| During the process of pollen development,thousands of genes take part in activities as microspore development,pollen wall construction,mitosis,meiosis and synthesis of pollen materials.Spatiotemporal expression of these genes is essential for the formation of fertile pollen grains.Changes of environmental factors or gene mutation may affect pollen development or even cause male sterility.We adopted cDNA amplified fragment length polymorphism(cDNA-AFLP)technique and Affymetrix GeneChip to learn about genes expressed during flower bud development among three Chinese cabbage-pak-choi(Brassica campestris L.ssp.chinenesis Makino,syn.B.rapa ssp.chinenesis)materials,Polima cytoplasmic male sterile(CMS)line 'Bcpol97-05A',Ogura CMS line 'Bcogu97-06A'and their co-maintainer line 'Bcajh97-01B'.On the basis of cDNA-AFLP and GeneChip results,we isolated some important genes from 'Aijiaohuang' Chinese cabbage-pak-choi and its relative species by means of rapid-amplification of cDNA ends(RACEs)and homology-based cloning method.(1)By means of cDNA-AFLP,98 transcript-derived fragments(TDFs)were obtained.Their homologous genes participated in cell wall metabolism,defense response,electron transport or energy pathway,metabolism,protein metabolism,signal transduction,transcription and transport.61 TDFs expressed especially in flower buds of 'Bcpol97-05A' and "Bcajh97-01B',and 30 TDFs silenced in "Bcpol97-05A'.12 TDFs were detected in 'Bcpol97-05A' and no expression in other two materials.There were 75 TDFs difference between 'Bcogu97-06A' and "Bcajh97-01B' and 40 were found silent in 'Bcogu97-06A'.8 TDFs expressed only in flower buds of "Bcogu97-06A'.(2)Total 688 differential expressed genes were obtained from three Chinese cabbage-pak-choi materials, 'Bcpol97-05A','Bcogu97-06A' and 'Bcajh97-01B',by Arabidopsis whole genomie ATH1 genechip.388 of them were down-regulated and 352 up-regulated in two male sterile lines.In those up-regulated genes,the two CMS line shared 63 genes and there are amount of mitochondrial and chloroplast genes.94 genes especially expressed in flower buds of "Bcpol97-05A' and 24 of them were chloroplast and mitochondrial genes.Among 338 down-regulated genes,the two CMS materials shared 75 genes.There were 93 genes specially down-regulated in "Bcpol97-05A" flower buds and 109 genes in "Bcogu97-06A'.(3)By means of cDNA-AFLP,a fragment P1708 was amplified from cytoplasmic male sterile Chinese cabbage 'Bpol97-05A' in Chinese cabbage-pak-choi,RT-PCR showed that this fragment was specifically expressed in male sterile material.Sequencing and Blast indicated that P1708 had 100%homolog with chloroplast gene between tree and ndhJ genes except a 54-bp repeat sequence.We designed the full length primers according to trnL and ndhJ gene region and a 2000-bp fragment was amplified using cDNA as templates.The sequencing results showed that the gene region trnL-ndhJ of cytoplasmic male sterile Chinese cabbage-pak-choi had a 54-bp repeat sequence and some sites variation.(4)A novel anther-specific proline rich protein gene,designated BcAPG(Brassica campestris anther-specific proline-rich protein gene),was isolated from male fertile Chinese cabbage-pak-choi 'Bajh97-05B' by means of cDNA-AFLP and RACEs.BcAPG gene contained an open reading frame of 1,731 bp encoding 576 amino acids with high level of proline residue(22.4%).RT-PCR indicated that BcAPG expressed specifically in anthers of 'Bajh97-O5B' but no detection in all tissues of two CMS lines, Polima CMS line 'Bpol97-05A' and Ogura CMS line 'Bogu97-06A'.Homologous genes of BcAPG were isolated from other six colewort crops and sequence comparison was performed.(5)A male sterility gene homolog,designated BcMS2(Brassica campestris Male Sterility 2 gene),was isolated from flower buds using gene-specific primer pairs and was submitted to GenBank under accession number EF093533.Comparison of BcMS2 gene with MS2 from Arabidopsis thaliana and MS2Bnap from B. napus revealed some differences in gene structure and evolution.The full genornic DNA sequence of BcMS2 was 2,576 bp in length containing 8 exons and 7 introns,more than those of MS2Bnap but less than MS2. RT-PCR showed that BcMS2 gene expressed only in stageâ…¢flower buds of male fertile Chinese cabbage-pak-choi 'Bajh97-01B' and there were no detection in all organs of Polima CMS line 'Bpol97-05A' and Ogura CMS line 'Bogu97-06A'.Furthermore,RT-PCR revealed that BcMS2 expressed only in anthers of male fertile material and there were no expression in sepals,petals,filaments and pistils.These results suggested that BcMS2 was an anther-specific gene and might be essential for the fertility of Chinese cabbage-pak-choi.(6)Actin is an important material for construction of cell cytoskeleton microfibre systems.During pollen development and pollen tube elongation,the higher activity of actin protein is essential for both biological processes.We designed full length primer pairs according to known sequences and successfully isolated the gene which was assigned BcACT(Brassica campestris actin protein gene)from both mixed flower buds cDNA and genomic DNA of Chinese cabbage-pak-choi.BcACT is 1,704 bp in length with 3 introns.The complete cds of BcACT is 1,134 bp in length encoding 377 amino acids.RT-PCR results showed BcACT express in flower buds of both maintainer "Bajh97-05B'and Polima CMS line'Bpol97-05A'. Further studies showed that BcACT gene was an anther-specific one and up-regulated during anther development.(7)A gene,designated BcPRO(Brassica campestris profilin protein gene),was isolated from flower buds using gene-specific primer pairs and was submitted to GenBank under accession number EF492988. The complete coding DNA sequence of BcPRO was 405 bp in length encoding 134 amino acids and shared more than 90%identity with those of B.napus and A.thaliana.RT-PCR showed that BcPRO gene expressed in stageâ…¢,â…£andâ…¤flower buds of maintainer and in stageâ…¢andâ…£flower buds of Polima CMS. However,there was no detection in all tissues of Ogura CMS.RT-PCR revealed BcPRO gene expressed only in anthers,not in sepals,petals,filaments and pistils.These results suggested that BcPRO gene was an anther-specific one and might play important roles in pollen development.(8)Chalcone synthase(CHS)probe indicated different expression pattern among three Chinese cabbage-pak-choi materials.We designed gene-specific primer pairs according reported sequences of CHS and isolated BcCHS(Brassica campestris chalcone synthase gene)from mixed flower buds of male fertile line.The gene was designated BcCHS and submitted to NCBI under the accession number of EF101136. BcCHS gene is 1,236 bp in length with a sole 75 bp intron.The complete cds of BcCHS is 1,188 bp in length encoding 395 amino acids.BcCHS gene expressed in gradeâ…£,â…¤and open flowers of 'Bcajh97-01B' and 'Bcogu97-06A'.Further studies showed BcCHS express in anthers and petals.Additionally,a spontaneous white-flower mutant of Chinese cabbage-pak-choi was found in our test fields,and all the plant characters except flower color were identical with wild type ones.We isolated BcCHS-wf gene using the same primers and PCR procedure as cloning BcCHS.Comparison of the genomic sequences revealed two mutations in BcCHS-wf,both with A to G transitions,one at position +37 bp and the other at +970 bp.Both nucleotide substitutions occurred in AGA codes for arginine into GGA for glycin at residue +13 and into AGC coding for serine at residue +229,respectively.
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