TRV介导的观赏花烟草(Nicotiana sanderae)VIGS体系的构建
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- 英文论文题名：Establishment of TRV-mediated VIGS System in Nicotiana sanderae
- 论文作者：丁海琴 ; 张茜 ; 曾祥玲 ; 王艳丽 ; 郑日如 ; 罗靖 ; 王彩云
- 英文论文作者：DING Hai-qin ; ZHANG Qian ; ZENG Xiang-ling ; WANG Yan-li ; ZHENG Ri-ru ; LUO Jing ; WANG Cai-yun ; Key Laboratory for Biology of Horticultural Plants of Ministry of Education ; Huazhong Agricultural University ; Wuhan Nusun Landscape Co.Ltd
- 年：2016
- 作者机构：华中农业大学园艺植物生物学教育部重点实验室;武汉农尚环境股份有限公司;
- 论文关键词：观赏花烟草 ; 病毒诱导的基因沉默 ; 烟草脆裂病毒 ; 不依赖连接的克隆 ; 花色花香
- 英文论文关键词：Nicotiana sanderae ; Virus-induced gene silencing ; Tobacco rattle virus ; Ligation-independent cloning ; Flower color and floral
- 会议召开时间：2016-07-19
- 会议录名称：中国观赏园艺研究进展2016
- 语种：中文
- 分类号：S682.19
- 学会代码：EGYP
- 会议名称：2016年中国观赏园艺学术研讨会
- 会议地点：中国湖南长沙
- 主办单位：中国园艺学会观赏园艺专业委员会、国家花卉工程技术研究中心
- 学会名称：中国园艺学会
- 页数：5
- 文件大小：668k
- 原文格式：O
- 会议级别：全国
摘要
本研究以八氢番茄红素脱氢酶(Phytoene desaturase,PDS)基因和查尔酮合酶(Chalcone synthase,CHS)基因为报告基因,以烟草脆裂病毒(Tobacco rattle virus,TRV)为载体建立基于病毒介导的观赏花烟草(Nicotiana sanderae)基因沉默体系(Virus-induced gene silencing,VIGS)。结果表明,根吸收法不适用于观赏花烟草的基因沉默体系,而利用叶背注射渗透法对观赏花烟草进行基因沉默的效果较好。在叶背注射渗透法中,用PDS基因作为报告基因时,处理组植株在全黑暗条件下培养1天后进行常规养护,25天后80%植株表现出叶片光漂白现象。在用花青素合成代谢路径上关键基因CHS作为报告基因时,60%的植株在开花后表现出预期花冠褪红斑白的现象,半定量RT-PCR结果显示被沉默植株的CHS基因表达量明显下调,观赏花烟草内源CHS基因被有效沉默。综上所述,本研究初步建立了观赏花烟草VIGS体系。
Explored and established an appropriate TRV-mediated virus-induced gene silencing system in Nicotiana sanderae,making Phytoene desaturase(PDS) and Chalcone synthase(CHS) gene as reporter genes,to establish an effective system for the research of gene function and provide new reference for the ornamental plant gene functional verification.The results showed that the root absorption method does not apply to gene silencing system of tobacco flower,while the dorsal injection infiltration method is more effective.When using PDS gene as a reporter gene in the dorsal injection infiltration method,the plants were treated in darkness for one day first.Then after 25 days of routine maintenance,80%of the plants showed blade photo bleaching which means the PDS gene was silenced.When using the anthocyanin synthesis metabolic pathway key genes CHS as a reporter gene,60%of the plants exhibited erythema faded white corolla after flowering.Semi-quantitative RT-PCR showed that the CHS gene expression reduced significantly in gene silenced plants,suggesting tobacco endogenous CHS gene is effectively silenced.In summary,this study initially established an effective VIGS system in Nicotiana sanderae.
Explored and established an appropriate TRV-mediated virus-induced gene silencing system in Nicotiana sanderae,making Phytoene desaturase(PDS) and Chalcone synthase(CHS) gene as reporter genes,to establish an effective system for the research of gene function and provide new reference for the ornamental plant gene functional verification.The results showed that the root absorption method does not apply to gene silencing system of tobacco flower,while the dorsal injection infiltration method is more effective.When using PDS gene as a reporter gene in the dorsal injection infiltration method,the plants were treated in darkness for one day first.Then after 25 days of routine maintenance,80%of the plants showed blade photo bleaching which means the PDS gene was silenced.When using the anthocyanin synthesis metabolic pathway key genes CHS as a reporter gene,60%of the plants exhibited erythema faded white corolla after flowering.Semi-quantitative RT-PCR showed that the CHS gene expression reduced significantly in gene silenced plants,suggesting tobacco endogenous CHS gene is effectively silenced.In summary,this study initially established an effective VIGS system in Nicotiana sanderae.
引文
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11.Bennypaμl HS,Mutti JS,Rustgi S et al.Virus-induced gene silencing(VIGS)of genes expressed in root,leaf,and meiotic tissues of wheat[J].Function Integrate Genomics,2012,12:143-156.
12.崔艳红,贾芝琪,李颖,等.利用VIGS研究马铃薯晚疫病抗性基因R3a和RB的信号传导[J].园艺学报,2009,36(7):997-1004.
13.Liu YL,Schiff M,Czymmek K et al.Autophagy regμlates programmed cell death during the plant innate immune response[J].Cell,2005,121:567-577.
14.Burch-Smith TM,Anderson JC,Martin GB and Dinesh-Kumar S P.Applications and advantages of virus-induced gene silencing for gene function studies in plants[J].Plant Journal,2004,39(5):734-746.
15.Doroszewska T,Depta A,Czubacka A.Album of Nicotiana species.Institute of Soil Science and Plant Cμltivation National Reseach Institute,2009,Pμlawy.
16.宋震,李中安,周常勇.病毒诱导的基因沉默(VIGS)研究进展[J].园艺学报.2014,41(9):1885-1894.
17.Zheng S J,Snoeren T A L,Hogewoning S W et al.Disruption of plant carotenoid biosynthasis through virusinduced gene silencing affects oviposition behaviour of the butterfly Pieris rapae[J].New Phytology,2010,186(3):733-745.
18.Spitzer B,Zvi MMB,Ovadis M et al.Reverse Genetics of floral scent;application of Tobacco rattle virus-based gene silencing in Petunia[J].Plant Physiology,2007,145;1241-1250.
19.Nagamatsu A,Masuta C,Senda M et al.Functional analysis of soybean genes involved in flavonoid biosynthesis by virusinduced gene silencing[J].Plant Biotechnology Journal,2007,5:778-790.
20.Deng XB,Elomaa P,Nguyen CX et al.Virus-induced gene silencing for Asteraceae—a reverse genetics approach for functional genomics in Gerbera hybrida[J].Plant Biotechnology Journal,2012,10:970-978.
2.Scofield S R,Huang L,Brandt A S et al.Development of a virus-induced gene-silencing system for hexaploid wheat and its use in functional analysis of the Lr21-mediated leaf rust resistance pathway[J].Plant Physiology,2005,138:2165-2173.
3.Ratcliff F,Martin-Hernandez A M,Baμlcombe D C.Tobacco rattle virus as a vector for analysis of gene function by silencing[J].The Plant Journal,2001,25(2):237-245.
4.Kumagai MH,Donson J,Della-Cioppa G et al.Cytoplasmic inhibition of carotenoid biosynthesis with virus-derived RNA[J].Proceedings of the National Academy of Sciences,1995,92:1679-1683.
5.Burch-Smith T M,Schiff M,Liu Y,Dinesh-Kumar S P.Efficient virus-induced gene silencing in Arabidopsis[J].Plant Physiology,2006,142:21-27.
6.Ryu C M,Anand A,Kang L et al.Agrodrench:a novel and effective agroinoculation method for virus-induced gene silencing in roots and diverse Solanaceous species[J].Plant Journal,2004,40:322-331.
7.Senthil-Kumar M,Hema R,Anand A et al.A systematic study to determine the extent of gene silencing in Nicotiana benthamiana and other Solanaceae species when heterologous gene sequences are used for virus-induced gene silencing[J].New Phytology,2007,176:782-791.
8.Valentine T,Shaw J,Blok V C et al.Efficient virus-induced gene silencing in roots using a modified tobacco rattle virus vector[J].Plant Physiology,2004,136:3999-4009.
9.LiuHai-ping,Fu Da-qi,Zhu Ben-zhong et al.Virus-induced gene silencing in eggplant(Solanum melongena)[J].Journal of Integrative Plant Biology,2012,54(6):422-429.
10.Zhu H L,Zhu B Z,Shao Y et al.Tomato fruit development and ripening are altered by the silencing of LeEIN2 gene[J].Journal of Integrate Plant Biology,2006,48:1478-1485.
11.Bennypaμl HS,Mutti JS,Rustgi S et al.Virus-induced gene silencing(VIGS)of genes expressed in root,leaf,and meiotic tissues of wheat[J].Function Integrate Genomics,2012,12:143-156.
12.崔艳红,贾芝琪,李颖,等.利用VIGS研究马铃薯晚疫病抗性基因R3a和RB的信号传导[J].园艺学报,2009,36(7):997-1004.
13.Liu YL,Schiff M,Czymmek K et al.Autophagy regμlates programmed cell death during the plant innate immune response[J].Cell,2005,121:567-577.
14.Burch-Smith TM,Anderson JC,Martin GB and Dinesh-Kumar S P.Applications and advantages of virus-induced gene silencing for gene function studies in plants[J].Plant Journal,2004,39(5):734-746.
15.Doroszewska T,Depta A,Czubacka A.Album of Nicotiana species.Institute of Soil Science and Plant Cμltivation National Reseach Institute,2009,Pμlawy.
16.宋震,李中安,周常勇.病毒诱导的基因沉默(VIGS)研究进展[J].园艺学报.2014,41(9):1885-1894.
17.Zheng S J,Snoeren T A L,Hogewoning S W et al.Disruption of plant carotenoid biosynthasis through virusinduced gene silencing affects oviposition behaviour of the butterfly Pieris rapae[J].New Phytology,2010,186(3):733-745.
18.Spitzer B,Zvi MMB,Ovadis M et al.Reverse Genetics of floral scent;application of Tobacco rattle virus-based gene silencing in Petunia[J].Plant Physiology,2007,145;1241-1250.
19.Nagamatsu A,Masuta C,Senda M et al.Functional analysis of soybean genes involved in flavonoid biosynthesis by virusinduced gene silencing[J].Plant Biotechnology Journal,2007,5:778-790.
20.Deng XB,Elomaa P,Nguyen CX et al.Virus-induced gene silencing for Asteraceae—a reverse genetics approach for functional genomics in Gerbera hybrida[J].Plant Biotechnology Journal,2012,10:970-978.