Heterologous Expression Of Cry Genes For Enhanced Resistance Against Insect Pests In Cotton

Transgenic technology has helped a lot to develop insect-resistant plants resulting in the reduced application of pesticides.We reported here the expression of two cry proteins（cry3Bb1 and cry3）in cotton for enhanced resistance against chewing insect pests.The related genes were synthetically developed and were cloned under appropriate regulatory sequences followed by transformation into Eagle-2 genotype of cotton through shoot apex-cut Agro-infiltration.After shedding,cotton seeds underwent sterilization,during which the germination index and transformation efficiency were documented as 77.14%and 1.56%,respectively.The main findings are as follows:1.After embryo isolation,incision was performed and the embryos were co-cultured in Murashige and Skoog media with an Agrobacterium culture that contained the target gene.Candidates for transformation were selected in MS media containing hygromycin（25 mg/m L）,acclimated,and then transplanted to a greenhouse for further development and multiplication.Polymerase Chain Reaction（PCR）was used to verify transgene incorporation.PCR was used to confirm transgene incorporation.The integration of the transgene was validated by PCR using primers flanking the aforementioned cry genes.Transgene expression was assessed by q RT-PCR using GAPDH as a reference gene.At two weeks of seedlings,it was found that the relative expression of cry3,and cry3Bb1genes of transgenic plants M_1,M_2,and M₃was 4.5-,3.1-,and 3.0-folds higher respectively than that of the non-transgenic plants.The relative fold expression analyses at four weeks of seedling revealed that the highest expression of the transgene（s）in M1 plants which was a 4.6-fold expression（cry3+cry3Bb1）followed by M2（fold expression,3.2）（cry3Bb1）and M3（fold expression,3.0）（cry3）transformants of the cotton.The relative fold expression analyses at leaves stage revealed that the highest expression of the transgene（s）in M1 plants which was a 4.8-fold expression（cry3+cry3Bb1）followed by M2（fold expression,3.5）（cry3Bb1）and M3（fold expression,3.4）（cry3）transformants of the cotton.The relative fold expression analyses at cotton boll stages revealed that the highest expression of the transgene（s）in M1 plants which was a 2.56-fold expression（cry3+cry3Bb1）followed by M2（fold expression,2）（cry3Bb1）and M3（fold expression,2.1）（cry3）transformants of the cotton.2.The confirmed transgenic plants were exposed to insect pests,pink bollworm,and army bollworm.Bioassay at two weeks seedling results revealed that 50%of the mortality was observed against pink bollworm and 65%of the mortality was observed against army bollworm in transgenic plants containing both cry3Bb1 and cry3（M1 transgenic plants）.Bioassay at four weeks seedling results revealed that 55%of the mortality was observed against pink bollworm and 65%of the mortality was observed against army bollworm in transgenic plants containing both cry3Bb1 and cry3（M1 transgenic plants）.Bioassay at leaves stages results revealed that 60%of the mortality was observed against pink bollworm and 75%of the mortality was observed against army bollworm in transgenic plants containing both cry3Bb1 and cry3（M1 transgenic plants）.Non-significant results were observed among transgenic and non-transgenic plants for plant height,plant biomass,and for fiber quality traits.Bioassay at boll stages results revealed that 40%of the mortality was observed against pink bollworm and 55%of the mortality was observed against army bollworm in transgenic plants containing both cry3Bb1 and cry3（M1 transgenic plants）.Hence,the developed cotton transformants have improved resistance against chewing insect pests.3.We constructed a cassette containing two cry genes（cry1Fa and Cry32Aa）and two pme genes,one from Arabidopsis thaliana（At PME）,and other from Aspergillus.niger（An PME）in p CAMBIA1301plant expression vector using CAMV-35S promoter.This construction was transformed in Eagle-2cotton variety by using shoot apex-cut Agrobacterium-mediated transformation.Expression of cry genes and pme genes was confirmed by q PCR.At two weeks seedling stage,it was found that the relative expression of cry1Fa,cry32a,Atpme and Anpme genes of transgenic plants K₂was 3.1-,3.1-,and 3.1-and 3.0-folds higher respectively than that of the non-transgenic plants.Whereas at four weeks seedling,it was found that the relative expression of cry1Fa,cry32a,Atpme and Anpme genes of transgenic plants K₂was 3.7-,3.6-,and 3.66-and 4.56-folds higher respectively than that of the non-transgenic plants.At leaves stage,it was found that the relative expression of cry1Fa,cry32a,Atpme and Anpme genes of transgenic plants K₂was 4.68-,4.69-,and 4.5-and 4.6-folds higher as compared to non-transgenic plants.At cotton boll stage,the relative expression of cry1Fa,cry32a,Atpme and Anpme genes of transgenic plants K₂was 2.85-,2.91-,and 2.9-and 2.98-folds higher respectively as compared to non-transgenic plants.Methanol production was measured in control and in the cry and pme transformed plants showing methanol production only in transformed plants,in contrast to the non-transgenic cotton plants.4.Finally,insect bioassays performed with transgenic plants expressing cry and pme genes.At two weeks seedling stage,the mortality of K2 transgenic plants was higher than the K1,K3 and K4transgenic plants.It was observed to be 80%,60%and 75%against army boll,pink bollworm and spotted bollworm respectively in K2 transgenic plants.At four weeks seedling stage,the mortality of K2transgenic plants was higher than the K1,K3 and K4 transgenic plants.It was observed to be 85%,65%and 75%against army boll,pink bollworm and spotted bollworm respectively in K2 transgenic plants.At leaves stage,the mortality of K2 transgenic plants was higher than the K1,K3 and K4 transgenic plants.It was observed to be 100%,70%and 95%against army boll,pink bollworm and spotted bollworm respectively in K2 transgenic plants.At boll stage,the mortality of K2 transgenic plants was higher than the K1,K3 and K4 transgenic plants.It was observed to be 70%,55%and 60%against army boll,pink bollworm and spotted bollworm respectively in K2 transgenic plants.Non-significant results were observed among transgenic and non-transgenic plants for plant height,plant biomass,and for fiber quality traits.In summary,Bt.cry-genes coupled with pme genes is an effective strategy to improve the control of different insect pests.