绿僵菌黏附素MAD1体外表达及诱导花生响应的作用

doi:10.3864/j.issn.0578-1752.2021.04.007

摘要/Abstract

摘要：

【目的】绿僵菌具有昆虫致病性和植物共生性,其黏附素MAD1在绿僵菌侵染寄主昆虫过程中起重要作用。通过体外真核表达MAD1蛋白,明确MAD1在绿僵菌与植物共生中发挥的作用。【方法】以绿僵菌转录组的Unigenes作为参考序列,在GenBank中进行同源性检索比对,设计引物,以金龟子绿僵菌（Metarhizium anisopliae）IPPM010202菌株的cDNA作为模板,克隆得到mad1,通过DNAMAN对mad1序列进行蛋白翻译,利用ProtParam在线工具预测MAD1蛋白氨基酸组成及其理化特性,利用SMART在线程序分析其结构域。构建mad1重组真核表达载体并转化毕赤酵母,获得重组子,通过甲醇诱导表达得到MAD1蛋白;通过Ni-NTA亲和层析获得纯化蛋白。以纯化蛋白溶液（20 μg·mL^-1）浸没处理花生根0.5、6、12和24 h,通过qPCR检测花生根膜受体基因（CERK1、RPK）、免疫相关级联基因（MAPK、MMK1、CDPK）和转录因子基因（MYB86）、细胞壁整合基因（SCW1）及防御相关基因（PTi1、RML1A）的转录水平。【结果】克隆得到了绿僵菌黏附素基因mad1,全长2 136 bp,编码711个氨基酸,分子量约为74.8 kD;生物信息学分析表明,MAD1的N端有信号肽,C端有糖基磷脂酰肌醇（GPI）细胞壁锚定位点,且含有CFEM功能结构域,属于亲水性蛋白。成功构建了MAD1真核表达系统,诱导表达并纯化出具有生物活性的MAD1蛋白,对花生根进行不同时间段的处理,发现MAD1处理后0.5 h,根尖细胞感知并启动膜类识别受体基因CERK1表达;0.5—6 h,根膜受体基因CERK1、RPK转录水平均上调,膜整合基因SCW1转录由下调转为上调,免疫反应相关基因MAPK、MMK1、CDPK、MYB86的转录受到短暂抑制,防御基因PTi1、RML1A转录下调,植物根部免疫防御反应受到抑制;6—12 h,膜受体基因维持上调表达,而防御基因RML1A从下调逆转为强烈上调;12—24 h,膜受体类基因CERK1、RPK,防御基因PTi1、RML1A均上调,免疫相关级联基因MAPK、MMK1、CDPK,转录因子基因MYB86也出现微弱的上调,植物根部启动免疫防御反应。【结论】在绿僵菌与花生根的互作早期,绿僵菌MAD1蛋白 6 h时已激活花生根膜受体基因CERK1和RPK的识别响应,同时抑制花生免疫级联基因MAPK、CDPK、MMK1及防御相关基因如PTi1、RML1A的表达,有助于绿僵菌在花生根组织上定殖;随后诱导花生细胞壁整合基因SCW1上调表达,参与根上受损细胞壁的修复与重建,促进绿僵菌与花生共生关系的建立。诱导植物的免疫抑制和细胞壁重建整合可能是绿僵菌与植物建立共生关系的重要步骤。

关键词: MAD1, 金龟子绿僵菌, 共生, 植物免疫, 防御, 真核表达

Abstract:

【Objective】The fungus Metarhizium anisopliae has the characteristics of insect pathogenicity and plant symbiosis, and its adhesin MAD1 plays an important role in the infection to host insects. The objective of this study is to express MAD1 protein in eukaryotes in vitro, and to clarify the role of MAD1 in symbiosis between M. anisopliae and plants. 【Method】Unigenes of M. anisopliae transcriptome were used as the reference sequence to search and compare the homology in GenBank and design primers. The gene mad1 was cloned from the cDNA template of M. anisopliae IPPM010202. MAD1 protein sequence was translated by DNAMAN software. The online software ProtParam was used to predict the MAD1 protein composition of amino acid and its physicochemical characteristics. The protein structure was analyzed using SMART online program. The recombinant was constructed from eukaryotic expression vector with mad1 and Pichia pastoris transformation. The MAD1 protein was successfully expressed by methanol induction and the purified MAD1 was obtained by Ni-NTA affinity chromatography. The peanut roots were treated with immersion in the purified MAD1 solution (20 μg·mL^-1) for 0.5, 6, 12 and 24 h, and then the transcription levels of membrane receptor genes (CERK1, RPK), immune-related cascade related genes (MAPK, MMK1, CDPK) and transcription factor gene (MYB86), cell wall integrator gene (SCW1) as well as defense related genes (PTi1, RML1A) were detected by real-time fluorescence quantitative PCR (qPCR). 【Result】 The adhesive gene mad1 of M. anisopliae was cloned, with a total length of 2 136 bp, encoding 711 amino acids in molecular weight 74.8 kD. Bioinformatics analysis showed that the N-terminal of MAD1 has signal peptide, the C-terminal has a glycosylphosphatidyl inositol (GPI) anchor and contains CFEM functional domain and belongs to hydrophilic protein. The eukaryotic expression system with mad1 was successfully constructed and the active MAD1 protein was efficiently induced, expressed and purified. The results based on qPCR of peanut roots treated with purified MAD1 showed that, for 0.5 h, the root tip cells perceived the protein and activated the expression of membrane recognition receptor gene CERK1. In 0.5-6 h, the transcriptional levels of CERK1 and RPK were up-regulated, the transcription level of membrane-integrated gene SCW1 changed from down-regulation to up-regulation, while the transcriptional levels of MAPK, MMK1, CDPK and MYB86 were temporarily inhibited, and the transcriptional levels of defense genes PTi1 and RML1A were down-regulated, the root immune defense response was inhibited. In 6-12 h, the membrane recognition receptors maintained up-regulation, while the defensive gene RML1A reversed from down-regulation to strong up-regulation. In 12-24 h, CERK1 and RPK, PTi1 and RML1A were all up-regulated, MAPK, MMK1, CDPK and MYB86 appeared slightly up-regulated, the roots initiated immune defense response.【Conclusion】In the early stage of the interaction between M. anisopliae and peanut roots, the adhesin MAD1 protein activates the membrane receptor genes CERK1 and RPK recognition response in peanut at 6 h, while inhibits the expression of peanut immune cascade genes MAPK, CDPK, MMK1 and defense related genes such as PTi1 and RML1A, which is helpful for the colonization of M. anisopliae in peanut root tissue. Subsequently, it induces the up-regulation of expression of cell wall integrin gene SCW1, which participates in the repair and reconstruction of damaged cell wall on the root, and promotes the establishment of symbiotic relationship between M. anisopliae and peanut. Induction of plant immunosuppression and integral cell wall reconstruction may be important steps in the establishment of symbiotic relationship between M. anisopliae and plants.

Key words: MAD1, Metarhizium anisopliae, symbiosis, plant immunity, defense, eukaryotic expression

闫多子,蔡霓,王峰,农向群,王广君,涂雄兵,张泽华. 绿僵菌黏附素MAD1体外表达及诱导花生响应的作用[J]. 中国农业科学, 2021, 54(4): 744-753.

YAN DuoZi,CAI Ni,WANG Feng,NONG XiangQun,WANG GuangJun,TU XiongBing,ZHANG ZeHua. Expression in vitro of Metarhizium anisopliae Adhesin MAD1 and Its Effect on Inducing Response in Peanut[J]. Scientia Agricultura Sinica, 2021, 54(4): 744-753.

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https://www.chinaagrisci.com/CN/Y2021/V54/I4/744

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基因Gene	引物序列Primer sequence	基因Gene	引物序列Primer sequence
CERK1	5′-CTACTCAGCCATTGCCAGTG	RPK	5′-AAGGTGGAGTTTGTTATGCC
CERK1	5′-CAGTCCTAAGAGGGTATGTGATGA	RPK	5′-CCTAAGGACTTGAGACCCTG
PTi1	5′-TTTGACCTGGGCACAGAGAG	RML1A	5′-CAGTGAGCGTTGCGAGATAC
PTi1	5′-GACACGGGTGGAATGAAGAC	RML1A	5′-TTGGGTTTCTTTCAGCGACT
SCW1	5′-GAGTAAGCGATTGCGGACAG	CDPK	5′-CAAGACCATTGTTGAGGTTG
SCW1	5′-TGTTCTCTCCCAAGTTCCCA	CDPK	5′-CTCCACACATCTGATTCTGG
MMK1	5′-AACTCGGAGACGAATGAGCA	MAPK	5′-CTTCCACAATGTCCAAAGCA
MMK1	5′-TTGGTGAAGGTCAGTGTCCA	MAPK	5′-CCAGATGAGTTCCTTGATGTC
MYB86	5′-CCACAACAGACAACAGTTTACG	RPL7	5′-AGAAGAGGGAGGAGGAATGG
MYB86	5′-TAGAGCACCATCTCCTACCTC	RPL7	5′-GGATGCGGATGATAAACAGG