2022年7月7日 星期四
3 个芹菜品种Agnp-G3PDH 基因的克隆及其序列和表达特性分析
Cloning of Agnp-G3PDH gene from three cultivars of Apium graveolens and its sequence and expression characteristic analyses
2014年 第23卷 第2期 页码[1-10]    下载全文[2.3MB]  
摘要

从芹菜(Apium graveolens Linn.)品种‘六合黄心芹’(‘Liuhe Huangxinqin’)、‘津南实芹’(‘Jinnan Shiqin’)和‘文图拉’(‘Ventura’)中分别克隆获得非磷酸化甘油醛-3-磷酸脱氢酶基因Agnp-G3PDH。3 个品种的Agnp-G3PDH 基因序列均包含1 个全长1 491 bp 的开放阅读框(ORF),编码496 个氨基酸;存在84 个碱基位点差异,导致14 个氨基酸位点改变。由该基因编码的‘六合黄心芹’、‘津南实芹’和‘文图拉’的Agnp-G3PDH 蛋白的理论相对分子质量分别为53 201. 6、53 051. 4 和52 960. 3,理论等电点分别为pI 7. 49、pI 7. 86 和pI 8. 12,氨基酸组成中碱性氨基酸数量大于酸性氨基酸数量;该蛋白质具有亲水性与疏水性双重特性,均为疏水性蛋白。多重比对结果表明:3 个品种Agnp-G3PDH 基因编码的氨基酸序列同源性达到99. 09%,具有高度保守性;且与其他11 种植物np-G3PDH 的氨基酸序列的相似度也较高。在基于np-G3PDH 基因编码的氨基酸序列进化树上,3 个芹菜品种聚在同一分支中,并与杨柳科(Salicaceae)种类毛果杨(Populus trichocarpa Torr. & Gray)聚在一起,表明它们的np-G3PDH进化关系较近。实时定量PCR 分析结果表明:在3 个芹菜品种的不同组织中Agnp-G3PDH 基因的相对表达水平均有明显差异,其中,在‘津南实芹’的叶、‘文图拉’的花和‘六合黄心芹’的根中该基因的相对表达水平最高;经高温(38 ℃)、低温(4 ℃)和干旱(20%PEG)胁迫处理后3 个品种Agnp-G3PDH 基因的相对表达水平均极显著高于或低于对照,但经盐(0. 2 mol·L-1 NaCl)胁迫处理后仅品种‘津南实芹’Agnp-G3PDH 基因的相对表达水平显著高于对照,品种‘六合黄心芹’和‘文图拉’Agnp-G3PDH 基因的相对表达水平与对照无显著差异。表明该基因的表达具有组织特异性且与品种间的抗逆性差异有关。

Abstract

Non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase gene Agnp-G3PDH was cloned from cultivars ‘Liuhe Huangxinqin’, ‘Jinnan Shiqin’and ‘Ventura’of Apium graveolens Linn. ,respectively. The sequence of Agnp-G3PDH gene from three cultivars contains an open reading frame. (ORF) with the full length of 1 491 bp, which encodes 496 amino acids. There are 84 base site differences, leading to site changes of 14 amino acids. Theoretical relative molecular mass of protein encoded by Agnp-G3PDH gene from ‘Liuhe Huangxinqin’, ‘Jinnan Shiqin’ and ‘Ventura’ is 53 201. 6,53 051. 4 and 52 960. 3, respectively, and theoretical isoelectric point is pI 7. 49, pI 7. 86 and pI 8. 12,respectively. The number of basic amino acids is more than that of acidic amino acids in amino acid composition. And the proteins all possess double characteristics of hydrophilicity and hydrophobicity and all are the hydrophobic protein. The alignment result shows that homology of amino acid sequences encoded by Agnp-G3PDH gene from three cultivars reaches 99. 09%, which is highly conserved. Similarity of amino acid sequence among three cultivars and other eleven species is higher. Based on phylogenetic tree of amino acid sequence encoded by np-G3PDH gene, three cultivars are clustered in the same branch, and three cultivars and Populus trichocarpa Torr. & Gray in Salicaceae can be clustered together which shows that their np-G3PDH amino acid sequences have a closer evolutionary relationship. The result of quantitative real-time PCR shows that there are obvious differences in relative expression level of Agnp-G3PDH gene among different tissues of three cultivars, in which, relative expression level of the gene is the highest in leaf of ‘Jinnan Shiqin’ , in flower of ‘Ventura’ and in root of ‘Liuhe Huangxinqin’ . After treated by stress of high temperature (38 ℃), low temperature (4 ℃) and drought (20%PEG), relative expression level of Agnp-G3PDH gene from three cultivars is extremely significantly higher or lower than that of the control, but after treated by salinity (0. 2 mol·L-1NaCl) stress only that of ‘Jinnan Shiqin’is significantly higher than that of the control, and that of ‘Liuhe Huangxinqin’ and ‘Ventura’ has no significant difference with that of the control. It is indicated that the expression of Agnp-G3PDH gene has tissue specificity and is related to resistance difference among cultivars.

关键词芹菜; Agnp-G3PDH 基因; 序列分析; 同源性; 非生物胁迫; 表达特性
Key wordsApium graveolens Linn.; Agnp-G3PDH gene; sequence analysis; homology; abiotic stress; expression characteristics
作者田畅, 蒋倩, 王摇枫, 李梦瑶, 贾晓玲, 熊爱生
所在单位南京农业大学园艺学院作物遗传与种质创新国家重点实验室农业部华东地区园艺作物生物学与种质创制重点实验室, 江苏南京210095
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基金项目国家自然科学基金资助项目(31272175); 国家教育部“新世纪优秀人才支持计划"(NCET-11-0670); 江苏省自然科学基金资助项目 (BK20130027); 江苏高校优势学科建设工程一期项目(2011PAPD); 江苏省高层次创新创业人才引进计划专项基金(2011JSSC)