Expression Of An Arabidopsis Ankyrin Repeat-contain Protein 2A(AKR2A) In Cotton Affects Of The Components Of Cottonseed Oil

Cotton is an important economic crop in the world,and also one of China's top ten oil crops.The problem of small processing scale and undeveloped technology in cottonseed processing still exists,though China is a cotton-producing country.As a clean renewable energy,cottonseed oil is a good raw material of biodiesel,so it is important to improve the content and composition of cottonseed oil fatty acids in cotton breeding,without affecting the quality of cotton.In addition,the development of cotton industry in China has been blocked by the poor-quality cotton fibers,it is urgent to improve the quality of cotton fiber.Ankyrin repeat-contain protein 2A(AKR2A)is an efficient molecular chaperone protein,it interactes with KCS1 to regulate the synthesis of very-long-chain fatty acids(VLCFAs),which improve the oil content and fatty acid composition of cottonseed oil.While VLCFA is an important way to regulate the development of cotton fibers,and can improve the length of cotton fibers.AKR2A was transferred into cotton by genetic engineering technology,and the high expression of cotton transgenic plant lines AKR2A-2 and AKR2A-57 were obtained.Agronomic traits of transgenic cotton plants,the oil content and fatty acid composition of cottonseed oil were analyzed,as well as fatty acid synthesis related genes expression.AKR2A interacting protein KCS1 was screened by a yeast two hybridization experiment and model plant Arabidopsis thaliana.The changes of oil content,fatty acid composition and expression of fatty acid synthesis related genes of AKR2A,KCS1 single gene overexpression plants and co-overexpressing were also analyzed.The main test results are as follows:1.We dip cotton hypocotyls by Agrobaoterium tumefaciens to transfer AKR2A gene into cotton and produce regenerated cotton plants.Two higher expressing transgenic lines were obtained,named AKR2A-2 and AKR2A-57,by screening of PCR,real-time fluorescence quantitative PCR(RT-PCR)and Western blot.2.The changes of oil content of AKR2A-2 and AKR2A-57 transgenic cotton seeds at different seed developmental stages were analyzed by Soxhlet extraction method.It was found that the 20-30d-seed stage was the rapid increaseing period of seed oil content,compared with the control group.The oil content of transgenic cotton was significantly higher than that of wild type plants.The oil content of transgenic plants increased by 33%compared with wild type at the 30d-seed stage.The oil content of AKR2A-2 and AKR2A-57 mature seeds were 8.40%and 11.19%higher than that of wild type.It also showed that the mature seed oil in the transgenic plants was darker than that in wild type plants,indicatinga different properties.3.The changes of fatty acid content and composition of cotton seeds at different seed developmental stages were analyzed by gas chromatography(GC),the results showed that the content of long chain fatty acids in the transgenic plants was higher than that of the wild type in each seed development stage.At 15 days,transgenic plants were nearly doubled than the wild-type,and the ratio of long chain fatty acids and low chain fatty acids was also higher.4.The expression profile of fatty acid synthesis related genes in different seed development stages of cotton was analyzed by RT-PCR.The expression of fatty acid synthesis related genes was moderated and most of them were increased compared to wild type,15-20d,the gene expression reached the highest.The expression of ECR1 increased significantly in 15-20 days,and reached about 1.3 times that of control Coker at the highest level.The expression of FAD2-1 was significantly increased during the whole development of the seed,and maintained high expression status in AKR2A transgenic plants.5.Investigation of agronomic traits of transgenic cotton showed that the yield of cotton fiber and cottonseed was significantly higher than that in wild type.Compared with the wild type,the transgenic cotton fiber length at different cotton development stages increased in varying degrees,the fiber length of AKR2A transgenic cotton increased about 5 mm more than that of control at the 15d.6.AKR2A interacting protein KCS1 was screened and identified by a yeast two hybridization experiment.The AKR2A and KCS1 interaction was comfirmed in Vitro and in vivo exprements by immunoprecipitation verification.7.The expression profile of KCS familiy genes in AKR2A single gene overexpressing Arabidopsis plants was analyzed by RT-PCR.The expression of KCS familiy genes were up-regulated compared to wild-type Col-0.The oil content of the three transgenic plants:AKR2A/KCS1 single gene overexpressing plants or AKR2A and KCS1 co-overexpressing plants,were higher than that of the wild type.In the AKR2A and KCS1 co-overexpressing plants,the content of VLCFA increased by 24.7%compared with the wild type,and the ratio of VLCFA to total fatty acids increased by 25.97%.