Genetic Analysis On Resistance Of Bombyx Mori Against Bacillus Thuringiensis Toxin Cry1Ac And Positional Cloning Of Resistance Gene

Bt toxin is a kind of parasporal crystal that produced Bacillus thuringiensis(Bt), it has shown highly insecticidal activity to insects among some orders, it has been widely used in agricultural pest control. But as a large area of Bt crops planted, increasing resistance in insects has occurred inevitably, the research into mechanism of insect resistance underlies reasonable insect resistance manage strategy, providing an essential guarantee for the sustainable use of the Bt toxin.Lepidopteran insects are the second largest order that much widely distributed among insect and the vast majority of them are all pests in agriculture and forest, whose larvae do seriously damage to various types of cultivated plants. Silkworm(Bombyx mori) is one kind of insect that could produce silk with excellent economical value and abundant germplasm resources, playing a key role in the study of biology as a model organism in lepidopteran. In present research, the resistance level, resistance inheritance character and resistance gene of silkworm against Bt toxin Cry1 Ac was investigated and analyzed, explaining the resistance mechanism of silkworm against Bt toxin from a molecular point of view, providing a theory reference for the insect Bt toxin resistance manage. The main contents and results of the research are as following: 1. Resistivity difference among various silkworm strainsCry1Ac toxin was fed to the 2nd instar newly exuviated silkworm larvae of 100 strains, the mortality was investigated for the calculation of the LC50. It shows that the resistivity against Cry1 Ac differs greatly among different silkworm strains and represents pleomorphic. 2. Genetic analysis of resistance of silkworm against Bt toxin Cry1AcSingle moth area cross between resistant parent ACR1(R) and susceptible parent Nistari(S) was used to establish the F1 populations, F2 populations and BC1 populations. The resistivity of 2nd instar newly exuviated silkworm larvae of all the above populations are investigated, and the investigation results shows that the resistance of the resistant strain ACR1 to Bt toxin Cry1 Ac is controlled by a recessive single gene. 3. Linkage mapping analysis of resistance gene of silkworm against Bt toxin Cry1AcGenomic DNA from two parents and F1 crossing offspring are chosen as model, and the polymorphism markers were searched for among all the 28 linkages of silkworm. Polymorphism markers are then analyzed for genotyping with two parents and their offspring populations, and for the polymorphism markers on the 15 th linkage group, the banding patterns and phenotypes are consistent, suggesting that the resistance gene against Cry1 Ac toxin of ACR1 is on the 15 th linkage group.300 silkworm larvae were used to determine the order and genetic distance of the polymorphism markers linked with the Cry1 Ac resistance gene. The genetic linkage map of the resistance gene against Cry1 Ac in silkworm was finished and the genetic distance is 2.0 c M. 4. Cloning and sequencing of resistance gene of silkworm against Bt toxin Cry1AcWithin the position area according to the genetic linkage map, the gene BGIBMGA007792-93 was predicted to be the target gene. With primers designed from the sequence of Bombyx mori for ABC transporter gene complete cds(Gene Bank accession no. AB620075.1), and the c DNA from silkworm midgut was amplified as model, and the c DNA sequences of ACR1 and Nistari are acquired by cloning and sequencing. Sequence alignment was performed, and it appeared that three consecutive nucleotides inserted into the sequence of ACR1, which adds a tyrosine in the amino acid sequence. And in the amino acid sequences of another five resistant strains to Cry1 Ac toxin, the tyrosine insertion was also observed, but there’s no tyrosine insertion in the sequences of three susceptible strains, suggesting the additional tyrosine insertion is the primary cause leading to the sesistance of silkworm against Cry1 Ac toxin. 5. Preliminary prediction and functional verification of the resistance gene ofsilkworm against Bt toxin Cry1AcThe genome engineering in Bombyx mori mediated by CRISPR/Cas9 system was used to disturb the expression of the gene BGIBMGA007792-93 in the Nistari, which is susceptible to the Cry1 Ac toxin. Then Nistari became resistant to Cry1 Ac, showing that the BGIBMGA007792-93 gene is the key gene causing resistance to Cry1 Ac toxin in silkworm.