TRV介导的大豆基因瞬时沉默体系的建立

Establishment of TRV-mediated Transient Gene-Silencing System in Soybean

【目的】病毒诱导的基因沉默(virus-induced gene silencing,VIGS)技术已在植物基因功能研究领域得到广泛应用。建立以TRV为载体介导的大豆基因瞬时沉默体系,为将烟草脆裂病毒(Tobacco rattle virus,TRV)介导的基因沉默技术在大豆与大豆花叶病毒(Soybean mosaic virus,SMV)互作体系中对大豆基因功能进行研究提供基础。【方法】从大豆品种冀豆7号(J7)叶片中特异性扩增八氢番茄红素脱氢酶基因(Gm PDS)的部分片段,并将该基因片段插入质粒p TRV﹕RNA2中;向J7的第一位真叶注射携带有TRV或TRV﹕Gm PDS的农杆菌,注射后对上部未接种病毒的叶片进行表型观察,并通过半定量RT-PCR检测Gm PDS的表达量。为探讨接种TRV是否影响大豆对SMV的抗性表现,在大豆第一片真叶上预接种TRV病毒后10 d,再分别接种SMV株系N3和SC-8,以单独接种N3或SC-8作为对照。待接种SMV后第5天观察未接种的上位叶表型并检测SMV的外壳蛋白基因CP。【结果】大豆叶片注射携带有TRV﹕Gm PDS的农杆菌后25 d,上部未接种病毒的叶片出现了白化现象,通过半定量RT-PCR分析,发生白化的植株中Gm PDS的表达量明显降低。在此基础上,向大豆叶片预注射携带有TRV的农杆菌后再接种SMV,SMV株系N3和SC-8与J7分别组成不亲和与亲和组合。发现J7第一片真叶上预接种TRV后再接种SMV株系N3,与单独接种N3对上位叶的影响在表型上表现一致,对未接种的上位叶片进行病毒外壳蛋白基因CP检测,发现J7单独接种N3以及预接种TRV后再接种N3的上位叶片上均未能检测到CP表达。而J7单独接种SMV株系SC-8,以及预接种TRV后再接种SC-8均在上位叶上能够检测到CP。说明在J7上预接种TRV不影响J7对SMV的抗性。在感病品种南农1138-2上也获得了相似结果。【结论】建立了以TRV为载体介导的大豆基因瞬时沉默体系,并证明在大豆上先接种TRV不改变大豆对SMV的抗性表现。

【Objective】Virus-induced gene silencing（VIGS） technology has been widely applied in the field of functional gene research in plants. Tobacco rattle virus-mediated gene silencing technology was used in the interaction between soybean and soybean mosaic virus（SMV） for gene function evaluation, and a TRV-mediated gene transient silence system in soybean was established.【Method】Soybean cv. Jidou 7（J7） was used as material, and specifically amplified part of the Gm PDS gene fragment from J7 leaves, and then was inserted into the plasmid p TRV： RNA2; The Agrobacterium carried with TRV or TRV：Gm PDS were injected into the first true leaves of J7. After that, the upper non-inoculated leaves were observed and Gm PDS gene＇s expression level was confirmed by semi-quantitative RT-PCR analysis. In order to investigate the effect of inoculation with TRV on the resistance of soybean to SMV infection, the first true leaves of soybean inoculated with TRV for 10 days, then were inoculated with SMV strain N3 or SC-8. The control treatments were conducted by inoculating N3 or SC-8 alone. At 5 days after inoculating SMV, the phenotypes of upper uninoculated leaves were observed and SMV coat protein CP was detected. 【Result】At 25 days after injected with Agrobacterium suspension carrying TRV： Gm PDS into the first true leaves, the upper uninoculated leaves showed whitening phenotype and expression level of Gm PDS was decreased significantly by semi-quantitative RT-PCR analysis. Based on this, soybean leaves injected with Agrobacterium suspension（carrying TRV） were then inoculated with SMV. J7 and SMV strains N3 and SC-8 were used to constitute incompatible and compatible combinations. Inoculated with TRV and then with SMV strains N3 or SC-8, the phenotype of upper uninoculated leaves were consistent with the upper leaves after inoculated with SMV alone. Leaves were taken for viral coat protein CP test. It was found that when inoculated with N3 alone, or pre-inoculated with TRV and then inoculated with N3, the coat protein CP was not detected on the upper leaves. If inoculated with SC-8 alone, or pre-inoculated with TRV and then inoculated with SC-8, the viral coat protein CP was detected on the uninoculated upper leaves. These results showed that the TRV had no influence on the performance of soybean resistance to SMV. The similar results were got on susceptible cv. Nannong 1138-2. 【Conclusion】The TRV-VIGS system in soybean was established in this experiment. The TRV has no influence on the performance of soybean resistance to SMV.