| Seed weight is one of the important components of rapeseed yield.It is regulated by a complex gene network.The cloning of related genes and the analysis of their regulatory mechanisms are of great significance for understanding the genetic basis of rapeseed development and yield formation to improve yield.In our previous research,the seed weight QTL qSW.C9 and the number of seeds per silique QTL qSS.C9 were co-located in a DH population,and it was found that BnaC9.SMG7b is the gene that controls the number of seeds per silique,and the nearby BnaC9.RINGb may be the gene regulating seed weight.Further research found that BnaC9.SMG7b had no effect on seed weight,and it was preliminarily confirmed that BnaC9.RINGb has a potential regulatory effect on seed weight.In this study,on the basis of previous studies,the fine mapping of qSW.C9and the analysis of candidate genes were carried out,and the mechanism of qSW.C9regulating seed weight was analyzed.For the candidate gene BnaC9.RINGb,further construct transgenic complementary materials and gene editing materials were used to verify its function,and at the same time we explored its basic biological characteristics and the network involved in the regulation of seed weight.The main results obtained are as follows:1.Two BC3F2population confirmed that qSW.C9 regulates seed weight.In addition, qSW.C9 is also closely linked with the number of seeds per silique and the length of siliques.2.Based on the ZS11 reference genome,this study narrowed the qSW.C9 mapping interval to about 266 kb corresponding to the C9 chromosome.Sequence analysis of the candidate interval showed that there were 39 predictive genes in the range and a deletion of about 25 kb in HZ396 containing 7 predictive genes including BnaC9.RINGb and BnaC9.SMG7b,while the other 32 genes in the candidate interval did not differ between the parents.Expression analysis showed that only BnaC9.RINGb and BnaC09G0551700ZS were differentially expressed in 15 days after pollination seeds of HZ396 and NIL（Y106）in the localization interval.3.Observation of mature seed coat cells and hypocotyl cross-section cells of HZ396 and NIL（Y106）showed that there are significant differences in cell size and cell number between the two materials,indicating that qSW.C9 may regulate cell division and cell expansion by one or more pathways simultaneously,thereby regulating seed size.4.Transcriptome analysis showed that there were 1033 differentially expressed genes in seeds 15 days after pollination of HZ396 and NIL（Y106）,including some reported genes that regulate seed size and genes that may participate in seed size regulation. GO enrichment analysis indicated that qSW.C9 may regulate seed weight through various pathways,including biological processes such as anion homeostasis,ion transport,carbohydrate transport,ubiquitin protease activity,carbohydrate transmembrane transport activity,and methylation.Molecular functions such as transferase activity and monooxygenase activity.5.Using specific markers STC9-164 and XH14,505 inbred lines of Brassica napus were used for haplotype analysis,and the haplotype effect of each haplotype was analyzed based on the average value of thousand-seed weight（TSW）in three-year. The results showed that only 12 materials were consistent with the HZ396 haplotype, and this haplotype material had the highest TSW phenotype.This shows that the HZ396 haplotype is a rare haplotype with a high TSW phenotype and may has great potential for application in high-yield breeding.6.For the target gene BnaC9.RINGb,we constructed 16 separate complementary transformation events using HZ396 as the receptor and investigated the phenotypes of the T1 generation population and part of the T2 generation population of these transformation events.The results showed that there was no significant difference in the TSW of the positive and negative individual plants in the all the 5 segregatin populations.For the target gene BnaC9.RINGb and its homologous copy BnaC9.RINGc,we constructed multiple gene editing materials with 206R,NIL（Y106） and Y127 as receptors and investigated their representative types.The results showed that we were not able to isolate individual plants with increased TSW in accordance with the expected proportion among the progeny of the gene-edited materials.All of the results failed to prove that BnaC9.RINGb is a gene that regulates seed weight.7.The sequences of inbred lines AS11 and AS38 at BnaC9.RINGb are consistent with BnaC9.RINGc.We used both of them to construct F2 population with HZ396 and NIL（Y106）respectively and investigated the TSW of F2 population.The results showed that the TSW of the two F2 populations constructed by AS11（AS38）and HZ396 were separated,while the TSW of the F2 populations constructed by AS11 （AS38）and NIL（Y106）did not separate.The above results indicate that BnaC9.RINGb and BnaC9.RINGc have the same regulatory effect on seed weight,or both have no effect,which indirectly proves that BnaC9.RINGb is not a gene that regulates seed weight.8.In view of the basic biological characteristics of BnaC9.RINGb,predecessors have completed the exploration of the sequence and expression pattern.This study confirmed that BnaC9.RINGb has E3 ubiquitin ligase activity through the self- ubiquitination activity test.In addition,the subcellular localization results showed that BnaC9.RINGb and At TUB5 were co-localized on microtubules.9.In order to explore the regulatory network involved in BnaC9.RINGb,we constructed a seed development yeast library using ZS11 as the material.Based on this yeast library,we initially screened 100 genes that may interact with BnaC9.RINGb.The yeast two-hybrid experiment proved that at least 14 of the genes were truly interacting genes.Furthermore,we used the Split-luciferase experiment to verify 10 of the interacting genes.These interacting genes laid the foundation for studying the function and regulatory network of BnaC9.RINGb. |
