甘蔗NBS-LRR类抗病基因同源序列的分离与鉴定

doi:10.3724/SP.J.1006.2009.00631

摘要/Abstract

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)

阙友雄，许莉萍，林剑伟，陈如凯. 甘蔗NBS-LRR类抗病基因同源序列的分离与鉴定[J]. 作物学报, 2009, 35(4): 631-639.

QUE You-Xiong,XU Li-Ping,LIN Jian-Wei,CHEN Ru-Kai. Isolation and Characterization of NBS-LRR Resistance Gene Analogs from Sugarcane[J]. Acta Agron Sin, 2009, 35(4): 631-639.

参考文献

[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

相关文章 15

[1]	肖健, 陈思宇, 孙妍, 杨尚东, 谭宏伟. 不同施肥水平下甘蔗植株根系内生细菌群落结构特征[J]. 作物学报, 2022, 48(5): 1222-1234.
[2]	周慧文, 丘立杭, 黄杏, 李强, 陈荣发, 范业赓, 罗含敏, 闫海锋, 翁梦苓, 周忠凤, 吴建明. 甘蔗赤霉素氧化酶基因ScGA20ox1的克隆及功能分析[J]. 作物学报, 2022, 48(4): 1017-1026.
[3]	孔垂豹, 庞孜钦, 张才芳, 刘强, 胡朝华, 肖以杰, 袁照年. 不同施肥水平下丛枝菌根真菌对甘蔗生长及养分相关基因共表达网络的影响[J]. 作物学报, 2022, 48(4): 860-872.
[4]	杨宗桃, 刘淑娴, 程光远, 张海, 周营栓, 商贺阳, 黄国强, 徐景升. 甘蔗类泛素蛋白UBL5应答SCMV侵染及其与SCMV-6K2的互作[J]. 作物学报, 2022, 48(2): 332-341.
[5]	赵海涵, 练旺民, 占小登, 徐海明, 张迎信, 程式华, 楼向阳, 曹立勇, 洪永波. 水稻协优9308重组自交系群体白叶枯病抗性的全基因组关联分析[J]. 作物学报, 2022, 48(1): 121-137.
[6]	张海, 程光远, 杨宗桃, 刘淑娴, 商贺阳, 黄国强, 徐景升. 甘蔗PsbR亚基应答SCMV侵染及其与SCMV-6K2的互作[J]. 作物学报, 2021, 47(8): 1522-1530.
[7]	傅华英, 张婷, 彭文静, 段瑶瑶, 许哲昕, 林艺华, 高三基. 甘蔗新品种(系)苗期白条病人工接种抗性鉴定与评价[J]. 作物学报, 2021, 47(8): 1531-1539.
[8]	苏亚春, 李聪娜, 苏炜华, 尤垂淮, 岑光莉, 张畅, 任永娟, 阙友雄. 甘蔗割手密种类甜蛋白家族鉴定及栽培种同源基因功能分析[J]. 作物学报, 2021, 47(7): 1275-1296.
[9]	黄宁, 惠乾龙, 方振名, 李姗姗, 凌辉, 阙友雄, 袁照年. 甘蔗β-胡萝卜素异构酶基因家族的鉴定、定位和表达分析[J]. 作物学报, 2021, 47(5): 882-893.
[10]	王恒波, 陈姝琦, 郭晋隆, 阙友雄. 甘蔗抗黄锈病G1标记的分子检测及候选抗病基因WAK的分析[J]. 作物学报, 2021, 47(4): 577-586.
[11]	张荣跃, 王晓燕, 杨昆, 单红丽, 仓晓燕, 李婕, 王长秘, 尹炯, 罗志明, 李文凤, 黄应昆. 甘蔗新品种及主栽品种对褐锈病抗性与Bru1基因分子检测[J]. 作物学报, 2021, 47(2): 376-382.
[12]	仓晓燕, 夏红明, 李文凤, 王晓燕, 单红丽, 王长秘, 李婕, 张荣跃, 黄应昆. 甘蔗优良品种(系)对黑穗病的抗性评价[J]. 作物学报, 2021, 47(11): 2290-2296.
[13]	张海, 程光远, 杨宗桃, 王彤, 刘淑娴, 商贺阳, 赵贺, 徐景升. 甘蔗ScCRT1基因克隆及其应答SCMV侵染分子机制的研究[J]. 作物学报, 2021, 47(1): 94-103.
[14]	黄小芳,毕楚韵,石媛媛,胡韵卓,周丽香,梁才晓,黄碧芳,许明,林世强,陈选阳. 甘薯基因组NBS-LRR类抗病家族基因挖掘与分析[J]. 作物学报, 2020, 46(8): 1195-1207.
[15]	郑清雷,余陈静,姚坤存,黄宁,阙友雄,凌辉,许莉萍. 甘蔗Rieske Fe/S蛋白前体基因ScPetC的克隆及表达分析[J]. 作物学报, 2020, 46(6): 844-857.

Metrics

Viewed

Full text

Abstract

Cited

Shared

Discussed