利用基因沉默技术验证东方百合‘Justina’番茄红素β-环化酶基因的功能
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摘要
【目的】研究调控东方百合叶色的类胡萝卜素代谢途径关键酶番茄红素β-环化酶基因功能。【方法】利用基因沉默技术(Virus Induced Gene Silencing,VIGS)构建烟草脆裂病毒与东方百合番茄红素β-环化酶基因的重组载体,采用切割侵染法侵染东方百合‘Justina’植株叶片,分析对照组、烟草脆裂病毒空载体侵染组和携带有番茄红素β-环化酶基因的重组烟草脆裂病毒载体侵染组这些组中东方百合叶片的各种色素含量和番茄红素β-环化酶基因表达量变化。【结果】对照组叶片和空载体侵染组叶片未有明显的白化或黄化现象,重组载体侵染组叶片在刀口边缘出现明显黄化现象,番茄红素β-环化酶基因的表达量及类胡萝卜素含量均明显低于对照组。【结论】番茄红素β-环化酶基因沉默导致东方百合‘Justina’叶片的黄化,抑制类胡萝卜素的合成,表明番茄红素β-环化酶在东方百合‘Justina’叶片类胡萝卜素合成调控中起着重要作用。
【Objective】This study aims to analyse the function of LoLcyB gene,a key enzyme gene of carotenoid metabolism pathway which regulates the color of Lilium orientalis ‘Justina'leaves.【Method】TRV-LoLcyBvectors were constructed by(Virus Induced Gene Silencing)VIGS technique,and the leaves were infected by incision method.The changes of pigment contents and LoLcyBexpressions in leaves of control group,TRV group and TRV-LoLcyB group were analyzed.【Results】The incision on the leaves did not show obvious albinism and eitolation phenomenon between control and TRV groups.The eitolation phenomenon was found at the edges of the leaves of TRV-LoLcyB group.The expression of LoLcyB gene in leaves of TRV-LoLcyB group was significantly lower than that of control group,as well as the carotenoid content.【Conclusion】The eitolation at the incision and the depression of carotenoid content of leaves resulted fromLoLcyBgene silencing.The results indicate that LoLcyB gene involves in the metabolism regulation of carotenoids in leaves of L.orientalis ‘Justina'.
【Objective】This study aims to analyse the function of LoLcyB gene,a key enzyme gene of carotenoid metabolism pathway which regulates the color of Lilium orientalis ‘Justina'leaves.【Method】TRV-LoLcyBvectors were constructed by(Virus Induced Gene Silencing)VIGS technique,and the leaves were infected by incision method.The changes of pigment contents and LoLcyBexpressions in leaves of control group,TRV group and TRV-LoLcyB group were analyzed.【Results】The incision on the leaves did not show obvious albinism and eitolation phenomenon between control and TRV groups.The eitolation phenomenon was found at the edges of the leaves of TRV-LoLcyB group.The expression of LoLcyB gene in leaves of TRV-LoLcyB group was significantly lower than that of control group,as well as the carotenoid content.【Conclusion】The eitolation at the incision and the depression of carotenoid content of leaves resulted fromLoLcyBgene silencing.The results indicate that LoLcyB gene involves in the metabolism regulation of carotenoids in leaves of L.orientalis ‘Justina'.
引文
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[16]Liu Y,Michael S,Rajendra M,Dinesh-Kumar S P.Tobacco Rar1,EDS1and NPR1/NIM1like genes are required for N-mediated resistance to tobacco mosaic virus[J].Plant Journal,2010,30(4):415-429
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[19]Harjes CE,Rocheford TR,Bai L,Brutnell TP,Kandianis CB,Sowinski SG,Stapleton AE,Vallabhaneni R,Williams M,Wurtzel ET.Natural Genetic Variation in Lycopene Epsilon Cyclase Tapped for Maize Biofortification[J].Science,2008(319):330-333
[20]Liu E,Page J E.Optimized cDNA libraries for virusinduced gene silencing(VIGS)using tobacco rattle virus[J].Plant Methods,2008,4(1):5-5
[21]Robertson,Dominique.VIGS Vectors For Gene Silencing:Many Targets,Many Tools[J].Annual Review of Plant Biology,2004,55(1):495-519
[22]陶小荣,周雪平,崔晓峰,钱亚娟.病毒诱导的基因沉默及其在植物基因功能研究中的应用[J].生物化学与生物物理进展,2004,31(9):777-783
[23]张增艳,姚乌兰,辛志勇.植物基因功能鉴定新工具---病毒诱导基因沉默技术的研究进展[J].植物遗传资源学报,2006(1):100-105
[2]Kant R,Sharma S,Dasgupta I.Virus-Induced Gene Silencing(VIGS)for Functional Genomics in Rice Using Rice tungro bacilliform virus,(RTBV)as a Vector[M].New York:Plant Gene Silencing.Springer,2015
[3]Rtclff F,Martin-Herandez A M,Baulcombe D C,Technical sdvance.Tobacco rattle virus as a vector for analysis of gene function by silencing[J].The Plant Journal for Cell and Molecular,2001,25(2):237-245
[4]李建平,郝晓燕,李琴,高升旗,常晓春,陈勋基,足木热木·吐尔逊,陈果,黄全生.棉花VIGS病毒载体的构建及其在抗病基因功能鉴定的应用[J].新疆农业科学,2018,55(7):1203-1208
[5]季娜娜,闵德栋,邵淑君,李富军,张新华.VIGS载体在蔬菜作物中的应用研究进展[J].植物生理学报,2016,52(6):810-816
[6]Kumar P,Pandit SS,Baldwin IT.Tobacco rattle virus vector:a rapid and transient means of silencing Manduca sexta genes by plant mediated RNA interference[J].PLoSOne,2012,7(2):e31347
[7]傅达奇,朱本忠,赵晓丹,朱宏亮,谢远红,罗云波.病毒诱导烟草的基因沉默[J].西北植物学报,2005(8):1517-1520
[8]LU Y,Schiff M,Dlnesh-Kumar S P.Virus induced gene silencing in tomato[J].The Plent Journal for Cell and Molecular Biology,2002,31(6):777-786
[9]吕坤.棉花病毒诱导的基因沉默体系的建立及其在棉花抗黄萎病中的应用[D].南京:南京农业大学,2013
[10]Spitzer B,Zvi M M,Ovadis M,Marhevka E,Barkai O,Edel-baum O,Marton I,Masci T,Alon M,Morin S,Rogachev I,Aharoni A,Vainstein A.Reverse genetics of floral scent:application of tobacco rattle virus-based gene silencing in Petunia[J].Plant Physiology,2007,145:1241-1250
[11]朱长甫,陈星,王英典.植物类胡萝卜素生物合成及其相关基因在基因工程中的应用[J].植物生理与分子生物学学报,2004(6):609-618
[12]莫爱琼,文了,黎海燕,马丽,万小荣.番茄红素β-环化酶基因(LcyB)启动子调控LcyB RNAi双元载体构建[J].华南师范大学学报(自然科学版),2016,48(4):50-56
[13]龙雅宜,张金政,张兰年.百合-球根花卉之王[M].北京:金盾出版社,1999
[14]杨晶,商万有.百合的观赏价值研究[J].吉林农业,2011(9):171-171
[15]梁晶,王莉,丁榕,崔金腾,张克中.百合类胡萝卜素代谢途径关键酶基因的分析及LoLcyB的克隆[J].分子植物育种,2018,16(14):4520-4529
[16]Liu Y,Michael S,Rajendra M,Dinesh-Kumar S P.Tobacco Rar1,EDS1and NPR1/NIM1like genes are required for N-mediated resistance to tobacco mosaic virus[J].Plant Journal,2010,30(4):415-429
[17]黄秋婵,许元明,韦友欢,梁云贞.深色叶植物叶片类胡萝卜素含量比较[J].湖北农业科学,2013,52(11):2573-2574
[18]Cunningham FX,Pogson B,Sun Z,Mc Donald KA,Della Penna D,Gantt E.Functional analysis of the beta and epsilon lycopene cyclase enzymes of Arabidopsis reveals a mechanism for control of cyclic carotenoid formation[J].Plant Cell,1996(8):1613-1626
[19]Harjes CE,Rocheford TR,Bai L,Brutnell TP,Kandianis CB,Sowinski SG,Stapleton AE,Vallabhaneni R,Williams M,Wurtzel ET.Natural Genetic Variation in Lycopene Epsilon Cyclase Tapped for Maize Biofortification[J].Science,2008(319):330-333
[20]Liu E,Page J E.Optimized cDNA libraries for virusinduced gene silencing(VIGS)using tobacco rattle virus[J].Plant Methods,2008,4(1):5-5
[21]Robertson,Dominique.VIGS Vectors For Gene Silencing:Many Targets,Many Tools[J].Annual Review of Plant Biology,2004,55(1):495-519
[22]陶小荣,周雪平,崔晓峰,钱亚娟.病毒诱导的基因沉默及其在植物基因功能研究中的应用[J].生物化学与生物物理进展,2004,31(9):777-783
[23]张增艳,姚乌兰,辛志勇.植物基因功能鉴定新工具---病毒诱导基因沉默技术的研究进展[J].植物遗传资源学报,2006(1):100-105