元江普通野生稻中抗白叶枯病基因鉴定与分析

doi:10.7668/hbnxb.20193594

华北农学报 ›› 2023, Vol. 38 ›› Issue (2): 199-205. doi: 10.7668/hbnxb.20193594

所属专题： 水稻；植物保护；生物技术；热点文章；

• 资源环境·植物保护 • 上一篇下一篇

元江普通野生稻中抗白叶枯病基因鉴定与分析

卢源达¹^,², 钟巧芳¹, 王波¹, 张敦宇¹, 殷富有¹, 王玲仙¹, 程在全¹, 陈玲¹

1.云南省农业科学院生物技术与种质资源研究所,云南省农业生物技术重点实验室, 农业部西南作物基因资源与种质创制重点实验室,云南昆明 650205
2.云南农业大学植物保护学院,云南昆明 650224

收稿日期:2022-11-26 出版日期:2023-04-28
通讯作者:
程在全(1964-),男,重庆南川人,研究员,博士,主要从事稻种资源种质创新和基因挖掘研究。
陈玲(1985-),女,云南曲靖人,助理研究员,硕士,主要从事水稻白叶枯病抗病基因的挖掘及功能研究。
作者简介:
卢源达(1997-),男,云南曲靖人,在读硕士,主要从事水稻白叶枯病抗病基因的挖掘及功能研究。
基金资助:
云南省颜龙安院士专家工作站(202005AF150032); 云南省基础研究专项面上项目(202001AT070015); 云南省基础研究专项面上项目(202001AT070003); 云南省科技人才和平台计划项目(2019HB034); 云南省科技人才和平台计划项目(202005AM070029); 云南省青年拔尖人才专项(YNWR-QNBJ-2018-284); 国家自然科学基金地区项目(31960374); 中央引导地方科技发展资金计划(202207AB110012); 云南种子种业联合实验室项目(202205AR070001)

Identification and Analysis of Resistance Genes to Bacterial Blight in Yuanjiang Common Wild Rice

LU Yuanda¹^,², ZHONG Qiaofang¹, WANG Bo¹, ZHANG Dunyu¹, YIN Fuyou¹, WANG Lingxian¹, CHENG Zaiquan¹, CHEN Ling¹

1. Biotechnology and Germplasm Resources Institute,Yunnan Academy of Agricultural Sciences, Yunnan Provincial Key Laboratory of Agricultural Biotechnology,Key Laboratory of Southwestern Crop Gene Resources and Germplasm Innovation,Ministry of Agriculture,Kunming 650205,China
2. College of Plant Protection,Yunnan Agricultural University,Kunming 650224,China

Received:2022-11-26 Published:2023-04-28

摘要/Abstract

摘要：

元江普通野生稻(YP)是一份被称为植物活化石的种质材料,同时其也拥有大量的抗性(R)基因。为了鉴定元江普通野生稻中含有目前已知白叶枯病抗性基因的情况。以元江普通野生稻(YP)、渗入系后代(L214和G252)及其渗入系的感病亲本栽培稻合系35(HX35)作为供试材料,通过接菌鉴定、功能标记检测、同源克隆和实时荧光定量(qRT-PCR)技术来评价这些材料对白叶枯病的抗性。接菌鉴定结果显示,YP、L214、G252对供试的白叶枯病菌C1、C2、C3、C4、C5、C6、C7、C9和PXO99^A均表现出抗性且病斑长度均小于2 cm;而HX35对供试的9个菌株均表现出感病且病斑长度远超6 cm。白叶枯病抗性基因检测结果显示,YP中含有Xa10的同源基因,HX35、L214、G252中含有xa23的感病同源基因。序列比对结果显示,抗病基因Xa10与YP中的Xa10启动子的效应因子结合元件(EBE_AvrXa10)区域序列差异较大。同样地,隐性感病基因xa23与HX35、L214和G252中的xa23启动子的效应因子结合元件(EBE_AvrXa23)区域序列一致。qRT-PCR结果显示,在接了PXO99^A菌株24 h后,YP、HX35、L214和G252中的免疫反应被激活,而YP中的Xa10和HX35、L214、G252中的xa23在所有处理时间段内均未被诱导表达。由此推测,YP、L214和G252中可能含有新的抗白叶枯病基因。

关键词: 元江普通野生稻, 渗入系, 白叶枯病, 抗性基因

Abstract:

Yuanjiang common wild rice(YP)is a germplasm material known as a living fossil in plants,which also has a large number of resistance(R)genes.Therefore,knowing the R genes in YP will help to apply different R genes to cultivated rice.Here,we identified the bacterial blight(BB)R genes in Yuanjiang common wild rice(YP),progeny of introgression lines(L214 and G252)and its susceptible parent,cultivated rice line 35(HX35).Meanwhile,the resistance of YP and its introgression lines to several Xanthomonas oryzae pv.oryzae (Xoo)strains was determined at the late tillering stage of rice.Under indoor control conditions,YP and its introgression lines showed resistance to 9 different Xoo strains(C1,C2,C3,C4,C5,C6,C7,C9 and PXO99^A),indicating that they had broad-spectrum resistance to BB.The results of bacterial blight resistance gene detection showed that YP contained homologous genes of Xa10,while HX35,L214 and G252 contained susceptible homologous genes of xa23.The sequence alignment results showed that the sequence of the effector binding element (EBE_AvrXa10) region of the disease resistance gene Xa10 was significantly different from that of the Xa10 promoter in YP. Similarly, the recessive susceptible gene xa23 was consistent with the effector binding element (EBE_AvrXa23) region of the xa23 promoter in HX35, L214 and G252. The qRT-PCR results showed that the immune responses in YP,HX35,L214 and G252 were activated after inoculation with strain PXO99^A,but Xa10 in YP and xa23 in HX35,L214 and G252 were not induced to be expressed.It is thus hypothesized that YP,L214 and G252 may contain new genes for resistance to bacterial blight.

Key words: Yuanjiang common wild rice, Infiltration line, Bacterial blight, Resistance gene

卢源达, 钟巧芳, 王波, 张敦宇, 殷富有, 王玲仙, 程在全, 陈玲. 元江普通野生稻中抗白叶枯病基因鉴定与分析[J]. 华北农学报, 2023, 38(2): 199-205. doi: 10.7668/hbnxb.20193594.

LU Yuanda, ZHONG Qiaofang, WANG Bo, ZHANG Dunyu, YIN Fuyou, WANG Lingxian, CHENG Zaiquan, CHEN Ling. Identification and Analysis of Resistance Genes to Bacterial Blight in Yuanjiang Common Wild Rice[J]. Acta Agriculturae Boreali-Sinica, 2023, 38(2): 199-205. doi: 10.7668/hbnxb.20193594.

http://www.hbnxb.net/CN/Y2023/V38/I2/199

图/表 9

表1 9个Xoo菌株来源信息

Tab.1 9 Xoo strains information sources

菌株名称 Strain name	类型 Type
C1	中国1号代表菌株
C2	中国2号代表菌株
C3	中国3号代表菌株
C4	中国4号代表菌株
C5	中国5号代表菌株
C6	中国6号代表菌株
C7	中国7号代表菌株
C9	中国9号代表菌株
PXO99^A	菲律宾6号代表菌株

表2 本试验用到的引物

Tab.2 Primers used in this experiment

编号 Code	引物序列(5'-3') Primer sequence (5'-3')	目的 Purpose
Xa7-kelong-F1	TCTACCGGGGTTCAGT	目标片段扩增
Xa7-kelong-R1	GACCCTTCGTATGGAATTGA
Xa10-kelong-F1	CACCAAAAGGTCCAACAC
Xa10-kelong-R1	GCCGGTTTCTCTTTATTAAC
Xa23-kelong-F1	AATTATGCGGCATCACTAAC
Xa23-kelong-R1	TAACAATCAAACCGAGGAGA
Actin-F	CAGGCCGTCCTCTCTCTGTA	实时荧光定量PCR
Actin-R	AAGGATAGCATGGGGGAGAG
OsPR1a-F	TTCATCACCTGCAACTACTCG
OsPR1a-R	TGCATAAACACGTAGCATAGCAT
OsPR10-F	CACCATCTACACCATGAAGC
OsPR10-R	AGCACATCCGACTTTAGGAC
qRT-PCR-Xa10-F	TCTTCTACCTCTCTGCCGCT
qRT-PCR-Xa10-R	CGGCAGCTATACGGGCATAA
qRT-PCR-Xa23-F	CTGTTTGGCTCCATCATGCC
qRT-PCR-Xa23-R	AAGATGAAGGCGTAGGCGAG

图1 接种不同Xoo生理小种的表型

Fig.1 Phenotypes of different Xoo races inoculated

表3 抗白叶枯病基因的等位标记/同源片段情况

Tab.3 The presence of allelic markers/fragments of BB resistance genes

基因 Gene	元江普通野生稻(♂) YP	合系35 (♀) HX35	L214 (IL) L214	G252 (IL) G252
Xa1	-	-	-	-
Xa3/Xa26	-	-	-	-
Xa4	-	-	-	-
xa5	-	-	-	-
Xa7	+-	-	-	+-
Xa10	++	-	-	-
xa13	-	-	-	-
Xa21	-	-	-	-
Xa23	-	++	-	+-
Xa27	-	-	-	-

图2 白叶枯病抗性基因的PCR鉴定 O.rufipogon.海南文昌普通野生稻。

Fig.2 Identification of resistance genes to bacterial blight by PCR O.rufipogon.Hainan Wenchang common wild rice.

图3 Xa10 (YP)与Xa10 (IRBB10)的序列比对红色框区域的核苷酸序列分别代表Xa10基因启动子的EBE_AvrXa10位点序列。

Fig.3 Sequence alignment of Xa10 (YP)and Xa10 (IRBB10) The nucleotide sequences in the red box region represent the EBE_AvrXa10 locus sequence of the Xa10 gene promoter,respectively.

图4 Xa23同源基因序列比对红色框区域的核苷酸序列分别代表Xa23基因启动子的EBE_AvrXa23位点序列。

Fig.4 Represents the sequence alignment of Xa23 homologous allele The nucleotide sequences in the red box region represent the EBE_AvrXa23 locus sequence of the Xa23 gene promoter,respectively.

图5 XA23蛋白序列比对红色背景代表氨基酸序列的差异。

Fig.5 XA23 protein sequence alignment Red background represents the difference of amino acid sequence.

图6 PXO99^A 处理后Xa10和Xa23的表达分析内参基因Actin1;不同小写字母表示差异显著(P<0.05)。

Fig.6 Expression analysis of Xa10 and Xa23 under PXO99^A treatment The internal reference gene Actin1;Different lowercase letters indicate significant differences(P<0.05).

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元江普通野生稻中抗白叶枯病基因鉴定与分析

Identification and Analysis of Resistance Genes to Bacterial Blight in Yuanjiang Common Wild Rice

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