Data_Sheet_3_Construction of a heat-resistant strain of Lentinus edodes by fungal Hsp20 protein overexpression and genetic transformation.docx

The shiitake mushroom (Lentinus edodes) is the second most popular edible mushroom globally due to its rich nutritional value and health benefits associated with consumption. However, the characteristics of growing at low temperatures limit the area and time of its cultivating. We selected a low-temperature cultivar as the original strain. We proposed to construct a heat-shock protein expression vector to achieve genetic transformation in this low-temperature strain to improve the survivability of the strain against the heat-shock response. In this study, an overexpression vector pEHg-gdp-hsp20 for the heat shock protein 20 gene of A. bisporus was constructed using a homologous recombination method. This vector was transferred into dikaryotic and monokaryotic mycelia by the Agrobacterium tumefaciens-method. The integration of hygb and hsp20 into the genome of L. edodes mycelia was verified by growth experiments on resistant plates and PCR analysis. The expression of the reporter gene mgfp5 was verified by fluorescence microscopy analysis and statistically resulted in 18.52 and 26.39% positivity for dikaryon, and monokaryon, respectively. Real-time PCR analysis showed that the expression of the hsp20 gene was more than 10-fold up-regulated in the three transformants; the mycelia of the three overexpression transformants could resume growth after 24 h heat treatment at 40°C, but the mycelia of the starting strain L087 could not recover growth at 25°C indicating that strains that successfully expressed hsp20 had greater overall recovery after heat shock. According to the study, A. bisporus hsp20 gene overexpression effectively improves the defensive capability of low-temperature mushroom strains against heat shock, laying the foundation for breeding heat-resistant high-quality transgenic shiitake mushrooms.

香菇（Lentinus edodes）是全球第二大受欢迎的食用蘑菇，因其丰富的营养价值与食用带来的健康益处而备受青睐。然而，其低温生长的特性限制了其栽培的地域范围与栽培时段。本研究选取一株低温栽培品种作为出发菌株，拟构建热激蛋白表达载体，对该低温菌株进行遗传转化，以提升菌株应对热激胁迫的存活能力。本研究通过同源重组法，构建了双孢蘑菇（A. bisporus）的热激蛋白20基因（hsp20）过表达载体pEHg-gdp-hsp20。采用根癌农杆菌（Agrobacterium tumefaciens）介导法，将该载体转化至双核菌丝与单核菌丝中。通过抗性平板生长实验与PCR分析，验证了hygb与hsp20基因整合至香菇菌丝基因组的情况。通过荧光显微镜分析验证了报告基因mgfp5的表达，统计结果显示双核菌丝与单核菌丝的阳性转化率分别为18.52%与26.39%。实时荧光定量PCR分析显示，三株转化子中hsp20基因的表达量上调幅度均超过10倍；三株过表达转化子的菌丝在40℃热激处理24小时后，转移至25℃条件下可恢复生长，而出发菌株L087的菌丝在相同条件下无法恢复生长，表明成功表达hsp20的菌株在热激胁迫后的整体恢复能力更强。本研究结果表明，双孢蘑菇hsp20基因的过表达可有效提升低温食用菌菌株应对热激胁迫的防御能力，为培育耐热优质转基因香菇奠定了基础。