番茄茉莉酸缺失突变体灰霉菌侵染响应miRNA及其表达分析

为揭示miRNA对番茄灰霉菌胁迫的响应机制，以番茄茉莉酸（JA）缺失突变体def1、spr2及其野生型（CM）为试验材料，构建了3个材料接种灰霉菌前后（0 h、48 h）2个时期的miRNA文库，并采用Illumina平台测序，对测序数据进行生物信息分析，结合实时荧光定量检测目的miRNA及其预测的靶基因表达情况。结果表明灰霉菌侵染番茄后，JA缺失突变体def1和spr2的病情指数显著高于CM，H2O2含量低于CM。高通量测序鉴定了130个已知miRNA和811个新miRNA。进一步筛选出8个保守miRNA（Sly-miR156e-3p、Sly-miR166c-5p、Sly-miR171f、Sly-miR172b、Sly-miR319a、Sly-miR390b-5p、Sly-miR399b、Sly-miR482d-5p），其在6个样本中表达模式各异。预测差异miRNA靶基因122个，结合qRT-PCR技术分析了8个miRNA和8个靶基因的表达情况，与高通量测序结果基本一致。推测Sly-miR156e-3p、Sly-miR390b-5p、Sly-miR399b、Sly-miR482d-5p通过依赖JA信号途径参与番茄对灰霉病的抗性。

Expressied Analysis of miRNA with Tomato JA Deficient Mutant Reponse to Botrytis cinerea Infection

In order to investigate the mechanism of miRNA response to Botrytis cinerea in tomato，two JA deficient mutants def1 and spr2，and the wild type（Castlemart）plant had been used in this study. miRNA database were conducted following RNA-seq and bioinformatics analysis after B.cinerea inoculation on plant materials at 0 h and 48 h.The qRT-PCR was performed to examine the target miRNA and its predicted target gene expression levels.The results showed that JA defective mutants were more sensitive than wild type to B. cinerea. And H2O2 content was also lower compared to that of wild type. One hundred and thirty known miRNAs and 811 new miRNAs had been identified by RNA-seq，and 8 conserved miRNAs（Sly-miR156e-3p，Sly-miR166c-5p，Sly-miR171f，Sly-miR172b，Sly-miR319a，Sly-miR390b-5p，Sly-miR399b and Sly-miR482d-5p）showed different expression patterns which had been selected in 6 samples. Altogether 122 predicted target genes were obtained，and the expression level of 8 miRNAs and their target genes have been verified by qRT-PCR which showed consistent results with RNA-seq.Therefore，Sly-miR156e-3p，Sly-miR390b-5p，Sly-miR399b and Sly-miR482d-5p may involve in B. cinerea resistance through JA pathway.