黑龙江省和海南省PWL基因家族在稻瘟病菌中的分布及变异

doi:10.3864/j.issn.0578-1752.2023.02.005

摘要/Abstract

摘要：

【目的】了解不同稻瘟病菌（Magnaporthe oryzae）菌株群体中PWL基因家族的分布和变异特征，为研究不同稻瘟病菌群体的遗传多样性及特异性提供依据。【方法】通过参考NCBI中公布的无毒基因序列分别对PWL基因家族的启动子区和CDS区设计特异性引物共8对，对2020年采自黑龙江省和海南省不同地区的397个稻瘟病菌单孢分离菌株提取DNA，进行无毒基因的PCR扩增并通过琼脂糖凝胶电泳检测，从检测结果中分别选取囊括不同地区的代表菌株对扩增片段进行测序分析。测序结果与NCBI中相应无毒基因启动子区和CDS区的碱基与氨基酸序列进行比较分析。【结果】在PCR电泳检测结果中，PWL1在所有菌株中均未检测出；PWL2、PWL3和PWL4在黑龙江省和海南省中均扩增出特异性片段，说明这3种基因在两省中均有分布并分别存在不同的分布频率与变异类型。其中PWL2在黑龙江省和海南省中分布频率最高，分别为98.14%和100%；PWL3和PWL4在两省中的分布频率具有显著差异，这2种基因在黑龙江菌株中频率分别为89.30%和82.79%，而在海南菌株中均为5.49%。通过对无毒基因组合进行分析，结果显示，组合类型可分为6种，分别为PWL(f)、PWL2、PWL3、PWL2+PWL3、PWL2+PWL4、PWL2+PWL3+PWL4。其中黑龙江菌株含有所有组合类型，海南菌株仅含有2种，说明黑龙江菌株与海南菌株相比无毒基因型种类较为丰富。通过对PWL基因家族的PCR产物测序发现，PWL基因家族在启动子区和CDS区变异位点丰富，以点突变和缺失为主要变异类型划分为9种，且不同群体来源菌株的变异类型具有特异性和一致性。其中PWL2检测出5种变异类型PWL2-（1，2，3，4，5），碱基序列变化导致氨基酸序列发生错义突变；PWL3和PWL4分别检测出2种变异类型，分别为PWL3-（1，2）和PWL4-（1，2），均发生移码突变使后面氨基酸发生变化。【结论】不同群体来源的稻瘟病菌菌株中PWL基因家族的分布和变异类型具有地域差异性，且变异位点丰富。

关键词: 稻瘟病菌, PWL基因家族, 变异, 无毒基因, 黑龙江省, 海南省

Abstract:

【Objective】The objective of this study is to understand the distribution and variation characteristics of PWL gene family in different Magnaporthe oryzae strains, and to provide a basis for studying the genetic diversity and specificity of different M. oryzae strains. 【Method】A total of 8 pairs of specific primers were designed for the promoter region and CDS region of the PWL gene family by referring to the avirulence gene sequence published in NCBI. DNA was extracted from 397 single spore isolates of M. oryzae collected from different regions of Heilongjiang Province and Hainan Province in 2020. PCR amplification of avirulence genes was conducted and the genes were detected by agarose gel electrophoresis. Representative strains from different regions were selected from the test results to sequence and analyze the amplified fragments. The sequencing results were compared with the base and amino acid sequences of the corresponding avirulence gene promoter region and CDS region in NCBI. 【Result】In the results of PCR electrophoresis, PWL1 was not detected in all strains. The specific fragments of PWL2, PWL3 and PWL4 were amplified in Heilongjiang Province and Hainan Province, indicating that these three genes were distributed in both provinces and had different distribution frequencies and variation types. Among them, PWL2 had the highest distribution frequency in Heilongjiang Province and Hainan Province, which were 98.14% and 100%, respectively. The distribution frequencies of PWL3 and PWL4 in the two provinces were significantly different. The frequencies of these two genes in Heilongjiang strains were 89.30% and 82.79%, respectively, while in Hainan strains were 5.49%. Through the analysis of avirulence gene combinations, the results showed that the combination types could be divided into six types, namely PWL(f), PWL2, PWL3, PWL2+PWL3, PWL2+PWL4, PWL2+PWL3+PWL4. Among them, Heilongjiang strains contained all combination types, and Hainan strains contained only two, indicating that Heilongjiang strains were more abundant in avirulent genotypes than Hainan strains. By sequencing the PCR products of the PWL gene family, it was found that the PWL gene family had abundant variation sites in the promoter region and CDS region, and was divided into nine types with point mutations and deletions as the main variation types, and the variation types of strains from different populations were specific and consistent. Among them, five variant types PWL2- (1, 2, 3, 4, 5) were detected in PWL2, base sequence changes led to amino acid sequence missense mutations; two mutation types were detected in PWL3 and PWL4, which were PWL3- (1, 2) and PWL4- (1, 2), respectively. Both of them had frameshift mutations that caused changes in the following amino acids. 【Conclusion】The distribution and variation types of PWL gene family in M. oryzae strains from different populations have regional differences, and the variation sites are abundant.

Key words: Magnaporthe oryzae, PWL gene family, mutation, avirulence gene, Heilongjiang Province, Hainan Province

刘瑞, 赵羽涵, 付忠举, 顾欣怡, 王艳霞, 靳学慧, 杨莹, 吴伟怀, 张亚玲. 黑龙江省和海南省PWL基因家族在稻瘟病菌中的分布及变异[J]. 中国农业科学, 2023, 56(2): 264-274.

LIU RUI, ZHAO YuHan, FU ZhongJu, GU XinYi, WANG YanXia, JIN XueHui, YANG Ying, WU WeiHuai, ZHANG YaLing. Distribution and Variation of PWL Gene Family in Rice Magnaporthe oryzae from Heilongjiang Province and Hainan Province[J]. Scientia Agricultura Sinica, 2023, 56(2): 264-274.

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链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2023.02.005

https://www.chinaagrisci.com/CN/Y2023/V56/I2/264

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省份Province	采集地点Sampling location	菌株编号<BOLD>S</BOLD>train number	菌株数Number of strains
黑龙江省（215^a） Heilongjiang Province（215^a）	绥化北林区Beilin District, Suihua	BL20001-BL20006	6
	绥化庆安Qing’an, Suihua	QA20007-QA20009	3
	绥化青冈Qinggang, Suihua	QG20010-QG20012	3
	伊春铁力Tieli, Yichun	TL20013-TL20018	6
	哈尔滨方正Fangzheng, Harbin	FZ20019-FZ20044	26
	哈尔滨通河Tonghe, Harbin	TH20045-TH20069	25
	哈尔滨木兰Mulan, Harbin	ML20070-ML20081	12
	哈尔滨依兰Yilan, Harbin	YL20082-YL20085	4
	鹤岗绥滨Suibin, Hegang	SB20086-SB20102	17
	佳木斯富锦Fujin, Jiamusi	FJ20103-FJ20106	4
	佳木斯东风区Dongfeng District, Jiamusi	DF20199-DF20215	17
	鸡西虎林Hulin, Jixi	HL20107-HL20110	4
	鸡西密山Mishan, Jixi	MS20111-MS20169	59
	牡丹江东宁Dongning, Mudanjiang	DN20170-DN20198	29
海南省（182^b） Hainan Province（182^b）	东方感城Gancheng, Dongfang	DF20001-DF20004	4
	陵水Lingshui	LS20085-LS20094	10
	文昌铺前Puqian, Wenchang	WC20005-WC20038	34
	定安定城Dingcheng, Dingan	DC20039-DC20065	27
	定安雷鸣Leiming, Dingan	LM20066-LM20084	19
	澄迈金江Jinjiang, Chengmai	CM20095-CM20182	88

无毒基因 Avr-gene	引物序列 Primer sequence (5°-3°)	片段大小 Length of targeted fragments (bp)	登录号GenBank accession number
PWL1	F1：TTTCACGCCCATTAACGG	1262	AB480169.1
	R1：ACGCACAACACGAGGT	1262
	F2：GTCGCAAATGGACTAACA	395
	R2：AAACCCTATTCCAGCAGT	395
PWL2	F1：TTCGGGCACTCCGTTACT	521	U26313.1
	R1：CCTCTCTTTCGCCTTTG	521
	F2：ATGAAATGCAACAACATC	495
	R2：CCTCACACTTAAGTTAACAC	495
PWL3	F1：TGCGAGTAAAAGCCTGAA	548	U36995.1
	R1：GTGGGTGTTGTAATAGCGAT	548
	F2：GCTGGGAAGCCTGTCACCT	1412
	R2：GTTCACGCAACTGGAGGG	1412
PWL4	F1：TCCTCTGGAACATGACAATAGT	385	U36996.1
	R1：TCCGTGGGTGTTGTAGTAGC	385
	F2：CGGCTCATTGTCTGTCGTAAAGG	292
	R2：TTCGCTGGTCTTTACAAACTCCC	292

基因型 Genotype	黑龙江Heilongjiang		海南Hainan
基因型 Genotype	菌株数 Number of strains	频率Frequency (%)	菌株数 Number of strains	频率Frequency (%)
PWL(f)	1	0.47	0	0
PWL2	25	11.63	172	94.51
PWL3	1	0.47	0	0
PWL2+PWL3	14	6.51	0	0
PWL2+PWL4	3	1.40	0	0
PWL2+PWL3+PWL4	171	79.53	10	5.49

等位基因型 Allele genotype	碱基突变位点Base mutation site (bp)
等位基因型 Allele genotype	-307	-223	-169	-152	-129	-73	-51	268
PWL2	CGC	TAT	GGT	CAT	TGG	TCG	GAA	GAT
PWL2-1	--T	---	---	---	---	---	---	---
PWL2-2	---	---	---	---	---	---	---	A--
PWL2-3	---	---	---	---	---	---	A--	A--
PWL2-4	---	--G	--G	-T-	G--	--T	A--	A--

等位基因型 Allele genotype	碱基突变位点Base mutation site (bp)
等位基因型 Allele genotype	-187	-151	301	363	391	408	409，411
PWL3	AAA	AGT	TAT	GGG	AAG	GAC	GAC
PWL3-1	---	--C	*--	--A	C--	--A	T-G
PWL3-2	--*	--C	*--	--A	C--	--A	T-G