大蒜果聚糖合成相关基因As6G-FFT的克隆及表达分析

doi:10.16420/j.issn.0513-353x.2021-0403

摘要/Abstract

摘要：

果聚糖是大蒜的主要贮藏物质,其代谢与产量形成和逆境适应性密切相关。从‘乐都紫皮大蒜’中克隆得到果聚糖︰果聚糖6G-果糖基转移酶基因As6G-FFT的序列,其CDS全长为1 839 bp,编码612个氨基酸,分子量为68.64 kD,等电点（pI）为5.43,属于亲水性蛋白;结构域分析表明,该基因推导的氨基酸序列含有植物糖基水解酶32家族的NDPSG、RDP和EC保守结构域;在进化关系上,As6G-FFT氨基酸序列与洋葱最为接近。亚细胞定位发现,As6G-FFT蛋白集中分布于内质网中。荧光定量PCR分析表明,As6G-FFT在大蒜各组织中均有表达,其表达量表现为叶片 > 根 > 母瓣 > 假茎;另外,在低温（4 ℃）或干旱胁迫（基质相对含水量45% ~ 55%）不同阶段各组织中As6G-FFT的表达特性差异显著,干旱胁迫能够较早地诱导根中As6G-FFT的表达,而低温胁迫下母瓣As6G-FFT的响应更加敏感,说明不同胁迫下大蒜各组织As6G-FFT对逆境的响应机理有所差异。

关键词: 大蒜, 果聚糖, As6G-FFT, 基因克隆, 表达分析

Abstract:

Fructan is the main storage material of garlic,and its metabolism is closely related to yield formation and stress adaptability. In order to understand the sequence characteristics and expression patterns of fructan：fructan 6G-Fructosyltransferase gene（As6G-FFT）in different tissues and stress conditions of garlic,the As6G-FFT（fructan：fructan 6G-Fructosyltransferase）gene was cloned from ‘Ledu Purple Skin Garlic’. The CDS full length of As6G-FFT gene was 1 839 bp,encoding 612 amino acids with molecular mass of 68.64 and theoretical isoelectric point of 5.43. It was a hydrophilic protein and contained three important conserved domains of NDPSG,RDP and EC of plant glycosylhydrolase 32 family. In terms of evolutionary relationship,garlic As6G-FFT gene was close to onion. The result of subcellular localization showed that As6G-FFT protein was located in endoplasmic reticulum. qRT-PCR analysis showed that As6G-FFT was expression in all tissues examined,which expression level was leaf > root > mother petal > pseudostem. In addition,the expression characteristics of As6G-FFT gene in different tissues were also different after low temperature（4 ℃）or drought stress treatment（45%-55% relative water content of matrix）. Compared with other tissues,drought stress could induce the expression of As6G-FFT gene in roots earlier,whereas the expression of As6G-FFT in mother petal was more sensitive under low temperature stress,which indicated that the response mechanism of As6G-FFT in garlic tissues to stress signals was different.

Key words: garlic, fructan, As6G-FFT gene, gene cloning, expression analysis

中图分类号:

S633.4

田洁, 边海燕, 铁原毓. 大蒜果聚糖合成相关基因As6G-FFT的克隆及表达分析[J]. 园艺学报, 2021, 48(12): 2403-2413.

TIAN Jie, BIAN Haiyan, TIE Yuanyu. Cloning and Expression Analysis of As6G-FFT Gene Encoding Fructan Synthase from Garlic[J]. Acta Horticulturae Sinica, 2021, 48(12): 2403-2413.

图/表 8

图1 As6G-FFT的PCR扩增

Fig. 1 PCR amplification of As6G-FFT

图2 大蒜As6G-FFT的核苷酸序列及其预测的氨基酸序列

Fig. 2 Nucleotide sequences and predicted amino acid sequences of As6G-FFT from garlic

图3 大蒜As6G-FFT蛋白序列中各氨基酸的含量

Fig. 3 The contents of amino acids of As6G-FFT protein sequence from garlic

图4 大蒜与其他植物6G-FFT保守基序分析

Fig. 4 Analysis of 6G-FFT conserved motifs in garlic and other plants

图5 大蒜与其他植物6G-FFT氨基酸序列的比对分析红框表示NDPNG、RDP和EC等3个保守基序。

Fig. 5 Comparative analysis of 6G-FFT amino acid sequences between garlic and other plants The red box indicates three conserved motifs：NDPNG,RDP and EC.

图6 大蒜As6G-FFT蛋白的亚细胞定位

Fig. 6 Subcellular localization analysis of Allium sativum As6G-FFT protein

图7 As6G-FFT在大蒜不同组织中的相对表达水平

Fig. 7 Relative expression level of As6G-FFT gene in different garlic tissues P < 0.05.。。

图8 低温和干旱胁迫下As6G-FFT基因在不同大蒜组织中的相对表达水平

Fig. 8 Relative expression level of as6g FFT gene in different garlic tissues under low temperature and drought stress P < 0.05.

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