沉默2b基因获得抗黄瓜花叶病毒(CMV)的转基因烟草
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摘要
【目的】本研究利用dsRNA技术沉默2b基因,获得烟草品种云烟85的T_1代转基因抗CMV株系。【方法】构建含有CMV 2b部分序列反向重复的植物表达载体pBIN-2b(r)-In-2b(i),以农杆菌介导的叶盘法转化普通烟草(Nicotiana tabacum)品种云烟85。【结果】抗性筛选获得112株T_0代转基因阳性植株,接种CMV进行抗病性鉴定,发现有46株T_0代转基因植株表现为抗病,其他66株表现为感病。Tas-ELISA检测表明,T_0代抗病烟株的病毒积累量显著低于感病烟株。选取10个T_0代抗病株系繁殖,接种CMV鉴定T_1代抗病性,发现部分T_1代烟株发生一定比例的抗感分离,T_1代抗性株率为46.7%～100%。Real-time PCR检测T_1代烟株,发现T_1代抗病烟株CMV 2b基因RNA积累水平显著低于感病烟株。【结论】基于以上策略及实验,获得抗CMV转基因烟草株系。
Sense and antisense fragments of 5' end of gene 2b were ampli?ed respectively using gene 2b of Cucumber mosaic virus(CMV)as template. Inverted repeat plant expression vector was constructed, and tobacco(Nicotiana tabacum) cultivar Yunyan 85 was transformed via Agrobacterium tumefaciens mediation. One hundred and twelve T_0 generation transgenic plants were acquired by resistance screening. It was found that 46 transgenic tobacco plants were resistant and other 66 transgenic tobacco plants are susceptible to CMV via resistance identi?cation by inoculating CMV. It was indicated that virus accumulation amount of resistant plants was signi?cantly lower than that of susceptible plants by Tas-ELISA detecting. Ten T_0 resistance transgenic tobacco lines were randomly selected and propagated from the forty-six T_0 resistance transgenic tobacco plants. It was found that T_1 progeny of partial resistant lines generated a certain proportion of segregation of resistance to susceptibility by observing the symptoms after inoculation, and the proportion of resistant plants was 46.7%～100%. T_1 generation tobacco plants generating segregation of resistance to susceptibility were detected by real-time PCR. It was found that RNA accumulation level of gene 2b in resistant tobacco plants was signi?cantly lower than that in susceptible tobacco plants. Gene 2b was silenced by using dsRNA technique, and T_1 generation transgenic tobacco cultivar Yunyan 85 resistant to CMV was obtained.
Sense and antisense fragments of 5' end of gene 2b were ampli?ed respectively using gene 2b of Cucumber mosaic virus(CMV)as template. Inverted repeat plant expression vector was constructed, and tobacco(Nicotiana tabacum) cultivar Yunyan 85 was transformed via Agrobacterium tumefaciens mediation. One hundred and twelve T_0 generation transgenic plants were acquired by resistance screening. It was found that 46 transgenic tobacco plants were resistant and other 66 transgenic tobacco plants are susceptible to CMV via resistance identi?cation by inoculating CMV. It was indicated that virus accumulation amount of resistant plants was signi?cantly lower than that of susceptible plants by Tas-ELISA detecting. Ten T_0 resistance transgenic tobacco lines were randomly selected and propagated from the forty-six T_0 resistance transgenic tobacco plants. It was found that T_1 progeny of partial resistant lines generated a certain proportion of segregation of resistance to susceptibility by observing the symptoms after inoculation, and the proportion of resistant plants was 46.7%～100%. T_1 generation tobacco plants generating segregation of resistance to susceptibility were detected by real-time PCR. It was found that RNA accumulation level of gene 2b in resistant tobacco plants was signi?cantly lower than that in susceptible tobacco plants. Gene 2b was silenced by using dsRNA technique, and T_1 generation transgenic tobacco cultivar Yunyan 85 resistant to CMV was obtained.
引文
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[11]朱俊华,朱常香,温孚江,等.正向和反向重复RNA介导的抗马铃薯Y病毒基因工程比较研究[J].植物病理学报,2004,34(2):133-140.ZHU Junhua,ZHU Changxiang,WEN Fujiang,et al.Comparison of resistance to Potato virus Y mediated by direct and inverted repeats of the coat protein gene segments in transgenic tobacco plants[J].Acta Phytopathologica Sinica,2004,34(2):133-140.
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[2]DíAZ-PENDóN J A,DING Shouwei.Direct and indirect roles of viral suppressors of RNA silencing in pathogenesis[J].Annual Review of Phytopathology,2008,46:303-326.
[3]BAULCOMBE D C.Unwinding RNA silencing[J].Science,2000,290(5494):1108-1109.
[4]LINDBO J A,SILVA-ROSALES L,PROEBSTING W M,et al.Induction of a highly specific antiviral state in transgenic plants:Implications for regulation of gene expression and virus resistance[J].Plant Cell,1993,5(12):1749-1759.
[5]江彤,邓竹根,宋培培,等.利用dsRNA介导的抗病性获得抗马铃薯Y病毒(PVY)的转基因烟草[J].热带作物学报,2013,34(4):648-653.JIANG Tong,DENG Zhugen,SONG Peipei,et al.Transgenic tobacco plants resistant to Potato virus Y(PVY)via dsRNA-mediated virus resistance[J].Chinese Journal of Tropical Crops,2013,34(4):648-653.
[6]叶艳英,曾钢,曹鸣庆,等.HC-Pro基因片段介导的高抗TuMV[J].作物学报,2014,40(3):550-555.YE Yanying,ZENG Gang,CAO Mingqing,et al.HC-Pro gene segment mediated hyper-resistance to Turnip mosaic virus[J].Acta Agronomica Sinica,2014,40(3):550-555.
[7]郑璐平,林辰,谢荔岩,等.水稻条纹病毒NS2和NS3基因共干扰转基因水稻的培育及抗病性分析[J].病毒学报,2014,30(6):661-667.ZHENG Luping,LIN Chen,XIE Liyan,et al.Construction of Rice stripe virus NS2 and NS3 Co-RNAi transgenic rice and diseaseresistance analysis[J].Chinese Journal of Virology,2014,30(6):661-667.
[8]宋培培,李爽,檀根甲,等.沉默抑制子CMV 2b基因的克隆及其反向重复植物表达载体的构建[J].激光生物学报,2010,19(3):347-352.SONG Peipei,LI Shuang,TAN Genjia,et al.Cloning of silencing suppressor CMV 2b gene and its construction of plant expression vector containing inverted repeat fragments[J].Acta Laser Biology Sinica,2010,19(3):347-352.
[9]BRIGNETI G,VOINNET O,LI Wanxiang,et al.Viral pathogenicity determinants are suppressors of transgene silencing in Nicotiana benthamiana[J].EMBO Journal,1998,17(22):6739-6746.
[10]SMITH N A,SINGH S P,WANG M B,et al.Gene expression:total silencing by intron-spliced hairpin RNAs[J].Nature,2000,407(6802):319-320.
[11]朱俊华,朱常香,温孚江,等.正向和反向重复RNA介导的抗马铃薯Y病毒基因工程比较研究[J].植物病理学报,2004,34(2):133-140.ZHU Junhua,ZHU Changxiang,WEN Fujiang,et al.Comparison of resistance to Potato virus Y mediated by direct and inverted repeats of the coat protein gene segments in transgenic tobacco plants[J].Acta Phytopathologica Sinica,2004,34(2):133-140.
[12]CALLIS J,FROMM M,WALBOT V.Introns increase gene expression in cultured maize cells[J].Genes and Development,1987,1(10):1183-1200.
[13]TANNAKA A,MITA S,OHTA S,et al.Enhancement of foreign gene expression by a dicot intron in rice but not in tobacco is correlated with an increased level of mRNA and an efficient splicing of the intron[J].Nucleic Acids Research.1990,18(23):6767-6770.
[14]STOUTJESDIJK P A,SINGH S P,LIU Qing,et al.hpRNA-mediated targeting of the arabidopsis FAD2 gene gives highly efficient and stable silencing[J].Plant Ecophysiology.2002,129(4):1723-1731.