禾谷炭疽菌<i>CgRAB</i>4的功能研究

齐尧尧; 赵耀; 石晓玲

doi:10.19303/j.issn.1008-0384.2021.06.009

禾谷炭疽菌CgRAB4的功能研究

doi: 10.19303/j.issn.1008-0384.2021.06.009

齐尧尧^{1, 2,},
赵耀²,
石晓玲²

1.
临沂职业学院，山东　临沂　276017
2.
临沂大学农林科学学院，山东　临沂　276000

基金项目: 山东省自然科学基金项目（ZR2019PC001）；福建农林大学生物农药与化学生物学教育部重点实验室开放课题（keylab2018-03）；福建省高校重点实验室开放基金项目（PMI2018KF3）

详细信息

作者简介:
齐尧尧（1987−），女，博士，副教授，研究方向：植物与真菌的相互作用（E-mail：982440756@qq.com）

中图分类号: S 435.1
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出版历程
- 收稿日期: 2021-03-26
- 修回日期: 2020-04-26
- 网络出版日期: 2021-06-06
- 刊出日期: 2021-06-28

Functional Analysis on CgRAB4 of Colletotrichum graminicola

1.
Linyi Vocational College, Linyi, Shandong　276017, China
2.
College of Agriculture and Forestry Science, Linyi University, Linyi, Shandong　276000, China

摘要

摘要: 目的阐明禾谷炭疽菌（Colletotrichum graminicola）中CgRAB4的功能，为探究Rab蛋白在禾谷炭疽病菌中的保守性和特殊功能奠定理论基础。方法通过同源重组的方法对CgRAB4进行基因敲除，经潮霉素抗性筛选和PCR验证后获得缺失突变体，进而分析其在禾谷炭疽菌生长发育和侵染致病过程中的作用。结果成功获得CgRAB4基因的3个缺失突变体和1个异位整合突变体，对其表型分析发现CgRAB4基因缺失后促进了菌丝的生长，但不影响菌丝的形态，且不参与调控外界胁迫的响应过程，产孢量、孢子形态、附着胞的产生以及对玉米的致病性都没有明显变化。结论通过对CgRAB4基因进行敲除，表型分析后发现该基因可能参与调控禾谷炭疽菌的生长过程，为揭示Rab蛋白在禾谷炭疽菌生长发育和侵染致病过程的作用奠定理论基础，为防治玉米炭疽病提供理论指导。
- 禾谷炭疽菌 /
- CgRAB4 /
- 敲除 /
- 表型分析
Abstract: Objective Conserve and special functions of Rab4 protein of Colletotrichum graminicola (Cg) were studied. Method Based upon the principle of homologous recombination, CgRAB4 was knocked out from the DNA of Cg to obtain mutants with the target gene deleted. A hygromycin-resistance screening and PCR verification were performed on the mutants prior to a functional analysis on Cg development and pathogenesis. Result Three CgRAB4-deleted and one ectopic mutants were secured by the designed process. The phenotypic analysis revealed that the deletion promoted the hypha growth but did not affect the mycelial morphology, nor regulate the response to external stress or significant change in the spore production and morphology, appressorium germination or the pathogenicity on the mutants. Conclusion CgRAB4 was successfully removed from Cg to unveil the involvement of the gene in regulating the mycelial growth. Understanding on the roles of Rab proteins played in the development and pathogenesis of Cg provided a guiding direction for further research on the prevention and treatment of corn anthrax.
- Colletotrichum graminicola /
- CgRAB4 /
- knock out /
- phenotypic analysis

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图 1 同源重组的敲除策略（A）和SOE-PCR热循环体系（B）

Figure 1. Homologous recombination knockout (A) and SOC-PCR system (B)

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图 2 SOE-PCR扩增AH和HB

注： M: DL2000 marker。

Figure 2. SOE-PCR amplifications of AH and HB

Note: M: DL2000 marker.

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图 3 转化子的DNA提取与验证

注：M: DL2000 marker M2: 野生型基因组DNA。

Figure 3. DNA extraction and verification on transformants

Note: M: DL2000 marker; M2: DNA of wild type genome.

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图 4 禾谷炭疽菌CgRAB4缺失突变体的生长发育

注：①A：菌落形态；B：菌丝生长速率； C：菌丝形态。②不同小写字母表示差异达显著水平（ P ＜0.05），下同。

Figure 4. Development of CgRAB4-deleted mutants

Note: ①A: colony morphology; B: growth rate; C: mycelial morphology.② Data with different lowercase letters indicate significant difference at 0.05 level, the same as below.

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图 5 CgRAB4缺失突变体胁迫响应试验

注：A：含有不同添加物的CMII培养基上CgRAB4缺失突变的菌落形态；B：CMII培养基上的抑制率；C：含有不同添加物的PDA培养基上CgRAB4缺失突变的菌落形态；D：PDA培养基上的抑制率。

Figure 5. Stress responses of CgRAB4-deleted mutants

Note: A: colony morphology of CgRAB4-deleted mutants obtained on CMII media containing varied supplements; B: inhibition rate on CMII media; C: colony morphology of CgRAB4-deleted mutants obtained on PDA media containing varied supplements; D: inhibition rate on PDA media.

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图 6 CgRAB4缺失突变体产孢量和孢子形态

注：A：产孢量统计；B：孢子形态观察。

Figure 6. Conidiations and spore morphology of CgRAB4-deleted mutants

Note: A: conidiation; B: spore morphology.

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图 7 CgRAB4缺失突变体的孢子萌发和附着胞形态

注：A：24 h孢子萌发产生附着胞；B：48 h 附着胞形态。

Figure 7. Spore germination and appressorium morphology of CgRAB4-deleted mutants

Note: A:the spores germinate to produce appressorium on 24 h; B: morphology of appressorium on 48 h.

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图 8 CgRAB4缺失突变体对玉米的致病性

注：A：造伤接种；B：喷雾接种。

Figure 8. Pathogenesis of CgRAB4-deleted mutants on corn

Note: A: injury inoculation; B: spray inoculation.

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表 1 供试培养基

Table 1. Culture media applied

名称 Name	配方 Ingredient	添加物 Supplement	用途 Application
MK	40 g米糠		不含添加物的固体培养基用于禾谷炭疽菌野生型以及缺失突变体的生长速率的测定
	20 g琼脂粉		不含添加物的固体培养基用于禾谷炭疽菌野生型以及缺失突变体的生长速率的测定
CMII	50 mL N源	氯化钠 NaCl	含添加物的固体培养基用于禾谷炭疽菌野生型以及缺失突变体胁迫敏感性测定
	1 mL 微量元素 Trace elements	氯化钾 KCl	含添加物的固体培养基用于禾谷炭疽菌野生型以及缺失突变体胁迫敏感性测定
	1 mL维生素 Vitamin solution	山梨醇 Sorbitol
	10 g D-葡萄糖	十二烷基硫酸钠 SDS
	2 g 蛋白胨 Peptone	荧光增白剂 CFW
	1 g 酵母提取物 Yeast extraction	刚果红 CR
	1 g（酸水解酪蛋白） Casamino acids
	pH6.5
PDA		氯化钠 NaCl	液体培养基用于禾谷炭疽菌野生型以及缺失突变体生物量测定
	46 g·L⁻¹ 马铃薯葡萄糖 Potato dextrose	氯化钾 KCl	液体培养基用于禾谷炭疽菌野生型以及缺失突变体生物量测定
	15 g·L⁻¹ 琼脂粉 Agar	山梨醇 Sorbitol
		十二烷基硫酸钠 SDS
		荧光增白剂 CFW
		刚果红 CR
TB3	200 g·L⁻¹ 蔗糖 Sucrose		用于原生质体再生
	6 g·L⁻¹ 酸水解酪蛋白 Casamino acids
	6 g·L⁻¹ 酵母提取物 Yeast extract
	15 g·L⁻¹ 琼脂粉 Agar

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表 2 供试引物序列

Table 2. Sequence of primer employed

引物名称 Primer	引物序列 Sequence	引物用途 Application
CgRAB4 AF	CGAATGGGAGTTAGGGTG	扩增A片段 Applification of A fragment
CgRAB4 AR	TTGACCTCCACTAGCTCCAGCCAA GCCACAGCAGTCGGTTGGTT
CgRAB4 BF	GAATAGAGTAGATGCCGACCGCGG GTTTTTGGTTTACCTGACGAT	扩增B片段 Applification of B fragment
CgRAB4 BR	CTTCACCTCTTGCTCCTG
CgRAB4 OF	CTCCCAACTTGTCTTGCCTTCT	敲除突变体的验证 Verification of knockout mutants
CgRAB4 OR	TCGCCTGCCCTTTCGTCT
CgRAB4 UAF	CGCAGAGTCAGCGTCAAT
H853	GACAGACGTCGCGGTGAGTT
YG/F	GATGTAGGAGGGCGTGGATATGTCCT	扩增潮霉素基因全长 Amplification of full length of hygromycin gene
HY/R	GTATTGACCGATTCCTTGCGGTCCGAA
HYG/F	GGCTTGGCTGGAGCTAGTGGAGGTCAA
HYG/R	AACCCGCGGTCGGCATCTACTCTATTC

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