Establishment Of Automatic Deletion System Of Marker Gene Based On Cre/LoxP Recombination System

Transgenic crops have been widely planted in the past 30 years.In 2017,the global planting area of transgenic crops reached 189.8 million hectares,which has become a huge industry.The emergence of transgenic technology has brought new hope to solve the food crisis and ecological crisis for human beings.Transgenic technology can be used to obtain new products with high yield,high quality and resistance to adversity,so as to meet the needs of human beings for food and other necessities of life.In the transgenic operation,resistance genes from microorganisms are often used as marker genes to screen the transformed cells or plants.This is a necessary means to obtain transgenic plants.However,at present,the marker genes mainly come from non food source microorganisms,which brings consumers the fear of food safety.Moreover,after obtaining transgenic plants,the marker genes are no longer needed.Therefore,it is necessary to remove these marker genes from transgenic plants.At present,the methods of deleting marker genes in transgenic plants have been established by transposon technology,CO transformation technology,site-specific recombinase technology and homologous recombination technology.Among them,Cre/LoxP recombinase system is the most in-depth study.Although the Cre/LoxP system can be controlled by heat induced promoters and pollen tissue-specific expression promoters to obtain the transgenic plants without marker genes,but the efficiency of deletion is still low.This study intends to use the meiotic promoter(M1P),pollen/egg specific expression hybrid promoter(PolEM),pistil/stamen specific expression hybrid promoter(AGiM)and pollen/seed specific expression promoter(PAB5)Control the Cre/LoxP recombinase system separately,analyze the deletion efficiency of marker genes after transforming tobacco,and screen out efficient marker gene automatic deletion system,which will provide technical reference for obtaining transgenic plants without marker genes in the future.The main results are as follows:1.The vector pVCT2423 with M1P::Cre/LoxP structure was constructed,and it was confirmed that the Cre/LoxP system controlled by the meiosis promoter M1P could not effectively delete the marker genesThe expression vector pVCT2423 containing meiosis promoter(M1P)controlling Cre/loxP recombination system was constructed and transferred to tobacco for validation.Using different primers to detect the genome DNA of T1 generation seedlings of positive plants,it was found that only two of 10 positive transgenic lines 2423-1 and 2423-21 had partial recombination of Cre gene.56 individual plants were detected in 2423-1 and 24 in 2423-21.The efficiency of recombination was 8.92%and 8.33%respectively.However,there was no complete recombination in the two transgenic lines.In T2 generation,2423-1-1 seedlings were detected,and the efficiency of Cre gene recombination driven by meiosis promoter was 12.5%,which was quite different from the expected deletion efficiency.The effect of meiosis promoter M1P on Cre gene expression was not as expected.2.The vector pVCT2437 with the structure of PolEM::Cre/LoxP was constructed,and it was confirmed that the Cre/LoxP system controlled by PolEM could automatically delete the marker genesThe expression vector pVCT2437 containing the Cre/LoxP recombination system controlled by PolEM was constructed and introduced into the tobacco genome.Using PCR technology to amplify the genomic DNA of the obtained T1 generation transgenic lines,it was found that only one line 2437-28 genomic DNA of the transgenic lines was recombined,and then the obtained T2 generation transgenic single plant genomic DNA was amplified.Among the 36 seedlings tested by 2437-28-3,2437-28-5 and 243728-8,the efficiency of Cre gene recombination was 47.22%,75%and 100%respectively,and the efficiency of Cre gene Complete reorganization was 16.66%,16.66%and 25%respectively.The sequencing results also confirmed the complete recombination.The results showed that the expression of Cre gene could be effectively regulated by the PolEM promoter,and obtained the transgenic plants with marker gene deleted automatically.3.The vector pVCT2424 with AGiM::Cre/LoxP structure was constructed,and it was confirmed that the hybrid promoter AGiM controlled Cre/LoxP system could effectively delete the marker genesA pistil/stamen transgenic deletion system based on AGiM::Cre/LoxP structure was constructed.After the system was integrated into the tobacco genome,11 positive transgenic tobacco lines were obtained.During the whole growth process,there was no significant difference in the number of pollen per flower,the number of seeds per fruit and the seed germination rate between the transgenic tobacco plants and the wild type,indicating that the introduction of AGiM::Cre/LoxP structure into the tobacco genome had no effect on its growth and reproduction.Among the three seedlings of 2424-37,the efficiency of Cre recombination was 100%,83.33%and 100%in 242437-3,2424-37-7 and 2424-37-10,and the efficiency of Cre complete recombination was 8.33%,8.33%and 16.67%respectively.The results showed that the constructed hybrid promoter of AGiM could effectively regulate the expression of Cre gene,and the transgenic plants with marker gene deleted automatically were obtained.The results showed that the recombination efficiency of Cre driven by AGiM was higher than that by PolEM.4.The vector pCVT2446 containing the structure of PAB5::Cre/LoxP was constructed,and it was confirmed that the Cre/LoxP system controlled by the pollen/seed specific expression promoter PAB5 could effectively delete the marker genesThe vector pVCT2446 containing PAB5::Cre/LoxP structure was transformed into tobacco and verified.12 positive transgenic tobacco lines were screened by PCR.In the inbred progenies of 12 positive T0 plants,the genomic DNA of T1 transgenic tobacco lines was extracted by CTAB method and amplified by PCR technology.It was found that most of the genomic DNA of the transgenic tobacco lines were recombined,and some of the individual lines were completely recombined.Among them,the recombination efficiency of Cre gene in 2446-9,2446-11 and 2446-12 was 75%,66.67%and 75%respectively,and the complete recombination efficiency of Cre gene was 25%,16.67%and 33.33%respectively.By comparing the recombination efficiency of Cre gene driven by meiosis promoter,pollen/egg heterozygous promoter and pistil/stamen heterozygous promoter,it was found that only pollen/seed heterozygous promoter could completely recombine Cre gene in T1 generation transgenic plants.The results showed that the Cre/loxP system expression regulated by PAB5 promoter had higher deletion efficiency.