白菜多聚半乳糖醛酸酶基因B cM F6的克隆、序列分析及其表达

 从白菜(Brassica campestris L. ssp. chinensis Makino) 核隐性不育两用系‘Bajh97201A /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1 194 bp, 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(²²⁷RVTCGPGHGIS1 IGS²⁴⁰ ) 等。将B cM F6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。

Cloning, Characterization and Expression of a Polygalacturonase Gene BcMF6 from Chinese Cabbage-pak-choi(Brassica campestris L. ssp. chinensis Makino)

On the basis of one cDNA-AFLP differential fragment isolated from the fertile Bline ofChinese cabbage-pak-choi (Brassica campestris L. ssp. chinensis Makino) nuclear recessive sterile A /B line(Bajh97201A /B) , the full length DNA and cDNA of BcMF6 gene encoding pollen2specific polygalacturonasewere cloned by rapid amplification of cDNA ends (RACE). The gene consisted of 1 194 bp encoding aprotein of 397 amino acids and was interrup ted 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 Cphosphorylation site,five casein kinaseⅡphosphorylation site, ten N-myristoylation sites and one polygalacturonase active position(²²⁷RVTCGPGHGIS1 IGS²⁴⁰ ). And the first 22 amino acids of the predicted BcMF6 protein form a N-terminalhydrophobic domain which disp layed the p roperties of a signal peptide and four parallel beta-helix repeatsfollowed behind. Four domains which were highly conserved in all p lant and fungal PGs was p resent inBcMF6. Phylogenetic analysis showed that BcMF6 falls into the category of clade2C, which included PGrelated to pollen. These results showed that BcMF6 acts as pollen-specific polygalacturonase. Furthermore,the exp ression using RT-PCR discovered that BcMF6 was exclusively exp ressed 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.