中国水稻科学2025，Vol.39Issue(3)：352-364,13.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
Cloning and Functional Verification of Rice-Blast Resistance Gene Pi-kf2(t)in Kangfeng B
摘要
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.关键词
水稻/稻瘟病/Pi2/Pi9/抗性基因/NBS-LRRKey words
rice/rice blast/Pi2/Pi9/resistance gene/NBS-LRR引用本文复制引用
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):352-364,13.基金项目
国家现代农业产业技术体系专项(CARS-01) （CARS-01）
福建省自然科学基金资助项目(2021J01535) （2021J01535）
三明市科技计划资助项目(2022-N-7). （2022-N-7）
