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Acta Agronomica Sinica ›› 2020, Vol. 46 ›› Issue (12): 1884-1893.doi: 10.3724/SP.J.1006.2020.02011

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

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 Online:2020-12-12 Published:2020-07-17
  • Contact: CHEN Xi-Jun E-mail:xjchen@yzu.edu.cn
  • Supported by:
    National Key Research and Development Program of China(2018YFD0300800);National Major Project for Developing New GM Crops(2016ZX08001002)

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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

Table 1

Primers used to amplify OsPGIP genes"

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

Fig. 1

Phylogenetic relationship among OsPGIPs and PGIPs from other plant species OsPGIPs is the abbreviations of polygalacturonase-inhibiting protein from Oryza sativa, the others are the NCBI accession numbers of the corresponding sequences. A: phylogenetic tree; B: subtree."

Table 2

Secondary structure of OsPGIPs predicted by HNN method"

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

Fig. 2

Primary structure of OsPGIPs"

Fig. 3

3D structure of OsPGIPs Red, yellow, and green in the cartoon figures mean α-helix, β-sheet, and random coil, respectively. A: 3D structure of OsPGIPs with typical spatial structure of PGIP. B: 3D structures of OsPGIP6 and OsPGIP7 constructed with homologous modeling methods of SWISS-MODEL, CPH model and SCRATCH. C: Protein docking of OsPGIP7 and RsPG2. Mesh and cartoon figures mean OsPGIP7 and RsPG2, respectively."

Table 3

Functions and Characterics of OsPGIPs predicted by online software"

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

Fig. 4

Expression levels of OsPGIP genes in rice cultivars with different resistance to sheath blight at different growth stages A: seedling stage; B: adult-plant stage; C: spike stage."

Fig. 5

Expression levels of OsPGIP genes in rice cultivars with different resistance to sheath blight under different stress condition. A: low temperature; B: dark; C: inoculation."

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