Priliminary Functional Dissection Of A Seed Weight Gene BnC08.JAZ1-1 In Rapeseed

Seed weight is an important trait of yield components and targets for selection in rapeseed breeding.In rapeseed germplasm,seed weight showed a very wide variation?thousand seed weight 2-8g?,which harboured rich alleles for large seeds.However,the regulatory genes and mechanisms underlying the natural variation of seed weight are poorly known in rapeseed,which limites the further improvement of seed weight and yield.Therefore,it's very necessary to explore the key regulatory genes for seed weight and further elucidate its molecular mechanisms,which will provide both theoretical guidance and technical support to breed large-seed and high-yield cultivar in rapeseed.In our previous study,several repeatable seed weight QTLs were detected using the BnaZNF₂ and the corresponding F_2:3:3 and F_2:4:4 populations,which were derived from two sequenced cultivars Zhongshuang11?de-novo sequencing?and No.73290?re-sequencing?.Then,the seeds at 15 DAF of two bulks from the F₆ RIL with extreme seed-weight were subjected to transcriptome sequencing and expression profiling analysis.Integration the results of QTL mapping and DEGs,a new candidate gene?Bna085006?for seed weight was screened for one seed weight QTL-qSW.C08.In the current study,the function of Bna085006 is preliminarily validated in A.thaliana,the main results and conclusions were as follow:1.The bioinformatics analysis showed that Bna085006 is highly homologous with JAZ1 gene in Arabidopsis?Amino acid sequence similarity 73.23%?,which encodes a nuclear-localized transcript factor involving in jasmonate signaling.In addition,JAZ1 has three copies in reference genome of B.rapa?A06,A08 and A09?and B.oleracea?C05,C08,C08?,respectively,as well as six copies in B.napus.Because Bna085006 should be derived from the C08 chromosome and then was designated as BnC08.JAZ1-1.2.The CDS sequence of BnC08.JAZ1-1 was cloned from Zhongshuang11 and over-expressed in A.thaliana.The seed weight of more than ten independent transgenic lines in the T₂ generation increased significantly,and the proportion of one line is?20%.Cytological observation showed that the increase of seed size of this line was due to the increase in cell number rather than size.3.The spatial and temporal expression pattern of BnC08.JAZ1-1 was analysed by GUS staining.The results showed that BnC08.JAZ1-1 expression was strong in the young petals and decreased gradually as time,but not found in the leaf,stem,silique and seed.So,the expression level of BnC08.JAZ1-1 was supposed to associate with the developmental degree of organ.4.The subcellular localization of BnC08.JAZ1-1 was analysed using a fusion protein of 35S:BnC08.JAZ1-1-GFP,which was expressed in the cell nucleus of tobacco leaf.5.The developing seeds at 15 DAF for the control and BnC08.JAZ1-1 over-expression line was subjected to transcriptome analysis and a total of 582 DEGs were identified.These DEGs were enriched mainly in the three pathways:plant-pathogen interaction,ubiquitin mediated proteolysis and circadian rhythm.Of which,ubiquitin-proteasome pathway is highly related with the seed size regulation.