【摘要】：【目的】克隆橡膠樹磷酸烯醇式丙酮酸羧化酶(PEPC)基因的全長cDNA,通過生物信息學(xué)和基因表達分析,為基因功能研究奠定基礎(chǔ)�！痉椒ā扛鶕�(jù)橡膠樹PEPC基因片段序列設(shè)計引物,通過RACE獲得cDNA全長,用DNAMAN軟件預(yù)測編碼多肽序列,用Prot Param工具分析多肽基本理化性質(zhì),用NCBI-Blast工具搜索同源的核酸及多肽序列,用Clustal X2.0軟件比對不同植物來源的多肽序列,并用MEGA 6.06軟件構(gòu)建系統(tǒng)進化樹,用Real-time RT-PCR分析基因表達模式�！窘Y(jié)果】從橡膠樹膠乳中克隆到一個新的PEPC基因的全長cDNA,其長度為3206 bp,包含一個2898 bp的開放讀碼框(ORF),112 bp的5’UTR和196 bp的3’UTR。預(yù)測該基因編碼965個氨基酸的多肽(HbPPC2),其分子量為110.35 kDa,等電點為5.76;該基因的編碼區(qū)核苷酸序列和其編碼多肽氨基酸序列分別與早期克隆的橡膠樹HbPPC1基因(KJ721228)的編碼區(qū)和編碼多肽序列有81.3%和92.1%的同源性。預(yù)測HbPPC2定位于細胞質(zhì),為不穩(wěn)定蛋白,其活性受多種因素調(diào)控。預(yù)測HbPPC2酶活性中心是其第172位殘基His,磷酸化位點是其第11位殘基Ser,單泛素化位點是其第628位殘基Lys。其第178、179、226和366位殘基Arg在三維結(jié)構(gòu)中靠近,構(gòu)成葡萄糖-6-磷酸(G6P)的結(jié)合位點;其第450、641、753、767位殘基Arg在三維結(jié)構(gòu)中靠近,構(gòu)成PEP的結(jié)合位點。HbPPC2基因在葉片、樹皮、膠乳、雄花、雌花均有表達,在膠乳表達量最高;乙烯利處理顯著下調(diào)該基因在膠乳的表達。【結(jié)論】本研究可為橡膠樹PEPC功能研究提供理論基礎(chǔ)。
[Abstract]:[objective] to clone the full-length cDNAof Hevea phosphoenolpyruvate carboxylase (PEPC) gene and lay a foundation for the study of gene function by bioinformatics and gene expression analysis. [methods] Primer was designed according to the sequence of PEPC gene fragment in Hevea. The full-length cDNA was obtained by race, the coding peptide sequence was predicted by DNAMAN software, the basic physicochemical properties of the peptide were analyzed by Prot Param tool, the homologous nucleic acid and peptide sequences were searched by NCBI-Blast tool, and the peptide sequences from different plant sources were compared with Clustal X2.0 software. The phylogenetic tree is constructed with MEGA6.06 software. Real-time RT-PCR was used to analyze the gene expression pattern. [results] A new full-length cDNAof PEPC gene was cloned from rubber latex with a length of 3206 BP, containing a 2898 BP open reading frame (ORF) of 112bp 5UUTR and 196bp 3UUTR. The peptide encoding 965 amino acids (HbPPC2) was predicted, with a molecular weight of 110.35 kDaand a isoelectric point of 5.76.The coding region of HbPPC1 gene (KJ721228) and the amino acid sequence of HbPPC1 gene (KJ721228) cloned in the early stage of HbPPC1 were compared with those of HbPPC1 gene (KJ721228). There were 81.3% and 92.1% homology with the coding polypeptide sequence. HbPPC2 was predicted to be an unstable protein located in the cytoplasm, and its activity was regulated by many factors. It is predicted that HbPPC2 enzyme activity center is its 172nd residue His, phosphorylation site is its 11th residue Seran, and monoubiquitin site is its 628th residue Lys. The binding site of glucose-6-phosphoric acid (G6P) was formed by the Arg at positions 178179226 and 366.The Arg at position 450641753767 was close to the three-dimensional structure, and the binding site of HbPPC2 gene was found in leaves, bark, latex and male flowers. Ethephon treatment significantly down-regulated the expression of the gene in latex. [conclusion] this study may provide a theoretical basis for the study of PEPC function of rubber tree.
【作者單位】： 中國熱帶農(nóng)業(yè)科學(xué)院橡膠研究所"農(nóng)業(yè)部橡膠樹生物學(xué)與遺傳資源利用重點實驗室;
【基金】：中國熱帶農(nóng)業(yè)科學(xué)院基本科研業(yè)務(wù)費專項資金(1630022012011,1630022017023)
【分類號】：S794.1

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