Preliminary Molecular Mechanism On QSW.C9 Regulating Silique Length And Seed Weight In Rapeseed

Rapeseed is one of the most important oil crops in China,and it is a persistent goal to improve its yield by genetic breeding.Seed weight is one of the three factors that constitute the yield per plant of rapeseed and plays an important role in improving rapeseed yield.In our previous research,a seed weight QTL q SW.C9 was identified by a DH population derived from the F1 cross between HZ396 and NIL（Y106）hybrid.Genetic analysis and complementary verification preliminarily showed that Bna C9.RINGb is likely the candidate gene of q SW.C9,and a Brassica napus protein orthologous to HY35（AT4G16360）was identified to be interacting with Bna C9.RINGb by yeast two-hybrid experiments,HY35 encodes a component of the regulatory subunit of the plant Sn RK1 protein kinase.In addition,some differentially expressed genes during the silique development stage were revealed between HZ396 and NIL（Y106）,including CWINV5,SUC2,KLU and EOD3.Therefore,this study is to verify the potential biological function of HY35 in controlling silique length and seed weight of Brassica napus and investigate whether the above differential expression genes are involved in the determination of rapeseed seed size.The main results obtained are as follows:1.A comprehensive analysis including evolutional trees,conservative domains and tissue expression showed that the three HY35 copies,Bna A03.HY35,Bna A08.HY35 and Bna C08.HY35,have higher similarity and higher expression levels during silique development,suggesting that they may play roles in silique development of rapeseed.Therefore,the CRISPR/Cas9 gene editing vectors were designed for the conservative sequence of the above three copies and transformed into the recipient Xiaoyun,three transgenic edited seedlings were obtained.2.The three yield-related traits of homozygous mutant hy35 were investigated in the greenhouse and the transgenic fields.The results of the greenhouse phenotype study showed that compared with Xiaoyun,the thousand-seed weight of the hy35 T3 families was significantly increased by 30.5%,the silique length was shortened by 11.9%-30.0%,and the number of seeds per silique was reduced by 31.0%-48.7%.The hy35 T₄ families showed a consistent tendency with the hy35 T3 generation.The results of the transgenic fields phenotype study showed that compared with the control,the thousand-seed weight of the hy35 mutants was significantly increased by 20.8%-49.0%,the silique length was shortened by 16.4%-21.6%,and the number of seeds per silique was reduced by 29.0%-45.2%.These results indicating that HY35 participates in the silique development of rapeseed.3.The number of ovules between hy35 and Xiaoyun were comparatively counted from the 3-4 mm and 4-5 mm ovaries before flowering,and 1-day,3-day,5-day and 7-day ovaries after pollination,respectively.It was found that the degeneration of some ovules could be clearly observed in the ovary of hy35 on the 3rd day after pollination,and the ovule abortion rate was between 22.3% and 31.0%.However,the abortion rate of ovules of Xiaoyun was only 3.8%,indicating that HY35 gene may be involved in the early developmental regulation of fertilized embryos of Brassica napus.4.Compared with Xiaoyun,the surface area of hy35 seeds was significantly increased by 30.0%,the size of epidermal cells was increased by 18.3%,and the number of epidermal cells was increased by 9.1%,indicating that HY35 simultaneously regulates cell proliferation and expansion of seed epidermal cells to affect the seed size.The cell length of hy35 silique epidermis was clearly shortened by 25.0%,and the cell width was evidently narrowed by 34.2%,indicating that HY35 affect silique development by conditioning the elongation and expansion of silique epidermal cells.5.Compared with Xiaoyun,the expression levels of EOD3,KLU and SUC2 related to seed development or sugar metabolism were significantly down-regulated in the early stage of silique development of hy35,and the expression levels of CWINV5 and FUS3 were significantly up-regulated in the early stage of silique development of hy35,indicating that these genes may be involved in early silique developmental pathways regulated by HY35.6.CRISPR/Cas9 vectors were designed for the differentially expressed genes EOD3,KLU,CWINV5 and SUC2,respectively,and the above genes were knocked out in Xiaoyun.The three yield-related traits of the transgenic T1 generation were investigated,and it was found that only the eod3 mutant plants had significantly shorter silique length,lower seed weight,and reduced number of seeds per silique.No significant changes on the three yield-related traits was observed for the other three mutants.The phenotypic trend of the transgenic T₂ generation was consistent with that of the T₁ generation.