苎麻果胶合成关键酶GalAT基因的克隆及表达

 【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986 bp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Galacturonosyltransferase 4同源性最高，分别为77%和83%;推定的氨基酸序列与拟南芥的Galacturonosyltransferase 4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。

Cloning and Expression of Key Enzyme Gene GalAT in Ramie Pectin Biosynthesis

【Objective】 The aim of this study was to isolate the cDNA partial sequence of key enzyme gene GalAT for pectin biosynthesis in ramie Boehmeria nivea (L.) Gaud], and understanding of the expression of GalAT gene in different tissues of ramie. 【Method】 Degenerate primer RT-PCR method was used to clone GalAT gene, bioinformatics methods were used to analyze cDNA sequence obtained and putative amino acid sequence, and fluorescence real time quantitative PCR method were used to study the expression of GalAT gene in different tissues. 【Result】 The cDNA partial sequence of GalAT gene from ramie variety Zhongzhu 1 was cloned. The cloned cDNA was 986 bp in length which encoded 328 amino acid sequences (GenBank accession number: EU 131377). This is the firstly reported GalAT gene in ramie. The cDNA sequence and putative amino acid sequence of GalAT shared high identity with previously reported Arabidopsis thaliana galacturonosyltransferase 4: 77 % and 83%, respectively; and the putative amino acid sequence and A. thaliana's galacturonosyltransferase 4 was gather to a same group; GalAT can be expressed in different kinds of ramie tissues, and its mRNA accumulated most abundantly in root. 【Conclusion】 It was inferred that the sequence obtained is cDNA partial sequence of GalAT gene, and can express in any tissue of ramie. GalAT mRNA accumulated abundantly most in root and GalAT expression in ramie tissues was root>leaf>bast>or≈xylem.