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摘 要 前期利用cDNA-AFLP技术分离获得了一个与橡胶树抗棒孢霉落叶病反应相关的差异表达基因片段EST-IAN-188，通过Blast比对分析发现，该基因片段与植物抗病相关NPR基因家族中的NPR1具有高度的同源性。将该片段与橡胶树基因组序列进行比对，设计引物进行PCR扩增，得到了一个橡胶树NPR1基因的cDNA和基因组序列，命名为HbNPR1基因。基因序列分析显示，该基因编码区全长（CDS）1 374 nt，含有两个外显子和一个内含子，编码457个氨基酸，蛋白分子量为51.0 ku，等电点5.82，具有BTB/POZ、ANK 锚蛋白重复序列、DUF和NPR1-like C等4个结构域。qRT-PCR 定量分析发现，HbNPR1基因在橡胶叶片中的表达丰度最高，特别是古铜期叶片；多主棒孢病菌（Corynespora cassiicola）诱导下HbNPR1基因在抗病品种（IAN873）的表达丰度明显高于感病品种（PR107）；另外，SA、MeJA、ET处理下均能诱导HbNPR1基因的表达，SA处理后的表达量丰度最高。本研究初步表明，HbNPR1基因可能参与橡胶树抗病信号途径的调控和寄主对病原菌侵染的防御反应。
关键词 橡胶树；HbNPR1基因；克隆；表达分析
中图分类号 S794.1；Q78 文献标识码 A
Cloning and Expression Analysis of Resistance-related
Gene HbNPR1 from Hevea brasiliensis
LI Boxun，SHI Tao，LIN Chunhua，LIU xianbao，CAI Jimiao，HUANG Guixiu*
1 Agronomy college， Hainan University， Haikou， Hainan 570228，Chiana
2 Environment and Plant Protection Institute， CATAS/Key Laboratory of Integrated Pest Management on
Tropical Crops， Ministry of Agriculture/Hainan Key Laboratory for Monitoring and
Control of Tropical Agricultural Pests，Haikou， Hainan 571101，Chiana
Abstract A resistance related differential expression fragment of rubber tree to Corynespora leaf fall disease was obtained in pre-work， and named as EST-IAN-188. Blast showed that the fragment had high homology with NPR1， a plant resistance NPR gene family member. Based on the Hevea brasiliensis genome database， the long fragment including EST-IAN-188 was gained and Hevea NPR1 gene was cloned， named as HbNPR1. Sequence analysis revealed that the CDS length of HbNPR1 was 1 374 nt， encoding a protein of 457 amino acids with an average molecular weight of 51.0 ku， and a pI value of 5.82. Exon/intron structure analysis revealed 2 exons and 1 introns in HbNPR1 genomic sequence. The protein had four conserved domains of BTB/POZ， ANK anchored protein repeat sequences， DUF and NPR1-like C. The expression pattern of HbNPR1 in rubber plants was assessed by qRT-PCR. The tissue-specific expression analysis indicated that the HbNPR1 was mainly expressed in leaves， especially in the bronze blade. When C. cassiicola infected， the expression level of HbNPR1 was significantly higher in resistant rubber germplasm（IAN873） than in susceptible rubber germplasm（PR107）. The expressions of HbNPR1 were up-regulated by ethylene、salicylic acid and Methyl jasmonate treatment， especially by salicylic acid. The results preliminarily show that HbNPR1 gene may be involved in rubber tree resistance signal pathway. [7] Philippe B， Valérie P R， Frédéric B， et al. Structural Analysis of Cassiicolin， a Host-selective Protein Toxin from Corynespora cassiicola[J]. J， Mol， Biol， 2007， 367： 89-101.
[8] 方中达.植病研究方法（第三版）[M].北京：中国农业出版社，1998.
[9] 时 涛， 蔡吉苗， 李超萍，等. 橡胶树多主棒孢菌室内产孢条件的优化[J]. 热带作物学报， 2010， 31（1）： 98-105.
[10] Cai Zhiying， Li Guohua， Lin Chunhua， et al. Identifying pathogenicity genes in the rubber tree anthracnose fungus Colletotrichum gloeosporioides through random insertional mutagenesis[J]. Microbiological Research， 2013， 168： 340-350.
[11] 许 玲， 孙晓波， 张 旭，等. 荆州黑麦NPR1同源基因ScNPR1的克隆与特性分析[J]. 麦类作物学报， 2011， 31（2）： 209-216.
[12] Cai X Z， Zheng Z. Mechanism and pathways of plant systemic acquired resistance[J]. Acta Phytoecologica Sinica， 1999， 26（1）： 83-90.
[13] Yuan Y， Zhong S， Li Q， et al. Functional analysis of rice NPR1-like genes reveals that OsNPR1/NH1 is the rice orthologue conferring disease resistance with enhanced herbivore susceptibility[J]. Plant Biotechnology Journal， 2007， 5（2）： 313-324.
[14] Loake G， Grant M. Sallicylic acid in plant defence the players and protagonists[J]. Current Opinion in Plant Biology， 2007， 10： 466-472.
[15] Yu D， Chen C， Chen Z. Evidence for an important role of WRKY DNA binding proteins in the regulation of NPR1 gene expression[J]. Plant Cell， 2001， 13： 1 527-1 539.
[16] Alvarez M E， Pennenll R I， Meijer P J， et al. Reactive oxygen intermediates mediate a systemic signal network in the establishment of plant immunity[J]. Cell， 1998， 92： 73-784.
[17] Spoel S H， Koornneef A， Claessens S M C， et al. NPR1 modulatescross talk between salicylate and jasmonate dependent defense pathways through a novel function in the cytosol[J]. Plant Cell， 2003， 15： 760-770.
责任编辑：凌青根
关键词 橡胶树；HbNPR1基因；克隆；表达分析
中图分类号 S794.1；Q78 文献标识码 A
Cloning and Expression Analysis of Resistance-related
Gene HbNPR1 from Hevea brasiliensis
LI Boxun，SHI Tao，LIN Chunhua，LIU xianbao，CAI Jimiao，HUANG Guixiu*
1 Agronomy college， Hainan University， Haikou， Hainan 570228，Chiana
2 Environment and Plant Protection Institute， CATAS/Key Laboratory of Integrated Pest Management on
Tropical Crops， Ministry of Agriculture/Hainan Key Laboratory for Monitoring and
Control of Tropical Agricultural Pests，Haikou， Hainan 571101，Chiana
Abstract A resistance related differential expression fragment of rubber tree to Corynespora leaf fall disease was obtained in pre-work， and named as EST-IAN-188. Blast showed that the fragment had high homology with NPR1， a plant resistance NPR gene family member. Based on the Hevea brasiliensis genome database， the long fragment including EST-IAN-188 was gained and Hevea NPR1 gene was cloned， named as HbNPR1. Sequence analysis revealed that the CDS length of HbNPR1 was 1 374 nt， encoding a protein of 457 amino acids with an average molecular weight of 51.0 ku， and a pI value of 5.82. Exon/intron structure analysis revealed 2 exons and 1 introns in HbNPR1 genomic sequence. The protein had four conserved domains of BTB/POZ， ANK anchored protein repeat sequences， DUF and NPR1-like C. The expression pattern of HbNPR1 in rubber plants was assessed by qRT-PCR. The tissue-specific expression analysis indicated that the HbNPR1 was mainly expressed in leaves， especially in the bronze blade. When C. cassiicola infected， the expression level of HbNPR1 was significantly higher in resistant rubber germplasm（IAN873） than in susceptible rubber germplasm（PR107）. The expressions of HbNPR1 were up-regulated by ethylene、salicylic acid and Methyl jasmonate treatment， especially by salicylic acid. The results preliminarily show that HbNPR1 gene may be involved in rubber tree resistance signal pathway. [7] Philippe B， Valérie P R， Frédéric B， et al. Structural Analysis of Cassiicolin， a Host-selective Protein Toxin from Corynespora cassiicola[J]. J， Mol， Biol， 2007， 367： 89-101.
[8] 方中达.植病研究方法（第三版）[M].北京：中国农业出版社，1998.
[9] 时 涛， 蔡吉苗， 李超萍，等. 橡胶树多主棒孢菌室内产孢条件的优化[J]. 热带作物学报， 2010， 31（1）： 98-105.
[10] Cai Zhiying， Li Guohua， Lin Chunhua， et al. Identifying pathogenicity genes in the rubber tree anthracnose fungus Colletotrichum gloeosporioides through random insertional mutagenesis[J]. Microbiological Research， 2013， 168： 340-350.
[11] 许 玲， 孙晓波， 张 旭，等. 荆州黑麦NPR1同源基因ScNPR1的克隆与特性分析[J]. 麦类作物学报， 2011， 31（2）： 209-216.
[12] Cai X Z， Zheng Z. Mechanism and pathways of plant systemic acquired resistance[J]. Acta Phytoecologica Sinica， 1999， 26（1）： 83-90.
[13] Yuan Y， Zhong S， Li Q， et al. Functional analysis of rice NPR1-like genes reveals that OsNPR1/NH1 is the rice orthologue conferring disease resistance with enhanced herbivore susceptibility[J]. Plant Biotechnology Journal， 2007， 5（2）： 313-324.
[14] Loake G， Grant M. Sallicylic acid in plant defence the players and protagonists[J]. Current Opinion in Plant Biology， 2007， 10： 466-472.
[15] Yu D， Chen C， Chen Z. Evidence for an important role of WRKY DNA binding proteins in the regulation of NPR1 gene expression[J]. Plant Cell， 2001， 13： 1 527-1 539.
[16] Alvarez M E， Pennenll R I， Meijer P J， et al. Reactive oxygen intermediates mediate a systemic signal network in the establishment of plant immunity[J]. Cell， 1998， 92： 73-784.
[17] Spoel S H， Koornneef A， Claessens S M C， et al. NPR1 modulatescross talk between salicylate and jasmonate dependent defense pathways through a novel function in the cytosol[J]. Plant Cell， 2003， 15： 760-770.
责任编辑：凌青根