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

Abstract

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.

Genomic characterization of Bacillus cereus isolated from food poisoning cases revealed the mechanism of toxin production

“…As illustrated in Figure 1 , the close clustering pattern between LY01-LY09 and B. anthracis suggests that LY01-LY09 may be pathogens that are harmful to humans as well. Previous studies have demonstrated that HSP20 exhibits chaperone activity, which can enhance cellular tolerance to damage and maintain normal cellular metabolism under various stresses, thus improving cell survival rates ( Kranzler et al, 2016 ; Ling et al, 2022 ; Niu et al, 2022 ). LY07 and LY09 had more than one Hsp20 genes, some of which undergone point mutations and loss of long amino acids.…”

Two Strains of Lentinula edodes Differ in Their Transcriptional and Metabolic Patterns and Respond Differently to Thermostress

“…The optimum growth temperature for L. edodes mycelium ranges from 24 to 27 °C and the lethal temperature could be 38 °C or above, depending on the strain [ 26 ]. Heat stress appears to be the major abiotic constraint inhibiting mycelium growth, disease resistance, and fruiting body development, thereby seriously reducing fruiting body productivity and the quality of L. edodes [ 24 , 53 , 54 , 55 ]. Therefore, it is of great significance to decipher the mechanisms by which L. edodes addresses thermostress.…”

“…A considerable number of studies have accumulated over the years addressing the topic of heat resistance in the mycelium of L. edodes [ 15 , 26 , 55 ]. Previous studies have documented that heat shock proteins (HSPs), indoleacetic acid (IAA), catalase, and trehalose play crucial roles in the thermotolerance of L. edodes [ 15 , 29 , 30 ].…”

“…In this paper, we found that 13 Hsp20 genes were significantly up-regulated in response to heat stress in both the H- and L-type L. edodes strains, highlighting the importance of HSPs for L. edodes to cope with heat stress. More recently, it was reported that overexpression of the Agaricus bisporus Hsp20 gene enhanced the mycelial thermotolerance of L. edodes [ 55 ]. Moreover, the HSP40 protein LeDnaJ07 was also proven to be integral to conferring heat resistance on L. edodes [ 15 , 29 , 30 ].…”

Transcriptome Metabolic Characterization of <em>Tuber borchii</em> SP1, a New Spanish Strain for<em> In Vitro</em> Studies of the Bianchetto Truffle

“…However, the search for BlastP hits of this protein reveals its presence in T. brumale, T. aestivum, T. indicum, T. magnatum, and many other ascomycetes. This protein has been used to construct a temperature-tolerant strain of Lentinula edodes [38] and found in tandem repeats in a temperature-adapted Coriolopsis trogii. The model identified in T. borchii corresponds to a secreted protein.…”