| Housefly,Musca domestica(Diptera: Muscidae) is an important resources insect, also an important vector insect. Housefly larvae are saprophagous and rich in proteins. They were widely used in the bioconversion of manure and food waste, as well as in the farming of chicken,fish,shrimp,crab and frogs and so on. In the present stduy,we aimed to obtain the gene transcription information by transcriptome sequencing technology,to select stable reference genes under special conditions which can be used in subsequent molecular biology sduties,and to identifiy metallothioneins and evaluate its respond to zinc. The main findings are as follows:Totally 33,810,314 clean reads in our transcriptome data were annotated into 20073 unigenes in known databases. In NR database,12,079 unigenes were annotated; in NT database,3,727 unigenes were annotated; in GO database,26,030 unigenes were annotated; in PFAM database,10,697 unigenes were annotated; in KOG/COG database,7,437 unigenes were annotated; in KO database,3,245 unigenes were annotated.The expression stability of nine reference genes were tested under different experimental conditions. The results showed that in different developmental stages,comprehensive ranking order was: RPL8 > Actin > HSP70 > TUB > RPS18 > GAPDH > EF-1 > 18 S > AK; in different diet treatment, RPL8 > AK > RPS18 > Actin> TUB > GAPDH > EF-1 > 18 S > HSP70; confronting starvation-stress, EF-1 > TUB > RPS18 > HSP70 > RPL8 > Actin > GAPDH > AK > 18S; in different temperature, EF-1 > TUB > Actin > GAPDH > RPS18 > AK > 18 S > RPL8 > HSP70; in different tissues, EF-1 > TUB > AK > RPS18 > Actin > HSP70 > RPL8 > GAPDH > 18S; in different concentrations of Zn Cl2 treatment,EF-1 > HSP70 > TUB > RPL8 > Actin> 18 S > RPS18 > GAPDH > AK.Three metallothionein genes were identified in our transcriptome data. Submitting them into NCBI, three accession numbers were obtained(KP783493,KP783494 and KP783495).When exposure to 5333.33 ug g-1 Zn2+,the expression level of metallothionein significantly higher than that of control and 10000 ug g-1 Zn2+ stress. There is on significant difference between 10000 ug g-1 Zn2+ stress and the control.We obtained a comprehensive transcriptome of housefly larvae,which provides a foundation for the further study of this important insect; investigated the expression stability of 9 reference genes under different conditions which provides a scientific reference and foundation for the accurate quantitative analysis in reference genes selecting; identified three metallothioneins and analyzed the expression of the metallothionein KP783493 when exposure to different doses zinc in diet which is significant important for the further study of metallothioneins respond to heavy metals. |
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