Cloning and Expression of Pathogenesis-related Protein Gene, BoPR1, in Brassica oleracea var. italica
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摘要: 病程相关蛋白（Pathogenesis-related protein，PR）是参与植物抗病性的重要物质，在诱导系统抗性过程中起着重要作用。本研究以青花菜为材料，在克隆BoPR1基因的基础上，利用荧光定量PCR技术研究它们在根肿菌和核盘菌侵染下的表达模式。序列分析结果表明，BoPR1基因组全长为489 bp，无内含子，编码162个氨基酸，具1个信号肽和1个SCP结构域。系统发育分析的结果表明，BoPR1与甘蓝型油菜和白菜的PR1遗传距离最小，亲缘关系最近，在进化树上聚为一组；与醉蝶花PR1遗传距离最大，亲缘关系最远。荧光定量PCR结果显示，BoPR1基因的表达受根肿菌诱导，在接种5 d时的表达量最高，为对照的11.84倍；BoPR1基因的表达则不受核盘菌诱导。Abstract: Pathogenesis-related proteins (PRs) are crucial in the induced systemic disease resistance for plants. Using real-time fluorescence quantitative PCR, this study isolated a gene, designated as BoPR1, frombroccoli to examine its expression patterns after inoculated by pathogens, Plasmodiophora brassicae and Sclerotinia sclerotiorum. A sequence analysis indicated that the full genome DNA of BoPR1 was 489 bp in length encoding 162 amino acids and containing no intron. The deduced protein consisted of a signal peptide and a SCP domain. The results from aphylogenetic analysis showed thatBoPR1 had a minimum genetic distance, clustering on a same clade, with the PR1 proteins from Brassica napus and B. rapa, indicating a close relationship among them. On the other hand, it was remotely related to Tarenaya hassleriana, as the genetic distance between the two PR1swas the greatest among the tested samples. Theq RT-PCR results suggested that the expression of BoPR1 was induced by P. brassicae, with the highest level observed 5 d after inoculationwhich was 11.84-fold of control. But, BoPR1 expression was not affected by S. sclerotiorum. Theobtained isolation and expression information on BoPR1 would be useful for futureresearch on thedisease resistance mechanism as well as the molecular breeding programs on B. oleracea.
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Key words:
- Brassica oleracea var. italica /
- BoPR1 /
- gene cloning /
- expression analysis
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图 2 BoPR1基因及其编码蛋白序列
注：阴影部分为信号肽；划线部分为SCP结构域。
Figure 2. Sequences of BoPR1 gene and deduced protein
图 3 BoPR1及其同源序列的比较
Figure 3. Comparison between BoPR1 and counterparts with homologous sequence
图 4 BoPR1及其同源序列的系统发育树
Figure 4. Phylogenetic tree of BoPR1 and counterparts with homologous sequence
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[1] 林俊. 青花菜标准化栽培关键技术研究[D]. 杭州: 浙江大学, 2008. http://cdmd.cnki.com.cn/Article/CDMD-10335-2009156642.htm [2] 陈晓峰. 不结球白菜抗真菌病基因的克隆与表达分析[D]. 扬州: 南京农业大学, 2008. http://cdmd.cnki.com.cn/Article/CDMD-10307-2009076976.htm [3] 崔香仙.西兰花主要病害及其综合防治[J].特种经济动植物, 2005, 8(3):42-42. http://www.cqvip.com/QK/92994A/200503/15162748.html [4] 陈海平. 青花菜霜霉病病原和流行因素研究[D]. 杭州: 浙江大学, 2012. http://cdmd.cnki.com.cn/Article/CDMD-10335-1013186946.htm [5] 高婷婷. 大白菜抗霜霉病防御信号途径中相关基因的表达分析[D]. 扬州: 扬州大学, 2013. http://www.wanfangdata.com.cn/details/detail.do?_type=degree&id=y2418919 [6] SEKHWAL M K, LI P, LAM I, et al. Disease resistance gene analogs (RGAs) in plants[J]. International Journal of Molecular Sciences, 2015, 16(8):19248-19290. doi: 10.3390/ijms160819248 [7] GOVRIN E M, LEVINE A. The hypersensitive response facilitates plant infection by the necrotrophic pathogen Botrytis cinerea[J]. Current Biology, 2000, 10(13):751-757. doi: 10.1016/S0960-9822(00)00560-1 [8] 刘利华, 林奇英, 谢华安, 等.病程相关蛋白与植物抗病性研究[J].福建农业学报, 1999, 14(3):53-58. http://www.cnki.com.cn/Article/CJFDTOTAL-FJNX199903011.htm [9] 杨瑞瑞, 易小娅, 曾幼玲. PR10的结构、信号转导以及功能的研究进展[J].中国农学通报, 2014, 30(4):251-258. doi: 10.11924/j.issn.1000-6850.2013-2152 [10] 王勇刚, 曾富华, 吴志华, 等.植物诱导抗病与病程相关蛋白[J].湖南农业大学学报:自然科学版, 2002, 28(2):177-182. http://www.cnki.com.cn/Article/CJFDTOTAL-SJNY199108016.htm [11] 杨德翠, 张玉喜, 郑国生.牡丹病程相关蛋白1基因的克隆及表达分析[J].园艺学报, 2013, 40(8):1583-1590. http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=yyxb201308019 [12] 王燕华, 何水林.植物诱导抗病性的分子生物学研究进展[J].应用与环境生物学报, 2004, 10(6):811-815. http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=yyyhjswxb200406029 [13] 罗红丽, 秦云霞, 闫志烨, 等.巴西橡胶树病程相关蛋白基因(HbPR1)的克隆及分析[J].热带作物学报, 2011, 32(8):1499-1502. http://www.cnki.com.cn/Article/CJFDTotal-CYKE201506003.htm [14] ZHOU J M, TRIFA Y, SILVA H, et al. NPR1 differentially interacts with members of the TGA/OBF family of transcription factors that bind an element of the PR-1 gene required for induction by salicylic acid[J]. Molecular Plant-microbe Interactions, 2000, 13(2):191-202. doi: 10.1094/MPMI.2000.13.2.191 [15] 张小丽, 刘玉梅, 方智远, 等.青花菜及近缘种属种质资源抗根肿病鉴定[J].植物遗传资源学报, 2016, 17(6):1106-1115. http://www.cnki.com.cn/Article/CJFDTOTAL-ZNYK200810015.htm [16] DELLAPORTA S L, WOOD J, HICKS J B. A plant DNA minipreparation:version Ⅱ[J]. Plant Molecular Biology Reporter, 1983, 1(4):19-21. doi: 10.1007/BF02712670 [17] 张凯敏, 王玉成, 杨桂燕, 等.柽柳ThPR1基因的克隆与表达分析[J].南京林业大学学报:自然科学版, 2013, 37(2):45-49. http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=njlydxxb201302008 [18] 马立功, 张匀华, 孟庆林, 等.向日葵病程相关蛋白HaPR1基因的克隆与功能[J].作物学报, 2015, 41(12):1819-1827. http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=zuowxb201512006 [19] 姚红燕, 皇甫伟国, 赵海棠, 等.青花菜几种病害的初步调查[J].宁波农业科技, 2004, (4):2-4. http://cpfd.cnki.com.cn/Article/CPFDTOTAL-IGSS200400001016.htm [20] 王汉荣. 青花菜褐茎病病原鉴定及其防治技术研究[D]. 杭州: 浙江大学, 2010. http://cdmd.cnki.com.cn/article/cdmd-10335-1011060984.htm [21] LOON L C V, REP M, PIETERSE C M J. Significance of inducible defense-related proteins in infected plants[J]. Annual Review of Phytopathology, 2006, 44(1):135-162. doi: 10.1146/annurev.phyto.44.070505.143425 [22] 侯丽霞, 高超, 车永梅, 等.葡萄病程相关蛋白1基因的克隆和表达分析[J].植物生理学报, 2012, 48(1):57-62. http://www.cnki.com.cn/Article/CJFDTOTAL-ZWSL201201011.htm [23] AGRAWAL G K, NAMSOO J, RAKWAL R. A novel rice (Oryza sativa L.) acidic PR1 gene highly responsive to cut, phytohormones, and protein phosphatase inhibitors[J]. Biochemical & Biophysical Research Communications, 2000, 274(1):157-165. https://www.sigmaaldrich.com/catalog/papers/10903912 [24] 赵淑清, 郭剑波.植物系统获得抗性及信号转导途径[J].中国农业科学, 2003, 36(7):781-787. http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=zgnykx200307009 [25] ALEXANDER D, GOODMAN R M, GUT-RELLA M, et al. Increased tolerance to two oomycete pathogens in transgenic tobacco expressing pathogenesis-related protein 1a[J]. Proceeding of the National Academy of Science of America, 1993, 90(15):7327-7331. doi: 10.1073/pnas.90.15.7327 [26] SAROWAR S, KIM Y J, KIM E N, et al. Overexpression of a pepper basic pathogenesis-related protein 1 gene in tobacco plants enhances resistance to heavy metal and pathogen stresses[J]. Plant Cell Reports, 2005, 24(4):216-224. doi: 10.1007/s00299-005-0928-x [27] 赵长江, 鲁国东, 杜晓昱, 等.水稻纹枯病发病过程PR1和PBZl的表达动态[J].植物病理学报, 2006, 26(4):317-321. http://mall.cnki.net/magazine/Article/ZWBL200604006.htm -
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