The Study Of Genetic Transformation Of Deltocdes Cv ’55/56’× Deltocdescv ’2KEN8’ By Two Double Bt Gene Plant Transformation Vector

Deltocdes CV’55/56’x deltocdescv is a good hybrid varieties of poplar cultivation,by the Chinese academy of agricultural sciences,and is the only Chinese varieties named with“super speed”.In recent years,the expression of exogenous gene silence had been reported,and as the large area around the plant insect-resistant genes,insects,there gradually developed a tolerance and resistance of transgenic plants.To improve the efficiency of genetically modified（gm）polar Bt gene expression and expand the insect-resistant spectrum,the two different types of Bt gene Cry1Ac and Cry3A,were simultaneously built on two different types of plant transformation vector.Using agrobacterium mediated method,the two kinds carrier of American giant bully black fine hybrid clones polar were chosen and tested and 4 transgenic resistant strains were obtained.The results are as follows:1 The choice of carrier p71A68Y71 marker gene was equipped with the resistant neomycin phosphoric acid transferase Gene（npt II）for kanamycin r,target gene Cry1Ac is in front of Cry3A,the promoter of Cry1Ac gene is CAMV35S,promoter of Cry3A gene is Co YMV,and the MAR from tobacco was added on both sides of purpose gene（Matrix Attachment Region）structure;The choice of carrier p CAMBIA1305 marker genes was equipped with the resistant hygromycin phosphoric acid transferase gene（HPT）for hygromycin,target gene Cry3A was in front of Cry1Ac,two gene promoters were all CAMV35S and there was no MAR structure.2 Using agrobacterium mediated method,the two kinds of carrier American giant bully black fine hybrid clones Yang Yang were choisen and screened by the kanamycin and hygromycin,4 strains resistant plants were obtained;All purpose stripes were detected by PCR identification,which meant that the purpose gene has been integrated into the genome in the giant Yang.3 Using the fluorescent quantitative PCR technique to detect the eight gm strains Bt gene transcription abundance in the leaves,the results revealed that the difference existed between the two Bt gene transcription abundance in different strains:the transcription abundance Cry1Ac gene from carrier p71A68Y71 K series strain was significantly higher than the H series strain from another carrier,8.03 x 10³ and 3.59 x 10²,respectively,but there was no significant difference between two vectors for Cry3A gene transcription abundance,among these,the H4 and K1 strain transcription abundance was highest of 7.42x 10⁶ and 5.15 x 10⁶;Cry3A gene transcription abundance was significantly higher than Cry1Ac.4 Using ELISA to detect the eight gm strains Bt toxic protein gene expression content in the leaves,the results indicated that two kinds of Bt toxic protein expression quantity were significantly different between each strain but H and K series.Cry3A toxic protein levels in each strain were significantly higher than Cry1Ac,and the Cry1Ac’s expression was extremely low,only 0.13 to 0.13 ng g^-1;the expression of H4 and K1 strain quantity were the highest,2365.65±14.9 ug·g^-1 and 236.91±25.7 ug·g^-1,respectively.5 Iinsect-resistant tests were conducted for each gm strains,the results showed that each strains’insect-resistant effect to lepidoptera pests,larvae（H.cunea）was not obvious,there was no significant difference between different strain and different vehicle types.The strains showed very high insect-resistant effect to coleoptera pests,blue plagiodera to a instars and second instars,the fatality rate reached 90%,to the three instars,the fatality rate reached 70%,and were significantly higher than that of single gene Cry3A poplars high strain CC84.