橡胶树胶孢炭疽菌Zn2Cys6型转录因子CgAswA的生物学功能

doi:10.13560/j.cnki.biotech.bull.1985.2021-0047

摘要/Abstract

摘要：

Zn₂Cys₆型转录因子是真菌特有的调控因子,在植物病原真菌生长发育及致病过程中起着重要的调节作用,目前关于胶孢炭疽菌的转录因子研究报道较少。利用 PCR 技术扩增CgaswA的基因并进行生物信息学分析,利用同源重组的方法获得CgaswA基因的敲除突变株,在突变株的基础上获得互补株,并通过营养生长、分生孢子产生、附着胞形成及致病性等方面的表型分析,确定该基因的生物学功能。结果显示,通过 PCR 扩增获得了CgaswA的基因,其编码一个 713个氨基酸的蛋白。敲除突变株与野生型菌株相比,其营养生长缓慢,分生孢子产量降低且孢子萌发和附着胞的形成滞后,致病性减弱等。CgAswA蛋白参与调控胶孢炭疽菌的营养生长、分生孢子的产生和萌发、附着胞的形成、侵入及致病性。

关键词: 胶孢炭疽菌, 转录因子, 基因功能, 致病性

Abstract:

Zn₂Cys_6-type transcription factor is a unique regulation factor in fungi and plays an important regulatory role in the growth,development and pathogenicity of plant pathogenic fungi. Currently,there are few reports on the transcription factor of Colletotrichum gloeosporioides. The gene of CgaswA was amplified by PCR and bioinformatics analysis was conducted. The CgaswA knock-out mutant was obtained by homologous recombination method,and the complementary strain was constructed on the basis of the mutant strain. Further,the biological function of CgaswA was determined by the phenotypic analyses of vegetative growth,conidia,production,appressorium formation and pathogenicity,etc. Results showed that gene CgaswA was acquired by PCR amplification,and it encoded a 713-amino acids protein. Compared with the wild-type strain,the knock-out mutant showed slow vegetative growth,low conidial yield,delayed spore germination and appressorium formation,and reduced pathogenicity. In conclusion,CgAswA is involved in the regulation of vegetative growth,conidiation,germination,appressorium formation and invasion,and pathogenicity in C. gloeosporioides.

Key words: Colletotrichum gloeosporioides, transcription factor, gene function, pathogenicity

刘沙玉, 曹健, 李蒙, 柳志强, 李晓宇. 橡胶树胶孢炭疽菌Zn₂Cys₆型转录因子CgAswA的生物学功能[J]. 生物技术通报, 2021, 37(9): 161-170.

LIU Sha-yu, CAO Jian, LI Meng, LIU Zhi-qiang, LI Xiao-yu. Biological Function of a Zn₂Cys₆ Transcription Factor CgAswA in Colletotrichum gloeosporioides[J]. Biotechnology Bulletin, 2021, 37(9): 161-170.

图/表 8

图1 CgaswA基因的同源重组原理 Sur为氯嘧磺隆抗性基因,黑色三角表示验证敲除突变体所用的引物

Fig. 1 Homologous recombination principle of the gene CgaswA Sur is the chlorimuron-ethyl resistance gene,and black triangles indicate the primers used to verify gene-knockout mutants

表1 引物名称及序列

Table 1 Primer and sequence

Primer	Primer sequence（5'-3'）
CgaswAupF	CGGAATTCTCAGAAGAGGCACCGCAACGCAT
CgaswAupR	CCCAAGCTTATCTCCTTCATCCGACACGACTG
CgaswAF	ATGAACGACAACATACTCTACC
CgaswAR	TCAAGTGCACAAGAAGTCGAGG
CgaswAdownF	GCTCTAGACGCTCAACCACCTCATCGCTACC
CgaswAdownR	CGGAATTCCCGTCCGTTGTCTTGACCTTTGC
CgaswAUU	CTCCTTCGCTCACTTGCTCGCTC
PI	CAGGGTTTTCCCAGTCACGACGTTG
PI1	GTATGTTGTGTGGAATTGTGAGCGG
CgaswADD	GCACAATGGAGATCTTCGCCCTT
CgaswAhbF	CGGAATTCTGCTTTCGCTCCCTCTGTTTCTT
CgaswAhbR	GCTCTAGAGTCCGTTGTCTTGACCTTTGCTG

图2 CgAswA蛋白结构域和系统发育树 A：CgAswA的蛋白质结构域分析;B：CgAswA的系统进化分析;序列ID位于括号中;水平线上的数字代表进化距离

Fig. 2 Protein domain and phylogenetic tree of CgAswA protein A：Protein domain analysis of CgAswA. B：Phylogenetic analysis of CgAswA. The sequence ID is in parentheses;the numbers on the horizontal lines represent evolutionary distances

图3 CgaswA 基因的敲除和互补验证 A：引物CgaswAF / CgaswAR的PCR扩增结果;B：引物CgaswAUU / PI的PCR扩增结果;C：PI1 / CgaswADD的引物的PCR扩增结果。其中M：DL2 000 DNA marker;1：野生型;2：ΔCgaswA-3;3：ΔCgaswA-8;4：ΔCgaswA-11;5：ΔCgaswA/ aswA

Fig. 3 Verification of CgaswA gene knockout and com-plementation A：PCR results of CgaswAF / CgaswAR;B：PCR results of CgaswAUU / PI;C：PCR results of PI1 / CgaswADD. M：DL2 000 DNA marker;1：wild type;2：ΔCgaswA-3;3：ΔCgaswA-8;4：ΔCgaswA-11;5：ΔCgaswA/aswA

图4 菌株在4种培养基上生长情况比较 A：菌株在4种培养基上的生长;B：菌落直径统计分析;a,b表示显著水平,P<0.05

Fig. 4 Comparison of strains’ growth on four media A：Growth of strains on four media. B：Statistical analysis of colony diameter. a,b：Significant level,P<0.05

图5 野生型、ΔCgaswA与ΔCgaswA/aswA分生孢子的产生、萌发和附着胞的形成 A：分生孢子产量统计结果;B：分生孢子萌发率统计结果;C：附着胞形成率统计结果;D：在4 h、8 h和12 h分生孢子萌发和附着胞的形成情况;co：分生孢子,gt：芽管,ap：附着胞;a,b,c,d表示显著水平,P <0.05;Scale bars = 20 μm

Fig. 5 Sporulation,germination and appressorium formation of wild type,ΔCgaswA and ΔCgaswA/aswA A：Statistical results of conidia yield. B：Statistical results of conidia germination rate. C：Statistical results of appressorium formation rate. D：Conidial germination and appressorium formation at 4 h,8 h and 12 h;co：conidium,gt：germ tube,ap：appressorium;a,b,c,d：significant level,P<0.05. Scale bars = 20 µm

图6 洋葱表皮侵染和玻璃纸穿透试验 A：不同菌株在洋葱表皮上附着胞的侵染试验;B：分生孢子悬液的玻璃纸穿透试验;C：菌饼的玻璃纸穿透试验。co：分生孢子;ap：附着胞;ih：侵染结构。a,b,c,d表示显著水平,P <0.05;scale bars = 20 μm

Fig. 6 Onion epidermis infection and cellophane penetration assays A：Appressorium infection assay of different strains on onion epidermis. B：Cellophane penetration assay of conidia suspensions. C：Cellophane penetration assay of mycelial plugs;co：conidium,ap：appressorium,ih：invasive hyphae;a,b,c,d：significant level,P<0.05;scale bars = 20 µm

图7 野生型、ΔCgaswA与ΔCgaswA/aswA致病性分析 A：菌饼接种橡胶叶片4 d后的症状及病斑直径统计结果;B：分生孢子悬液接种橡胶叶片5 d后的症状及病斑直径统计结果。1：CK;2：WT;3：ΔCgaswA/ aswA;4：ΔCgaswA-3;5：ΔCgaswA-8;6：ΔCgaswA-11;a,b,c,d表示显著水平,P <0.05

Fig. 7 Pathogenicity analyses of wild type,ΔCgaswA and ΔCgaswA/aswA A：Symptoms on rubber leaves at 4 d post-inoculation using mycelial plugs and statistical analyses of the diameters of disease spots. B：Symptomss on rubber leaves at 5 d post-inoculation using conidial suspensions and statistical analyses of the diameters of disease spots. 1：CK;2：WT;3：ΔCgaswA/ aswA;4：ΔCgaswA-3;5：ΔCgaswA-8;6：ΔCgaswA-11;a,b,c,d：significant level,P<0.05

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橡胶树胶孢炭疽菌Zn₂Cys₆型转录因子CgAswA的生物学功能

Biological Function of a Zn₂Cys₆ Transcription Factor CgAswA in Colletotrichum gloeosporioides

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