VIGS沉默番茄SlDCL2和SlDCL4破坏Ty-1/Ty-3对TYLCV的抗性

VIGS Silencing SlDCL2 and SlDCL4 Destroy Tomato Ty-1/Ty-3 Resistance to Tomato Yellow Leaf Curl Virus(TYLCV)

番茄黄化曲叶病毒(TYLCV)严重威胁茄科蔬菜作物的生产。抗性标记Ty-1和Ty-3是一对等位基因,在番茄抗TYLCV育种中应用较为广泛。为了探究Ty-1/Ty-3抗病毒分子机制,本研究以携带Ty-1/Ty-3抗性标记的番茄Y19为材料,利用病毒诱导基因沉默(VIGS)技术,沉默RNA干扰(RNAi)机制关键基因,即番茄核糖核酸内切酶编码基因2a/b/c/d(SlDCL2)和番茄核糖核酸内切酶编码基因4(SlDCL4),初步分析其在Ty-1/Ty-3抗TYLCV中的功能。PCR与测序结果发现SlDCL2、SlDCL4的VIGS沉默载体pTRV2∶SlDCL2和pTRV2∶SlDCL4构建成功。以沉默番茄八氢番茄红素脱氢酶(SlPDS)基因为标记植株,定量PCR检测SlDCL2、SlDCL4沉默株系中SlDCL2、SlDCL4基因的沉默效率,结果显示SlDCL2、SlDCL4沉默株系中对应基因的表达均小于对照植株的50%,表明SlDCL2、SlDCL4沉默载体确实可以降低对应基因的表达,且不影响其他DCL基因的表达。VIGS沉默植株SlDCL2、SlDCL4基因后接种TYLCV,接种TYLCV 30dpi结果显示,Y9材料表现出明显的TYLCV病毒症状,通过比较植株发病严重度发现,SlDCL2、SlDCL4沉默株系发病严重度分别为1.97、2.35,显著高于空载体注射株系(0.14)和对照株系(0.07)。本研究结果表明RNAi通路关键基因SlDCL2、SlDCL4在Ty-1/Ty-3抗TYLCV中发挥着重要作用,这为利用Ty-1/3抗性标记基因育种奠定了基础。

Tomato yellow leaf curl virus(TYLCV) seriously threatens the production of Solanaceae vegetable crops. Resistance markers Ty-1 and Ty-3 are a pair of allele genes, which are widely used in tomato breeding for TYLCV resistance. In order to explore the antiviral molecular mechanism of Ty-1/Ty-3, tomato ‘Y19’ with the Ty-1/Ty-3 resistance marker was used as the material, the key genes SlDCL2a/b/c/d(SlDCL2) and SlDCL4 involved in RNA interference(RNAi) were silenced by virus induced gene silencing(VIGS) technology, and their function in resistance of Ty-1/Ty-3 to TYLCV was study. The targeted fragments of SlDCL2 and SlDCL4 were amplified and sequenced, results showed that the silencing vector, pTRV2∶SlDCL2 and pTRV2∶SlDCL4 were constructed successfully. The silencing of tomato PDS(SlPDS) gene was used as a marker for successful silencing in plant, and the silencing efficiency of the SlDCL2 or SlDCL4 genes in the SlDCL2 or SlDCL4 silencing lines was detected by quantitative PCR(qPCR). The results showed that the expressions of corresponding gene in the SlDCL2 and SlDCL4 silencing lines were 50% less than that in the control plants, which indicated that SlDCL2 and SlDCL4 silencing vectors can indeed reduce the expression of corresponding genes. SlDCL2-silenced and SlDCL4-silenced plants were inoculated with TYLCV respectively. The results showed that the ‘Y9’ material had obvious symptoms of TYLCV, the disease severity of SlDCL2-and SlDCL4-silenced plants were 1.97±1.01, 2.35±0.75 respectively, significantly higher than that of the plants transformed with empty vector(0.14±0.24) and no vector(0.07±0.19). This result indicates that SlDCL2 and SlDCL4 play important roles in the resistance of Ty-1/Ty-3 to TYLCV, which laid a foundation for tomato breeding with resistance marker genes of Ty-1/3.