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摘要 本文旨在探討核苷酸结合位点和富含亮氨酸重复（NBS-LRR）类基因参与甘蔗抗梢腐病菌侵染应答机制，为后续克隆关键抗病基因以及研究抗病机理提供理论依据。试验利用Illumina高通量转录组测序技术检测高抗梢腐病品种‘粤糖94-128’和高感梢腐病品种‘桂糖37号’接种前后NBS-LRR类抗梢腐病基因的表达情况，然后设计引物对显著差异表达基因进行不同接种时期下的荧光定量PCR验证。结果表明，16个NBS-LRR类基因在甘蔗叶片受到梢腐病菌侵染后持续上调表达，但表达趋势存在两种情况，13个基因在诱导后7 d显著或极显著上调表达，3个基因在诱导7 d后上调表达不显著，在诱导14 d后才显著上调表达。据此可将其分为瞬时基础防御基因（0～7 d）和滞后特异性防御基因（14～21 d），确定NBS-LRR类基因参与甘蔗防御梢腐病菌的侵染。
关键词 甘蔗; 梢腐病; 转录组; NBS-LRR
中图分类号： S 556.1， S 435.661
文献标识码： A
DOI： 10.16688/j.zwbh.
2020301
Screening and quantitative analysis of NBS-LRR gene against pokkah boeng disease in sugarcane
WANG Zeping1， SONG Xiupeng1， YAN Meixin1， LI Yijie1， LI Xiang1，ZHANG Xiaoqiu1， TANG Hongqin2*， LONG Shengfeng3*
（1. Sugarcane Research Institute， Guangxi Academy of Agricultural Sciences， Sugarcane Research Center，
Chinese Academy of Agricultural Science， Key Laboratory of Sugarcane Biotechnology and Genetic
Improvement （Guangxi）， Ministry of Agriculture and Rural Affairs， Nanning 530007， China;
2. Institute of Agricultural Resources and Environment， Guangxi Academy of Agricultural Sciences，
Nanning 530007， China; 3. Guangxi Academy of Agricultural Sciences， Nanning 530007， China）
Abstract
The molecular mechanism of nucleotide binding sites and leucine-rich replication （NBS-LRR） genes resistant to pokkah boeng disease （PBD） in sugarcane was explored to provide a theoretical basis for cloning resistant genes. This study firstly used Illumina high-throughput transcriptome sequencing technology to detect NBS-LRR genes against PBD with typical resistant and susceptive sugarcane materials， and then designed primers to verify the expression characteristics of significant differentially expressed NBS-LRR genes under different inoculation periods using real-time quantitative PCR （qRT-PCR）. The results showed that 16 NBS-LRR-like genes steadily increased expression when sugarcane leaves were infected with the PBD pathogen， including two trends in expression： 13 genes significantly or extremely significantly increased expression at 7 d post-inoculation， but three genes did not significantly increased expression at 7 d post-inoculation， but significantly increased expression at 14 d post-inoculation. Therefore， these genes could be divided into instantaneous basic defense genes （0 d to 7 d） and specific lagging defense genes （14 d to 21 d）， suggesting that NBS-LRR genes are actively involved in resistance against the PBD pathogen. 综上所述，本文在获得差异基因表达谱的基础上，根据已知植物抗病基因保守区设计简并引物，成功验证了NBS-LRR表达规律，可为最终克隆获得甘蔗抗梢腐病基因奠定基础。
参考文献
[1] 王泽平， 陈奕， 孙海军， 等. 广西甘蔗主栽品种梢腐病田间抗性初步评价[J]. 热带作物学报， 2016， 37（5）： 952-957.
[2] 兰冬雪，汤丽影，李佳，等. 禾本科植物NBS-LRR类抗病基因结构、功能和进化研究进展[J].中国农学通报，2019， 35（15）：124-127.
[3] ROSSI M， ARAUJO P G， PAULET F， et al. Genomic distribution and characterization of EST-derived resistance gene analogs （RGAs） in sugarcane [J]. Molecular Genetics and Genomics， 2003， 269（3）： 406-419.
[4] MCINTYRE C L， CASU R E， DRENTH J， et al. Resistance gene analogues in sugarcane and sorghum and their association with quantitative trait loci for rust resistance [J]. Genome， 2005， 48（3）： 391-400.
[5] WANDERLEY-NOGUEIRA A C， SOARES-CAVALCANTI N M， MORAIS D A L， et al. Abundance and diversity of resistance genes in the sugarcane transcriptome revealed by in silico analysis [J]. Genetics and Molecular Research， 2007， 6（4）： 866-889.
[6] 阙友雄， 许莉萍， 张木清， 等.甘蔗中一个NBS-LRR类基因的全长克隆与表达分析[J]. 作物学报， 2009， 35（6）： 1161-1166.
[7] 贺尔奇， 李向勇， 彭仕荣， 等. 果蔗NBS类抗病基因同源序列的克隆与分析[J]. 分子植物育种， 2016， 14（2）： 352-358.
[8] KRASILEVA K V， DAHLBECK D， STASKAWICZ B. Activation of an Arabidopsis resistance protein is specified by the in planta association of its leucine-rich repeat domain with the cognate oomycete effector [J]. The Plant Cell， 2010， 22（7）： 2444-2458.
[9] 王泽平， 段维兴， 李毅杰， 等. 甘蔗梢腐病田间抗性评价体系的建立[J]. 华南农业大学学报， 2016， 37（3）： 67-72.
[10]王泽平， 林善海， 梁强， 等. 甘蔗叶冠形态与抗梢腐病相关性探讨[J]. 中国农业大学学报， 2017， 22（7）： 40-46.
[11]WANG Zeping， SUN Haijun， GUO Qiang， et al. Artificial inoculation method of pokkah boeng disease of sugarcane and screening of resistant germplasm resources in subtropical China
关键词 甘蔗; 梢腐病; 转录组; NBS-LRR
中图分类号： S 556.1， S 435.661
文献标识码： A
DOI： 10.16688/j.zwbh.
2020301
Screening and quantitative analysis of NBS-LRR gene against pokkah boeng disease in sugarcane
WANG Zeping1， SONG Xiupeng1， YAN Meixin1， LI Yijie1， LI Xiang1，ZHANG Xiaoqiu1， TANG Hongqin2*， LONG Shengfeng3*
（1. Sugarcane Research Institute， Guangxi Academy of Agricultural Sciences， Sugarcane Research Center，
Chinese Academy of Agricultural Science， Key Laboratory of Sugarcane Biotechnology and Genetic
Improvement （Guangxi）， Ministry of Agriculture and Rural Affairs， Nanning 530007， China;
2. Institute of Agricultural Resources and Environment， Guangxi Academy of Agricultural Sciences，
Nanning 530007， China; 3. Guangxi Academy of Agricultural Sciences， Nanning 530007， China）
Abstract
The molecular mechanism of nucleotide binding sites and leucine-rich replication （NBS-LRR） genes resistant to pokkah boeng disease （PBD） in sugarcane was explored to provide a theoretical basis for cloning resistant genes. This study firstly used Illumina high-throughput transcriptome sequencing technology to detect NBS-LRR genes against PBD with typical resistant and susceptive sugarcane materials， and then designed primers to verify the expression characteristics of significant differentially expressed NBS-LRR genes under different inoculation periods using real-time quantitative PCR （qRT-PCR）. The results showed that 16 NBS-LRR-like genes steadily increased expression when sugarcane leaves were infected with the PBD pathogen， including two trends in expression： 13 genes significantly or extremely significantly increased expression at 7 d post-inoculation， but three genes did not significantly increased expression at 7 d post-inoculation， but significantly increased expression at 14 d post-inoculation. Therefore， these genes could be divided into instantaneous basic defense genes （0 d to 7 d） and specific lagging defense genes （14 d to 21 d）， suggesting that NBS-LRR genes are actively involved in resistance against the PBD pathogen. 综上所述，本文在获得差异基因表达谱的基础上，根据已知植物抗病基因保守区设计简并引物，成功验证了NBS-LRR表达规律，可为最终克隆获得甘蔗抗梢腐病基因奠定基础。
参考文献
[1] 王泽平， 陈奕， 孙海军， 等. 广西甘蔗主栽品种梢腐病田间抗性初步评价[J]. 热带作物学报， 2016， 37（5）： 952-957.
[2] 兰冬雪，汤丽影，李佳，等. 禾本科植物NBS-LRR类抗病基因结构、功能和进化研究进展[J].中国农学通报，2019， 35（15）：124-127.
[3] ROSSI M， ARAUJO P G， PAULET F， et al. Genomic distribution and characterization of EST-derived resistance gene analogs （RGAs） in sugarcane [J]. Molecular Genetics and Genomics， 2003， 269（3）： 406-419.
[4] MCINTYRE C L， CASU R E， DRENTH J， et al. Resistance gene analogues in sugarcane and sorghum and their association with quantitative trait loci for rust resistance [J]. Genome， 2005， 48（3）： 391-400.
[5] WANDERLEY-NOGUEIRA A C， SOARES-CAVALCANTI N M， MORAIS D A L， et al. Abundance and diversity of resistance genes in the sugarcane transcriptome revealed by in silico analysis [J]. Genetics and Molecular Research， 2007， 6（4）： 866-889.
[6] 阙友雄， 许莉萍， 张木清， 等.甘蔗中一个NBS-LRR类基因的全长克隆与表达分析[J]. 作物学报， 2009， 35（6）： 1161-1166.
[7] 贺尔奇， 李向勇， 彭仕荣， 等. 果蔗NBS类抗病基因同源序列的克隆与分析[J]. 分子植物育种， 2016， 14（2）： 352-358.
[8] KRASILEVA K V， DAHLBECK D， STASKAWICZ B. Activation of an Arabidopsis resistance protein is specified by the in planta association of its leucine-rich repeat domain with the cognate oomycete effector [J]. The Plant Cell， 2010， 22（7）： 2444-2458.
[9] 王泽平， 段维兴， 李毅杰， 等. 甘蔗梢腐病田间抗性评价体系的建立[J]. 华南农业大学学报， 2016， 37（3）： 67-72.
[10]王泽平， 林善海， 梁强， 等. 甘蔗叶冠形态与抗梢腐病相关性探讨[J]. 中国农业大学学报， 2017， 22（7）： 40-46.
[11]WANG Zeping， SUN Haijun， GUO Qiang， et al. Artificial inoculation method of pokkah boeng disease of sugarcane and screening of resistant germplasm resources in subtropical China