炭疽菌诱导的枇杷病程相关基因EjPR1的克隆及其表达分析

doi:10.16420/j.issn.0513-353x.2020-0141

园艺学报 ›› 2021, Vol. 48 ›› Issue (2): 367-376.doi: 10.16420/j.issn.0513-353x.2020-0141

炭疽菌诱导的枇杷病程相关基因EjPR1的克隆及其表达分析

邹晖¹^,², 赵亮¹, 袁婷¹^,², 郭启高¹, 吴頔²^,^*(), 梁国鲁¹^,^*()

¹西南大学园艺园林学院,南方山地园艺学教育部重点实验室,重庆 400715
²西南大学农业科学研究院,西南大学南方山地作物逆境生物学国家级培育基地,重庆 400715

收稿日期:2020-05-29 修回日期:2020-09-06 出版日期:2021-02-25 发布日期:2021-03-09
通讯作者: 吴頔,梁国鲁 E-mail:wudisuper610@126.com;lianggl@swu.edu.cn
基金资助:
国家自然科学基金青年基金项目(31701876);重庆市西南大学博士基金项目(SWU118046);重庆市高校创新研究群体项目资助(CXQT19005)

Cloning and Expression of a Pathogenesis Related Protein Gene EjPR1 from Loquat Induced by Colletotrichum gloeosporioides

ZOU Hui¹^,², ZHAO Liang¹, YUAN Ting¹^,², GUO Qigao¹, WU Di²^,^*(), LIANG Guolu¹^,^*()

¹Key Laboratory of Horticulture Science for Southern Mountains Regions,Ministry of Education,College of Horticulture and Landscape Architecture;Southwest University,Chongqing 400715,China
²Academy of Agricultural Sciences of Southwest University;State Cultivation Base of Crop Stress Biology for Southern Mountainous Land of Southwest University,Chongqing 400715,China

Received:2020-05-29 Revised:2020-09-06 Online:2021-02-25 Published:2021-03-09
Contact: WU Di,LIANG Guolu E-mail:wudisuper610@126.com;lianggl@swu.edu.cn

摘要/Abstract

摘要：

以炭疽病病原菌（Colletotrichum gloeosporioides）处理的枇杷高抗品种‘Peluches’的叶片为材料,利用RT-PCR和RACE克隆技术,获得枇杷病程相关蛋白1基因的cDNA全长序列,命名为EjPR1。该基因全长为731 bp（GenBank登录号为MN92775）,5′端非编码区79 bp,3′端非编码区91 bp。其中ORF阅读框为561 bp,编码186个氨基酸,蛋白质等电点为5.63,分子量为21 485.34,其预测结果的理论值与原核表达结果一致。序列分析结果表明,该基因编码的氨基酸序列与其他6种蔷薇科植物PR1蛋白的氨基酸序列相似性在76.41% ~ 94.36%之间。接种炭疽菌后时序表达分析结果表明,该基因在高抗品种‘Peluches’中受病原菌诱导后明显上调表达,在接种后96 h最高,但在高感品种‘森尾早生’中无明显变化,说明EjPR1可能参与了枇杷的抗病防御过程。

关键词: 枇杷, EjPR1, 基因克隆, 原核表达, 表达分析

Abstract:

A full length cDNA of pathogenesis-related protein 1 gene named EjPR1 was obtained from the leaves of‘Peluches’which was a loquat cultivar with high resistance to anthracnose treated by Colletotrichum gloeosporioides using RT-PCR and RACE technology. Bioinformatics analysis indicated that the full length of EjPR1 was 731 bp（GenBank number：MN92775）,the length of 5′ UTR and 3′ UTR were 79 bp and 91 bp,respectively. The length of open reading frame（ORF）was 561 bp encoding 186 amino acids with isoelectric point 5.63 and molecular weight 21 485.34 D which was consistent with the results of prokaryotic expression. Multiple alignment analysis based on the amino acids encoded by different PR1 genes from six other species of Rosaceae showed that EjPR1 was conserved among them with the similarity of 76.41%-94.36%. The results of time series expression analysis after inoculation with anthrax indicated that EjPR1 expression was up-regulated by infection and the highest expression was observed 96 h after inoculation in highly resistant cultivar while there was no significant difference in highly susceptible cultivar. It was speculated that EjPR1 might be involved in disease defense of loquat.

Key words: Eriobotrya japonica, EjPR1, gene clone, prokaryotic expression, expression analysis

中图分类号:

S667.3

邹晖, 赵亮, 袁婷, 郭启高, 吴頔, 梁国鲁. 炭疽菌诱导的枇杷病程相关基因EjPR1的克隆及其表达分析[J]. 园艺学报, 2021, 48(2): 367-376.

ZOU Hui, ZHAO Liang, YUAN Ting, GUO Qigao, WU Di, LIANG Guolu. Cloning and Expression of a Pathogenesis Related Protein Gene EjPR1 from Loquat Induced by Colletotrichum gloeosporioides[J]. Acta Horticulturae Sinica, 2021, 48(2): 367-376.

图/表 7

表1 本研究中所用的引物序列

Table 1 The sequences of primer used in this study

用途
Primer funtion

序列
Sequence

EjPR1
3′RACE
5′RACE
EjPR1-CDs
pET-EjPR1
EjPR1D
EjActin

F：GGTTGCATAGGAGAGTCCTTG;R：GCACATTTGACCAGCCTCGC
Outer：GATGTGGGACTTTCAACTCG;Inner：CAAGCGACGCAGTGAAGACA
Long：TGTCTTCACTGCGTCGCTTG;Short：CGAGTTGAAAGTCCCACATC
F：ATGAAGGCACAATTCCTCTTTATCC;R：CTAGTATGGCCTCTCACCAATATAA
F：CGGGATCCATGAAGGCACAATTCCTCTTTATCC;R：CGAGCTCCTAGTATGGCCTCTCACCAATATAA
F：AAGCGACGCAGTGAAGACA;R：ACAACCCTAGCACACCCGA
F：AATGGAACTGGAATGGTCAAGGC;R：TGCCAGATCTTCTCCATGTCATCCCA

图1 枇杷中分离的EjPR1电泳图

Fig. 1 Result of amplification product of EjPR1 from loquat

图2 EjPR1基因ORF全长及编码的氨基酸序列

Fig. 2 The full length of EjPR1 ORF and deduced amino acid sequences

图3 EjPR1蛋白多序列比对

Fig. 3 Multiple sequence alignment of EjPR1 protein

图4 植物PR1蛋白系统发育树进化树分支处的数字表示该分支的置信度。

Fig. 4 Phylogenetic tree of PR1 protein from plants The number at the branch of evolutionary tree indicate the confidence of the branch.

图5 接种病原菌后‘Peluches’和‘森尾早生’中EjPR1基因表达分析 * 代表相同时刻不同处理存在显著差异（P < 0.05）。

Fig. 5 Analysis of EjPR1 gene expression in‘Peluches’and‘Senwei Zaosheng’at different hpi * shows that there are significant differences between different treatments at the same time point（P < 0.05）.

图6 pET-EjPR1在大肠杆菌中表达蛋白SDS-PAGE分析

Fig. 6 SDS-PAGE analysis of protein expressed in Escherichia coli by pET-EjPR1

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炭疽菌诱导的枇杷病程相关基因EjPR1的克隆及其表达分析

Cloning and Expression of a Pathogenesis Related Protein Gene EjPR1 from Loquat Induced by Colletotrichum gloeosporioides

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炭疽菌诱导的枇杷病程相关基因EjPR1的克隆及其表达分析

Cloning and Expression of a Pathogenesis Related Protein Gene EjPR1 from Loquat Induced by Colletotrichum gloeosporioides

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