Isolation And Functional Confirmation Of A Male Sterile Related Gene BcMF15 And Its Promoter In Chinese Cabbage-pak-choi

Brassica crops is a kind of important crops in agricultural production.The male sterile line was widely utilized in practice to produce F₁ hybrid seeds.Much attention was paid to research on the plant breeding of the male sterile line and the basis for application in Brassica crops.The research on male sterility mechanism of Brassica contributed to the understanding of microspore and pollen development process and provided theory direction to create man-made male sterile line.In this investigation a novel preferentially expressed cDNA sequence from B.campestris ssp.chinensis was isolated and characterized. The corresponding full-length cDNA and DNA was subsequently amplified by Rapid amplification of cDNA ends(RACE),named BcMF15(Brassica campestris Male Fertility 15,GenBank accession no.EF600901). The gene sequence was analyzed and proteins functions were predicted.Spatial and temporal expression patterns were analysed by RT-PCR during the different stages of plant development.On this base the plant RNAi expression vector of BcMF15 was constructed regulated by the constitutive promoter CaMV35S. flowering Chinese cabbage[B.campestris L.ssp.chinensis Markino var.parachinensis(Bailey)Tsen et Lee] was transformed by agrobacterium-mediated method to obtain their loss-of-function mutant.Lastly,their function in pollen development process was analyzed,The major study results as follows:(1)The full-length cDNA and DNA has been cloned from Chinese cabbage using RACE.The full-length cDNA was 768 bp.A comparison of the cDNA and DNA sequence by DNAStar software showed that BcMF15 was composed of a 312 bp open reading frame,encoding an 11.36 kDa protein of 103 amino acids.This amino acid sequence was a membrane protein with a signal peptide at the N-terminus.which has an obvious transmembrane helix.The GenBank BLAST results,showed that the BcMF15 sequence that produced significant alignments had 88%nucleotide similarity to a LTP-like mRNA sequence from Arabidopsis(Columbia).Meanwhile,six domains were predicted in the deduced BcMF15 protein,such as the AAI domain existing in some crucial proteins of pollen development-preferential,signal peptide, transmembrane domain,vWF domain,ZnF_C4 domain,and Tryp_alpha_amyl domain.The expression of its spatial and temporal expression patterns by comparing RNA isolated from various tissues of the wild-type plant with those of the mmc mutant(ZUBcajh97-01A)by RT-PCR.The transcript of the BcMF15 was undetectable in sporophytic tissues of fertile line such as scapes and leaves of seedling.Moreover,the expression signal of BcMF15 was detected from flower buds of stage 1 to stage 5,the flower and germinal siliques In contrast,the expression signal of BcMF15 was undetectable in any tissues of the mmc mutant. This indicated that BcMF15 was a crucial gene in microspore development.(2)Gene analogues from 10 species of six genera in Cuciferae were obtained by PCR strategy using specific primers designed from the full length of BcMF15.Homologous sequences of BcMF15 comparison indicated that the similarities among the genes at nucleotide and amino acid levels were over 80%and 53%, respectively.The conserved region of amino-acid sequences of BcMF15 orthologs included the transmembrane region in N-terminal,however there was some variation in C-terminal.The maximum parsimony(MP)trees showed that Brassica was closely related to Raphanus,followed by Capsella Medic, Arahidopsis Heynh and Barbarea,most distantly related to Orychophrogmus.These results.showed in Cruciferae the BcMF15 was relative conservative in evolution,and BcMF15 may play an important role in pollen development.(3)The RNAi expression vector of BcMF15 containing cons(?)tutive promoter CaMV35S was constructed,by agrobacterium-mediated method,their flower Chinese cabbage transgenic plantlets were obtained.(4)Molecular,morphological and cytological characterization of flower Chinese cabbage transgenic Kan^R plantlets transformed from pBI35S-RMF15 was carried out Real time quantitative RT-PCR on cDNA of small,middle,big floral buds,flower and-germinal siliques to quantify gene expression in plantlets transformed from pBI35S-RMF15 and nontransgenic plantlets.The results showed the expression of BcMF15 was caught and decreased in comparison to positive CK,and the expression of BcMF15 in middle, big floral buds of pBI35S-RMF15 were sharply inhibited,which proved that BcMF15 was a pollen-expressed LTP.The morphology of flowers in flowering Chinese cabbage transgenic plants for the RNAi BcMF15 revealed there were short and white flower filaments and little pollen in plants.As far as pBI35S-RMF15 was concerned the pollen germination was 35.94%in vitro,germination ratio of pollen decreased distinctly than 74.40%in the transgenic plants containing pBI121.The comparison of microspore morphology detected by scan electron microscope in transgenic plants Containing pBI35S-RMF15,When BcMF15 was inhibited,the abnormal development of pollen would be captured.The result proved that it was essential for BcMF15 to keep normal shape of pollen.Combining resin semithin sections in light microscope with Ultrathin sections of flower buds in transmission electron microscope,the observations for pollen development reveal at late uninucleate microspore stage the tapetum degenerated ahead of schedule,and the pollen was aborted.The result indicated that the expression of BcMF15 was inhibited by pBI35S-RMF15 which resulted in germination ratio of pollen decreased and abnormal pollen.So it was presumed the LTP encoded by BcMF15 was anther-specific,it may be responsible for lipid metabolism in anther through regulating the development of tapetum,and has an effect on the formation of pollen coat,pollination and elongation of pollen tube during microspore development.(5)Promoter of BcMF15 was isolated by TAIL-PCR for the first time.The GenBank BLAST results showed that the 5'-flanking regions of BcMF15 gene,named pBC15,produced significant alignments had 99%nucleotide similarity to genomic DNA from Brassica rapa subsp,pekinensis(Accession:AC189225). The sequence was abundant A/T,which consistent to the composition of bases in promoter sequence.A number of potential regulatory motifs corresponding to known cis-regulatory signals of eukaryotic genes were found to be present in the sequence.The cloned Sequence analysis showed that TATA box elements were found in the sequence,GATA motif in CaMV 35S promoter,also some pollen-specific function elements were present in promoter of BcMF15,such as GGTT and GTGA motif,which were pollen-specific enhancer sequences and shared regulatory elements;POLLEN1LELAT52 motif,which was One of two co-dependent regulatory elements responsible for pollen specific activation of tomato lat52_gene.Some elements to respond to environment were also found in the 5'-flanking regions of BcMF15 gene.These preliminary results indicate that this DNA fragment upstream of the BcMF15 coding sequence could very possibly be a promoter with pollen specificity,the expresesion patterns may be influenced by environmental factors such as light,dehydration and other stress factors.(6)Construction of pBc15::GUS fusing vector was achieved.Recombinant plasmid was designated as pBI-pBc15.Plant expression vector was constructed by ligating this promoter and with gus gene then transferred into epidermis cell of onion by microprojectile bombardment.Histochemical GUS assay showed cell transformed with fused plasmid pBI=pBc15 of BcMF15 putative promoter may directed the expression of pBI-pBc15,and fused proteins were localized in the nuceil of transformed cells.It revealed the promoter region is functionally active.(7)The BcMF15 promoter-GUS fusion construct was stably transformed into wild type(Columbia) Arabidopsis plants by floral dip and homozygous transgenic plants were developed.Screening Kan^R plantlets and PCR analysis of T1 generation transgenic seedling were carried.Histochemical assays of gus activity and subsequent resin section analysis on promoter transformant plant showed that expression of pBclS::GUS was detected in anthers.