The Effect Of Grubs Environment In Vivo On The Insecticidal Activity Of Bacillus Thuringiensis

Bacillus thuringiensis is a gram-positive bacterium that is insecticidal against kinds of agroforestry pests and.It is widely used as a biopesticide.For the important underground pests,some grubs-specific Bt strain was isolated from our laboratory.Bt strains infection pRecent research showed that some insecticidal including Cry8 Ga proteins could destroy the midgut efficiently,but the further infection efficiency of Bt was low.On one hand in this study,In order to investigate the mechanism of grubs against Bt infection,we sequencing the transcriptome of grubs' the midgut issue,analysis grubs' immune related genes in midgut.On the other hand,for the immune factors in gut and haemolymph,screening resistant strains.All of these provide ideas for the further development of grubs efficient Bt insecticides,the relevant research results are as follows:Firstly,the analysis of the midgut transcriptome of the third instar larvae of Holotrichia oblita was completed.1,Gut expresses peptidoglycan recognition proteins(GPRP)and other receptor protein,these receptors are important proteins to identify bacteria,they can activate the grubs of the immune pathway and expression immune factors;2,the midgut expresses high abundance of the serine protease,which is mainly secreted in gut and may have an effect on Bt cells 3.Transcriptome data also show that midgut also expresses immune effects such as defincin,which are usually present in insect haemolymph,they may be involved in the invasion of haemolymph Bt inhibition and death.Secondly,the midgut immune-recognition-related receptor gene cloning was completed.1,we completed recognition of Gram-positive bacteria receptor peptidoglycan recognition proteins(gprp)gene cloning,obtained a full-length gene sequence,BLSAT analysis showed that show highest similarity of the Holotrichia diomphalia gprp-2 gene.2.Receptor lectin gene cloning was completed,obtained 4 full-length gene sequence,BLSAT analysis with other lectin gene in ncbi.The results showed that they have highest similarity of the endoplasmic reticulum lectin 1 gene of Zootermopsis nevadensis,the Malectin-B gene and Galectin-4 gene in Zootermopsis nevadensis.The similirity is 53%,62%,46%,respectively.3.Receptor defensin gene cloning was completed,obtained 2 full-length gene sequence,BLSAT analysis with other lectin gene in ncbi.The results showed that they have highest similarity of thedefensin B of Anomala cuprea.The similirity is 69%,62%,respectively.And the cloning of the serineprotease gene of the digestive enzyme with high expression rate in the midgut was completed.Six full-length gene sequences were obtained.BLSAT analysis showed that,The results showed that they have highest similarity of the trypsin-5 gene of Culex quinquefasciatus,trypsin-5 gene of Culex quinquefasciatus,the Tribolium castaneum serine protease-P76 gene,serine protease-3 gene in Costelytra zealandica,Holotrichia oblita serine protease 1,serine protease-4 gene of Costelytra zealandica,the serine protease of Eupolyphaga sinensis,respectively.The similarity is 30%,46%,82%,68%,48%,49%,respectively.This part of the work for the further design of RNA interference insecticide provides a new target.Thirdly,161 strains of Bt strains were tested for the intestine tolerance of grubs.Test insects are three instar larvae of Holotrichia oblita.74 Bt strains were significantly tolerated in the midgut of the larvae.The above results show that Bt is resistant to the immune effector in the intestinal fluid of grubs,and can further screen for highly tolerant Bt strains of intestinal fluid for the development of anti-grubs Bt insecticides.Fourthly,161 strains of Bt strains were tested for the haemolymph tolerance of grubs.87 strains of Bt strains were significantly tolerated in the hemolymph of the larvae of Holotrichia oblita,and the larvae of the larvae of the Protaetia brevitarsis larvae were tested.55 Bt strains were significantly tolerated in the haemolymph of the larvae of the Protaetia brevitarsis.The above results showed that the tolerance of Bt to the immune factor in the haemolymph of the grubs was varied,which can be further developed by screening Bt strains with highly tolerant haemolymph for the development of anti-grubs Bt insecticides.The results of this study show that the grubs have a variety of microbial immune recognition receptors and a variety of immune factors,the cloning of the receptor gene and immune effector gene can provide a new target for RNAi insecticide.Further studies on the immunoreactivity tolerance test of Bt to grubs showed that there was a strain that was highly tolerant to its immune factors,and these kinds of strain could be used for further development of anti-grubs Bt insecticides.The results of this study are important for the biological control of grubs.