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作物学报 ›› 2020, Vol. 46 ›› Issue (12): 1884-1893.doi: 10.3724/SP.J.1006.2020.02011

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

水稻多聚半乳糖醛酸酶抑制蛋白家族OsPGIP结构及基因表达特征
分析

陈夕军1,2,*(), 唐滔1, 李丽丽1, 陈宸1, 陈煜文1, 张亚芳2, 左示敏2   

  1. 1扬州大学园艺与植物保护学院, 江苏扬州 225009
    2江苏省作物遗传生理重点实验室 / 教育部功能基因组学重点实验室 / 扬州大学, 江苏扬州 225009
  • 收稿日期:2020-02-16 接受日期:2020-07-02 出版日期:2020-12-12 网络出版日期:2020-07-17
  • 通讯作者: 陈夕军
  • 基金资助:
    国家重点研发计划项目(2018YFD0300800);国家转基因生物新品种培育重大专项(2016ZX08001002)

Analysis on the structures of polygalacturonase-inhibiting proteins and the expression profile of its encoding genes in rice

CHEN Xi-Jun1,2,*(), TANG Tao1, LI Li-Li1, CHEN Chen1, CHEN Yu-Wen1, ZHANG Ya-Fang2, ZUO Shi-Min2   

  1. 1Horticultrue and Plant Protection College, Yangzhou University, Yangzhou 225009, Jiangsu, China
    2Key Laboratory of Crop Genetics and Physiology of Jiangsu Province / Key Laboratory of Plant Functional Genomics of the Ministry of Education / College of Agriculture, Yangzhou University, Yangzhou 225009, Jiangsu, China
  • Received:2020-02-16 Accepted:2020-07-02 Published:2020-12-12 Published online:2020-07-17
  • Contact: CHEN Xi-Jun
  • Supported by:
    National Key Research and Development Program of China(2018YFD0300800);National Major Project for Developing New GM Crops(2016ZX08001002)

摘要:

植物多聚半乳糖醛酸酶抑制蛋白(polygalacturonase-inhibiting protein, PGIP)可特异性识别病原菌PG (polygalacturonase), 从而提高植物的抗病能力。研究表明水稻中共存在7个OsPGIP基因, 为明确OsPGIP家族的蛋白质结构及基因表达特征, 从水稻cDNA中扩增各OsPGIP基因序列, 经克隆、测序后进行生物信息学分析与蛋白质结构模拟, 并测定其在生物逆境与非生物逆境胁迫下的表达量变化。经多序列比较与系统发育进化分析发现, 相同或相近物种PGIP往往具有较高的相似性, 虽然多数OsPGIP亲缘关系较近, 但它们并不能完全聚类在一起。7个OsPGIP蛋白均具有一个信号肽和9~11个LRR片段, 各LRR片段中均含有PGIP的特征结构域xxLxLxx。二级结构由α-螺旋、β-折叠和随机卷曲组成, 且多以随机卷曲为主, 这些二级结构以重复的随机卷曲—α-螺旋—随机卷曲—β-折叠组成线圈状结构, 并按右手螺旋规则形成一个特定的凹面, 负责OsPGIP与有害生物PG的互作。7个OsPGIP蛋白多较稳定, 且均为疏水蛋白、脂溶性好、具有跨膜结构、定位于细胞外、有1到多个N-糖基化位点、在大肠杆菌中原核表达后基本不溶。经生物和非生物逆境处理后, 水稻中不同OsPGIP基因的表达量上下调差异较大, 但表达量总和明显上调, 说明在逆境条件下水稻可通过调节自身OsPGIP基因的表达量, 从而提高其抗逆性。

关键词: 表达特征, 蛋白结构, 分析, 多聚半乳糖醛酸酶抑制蛋白, 水稻

Abstract:

Polygalacturonase-inhibiting protein, the extracellular leucine-rich repeat protein, specially recognizing and inhibiting polygalacturonase (PG) from pathogenic organism, can improve the resistance of plant against the pathogen. In order to clarify the structures of OsPGIPs and the expression profile of its encoding genes in rice, seven OsPGIP (polygalacturonase-inhibiting protein) genes were amplified from rice cDNA by PCR. Bioinformatics analysis and structural prediction of OsPGIPs were performed and expression profile of its encoding genes under biotic and abiotic stresses was determined after these genes being cloned and sequenced. The results showed that PGIPs from the same or similar species had high similarity. Through multi-sequence alignment and phylogenetic analysis, it was found that most OsPGIPs had closer genetic relationship, but all of them could not be grouped in one group. All OsPGIPs had a signal peptide and 9 to11 LRR fragments included the typical PGIP’s motif of xxLxLxx. Secondary structure prediction indicated that all OsPGIPs consist of ɑ-helix (H), extended strand (ES) and random coil (RC), which construct repeated RC-H-RC-ES- and form a typical concave coinciding with the right-hand helix rule. The concave might be responsible for the interaction between OsPGIPs and PGs from different agents. Most of the seven OsPGIPs were stable, and they all were hydrophobic proteins, good lipid solubility, with transmembrane structure, extrcellular localization, one or more N-glycosylation sites, and basically insoluble after expression in E. coli. After being treated with biotic and abiotic stress factors, the expression levels of different OsPGIP genes in rice were significantly up-regulated or down-regulated, but the total expression levels were significantly up-regulated, which indicated that rice could improve its own ability against stresses by regulating the expression levels of OsPGIP genes under stress conditions.

Key words: expression profile, protein structure, analysis, PGIP, rice

表1

扩增各OsPGIP基因所用引物"

基因
Gene
正向引物
Forward primer (5′-3′)
反向引物
Reverse primer (5′-3′)
OsPGIP1 TGACTCGCTATTGCATGCG TGGGAGCTTAATTGCAGGGA
OsPGIP2 ATACACGGCATTGCATGCAC CTTACACTCGTTCTCCGTAC
OsPGIP3 TAGAAGAGAGGAAGCACGCA TTGGTGGCCTGAGATAGGT
OsPGIP4 TGTCGTGCACTTGTGTTCAA GCATTAGCTGGTTGCTTC
OsFOR1 TTCAGGTAGATACAATGGCG ATGGATGGATGGATGCTC
OsPGIP6 GAGCCGAGACGAGACGA ATATGTACCCAAGCCCAAA
OsPGIP7 TCCTGCACGGATTTGAGC TAACAACAGCCAGTCAGCAAT

图1

OsPGIP和其他物种来源的PGIP系统发育树 图中除OsPGIP为名称缩写外, 其他均为对应PGIP的NCBI序列号。A: 总进化树; B: 子树。"

表2

OsPGIP二级结构预测结果"

蛋白
Protein
α-螺旋
α-helix
延伸链
Extended strand
随机卷曲
Random coil
半胱氨酸数
Number of cysteine
二硫键位置
Position of disulfide bond
OsPGIP1 36.25 10.68 53.07 8 56-63, 278-298, 300-308
OsPGIP2 46.20 10.23 43.57 9 34-64, 65-72, 310-323, 331-339
OsPGIP3 32.74 13.86 53.39 10 17-27, 56-64, 322-328, 330-337
OsPGIP4 44.70 9.74 45.56 9 33-63, 64-71, 333-339, 341-348
OsFOR1 30.12 10.24 59.64 10 27-58, 59-66, 312-320
OsPGIP6 49.21 4.47 46.32 10 64-73, 114-137, 348-370, 372-379
OsPGIP7 27.19 10.82 61.99 13 16-65, 25-34, 66-73, 322-328, 330-337

图2

7个OsPGIP一级结构"

图3

OsPGIP蛋白质3D结构模拟图 卡通图中红色、黄色和绿色分别表示α-螺旋、β-折叠和随机卷曲。A: 具有典型PGIP空间结构的各OsPGIP3D模型; B: 分别采用SWISS-MODEL、CPHmodels和SCRATCH方法模拟的OsPGIP6和OsPGIP7空间结构模型; C: OsPGIP7与RsPG2的蛋白分子对接, 网眼图为OsPGIP7, 卡通图为RsPG2。"

表3

在线软件预测的OsPGIP蛋白性质与功能"

蛋白
Protein
染色体
位置Location
分子量
Molecular weight
(kD)
等电点
pI
不稳定
指数
Instability index
脂溶性
指数
Aliphatic
index
总平均
亲水性
Grand average of hydropathicity
跨膜域
Trans-
membrane
亚细胞定位
Subcellular localization
N-糖基化
位点数
Number of N-glycosylation sites
大肠杆菌中表达的溶解性
Chance of
solubility
expressed in
E. coli (%)
OsPGIP1 Chr. 5 32.75 6.98 31.82 98.58 0.183 Yes Extracellular 4 0
OsPGIP2 Chr. 5 36.97 4.73 40.16 103.33 0.080 Yes Extracellular 8 0
OsPGIP3 Chr. 5 36.14 5.94 34.36 95.63 0.181 Yes Extracellular 5 0
OsPGIP4 Chr. 5 37.22 8.37 33.98 101.58 0.203 Yes Extracellular 4 0
OsFOR1 Chr. 8 35.46 7.09 36.08 99.94 0.041 Yes Extracellular 1 0
OsPGIP6 Chr. 8 38.79 5.86 35.69 106.42 0.286 Yes Extracellular 1 0
OsPGIP7 Chr. 9 35.88 5.90 33.26 101.87 0.265 Yes Extracellular 3 0.7

图4

不同生育期抗、感纹枯病水稻品种叶鞘中各OsPGIP基因的表达量 A: 苗期; B: 成株期; C: 穗期。"

图5

不同逆境条件下抗、感纹枯病水稻品种叶鞘中各OsPGIP基因的表达量 A: 低温; B: 遮光; C: 接种。"

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