小黑杨PsnAP1-1及PsnAP1-2基因的克隆及原核表达分析

Cloning and Prokaryotic Expression of PsnAP1-1 and PsnAP1-2 Genes in Poplar(Populus simonii×Populus nigra)

植物由营养生长向生殖生长的过程中,APETALA1(AP1)基因在其中起重要作用。我们采用RT-PCR方法克隆了2个杨树AP1同源基因全长cDNA,暂时命名为PsnAP1-1(GenBank No.KC866354)和PsnAP1-2(GenBank No.KC866355)。PsnAP1-1编码241个氨基酸,开放阅读框长度为726 bp,蛋白质分子量为28.1 kD,等电点为8.19;PsnAP1-2编码249个氨基酸,开放阅读框长度为750 bp,蛋白质分子量为28.7 kD,等电点为9.07。同源性分析表明,PsnAP1-1的核苷酸序列与拟南芥AP1同源基因的一致性为71%,而PsnAP1-2的同源性为67%。半定量RT-PCR分析表明,杨树PsnAP1-1和PsnAP1-2基因在根、茎、叶中均不表达,仅仅在花芽组织中表达。分别构建了原核表达质粒pET-PsnAP1-1和pET-PsnAP1-2,并转化大肠杆菌BL21,以IPTG诱导该融合蛋白体外表达,结合SDS-PAGE分析,证实这2个基因均表达了约35 kD的蛋白,该结果为深入研究AP1与其他MADS-box蛋白的互作机制及花分生组织的分子调控奠定了基础。

Flowering is the transition from vegetative to reproductive phase in plants, and it is regulated by APETALA1 （AP1） gene. We cloned two full-length cDNA sequences of homologous gene AP1 from Populus simonii x Populus nigra by RT-PCR, named as PsnAPI-1 （ GenBank No. KC866354） and PsnAP1-2 （ GenBank No. KC866355 ）. PsnAPI-1 contains an 726 bp open reading frame （ORF） corresponding to a deduced protein of 241 amino acids, while the estimated molecular weight and the isoelectric point of the putative protein were 28.1 kD and 8.19. PsnAP1-2 with a open reading frame（ORF） of 750 bp, encoding 249 amino acid with a predicted molecular mass of 28.7 kD and a pI of 9.07. A comparison of the deduced amino acid residues indicated that PsnAPI-1 and PsnAP1-2 nucleotide sequences were 71% and 67% identities with AP1 gene homologues of Arabidopsis thaliana. With expression analysis by RT-PCR, the PsnAPI-1 and PsnAP1-2 genes only expressed in flower buds, but not expressed in root, leaf, and stem tissues. Furthermore, we constructed recombinant plasmid pET-PsnAPI-1 and pET-PsnAP1-2, transformed to E. coli（ BI21 ）, and then induced proteins expression by IPTG. Two 35 kD recombinant proteins were expressed and separated by SDS-PAGE electrophoresis. Our results will provide theoretical and technical bases for analyzing the interaction mechanism of AP1, other MADS-box protein, and the molecular regulation of floral meristem in poplar.