基于病毒诱导的基因沉默(VIGS)方法的LsRGL1基因在生菜中的功能分析

Function analysis of LsRGL1 gene in lettuce(Lactuca sativa L.)based on virus-induced gene silencing(VIGS)

为了确定LsRGL1基因对生菜抽薹的影响,以S39生菜品种为试验材料,通过逆转录聚合酶链反应(RT-PCR)从S39生菜品种中克隆得到LsRGL1基因;采用病毒诱导基因沉默(VIGS)方法,利用烟草脆裂病毒(TRV)载体对目的基因进行沉默;采用表型观察、石蜡切片、实时荧光定量PCR(qPCR)方法、酶联免疫吸附法(ELISA)分别研究空白对照组(无TRV载体)、阴性对照组(按质量比1∶1同时注射TRV1空载体和TRV2空载体)和试验组(按质量比1∶1同时注射TRV1空载体和TRV2-LsRGL1重组载体)中植株表型、花芽分化、LsRGL1基因的相对表达量和内源激素质量分数的变化。结果表明:病毒侵染后,试验组植株的茎长相对于空白对照组和阴性对照组的茎长明显增大,且试验组各项表型数据均大于2个对照组的表型数据,而空白对照组与阴性对照组各项表型数据之间并未出现明显差异;石蜡切片结果表明试验组植株已经开始花芽分化,相较于对照组提前进入生殖生长时期;qPCR分析结果显示,试验组中LsRGL1基因的相对表达量相较于空白组和阴性对照组的显著下调,且下调幅度均超过50%。试验组中内源激素赤霉素(GA3)、脱落酸(ABA)、生长素(IAA)的含量显著高于对照组的含量,而茉莉酸(JA)含量显著下降。综上可知,沉默LsRGL1基因加快了生菜的抽薹。

To determine the influence of the LsRGL1 gene on lettuce bolting,the S39 lettuce variety was used as the experimental material,and the LsRGL1 gene was cloned from the S39 lettuce variety using reverse transcription polymerase chain reaction(RT-PCR).The goal gene was silenced using a virus-induced gene silencing(VIGS)method with a tobacco rattle virus(TRV)vector.Methods of phenotypic observation,paraffin section,real-time fluorescence quantitative PCR(qPCR)and enzyme-linked immunosorbent assay(ELISA)were used to study the phenotypes,bud differentiation,relative expression levels of the LsRGL1 gene,and endogenous hormone mass fractions changes in the blank control group(without TRV vector),negative control group(injection of TRV1 empty vector and TRV2 empty vector in a mass ratio of 1∶1),and experimental group(injection of TRV1 empty vector and TRV2-LsRGL1 recombinant vector in a mass ratio of 1∶1).The results showed that after virus infection,the stem length of the experimental group was significantly larger than that of the blank control group and the negative control group,and the data of all traits in the experimental group were all greater than those of the two control groups.There was no obvious difference in the traits of the blank control group and the negative control group.The paraffin section results showed that the experimental group had begun bud differentiation,entering the reproductive growth period earlier than the control group.The qPCR analysis results showed that compared with the blank group and the negative control group,the relative expression level of the LsRGL1 gene in the experimental group was significantly downregulated with 50%more in the amplitude.The mass fractions of endogenous hormones gibberellin(GA3),abscisic acid(ABA)and auxin(IAA)in the experimental group were significantly higher than those of the control group,while the mass fraction of jasmonic acid(JA)was significantly lower.In summary,silencing the LsRGL1 gene could accelerate bolting in lettuce.