Genbank were 98.58%, 99.18%, 97.86%, 99.89%, 98.02%, 99.19%, 99.32%, 98.46%, 99.30%, 98.05%, 96.97%, 98.33%, 99.46%, 99.60%, 99.03%, 99.45%, 99.01%, 99.52% respectively. All the 16S rDNA sequeeces abtained were logged in Genbank and the accession number were from EU554427 to EU554444.(3) The Escherichia coli - Bacillus thuringiensis shuttle plasmid pHT305a and pHT7911, which contained anti- Erythromycin gene (Erythromycinr) and cry3A gene, had been transformed into predominant bacteria strains S. haemolyticus and S. homis and resident bacteria strains B. brevis and B. thuringiensis respectively. Further examinations by the electron microscope observation of the crystal protein and the protein SDS-PAGE analysis approved that the newly transgenic engineering bacterial strains have been constracted successfrlly. Four engineering bacteria were obtained which can colonize and express the target pesticide gene Bt cry3A perfectly in A. germari larvae, which may be developed a newly pesticide to control the pests.
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