苹果转录因子MdWRKY40b抗白粉病的机理

doi:10.3864/j.issn.0578-1752.2021.24.005

摘要/Abstract

摘要：

【目的】探究苹果白粉病相关基因MdWRKY40b调控苹果抗白粉病的机理,为苹果白粉病的抗性育种提供理论依据。【方法】以苹果品种‘嘎拉’叶片为材料提取总RNA,并以cDNA为模板克隆MdWRKY40b 552 bp特异性片段,采用Gateway技术将该基因片段的正反向序列构建入RNAi表达载体pB7GWIWG2(Ⅱ)中,然后利用农杆菌介导法对苹果叶片进行转化获得转基因植株,苹果叶片选用组培苗茎尖处幼嫩叶片;利用RT-qPCR技术对转基因植株中MdWRKY40b、超氧化物歧化酶基因（SOD）、过氧化氢酶基因（CAT）、过氧化物酶基因（POD）以及β-1,3-葡聚糖酶基因（β-1,3-glucanase）的表达量进行分析;进一步以1年生植株为材料,通过白粉病菌（Podosphaera leucotricha）接种试验,分析转基因植株对白粉病的抗病性;然后以接种白粉病菌后0、5、10、15、20 d的转基因植株及其对照植株叶片为材料,进行氯化硝基四氮唑蓝液（NBT）和二氨基联苯胺法（DAB）染色来分析病菌侵染期间植物材料中超氧阴离子和过氧化氢的积累量,进一步对病菌侵染期间植株中SOD、POD、CAT和β-1,3-葡聚糖酶等酶活性进行测定。【结果】经过PCR检测,确定获得3个MdWRKY40b基因沉默株系,分别为RNAi-1、RNAi-2和RNAi-3;RT-qPCR结果显示3个转基因株系MdWRKY40b基因沉默效率分别为95.2%、92.2%、79.8%,转基因植株中SOD、CAT、β-1,3-glucanase的表达量相较野生型显著上调;人工接种白粉病菌后发现,与野生型植株相比,转基因植株叶片中的粉状病斑显著减少,超氧阴离子和过氧化氢的积累量显著降低,SOD、CAT、POD等调节植物超氧阴离子代谢的抗氧化酶以及抗病相关的β-1,3-葡聚糖酶的活性显著增强。【结论】MdWRKY40b的沉默使得苹果植株对白粉病的抗病性增强,推测转基因植株中SOD、CAT、β-1,3-glucanase的上调表达提高了苹果植株对白粉病的基础抗性,导致植物对超氧阴离子的清除能力增加,维持了植物体在响应病菌侵染过程中的低浓度超氧阴离子含量,从而减少高浓度超氧阴离子对植物的损伤。

关键词: 苹果, 白粉病, MdWRKY40b, 转基因, 活性氧, 抗氧化酶

Abstract:

【Objective】The research aims to explore the mechanism of apple resistance to powdery mildew regulated by transcription factor gene MdWRKY40b, and to provide a theoretical basis for apple powdery mildew resistance breeding. 【Method】The 552 bp specific fragment of MdWRKY40b was cloned using the cDNA obtained from the ‘Gala’ leaf tissue, the forward and reverse sequence of this gene specific fragment was constructed into the RNAi expression vector pB7GWIWG2(Ⅱ) by the Gateway technique, and then transgenic plants were obtained by an Agrobacterium-mediated method. Notably, the young leaves at the stem tips of tissue culture seedlings were chosen as the materials for Agrobacterium-mediated infection experiments. The expression levels of MdWRKY40b, superoxide dismutase gene (SOD), catalase gene (CAT), peroxidase gene (POD), and β-1,3-glucanase in transgenic plants were analyzed by RT-qPCR technology. Furthermore, using one-year-old plants as materials, the resistance of transgenic plants to powdery mildew was analyzed through the inoculation test of the powdery mildew pathogen (Podosphaera leucotricha). The leaves of transgenic and control plants at 0, 5, 10, 15, and 20 d after inoculation with P. leucotricha were used as materials, and the accumulation of superoxide anion and hydrogen peroxide in plant materials during P. leucotricha infection was analyzed by staining with nitrotetrazolium chloride solution (NBT) and diaminobenzidine (DAB). Furthermore, the activities of SOD, POD, CAT, and β-1,3-glucanase in plants were determined during P. leucotricha infection. 【Result】Three MdWRKY40b gene silencing lines were identified by PCR detection. The RT-qPCR results showed that the gene silencing efficiency of RNAi-1, RNAi-2, and RNAi-3 was 95.2%, 92.2%, and 79.8%, respectively. The expression level of SOD, CAT, and β-1,3-glucanase in transgenic plants was significantly up-regulated compared with the wild-type. Under the condition of P. leucotricha infection, compared with wild-type plants, the area of powdery disease spots in the leaves of transgenic plants decreased significantly, the accumulation of superoxide anion and hydrogen peroxide was reduced significantly, and the activities of antioxidant enzymes such as SOD, CAT, and POD regulating superoxide anion metabolism and β-1,3-glucanase related to disease resistance were significantly enhanced. 【Conclusion】The silencing of MdWRKY40b enhances the resistance of apple plants to powdery mildew. It is speculated that the up-regulated expression level of SOD, CAT, and β-1,3-glucanase in transgenic plants improves the basic resistance of apple plants to powdery mildew, and increases the scavenging ability of plants to superoxide anions. It can maintain a low concentration of superoxide anion content in response to P. leucotricha infection, so as to reduce the damage of high concentrations of superoxide anion to plants.

Key words: apple, powdery mildew, MdWRKY40b, transgene, reactive oxygen species (ROS), antioxidant enzyme

沙仁和,兰黎明,王三红,罗昌国. 苹果转录因子MdWRKY40b抗白粉病的机理[J]. 中国农业科学, 2021, 54(24): 5220-5229.

SHA RenHe,LAN LiMing,WANG SanHong,LUO ChangGuo. The Resistance Mechanism of Apple Transcription Factor MdWRKY40b to Powdery Mildew[J]. Scientia Agricultura Sinica, 2021, 54(24): 5220-5229.

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https://www.chinaagrisci.com/CN/Y2021/V54/I24/5220

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引物名称 Primer name	引物序列 Primer sequence
MdWRKY40b	F: 5′-CACCAGCGAAAGGCAGACAGTGAAG-3′ R: 5′-CGTCGGAGCTTGAGTTTCTGG-3′
LOCUS1	F: 5′-GTGATTTTTGCGGACTCTAGCAT-3′ R: 5′-CAGGTCAGCTTGACACTGAACAT-3′
LOCUS2	F: 5′-CCATGAGGTTGCTTCTGATCC-3′ R: 5′-CAACATTACAATTTACTATTCTAGT-3′
Bar	F: 5′-CCGTTCTTCCGAATAGCA-3′ R: 5′-TCATACATGAGAATTAAGGGAG-3′

引物名称 Primer name	引物序列 Primer sequence
Ubiquitin	F: 5′-AAATCCAGGACAAGGAGGGC-3′ R: 5′-CACCACGGAGACGCAACAC-3′
MdWRKY40b	F: 5′-TGCTCAACTCCAAGGCAATG-3′ R: 5′-TTCACTGTCTGCCTTTCGCT-3′
CAT	F: 5′-GCAGCCAGTTGGACGTTTG-3′ R: 5′-CTGCAGATAGTTCGGGCCAA-3′
SOD	F: 5′-GTTCGATCCATCAAACGCCG-3′ R: 5′-ATGAAGTCCAGGCTTGAGGC-3′
POD	F: 5′-TGAAGAAACTGCTGGGGGTC-3′ R: 5′-AACGTCCAAATTCGCATTGAT-3′
β-1,3-glucanase	F: 5′-GGGGTGAGGCTATTGGTGAG-3′ R: 5′-TCTGCCCCATCCCTTATTGC-3′