灰葡萄孢BcPDR1与MAPK途径基因BcBMP1和BcBMP3的关系

doi:10.7668/hbnxb.20192641

摘要/Abstract

摘要：

为了确定灰葡萄孢致病基因BcPDR1与病菌MAPK途径基因BcBMP1和BcBMP3之间的关系,利用实时荧光定量PCR技术,分析BcPDR1基因突变体中BcBMP1、BcBMP3的表达情况以及BcBMP1、BcBMP3基因RNAi突变体中BcPDR1的表达情况;在突变体ΔBcpdr1背景下,构建了BcBMP1和BcBMP3过表达的菌株ΔBcpdr1/BcBMP1-OE和ΔBcpdr1/BcBMP3-OE,并对过表达菌株的表型特征和致病能力进行分析。结果发现,BcPDR1基因突变体BCt89和ΔBcpdr1中BcBMP1、BcBMP3的表达水平显著优于野生型菌株和回复菌株,BcBMP1、BcBMP3基因RNAi突变体中BcPDR1的表达水平显著低于野生型;过表达菌株ΔBcpdr1/BcBMP1-OE和ΔBcpdr1/BcBMP3-OE的菌落形态、菌丝形态、生长速率、产孢量和致病力均与突变体ΔBcpdr1表现明显的不同,而更接近野生型BC22。结果表明,灰葡萄孢BcPDR1与BcBMP1、BcBMP3的表达水平密切相关,BcPDR1负调控BcBMP1、BcBMP3的表达,而BcBMP1、BcBMP3正调控BcPDR1的表达。

关键词: 灰葡萄孢, BcPDR1, MAPK途径基因, BcBMP1, BcBMP3

Abstract:

To clarify the relationship between BcPDR1 gene and MAPK pathway genes BcBMP1 and BcBMP3 of Botrytis cinerea.We analyzed the expression levels of BcBMP1 and BcBMP3 in mutants of BcPDR1 by Real-time PCR and the expression levels of BcPDR1 in RNAi mutants of BcBMP1 and BcBMP3.In the background of mutant ΔBcpdr1,strains ΔBcpdr1/BcBMP1-OE and ΔBcpdr1/BcBMP3-OE over-expressing BcBMP1 and BcBMP3 were constructed,and the phenotype and pathogenicity of the over-expressing strains were analyzed.It was found that the expression levels of BcBMP1 and BcBMP3 in BCt89 and ΔBcpdr1,gene mutants of BcPDR1,were significantly better than those of the wild-type strain and the revertant strain,and the expression levels of BcPDR1 in the RNAi mutants of BcBMP1 and BcBMP3 gene were obviously lower than those of the wild-type strain.The colony morphology,hyphal morphology,growth rate,sporulation,and pathogenicity of the over-expression isolates ΔBcpdr1/BcBMP1-OE and ΔBcpdr1/BcBMP3-OE were obviously different from ΔBcpdr1,and closer to the wild-type BC22.These results indicated that the BcPDR1 was closely related to the expression levels of BcBMP1 and BcBMP3 in B.cinerea.The BcPDR1 gene negatively regulates the expression of BcBMP1 and BcBMP3,while BcBMP1 and BcBMP3 genes positively regulate the expression of the BcPDR1.

Key words: Botrytis cinerea, BcPDR1, MAPK pathway genes, BcBMP1, BcBMP3

刘晓颖, 魏雅迪, 李白, 曹宏哲, 藏金萍, 张康, 邢继红, 董金皋. 灰葡萄孢BcPDR1与MAPK途径基因BcBMP1和BcBMP3的关系[J]. 华北农学报, 2022, 37(S1): 318-323. doi: 10.7668/hbnxb.20192641.

LIU Xiaoying, WEI Yadi, LI Bai, CAO Hongzhe, ZANG Jinping, ZHANG Kang, XING Jihong, DONG Jingao. Relationship Between BcPDR1 Gene and MAPK Pathway Genes BcBMP1 and BcBMP3 in Botrytis cinerea[J]. Acta Agriculturae Boreali-Sinica, 2022, 37(S1): 318-323. doi: 10.7668/hbnxb.20192641.

http://www.hbnxb.net/CN/Y2022/V37/IS1/318

图/表 6

表1 Real-time PCR引物设计

Tab.1 Real-time PCR primers design

基因名称 Gene name	引物名称 Primer name	序列 (5'-3') Sequence(5'-3')
BcBMP1	rtBcBMP1-F	ATCTCTTTCAATGTCAGCGAGCAAT
	rtBcBMP1-R	ATGGAGTAATCTTCTTGATAGCGAC
BcBMP3	rtBcBMP3-F	ACAACATGTCCGTGTTCAACCTG
	rtBcBMP3-R	CACGCCAGCTTGCTTGTTCT
BcPDR1	rtBcPDR1-F	CATTCCGGGGTGCCAAGACTCCTGA
	rtBcPDR1-R	ACATGATTTTCATCATAAGGCTTCG
Tubulin	Tubulin-F	TCTGGCGAGCACGGTCTTGACGGTT
	Tubulin-R	TGGCTCCAAATCGACGAGGACGGCA
BcBMP1	BcBMP1-F	ATGACAGCTCGTGCGCCTAATCC
	BcBMP1-R	TCTCATGATCTCATCATAAATC
BcBMP3	BcBMP3-F	ATGGCAGACCTGCAAGGAAGAAAGG
	BcBMP3-R	CGATCGCATAGCATCCAAACCACCT

图1 BcPDR1基因突变体中BcBMP1和BcBMP3基因的表达分析 A.BcPDR1基因突变体中BcBMP1的表达;B.BcPDR1基因突变体中BcBMP3的表达。图中不同的小写字母表示显著性差异(P<0.05)。图2-5同。

Fig.1 Expression levels of BcBMP1 and BcBMP3 in BcPDR1 gene mutants A.Expression of BcBMP1 in BcPDR1 gene mutant;B.Expression of BcBMP3 in BcPDR1 gene mutant.The different lowercase letters in the figure show significant differences(P<0.05).The same as Fig.2-5.

图2 BcBMP1(A)和BcBMP3(B)基因突变体中BcPDR1基因的表达分析 A.BcBMP1基因RNAi突变体中BcPDR1的表达;B.BcBMP3基因RNAi突变体中BcPDR1的表达。

Fig.2 Expression levels of the BcPDR1 in RNAi mutants of BcBMP1 (A)and BcBMP3 (B)genes A.Expression of BcPDR1 in RNAi mutants of the BcBMP1 gene;B.Expression of BcPDR1 in RNAi mutants of BcBMP3.

图3 突变体ΔBcpdr1背景下BcBMP1、BcBMP3过表达菌株的构建 A.菌株中BcBMP1的表达分析;B.菌株中BcBMP3的表达分析;C.菌株的GFP荧光检测。

Fig.3 Construction of over-expression isolates of BcBMP1 and BcBMP3 in ΔBcpdr1 background A.Expression analysis of BcBMP1 in strains;B.Expression analysis of BcBMP3 in strains;C.GFP fluorescence detection of strains.

图4 突变体ΔBcpdr1背景下BcBMP1、BcBMP3过表达菌株的表型分析 A.菌落形态观察;B.菌丝形态观察;C.生长速率测定;D.分生孢子产量分析。

Fig.4 Phenotypic analysis BcBMP1 and BcBMP3 over-expressing strains in the background of mutant ΔBcpdr1 A.Colony morphology observation; B.Observation of hyphal morphology; C.Growth rate assay; D.Conidial yield analysis.

图5 突变体ΔBcpdr1背景下BcBMP1、BcBMP3过表达菌株的致病力分析 A.过表达菌株的致病力分析;B.病斑面积统计。

Fig.5 Pathogenicity analysis of BcBMP1 and BcBMP3 over-expressing strains in the background of mutant ΔBcpdr1 A.Analysis of the pathogenicity of the overexpression strains;B.Statistics on lesion area.

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