Fine Mapping And Cloning The Multilocular Gene Bjln2 In Brassica Juncea

Multilocular traits have an important effect on plant genetic improvement and may provide new ideas for rapeseed breeding.Therefore,it is necessary to study the mechanism of multilocular trait.The parental materials used in this study were Qinghai Duoshi and Xinjie.Previous genetic studies have shown that multilocular trait of the Brassica juncea cultivar Duoshi is controlled by two recessive genes,Bjln1and Bjln2.The Bjln1 gene was located on A07 chromosome and has been cloned and the Bjln2 gene was located on E Block of B07 chromosome.Based on previous studies and genome sequencing results of Brassica juncea,the objective of the present study is to map,clone and transform the Bjln2 gene,and to understand the function of the Bjln2 gene.The main conclusions are summarized as follows:1.Fine mapping and prediction candidate gene of the BjLn2:9 markers were closely linked to multilocular gene Bjln2 were identified.The most closest markers on both sides were 8-221 and 11-46?and 11-52?,with genetic distance of 0.42 cM and0.21 cM,respectively.The Bjln2 gene was restricted to a 0.63 cM interval.BLASTn analysis all markers and Bjln2 gene was located within a 11.81-16.65 Mb region on chromosome B07.Combining fine mapping results with comparative genomic information,At1g75820?CLV1?in Arabidopsis thaliana was presumed to be a candidate gene for BjLn2 gene,named BjuB07.CLV1.2.Cloning and analysis of the candidate gene:The full gDNA and cDNA length of BjuB07.CLV1 and BjuB07.clv1 gene cloned respectively from bilocular and multilocular materials of near-isogenic lines.Subsequent sequence analysis indicated that BjuB07.CLV1 gene was composed of one intron and two exons.A 4961bp LTR transposon at 2346 bp of the coding region in multilocular materials insertion disrupted the normal transcription of BjuB07.CLV1 gene in the multilocular material,thus showing a multilocular phenotype.3.Protein structure analysis of the candidate gene:The results of protein structure prediction for candidate gene showed that BjuB07.CLV1 candidate gene protein consisting of 987 amino acids and BjuB07.clv1 protein consisting of 782amino acids.Both BjuB07.CLV1 and BjuB07.clv1 gene encode a leucine-rich repetitive receptor protein kinase?LRR-RLK?,while the protein kinase domain of BjuB07.clv1 gene is an incomplete serine/threonine protein kinase domain.It is inferred that incomplete serine/threonine protein kinase domains inhibit the expression of BjuB07.CLV1 gene,presenting as multilocular traits.4.Phylogeny analysis of BjuB07.CLV1:The phylogenetic tree analysis of the amino acid sequence encoded by the CLV1 gene of Brassicaceae showed that the BjuB07.CLV1 gene had a close relationship with Brassica nigra and had a far relationship with Brassica rapa.It is speculated that the BjuB07.CLV1 gene may be come from Brassica nigra,but not Brassica rapa.5.Genetic transformation of BjuB07.CLV1 gene:The structure of pBjuB07.CLV1:BjuB07.CLV1 was constructed and cloned into pCAMBIA2300 vector to make a recombinant vector.Thirty T₁ Arabidopsis-positive plants were obtained via kanamycin-resistant transformant screening into Arabidopsis clv1 mutant?CS45?.T₂ generations of transgenic plants from the same high-recovery T₁ plant exhibited a3:1?bilocular plants:multilocular plants?segregation ratio.These results indicated that the BjuB07.CLV1 gene has the same function as the clv1 gene of Arabidopsis and can be stably inherited between generations.The pBjuB07.CLV1:BjuB07.CLV1 construct was transformed into multilocular plants via Agrobacterium-mediated transformation simultaneously,and 14 T₀ B.juncea-positive plants were obtained.These results indicated that the BjuB07.CLV1 was BjLn2.6.Expression analysis of BjuB07.CLV1 gene:qRT-PCR was used to analyze the expression of roots,leaves,stems,SAMs,buds,flowers,pistils,stem leaves,and siliques between homozygous bilocular and multilocular materials.The BjuB07.CLV1gene was expressed in all tissues,and the expression level in bilocular plants was significantly higher than that in multilocular plants.This indicates that the BjuB07.CLV1 gene was ubiquitously expressed in all studied tissues.Cytological observation:Paraffin section and scanning electron microscopy analysis showed that the differences of siliques between homozygous bilocular and multilocular materials occurred before the primary inflorescence meristem.7.Cytological observation:Paraffin section and scanning electron microscopy analysis showed that the differences of siliques between homozygous bilocular and multilocular materials occurred before the primary inflorescence meristem.Longitudinal section of buds showed that the ovaries of multilocular plants were thicker and the pistils was larger.After transverse cutting the flower buds,the ovaries of bilocular materials were divided into two equal-sized locules by a fake diaphragm on average.With the growth of plants,the two fake diaphragms of multilocular materials gradually changed from"+"to"II",and the locules gradually changed from four equal-sized locules to three locules,with the middle locules larger than the bilateral locules.8.Effect of illumination duration on multilocular siliques rate:Multilocular traits can be inherited steadily,but they are significantly affected by environmental conditions.The chimerism rate of multilocular siliques under different illumination duration was analyzed.With the prolongation of illumination time,the percentage of multilocular siliques increased gradually.The rate of three carpels decreased gradually with the increase of the illumination duration.Conversely,the rate of the four carpels gradually increased with the increase of the illumination duration.