抗感菌核病甘蓝型油菜近等基因系盛花期叶片转录组比较分析

doi:10.7668/hbnxb.20190761

摘要/Abstract

摘要： 为了研究甘蓝型油菜与菌核病病原核盘菌互作的分子机制，挖掘分别与甘蓝型油菜抗、感病相关的基因，以高抗菌核病品种湘油15（Xiangyou 15）及其感病的近等基因系98C40NL为材料，利用转录组测序技术（RNA-Seq）对2个品种盛花期叶片接种核盘菌前及接种后12，24 h差异表达基因进行了分析，并采用实时荧光定量PCR技术对部分基因表达进行验证。测序得到34.16 G Clean bases，6个样本中共鉴定出78 582个基因。相对于高感品种，湘油15在3个时间点共有1 296个基因下调表达，1 495个基因上调表达；54个基因在3个时间点都表现显著差异，其中18个基因在3个时间点都下调，27个基因在3个时间点都上调。差异基因主要富集在氧化还原、植物激素信号转导、钙信号通路、苯丙烷类代谢、次生代谢产物合成等途径。通过荧光定量PCR验证，BnaA03g09060D、BnaA01g26640D、BnaA03g09090D、BnaA04g12120D、BnaA01g26920D 5个基因在高抗品种的表达显著高于高感品种，可能与寄主植物防御核盘菌的侵染有关；BnaA06g10010D、BnaA09g16180D、BnaAnng19580D、BnaC01g40400D、和BnaC06g05340D 5个基因则在高感品种中表达显著高于高抗品种，可能与核盘菌感病受体相关。研究结果为解析油菜与核盘菌互作机理及挖掘菌核病抗性和受体基因提供参考依据。

关键词: 甘蓝型油菜, 菌核病, 转录组, 差异表达基因, 功能

Abstract: A transcriptome comparisons of two genotypes of Brassica napus with different resistant ability against Sclerotinia sclerotirium, Xiangyou 15 with high resistance and its near-isogenic line 98C40NL with high susceptibility, were conducted by RNA-Seq in order to study the molecular mechanism of the interaction between B. napus and S. Sclerotirium and find out genes relevant with resistance and susceptibility respectively. The differentially expressed genes before inoculation with S.Sclerotirium,and 12,24 h after infection were analysed, followed by a qRT-PCR verification of gene expression. Totally, 34.16 G Clean bases and 78 582 genes were obtained through RNA-Seq. The results showed that 1 296 genes were down regulated while 1 495 genes were up regulated in Xiangyou 15 compared to 98C40NL. 54 genes expressed differentially in all of the three time points, 18 of which were down regulated and 27 genes were up regulated. The differentially expressed genes enriched in pathways related to oxidation-reduction, plant hormone signal transduction, calcium signal transduction,phenylpropane metabolism and synthesis of secondary metabolites. qRT-PCR results revealed that the genes with abundant expression in high resistant cultivar including BnaA03g09060D, BnaA01g26640D, BnaA03g09090D, BnaA04g12120D, and BnaA01g26920D could be related to resistance. However, genes BnaA06g10010D, BnaA09g16180D, BnaAnng19580D, BnaC01g40400D, and BnaC06g05340D might be connected with susceptibility, showing high expression in 98C40NL. The results can provide clue for the mechanism analysis of interactions between B. napus and S.sclerotirium and provide candidate genes for screening related resistance and receptor genes.

Key words: Brassica napus, Sclerotinia sclerotirium, Transcriptome, Differentially expressed genes, Function

中图分类号:

S432.4

张秋平, 文李, 张振乾, 王峰, 官春云. 抗感菌核病甘蓝型油菜近等基因系盛花期叶片转录组比较分析[J]. 华北农学报, 2020, 35(3): 168-177. doi: 10.7668/hbnxb.20190761.

ZHANG Qiuping, WEN Li, ZHANG Zhenqian, WANG Feng, GUAN Chunyun. Comparative Transcriptomic Analysis of Brassica napus Near-isogenic Line Resistant to Sclerotinia sclerotirium at Flowering Stage[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2020, 35(3): 168-177. doi: 10.7668/hbnxb.20190761.

http://www.hbnxb.net/CN/Y2020/V35/I3/168

参考文献

[1] 王汉中. 以新需求为导向的油菜产业发展战略[J]. 中国油料作物学报, 2018, 40(5):613-617.doi:10.7505/j.issn.1007-9084.2018.05.001.
Wang H Z.New demand oriented oilseed rape industry developing strategy[J]. Chinese Journal of Oil Crop Sciences, 2018, 40(5):613-617.
[2] Food and agriculture organization of the United Nations Production, Crops. FAOSTAT[DB/OL]. 2017-9-26.http://faostat3.fao.org/browse/Q/QC/E.
[3] Liang X F, Rollins J A. Mechanisms of broad host range necrotrophic pathogenesis in Sclerotinia sclerotiorum[J]. Phytopathology, 2018, 108(10):1128-1140. doi:10.1094/PHYTO-06-18-0197-RVW.
[4] 官春云, 李方球, 陈社员. 双低油菜湘油15号对菌核病抗性研究简报[J]. 作物研究, 2001,15(3):33. doi:10.3969/j.issn.1001-5280.2001.03.012.
Guan C Y, Li F Q, Chen S Y. Brief report of characterization of stem rot resistance in the double-low rapeseed cultivar Xiangyou 15[J]. Crop Research,2001,15(3):33.
[5] 齐绍武,官春云,刘春林.甘蓝型油菜品系一些酶的活性与抗菌核病的关系[J].作物学报,2004,30(3):270-273. doi:10.3321/j.issn:0496-3490.2004.03.015.
Qi S W, Guan C Y, Liu C L. Relationship between some enzyme activity and resistance to Sclerotinia sclerotiorum of rapeseed cultivars[J]. Acta Agronomica Sinica,2004, 30(3):270-273.
[6] Wen L, Tan T L, Shu J B,Chen Y, Liu Y, Yang Z F, Zhang Q P, Yin M Z, Tao J, Guan C Y. Using proteomic analysis to find the proteins involved in resistance against Sclerotinia sclerotiorum in adult Brassica napus[J]. European Journal of Plant Pathology, 2013,137(3):505-523. doi:10.1007/s10658-013-0262-z.
[7] Wang Z R, Wan L L, Xin Q, Chen Y, Zhang X H, Dong F M, Hong D F, Yang G S. Overexpression of OsPGIP2 confers Sclerotinia sclerotiorum resistance in Brassica napus through increased activation of defense mechanisms[J]. Journal of Experimental Botany, 2018,69(12):3141-3155. doi:10.1093/jxb/ery138.
[8] 贾承国,向珣,王思周,汪俏梅.茉莉酸类化合物在植物防卫反应中的作用[J]. 细胞生物学杂志,2006,28(1):57-60. doi:10.3969/j.issn.1674-7666.2006.01.013.
Jia C G, Xiang X,Wang S Z,Wang Q M.The role of jasmonate in plant defense response[J]. Chinese Journal of Cell Bio1ogy, 2006,28(1):57-60.
[9] 刘庆霞,李梦莎,国静. 茉莉酸生物合成的调控及其信号通路[J]. 植物生理学报,2012,48(9):837-844.doi:10.13592/j.cnki.ppj.2012.09.013.
Liu Q X, Li M S,Guo J.Regulation of jasmonic acid biosynthesis and jasmonic acid signaling pathway[J]. Plant Physiology Journal, 2012, 48(9):837-844.
[10] Wu J, Zhao Q, Yang Q Y, Liu H, Li Q Y, Yi X Q, Cheng Y, Guo L, Fan C C, Zhou Y M. Comparative transcriptomic analysis uncovers the complex genetic network for resistance to Sclerotinia sclerotiorum in Brassica napus[J]. Sci Rep,2016(6):19007. doi:10.1038/srep19007.
[11] Wang Z, Tan X L, Zhang Z Y, Gu S L, Li G Y, Shi H F. Defense to Sclerotinia sclerotiorum in oilseed rape is associated with the sequential activations of salicylic acid signaling and jasmonic acid signaling[J]. Plant Sci, 2012, 184(9):75-82. doi:10.1016/j.plantsci.2011.12.013.
[12] Liu F, Li X X, Wang M R, Wen J, Yi B, Shen J X, Ma C Z, Fu T D, Tu J X.Interactions of WRKY15 and WRKY33 transcription factors and their roles in the resistance of oilseed rape to Sclerotinia infection[J]. Plant Biotechnol J, 2018, 16(4):911-925. doi:10.1111/pbi.12838.
[13] 郝向阳, 孙雪丽, 王天池, 吕科良,赖钟雄,程春振. 植物PAL基因及其编码蛋白的特征与功能研究进展[J]. 热带作物学报, 2018, 39(7):1452-1461. doi:10.3969/j.issn.1000-2561.2018.07.028.
Hao X Y,Sun X L, Wang T C, Lü K L, Lai Z X, Cheng C Z.Characteristics and functions of plant phenylalanine ammonia lyase genes and the encoded proteins[J]. Chinese Journal of Tropical Crops, 2018, 39(7):1452-1461.
[14] Erdemoglu N,Ozkan S,Tosun F. Alkaloid profile and antimicrobial activity of Lupinus angustifolius L. alkaloid extract[J]. Phytochemistry Reviews, 2007,6(1):197-201. doi:10.1007/s11101-006-9055-8.
[15] Erdemoglu N,Ozkan S,Duran A,Tosun F. GC-MS analysis and antimicrobial activity of alkaloid extract from Genista vuralii[J]. Pharmaceutical Biology, 2009, 47(1):81-85. doi:10.1080/13880200802448674.
[16] Zarinpanjeh N, Motallebi M, Zamani M R, Ziaei M.Enhanced resistance to Sclerotinia sclerotiorum in Brassica napus by co-expression of defensin and chimeric chitinase genes[J]. Journal of Applied Genetics, 2016, 57(4):417-425. doi:10.1007/s13353-016-0340-y.
[17] Wippich C,Wink M. Biological properties of alkaloids. Influence of quinolizidine alkaloids and gramine on the germination and development of powdery mildew, Erysiphe graminis f. sp. Hordei[J]. Experientia,1985,41(11):1477-1479. doi:10.1007/bf01950046.
[18] Zhao W M, Wolfender J L,Hostettmann K,Xu R S, Qin G W. Antifungal alkaloids and limonoid derivatives from Dictamnus dasycarpus[J]. Phytochemistry,1998,47(1):7-11. doi:10.1016/s0031-9422(97)00541-4.
[19] Cheng S H, Willmann M R, Chen H C, Sheen J. Calcium signaling through protein kinases. The Arabidopsis calcium-dependent protein kinase gene family[J]. Plant Physiolog, 2002, 129(2):469-485. doi:10.1104/pp.005645.
[20] Harmon A C, Gribskov M, Harper J f, Harmon A C, Gribskov M, Harper J F. CDPKs a kinase for every Ca ²⁺ signal?[J]. Trends in Plant Science, 2000,5(4):154-159. doi:10.1016/S1360-1385(00)01577-6.
[21] Yamniuk A P, Vogel H J. Structural investigation into the differential target enzyme regulation displayed by plant calmodulin isoforms[J]. Biochemistry, 2005, 44(8):3101-3111. doi:10.1021/bi047770y.
[22] Lee S H, Johnson J D, Walsh M P, Van Lierop J E, Sutherland C, Xu A,Snedden W A, Kosk-Kosicka D, Fromm H, Narayanan N, Cho M J. Differential regulation of Ca²⁺/calmodulin-dependent enzymes by plant calmodulin isoforms and free Ca²⁺ concentration[J]. Biochem Journal, 2000, 350(1):299-306. doi:10.1042/0264-6021:3500299.
[23] Batistic O, Kudla J. Integration and channeling of calcium signaling through the CBL calcium sensor/CIPK protein kinase network[J]. Planta, 2004, 219(6):915-924.doi:10.1007/s00425-004-1333-3.
[24] Batisti O, Kudla J. Analysis of calcium signaling pathways in plants[J]. Biochimica et Biophysica Acta (BBA)-General Subjects, 2012, 1820(8):1283-1293. doi:10.1016/j.bbagen.2011.10.012.
[25] 吕琳慧, 徐幼平, 任至玄,康冬,王继鹏,蔡新忠. Ca²⁺信号通路对本氏烟叶位介导的核盘菌抗性的影响[J]. 浙江大学学报(农业与生命科学版), 2014, 40(6):605-610. doi:10.3785/j.issn.1008-9209.2014.03.131.
Lü L H, Xu Y P, Ren Z X, Kang D, Wang J P, Cai X Z. Effect of Ca²⁺ signaling pathway on leaf position-associated resistance to Sclerotinia sclerotiorumin in Nicotiana benthamiana[J]. Journal of Zhejiang University(Agriculture and Life Sciences),2014, 40(6):605-610.
[26] 张秋平, 文李, 王峰, 廖志强,李慧,刘睿洋,官春云.油菜钙依赖蛋白激酶BnCDPK1 的克隆和表达分析[J]. 植物遗传资源学报, 2014(6):1320-1326. doi:10.13430/j.cnki.jpgr.2014.06.021.
Zhang Q P, Wen L, Wang F, Liao Z Q, Li H, Liu R Y, Guan C Y. Molecular cloning and expression analysis of calcium-dependent protein kinase BnCDPK1 in Brassica napus[J]. Journal of Plant Genetic Resources, 2014(6):1320-1326.
[27] Ding Y J, Mei J Q, Chai Y R, Yu Y, Shao C G, Wu Q N, Disi J O, Li Y H, Wan H F, Qian W.Simultaneous transcriptome analysis of host and pathogen highlights the interaction between Brassica oleracea and Sclerotinia sclerotiorum[J]. Phytopathology, 2019,109(4):542-550. doi:10.1094/PHYTO-06-18-0204-R.

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