Functional Analysis Of Anthranilate Synthase LetrpE Related To Lentinula Edodes Heat-Tolerance

Lentinula edodes(Berk.)Pegler is one of the most important and commercialized cultivation mushrooms.During cultivation process,high temperature resulted in the slow growth and development for hypha,even rot logs,causing a heavy loss in economy.Anthranilate synthase TrpE(EC:4.1.3.27),an important stress-induced protein in organisms,was important for the survival capacity of Thermus thermophiles subjected to high temperature.Previous study showed that after 24 hours of 40℃ heat stress,LetrpE displayed a significant increase for protein level but an obvious decrease for transcript level in L.edodes strain S606.Additionally,exogenous anthranilate acid in sawdust medium improved the thermotolerance of L.edodes,suggesting that LetrpE could play an important role in response to heat stress for L.edodes mycelia(unpublished).This study aims at exploring the function of LetrpE gene by RNAi and over-expression methods in L.edodes.L.edodes cultivation strain S606 was treated as the tested strain.Coding sequence of LetrpE was amplified in the template of S606 DNA.395 bp antisense fragment from the conserved domain of LetrpE was promoted dually via Legpd and Leactin promotors to generate RNAi vector.Coding sequence of LetrpE was promoted via Leactin promotor to construct over-expression vector.The vector was transferred to L.edodes(S606)mycelia via Agrobacterium tumefaciens mediation method.The stable transformants were obtained via five-time selection on MYG medium containing 6μg/mL hygromycin.Special primers were used to analyze all transformant,and 11 RNAi and 20 over-expression transformants were detected.According to qRT-PCR results,it was found that compared with wild type S606,LetrpE expression level in LetrpE-i-37 and LetrpE-i-53 had lowered to 29.327% and42.779%,respectively,indicating that those two strains were LetrpE silence transformants;the expression levels of LetrpB,LeTam-1 and LeYUCCA related to IAA biosynthesis pathway had down-regulation when subjected to 40℃ heat stress for 24 h.Thermotolerance assay showed that wild type could regrow after 24 h heat stress at 40℃,while two transformants could not regrow.Based on the qRT-PCR analysis of over-expression transformants,we found that LetrpE expression levels in three transformants(LetrpE-o-11,LetrpE-o-13,LetrpE-o-14)had two to three fold upregulation,indicating that those three strains were LetrpEover-expression transformants;compared with wild type,LetrpB and LeTam-1expression levels had about 2-fold increase,and expression level of LeYUCCA,a last enzyme in IAA biosynthesis pathway,had about 10-fold up-regulation.Mycelia of the over-expression transformants subjected to 41℃ heat stress for 24 h could regrow at25℃,but wild type could not regrow.These results indicated that LetrpE,the upstream gene involved in IAA biosynthesis pathway,performed an important function on L.edodes thermotolerance.This study will provide the important theoretical significance and practical value on molecular mechanism and genetic basis as well as the new variety selection of L.edodes thermotolerance.