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Acta Agronomica Sinica ›› 2018, Vol. 44 ›› Issue (6): 824-835.doi: 10.3724/SP.J.1006.2018.00824

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

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 Online:2018-06-12 Published:2018-03-19
  • Contact: You-Xiong QUE E-mail:queyouxiong@126.com
  • 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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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

Table 1

Material processing for Real-time PCR"

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

Table 2

Primers used in ScSEC14 gene cloning and expression analysis"

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

Fig. 1

RT-PCR amplification of ScSEC14 gene in sugarcane M: DNA marker, D2000 bp; 1: Target fragment."

Fig. 2

Nucleotide sequence and deduced amino acid sequence of sugarcane ScSEC14 gene (* stop codon) The sequence fragment complementary to primer is highlighted in the box."

Fig. 3

Predicted hydrophobicity of the amino acid sequence of sugarcane ScSEC14 protein"

Table 3

Secondary structure prediction of sugarcane ScSEC14 protein"

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

Fig. 4

Predicted tertiary structure of SEC14 protein in Sugarcane officinarum, Sorghum bicolor, Zea mays, Setaria italic, and Oryza sativa"

Fig. 5

Conserved domain prediction of sugarcane ScSEC14 protein"

Fig. 6

Deduced amino acid sequence and the phylogenic tree of ScSEC14 protein A: Amino acid sequence alignment of the ScSEC14 protein with SEC14 proteins of other species; B: Phylogenetic tree analysis of ScSEC14 protein and SEC14 protein from other species; C: Domain analysis of ScSEC14 protein and SEC14 proteins of other species."

Fig. 7

Subcellular localization of ScSEC14 in Nicotiana benthamiana leaves Red arrow represents nucleus; white arrow represents plasma membrane; blue arrow represents cytoplasm."

Fig. 8

SDS-PAGE analysis of the expression of pEZY19-ScSEC14 and pEZY19(+) 1: marker; 2: empty bacteria induced for 0 h; 3: empty bacteria induced for 8 h; 4: empty vector induced for 0 h; 5: empty vector induced for 8 h; 6-12: recombinant bacteria induced for 0, 1.5, 1, 2, 4, 6, 8 h; white arrow represents the target protein induced."

Fig. 9

Relative expression of ScSEC14 gene in different tissues of sugarcane Error bars represent the standard error of each treating group (N = 3)."

Fig. 10

Relative expression of ScSEC14 in sugarcane under different exogenous stresses Error bars represent the standard error of each treating group (N = 3)."

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