Expression Of Cry1Ac Gene And Methylation Of Promoter In Stacked Transgenic Cotton

Bt cotton,the largest transgenic crop,has been approved for commercial cultivation for 20 years in China.While the expression of exogenous gene is an important part of transgenic genetic study and molecular characterization.In order to clarify the genetic stability of the cotton after the superposition of multiple transformants,MON531,7S Non-functional insertion(called 7S),and MON757 from same transformed event with MON531 were used as experimental materials in this study.The differences in the expression of exogenous insecticidal proteins,mRNA transcription levels and promoter methylation levels were analyzed between the single transformants and stacked transformant.The main study as followed:1.We have established the qualitative and quantitative detection methods for 7S Non-functional insertion based on 5?flanking cotton genome sequence and adjacent sequence.The 7S Non-functional insertion had been detected in eight of forty eight varieties collected from Hubei province in 2014.The content of 7S Non-functional insertion of those positive samples are 0.46% to 34.39%.At the further study,we found 7S Non-functional insertion and functional insertion of insect resistance are inherited independently in Bollgard 531.2.The expression of Cry1 Ac protein in MON531,MON757,7S Non-functional insertion and its stacked transformants was determined by Enzyme Linked Immunosorbent Assay(ELISA).Three varieties of transgenic cotton from market were used as experimental materials including Ezamian26F1,Xiangzamian13F1,Yazamian1F1;There was no significant interaction between MON531 and 7S Non-functional insertion.The expression of Cry1 Ac protein in five stages(parental seed,seedling,bud stage,boll stage and progeny seed)of homozygous MON531,MON757,MON531/MON757 with the same genetic background has been tracked.MON757 was significantly higher than MON531 except for boll stage,and the stacked transformant is significantly lower than the single transformant at any stages but offspring3.The RNA was extracted from cotton leaves in three periods(seeding,bud stage and boll stage),the specific primers and the real-time PCR was used to analyze the transcription of cry1 Ac gene in homozygous MON531,MON757,MON531/MON757.It showed a general trend of first rise and fall in three experimental materials.The transcription of cry1 Ac in MON757 remained at a high level,with approximately twice than MON531.Although The copies of target gene in the stacked transgene MON531 / MON757 is doubled compared with the single transgene,the mRNA transcription was significantly lower than that of the single transgene(p <0.05).4.The methylation level of 35 S promoter of full-length cry1 Ac gene were studied by bisulfite sequencing PCR(BSP).There is no significant difference between the CG,CHG,CHH and total methylation of MON531 in different periods;The parent was 1.0%-1.7%,the seedling stage was 4.2%-5.0% and the offspring was 0.8%-1.3%.The methylation rate of MON757 in CG and CHG is about twice as much as MON531,but the difference between CHH region and total methylation level is small except for offspring.The methylation of the stacked transgenic promoter was mainly focused on CG and CHG,and the methylation rate of different generations was as high as 55.0% about 20 times than MON531,which was significantly higher than that of the single transgene.CpG region indicated that the promoter of the MON531 and MON757 were at low level of methylation,while the combination of the two single transformants showed significantly increase.In this study,we established a specific qualitative and quantitative PCR method for the 7S of Bollgard 531,and made a preliminary test for the cotton on sale.This study will provide the detection technique for the safety management of transgenic insect-resistant cotton.The expression of cry1 Ac protein have been studied in MON531,MON757,7S and its stacked transgenic.It found that there was interaction of Cry1 Ac protein in stacked transgene.we revealed the gene silencing after multi-gene stacked from the mRNA transcription and the methylation level of promoter.This study not only provide reference for cultivation of new varieties,but also put forward higher requirements for safety evaluation of stacked transgene : not only limited to a single transformant,should be more concerned about the epigenetic changes arise from gene interaction.