Insect Resistance Assay Of Poplar Transformated With Chitinase DsRNA From Clostera Anastomosis

The Chitinase plays an importmit role in the decrustation, the growth and development process in insects. RNAi (RNA interence) as an important way of gene silencing widely used and researched in gene function and genetic improvement.we cloned the Chitinase and according to the genetic sequences analysised bioinformatics.The leaves of Populus davidia-na×Rbolleana as explants constructed chitinase gene expression vector Chi interference pBI121-Chif-intron-Chir, the use of Agrobacterium-mediated method Populus davidiana×P.bolleana genetic transformation, and the transformed strain lines were screened, PCR testing, test gene expression level Chi and insect resistance was carried out research, the main results obtained are as follows:1.To extract Clostera anastomosis RNA by STE, and reverse transcription obtained by cDNA, The Chitinase genes of Clostera anastomosis were cloned by PCR, and get its gene sequence according to their sequence of sequence analysis,understand its amino acid composition and content, predict conserved regions and neuropeptides, and its 27 kinds of amino acid sequence similarity to Chitinase genes were multiple sequence comparison and make a phylogenetic tree constructed chitinase protein tertiary structure model qualitative gene.2.To extract Genomic DNA of Clostera anastomosis, cloned by PCR that got the third intron sequences Clostera anastomosis PBAN neuropeptide genes. In Clostera anastomosis RNA as a template, was cloned by PCR gene partial sequence Chi conserved regions, and Connected Positive and reverse with PBAN gene intron gene fragment ligated transformed by a series of operations to build to the intermediate vector pUC19, and the antibiotic kanamycin gene as a screening interference expression vector pBI121-Chif-intron-Chir. And the recombin-ant vector is introduced into Agrobacterium tumefaciens EHA105.3. Populus davidianaxRbolleana as explants, its antibiotic sensitivity determination and the establishment of the best genetic transformation system, by choosing cultured transgenic plants with a fragment of the Populus davidiana×P.bolleana, and finally by PCR determined to get a new transgenic Populus davidiana×P.bolleana has the purpose of fragments, determ-ined by quantitative PCR seven kinds of Populus davidiana×P.bolleana of transgenic expre-ssion T4> T7> T2> T6> T5> T3> T1.4.Containing Chi RNAi transgenic Populus davidiana×P.bolleana tissue culture to ex-pand breeding, feeding it of Clostera anastomosis,found determined by quantitative PCR.The contents of Chi gene were decreased, the change of T3 gene was the most obvious, and the change of T6 gene was the smallest.Lymantria dispar, found no significant effect of transgenic Populus strains developmental stage, weight and mortality of Lymantria dispar.