The Highly Effective Approach Of Copy Number Detection With Quantitative Real-Time PCR For Transgenic Cotton

Cotton(Gossypium hirsutum)is an important cash crop.By the end of 2019,76%of the total cotton planting area is transgenic cotton worldwide.For transgenic plants,the copy number of foreign genes is the key factor affecting their own expression level and genetic stability.It is necessary to apply a high-throughput detection method of foreign gene copy numbers to transgenic breeding.So far,Southern blot has been generally used to detect the copy number of inserted foreign genes.However,the quality of DNA extracted in large quantities is difficult to meet the requirements for Southern blot because of the abundant secondary metabolites in cotton.In addition,the large genome of cotton caused that it is difficult to ensure that there is a relatively large amount of DNA on nylon membrane after complicated links such as DNA digestion,electrophoresis and membrane transfer,so the hybridization signal is always weak.So it is difficult to repeat the Southern blot of cotton in different laboratories,especially for batches.Although our laboratory has mature conditions for Southern blot,the poor repeatability of this experiment among graduate students leads to a great waste of manpower and material resources,and seriously lagged the experimental progress.It is necessary to develop a new and efficient method to detect foreign gene copy numbers in cotton.Therefore,our study aims to develop a convenient and efficient copy number detection kit for foreign genes in the form of collaboration with biotechnology companies.In this study,a copy number detection method of exogenous genes based on a relative quantitative method was developed,which used the SYBR Green quantitative real-time PCR technology with cotton endogenous ubiquitinase gene Gh UB7 as the endogenous reference gene.The method takes the constructed standard plasmid that cloned the endogenous reference gene UB7 and exogenous reference genes Cas9,NPTⅡ,35S and Ubi as reference,and detects the copy number of exogenous genes Cas9,NPTⅡ,35S and Ubi in transgenic cotton.The results showed that the correlation coefficient(R~2)of the standard curves of endogenous reference gene UB7 and exogenous reference genes Cas9,NPTⅡ,35S and Ubi were all close to 1,and their amplification efficiency was close to 100%.In addition,the amplification efficiency of endogenous reference gene UB7 was 101%.And the amplification efficiency of exogenous reference genes Cas9,NPTⅡ,35S and Ubi were 99%,98%,102%and 102%respectively.The results of Southern blot verified that,the correlation coefficient of foreign genes copy number which was detected by quantitative real-time PCR method and Southern blot was 0.8962.Our study established a fast and efficient copy number detection method of exogenous genes Cas9,NPTⅡ,35S and Ubi in transgenic cotton,which simplified the copy number identification process of transgenic cotton and improved throughput of copy number detection.To date,our production has been applied for a patent and the kit for trial use is being produced.This method can also be applied and extended to other crops.