【摘要】 从白菜(Brassica campestris L.ssp.chinensis Makino)核隐性不育两用系‘Bajh97-01A/B’可育株中分离得到的一个cDNA-AFLP差异片段入手,利用RACE技术成功克隆了一个花粉特异的多聚半乳糖醛酸酶(PG)基因BcMF6的DNA和cDNA全长序列,并对其序列进行了分析。结果表明,该基因包含4个外显子和3个内含子,最大开放阅读框为1194bp,编码397个氨基酸序列,对推导的氨基酸序列进行分析,1到22个氨基酸是1信号肽序列,其后有4个平行β螺旋重复(PbH1)结构,该序列包含3个N-糖基化位点,4个蛋白激酶c磷酸化位点,5个酪蛋白激酶Ⅱ磷酸化位点,10个N-豆蔻酰化位点,1个PG活性位点(227RVTCGPGHGIS.IGS240)等。将BcMF6基因氨基酸序列与数据库中的其他PGs基因进行同源序列比对,构建系统进化树,发现该基因具有所有PGs基因特有的4个保守结构域,并且与花粉中特异表达的PG基因聚为一类,表明该基因是与花粉发育相关的多聚半乳糖醛酸酶基因。RT-PCR对其表达的研究表明,该基因在可育株(野生型)的中大蕾、开放花和短角果中特异表达,进一步表明该多聚半乳糖醛酸酶基因与白菜的花粉发育相关。更多还原

【Abstract】 On the basis of one cDNA-AFLP differential fragment isolated from the fertile B line of Chinese cabbage-pak-choi(Brassica campestris L.ssp.chinensis Makino)nuclear recessive sterile A/B line(Bajh97-01A/B),the full length DNA and cDNA of BcMF6 gene encoding pollen-specific polygalacturonase were cloned by rapid amplification of cDNA ends(RACE).The gene consisted of 1 194 bp encoding a protein of 397 amino acids and was interrupted by three introns of 81 bp,95 bp and 127 bp in length.Sequence analysis revealed that it has three N-glycosylation sites,four protein kinase C phosphorylation site,five casein kinaseⅡphosphorylation site,ten N-myristoylation sites and one polygalacturonase active position(227RVTCGPGHGIS.IGS240).And the first 22 amino acids of the predicted BcMF6 protein form a N-terminal hydrophobic domain which displayed the properties of a signal peptide and four parallel beta-helix repeats followed behind.Four domains which were highly conserved in all plant and fungal PGs was present in BcMF6.Phylogenetic analysis showed that BcMF6 falls into the category of clade-C,which included PG related to pollen.These results showed that BcMF6 acts as pollen-specific polygalacturonase.Furthermore,the expression using RT-PCR discovered that BcMF6 was exclusively expressed in middle and big flower bud,opened flower and slow pod of fertile line(wild type),which showed BcMF6 closely related with the development of flower.更多还原