Functional Study Of WS1/WS2 On Seed Size Regulation

In the context of the global yield reduction of rapeseed and the serious shortage of oil crops in China,raising the rapeseed production,promoting and optimizing the steady development of rape industry,are of great significance for increasing the self-sufficiency rate of edible oil,maintaining food security,and increasing peasants' income in China.Some researchers have shown that selecting the materials with higher 1000-grain weight is one of the main objectives of high yield breeding of Brassica napus.Previous studies have shown that ZmWS1 and ZmWS2 may be invloved in the regulation of seed size and increased the 1000-grain weight in crops.Therefore,we carried out a series of studies on these two genes.The main results are as follows:1.Overexpression constructs of ZmWSl and ZmWS2 were introduced into B.napus receptor"Yangyou 9" by Agrobacterium-mediated transformation method.The molecular and genotype identification of T0 and T1 generation transgenic plants were carried out,and the results suggested that ZmWSl and ZmWS2 might regulate the seed size.2.Further histological analysis of the mature seeds of T1 generation showed that ZmWS1 and ZmWS2 regulated seed size by regulating cell size.3.Subcellular localization of ZmWS1 and ZmWS2 proteins was analyzed by transient expression in tobacco epidermal cells.It was found that BnWS1 and BnWS2 were localized to the cell membrane.4.Yeast two-hybrid screening was performed to analyze the putative interacting proteins of ZmWSl and ZmWS2 using the B.napus seed library,and 3 putative interacting protins of ZmWSl and 9 putative interacting protins of ZmWS2 were obtained.Some of these proteins may be associated with embryonic development.5.In order to investigate whether the homologous genes of ZmWS1 and ZmWS2 have the similar functions,we isolated the homologous genes of ZmWS1 and ZmWS2 in B.napus(BnWSl and BnWS2)and studied their function.We constructed the overexpression vectors of BnWS1 and BnWS2.The constructs were transformed into B.napus,and obtained four positive transgenic plants of each construct.6.Subcellular localization of BnWS1 and BnWS2 proteins was analyzed by transient expression in tobacco epidermal cells.It was found that BnWS1 and BnWS2 were localized to the cell membrane.7.Yeast two-hybrid screening was performed to analyze the putative interacting proteins of BnWS1 using the B.napus seed library,and 6 putative interacting protins of BnWS1 were obtained.Some of these proteins may be associated with embryonic development,proteasome complex,and cell differentiation.8.The pathway in which WS1 and WS2 are involved in regulating seed size was analyzed using the software Cytoscape,and the results suggested that WS1 and WS2 were likely to participate in regulating the size of seeds via proteasome degradation pathways.In summary,molecular and phenotype identification was performed on the transgenic B.napus overexpressing ZmWS1 and ZmWS2.The results suggested that ZmWS1 and ZmWS2 were involved in the regulation of seed size.The overexpression of ZmWS1 and ZmWS2 in B.napus increased the seed size via increasing the size of the cells.ZmWS1 and ZmWS2 were localized on the cell membrane and might interact with some embryonic development associated proteins.We further investigated the function of the homologous gene of ZnWS1 and ZmWS2 in B.napus,BnWS1 and BnWS1.The results showed that BnWS1 and BnWS2 were localized on the cell membrane and might interact with some embryonic development and proteasome related proteins.Bioinformatics analysis revealed that WS1 and WS2 were likely to participate in proteasome degradation pathways to regulate seed size.