Positional Cloning And Functional Study Of Multilocular Gene Bjln1 In Brassica Juncea L.

Multilocular siliques with spontaneous mutation were existed in Brassica juncea,due to the characteristics of multi-locules（3–5）,which could increase the number of grains per silique to enhance yield.The multilocular traits were controlled by two recessive nuclear genes（Bjln1 and Bjln2）in B.juncea of Qinghai province.To reveal the formation mechanism of multilocular traits,the multilocular gene Bjln1was isolated from Qinghai multilocular rapeseed through the method of map-based cloning,the formation mechanism of multilocular traits and the function of Bjln1gene in multilocular B.juncea of Qinghai province were elaborated using ctology and molecular biology approaches.The main studies results were as follows:1.Fine mapping of BjLn1 to an interval of 85 Kb:The multilocular gene Bjln1was mapping to an approximately 85 Kb syntenic interval of A07 in the B.rapa genome by map-based cloning approach through the BC₃F₅ population derived from the cross of Qinghai multilocular rapeseed and Tayou2（bilocular parent）.Combined with the information analysis of comparative genomics,BjuA07.CLV1 as a candidate gene of BjLn1,which is homologous with CLV1 of Arabidopsis thaliana.2.BjuA07.CLV1 has the ability to recover the bilocular traits:Using homozygous bilocular parental DNA as template,full-length amplified primers were designed to obtain a 5365 bp gDNA sequence of BjuA07.CLV1.The sequence was introduced into the expression vector of pCAMBIA2300 and transformed into the Qinghai multilocular rapeseed,58 transgenic positive plants（T₀）were obtained,25 plants showed varying degrees recovery of bilocular siliques,and 6 plants exhibited a higher bilocular silique percentage（>80%siliques）.The T26 with the highest recovery rate was self-pollinated.There were 42 individual plants in T₁ generation,and the separation ratio of bilocular plants and multilocular plants was nearly 3:1,indicating that only one copy of T26 has the insertion of the target fragment.Specific primers were used to detect the insertion of exogenous fragments of BjuA07.CLV1 in 42individual strains,corresponding to their phenotypes.Complete cosegregation between genotypes and phenotypes of 42 T₁ plants were detected by closely linked molecular markers.This indicated that the exogenous vector of BjuA07.CLV1 was successfully integrated into Qinghai multilocular rapeseed,which could restore the multilocular silique to bilocular steadily.Therefore,the BjuA07.CLV1 was experimentally confirmed to correspond to the BjLn1 gene,and the allele Bjln1 was named as BjuA07.clv1.3.Amino acid changes at positions 28 and 63 of BjuA07.CLV1 affected the carpel numbers of silique:Through the comparative analysis of the cDNA and gDNA sequences of BjuA07.CLV1 and BjuA07.clv1,the coding region of BjuA07.CLV1 was composed of two exons and one intron,and the CDS sequence is 2964 bp,encoding a total of 987 amino acids.There are fifty-nine SNPs in the CDS sequence of BjuA07.CLV1and BjuA07.clv1.Wherein,five SNPs at positions 83,119,151,187 and1014 caused five amino acid mutations at positions 28,40,51,63 and 338.To detect whether the five non-synonymous SNPs causing amino acid changes in BjuA07.clv1affect its function,the transformation vectors of pBjuA07.CLV1::BjuA07.CLV1（vector 1）and pBjuA07.CLV1::BjuA07.clv1（vector 2）were constructed,and were transformed into CS45 mutant,respectively.The results showed that the recovery rate of bilocular siliques in CS45 after the transfer of vector 2 was significantly lower than that after the transfer of vector 1,suggesting that five non-synonymous SNPs in the CDS of BjuA07.CLV1 affected the number of carpels.To further detect which of the five non-synonymous SNPs correlated with the changes of the carpel number,five SNP mutation detection vectors of pBjuA07.CLV1::BjuA07.clv1（83 T→C）（vector 3）,pBjuA07.CLV1::BjuA07.clv1（119 A→C）（vector 4）,pBjuA07.CLV1::BjuA07.clv1（151 C→A）（vector 5）,pBjuA07.CLV1::BjuA07.clv1（187 T→G）（vector 6）and pBjuA07.CLV1::BjuA07.clv1（1014 G→C）（vector 7）were constructed,and were transformed into CS45 mutant,respectively.Transgenic plants of 5 vectors all showed partial recovery of bilocular siliques.The recovery rate of bilocular siliques of transgenic plants with vector 4,5 and 7 was not significantly different from that of transgenic plants with vector 2,while the recovery rate of bilocular siliques of transgenic plants with vector 3 and vector 6 was significantly higher than that of transgenic plants with vector 2.In addition,there was no significant difference between the recovery rate of bilocular siliques of transgenic plants with vector 3 and vector 6 and that with vector 1,indicating that the multilocular siliques of CS45mutant was restored to be bilocular,which was closely related to the transfer of vector3 and vector 6.Finally,it was confirmed that the two SNPs at positions 83 and 187 in the CDS of BjuA07.clv1 resulted in the variation of amino acids at positions 28 and63,which affected the function of BjuA07.clv1 gene and caused the increase of the carpel numbers of silique.4.The 702 bp deletion in the promoter region of BjuA07.clv1 affects the number of carpel:Compared with the bilocular gene BjuA07.CLV1,the multilocular gene BjuA07.clv1 has a 702 bp deletion at position 1015 in the upstream of the starting codon.To tetect whether this 702 bp deletion influence promoter function,plant binary expression vector pCAMBIA2300 was selected to construct the vector of pBjuA07.clv1（702 bp deletion at position-1015）::BjuA07.CLV1（vector 8）,and was transformed into CS45 mutant.A total of 11 transgenic positive plants were obtained in T₁ generation,partial multilocular siliques of 11 plants were restored to bilocular siliques.The recovery rate of bilocular siliques of transgenic plants with pBjuA07.clv1（702 bp deletion at position-1015）::BjuA07.CLV1（vector 8）was significantly lower than that of transgenic plants with pBjuA07.CLV1::BjuA07.CLV1（vector 1）,implying that the 702 bp deletion in the promoter region of BjuA07.clv1affected the promoter function and led to the increase of the carpel numbers.5.The Comparative analysis on flower bud differentiation of multilocular and bilocular traits in B.juncea:In order to disclose the formation process of multilocular traits,we constructed a BC₃F₅ population from a cross between Qinghai multilocular rapeseed and Tayou2（bilocular parent）to compare the differentiation of different tissues of homozygous bilocular plants and multilocular plants through paraffin section along with scanning electron-microscopy observation.The results indicate that the flower bud differentiation in Qinghai multilocular rapeseed took place at about 22 days after seedling emergence,with an average leaf number of 5,and whole differentiation was divided into five stages.There were differences in the SAM,FM,carpel,stigma,and seed rows between multilocular rapeseed and bilocular rapeseed.6.BjuA07.CLV1 affects the diameter of SAM and FM:In the middle and late stage of flower bud differentiation,the diameter of SAM in multilocular rape was remarkably larger than that in bilocular rape,SAM was considered to be the earliest tissue that emerged differentiation difference between bilocular rapeseed and multilocular rapeseed,and the difference was maintained until the emergence of flower bud primordium;Furthermore,the diameter of FM of multilocular plants was significantly greater than that in bilocular plants through the microscopical comparison of the longitudinal section of the flower bud primordium.These results show that BjuA07.CLV1 mainly affects the diameter of SAM and FM.7.BjuA07.CLV1 affects carpel numbers of siliques and seeds rows:At the stage of carpel differentiation,the floral meristem has structural changes.The center of growing cone of the bilocular was deeply recessed into an“I”shape,forming half and half regions with two carpels,in the middle is a row of pseudoseptum with a row of seeds on each side,eventually forming a bilocular silique with two carpels and two rows of seeds;while that of the multilocular into an“X”shaped region with four carpels,the central growth cone of the flower bud is divided into four regions,and the corresponding four ovary locules are gradually formed,accompanied by two parallel diaphragms and bearing four rows of seeds,eventually forming a four locules silique with four carpels and four rows of seeds.The increase of grain number per silique of multilocular materials was mainly due to the increase of seeds rows.8.BjuA07.CLV1 and BjuA07.clv1 exhibit significant differences in transcription level:To understand the expression patterns of BjuA07.CLV1 and BjuA07.clv1,we systematically studied the expression quantity of both in leaf,root,stem,cauline leaf,SAM,bud,flower,pistil and silique through the method combining quantitative real-time PCR with GUS staining.qRT-PCR analysis revealed that BjuA07.CLV1 was expressed in various organs containing root,leaf,cauline leaf,stem,SAM,bud,flower and silique.Transforming the Pro _BjuA07.CLV1:GUS fusion vector into Columbia wild plant of A.thaliana,GUS staining was detected in different tissues of transgenic T₁ generation plants,different tissue expression pattern was consistent with the results of qRT-PCR.Compared with BjuA07.CLV1,the expression quantity of BjuA07.clv1in stem,SAM and bud was significantly decreased.This suggests that the 702 bp deletion in the promoter region of BjuA07.clv1 affects the expression of this gene.9.BjuA07.CLV1 encodes a receptor kinase located in the cytoplasmic membrane:The amino acid sequence of BjuA07.CLV1 was submitted to the NCBI for Blast P analysis,a total of 14 protein sequences with high homology were retrieved,MEGA4.0 software was used for phylogenetic tree analysis among the protein sequences of BjuA07.CLV1 and its fourteen homologous sequences,results show that the protein sequences of BjuA07.CLV1 had considerably higher sequence identity（99%）with the protein sequences of BnCLV1 in B.napus and BrCLV1 in B.rapa,these results suggest that the evolution of BjuA07.CLV1 was very close to BrCLV1.The amino acid sequence alignment of BjuA07.CLV1 and AtCLV1 showed higher sequence consistency and the two sequences were over 88%homologous.The protein structure of BjuA07.CLV1 was predicted by ExPASy website,which is consisting of an extracellular leucine-rich repeat sequence（LRRs）,a transmembrane domain,and an active site of serine/threonine protein kinase,and it is similar to the protein sequences of AtCLV1.In order to further verify whether BjuA07.CLV1 has the similar function to AtCLV1,the fusion expression vector of BjuA07.CLV1 and green fluorescent protein was constructed and transformed into the protoplast of A.thaliana,the green fluorescent signal of BjuA07.CLV1-GFP was observed on the cytoplasmic membrane.It is believed that BjuA07.CLV1 and AtCLV1 have similar protein functions,encoding a receptor kinase located in the cytoplasmic membrane and playing a role in cell signal transduction.10.BjuA07.clv1 interfered with the CLV/WUS pathway:We used SAM tissues of multilocular and homozygous bilocular plants in BC₃F₅ population as materials,investigating the dynamic expression differences of BjuA07.CLV1 and the other eight related genes in the CLV/WUS regulatory pathway in the multilocular and bilocular SAM through the qRT-PCR approach,detecting the expressions of WUS,CLV3,RPK2 and POL in multilocular SAM were higher than those in bilocular SAM,while the expression of CLV2 and BjuA07.CLV1（CLV1）in multilocular SAM was significantly lower than that in bilocular SAM.It is believed that the increased expression level of POL gene in multilocular SAM weakens the signal transmission of CLV3,removes the expression restriction of WUS,produces more stem cells,and makes SAM swell.The results showed that the mutation of BjuA07.clv1 in multilocular SAM disrupted the CLV/WUS signaling pathway,leading to the increase of SAM and the formation of multilocular siliques.