基于柑橘叶斑驳病毒的表达载体构建及应用

doi:10.3864/j.issn.0578-1752.2022.22.006

摘要/Abstract

摘要：

【目的】 构建基于柑橘叶斑驳病毒（citrus leaf blotch virus，CLBV）的表达载体，通过系统表达抗菌肽提高植物抗病性，为柑橘溃疡病、柑橘黄龙病等病害的防控提供新型技术手段。【方法】 基于前期构建的侵染性克隆pCY-CLBV201，在外壳蛋白基因终止子后插入亚基因组启动子序列及多克隆位点，构建病毒表达载体pCLBV202。在多克隆位点插入绿色荧光蛋白（green fluorescent protein）基因（gfp），通过农杆菌介导接种、荧光观察验证pCLBV202-GFP表达GFP的情况。克隆天蚕的抗菌肽（cecropin B，CB）基因并构建重组载体pCLBV202-CB，通过农杆菌介导分别注射接种本氏烟和真空浸润接种柑橘实生苗，筛选阳性植株并分别注射接种和根灌接种烟草青枯病菌及针刺离体叶片接种柑橘溃疡病菌，同时设空载体接种植株为对照，通过症状观察、发病率及病情指数评价接种植株的烟草青枯病抗性；通过柑橘叶片的病斑数量、发病率及菌落浓度评价其溃疡病抗性。【结果】 pCLBV202-GFP接种烟草和尤力克柠檬后，均可以在系统新叶上观察到绿色荧光，在烟草上表现更为明亮，说明基于CLBV的表达载体构建成功。接种青枯病菌后，处理组（pCLBV202-CB）较对照组（pCLBV202）发病时间延迟4 d。在接种后第24天（24 dpi），处理组发病率为14.3%，对照组发病率为100%，差异显著。处理组相对于对照组的抗性指数为-2.66，抗性评价为高抗，表明利用pCLBV202-CB系统表达CB增强了对烟草青枯病的抗性。尤力克柠檬叶片针刺接种柑橘溃疡病菌，7 dpi时，处理组病斑数为47个，发病率为43.5%，对照组病斑个数为73个，发病率为67.6%。菌群数变化检测发现，处理组菌群数小于对照组菌群数，表明利用pCLBV202-CB系统表达CB增强了尤力克柠檬的溃疡病抗性。【结论】 构建了基于CLBV的病毒表达载体pCLBV202。利用pCLBV202在本氏烟和柑橘中系统表达CB可以提高植株对细菌性病害的抗性，这为柑橘细菌性病害的防控提供了新技术。

关键词: 柑橘叶斑驳病毒, 病毒表达载体, 抗菌肽, 抗病性

Abstract:

【Objective】 The objective of this study is to construct an expression vector based on citrus leaf blotch virus (CLBV) and systematically express antimicrobial peptides to improve plant disease resistance, which will provide a new technical means for the prevention and control of citrus canker, citrus Huanglongbing (HLB) and other diseases.【Method】The subgenomic promoter sequence and multiple cloning sites were inserted after the terminator of the coat protein gene of CLBV to construct the viral expression vector pCLBV202 based on the previously constructed infectious clone pCY-CLBV201. Then green fluorescent protein gene (gfp) was inserted into the multiple cloning sites, and the expression of GFP was verified by Agrobacterium-mediated inoculation and fluorescence observation. The cecropin B (CB) gene of Antheraea pernyi was cloned and the recombinant vector pCLBV202-CB was constructed. Nicotiana benthamiana and citrus seedlings were inoculated by pCLBV202-CB using Agrobacterium-mediated injection and vacuum infiltration, respectively. Positive plants were screened and subjected to inoculation of Ralstonia solanacearum by injection and root irrigation or Xanthomonas citri subsp. citri (Xcc) by pricking the detached leaves. At the same time, the plants inoculated with the empty vector were set as the control. The resistance to tobacco bacterial wilt of the inoculated plants was evaluated by symptom observation, incidence rate and disease index. The citrus resistance to canker was evaluated by the detriment, incidence rate and colony concentration on leaves. 【Result】After inoculated by pCLBV202-GFP, green fluorescence could be observed on the systemic leaves of the N. benthamiana and Citrus limon, which was brighter in N. benthamiana, indicating that the expression vector based on CLBV was successfully constructed. After inoculation of R. solanacearum, the onset time of treatment group (pCLBV202-CB) was delayed by 4 days compared with the control group (pCLBV202). On the 24th day past inoculation (24 dpi), the incidence rate of the treatment group was 14.3%, and that of the control group was 100%. Compared with the control group, the resistance index of the treatment group was -2.66, and the resistance evaluation was high, indicating that the expression of CB by pCLBV202-CB enhanced the resistance to tobacco bacterial wilt. When C. limon leaves was inoculated by Xcc, the number of detriment in the treatment group at 7 dpi was 47, and the incidence rate was 43.5%, while that of the control group was 73, and 67.6%. The detection of colony concentration changes of Xcc showed that the number of Xcc in the treatment group was less than that in the control group, indicating that the expression of CB by pCLBV202-CB enhanced the resistance to citrus canker.【Conclusion】 The CLBV-based viral expression vector pCLBV202 was constructed. Using pCLBV202 to systematically express CB in N. benthamiana and citrus can improve the resistance of plants to bacterial diseases, which provided a new technology for the prevention and control of citrus bacterial diseases.

Key words: citrus leaf blotch virus (CLBV), viral expression vector, antimicrobial peptide, disease resistance

张琦,段玉,苏越,蒋琪琪,王春庆,宾羽,宋震. 基于柑橘叶斑驳病毒的表达载体构建及应用[J]. 中国农业科学, 2022, 55(22): 4398-4407.

ZHANG Qi,DUAN Yu,SU Yue,JIANG QiQi,WANG ChunQing,BIN Yu,SONG Zhen. Construction and Application of Expression Vector Based on Citrus Leaf Blotch Virus[J]. Scientia Agricultura Sinica, 2022, 55(22): 4398-4407.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2022.22.006

https://www.chinaagrisci.com/CN/Y2022/V55/I22/4398

图/表 8

表1

图1

图2

图3

图4

图5

图6

图7

参考文献 34

[1]	陈祝安, 曹光照, 许益伟, 黄基荣, 葛岚屏. 柑桔害虫病原真菌资源的考察和生测. 微生物学通报, 1985(5): 194-198.
	CHEN Z A, CAO G Z, XU Y W, HUANG J R, GE L P. Investigation and bioassay of citrus pest pathogenic fungi resources. Microbiology China, 1985(5): 194-198. (in Chinese)
[2]	BROWN K. Florida fights to stop citrus canker. Science, 2001, 292(5525): 2275-2276. pmid: 11423648
[3]	宋二玲. 三个病原物诱导启动子在转基因柑橘中受溃疡病菌和创伤诱导的表达分析[D]. 重庆: 西南大学, 2013.
	SONG E L. Canker bacterium- and wound-response characteristics of three pathogen-induced promoters in transgenic citrus[D]. Chongqing: Southwest University, 2013. (in Chinese)
[4]	NARDO A D, VITIELLO A, GALLO R L. Cutting edge: Mast cell antimicrobial activity is mediated by expression of cathelicidin antimicrobial peptide. Journal of Immunology, 2003, 170(5): 2274-2278. doi: 10.4049/jimmunol.170.5.2274 pmid: 12594247
[5]	ZASLOFF M. Antimicrobial peptides of multicellular organisms. Nature, 2002, 415(6870): 389-395. doi: 10.1038/415389a
[6]	郑启发, 陈大成, 黄自然, 胡桂兵. 人工合成柞蚕抗菌肽D基因转化沙田柚. 华南农业大学学报, 1999, 20(1): 103-106.
	ZHENG Q F, CHEN D C, HUANG Z R, HU G B. Transformation of the Shatian pummelo (Citrus grandis) with the synthetic oak silkworm antibacterial peptide D gene. Journal of South China Agricultural University, 1999, 20(1): 103-106. (in Chinese)
[7]	陈善春, 张进仁, 黄自然, 高峰, 陈凤珍, 隆有庆. 根癌农杆菌介导柞蚕抗菌肽D基因转化柑桔的研究. 中国农业科学, 1997, 30(3): 7-13.
	CHEN S C, ZHANG J R, HUANG Z R, GAO F, CHEN F Z, LONG Y Q. Studies on Agrobacterium-mediated antibacterial peptide D gene transfer in citrus. Scientia Agricultura Sinica, 1997, 30(3): 7-13. (in Chinese)
[8]	刘琦琦. 转cecropin B基因柑橘对溃疡病抗性的研究[D]. 重庆: 西南大学, 2014.
	LIU Q Q. In vitro resistance of transgenic plants with cecropin B gene to citrus canker caused by Xanthomonas axonopodis pv. citri[D]. Chongqing: Southwest University, 2014. (in Chinese)
[9]	SATYANARAYANA T, GOWDA S, BOYKO V P, ALBIACH- MARTI M R, MAWASSI M, NAVAS-CASTILLO J, KARASEV A V, DOLJA V, HILF M E, LEWANDOWSKI D J, MORENO P, BAR-JOSEPH M, GARNSEY S M, DAWSON W O. An engineered closterovirus RNA replicon and analysis of heterologous terminal sequences for replicatio. Proceedings of the National Academy of Sciences of the United States of America, 1999, 96(13): 7433-7438.
[10]	VIVES M C, MARTÍN S, AMBRÓS S, RENOVELL Á, NAVARRO L, PINA J, MORENO P, GUERRI J. Development of a full-genome cDNA clone of citrus leaf blotch virus and infection of citrus plants. Molecular Plant Pathology, 2008, 9(6): 787-797. doi: 10.1111/j.1364-3703.2008.00501.x pmid: 19019007
[11]	OHIRA K, NAMBA S, ROZANOV M, KUSUMI T, TSUCHIZAKI T. Complete sequence of an infectious full-length cDNA clone of citrus tatter leaf capillovirus: Comparative sequence analysis of capillovirus genomes. Journal of General Virology, 1995, 76(9): 2305-2309. doi: 10.1099/0022-1317-76-9-2305
[12]	GOWDA S, SATYANARAYANA T, ROBERTSON C J, GARNSEY S M, DAWSON W O. Infection of citrus plants with virions generated in Nicotiana benthamiana plants agroinfiltrated with a binary vector based citrus tristeza virus//Proceedings of the Sixteenth Conference of the International Organization of Citrus Virologist. Riverside: IOCV, 2005: 23-33.
[13]	SÁNCHEZ F, MARTINEZ-HERRERA D, AGUILAR I, PONZ F. Infectivity of turnip mosaic potyvirus cDNA clones and transcripts on the systemic host Arabidopsis thaliana and local lesion hosts. Virus Research, 1998, 55(2): 207-219. doi: 10.1016/S0168-1702(98)00049-5
[14]	周彦. 果树病毒载体研究进展. 中国农业科学, 2014, 47(6): 1119-1127.
	ZHOU Y. Progresses in study of virus-based vectors of fruit trees. Scientia Agricultura Sinica, 2014, 47(6): 1119-1127. (in Chinese)
[15]	GALIAKPAROV N, TANNE E, SELA I, GAFNY R. Infectious RNA transcripts from grapevine virus A cDNA clone. Virus Genes, 1999, 19(3): 235-242. pmid: 10595415
[16]	VELA L B, SKARIA M. Seven isolates of citrus tatter leaf virus induce varying levels of xylem tissue abnormalities in two citrange rootstocks//Proceedings of the Fifteenth Conference of the International Organization of Citrus Virologists. Riverside: IOCV, 2002: 371-372.
[17]	MIYAKAWA T, MATSUI C. A bud-union abnormality of Satsuma mandarin on Poncirus trifoliata rootstock in Japan//Proceedings of the Seventh Conference of the International Organization of Citrus Virologists. Riverside: IOCV, 1976: 125-131.
[18]	CAO M J, YU Y Q, TIAN X, YANG F Y, LI R H, ZHOU C Y. First report of citrus leaf blotch virus in lemon in China. Plant Disease, 2017, 101(8): 1561.
[19]	CHAVAN R R, BLOUIN A G, COHEN D, PEARSON M N. Characterization of the complete genome of a novel citrivirus infecting Actinidia chinensis. Archives of Virology, 2013, 158(8): 1679-1686. doi: 10.1007/s00705-013-1654-2
[20]	XUAN Z Y, XIE J X, YU H D, ZHANG S, LI R H, CAO M J. Mulberry (Morus alba) is a new natural host of citrus leaf blotch virus in China. Plant Disease, 2021, 105(3): 716.
[21]	VIVES M C, GALIPIENSO L, NAVARRO L, MORENO P, GUERRIL J. The nucleotide sequence and genomic organization of citrus leaf blotch virus: Candidate type species for a new virus genus. Virology, 2001, 287(1): 225-233. pmid: 11504557
[22]	AGÜERO J, VIVES M C, VELÁZQUEZ K, RUIZ-RUIZ S, JUÁREZ J, NAVARRO L, MORENO P, GUERRI J. Citrus leaf blotch virus invades meristematic regions in Nicotiana benthamiana and citrus. Molecular Plant Pathology, 2013, 14(6): 610-616. doi: 10.1111/mpp.12031
[23]	RENOVELLÁ, GAGO S, RUIZ-RUIZ S, VELÁZQUEZ K, NAVARRO L, MORENO P, VIVES M C, GUERRI J. Mapping the subgenomic RNA promoter of the citrus leaf blotch virus coat protein gene by Agrobacterium-mediated inoculation. Virology, 2010, 406(2): 360-369. doi: 10.1016/j.virol.2010.07.034
[24]	AGÜERO J, RUIZ-RUIZ S, VIVES M C, VELÁZQUEZ K, NAVARRO L, PEÑA L, MORENO P, GUERRI J. Development of viral vectors based on citrus leaf blotch virus to express foreign proteins or analyze gene function in citrus plants. Molecular Plant-Microbe Interactions, 2012, 25(10): 1326-1337. doi: 10.1094/MPMI-02-12-0048-R pmid: 22670755
[25]	崔甜甜. 柑橘黄化脉明病毒和柑橘叶斑驳病毒的侵染性克隆构建[D]. 重庆: 西南大学, 2018.
	CUI T T. Construction of infectious cDNA clones of citrus yellow vein clearing virus and citrus leaf blotch virus[D]. Chongqing: Southwest University, 2018. (in Chinese)
[26]	段玉, 许建建, 马志敏, 宾羽, 周常勇, 宋震. 柑橘叶斑驳病毒的逆转录重组酶聚合酶扩增检测. 中国农业科学, 2021, 54(9): 1904-1912.
	DUAN Y, XU J J, MA Z M, BIN Y, ZHOU C Y, SONG Z. Detection of citrus leaf blotch virus by reverse transcription-recombinase polymerase amplification (RT-RPA). Scientia Agricultura Sinica, 2021, 54(9): 1904-1912. (in Chinese)
[27]	任广伟, 孔凡玉, 王凤龙. 烟草病虫害分级及调查方法: GB/T 23222—2008[S]. (2008-12-31) [2022-07-04].
	REN G W, KONG F Y, WANG F L. Grade and investigation method of tobacco diseases and insect pests: GB/T 23222—2008[S]. (2008- 12-31) [2022-07-04]. (in Chinese)
[28]	DUAN S, JIA H G, PANG Z Q, TEPER D, WHITE F, JONES J, ZHOU C Y, WANG N. Functional characterization of the citrus canker susceptibility gene CsLOB1. Molecular Plant Pathology, 2018, 19(8): 1908-1916. doi: 10.1111/mpp.12667
[29]	李乃坚, 袁四清, 蒲汉丽, 周会光, 戴冕, 陈俊标, 罗战勇, 崔植琳, 黄自然, 李传瑛, 陈凤珍. 抗菌肽B基因转化烟草及转基因植株抗青枯病的鉴定. 农业生物技术学报, 1998, 6(2): 178-184.
	LI N J, YUAN S Q, PU H L, ZHOU H G, DAI M, CHEN J B, LUO Z Y, CUI Z L, HUANG Z L, LI C Y, CHEN F Z. Transfer of cecropin B gene into tobacco plant and confer the bacterial wilt resistance. Journal of Agricultural Biotechnology, 1998, 6(2): 178-184. (in Chinese)
[30]	黎观红, 洪智敏, 贾永杰, 瞿明仁. 抗菌肽的抗菌作用及其机制. 动物营养学报, 2011, 23(4): 546-555.
	LI G H, HONG Z M, JIA Y J, QU M R. Activities and mechanisms of action of antimicrobial peptides. Chinese Journal of Animal Nutrition, 2011, 23(4): 546-555. (in Chinese)
[31]	王志兴, 贾士荣. 抗菌肽分泌型载体的构建及转基因马铃薯中蛋白的胞外分泌. 农业生物技术学报, 1996, 4(3): 277-286.
	WANG Z X, JIA S R. Construction of vectors harboring a barley α-amylase signal sequence and a cecropin B or Shiva A gene that resulted in the extracellular protein secretion in potato transgenic plants. Journal of Agricultural Biotechnology, 1996, 4(3): 277-286. (in Chinese)
[32]	贾士荣, 屈贤铭, 冯兰香, 唐惕, 唐益熊, 刘坤, 赵艳丽, 白永延, 蔡敏莺. 转抗菌肽基因提高马铃薯对青枯病的抗性. 中国农业科学, 1998, 31(3): 5-12.
	JIA S R, QU X M, FENG L X, TANG T, TANG Y X, LIU K, ZHAO Y L, BAI Y Y, CAI M Y. Development of potato clones with enhanced resistance to bacterial wilt by introducing antibacterial peptide gene. Scientia Agricultura Sinica, 1998, 31(3): 5-12. (in Chinese)
[33]	KURTH E G, PEREMYSLOV V V, PROKHNEVSKY A I, KASSCHAU K D, MILLER M, CARRINGTON J C, DOLJA V V. Virus-derived gene expression and RNA interference vector for grapevine. Journal of Virology, 2012, 86(11): 6002-6009. doi: 10.1128/JVI.00436-12 pmid: 22438553
[34]	ZHOU Y, ZHOU C Y, SONG Z, LIU K H, YANG F Y. Characterization of citrus tristeza virus isolates by indicators and molecular biology methods. Agricultural Sciences in China, 2007, 6(5): 573-579. doi: 10.1016/S1671-2927(07)60085-6

引物rimer	引物序列Primer sequence (5′-3′)
CLBV1F	AGCCATAGTTGAACCATTCCTC
CLBV5R	GCAGATCATTCACCACATGC
(3+1)F	AACAAAGCAAAACTCTTAGAAATGTAGCCC
(3+1)R	CTTTAAGAGTGTAAAGTCCTGGCCCACCCA
5-F	TAGATACTAGCAGAACTCGAGCCGAAACAGCTGTTATT
5-R	ACACATAATCCTTCACTACATTTCTAAGAGTTTTGC
3-F	GAGCTGTACAAGTGACCCGGGTCCCGAATTCTGGCATGGGC
3-R	AAATGTTTGAACGATATCGGGGAAATTCGAGCTCT
92bpf1	CAAAACTCTTAGAAATGTAGGAAGGATTATGTGTCTCATGTTAAGTCAG
92bpr1	GAGATTGGGGTAATGATAGATTGAGAAAATCCCGGGATGAGTAAAGGAGAAGAACTTTTCAC
gfpf1	GTGAAAAGTTCTTCTCCTTTACTCATCCCGGGATTTTCTCAATCTATCATTACCCCAATCTC
gfpr1	GCCCATGCCAGAATTCGGGATCCCCCGGGTTATTTGTATAGTTCATCCATGCCATGT