玉米受伤诱导基因Wip1的启动子克隆及表达分析

doi:10.3864/j.issn.0578-1752.2014.14.019

摘要/Abstract

摘要： 【Objective】 Protease inhibitor is an important defense protein in plant for resistance to infection or feeding by pathogen and animals. Maize Wip1(wound-induced protein) is the member of Bowman-Birk inhibitor(BBI) gene family. To provide new information for the application of resistance gene to insect in plant, this experiment was designated to understand the activity of Wip1 promoter, assay spatial and temporal expression of Wip1 gene and its response to phytohormones and abiotic stresses.【Method】A clone containing the upstream region of Wip1 was isolated and sequenced from maize leaf cDNA. The promoter sequences of Wip1 gene was inserted into binary expression vector p1300 to produce recombinant plasmids p1300-Wip1-GUS, which was transferred into Agrobacterium LBA4404 by freeze thawing method. Functional analysis of the promoter was conducted through heterologous expression in maize callus by agro-infiltration method. Total RNA was isolated from maize tissues using hot phenol method. Purified RNA was subjected to formaldehyde-containing-agarose gel, blotted onto a nylon membrane, and Northern blotting was performed with [α-32P] dCTP-labeled probe to characterized tissue-speci?c location, phytohormones and abiotic stresses analysis of Wip1. 【Result】 Promoter region was 1 737 bp and coding sequence was 512 bp. The result showed that GUS activity driven by the Wip1 promoter was detected in maize callus tissues after infection by Agrobacterium haboring recombinant plasmids p1300-Wip1-GUS. The expression of Wip1 was assayed at various developmental stages. Northern blotting showed that Wip1 gene abundantly expressed in injured coleoptile, root, stem, leaf, ear, tassel, silk and husk, while expressed little in uninjured coleoptile and not expressed in other uninjured detection tissues. The results also showed that Wip1 was not affected by submergence, cold, dehydration, PEG, ABA, NaCl and IAA treatments. The dynamic expression of Wip1 gene under injure stress from 0 h to 24 h with 8 time points, was analyzed in maize leaf tissue. Wip1 gene was detected after stressing for 2 h, the expression level increased rapidly, and with a continually increasing trend from 4 h to 24 h. 【Conclusion】 Wip1 gene is induced by injure-specific stress. Wip1 promoter is an effective injure-specific promoter to express the desired genes in plants.

关键词: maize , wound induced , Wip1 , serine protease inhibitor , bowman-birk

Abstract: 【Objective】 Protease inhibitor is an important defense protein in plant for resistance to infection or feeding by pathogen and animals. Maize Wip1(wound-induced protein) is the member of Bowman-Birk inhibitor(BBI) gene family. To provide new information for the application of resistance gene to insect in plant, this experiment was designated to understand the activity of Wip1 promoter, assay spatial and temporal expression of Wip1 gene and its response to phytohormones and abiotic stresses.【Method】A clone containing the upstream region of Wip1 was isolated and sequenced from maize leaf cDNA. The promoter sequences of Wip1 gene was inserted into binary expression vector p1300 to produce recombinant plasmids p1300-Wip1-GUS, which was transferred into Agrobacterium LBA4404 by freeze thawing method. Functional analysis of the promoter was conducted through heterologous expression in maize callus by agro-infiltration method. Total RNA was isolated from maize tissues using hot phenol method. Purified RNA was subjected to formaldehyde-containing-agarose gel, blotted onto a nylon membrane, and Northern blotting was performed with [α-32P] dCTP-labeled probe to characterized tissue-speci?c location, phytohormones and abiotic stresses analysis of Wip1. 【Result】 Promoter region was 1 737 bp and coding sequence was 512 bp. The result showed that GUS activity driven by the Wip1 promoter was detected in maize callus tissues after infection by Agrobacterium haboring recombinant plasmids p1300-Wip1-GUS. The expression of Wip1 was assayed at various developmental stages. Northern blotting showed that Wip1 gene abundantly expressed in injured coleoptile, root, stem, leaf, ear, tassel, silk and husk, while expressed little in uninjured coleoptile and not expressed in other uninjured detection tissues. The results also showed that Wip1 was not affected by submergence, cold, dehydration, PEG, ABA, NaCl and IAA treatments. The dynamic expression of Wip1 gene under injure stress from 0 h to 24 h with 8 time points, was analyzed in maize leaf tissue. Wip1 gene was detected after stressing for 2 h, the expression level increased rapidly, and with a continually increasing trend from 4 h to 24 h. 【Conclusion】 Wip1 gene is induced by injure-specific stress. Wip1 promoter is an effective injure-specific promoter to express the desired genes in plants.

Key words: maize , wound induced , Wip1 , serine protease inhibitor , bowman-birk

练云1, 2, 刘允军1, 王国英1. 玉米受伤诱导基因Wip1的启动子克隆及表达分析[J]. 中国农业科学, 2014, 47(14): 2889-2896.

LIAN Yun-1, 2 , LIU Yun-Jun-1, WANG Guo-Ying-1. Isolation of a Maize Wound-Induced Gene Promoter and Characterization of the Gene Expression in Maize[J]. Scientia Agricultura Sinica, 2014, 47(14): 2889-2896.

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