黑龙江省稻瘟病菌无毒基因AVR-Pita及其同源基因的检测与分析

doi:10.16819/j.1001-7216.2020.9085

摘要/Abstract

摘要：

【目的】为了检测黑龙江省稻瘟病菌无毒基因AVR-Pita及其同源基因分布情况与变异机制,了解其变异类型的致病表型。【方法】采用3个无毒基因AVR-Pita1、AVR-Pita2和AVR-Pita3的特异性引物,对202个采自黑龙江省各稻区的稻瘟病菌单孢分离菌株的DNA进行PCR扩增,通过琼脂糖凝胶电泳检测分析,挑选不同带型和不同地区代表菌株的PCR产物进行测序。测序结果与相应无毒基因序列进行碱基与氨基酸序列的比较分析,并利用水稻抗性单基因系,对不同变异类型的稻瘟病菌株进行功能验证。【结果】AVR-Pita1的出现频率为36.14％,AVR-Pita3出现频率为59.41％。AVR-Pita2在黑龙江省202个菌株DNA中未扩增出目的条带。对AVR-Pita1和AVR-Pita3的部分PCR产物进行序列分析,检测出AVR-Pita1有5种变异类型,它们是AVR-Pita1-1、AVR-Pita1-A、AVR-Pita1-B、AVR-Pita1-C和AVR-Pita1-D。经功能验证,AVR-Pita1-1、AVR-Pita1-A、AVR-Pita1-B和AVR-Pita1-D无毒功能丧失。而无毒基因AVR-Pita3未检测出变异菌株。【结论】AVR-Pita1变异能力较强,导致大多数菌株无毒功能丧失,需与其他抗性基因搭配使用。在黑龙江省稻瘟病菌生理小种中未发现AVR-Pita2基因。AVR-Pita3基因序列在菌株中比较稳定。

关键词: 稻瘟病菌, AVR-Pita及其同源基因, 功能验证

Abstract:

【Objective】We aim to investigate the distribution and variation of avirulent AVR-Pita family of Magnaporthe oryzae from Heilongjiang Province, and to understand the pathogenic phenotypes of avirulence alleles.【Methods】Specific primers of the three avirulent genes AVR-Pita1, AVR-Pita2 and AVR-Pita3 were used. 202 M. oryzae single-spore isolates were obtained from different regions of Heilongjiang Province, their DNA were PCR-amplified by using avirulent genes primers in 2017. PCR products of representative strains falling into different types and from different aresa were selected and sequenced by using agarose gel electrophoresis detection. The base and amino acid sequences were compared with those of the corresponding avirulent genes, and the rice resistant single gene line was used to verify the function of different strains of Magnaporthe oryzae.【Results】The frequency of AVR-Pita1 is 36.14%, and that of AVR-Pita3 is 59.41%. The target band of AVR-Pita2 could not be amplified from the DNA of 202 strains from Heilongjiang Province. Sequence analysis of some PCR products of AVR-Pita1 and AVR-Pita3 were carried out. Five variation types of AVR-Pita1 were detected; they were AVR-Pita1-1, AVR-Pita1-A, AVR-Pita1-B, AVR-Pita1-C and AVR-Pita1-D. Functional verification showed that AVR-Pita1-1, AVR-Pita1-A, AVR-Pita1-B and AVR-Pita1-D lost their function. No mutant strain was detected in the virulent gene AVR-Pita3. 【Conclusion】 AVR-Pita1 had strong mutation ability, which led to the loss of avirulent function of most strains, this should be used in conjunction with other resistant genes. AVR-Pita2 was not found in Heilongjiang Province. The sequence of AVR-Pita3 gene was stable in the strain.

Key words: Magnaporthe oryzae, AVR-Pita gene family, functional verification

中图分类号:

S435.111.4⁺1

孟峰, 张亚玲, 靳学慧. 黑龙江省稻瘟病菌无毒基因AVR-Pita及其同源基因的检测与分析[J]. 中国水稻科学, 2020, 34(2): 143-149.

Feng MENG, Yaling ZHANG, Xuehui JIN. Detection and Analysis of Magnaporthe oryzae Avirulent Gene AVR-Pita and Its Homologous Genes in Heilongjiang Province[J]. Chinese Journal OF Rice Science, 2020, 34(2): 143-149.

图/表 8

表1 用于扩增稻瘟病菌无毒基因AVR-Pita的引物

Table 1 Primers for Magnaporthe oryzae avirulent gene AVR-Pita amplification.

无毒基因 Avr-gene	前引物序列 Forward primer (5′-3′)	后引物 Reverse primer (5′-3′)	目的片段长度 Length of target fragment/bp
AVR-Pita1	TGCCAATAGACTAGCTTCCG	ATTCCCTCCATTCCAACACT	1957
AVR-Pita2	TTTCGGCCCAACTCCGGTCC	TAAAGGGTCCACTGACCCCG	1642
AVR-Pita3	AAATATTACCTGCCAGCTGG	CTAAACGAATCGACGCTCCC	1487

图1 稻瘟病菌供试菌株无毒基因AVR-Pita及其同源基因的PCR扩增 A-AVR-Pita1的部分扩增结果; B-AVR-Pita3的部分扩增结果。M1-分子标记DL2000;+：存在;-：缺失;1：低带;2：双带;3：三带。

Fig. 1. PCR detection of AVR-Pita and its homologous genes amplification to the tested strains. A, Amplification result of AVR-Pita1; B, Amplification result of AVR-Pita3; M1, Marker DL2000; +, Presence; -, Absence; 1, Low band; 2, Double bands; 3, Three bands.

表2 2017年黑龙江省稻瘟病菌无毒基因AVR-Pita1及其同源基因分布情况

Table 2 Distribution of AVR-Pita1 and its homologous genes of Magnaporthe oryzae in Heilongjiang Province in 2017.

地区Area	供试菌株数 Number of strains		无毒基因检出率 Detection rate of avirulence genes /％
地区Area	供试菌株数 Number of strains		AVR-Pita1	AVR-Pita2	AVR-Pita3
齐齐哈尔甘南 Gannan, Qiqihar	16	6.2		0	81.3
大庆杜蒙Dumeng, Daqing	19	0		0	73.7
大庆肇源 Zhaoyuan, Dayuan	11	0		0	100.0
绥化望奎 Wangkui, Suihua	7	42.9		0	28.6
绥化庆安 Anqing, Suihua	14	42.9		0	71.4
哈尔滨通河 Tonghe, Harbin	16	31.2		0	31.2
哈尔滨依兰Yilan, Harbin	23	52.2		0	52.2
哈尔滨方正Fangzheng, Harbin	4	0		0	50.0
哈尔滨尚志 Shangzhi, Harbin	10	0		0	80.0
佳木斯桦川 Huachuan, Jiamusi	10	90.0		0	80.0
佳木斯汤原 Tangyuan, Jiamusi	22	22.7		0	50.0
佳木斯桦南 Huanan, Jiamusi	15	86.7		0	53.3
鹤岗绥滨 Suibin, Hegang	16	68.7		0	62.5
鸡西虎林 Hulin, Jixi	9	33.3		0	44.4
鸡西密山 Mishan, Jixi	10	50.0		0	20.0
合计	202	36.1		0	59.4

图2 稻瘟病菌供试菌株AVR-Pita1-A、AVR-Pita1-B、AVR-Pita1-C和AVR-Pita1-D碱基序列比对

Fig. 2. Base sequence comparison of AVR-Pita1-A, AVR-Pita1-B, AVR-Pita1-C and AVR-Pita1-D of the tested strains.

图3 稻瘟病菌AVR-Pita1-A、AVR-Pita1-B和AVR-Pita1-C氨基酸序列对比分析

Fig. 3. Comparison and analysis of amino acid sequence of AVR-Pita1-A, AVR-Pita1-B and AVR-Pita1-C genotypes.

图4 稻瘟病菌AVR-Pita1-D 氨基酸序列对比分析

Fig. 4. Comparison and analysis of amino acid sequence of AVR-Pita1-D genotypes of Magnaporthe oryzae.

图5 稻瘟病菌AVR-Pita1变异类型比例

Fig. 5. Proportion of AVR-Pita1 variant types.

图6 稻瘟病菌供试菌株AVR-Pita1-1、AVR-Pita1-A、AVR-Pita1-B、AVR-Pita1-C和AVR-Pita1-D致病症状

Fig. 6. Symptoms after infected by the strains with AVR-Pita1-1, AVR-Pita1-A, AVR-Pita1-B, AVR-Pita1 -C and AVR-Pita1-D.

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