Expressional Vertification And Safety Assessment Of Cry5b Transgenic Cotton

The Cry5b protein was derived from Bacillus thuringiensis,a gram-positive strain,which was different from the insecticidal protein gene contained in Bt cotton varieties（Bt cry1a）.The cotton research institute of Hunan Agricultural University adopted modern transgenic technology and pollen tube passage method to transferred the Cry5b into Jin1middle of Daizi cotton line,and produced JX0010 and JX0020 lines.for the identification of insect resistance of the bred lines,the expression validation and biosafety evaluation of the Cry5b transgenic cotton lines were carried out,and rapid detection techniques were developed to provide theoretical basis and rapid verification techniques for the application and production practice of new insect resistance genes in breeding.The main results of this study were as follows:（1）Using bioinformatics methods,the results of comparison NCBI databases and Uni Prot databases showed that the Cry5b protein had no higher homology with known toxic proteins and anti-nutritional factors,and was less likely to be potentially toxic and anti-nutritional;the comparison was found in the online allergenic database and the allergenic protein structure database,which showed that the protein was less likely to be potentially sensitized.（2）Rapid detection of recombinant polymerase amplification（RPA）of Cry5b genes:using puc57-Cry5b plasmid as template,copies/μL,4×103 And optimize the RPA reaction system,and the optimal amplification temperature was 35℃,best reaction time 15 min,RPA detection sensitivity is limited to 40 copies,with strong specificity.Two pairs of primer Cry5b-1-RPA and Cry5b-2-RPA.were screened.（3）Spatiotemporal expression of Bt toxin in cotton plant.Using p GEX-4T-3 as vector to construct Cry5b gene prokaryotic expression plasmid,transforming plasmids into escherichia coli BL21（DH3）to get Cry5b proteins,and Cry5b protein was injected into New Zealand rabbits to obtain Cry5b gene-specific polyclonal antibody,content of toxic protein（fresh weight,dry weight）in different organs and tissues of transgenic Cry5b cotton（strain）was determined.It turns out,highest seed content 2241.11 ng/g,After that,the leaf content reached 1552.22 ng/g,.bud content reached 1224.44 ng/g,flowers reache 1077.78 ng/g,cotton boll was the lowest 888.89 ng/g.The spatial distribution of toxic proteins showed that the upper and lower parts were basically the same,no significant difference（P>0.05）.The results of time expression showed that bud stage was the highest,Next to the biooming stage,The boll opening stage was the lowest.（4）Genetic stability JX0020 cotton lines with Cry5b genes:expression of Cry5b in cotton plants:combined with field sex comparison,kanamycin assay,the positive strain rate reached 99.2%.Random sampling,group detection,eight plants per group,PCR molecules,each sample has specific bands.After years of observation of 44 agronomic traits,the results showed that,except for plant height,node position of first fruit branch and length of first fruit node,the coefficient of variation was 10-20%,the interannual variation of the other agronomic characters was very small,the coefficient of variation was around 5%,good stability;at the same time,the results of environmental adaptability test showed JX0020adaptability to wasteland and cultivated land,survival competition ability and self-generating ability,there was no significant difference compared with control Xiangmian No.10.（5）Studies on the insect resistance JX0020 cotton lines of transgenic Cry5b gene:the separation rule of genetic characteristics of hybrid progeny F₁ all generations were resistant individuals,the proportion of anti-insect separation of F₂ obverse and inverse crisess groups was found to be 3:1（P<0.05）,indicating that exogenous groups were inserted into the genome and were stable dominant genes.Field tests showed that JX0020 strains had high purity of insect-resistant traits,good character consistency.JX0020 correct mortality rate of cotton bollworm during the second,third and fourth generation of cotton bollworm was as good as that of control GK19,and there was no difference between the two（P>0.05）.（6）Studies on the effects of cotton lines on non-target pests and gene drift of Cry5b genes showed that JX0020 had no toxic effect on non-target pests of cotton aphid,bug,red spider and thrips,there is no risk of mating and gene drift with related species as JX0010 and JX0020 of transgenic lines and conventional non-insect cotton.For the first time in this paper,the temporal and spatial expression,genetic law,insect resistance,gene drift and the dynamic effects of non-target pests on Cry5b cotton lines were studied.the rapid detection technique of recombinant polymerase amplification（RPA）of Cry5b genes was developed.the on-line search comparison of NCBI database and Uni Prot database showed that there was no high homology,potential toxicity,anti-nutritional and sensitization possibility between the Cry5b anti-insect cotton lines and known toxin proteins and anti-nutritional factors.This study has important theoretical and practical significance for the cultivation and production of transgenic new insect-resistant cotton varieties.