林业科学 ›› 2021, Vol. 57 ›› Issue (8): 82-93.doi: 10.11707/j.1001-7488.20210808
李雪燕,熊典广,田呈明*
收稿日期:
2020-11-10
出版日期:
2021-08-25
发布日期:
2021-09-30
通讯作者:
田呈明
基金资助:
Xueyan Li,Dianguang Xiong,Chengming Tian*
Received:
2020-11-10
Online:
2021-08-25
Published:
2021-09-30
Contact:
Chengming Tian
摘要:
目的: 对杨树腐烂病菌金黄壳囊孢胞外分泌复合体亚基CcExo70进行功能分析,探究其在杨树腐烂病菌生长发育及致病过程中的生物学功能,为深入了解杨树腐烂病菌致病机制以及制定病害防控策略提供科学依据。方法: 1)利用PCR技术克隆得到CcExo70基因的DNA序列;2)通过生物信息软件对其编码的氨基酸序列进行特征分析并构建其同源基因的系统发育树;3)通过PEG介导的遗传转化法得到CcExo70基因的敲除突变体及回补菌株;4)通过PDA平板生长试验和PDB摇培试验,分析敲除CcExo70基因对该病菌生长发育、菌丝形态及H2O2胁迫响应的影响;通过对1年生欧美杨健康枝条烫伤接种的方法,测定CcExo70基因对该病菌致病性的影响;5)利用荧光增白剂(CFW)、二氨基联苯胺(DAB)和FM4-64染剂探究各菌株中几丁质沉积、活性氧积累及内吞作用情况;6)通过蛋白质组测序分析,筛选可能受CcExo70调控分泌的外泌蛋白。结果: 1)在杨树腐烂病菌中克隆得到了一个CcExo70基因,该基因全长1 992 bp,包含1个内含子,编码636个氨基酸。2)系统发育和序列比对分析表明,CcExo70与其他病原真菌的同源基因在进化上是高度保守的。3)利用遗传转化法筛选得到2个敲除突变体(ΔCcExo70-8、ΔCcExo70-9),并获得了回补菌株(ΔCcExo70/C)。4)表型分析发现,与野生型和回补菌株相比,CcExo70敲除突变体在菌丝生长、形态发生、氧化应激反应和致病力等方面存在显著的缺陷。5)CFW染色观察显示,野生型和回补菌株菌丝尖端存在明显的几丁质沉积,而敲除突变体菌丝尖端未见明显的几丁质沉积但菌丝隔膜异常增加。此外,共聚焦显微镜观察发现,与野生型和回补菌株相比,CcExo70敲除突变体的内吞作用显著延迟。6)蛋白组测序分析发现,8个候选的外泌蛋白在CcExo70敲除突变体胞外培养滤液中的含量显著低于在野生型胞外培养滤液中的含量(降低了50%以上),其中有4个候选的外泌蛋白(CcSP2、CcSP3、CcSP6、CcSP7)在胞外培养滤液中的含量显著高于其对应菌株的胞内含量,其中包括1个候选的效应分子(CcSP2)和2个糖苷水解酶(CcSP6和CcSP7),表明这4个外泌蛋白的分泌受到了CcExo70的调控。结论: 胞外分泌复合体亚基CcExo70在杨树腐烂病菌的生长、胁迫响应、内吞作用和致病性等方面起着重要的调控作用。
中图分类号:
李雪燕,熊典广,田呈明. 杨树腐烂病菌胞外分泌复合体亚基CcExo70的功能[J]. 林业科学, 2021, 57(8): 82-93.
Xueyan Li,Dianguang Xiong,Chengming Tian. Functional Analysis of the Exocyst Subunit CcExo70 in Cytospora chrysosperma[J]. Scientia Silvae Sinicae, 2021, 57(8): 82-93.
表1
引物序列"
引物 Primer name | 引物序列 Primer sequence(5′—3′) | 扩增片段 Amplified fragment |
CcExo70-5Ffor | TGAGGTCATGTCGGGAATGG | CcExo70 5’侧翼序列 CcExo70 5’ flanking sequence |
CcExo70-5Frev | CGGAGAACTAACGGCACTTG | |
CcExo70-3Ffor | GGATCGCAGGAGTAGAGGTT | CcExo70 3’侧翼序列 CcExo70 3’ flanking sequence |
CcExo70-3Frev | CCGGCTTGGCTTTCTTCTAT | |
Hygromycinfor | CGCCAGGGTTTTCCCAGTCACGAC | 潮霉素片段 Hygromycin cassette |
Hygromycinrev | AGCGGATAACAATTTCACACAGGA | |
HY-R | GTATTGACCGATTCCTTGCGGTCCGAA | 2/3潮霉素片段 The 2/3rd portion of the hygromycin cassette |
YG-F | TCCTGTGTGAAATTGTTATCCGCT | |
External-Exo70for | TGCCGCTGACATCTATCCAA | 用于验证突变体的外部序列 External sequence used for validation of mutant |
External-Exo70rev | GCTTTCCTGCTCATCCCAAC | |
Internal-Exo70for | GTTGTCCCACTACCTGAGCT | 用于验证突变体的内部序列 Internal sequence used for validation of mutant |
Internal-Exo70rev | GCTCGACATGACCTCTACGA | |
Exo70-Compfor | AAAGGGTAGCGGGTGAGTAC | 回补序列 Complementary sequence |
Exo70-Comprev | GCTTTCCTGCTCATCCCAAC |
表2
ΔCcExo70-F与WT-F中蛋白质丰度比值"
基因名称 Gene name | 蛋白长度 Protein length/bp | 半胱氨酸的数量 Number of cysteine | 突变体/野生型 ΔCcExo70-F/WT-F | 结构域预测 Interproprediction |
CcSP1 | 514 | 4 | 0.29 | 热激蛋白HSP70家族(IPR013126) Heat shock protein 70 family(IPR013126) |
CcSP2 | 278 | 7 | 0.48 | 肽酶G1(IPR000250) Peptidase G1(IPR000250) |
CcSP3 | 401 | 0 | 0.39 | 乳糖酶,7叶片β-螺旋(IPR019405) Lactonase, 7-bladed β-helix(IPR019405) |
CcSP4 | 501 | 6 | 0.26 | 蛋白二硫化物异构酶(IPR005792) Protein disulphide isomerase(IPR005792) |
CcSP5 | 379 | 2 | 0.34 | L-赖氨酸6-单氧酶/L-鸟氨酸5-单氧酶(IPR025700) L-lysine 6-monooxygenase/L-ornithine 5-monooxygenase(IPR025700) |
CcSP6 | 956 | 10 | 0.07 | 糖苷水解酶家族31(IPR000322) Glycoside hydrolase family 31(IPR000322) |
CcSP7 | 505 | 8 | 0.39 | α-L-阿拉伯呋喃糖苷酶B(IPR038964) α-L-arabinofuranosidase B(IPR038964) |
CcSP8 | 1 021 | 3 | 0.20 | 热激蛋白HSP70家族(IPR013126) Heat shock protein 70 family(IPR013126) |
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