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作物学报 ›› 2009, Vol. 35 ›› Issue (4): 631-639.doi: 10.3724/SP.J.1006.2009.00631

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

甘蔗NBS-LRR类抗病基因同源序列的分离与鉴定

阙友雄，许莉萍*，林剑伟，陈如凯   

  1. 福建农林大学/农业部甘蔗遗传改良重点开放实验室，福建福州350002
  • 收稿日期:2008-08-28 修回日期:2008-12-13 出版日期:2009-04-12 网络出版日期:2009-02-13
  • 基金资助:

    本研究由国家高技术研究发展计划（863计划）项目（2007AA100701），农业部引进国际先进农业科学技术计划（948计划）项目（2006-G37），国家自然科学基金项目（30170639）；福建省科技厅国家科技项目备案（F2007AA100701）资助。

Isolation and Characterization of NBS-LRR Resistance Gene Analogs from Sugarcane

QUE You-Xiong,XU Li-Ping*,LIN Jian-Wei,CHEN Ru-Kai   

  1. Fujian Agriculture and forestry University,Fuzhou 350002,China
  • Received:2008-08-28 Revised:2008-12-13 Published:2009-04-12 Published online:2009-02-13

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2929

被引次数

118

摘要：

根据已知植物(拟南芥、烟草和亚麻)抗病基因(RGAs)保守序列设计简并引物, 从甘蔗高抗黑穗病品种NCo376的基因组DNA和cDNA中扩增出11条抗病基因同源序列, 其中5条来自基因组DNA(EF059973, EF059974, EF059975, EF059976和EF059977), 6条来自cDNA(EF155648、EF155649、EF155650、EF155651、EF155652和EF155653)。序列分析表明, 这些RGAs均含有典型的NBS-LRR类抗病基因所拥有的保守结构域P-loop, Kinase-2a, Kinase-3a 和疏水结构域。聚类分析表明, 11条RGA同RPS2和XA1聚为一类, 而N和L6则单独聚为一类。所有11条抗病基因同源序列中, kinase-2(LLVLDDVW/D)最后一个氨基酸皆为色氨酸。定量PCR分析表明, 编号为EF059974的PIC基因的表达不仅受黑穗病菌胁迫的影响, 而且受水杨酸的诱导和过氧化氢的抑制, 也具有抗病基因组织特异性和组成型表达特性。

关键词: 甘蔗, NBS-LRR, 抗病基因同源序列

Abstract:

The large group of plant disease resistance (R) genes that share similar structures possesss a predicted nucleotide-binding site (NBS) domain. NBS domains of this class of R genes show highly conserved amino acid motifs, which makes it possible to isolate resistance gene analogs (RGAs) by PCR with degenerate primers. According to the conserved motifs in the NBS regions of the three typical NBS-LRR type resistance genes (RPS2, N, and L6), five degenerate and one non-degenerate primers were designed to correspond to the P-loop motif in the sense direction, while nine degenerate plus one non-degenerate primers were made corresponding to the HD motif in the anti-sense direction. Then, the homologous PCR was used to amplify NBS sequences from genomic DNA and cDNA using sugarcane variety NCo376 with smut resistance. In all, eleven RGAs were obtained, five from DNA (EF059973, EF059974, EF059975, EF059976, and EF059977) and six from cDNA (EF155648, EF155649, EF155650, EF155651, EF155652, and EF155653). Sequence analysis showed that RGAs comprised the conserved domains P-loop, Kinase-2a, Kinase-3a and HD, which was conserved in NBS-LRR type disease resistance gene. Cluster analysis showed that eleven RGAs and RPS2 and XA1 were clustered into one group, and N and L6 were divided into another group. Further, amino acid sequences showed that their last amino acid in alignment was residue W in LLVLDDV(W/D) motif, which is typical to non-TIR-NBS-LRR type gene. It was suggested that only non-TIR-NBS-LRR but not TIR-NBS-LRR type resistance genes existed in sugarcane genome. One RGA termed PIC (EF059974) was selected randomly for function validation through Real-time PCR. The result showed that expression of PIC gene could to some extent be influenced by U.scitaminea, SA and H2O2, and had the characteristics of constitutive expression and tissue-specific. The RGA cloned in this experiment may provide the shortcut for cloning of sugarcane disease resistance gene.

Key words: Saccharum officinarum, NBS-LRR, RGA(resistance gene analogs)

[1] Hulbert S H, Webb C A, Smith S M, Sun Q. Resistance gene complexes: Evolution and utilization. Annu Rev Phytopathol, 2001, 39: 285–312
[2] Baker B, Zambryski P, Staskawicz B, Dinesh-Kumar S P. Signaling in plant-microbe interaction. Science, 1997, 276: 726–733
[3] Albert H H, Schenck S. PCR amplification from a homolog of the bE mating-type gene as a sensitive assay for the presence of Ustilago scitaminea DNA. Plant Dis, 1996, 80: 1189–1192
[4] Xu L-P(许莉萍), Chen R-K(陈如凯). Identification of RAPD marker linked to smut resistance gene in sugarcane. Chin J Appl Environ Biol (应用与环境生物学报), 2004, 10(3): 263–267
[5] Que Y X, Li W, Xu J S, Xu L P, Zhang M Q, Chen R K. A simple and versatile protocol for isolation of RNA from plant, fungi and animal. J Agric Sci Technol, 2008, 2: 63–63
[6] Yao W(姚伟), Geng G-L(耿广良), Yu A-L(余爱丽), Zhang M-Q(张木清), Chen R-K(陈如凯). An improved method for isolation of transgenic sugarcane genomic DNA. J Tropic Subtropic Bot (热带亚热带植物学报), 2004, 12(3): 257–260
[7] Huang D-Q(黄代青), Wang P(王平), Lü L-X(吕柳新). Isolation of NBS-LRR class resistance gene analogs from stigma cDNA of pomelo (Citrus grandis cv. guanxi). Sci Agric Sin (中国农业科学), 2004, 37(10): 1580–1584
[8] Qilin P, Jonathan W, Robert F. Divergent evolution of plant NBS-LRR resistance gene homologous in dicot and cereal genomes. J Mol Evol, 2000, 50: 203–213
[9] Brody J D, Roger W I. Plant NBS-LRR proteins in pathogen sensing and host defense. Nature Immunol, 2006, 7: 1243–1249
[10] He C-Y(贺超英), Zhang Z-Y(张志永), Chen S-Y(陈受宜). Cloning and analysis of a disease resistance gene homolog from soybean. Chin Sci Bull (科学通报), 2001, 46(12): 1017–1021
[11] Traut T W. The functions and consensus motifs of nine types of peptide segments that form different types of nucleotide binding-sites. Eur J Biochem, 1994, 222(1): 9–19
[12] Ajith A, Srinivasa R U, Choong-Min R, Stacy N A, Li K, Yuhong T, Kirankumar S M, Kumar D, Klessig D F. Salicylic acid and systemic acquired resistance play a role in attenuating crown gall disease caused by Agrobacterium tumefaciens. Plant Physiol, 2008, 146: 703–715
[13] Averyanov A A, Lapikova V P, Djawakhia V G. Active oxygen mediates heat-induced resistance of rice plant to blast disease. Plant Sci, 1993, 92: 27–34
[14] Wu G, Shortt B J, Lawrence E B, Levine E B, Fitzsimmons K C, Shah D M. Disease resistance conferred by expression of a gene encoding H2O2-generating glucose oxidase in transgenic potato plants. Plant Cell, 1995, 7: 1357–1368
[15] Liu X, Lin F, Wang L, Pan Q. The in silico map-based cloning of Pi36, a rice coiled-coil nucleotide-binding site leucine-rich repeat gene that confers race-specific resistance to the blast fungus. Genetics, 2007, 176: 2541–2549
[16] Rossi M, Araujo P G, Paulet F, Garsmeur O, Dias V M, Chen H, Van Sluys M A, D’Hont A. Genomic distribution and characterization of EST-derived resistance gene analogs (RGAs) in sugarcane. Mol Gen Genom, 2003, 269: 406–419
[17] McIntyre C L, Casu R E, Drenth J, Knight D, Whan V A, Croft B J, Jordan D R, Manners J M. Resistance gene analogues in sugarcane and sorghum and their association with quantitative trait loci for rust resistance. Genome, 2005, 48: 391–400
[18] Hermann S, Brumbley S, McIntyre C L. Analysing diversity in sugarcane resistance gene analogues. Australasian Plant Pathol, 2005, 35: 631–641
[19] Wanderley-Nogueira A C, Soares-Cavalcanti N M, Morais D A L, Belarmino L C, Barbosa-Silva A, Benko-Iseppon A M. Abundance and diversity of resistance genes in the sugarcane transcriptome revealed by in silico analysis. Genet Mol Res, 2007, 6: 866–889
[20] Que Y X, Lin J W, Zhang J S, Ruan M H, Xu L P, Zhang M Q. Molecular cloning and characterization of a non-TIR-NBS-LRR type disease resistance gene analogue from sugarcane. Sugar Tech, 2008, 10: 71–73
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