Cloning And Functional Confirmation Of A New Male Sterility-related Gene BcMF1 In Brassica Campestris L.

Brassica is one of the most important genus of Cruciferae,and is one of the vegetable crops culturedmost widely in China.Chinese cabbage pak-choi(Brassica campestris L.ssp.chinensis Makino),asubspecies of Brassica campestris L,is a typical allogamy plant with significant heterosis,and thepopularization and application of its F₁ hybrid seeds can improve the level of production in Chinese cabbage.The male sterility lines are very important for producing F₁ hybrid seeds in Chinese cabbage,and thebreeding and utilization of new male sterility lines are paid more attention by researchers.The isolation andfunctional confirmation of male sterility-related genes based on male sterility lines,as well as theinvestigation of behavior characteristics of these genes in chinese cabbage,might be useful to elucidate tothe possible molecular mechanism of plant male sterility,while improving breedings of male sterility lines.Earlier study in our laboratory has obtaind the preliminary full-length cDNA sequence of a newgene named Brassica campestris Male Fertility 1(BcMF1),by assembling the 5' end,and the 3' end fromRACE with a transcript-derived fragment(TDF) from cDNA-AFLP.In this paper,from the genic malesterile-fertile line'Bajh97-01AB'of Chinese cabbage taken as a research object,the real full-lengthcDNA and DNA sequences of BcMF1 were cloned by PCR amplification,firstly;the new promotersequences were isolated by TAIL PCR,secondly;the sequence characteristics of BcMF1 and its promoterwere analysed and the function of the hypothetical protein BcMF1 was predicted,thirdly;then RNAinterference(RNAi) expression vector and anti-sense expression vector of BcMF1 were constructed,andtransformed into flowering chinese cabbage(B.campestris ssp.chinensis var.parachinensis Tsen et Lee) byagrobacteriurn-mediated method to obtain their loss-of-function mutants for confirming the function ofBcMF1 in pollen development process;homologous genes of BcMF1 were cloned from deferent species inBrassica by PCR,and the phylogenetic trees of BcMF1 were constructed based on the alignment of thesehomologous genes,lastly.The major study results were as follows:(1) On the basis of cDNA-AFLP differential fragment BcMF1-A15T17 and its two RACE ends,the real full-length cDNA and DNA sequences of BcMF1 were cloned by PCR.BcMF1 was 1 684-bp long incDNA and 1 985-bp long in DNA,including two introns and three extrons in its DNA sequence,and itslargest open reading frame consisted of 1 416 bp,encoding 471 amino acid residues.The preliminaryfull-length cDNA sequence obtained from earlier study in our laboratory,was 1 613 bp long,notincluding a 70-bp AT-rich sequence and polyT tail,and its largest open reading frame consisted of 1 344 bp,coding 447 amino acid residues.The result in this paper corrected the errors of the cDNA and deducedamino acid sequence of BcMF1 from earlier study.Homology analysis showed that the deduced protein ofBcMF1 with no significant similarity to any known genes,had higher amino acids sequence similarity to aunknown protein family DUF1216 in Arabidopsis thaliana.BcMF1,encoding a extracellular protein inSecretory pathway,is the first expressed gene which had been reported in DUF1216 family.(2) The expression patterns of BcMF1 in the genic male sterile-fertile line'Bajh97-01AB'ofChinese cabbage were analyzed by Northern blot.The results showed that BcMF1 specially expressed inmiddle and big flower buds(≥1.0 mm in diameter),open flowers and stamens of the fertile lineBajh97-01B,and didn't express at any stage of the sterility'Bajh97-01A'line.The results suggest thatBcMF1 is a new gene related with genic male sterility in Chinese cabbage.(3) The DNA sequence of BcMF1 was amplified by PCR in the sterility line'Bajh97-01A',and thepromoter sequences of BcMF1 were isolated simultaneously by TAIL PCR BcMF1 from the fertile line'Bajh97-01B'and the sterility line'Bajh97-01A'.The sequence alignment was performed for finding out themolecular cause,in DNA level,why BcMF1 expressed differently between the fertile line'Bajh97-01B'and the sterility line'Bajh97-01A'.The results showed that no difference in BcMF1 and its promotersequences was found between the fertile line'Bajh97-01B'and the sterility line'Bajh97-01A',andthe results suggested that the expression difference was not caused by sequence mutantion of BcMF1and its promoter between the fertile line'Bajh97-01B'and the sterility line'Bajh97-01A'.Thus,it wasdeduced that another gene or some genes might regulate the expression of BcMF1.In the promotersequence of BcMF1,various cis-elements responding to light inducement,hormone inducement andstress condition were present,so it was predicted that the expression of BcMF1 might be influencedby light,hormone and stress condition.(4) BcMF1 Homologous genes were isolated from 7 species in Brassica by PCR,and the identification ratio of nucleotide and amino acid sequence were 97.8%～99.6% and 95.3%～98.9% in 7 specises,respectively.The phylogenetic trees were produced on the basis of homologous sequences alignment ofBcMF1 from different seven species in Brassica,and the evolution of homologous BcMF1 in these specieswas demonstrated by the phylogenetic trees.(5) It was proved that flowering Chinese cabbage is fit to confirm the function of BcMF1,by cloningand expression analysis of the homologous BcMF1 in flowering Chinese cabbage.Anti-sense plantexpression vector pBcMF1-AS and RNAi plant expression vector pBcMF1-RNAi of BcMF1 wereconstructed,and transformed into flowering Chinese cabbage by agrobacterium-mediated method.Themolecular assay showed that pBcMF1-AS and pBcMF1-RNAi constructs were integrated into the genomesof BcMF1-AS and BcMF1-RNAi transgenic plants with Kanamycin resistance respectively,and theexpression level of BcMF1 was down-regulated in BcMF1-AS and BcMF1-RNAi transformants.Growthperformance,floral organ development,pollen morphology,pollen germination in vitro and pollendevelopment of of transgenic plants were observed,these results showed that pollen development wereinfluenced by down-regulated expression of BcMF1 in transgenic plants.These results suggest that BcMF1is a new pollen development-related gene in Chinese cabbage.(6) Recombinant plant expression vectors constructed on the basis of the the binary vector pBI121,andtheir transgenic plants,were amplified by PCR with a universal primer pair designed according to the flanksequence of multiple clone site in pBI121;Further,the PCR products with predicted sizes were verified bydigestion with the restriction enzyme BamHâ… .The detecting results by PCR-Digestion were consistent withthese results by the traditional means,therefore a rapid and effective method to identify the expressionvectors and their transgenic plants containing pBI121 plasmid was developed in this paper.