稗草磷酸烯醇式丙酮酸羧化酶(PEPCase)基因的克隆与分析

为揭示C₄野生植物稗草（Echinochloa crusgalli）PEPCase的结构和功能特点，探索改善作物高光效新途径，本研究首次克隆了稗草（E. crusgalli）磷酸烯醇式丙酮酸羧化酶基因(ppc)的cDNA全长，测序及同源性比较分析结果证实，稗草ppc的cDNA全长为2 886 bp,编码961个氨基酸（GenBank登录号：AY251482）；核苷酸序列与谷子(Setaria italica)C₃型、高粱(Sorghum bicolor) C_3-2型、玉米(Zea mays) C_3-2型、水稻(Oryza sativa)C₃型磷酸烯醇式丙酮酸羧化酶基因的同源率分别达94.8%、93.2%、93.0%和89.7%。推导的氨基酸序列中C末端第771位氨基酸是丙氨酸（A），表明是一个C₃型基因。与其他植物几种不同形式PEPCase进行的多重序列比对与系统进化分析结果也证实，此基因的核苷酸序列及其编码的氨基酸序列与所有参与对比的C₃型序列的同源性均远远高于与C₄型的同源性。与玉米、高粱C_3-2型PEPCase的同源性分别高达到96.5%、96.4%，与高粱、玉米的C_3-1型PEPCase的同源性分别为84.3%、83.8%，而与谷子、玉米、甘蔗和高粱的C₄型PEPCase的一致性相对较低，分别为82.2%、79.1%、77.1%、76.6%。因此进一步推论新克隆的稗草ppc基因属于C_3-2型。对其编码的蛋白序列进行了结构域、活性位点和功能位点预测。

Cloning and Characterization of Phosphoenolpyruvate Carboxylase Gene from Echinochloa crusgalli

Phosphoenolpyruvate carboxylase(PEPCase) has a variety of functions in plants. In order to elucidate structural and functional characters of PEPCase from Echinochloa crusgalli,and search a new approach to improve crops photosynthesis,a full-length cDNA for PEPCase was isolated from E.crusgalli.Using the program of Blast on NCBI GenBank database,the sequence presented a very high match with the genes from other plants.After alignment on ClustalW program,the identities of the cloned fragment with PEPCase genes from Setaria italica C_3-form,Sorghum valgare C_4-form,Zea mays C_4-form and Oryza sativa C_3-form were about 94.8%,93.2%,93.0% and 89.7%,respectively.E.crusgalli ppc ORF is 2 886 bp,encoding 961 amino acid residues.The sequence has been submitted to the GenBank database,the accession number is AY251482.Alignment and phylogenetic analysis of the amino acid sequence deduced from the fragment and the PEPCase sequences of other plants retrieved from GenBank were carried out by ClustalW program,which showed that the sequences homology of E.crusgalli with C_3-form was higher than with C_(4-) form.To identify amino acid residues and/or domains responsible for C_4/ C_3-specific properties,we found the putative amino acid sequence contains a C_3 conserved alanine at position 771,and the sequence shared a homology of 96.5%,96.4% with the C_(3-2)-form PEPCase of Zea mays and Sorghum valgare,83.8%,84.3% with the C_(3-1)-form PEPCase of Zea mays and Sorghum valgare,and 82.2%,79.1%,77.1%,76.6% with the C_(4-) form PEPCase of S.italica,Z.mays,S.spontaneum and Sorghum valgare. In conclusion,the cloned sequence is a C_(3-2)-form of PEPCase gene from E.crusgalli.The domain,active sites and fuction sites of Echinochloa crusgalli PEPC protein are predicted.