Construction Of Two Chitinase-Overexpression Vectors And Their Genetic Transformation In Lecanicillium Lecanii

Dialeurodes citri [Ashmead, (Hemiptera:Aleyrodidae)], the citrus whitefly, is one of the important economical and widely distributed pests all over the world, feeding on various of host plants, such as citrus, gardenia, privet, clove and persimmon. In the past ten years, the citrus whitefly quickly spread and caused serious damage in all citrus growing regions of China. The nymphs of D. citri feed on the new shoots, secrect honeydews, induce the sooty mould, which will cause leaf drop and deduce tree vigor and growth rate, even yield reduction. For now, chemical pesticides are still regarded as the main strategy in D. citri control, due to the complexity of its occurrence in the field.Lecanicillium lecanii is considered to be one of the important Entomopathogenic fungi, and to be an outstanding and eco-friendly agent in controlling aphids, whiteflies, coccids and mites, which causes epidemic disease in those pests. It has been proven that chitinase has a particular function in degarding the integument of insect, which is synthesized by entomopathogenic fungi and considered to be an important virulence factor in screening of entomopathogenic fungi. More and more evidence have indicated that, the virulence factors of entomopathogenic fungi could be enhanced by over-expression technology by shortening the knockdown time, making it possible to be a biological agent in pest control.This dissertation focused on the strain of L. lecanii, and successfully constructed the chitinase-overexpression vectors of L. lecanii and Beauveria. bassiana, pRCyl::Tchitl and pRCy1::Bchit2, by means of ClonExpress technology. In order to improve the virulence of the fungi to the citrus whitefly, the chitinase genes were transferred into L. lecanii by Agrobacterium tumefaciens-mediated method. All these results will not only contribute to improve the virulence of the fungus to the citrus whitefly, but also provide theoretical basis for biological control of agricultural production. The main results were as follows:1. Construction of two chitinase-overexpression vectorsFirstly, the total RNA of L. lecanii was extracted and then reversed as cDNA. Primers were designed with restriction sites (XhoI and XbaI) respectively on each site to amplify the complete open reading frame (ORF) of gene Tchit1. The result showed that gene Tchit1 shared 100% identity with gene chitl of L. lecanii from NCBI GenBank. Then, the ORF of gene Tchit1 was directively inserted into expression vector of filamentous fungi pRCyl to construct the chitinase-overexpression vector of L. lecanii, pRCy1:Tchit1. The chitinase-overexpression vector of B. bassiana, pRCy1::Bchit2, was constructed in the same way. And gene Bchit2 shared 99% identity with gene chit2 of B. bassiana from NCBI GenBank.2. Agrobacterium tumefaciens-mediated genetic transformation in L. lecanii and its verificationThe two chitinase-overexpression vectors, pRCy1:Tchit1 and pRCy1::Bchit2, were respectively transferred into agrobacterium cells AGL1, and verified by PCR amplification and double digestion of plasmid. Then, the chitinase genes Tchit1 and Bchit2 were transferred into L. lecanii TL001 by Agrobacterium tumefaciens-mediated method. PCR and RT-PCR amplification indicated that the chitinase genes Tchitl and Bchit2 had been successfully transferred into strain TL001, and the transcription of positive transformants was normal. Analysis of genetic stability indicated that the chitinase genes could inherit stably.3. The determination of the activity of chitinase in positive transformants of L. lecaniiPositive transformants and wild strains of L. lecanii were cultivated on the cicada slough medium under 28℃, and then the crude enzyme of the second generation was extracted. The activity of chitinase of the samples was calculated on the basis of the standard curve of ELISA and the OD value. The results showed that the activity of chitinase in TTL001, BTL001and TL001 were respectively 10.21 U/L,2.69 U/L and 0.25 U/L. Compared with the control group, there was the over-expression of chitinase with enzyme activity in transgenic strains.