Preliminary Functional Verification Of Candidate Gene SWH On Seed Weight In Brassica Napus L.

Rapeseed(Brassica napus L.)is an important oilseed crop in China.As the planting area decreases every year,improving the yield per plant has became an important way to enhance yield.Seed weight,which is one of the yield factors,is usually selected for high-yield breeding in rapeseed because of its high heritability.Therefore,it is a great significance to improve thousands seed weight for high-yield breeding of rapeseed.Since seed weight is a complex quantitative trait regulated by multiple genes and the rapeseed genome is complex,it is a great challenge to dissect the molecular mechanism of seed weight in rapeseed.In our previous study,we collected the loci from genome-wide association analysis(GWAS)and QTL mapping on seed weight and extracted genes within the confidence interval as unknown genes.Meanwhile,we gathered known genes which controlled seed weight in different species and used their homologs in rapeseed as "bait" to construct gene co-expression network using 15 day after pollination siliques transcriptome data of 71 rapeseed inbred lines.In this study,we selected one hub gene Bna A09.SWH with high connectivity from Module1 as a candidate gene for preliminary functional verification and the possible molecular mechanism dissection.The results of the research are summarized as follows:(1)Co-expression analysis of candidate genes.The gene co-expression network of seed weight contains 3,242 genes and consised of 10 modules.Module1 was regarded as a core module due to its large number of known genes(73/776).We selected a hub gene Bna A09.SWH from Module1 for functional analysis.This gene was located in the q SW9-3 that is a major seed weight QTL reported before.There are 323 genes directly associated with it in Module1,of which 37 genes are known,and these genes are mainly involved in nutrient synthesis and transportation,and seed maturation progress.Through aligning homologous gene,we found that there were 5 homologous copies on Bna A09.SWH in rapeseed genome,and their protein sequence similarity was more than80%.The homologous gene in Arabidopsis thaliana was At SWH,encoding a transmembrane protein,but its function is unknown.(2)Functional verification of At SWH on seed weight in Arabidopsis thaliana.Seed weight and seed number per silique in a T-DNA loss-function mutant swh-1 were significantly higher than wild-type in Arabidopsis thaliana.Conversely,silique length was decreased.However,the over-expression of At SWH resulted in decreasing on seed weight and seed number per silique,yet silique length was increased.We transformed the over-expression vector of At SWH into T-DNA mutant swh-1,and restored the phenotype of swh-1.There were no significant differences in seed weight,seed number per silique and silique length between complemental lines and wild-type.At the same time,we found that the flowering time in swh-1 was significantly earlier than that in wild-type,but significantly delayed in over-expression lines.(3)Marker development and functional complementarity.First,we sequenced the full gene length of Bna A09.SWH and found that there were 19 bp deletions and 262 bp insertions in the promoter regions-633 bp and-241 bp between Express and SWU07,respectively.An In Del marker was developed according to the variation and validated in a natural population which containing 157 inbred lines for Brassica napus L..The results showed that there were two haplotypes and seed weight and seed number per silique between the two haplotypes were significantly different in the 6-year and 4-year field experiments,respectively.We ligated the full-length Bna A09.SWH of the two parents into the p Bin35SRed＿Hyg vector,respectively,and constructed a complementary vector to transform the swh-1.The results showed that the seed weight and seed number per silique of p Express::Bna A09.SWH complementary lines driven by the promoter of Bna A09.SWH in the small-seed parent Express were significantly lower than those in the mutant,while p SWU07::Bna A09.SWH complementation line driven by the promoter of Bna A09.SWH in the large-seed parent SWU07 were not significantly different from the mutant.(4)Subcellular localization of At SWH.35S::At SWH-GFP vector was transient expressed in N.benthamiana.We found that 35S::At SWH-GFP fluorescence signal was coincided with the ER marker,indicating that At SWH was located in the ER.(5)Transcriptome analysis.Transcriptome sequencing was performed on 10 DAP siliques from different materials in Arabidopsis thaliana.GO annotation of up-regulated DEGs in swh-1 and down-regulated DEGs in over-expression lines showed that they were both involved in the nutrient reservoir activity.Further analysis revealed that this process was related to seed storage proteins during maturation in Arabidopsis thaliana.Enrichment analysis for up-regulated and down-regulated DEGs in swh-1 and over-expression lines showed that they were significantly enriched in the process of seed development and maturation,response to different hormones,response to light and hypoxia in different materials.(6)Prediction and validation of interaction gene.Functional enrichment analysis on transcriptome sequencing was used to predict the DEGs that might interact with At SWH.The results of yeast two-hybrid experiment showed that At SWH had strong interaction with At LEA30 and At ERF019.In conclusion,we predicted a total of 3,242 candidate genes for controlling seed weight by co-expression analysis in Brassica napus L..We selected SWH gene for further functional validation.The results demonstrated that At SWH and Bna A09.SWH had negative regulation on seed weight and seed number per silique,whereas At SWH had positive regulation on silique length.At SWH might be interact with At LEA30 and At ERF019,the regulatory mechanisms need to be further explored.