Detection And Preliminary Analysis Of The Exogenous Genes Of Genetically Modified Cotton MON757

The expression of exogenous insecticidal protein is the most important indicator for the safety assessment of genetically modified cotton(Gossypium hirsutum) targeting Lepidoptera pests. GM cotton MON757 expressing Cry1 Ac protein was developed by U.S. Monsanto Company. It has already been approved for planting or using for processing ingredients in the United States, but has not yet been permitted for planting or application in China. Our lab early detected MON757 in the cotton seeds from the market of our country, and further got the materials whose Cry1 Ac protein expression has significant difference by breeding and screening. We took GM cotton MON757 which had significant difference in Cry1 Ac protein expression as materials, established event-specific PCR method, preliminary studied the expression of Cry1 Ac protein, and primarily analyzed the integrated structure and the transcriptional expression of exogenous genes. The main results were summarized as follows:1. We established the specific homozygous/heterozygous qualitative PCR method and the event-specific quantitative real-time PCR(qRT-PCR) method for MON757 based on the DNA sequences of the junctions between exogenous DNA fragments and cotton genomic DNA. The qualitative PCR method could accurately identify homozygous and heterozygous states of MON757 and the qRT-PCR method could specifically detect the samples of MON757 with the limit of detection(LOD) about 11 copies and the limit of quantitative(LOQ) about 44 copies.2. We tested the content of Cry1 Ac protein in the leaves of types of MON757H(Cry1Ac protein expressing high) and MON757L(Cry1Ac protein expressing low) by enzyme linked immunosorbent assay(ELISA). The results showed that Cry1 Ac protein in MON757 wasn’t significantly differently expressed between homozygous and heterozygous MON757. The content of Cry1 Ac protein in the leaves of MON757 H maintained high and was trending down with the development of cotton growth stages, while the content of Cry1 Ac protein in the leaves of MON757 L kept a low level throughout the life course.3. We analyzed the full sequence of inserted exogenous fragment in MON757 H and MON757 L using common PCR and LD–PCR. The results showed that the structure and sequence of the exogenous fragments in the two kinds of MON757 agreed and the significantly difference of Cry1 Ac protein was not caused by exogenous DNA structure.4. We studied transcriptional levels of foreign genes in MON757 H and MON757 L by reverse transcriptional qPCR. The results showed that the transcriptional level of full cry1 Ac gene is significantly higher than truncated cry1 Ac while nptⅡ showed no difference.The event-specific PCR detection methods built in this study provide important technological means for variety breeding and safey regulation of GM cotton. To study and analysis the Cry1 Ac protein expressing, the integrated structure of exogenous fragment and the transcriptional levels of foreign genes in different kinds of MON757 makes sense to understand the expression law of foreign genes and its influence mechanism as well as the case security analysis.