Functional Research Of BnaGRF7.C02 Gene Which Involved Seed Weight And Size Regulation In Brassica Napus L.

Brassica napus L.(rapeseed)is the first-leading oil crop worldwide,and the main source of edible vegetable oil and biodiesel,accounting for 57.2% of oil crops production in China.Although the annual rapeseed yield is high,the supply of rapeseed is insufficient as the acreage used for rapeseed production is continuously declining.In China,the foreign dependence ratio of rapeseed oil has exceeded 60%,and there is a potential to keep increasing.Therefore,yield enhancement remains one of the most important breeding goals for rapeseed improvement.Seed size is one of the three agronomic traits of rapeseed,and it is also an important factor affecting the rapeseed yield.Finding and studying genes that affect seed size and their functional SNPs and In Dels,and base on this developing practical molecular is of great significance for developing molecular marker assisted selection in crop breeding and increasing rapeseed yield.In this study,the grain weight-related gene BnaGRF7.C02,which has been obtained in our laboratory,was used as the starting point to study its function.The main result of this study are as follows:(1)To explore the expression pattern of BnaGRF7.C02 gene,fusion of BnaGRF7.C02 promoter with GUS gene,and transform B.napus K407 using Agrobacterium-mediated transformation hypocotyl rapeseed.The results showed that BnaGRF7.C02 had a higher expression level in vigorously growing tissues,such as cotyledons and stems during germination,growing leaves,veins,petioles and the junctions between petioles and stems,siliques wall and seeds.(2)The expression levels of the BnaGRF genes in different developmental stages of rape seeds showed that the BnaGRF7.C02 has a higher expression level in the later stage(35-46 days after flowering)of seed development.(3)Transformed BnaGRF7.C02-e GFP vector into rapeseed K407 and prepare leaf protoplasts of positive transformants to observe the subcellular localization of BnaGRF7.C02 protein.The results showed that the protein is located in the nucleus.(4)Overexpression BnaGRF7.C02 in Brassica napus K407,compared with the wild type,the cotyledon area,leaf area,plant height,siliques length,seed size and leaf cell size of the overexpression line increased significantly,indicating that the BnaGRF7.C02 affect the organs size by regulating cell expansion.In order to further verified the function of the BnaGRF7.C02,the RNA interference and CRISPR plants of the BnaGRF7.C02 gene have been obtained,and used for further study.(5)The plant hormone metabolism pathway-related gene expression levels in BnaGRF7.C02 overexpression and RNA interference plants were analyzed using q RTPCR.The results indicated that BnaGRF7.C02 affect plant growth and development by positively regulating expression levels of genes involved in the GA signaling pathway,while negatively regulating expression levels of genes involved in cytokinin and auxin signaling pathway.(6)Sequencing analysis of the BnaGRF7.C02 in 42 rapeseed varieties,founding that a type of 15 bp deletion in the coding region of BnaGRF7.C02,this deletion was related to the thousand-seed weight trait of rapeseed.Furthermore,developing molecular markers based on the 15 bp deletion used for obtain the high yield varieties.