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【目的】分离和克隆苎麻果胶合成关键酶GalAT基因部分序列,了解其在不同组织部位的表达情况。【方法】采用简并引物RT-PCR法克隆基因,用生物信息学方法对获得的cDNA序列及推定氨基酸序列进行分析,并用荧光实时定量PCR法研究GalAT基因在不同组织中的表达。【结果】克隆了986bp的GalAT基因部分序列(GenBank注册号:EU131377),可编码328个连续的氨基酸序列;核苷酸序列和推定的氨基酸序列与拟南芥的Galacturonosyltransferase4同源性最高,分别为77%和83%;推定的氨基酸序列与拟南芥的Galacturonosyltransferase4可聚为一类;GalAT基因在苎麻各个组织中都有表达,其中根部的GalAT表达量占大部分。【结论】确定所获得的序列是GalAT基因的cDNA序列;GalAT表达量为根部>叶片>韧皮部>或≈木质部,在根部的表达量占优势。
【Objective】 The objective of this study is to isolate and clone partial sequence of GalAT gene, a key enzyme in synthesis of ramie pectin, and to investigate its expression in different tissues. 【Method】 The gene was cloned by RT-PCR with degenerate primers. The cDNA sequence and deduced amino acid sequence were analyzed by bioinformatics method. The expression of GalAT gene in different tissues was detected by real-time quantitative PCR. 【Result】 The 986bp GalAT gene partial sequence (GenBank accession number: EU131377) was cloned and it can encode 328 contiguous amino acid sequences. The nucleotide and putative amino acid sequences shared the highest homology with Galacturonosyltransferase4 in Arabidopsis, which were 77% and 83%, respectively. The deduced amino acid sequence was similar to that of Galacturonosyltransferase4 in Arabidopsis thaliana. GalAT gene was expressed in all tissues of ramie, and the majority of GalAT mRNA was found in roots. 【Conclusion】 The cDNA sequence of GalAT gene was confirmed. The expression level of GalAT was root> leaf> phloem> or ≈ xylem, and the expression level in root was dominant.