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作物学报 ›› 2018, Vol. 44 ›› Issue (6): 824-835.doi: 10.3724/SP.J.1006.2018.00824

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

甘蔗磷脂酰肌醇转运蛋白基因ScSEC14响应干旱和盐胁迫

毛花英,刘峰,苏炜华,黄宁,凌辉,张旭,王文举,李聪娜,汤翰臣,苏亚春,阙友雄()   

  1. 福建农林大学农业部福建甘蔗生物学与遗传育种重点实验室 / 国家甘蔗工程技术研究中心, 福建福州 350002
  • 收稿日期:2017-12-10 接受日期:2018-03-15 出版日期:2018-06-12 网络出版日期:2018-03-19
  • 通讯作者: 阙友雄
  • 基金资助:
    本研究由国家自然科学基金项目(31671752);福建省杰出青年基金项目(2015J06006);国家农业产业技术体系建设专项资助(CARS-17)

A Sugarcane Phosphatidylinositol Transfer Protein Gene ScSEC14 Responds to Drought and Salt Stresses

Hua-Ying MAO,Feng LIU,Wei-Hua SU,Ning HUANG,Hui LING,Xu ZHANG,Wen-Ju WANG,Cong-Na LI,Han-Chen TANG,Ya-Chun SU,You-Xiong QUE()   

  1. Key Laboratory of Sugarcane Biology and Genetic Breeding (Fujian), Ministry of Agriculture, Fujian Agriculture and Forestry University / Sugarcane Research & Development Center, China Agricultural Technology System, Fuzhou 350002, Fujian, China
  • Received:2017-12-10 Accepted:2018-03-15 Published:2018-06-12 Published online:2018-03-19
  • Contact: You-Xiong QUE
  • Supported by:
    This study was supported by the National Natural Science Foundation of China(31671752);the Natural Science Foundation of Fujian Province for Distinguished Young Scholars(2015J06006);the China Agriculture Research System(CARS-17)

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摘要：

Sec14-like磷脂酰肌醇转运蛋白(Sec14-like phosphatidylinositol transfer proteins, PITPs), 广泛存在于真核生物细胞中, 参与肌醇磷酸代谢、膜运输、极性生长、信号转导、逆境胁迫等多种重要的生命过程。甘蔗中响应干旱和盐胁迫的Sec14-like基因尚未见报道。本研究从甘蔗受黑穗病胁迫的转录组数据库中获得一条SEC14基因序列, 并利用RT-PCR技术克隆得到甘蔗SEC14基因cDNA全长序列, 命名为ScSEC14 (GenBank登录号为MG571103)。生物信息学分析显示, ScSEC14基因全长1617 bp, 包含一个1008 bp的完整开放阅读框, 编码335个氨基酸; ScSEC14为不稳定的亲水性蛋白, 不存在信号肽; 蛋白二级结构元件多为α-螺旋, 具有典型的SEC14结构域和CRAL_TRIO_N结构域。此外, 系统进化树分析揭示, 该蛋白属于Sec14-like蛋白家族的SSH (soybean Sec14 homolog group)亚家族。亚细胞定位结果表明, ScSEC14蛋白主要定位于细胞膜。实时荧光定量PCR分析发现, ScSEC14基因在甘蔗中组成型表达, 在蔗皮中的表达量最低, 蔗叶中的表达量最高, 约为蔗皮的4.9倍; 该基因在PEG、NaCl、CaCl2和水杨酸(SA)胁迫下的表达量均上调。因此, 甘蔗ScSEC14基因可能参与Ca 2+和SA介导的抗逆信号通路, 积极响应逆境胁迫, 尤其调节了干旱和高盐环境下的抗逆性。

关键词: 甘蔗, Sec14-like磷脂酰肌醇转运蛋白, ScSEC14, 干旱, 盐胁迫

Abstract:

Sec14-like phosphatidylinositol transfer proteins are present in all eukaryotic genomes and involved in a variety of biological activities, such as metabolism of inositol phosphate, membrane transportation, polar growth, signal transduction and stress responses. The responses of Sec14-like gene to drought and salt stresses have not been reported in sugarcane. In this study, a SEC14 gene sequence was obtained in the sugarcane transcription database infected by Sporisorium scitamineum, and the full length cDNA sequence of a sugarcane SEC14 gene was obtained by RT-PCR technology and named as ScSEC14 (GenBank accession number: MG571103). Bioinformatics analysis showed a full length of 1617 bp in ScSEC14 gene containing a complete open reading frame of 1008 bp and encoding 335 amino acid residues. ScSEC14 is an unstable hydrophilic protein with no signal peptide. The secondary structure of ScSEC14 protein is mostly α-helices, with a typical SEC14 domain and a CRAL_TRIO_N domain. Phylogenetic tree analysis showed that ScSEC14 belonged to SSH (soybean Sec14 homolog group) subfamily of Sec14-like protein family. Subcellular localization experiment showed that ScSEC14 protein was mainly localized in the plasma membrane. Real-time quantitative PCR analysis showed that ScSEC14 gene was constitutively expressed in sugarcane, with the lowest expression level in skin, and the highest in leaf, which was 4.9 times of that in skin. The expression of ScSEC14 gene was up-regulated under the stresses of PEG, NaCl, CaCl2 and salicylic acid (SA). We speculate that ScSEC14 plays an important role in response to drought and salt stresses, and may be involved in the stress response signaling pathway mediated by Ca 2+ and SA.

Key words: sugarcane, Sec14-like phosphatidylinositol transfer protein, ScSEC14, drought, salt stress

表1

实时荧光定量材料处理"

处理条件
Treatment condition		 取样时间Sampling time (h)
取样点1 Site 1 取样点2 Site 2 取样点3 Site 3
5 mmol L-1 SA 3 6 12
25.0% PEG模拟干旱 25.0% PEG simulated drought 6 12 24
50 μmol L-1 CaCl2 3 6 12
250 mmol L-1 NaCl 6 12 24
500 mmol L-1 CuCl2 12 24 48
500 mmol L-1 CdCl2 12 24 48

表2

ScSEC14基因克隆与表达所用引物"

引物
Primer		 引物序列
Sequence information (5'-3')		 用途
Purpose
SEC14
 F: AGGAAGCGCACAAGAACAGA 基因克隆
Gene cloning
R: GGGAGTACAAGTCTCCTTGCATA
qSEC14
 F: CCACGAGTCACTTCCACACT 荧光定量Real-time-qPCR
R: TGGGACCAAGAGAGTCCTGA
CUL
 F: TGCTGAATGTGTTGAGCAGC 内参基因
Reference genes
R: TTGTCGCGCTCCAAGTAGTC
CAC
 F: ACAACGTCAGGCAAAGCAAA 内参基因
Reference genes
R: AGATCAACTCCACCTCTGCG
G-SEC14
 F: GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGGCGGCCACCTCCGGAAGG 载体构建
Vector construction
R: GGGGACCACTTTGTACAAGAAAGCTGGGTCTGGACCTTCGATCTGTATGCTG

图1

甘蔗ScSEC14基因的RT-PCR扩增 M: DNA marker, D2000 bp; 1:目的条带。"

图2

甘蔗ScSEC14基因的cDNA序列及其推导的氨基酸序列(*终止密码子) 方框部分为特异性引物在基因序列中的位置。"

图3

甘蔗ScSEC14蛋白氨基酸疏水性/亲水性预测"

表3

甘蔗ScSEC14蛋白二级结构预测分析"

二级结构类型
Secondary structure type		 氨基酸残基数目
Amino acid residue number		 百分比
Percentage (%)
α-螺旋Alpha-helix 145 43.28
延伸链Extended strand 52 15.52
无规则卷曲Random coil 138 41.19

图4

甘蔗、高粱、玉米、粟和水稻SEC14蛋白三级结构预测"

图5

甘蔗ScSEC14蛋白的保守结构域分析"

图6

ScSEC14 蛋白的氨基酸序列同源性分析和系统进化树分析 A: ScSEC14 蛋白与其他物种的SEC14 蛋白的氨基酸序列比对; B: ScSEC14 蛋白与其他物种SEC14 蛋白的系统进化树分析; C: ScSEC14 蛋白与其他物种SEC14 蛋白的结构域分析。"

图7

甘蔗ScSEC14蛋白在烟草叶片中的亚细胞定位 红色箭头代表细胞核; 白色箭头代表细胞膜; 蓝色箭头代表细胞质。"

图8

pEZY19-ScSEC14和pEZY19(+)表达产物的SDS-PAGE分析 1: marker; 2: 空菌诱导0 h; 3: 空菌诱导8 h; 4: 空载诱导0 h; 5: 空载诱导8 h; 6~12: 重组菌诱导0、1.5、1、2、4、6、8 h; 白色箭头代表被诱导的目标蛋白。"

图9

甘蔗ScSEC14基因在不同组织中的表达 误差线为每组处理的标准误差(N = 3)。"

图10

甘蔗ScSEC14基因在不同外源胁迫下的表达特性 误差线为每组处理的标准误差(N = 3)。"

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