Comparison Of Biological Diversities And Cloning Of The Genes Related To CMS Between Isonuclear Alloplasmic CMS And Maintainer In Brassica Campestris L.

Chinese cabbage-pak-choi(Brassica carapestris L.ssp.chinensis Makino)is typical allogamy plants and has obvious heterosis.Cytoplasmic male sterility line is a ideal system to product hybrid in cabbage and other Brassica plant.CMS is an important tool for groups improved and the heterosis advantages,but also an ideal material to investigate pollen development and nuclear-cytoplasmic interaction.Therefore,it is a great theoretical significance and production value to select and apply Chinese cabbage CMS.In this thesis,a novel alloplasmic CMS system is produced from B.napus(with OguraCMS and PolimaCMS)(+)B.campestris ssp.chinensis cv Aijiaohuang fertile strain,and continuous backcross with the maintainer.Chinese cabbage "Aijiaohuang" PolimaCMS(P)and OguraCMS(G)are isonuclear alloplasmic CMS lines to share the common maintainer line(B).We intend to do comparative analysis of biological diversities and cloning of differentially expressed genes related to male sterility between isonuclear alloplasmic CMS and maintainer in B.campestris L.The mechanism of CMS will be proven by the results.The main results are as follow:(1)Comparative study of sterility traits had been made among isonuclear alloplasmic CMS and maintainer.The results indicated the isonuclear alloplasmic CMS lines had the filament displayed shorter significantly,anther white and indehiscent,and the pistil and nectary were normal.The color of P and G anther within bud at 3.5mm was not yellow,compared with B.The results indicated that the isonuclear alloplasmic CMS lines could be used to produce F₁ hybrids.(2)Cytomorphological analysis indicated that the abortion of anther development in PolimaCMS lines of B.campestris may occur at tetrad stage.The programmed cell death of tapetum cell occurred early.The tapetum cytoplasm is degenerated with staining heavily,Tapetal cells were persistent and become small,the anther sac shriveled,and the continuous and senescing tapetum surrounds the aberrant pollen in the shriveled anther bounded by an intact endothecium and epidemis.The microspore has large vacuoles and mostly unstructured cytoplasm,lack of engorgement in microspore.The pollen plasmolysis and the micorspores show severe inability to dissociate,and collapsed,their cytoplasm degenerates.The abortion of anther development in OguraCMS lines of B.campestris may occur at uninucleic-vacuoles stage.The tapetum has become abnormally expanded with large vacuoles,The microspores cytoplasm detaches from the exine wall and undergoes degeneration;only the sporopollenin material of the exines is present in the collapsed and indehiscent anther (3)Content of 4 phytohormones,IAA,GA₃,ZR and JA,were measured by enzyme-linked immunosorbent assay(ELISA)in buds of the isonuclcar alloplasmic CMS lines and maintainer to study the relationship between male sterility and phytohormone changes.The results showed that the isonuclear alloplasmic CMS buds were lower in IAA,ZR and JA contents,but higher in GA₃ content than the maintainer buds,especially at the middle buds(about uninucleic-vacuoles stage.).The phytohormone equilibrium was broken between IAA,GA₃,ZR and JA.All results suggested that the ultra-expansion of OguraCMS tapetal cells might be accelerated by high GA₃ accumulation,and ZR and JA deficiency might be associated respectively with the inhibition of cell division in tapetum and microsporc cell,anther indehiscent and the filament displayed shorter significantly in the isonuclear alloplasmic CMS lines.(4)Gene expressions of the isonuclear alloplasmic CMS lines and maintainer at bud stage were analyzed by means of cDNA-AFLE Three patterns(seven kinds)of gene expression in maintainer were observed,which include:(â… )enhanced expression(three kinds);(â…¡)expression of weakening(three kinds); (â…¢)the expression identically(one kind);There were three patterns(seven kinds)of gene expression the isonuclear alloplasmic CMS lines,including gone expression trend unaffected by nuclear-cytoplasmic interaction and gene expression trend affected by nuclear-cytoplasmic interaction(four kinds).Nine different expression bands were cloned,sequenced,blasted in GenBank and function prediction.(5)In the attempt to elucidate the molecular mechanism of CMS.OguraCMS lines were obtained in Chinese cabbage after interspecific hybridization between B..napus L.OguraCMS and B.campestris ssp. chinensis followed by recurrent backcross with B.campestris ssp.chinensis as the pollen donor.The CMS lines were significantly characterized by the whitish anther and indehiscence of anther.The tapetal hypertrophy with excess vacuolation was the first observed defective soon after the tetrad stage, subsequently the microspores defected in pollen wall formation,and later the cytoplasm detached from the exine wall and underwent degeneration.With aid of cDNA-AFLP and RACE approaches,we cloned the BcMYBogu(GenBank accession No:EF127861)in Chinese cabbage,which is premature expressed in early and middle stage floral buds of G,and predicted to encode a novel protein with a DNA binding domain: SH[AL]QKY[RF]motif at the N-terminus.Phylogenetic comparison revealed that the BcMYBogu was clustered with AtMYB32,AtMYB26 and AtMYB4,which were indicated to be involved in male sterility in Arabidopsis thaliana.The BcMYBogu transcript was detected in rosette leaves,floral buds and stems by RT-PCR analysis.Compared with the maintainer,the expression level of BcMYBogu was increased in these organs,especially in floral buds of OguraCMS lines.The expression patterns of nine key enzymes in the phcnylpropanoid pathway were changed(PAL2,OMT1,4CL3,CHS,F3H,F3'H,FLS1,DFR,ANS). The buds flavone content of G was lower than B.The investigation suggests that BcMYBogu is a new member of the MYB family involved in male sterility in Chinese cabbage.The phenylpropanoid pathway might be related to the white anther of G. (6)This study was carried out in order to better understanding the mechanism of nuclear-cytoplasmic interactions in OguraCMS Chinese cabbage(B.campestris L.).Expreesion profiling was performed using cDNA-AFLP technology on floral buds of OguraCMS lines and maintainers in Chinese cabbage.A differentially expressed cDNA fragment was obtained,which was unexpressed in early-stage buds of maintainers,but expressed steabilely in OguraCMS floral buds.The full cDNA,designated as BcBHLogu (GenBank accession numbers:EF127860),was obtained by RACE.BcBHLogu coded 122 amino acids, contained a bHLH domain,and shared high homology with AtBHLH060.The phyogenetic analysis indicted BcBHLogu and AtBHLHs060/048 were orthologs genes.Its function was similar to BEEs1/2/3,which were three redundant Brassinostreriod early response genes.The filament of G displayed shorter significantly, anther white and indehiscent,and the pistil displayed longer than B.JA content of G was lower than B. BcBHLHogu was predicted a novel bHLH transcription factor in Chinese cabbage,which gene appears to be involved in developmental signaling events for floral processes and had a function related to Brassinosteriods signaling response.(7)To elucidate this network of interactions,we have applied a transcript profiling method to PolimaCMS lines and maintainer lines of Chinese cabbage subjected to early-,middle-and late-stage floral buds.The differential expressed cDNAs of buds were identified through cDNA-AFLP.Among them,the expressed sequence tag(EST)pA9T4,only expressed in late-stage buds of PoimalCMS lines,showed high sequence homology to the genes BoTMT2(B.oleracea).The cDNA and genomic clone of pA9T4 were subsequently amplified by PCR designated BcTMTpol(GenBank accession numbers:EF127862),.The BcTMTpol encoded polypeptides of identical lengths(227 amino acids)and predicted masses(ca.25.17 kDa).BcTMTpol contained the typical methyltransferase signatures motifâ… ,â…¡,â…¢.IAA content was upward and JA content was downward during middle to late-stage floral buds of P,respectively.IAA/JA was different also.The results furnishes BcTMTpol appears to be related with IAA-JA interaction and the interaction might be involved in male sterility.