生物技术通报 ›› 2021, Vol. 37 ›› Issue (9): 161-170.doi: 10.13560/j.cnki.biotech.bull.1985.2021-0047
收稿日期:
2021-01-11
出版日期:
2021-09-26
发布日期:
2021-10-25
作者简介:
刘沙玉,女,硕士研究生,研究方向:植物病原真菌学;E-mail: 基金资助:
LIU Sha-yu(), CAO Jian, LI Meng, LIU Zhi-qiang, LI Xiao-yu()
Received:
2021-01-11
Published:
2021-09-26
Online:
2021-10-25
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。
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
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 |
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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