Preliminary Analysis Of Truncated Cry1Ac Gene Expression In Transgenic Cotton MON531^R7569

Transgenic cotton is the largest transgenic crop in China.The MON531 transformant developed by Monsanto in the United States has been approved for commercial planting and application in China for more than 20 years.MON531^R7569 is a transgenic cotton material found in this laboratory that has the same flanking sequence as the MON531 transform but the cry1Ac gene is truncated.This study validated the molecular structure of MON531^R7569,established its detection method,analyzed the truncated cry1Ac gene transcription and protein expression levels,and preliminary analyzed the methylation status of the cry1Ac gene.The main results are as follows:1:The exogenous integration structure of MON531^R7569 was verified by PCR and a specific quantitative PCR detection method for MON531^R7569 was established.The results showed that the integration structure of MON531^R7569 was the same as that of MON531,but the deletion of the cry1Ac gene 3'terminal sequence and the terminator region.Quantitative detection methods were established based on the recombination region of cry1Ac gene.Quantitative analysis of 476 marketed transgenic cottons showed that MON531^R75697569 was detected in 4 samples with the highest content being 1.56%.2:The transcript and transcriptional levels of cry1Ac gene of MON531^R7569 were analyzed by RACE and RT-PCR.Transcript analysis revealed that the transcription termination site of the cry1Ac gene of MON531^R7569 was mainly concentrated at bases 161-173 and 233-265 downstream of the truncated position.Transcriptional level analysis showed that the transcription level of cry1Ac gene in leaves of MON531^R7569 and MON531 gradually decreased with seedling stage,bud stage,and boll stage.The transcription level of cry1Ac gene in leaves of MON531^R7569 at the seedling stage and bud stage was significantly higher than that of MON531.There are no significant differences in boll stage.3:The expression of Cry1Ac protein in MON531^R75697569 was determined by ELISA.The results showed that the expression of Cry1Ac protein in leaves gradually decreased with seedling stage,bud stage and boll stage,and the expression level of Cry1Ac protein in three stages was higher than that in MON531.4:The leaf blade resistance of MON531^R7569 to cotton bollworm was determined by the in vitro leaf method.The results showed that the corrected mortality rate of MON531^R7569 transgenic cotton against Helicoverpa armigera was 99.47%,the resistance level of transgenic insect resistant cotton MON531^R7569was high resistance,and the corrected mortality rate of MON531 transgenic cotton against Helicoverpa armigera was 95.21%.The resistance level of insect-resistant cotton MON531 is high resistance.5:Methylation-sensitive endonuclease quantitative PCR?MSRE-qPCR?and sulfite sequencing?BS-seq?were used to analyze the methylation levels of the cry1Ac gene expression cassettes in MON531^R7569and MON531.The results showed that 3 sites of MON531^R7569 in all 13 HpyCH4IV sites had high methylation levels outside the truncated cry1Ac gene reading frame.On the other hand,MON531 has 5sites with high methylation levels and is located in the 3'end region and adjacent regions of the cry1Ac gene.BS-seq results showed that the methylated region of MON531^R7569 was approximately 300 bp in length.The truncated cry1Ac gene transcription,expression,insect resistance and methylation of MON531^R7569 in this study showed that the cry1Ac gene can stably express exogenous insecticidal protein and has good insecticidal activity.This study provides important references and ideas for the case-by-case security analysis of the characteristics of transgenic molecules.