Identification Of Molecular Markers, Cloning And Expression Analysis Of Genes Related To Thermo-sensitive Cytoplasmic Male Sterility In Non-heading Chinese Cabbage

Cytoplasmic male sterility (CMS) not only plays an important role in the utilization ofhybrid vigor, but also acts as a useful model for study the nucleus- cytoplasm interaction.So a great deal of research was performed from its genetics, cytology and physiology, butlittle was known of thermo-sensitive(Pol) CMS mechanism in non-heading Chinesecabbage(Brassica campestris ssp. chinensis Makino). This paper systematicallyinvestigated its physiological and biochemical characteristics, nucleus, mitochondrial andchloroplast genome of Pol cytoplasmic male sterility in non-heading Chinese cabbage toexplore the sterility mechanism.The results are showed as flows:1 Studies on physiological and biochemical characteristics of thermo-sensitive CMSline and its maintainer line of non-heading Chinese cabbageThe studies indicated that the contents of soluble sugar, soluble protein and praline, andfree, conjugate, bound Polyamine in CMS were lower than those in its maintainer line.Theactivities of peroxidise(POD), superoxide dismumse(SOD) and catalase (CAT) of CMS linewere higher than those of its maintainer line. The isozyme patterns of POD in buds andflowers of CMS line were different from that of its maintainer line. These results suggestedthat the variation of the contents of soluble sugar, soluble protein, praline, free, conjugate,bound Put, Spd, Spm, and the activities of POD, SOD and CAT contributed to Pollenabortion in thermo-sensitive CMS of non-heading Chinese cabbage.2 Analysis of RAPD and AFLP and identification of molecular markers linked to thecytoplasmic male sterility genes of thermo-sensitive CMS line and its maintainer linein non-heading Chinese cabbageThe genomic DNA of CMS line and its maintainer line of non-heading Chinesecabbage was studied by means of RAPD. Among the 386 random primers, there were 346primers giving amplified products in the two lines. Among them 62 primers presentedpolymorphism. Further more, the specific amplified fragment OPAY1000 of CMS line wasfound. The result of the sequencing indicated that the length of the sequence was 1053 bp,the accession number of OPAY1000 in GenBank was DQ320101. According to the DNA sequence, one pair of primers was designed and the RAPD marker were converted intoSCAR marker.In order to screen the specific fragment related to the cytoplasmic male sterility, AFLPwas developed in eytoplamic male sterile line and its maintainer line of non-headingChinese cabbage with 72 pairs of AFLP primers combination, Through identification ofrepeating test, three specific amplified fragments, BrCMSA1,BrCMSA2 and BrCMSA3,were obtained, The results of cloning and sequencing indicate that BrCMSA1 was of 90%of homology with a reverse transcription gene of Brassica napa, BrCMSA2 was of 58% ofhomology with ten amino acids sequence of a cDNA clone of Japanese Oryza sativa,BrCMSA3 was of 93% of homology with contig A of Brassica rapa. The results ofSouthern blot were identical with the AFLP analysis. Their accession numbers in GenBankare DQ513189,DQ513190 and DQ513191, respectively. These results suggested that therewas difference in the nuclear DNA of eytoplamic male sterile line and its maintainer line innon-heading Chinese cabbage.3 Special fragments related to CMS in mitochondrial DNA of thermo-sensitive CMSand its maintainer line of non-heading Chinese cabbageIn order to research and obtain special fragments related to CMS in mitochondrialDNA, the mitoehondrial DNA of the Pol CMS and its maintainer line was analyzed byRAPD. With 285 RAPD random primers, two specific fragments, AY1200 and RD800were obtained in the CMS line. Sequence analysis showed that RD800 was of 47% ofhomology with 132 amino acids of Coxiella burnetii QpH1 plasmid genome, AY1200 wasof 33% of homology with 65 amino acids of ferredoxase-dependent GLU2 of Arabidopsisthaliana.4 SSR analysis of chloroplast DNA of thermo-sensitive cytoplasmic male sterile linein non-heading Chinese cabbageChloroplast simple sequence repeat (cpSSR) was developed and successfully used toanalyze chloroplast genome difference of Pol CMS line and its maintainer line ofnon-heading Chinese cabbage. Thirty-six previously reported cpSSR primer pairs fromtobacoo were tested in Non-heading Chinese cabbage, ten of which could amplify intensivePCR products by polyacrylamide gel electrophoresis. Genome of CMS line and itsmaintainer line was then analyzed by the ten cpSSR primer pairs, there were a fewpolymorphisms and one of the prescreened primer pairs showed polymorphisms bypolyacrylamide gel electrophoresis and obtained one special fragment in CMS line only.These results revealed the difference of the chloroplast genome between CMS line and its maintainer line, which likely related to CMS.5 Cloning and expression analysis of genes related to cytoplasmic male sterilityorf224c and atp6c genes were cloned from the Pol CMS line of non-heading Chinesecabbage by specific primers designed on the basis of orf224 and atp6 gene in GenBank.The sequence of orf224c and atp6c genes showed same to reported orf224 and atp6 genes.Access numbers of the orf224c and atp6c in GenBank were DQ400846,DQ412559,respectively. The Semi-Q RT-PCR analysis of orf224c expression in different organsrevealed that the transcribing of orf224c gene in＜0.5mm bud and anther was higher thanthat in the leaf of the Pol CMS line; but there is no difference among the leaf,＜0.5mm budand anther of the maintainer line. This result suggested that the up regulated expression oforf224c gene was related to the inhibiting of the differentiation of archesporial cell.6 Differential expression analyses of bud of thermo-sensitive CMS line and itsmaintainer line of non-heading Chinese cabbage through SRAP techniqueTo detect differential expression in buds of thermo-sensitive(Pol)CMS line and itsmaintainer line, cDNA was analysis by using a new DNA finger printing technique-SRAP.With 30 pairs of SRAP primers combination, two special fragments, BcCMS1 andBcCMS2, were obtained. The results of cloning and sequencing indicated that BcCMS1was of 63% of homology with parts of the amino acids of cytogenetic chromosome ofBrassica rapa, BcCMS2 was of 88% of homology with the flc3 gene for flowering proteinof Brassica oleracea. The technology of SRAP was firstly adopted in the research of genedifferential expression successfully.