籽粒苋C_4型磷酸烯醇式丙酮酸羧化酶基因的克隆和表达

Molecular Cloning and Expression Analysis of C_4 Phosphoenolpyruvate Car-boxylase Gene from A. hypochondriacus L.

磷酸烯醇式丙酮酸羧化酶(phosphoenolpyruvate carboxylase,PEPC)是C4途径的关键酶之一。本研究克隆了双子叶植物籽粒苋(A.hypochondriacus L.)C4型PEPC基因的cDNA和gDNA全长序列。该基因编码区cDNA全长2895bp(GenBank登录号为HQ186302),编码964个氨基酸残基。对应的gDNA全长为6785bp,含10个外显子和9个内含子,内含子总长3890bp,其中第一内含子最长,为1662bp,具GATA-motif、G-box等15个光应答元件,9个激素应答元件,29个CAAT box,72个TATA box。该特征在本研究范围内的植物中,为籽粒苋所独有。比较多种植物表明,不同植物之间的C3/C4型PEPC基因的外显子长度具有较高的一致性,而内含子的长度变化较大。不同植物PEPC基因对核苷酸具有一定的选择性,并且外显子和内含子的GC含量呈正相关。总的趋势是单子叶植物外显子和内含子的GC含量均高于双子叶植物。利用半定量RT-PCR分析表达模式,发现籽粒苋PEPC基因主要在叶片中表达,表达量随光照时间延长而增加。

Phosphoenolpyruvate carboxylase （PEPC） plays a crucial role in primarily fixing atmospheric CO2 in C4 and CAM plants. This paper aimed at cloning both cDNA and genomic DNA of PEPC gene from Amarnthus hypochondriacus in order to provide candidate genes for C4 genetic engineering. The cDNA was cloned by RT-PCR, and the corresponding gDNA was cloned by LA-PCR. The sequence characteristics were analyzed using bioinformatics softwares including Blast, ClustalX （1.8）, DNAMAN, PlantCARE. The gene ex-pression pattern was analyzed by using semi-quantitative RT-PCR. The results indicated that the cDNA with the complete coding region was 2 895 bp in length which encoded 964 putative amino acids. The amino acid at site 774 was the serine that is an invariant residue in all C4 phosphoenolpyruvate carboxylases regardless of their taxonomic. The corresponding gDNA was 6 785 bp in length which harbored ten exons and nine introns. The longest intron was the first intron with 1 662 bp in length. A computer scan disclosed that the first intron harbored 15 light-induced elements, nine hormone-induced elements, 29 TAAT motifs and 72 TATA motifs. In comparisons of PEPC genes between different species the exons are usually conserved but the introns have often diverged in both nucleotide sequences and lengths. The GC ratios of exons are positively correlated with those of introns in PEPC genes from different species. And the GC ratios of PEPC gene in dicotyledon A. hypochondriacus, potato, A. thaliana and ice plant are lower than those in monocotyledon maize, rice and wheat. The semi-quantitative RT-PCR analysis revealed that the cloned PEPC gene was light-inducible leaf-specifically expressed and classified as C4-type sub-family groups.