Map-based Cloning And Functional Analysis Of Trilocular Gene Bjmc1 In Brassica Juncea L.

It has been proved that the multiloculus is positively correlated with the high yield in rapeseed.The J163-4 whose trilocular phenotype is characteristic and stable is a desirable breeding material,and it has a great potential for beerding.The trilocular phenotype of J163-4 is controlled by two independent recessive genes,Bjmc1 and Bjmc2.This two genes have been isolated from the same plant by the molecular markers that are linked to the taget genes.In this study,to reveal the formation mechanism of trilocular silique in J163-4,map-based cloning was used to identify the bilocular gene BjMc1and trilocular gene Bjmc1,and the the evolutionary process of Mc1genes in land plants was analysed.Simultaneously,the gene function of BjMc1 in the development of carpels of B.juncea was discribed.the results were discribed as follows:1.The trilocular penotype of B.juncea J163-4The phenotypic observation of trilocular and bilocular siliques showed that the trilocular silique was wider and shorter.There are three locules in the trilocular silique while two locules in the bilocular silique.The results of scanning electron microscopy showed that although significantly difference was showed between the trilocular and bilocular silique,little difference was showed in the stem apical meristem and flower meristem.2.Fine mapping of the trilocular gene Bjmc1 in B.junceaIn this study,the AFLP,SSR and SCAR molecular markers linked to the BjMc1 gene was developed,and the BjMc1gene was mapped between the molecular marker EC14MC14 and SC20.Then,the C1-1 primer designed according to the sequence information of Bra015812 was used to scanning the BAC library and the positive clone,83D02,was obtained.According to the sequece of 83D02,SSR and SCAR markers was developed and three molecular markers,SC40,SC151and BSR52,were obtained.Ultimately,the candidate region of BjMc1 was found to be restricted between SC151 and EC14MC14 at a 1.14-c M region,and the SC40 was co-segregated with the target gene.Twenty-five open reading frames（ORFs）was predicted according to the 75kb sequence of 83D02 in which both the co-segregative SC40 and nearest marker SC151 located.The 25 ORFs were analyzed and annoting by the Arabidopsis genes,and the homologous gene of CLV1（AT1G75820）was predicted to be the candidate gene of BjMc1.3.The sequence ananlysis of candidate gene and the cloning of BjMc1geneComparetive sequencing between the bilocular and trilocular plants showed that there are two CLV1 homologous genes,Bj CLV1a and Bj CLV1b,in the B.juncea.No sequence variation was detected in Bj CLV1a between bilocular and trilocular plants,while an insertion of a 4961 bp Copia-LTR RTE1 was detected in Bj CLV1b of trilocular plants,and this Copia-LTR RTE1 interrupted the transcription of Bj CLV1b in trilocular plant.Finally,the genetic complementation suggested that Bj CLV1b was the target gene of BjMc1.4.Stucture analysis of BjMc1 geneFull-length complementary DNAs（c DNAs）of BjMc1 and Bjmc1 were identified by RACE technology.The results showed that the c DNAs of BjMc1 and Bjmc1 was 3,155 bp and 2583bp in length respectively.BjMc1 gene encodes a protein with 987 amino acid,consisting of the extracellular and intracellular domain.Compared with BjMc1,Bjmc1protein was derived from the truncation of BjMc1 at Gly⁷⁸² by Copia-LTR RTE1.5.The expression pattern and function study of BjMc1The results of q PCR analysis indicated that the highest expression level of BjMc1was detected in early inflorescence,but overall lower and variable expression levels were detected in different developmental periods of siliques,the SAM,the stem,the silique peel,the leaf and the root.Consistent with the q PCR analysis,the flower bud primordium in early inflorescence showed the highest GUS expression.The gene expression of GUS in the transgenic plants was also detected the SAM of early inflorescence and mainly in the vascular tissues of other organs,including cotyledons,rosette leaves,stems and roots.The subcellular localization of BjMc1 showed that the BjMc1-GFP fusion protein was co-localized with the marker in the plasma membrane,which was consistent with the previous study and suggested that BjMc1 could retain some conserved functions of its homologs in other species.6.The evolutionary process of Mc1 genes in land plantsMc1 genes were widespread in land plants.Structural analysis showed that the Mc1genes in land plants were conserved.Evolutionary analysis which was performed to detect the selective pressure for Mc1 genes revealed that although the result of site model suggested that the primary constraint for Mc1 genes in land plants was purifying selection,both the branch model and branch site model suggested that the ancestor of BjMc1 had undergone adaptive evolution.Two amino acids,the 139^th amino acid tryptophan（W）and899^th amino acid cysteine（C）,was detected to be the positive site by branch site model.The most reasonable explanation for these results was that in the evolutionary process of BjMc1 gene,the ancestor had undergone adaptive evolution during a short period of time before the divergence of dicot and monocot plants from their ancestors on the earth,and then purifying selection was the dominant force for the evolution of these amino acid sites.