绿僵菌mad2敲除株构建及其生物学和诱导植物响应的功能分析

doi:10.3864/j.issn.0578-1752.2021.22.008

摘要/Abstract

摘要：

【目的】昆虫病原真菌绿僵菌兼具植物内生性。已知黏附素MAD2是绿僵菌两种黏附蛋白之一,在实现绿僵菌与植物的黏附、定殖中起重要作用,但其作用机理知之甚少。本研究通过构建金龟子绿僵菌（Metarhizium anisopliae）mad2敲除突变株（Δmad2）,探究MAD2蛋白对绿僵菌生物学功能的影响。【方法】从NCBI中获取mad2前后基因组DNA序列,设计扩增mad2前后同源臂特异性引物,以绿僵菌基因组DNA为模板,扩增得到前后同源臂基因S1、S2;设计特异性引物Hyg-F/R,以pKH-KO载体为模板,扩增得到带有启动子序列的潮霉素基因hyg;再通过overlap PCR构建mad2的同源敲除盒S1H、S2H;最后利用PEG介导的原生质体转化,获得稳定遗传的mad2敲除株。通过对比敲除株与野生株（WT）的生长特性、黏附作用、杀虫毒力以及诱导花生共生基因转录水平的变化,分析MAD2蛋白的生物学功能。【结果】原生质体转化获得了敲除mad2的同源重组转化子;敲除株与野生株对比分析表明,敲除株的孢子萌发率显著低于野生株,萌发中时间比野生株延长5.47 h;培养12 h和14 h时,敲除株的菌丝长度显著均小于野生株,分别为野生株的77.8%和76.3%;培养12 d的产孢量也比野生株减少33.3%。敲除株对洋葱内表皮的黏附力明显降低,但对蝗虫后翅的黏附性无显著影响。敲除mad2并不影响绿僵菌对家蚕的毒力。敲除株处理花生12 h后,与野生株处理相比,花生共生受体SYMRK、钙信号解码相关基因（CaM、CCaMK和DELLA）、脂质氮素转运相关基因（LTP1、NRT24、ABCC2）的转录水平出现显著下调;而与空白对照相比,mad2缺失后SYMRK转录水平仍有一定的上调,CaM、CCaMK和DELLA的转录水平产生显著抑制,对ABCC2、LTP1、NRT24的转录无明显影响。【结论】金龟子绿僵菌黏附素MAD2影响菌株的孢子萌发、早期菌丝生长、产孢及对植物的黏附力,但对昆虫的黏附和杀虫毒力无影响;在菌株与花生互作早期,MAD2触发了花生共生基因的转录。

关键词: 金龟子绿僵菌, 黏附素MAD2, 基因敲除, 黏附性, 杀虫毒力, 生长特性

Abstract:

【Objective】 Metarhizium anisopliae, an entomopathogenic fungus, is found also an endophyte. MAD2 is known to be one of the two adhesin proteins of M. anisopliae, which plays a vital role in adhesion and colonization in plants, but its functional mechanism is poorly understood. The objective of this study is to explore the functional effect of MAD2 protein on the characteristics of growth, virulence, adhesion and inducing plant responses in M. anisopliae by construction of the mad2 mutant strain (Δmad2) of M. anisopliae strain Ma9. 【Method】 The genomic DNA sequences of mad2 anteroposterior were obtained from NCBI, and specific primers were designed to amplify mad2 homologous arm genes S1 and S2 by PCR based on genomic DNA template of M. anisopliae strain Ma9. Meanwhile, Hyg-F/R primer pair was designed to amplify hygromycin gene with promoter sequence based on pKH-KO vector DNA template. Then, homologous knockout boxes S1H and S2H of mad2 were constructed by overlap PCR. Finally, mad2-knockout strains with stable inheritance were obtained by PEG-mediated protoplast transformation of the homologous knockout boxes. By comparing the biological characteristics of the knockout strains (Δmad2) to the wild-type strains (WT), the effects of MAD2 protein on the characteristics of M. anisopliae growth, virulence, adhesion and inducing the peanut response of symbiosis genes were analyzed. 【Result】 Homologous recombination transformants with mad2 knockout were obtained by protoplast transformation. The spore germination rate of Δmad2 was significantly decreased and spore semi-germination time was significantly prolonged 5.47 h compare to WT, as well as the mycelium length of Δmad2 was significantly shorter than WT in 12 h and 14 h incubation, which occupied 77.8% and 76.3% of WT, respectively. The sporulation in 12-day incubation was reduced by 33.3% compared to WT. The ability of Δmad2 strain adhesion in onion was significantly decreased but showed no difference in adhere to the underwings of locust. In addition, mad2-knockout did not affect the virulence of M. anisopliae to silkworm. In the peanut inoculated mad2-knockout strain for 12 h, the transcription level of symbiosis receptor gene SYMRK, calcium signal decoding related genes (CAM, CCaMK, DELLA), lipid and nitrogen transfer related genes (LTP1, NRT24, ABCC2) was significantly down-regulated compared to the treatment of WT. While compared with blank control, Δmad2 still had certainly up-regulated SYMRK transcription level, significantly inhibited the transcription levels of CAM, CCAMK and DELLA, but had no effect on the transcription level of ABCC2, LTP1 and NRT24. 【Conclusion】 M. anisopliae adhesin protein MAD2 affects spore germination, initial growth of mycelium, sporulation quantity and plants adhesion, while has no effect on insect adhesion and virulence of strain, and MAD2 triggers the transcription of peanut symbiotic genes at the initial stage of interaction between the strain and peanut.

Key words: Metarhizium anisopliae, adhesin protein MAD2, gene knockout, adhesion, virulence, growth trait

蔡霓,闫多子,农向群,王广君,涂雄兵,张泽华. 绿僵菌mad2敲除株构建及其生物学和诱导植物响应的功能分析[J]. 中国农业科学, 2021, 54(22): 4800-4812.

CAI Ni,YAN DuoZi,NONG XiangQun,WANG GuangJun,TU XiongBing,ZHANG ZeHua. Adhesin Gene mad2 Knockout and Functional Effects on Biological Characteristics and Inducing Plant Responses in Metarhizium anisopliae[J]. Scientia Agricultura Sinica, 2021, 54(22): 4800-4812.

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引物名称Primer name	序列Sequence	片段长度Sequence length (bp)
Hyg-F	5′-ATGCCTCAGCGAATTCGATAACTG-3′	1426
Hyg-R	5′-GTTCCCGGTCGGCATCTACTCTA-3′	1426
mad2 S1-F	5′-CCCATTGTCTC CACGCACACT-3′	1046
mad2 S1-R	5′-GTTATCGAATTCGCTGAGGCATCGAAGGTATAAGGTGAGAGCGGTTG-3′	1046
mad2 S2-F	5′-TAGAGTAGATGCCGACCGGGAACGTTGACCGCACAGCATAT ATCCTGG-3′	1174
mad2 S2-R	5′-CCCAGCAACAAGCCCACATGCAGCAA-3′	1174
Hyg S2-F	5′-CAGCTTCGATGTAGGAGGGCGTGG-3′	1174 + 936
Hyg S1-R	5′-GGCAGTCCTCGGCCCAAAGCATCAG-3′	1046 + 945
OUT-mad2-F	5′-ATGAAGTCTTTCATTGCTGTTGCGG-3′	921
OUT-mad2-R	5′-CTAGATCAGGAGAGCGGCGATGAGG-3′	921

基因Gene	引物序列Primer sequence	基因Gene	引物序列Primer sequence
RPL7	5′-ACAGTTGGTCCTCACTTCAG-3′	LTP1	5′-CAACCAACCAATCACCCTC-3′
RPL7	5′-GCTCATTTATGTAAGCTTCCCT-3′	LTP1	5′-GGGCTAATCTTGTATGGGATG-3′
CCaMK	5′-ATCCGCCTTTCATTGCTC-3′	NRT24	5′-CCCACGGAGAAGAGAATCATC-3′
CCaMK	5′-GAACATCTTTGGCTACATCACC-3′	NRT24	5′-GGAATGGCAAACATCATAGCAC-3′
ABCC2	5′-CTCATAGGCATTGTCAGCAC-3′	DELLA	5′-CCTCCACCATCACAACTTCC-3′
ABCC2	5′-GTGAATCGGATGTTGTTGTC-3′	DELLA	5′-CATTCTCCTGCGAGTCAACC-3′
SYMRK	5′-AAGCAATGTGGAGAGTGGTG-3′	CaM	5′-TGATGGCTAAGTGAGGACC-3′
SYMRK	5′-AGATGAAGTAGAGGGCGGAA-3′	CaM	5′-AATGAAAGCAAGGGACAGAG-3′