Identification And Function Study Of Hermetia Illucens Metallothionein Gene

Black soldier fly(Hermetia illucens)larvae(BSFL)are outstanding insects for bioconversion of organic waste,which can convert separate or mixed waste,i.e.manure,kitchen waste and straw,into biomass rich in protein and fat.Studies found that BSFL can survive in high-concentration heavy metal environments,and BSFL can be used to treat heavy metal-contaminated organic waste,such as manure from livestock farms and crop straw from heavy metal-contaminated farmland.In order to explore the heavy metal tolerance of BSF,researches mainly focused on the effects of heavy metals on life cycle parameters of BSF,but the molecular mechanism was still unclear.In this study,metallothionein gene was used as the target gene to explore its important role in heavy metal tolerance in Hermetia illucens.The main results are as follows:1.Evaluation of internal reference gene stability in black soldier flyThe stability of reference genes were different under different experimental conditions,for example,EF1 and Actin would be selected under different tissues condition;Actin and RPL8 should be used at different developmental stages;Tubulin and RPL8 were recommended under different genders.By comprehensively analyzing the expression of reference genes under all experimental conditions,Ref Finder showed that Actin and RP49 were recommended for using in q PCR experiments.2.BSF MT genes sequences and expression pattern analysisThe c DNA sequences of three metallothionein genes(BSFMT1,BSFMT2 A,and BSFMT2B)were cloned from transcriptome of BSFL.The expression pattern analysis showed that BSFMT1 was not highly expressed throughout larval stage,but was highly expressed in adults and fat bodies.BSFMT2 A and BSFMT2 B were highly expressed in larval stage,and the expression increased with the development of the larvae.Under different concentrations of cadmium,the expression of MT genes in BSFL increased with the increase of cadmium concentration.3.Functional analysis of BSF MT genesThe prokaryotic expression results showed that three BSF MT genes could increase the tolerance of E.coli to Cd stress,but only BSFMT2 A and BSFMT2 B significantly increased the Cd accumulation of E.coli.After interfering with BSFL BSFMT2 B expression through RNAi,the mortality of larvae at 300 mg/kg Cd was 50.02%,which was significantly higher than that of the control group(18.06%),and the fresh weight of larvae(0.050 g)was significantly lower than the control group(0.067 g).Therefore,BSFMT2 B played an important role in the tolerance of black soldier fly to heavy metal stress.This study showed that MT genes were highly expressed in BSF,and the heavy metal tolerance of BSFL was inseparable from MT genes.This study clarified the function of the MT genes in black soldier fly,which was helpful to promote the application of black soldier fly bioadsorption of heavy metals.