香菇HMG-box转录因子lelcrp1基因的功能

Function analysis of HMG-box transcription factor lelcrp1 in Lentinula edodes

【目的】研究香菇(Lentinula edodes) HMG-box转录因子LELCRP1 (Lentinula edodes lignocellulase genes regulation protein 1)在木质纤维素降解相关酶基因表达中的功能与作用。【方法】通过double-joint及同源重组方法构建lelcrp1基因RNAi载体,采用根癌农杆菌介导转化的方法转入香菇异核菌株W1菌丝中,筛选得到RNAi转化子,通过Southern杂交检测插入片段在菌株W1基因组中的拷贝数量。采用荧光定量PCR检测RNAi转化子木质纤维素降解酶基因表达水平变化,并在含有3.5μg/mL潮霉素的MYG平板上测定RNAi转化子的菌丝生长速度。【结果】获得了4个lelcrp1基因表达水平与出发菌株W1相比显著下调6–7倍的RNAi转化子。Southern杂交结果显示,lelcrp1基因RNAi片段已成功整合至香菇菌株W1基因组内,并以单拷贝形式存在。对其中2个RNAi转化子的26个木质纤维素降解酶基因表达水平进行分析,发现其中9个纤维素酶基因、1个半纤维素酶基因、2个辅助酶AA9基因和1个锰过氧化物酶基因的表达水平均表现出明显的下调。平板生长试验表明,RNAi转化子菌丝生长速度均显著慢于出发菌株W1。【结论】通过RNAi技术成功抑制了香菇异核菌株中lelcrp1基因表达水平,并导致部分纤维素及木质素酶基因表达水平相应下调,首次发现HMG-box结构域的转录因子能调控木质纤维素降解相关酶基因表达。

[Objective]The objective of this study was to analyze the function of the HMG-box transcription factor LELCRP1(Lentinula edodes lignocellulase genes regulation protein 1)in the expression of lignocellulase genes. [Methods]The RNAi vector of lelcrpl was constructed by double-joint and homologous recombination,then transformed into the mycelia of Lentinula edodes heterotypic strain W1 by Agrobacterium tumefaciens-mediated transformation (ATMT).The copy number of the inserted fragment in the genome of strain W1 was detected by Southern blotting.Fluorescent quantitative PCR was used to detect the expression level of lignocellulase genes in RNAi transformants,and the mycelial growth rate was measured on MYG plates.[Results]We got four RNAi transformants which were significantly down-regulated by 6 to 7-fold compared with the original strain W1.The results of Southern blotting showed that the lelcrpl gene fragment had been successfully integrated into the genome of Lentinula edodes in a single copy.Analysis of expression profiles of 26 lignocellulase genes in two RNAi transformants revealed that 9 cellulase genes,1 hemicellulase gene,2 auxiliary enzymes AA9 gene and 1 manganese peroxide gene were down-regulated significantly,compared to the original strain.The mycelial growth rate of RNAi transformants were significantly slower than the original strain.[Conclusion]The expression of lelcrpl was silenced successfully by RNAi,resulting in down-regulation of the expression level of some cellulose and ligninase genes.The HMG-box domain transcription factor could regulate the expression of lignocellulase genes.