水稻康丰B抗稻瘟病基因Pi-kf2(t)的克隆与功能验证

doi:10.16819/j.1001-7216.2025.240502

中国水稻科学 ›› 2025, Vol. 39 ›› Issue (3): 352-364.DOI: 10.16819/j.1001-7216.2025.240502

水稻康丰B抗稻瘟病基因Pi-kf2(t)的克隆与功能验证

韦新宇¹^,²^,^#, 曾跃辉¹^,²^,^#, 肖长春¹^,², 黄建鸿¹^,², 阮宏椿⁴, 杨旺兴²^,³, 邹文广²^,³, 许旭明²^,³^,^*()

¹三明市农业科学研究院生物技术研究所, 福建三明 365500
²福建省(山区)作物遗传改良与创新利用重点实验室, 福建三明 365500
³三明市农业科学研究院水稻研究所, 福建三明 365500
⁴福建省农业科学院植物保护研究所, 福州 350013

收稿日期:2024-04-30 修回日期:2024-07-23 出版日期:2025-05-10 发布日期:2025-05-21
通讯作者: *email: fj63xxm@sina.com
作者简介:^#共同第一作者
基金资助:
国家现代农业产业技术体系专项(CARS-01);福建省自然科学基金资助项目(2021J01535);三明市科技计划资助项目(2022-N-7)

Cloning and Functional Verification of Rice-Blast Resistance Gene Pi-kf2(t) in Kangfeng B

WEI Xinyu¹^,²^,^#, ZENG Yuehui¹^,²^,^#, XIAO Changchun¹^,², HUANG Jianhong¹^,², RUAN Hongchun⁴, YANG Wangxing²^,³, ZOU Wenguang²^,³, XU Xuming²^,³^,^*()

¹Biotechnology Research Institute, Sanming Academy of Agricultural Sciences, Sanming 365500, China
²Fujian Key Laboratory of Crop Genetic Improvement and Innovative Utilization for Mountain Area, Sanming 365500, China
³Rice Research Institute, Sanming Academy of Agricultural Sciences, Sanming 365500, China
⁴Plant Protection Institute, Fujian Academy of Agricultural Sciences, Fuzhou 350013, China

Received:2024-04-30 Revised:2024-07-23 Online:2025-05-10 Published:2025-05-21
Contact: *email: fj63xxm@sina.com
About author:^#These authors contributed equally to this work

1. weixinyu table S1.doc(35KB)

摘要/Abstract

摘要：

【目的】稻瘟病是一种由丝状子囊菌稻瘟病菌(Magnaporthe oryzae)引起的世界性真菌病害，严重影响水稻的品质和产量，培育和种植抗性品种是控制稻瘟病发生最经济有效的措施，而抗性基因的挖掘和利用是选育抗病品种的基础和关键。本研究以三明市农业科学研究院选育且具有广谱抗性的优质籼型杂交稻保持系康丰B (KFB)为研究材料，对其抗稻瘟病基因Pi-kf2(t)进行抗谱分析、克隆和功能验证。【方法】利用来自全国不同地区的100个稻瘟病菌株对KFB[Pi-kf2(t)]、75-1-127 (Pi9)、C101A51 (Pi2)、IRBLzt-T (Piz-t)、EBZ (Pi50)和GM 4 (Pigm) 3~4叶期幼苗进行喷雾接菌；结合半定量PCR (RT-PCR)和实时荧光定量PCR (qRT-PCR)技术对Pi-kf2(t)两个候选基因Pi-kf2(t)-NBS2和Pi-kf2(t)-NBS4进行表达模式分析；采用同源克隆技术并构建系统进化树对Pi-kf2(t)基因进行克隆以及分析Pi-kf2(t)与Pi9、Pi2、Piz-t、Pi50和Pigm之间的进化关系；通过转基因互补试验对Pi-kf2(t)候选基因进行功能验证。【结果】Pi-kf2(t)与Pi9、Pi2、Piz-t、Pi50和Pigm存在明显的抗谱差异，其抗谱分别为93%、90%、91%、78%、95%和96%；候选基因Pi-kf2(t)-NBS2在KFB苗期叶片具有较高的表达水平且不受稻瘟病菌诱导，属于组成型表达，而Pi-kf2(t)-NBS4表达量极低；多重氨基酸序列比对分析表明，Pi-kf2(t)-NBS2与Pi9、Pi2、Piz-t、Pi50和Pigm蛋白分别存在45、31、34、2和2个氨基酸的差异；系统进化树分析表明，Pi-kf2(t)-NBS2与Pi50和Pigm具有更近的亲缘关系和更高的同源性；另外，转基因互补试验进一步验证了候选基因Pi-kf2(t)-NBS2即为Pi-kf2(t)抗稻瘟病基因。【结论】Pi-kf2(t)为Pi2/Pi9基因家族中一个新的稻瘟病抗性基因，本研究为Pi-kf2(t)后期抗病机理研究提供了理论基础，同时为选育抗稻瘟病水稻新品种提供了重要种质资源。

关键词: 水稻, 稻瘟病, Pi2/Pi9, 抗性基因, NBS-LRR

Abstract:

【Objective】Rice blast is a worldwide disease caused by filamentous ascomycete fungus Magnaporthe oryzae, which seriously affects the quality and yield of rice. At present, cultivating and planting disease-resistant varieties is the most economical and effective strategy to control rice blast, and the mining and utilization of blast-resistance genes is the key to disease-resistant rice breeding. In this study, Kangfeng B (KFB), an elite maintainer line of indica hybrid rice with broad-spectrum resistance derived from Sanming Academy of Agricultural Sciences was used as the test material, and the Pi-kf2(t) gene in which was used to conduct resistance spectrum analysis, cloning and functional verification.【Method】For resistance spectrum analysis, KFB (Pi-kf2(t)), 75-1-127 (Pi9), C101A51 (Pi2), IRBLzt-T (Piz-t), EBZ (Pi50) and GM-4 (Pigm) seedlings were inoculated with 100 rice blast isolates from different regions of China during 3-4 leaf stages; The expression analysis of two Pi-kf2(t) candidate genes, Pi-kf2(t)-NBS2 and Pi-kf2(t)-NBS4, were determined by semi-quantitative reverse-transcription PCR (RT-PCR) and quantitative real-time PCR (qRT-PCR); Pi-kf2(t) gene was cloned by homologous cloning strategy and a phylogenetic tree was constructed to investigate the possible relationships between Pi-kf2(t) and Pi9, Pi2, Piz-t, Pi50, and Pigm proteins in evolutionary history. Moreover, sequence and expression analyses revealed Pi-kf2(t)-NBS2 as the candidate Pi-kf2(t) gene, which was verified by transgenic complementation test.【Result】The resistance spectrum of Pi-kf2(t), Pi9, Pi2, Piz-t, Pi50 and Pigm were 93%, 90%, 91%, 78%, 95% and 96%, respectively. The candidate gene Pi-kf2(t)-NBS2 exhibited a high expression level in the leaves of KFB seedlings and was not induced by isolates of rice blast, which showed a constitutive expression, while Pi-kf2(t)-NBS4 exhibited an extremely poor expression. Multiple amino acid sequence alignment analysis revealed that there were 45, 31, 34, 2, and 2 amino acids differences between Pi-kf2(t)-NBS2 and Pi9, Pi2, Piz-t, Pi50, and Pigm proteins, respectively. Phylogenetic tree analysis showed that Pi-kf2(t)-NBS2 exhibited a closer genetic relationship and a higher homology with Pi50 and Pigm. Furthermore, genetic complementation test verified that the candidate gene Pi-kf2(t)-NBS2 was the Pi-kf2(t).【Conclusion】The rice blast-resistance gene Pi-kf2(t) is a novel member of the Pi2/Pi9 multigene family. This work provided a theoretical basis for further disease resistance mechanism study of Pi-kf2(t), and also provided an important germplasm resource for rice blast-resistance breeding.

Key words: rice, rice blast, Pi2/Pi9, resistance gene, NBS-LRR

韦新宇, 曾跃辉, 肖长春, 黄建鸿, 阮宏椿, 杨旺兴, 邹文广, 许旭明. 水稻康丰B抗稻瘟病基因Pi-kf2(t)的克隆与功能验证[J]. 中国水稻科学, 2025, 39(3): 352-364.

WEI Xinyu, ZENG Yuehui, XIAO Changchun, HUANG Jianhong, RUAN Hongchun, YANG Wangxing, ZOU Wenguang, XU Xuming. Cloning and Functional Verification of Rice-Blast Resistance Gene Pi-kf2(t) in Kangfeng B[J]. Chinese Journal OF Rice Science, 2025, 39(3): 352-364.

图/表 9

表1 来自全国不同地区的100个稻瘟病菌株

Table 1. 100 Magnaporthe oryzae isolates originated from different regions of China

编号Number	菌种名称Isolate	来源 Origin	编号Number	菌种名称Isolate	来源 Origin	编号Number	菌种名称Isolate	来源 Origin	编号Number	菌种名称 Isolate	来源 Origin
J01	2022075	福建福州	J26	2021146	广东惠州	J51	2021137	福建沙县	J76	2022027	福建武夷山
J02	2021024	福建武夷山	J27	2021021	广东惠州	J52	2022026	福建武夷山	J77	2021033	江西婺源
J03	2022048	湖北恩施	J28	2021119	福建连城	J53	2021048	福建将乐	J78	2022037	福建浦城
J04	2022036	湖北恩施	J29	2021013	福建尤溪	J54	2021018	江西婺源	J79	2021036	福建宁化
J05	2022024	福建建阳	J30	2021121	福建连城	J55	2021032	江西婺源	J80	2023020	湖北恩施
J06	2023090	福建柘荣	J31	2020117	安徽肥西	J56	2023112	福建宁化	J81	2022006	福建上杭
J07	2021017	江苏扬州	J32	2022072	安徽肥西	J57	2022005	福建上杭	J82	2023019	江苏扬州
J08	2020062	江苏扬州	J33	2021001	福建福州	J58	2020114	安徽肥西	J83	2020156	江苏扬州
J09	2021085	福建上杭	J34	2023104	江西婺源	J59	2020059	安徽肥西	J84	2022044	湖北宜昌
J11	2020010	福建宁化	J36	2021078	湖北宜昌	J61	2023131	福建将乐	J86	2022063	福建莆田
J12	2021068	福建莆田	J37	2022065	湖北恩施	J62	2020107	福建松溪	J87	2021031	福建松溪
J13	2021092	福建福安	J38	2022021	福建建阳	163	2023034	江苏扬州	J88	2020017	江西井冈山
J14	2022078	福建漳州	J39	2021037	福建宁化	J64	2022032	福建浦城	J89	2021016	江西井冈山
J15	2021122	福建连城	J40	2023018	江西婺源	J65	2020061	福建将乐	J90	2023035	江苏扬州
J16	2022074	福建福州	J41	2022049	福建福安	J66	2020041	福建上杭	J91	2021084	福建上杭
J17	2021034	江西井冈山	J42	2021138	福建沙县	J67	2022045	湖北宜昌	J92	2022085	福建南靖
J18	2022008	江西井冈山	J43	2021049	福建将乐	J68	2022081	福建漳州	J93	2021081	福建三明
J19	2021080	福建三明	J44	2021027	福建武夷山	J69	2023029	江苏徐州	J94	2022007	福建将乐
J20	2021040	福建将乐	J45	2021035	江西井冈山	J70	2023004	福建福州	J95	2023037	江西井冈山
J21	2021009	福建沙县	J46	2021020	江苏扬州	J71	2021141	福建沙县	J96	2020143	福建浦城
J22	2022046	湖北恩施	J47	2022015	江苏扬州	J72	2023003	江苏徐州	J97	2020105	福建松溪
J23	2022011	湖北宜昌	J48	2021014	福建尤溪	J73	2023031	江苏徐州	J98	2020039	福建上杭
J24	2023014	福建三明	J49	2023041	福建建阳	J74	2021149	广东惠州	J99	2022004	福建上杭
J25	2022018	福建三明	J50	2021072	福建莆田	J75	2021005	广东惠州	J100	2023042	江苏徐州

表1 来自全国不同地区的100个稻瘟病菌株

Table 1. 100 Magnaporthe oryzae isolates originated from different regions of China

编号Number	菌种名称Isolate	来源 Origin	编号Number	菌种名称Isolate	来源 Origin	编号Number	菌种名称Isolate	来源 Origin	编号Number	菌种名称 Isolate	来源 Origin
J01	2022075	福建福州	J26	2021146	广东惠州	J51	2021137	福建沙县	J76	2022027	福建武夷山
J02	2021024	福建武夷山	J27	2021021	广东惠州	J52	2022026	福建武夷山	J77	2021033	江西婺源
J03	2022048	湖北恩施	J28	2021119	福建连城	J53	2021048	福建将乐	J78	2022037	福建浦城
J04	2022036	湖北恩施	J29	2021013	福建尤溪	J54	2021018	江西婺源	J79	2021036	福建宁化
J05	2022024	福建建阳	J30	2021121	福建连城	J55	2021032	江西婺源	J80	2023020	湖北恩施
J06	2023090	福建柘荣	J31	2020117	安徽肥西	J56	2023112	福建宁化	J81	2022006	福建上杭
J07	2021017	江苏扬州	J32	2022072	安徽肥西	J57	2022005	福建上杭	J82	2023019	江苏扬州
J08	2020062	江苏扬州	J33	2021001	福建福州	J58	2020114	安徽肥西	J83	2020156	江苏扬州
J09	2021085	福建上杭	J34	2023104	江西婺源	J59	2020059	安徽肥西	J84	2022044	湖北宜昌
J11	2020010	福建宁化	J36	2021078	湖北宜昌	J61	2023131	福建将乐	J86	2022063	福建莆田
J12	2021068	福建莆田	J37	2022065	湖北恩施	J62	2020107	福建松溪	J87	2021031	福建松溪
J13	2021092	福建福安	J38	2022021	福建建阳	163	2023034	江苏扬州	J88	2020017	江西井冈山
J14	2022078	福建漳州	J39	2021037	福建宁化	J64	2022032	福建浦城	J89	2021016	江西井冈山
J15	2021122	福建连城	J40	2023018	江西婺源	J65	2020061	福建将乐	J90	2023035	江苏扬州
J16	2022074	福建福州	J41	2022049	福建福安	J66	2020041	福建上杭	J91	2021084	福建上杭
J17	2021034	江西井冈山	J42	2021138	福建沙县	J67	2022045	湖北宜昌	J92	2022085	福建南靖
J18	2022008	江西井冈山	J43	2021049	福建将乐	J68	2022081	福建漳州	J93	2021081	福建三明
J19	2021080	福建三明	J44	2021027	福建武夷山	J69	2023029	江苏徐州	J94	2022007	福建将乐
J20	2021040	福建将乐	J45	2021035	江西井冈山	J70	2023004	福建福州	J95	2023037	江西井冈山
J21	2021009	福建沙县	J46	2021020	江苏扬州	J71	2021141	福建沙县	J96	2020143	福建浦城
J22	2022046	湖北恩施	J47	2022015	江苏扬州	J72	2023003	江苏徐州	J97	2020105	福建松溪
J23	2022011	湖北宜昌	J48	2021014	福建尤溪	J73	2023031	江苏徐州	J98	2020039	福建上杭
J24	2023014	福建三明	J49	2023041	福建建阳	J74	2021149	广东惠州	J99	2022004	福建上杭
J25	2022018	福建三明	J50	2021072	福建莆田	J75	2021005	广东惠州	J100	2023042	江苏徐州

图1 康丰B（KFB）抗稻瘟病基因Pi-kf2(t)的抗谱分析 A: KFB、75-1-127、C101A51、IRBLzt-T、EBZ和GM-4对100个稻瘟病菌株的抗性反应; 其中, J01-J100代表来自全国不同地区100个稻瘟病菌株, R%代表抗谱, R、MR、MS、S和HS分别表示为抗、中抗、中感、感和高感稻瘟病; B, C, D: KFB、75-1-127、C101A51、IRBLzt-T、EBZ和GM-4对稻瘟病菌株J71 (B)、J91 (C)和J93 (D)的抗性反应。1: KFB; 2: 75-1-127; 3: C101A51; 4: IRBLzt-T; 5: EBZ; 6: GM-4。

Fig. 1. Resistance spectrum analysis of rice blast resistance gene Pi-kf2(t) in KFB A, Reaction of KFB, 75-1-127, C101A51, IRBLzt-T, EBZ, and GM-4 to 100 isolates of Magnaporthe oryzae. J01-J100, The 100 isolates were collected from different regions of China. R%, Resistance spectrum. R, MR, MS, S, and HS represent resistance, mid-resistance, mid-susceptibility, susceptibility, and high-susceptibility to rice blast, respectively; B, C, D, Reaction of KFB, 75-1-127, C101A51, IRBLzt-T, EBZ, and GM-4 to J71(B), J91(C), and J93(D), where 1, 2, 3, 4, 5, and 6 represent KFB, 75-1-127, C101A51, IRBLzt-T, EBZ, and GM-4, respectively.

表2 本研究所使用的引物序列

Table 2. Sequences of primers used in this study

引物名称 Primer name	引物序列 Primer sequence (5'-3')	用途 Usage	预测PCR产物大小 Excepted PCR product size(bp)
NBS2-Q-F	CTGCTAATGTACAGTGACCGA	NBS-LRR2基因qPCR检测	111
NBS2-Q-R	AGTGACTGATCCTTCGGCAT	qPCR detection for NBS-LRR2
NBS4-Q-F	TGATCACGACCTTGCTTGTG	NBS-LRR4基因qPCR检测	122
NBS4-Q-R	CTGACCTGTTGTTCGACGTC	qPCR detection for NBS-LRR4
HPT-F	ATTTGTGTACGCCCGACAGT	鉴定HPT	577
HPT-R	GTGCTTGACATTGGGGAGTT	PCR detection for HPT
NBS2Seq-01F	CCCAAGTGAGGATGCAAAACA	NBS-LRR2基因测序	803
NBS2Seq-01R	TGACATGCAAGAAACGGGTG	Sequencing for NBS-LRR2
NBS2Seq-02F	TGGCTAGGCTTACGTTTTGG	NBS-LRR2基因测序	2219
NBS2Seq-02R	ACAACGAAGAGTATGCAGACA	Sequencing for NBS-LRR2
NBS2Seq-03F	AGGATGTTACGGGTCTTGGA	NBS-LRR2基因测序	2107
NBS2Seq-03R	AACAACTGCAGGCCAAACAA	Sequencing for NBS-LRR2
OsActin-F	ACCTTCAACACCCCTGCTAT	内参基因OsActin qPCR检测	105
OsActin-R	CACCATCACCAGAGTCCAAC	qPCR detection for OsActin (as internal standard)

图2 Pi-kf2(t)候选基因的表达模式分析 A、C、E: 通过RT-PCR分析康丰B苗期叶片在接种稻瘟病菌株J71(A)、J91(C)和J93(E)后0、6、12、24、48和96 h候选基因Pi-kf2(t)-NBS2和Pi-kf2(t)-NBS4的表达模式；B、D、F: 通过qRT-PCR分析KFB苗期叶片在接种稻瘟病菌株J71（B）、J91（D）和J93（F）后0、6、12、24、48和96 h候选基因Pi-kf2(t)-NBS2的表达模式；G: 通过RT-PCR分析KFB苗期叶片在模拟接菌后0、6、12、24、48和96 h候选基因Pi-kf2(t)-NBS2和Pi-kf2(t)-NBS4的表达模式；H: 通过qRT-PCR分析KFB苗期叶片在模拟接菌后0、6、12、24、48和96 h候选基因Pi-kf2(t)-NBS2的表达模式。

Fig. 2. Expression pattern analysis of Pi-kf2(t) candidate gene A, C, E, Expression patterns of candidate genes Pi-kf2(t)-NBS2 and Pi-kf2(t)-NBS4 in KFB seedling leaves at 0, 6, 12, 24, 48, and 96 h after inoculation with the isolates J71(A), J91(C), and J93(E), determined by RT-PCR; B, D, F, Expression patterns of candidate genes Pi-kf2(t)-NBS2 in KFB seedling leaves at 0, 6, 12, 24, 48, and 96 h after inoculation with the isolates J71(B), J91(D), and J93(F), determined by qRT-PCR；G, Expression patterns of candidate genes Pi-kf2(t)-NBS2 and Pi-kf2(t)-NBS4 in KFB seedling leaves at 0, 6, 12, 24, 48, and 96 h after mock inoculation, determined by RT-PCR; H, The expression patterns of candidate genes Pi-kf2(t)-NBS2 in KFB seedling leaves at 0, 6, 12, 24, 48, and 96 h after mock inoculation, determined by qRT-PCR.

图3 Pi-kf2(t)-NBS2与Pi9、Pi2、Piz-t/Pizh、Pi50和Pigm氨基酸序列比对分析深蓝色突出显示的是相同氨基酸，粉红色和淡蓝色突出显示的是相似氨基酸，红色矩形表示LRR区域富含亮氨酸重复（xxLxLxx）基序。

Fig. 3. Amino-acid sequences alignment of Pi-kf2(t)-NBS2 with Pi9, Pi2, Piz-t/Pizh, Pi50, and Pigm Dark blue highlights identical residues, while similar residues are marked in pink or light blue, and red rectangles indicate the xxLxLxx motifs of LRR domain.

图4 Pi-kf2(t)-NBS2与Pi9-type1~Pi9-type13氨基酸序列比对分析深蓝色突出显示的是相同氨基酸，粉红色和淡蓝色突出显示的是相似氨基酸，红色矩形表示LRR区域富含亮氨酸重复（xxLxLxx）基序。

Fig. 4. Amino-acid sequences alignment of Pi-kf2(t)-NBS2 with Pi9-type1－Pi9-type13 Dark blue highlights identical residues, while similar residues are marked in pink or light blue, and red rectangles indicate the xxLxLxx motifs of LRR domain.

图5 Pi-kf2(t)-NBS2与Pi9、Pi2、Piz-t/Pizh、Pi50、Pigm和Pi9-type1~Pi9-type13蛋白序列系统进化树分析

Fig. 5. A phylogenetic tree representing the alignment of Pi-kf2(t)-NBS2 and protein sequences of Pi9, Pi2, Piz-t/Pizh, Pi50, Pigm, and Pi9-type1－Pi9-type13

图6 候选基因Pi-kf2(t)-NBS2的功能验证 A: 利用潮霉素抗性标记引物HPT-F/R鉴定T0代阳性转基因单株，其中“+”和“−”分别表示阳性对照和阴性对照；B: qRT-PCR分析过表达转基因株系中候选基因Pi-kf2(t)-NBS2的表达水平，其中“**”代表在0.01水平上的极显著差异，误差线表示标准差（SD）；C: 过表达转基因株系T1代单株接种稻瘟病菌株（J71，J91和J93）的抗性反应，其中“R”和“S”分别代表抗病和感病，Pi-kf2(t)-NBS2-OE2#、Pi-kf2(t)-NBS2-OE5#和Pi-kf2(t)-NBS2-OE7#株系分别接种稻瘟病菌株J71、J91和J93；D: RT-PCR分析T1代转基因株系单株中候选基因Pi-kf2(t)-NBS2的表达。

Fig. 6. Functional verification of candidate gene Pi-kf2(t)-NBS2 A, PCR identification of the positive transgenic plants in the overexpressed lines of T0 generation with HPT-F/R, where “+” and “−” indicate the positive control and negative control, respectively; B, Expression levels of Pi-kf2(t)-NBS2 candidate gene in the overexpressed transgenic lines, determined by qRT-PCR, where “**” indicates significant differences at 0.01 level and error bars represent the standard deviation (SD); C, Reaction of T1 transgenic lines inoculated with the isolates J71, J91, and J93, where “R” and “S” represent resistance and susceptibility to rice blast, respectively. Pi-kf2(t)-NBS2-OE2#, Pi-kf2(t)-NBS2-OE5#, and Pi-kf2(t)-NBS2-OE7# were inoculated with isolates J71, J91, and J81, respectively; D, Expression analysis of Pi-kf2(t)-NBS2 candidate gene in the individual plant of T1 transgenic lines, determined by RT-PCR.

表3 过表达转基因株系T1代植株抗感分离

Table 3 The segregation of resistant and susceptible individuals in the T1 population of three overexpressed transgenic lines

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水稻康丰B抗稻瘟病基因Pi-kf2(t)的克隆与功能验证

Cloning and Functional Verification of Rice-Blast Resistance Gene Pi-kf2(t) in Kangfeng B

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