Cloning Of Transcription Factor TcNrf2 From Tetranychus Cinnabarinus And Its Regulation On Antioxidant Enzymes

Cassava is a staple food and an important industrial raw material for nearly 800 million people in the world.T.cinnabarinus is one of the four major pests to cassava,and it is difficult to control by using pesticides.Cultivating and using insect-resistant varieties is the most economical,effective,and simple way to control pests.Studies showed that the activity of antioxidant enzymes in agricultural parasites is closely related to their host adaptability.Transcription factor Nrf2(Nuclear factor(erythroid-derived 2)-like 2)plays a key role in the regulation of antioxidant enzymes in vertebrates.However,up to now,no reports have been reported on the regulation of antioxidant enzymes by Nrf2 in pests(mites).Therefore,it is the first study to research that Nrf2 was cloned from T.cinnabarinus.The effects of inducing and inhibiting Nrf2 on the mortality of T.cinnabarinus,the expression of antioxidant enzymes genes and the activity of antioxidant enzymes were studied during feeding resistance and susceptibility to mite cassava germplasm.The preliminary analysis of the regulation function of Nrf2 on antioxidant enzymes of T.cinnabarinus was carried out,and the theoretical basis was provided for the molecular.The main findings are as follows:1.It is the first time that the transcription factor gene TcNrf2 of T.cinnabarinus was cloned.The full length of TcNrf2 gene of T.cinnabarinus was obtained by 5’and 3’RACE-PCR and sequence splicing.Sequencing results showed that the size of CDS region of TcNrf2 gene was 1950 bp.And further bioinformatics analysis confirmed that the cloned gene was TcNrf2,a transcription factor of T.cinnabarinus.2.The vertebrate Nrf2 inhibitor Brusatol(BA)and Retinoic acid(RA),Nrf2 activator Tert-Butylhydroquinone(TBHQ)and 3H-1,2-DITHIOLE-3-THIONE(D3-T)were found.T.cinnabarinus has a sublethal effect.The mortality of C.sinensis after Id,4d and 8d after feeding and resistance to cassava cultivars increased with the concentration of inhibitor and activator.The mortality of the T.cinnabarinus after treatment with the inhibitors BA,inhibitor RA,activator TBHQ,activator D3T and the resistance to cassava cultivar BRA900 and C1115 cultivar C1115 were 3.33%-20.96%,respectively.And 3.33%-64.76%,3.33%-9.85%and 3.33%-38.71%,3.33%-11.54%and 3.33%-51.06%,3.33%-9.92%and 6.67%-52.40%,both significantly higher than the control(P<0.05),the mortality of T.cinnabarinus leaves after treatment with 1.25μM BA,RA,TBHQ and D3T for 1d,4d and 8d was the smallest(P<0.05),which could be used as the most suitable treatment concentration for subsequent qPCR.3.It was found that the inhibitor BA treatment significantly inhibited the expression of TcNrf2 and antioxidant enzymes genes of T.cinnabarinus and susceptible cassava varieties,while the inhibitor RA had not.By contrast,the activator TBHQ and D3T treatment significantly increased the expression of TcNrf2 and antioxidant enzymes genes of T.cinnabarinus and susceptible cassava varieties.After BA treatment,TcNrf2 and antioxidant enzymes TcSOD,TcCAT,TcPOD and TcPPO of resistant and susceptible cassava varieties were significantly lower than those before feeding T.cinnabarinus(P<0.05),and the decrease was greater after feeding C1115,which was about three times lower than that of control.While after the treatment with RA inhibitor,there was no significant change in the expression of the above genes in resistant and susceptible cassava varieties with feeding T.cinnabarinus.When treated with activator TBHQ and D3T respectively,the expression of the above genes in resistant and susceptible cassava varieties,which was fed with T.cinnabarinus,increased significantly(P<0.05),and the increase was greater after feeding BRA900,which was between 2.36 and 3.92 times.4.It was found that BA treatment significantly inhibited the activity of antioxidant enzymes of T.cinnabarinus after feeding with resistant and susceptible cassava varieties,while RA treatment had not.However,TBHQ and D3T treatment significantly increased the activity of antioxidant enzymes of T.cinnabarinus after feeding with resistant and susceptible cassava varieties.After treatment with BA,SOD,CAT,POD and PPO,the resistant and susceptible cassava varieties were significantly decreased(P<0.05),compared with those not been fed.The decrease was greater after feeding C1115,and the decrease was between 3.04 and 4.15 times compared with untreated cassava varieties.The above antioxidant enzymes of resistant and susceptible cassava varieties,which fed with T.cinnabarinus,did not change significantly after treatment with RA inhibitor.When treated with activator TBHQ and D3T respectively,the above enzymes of resistant and susceptible cassava varieties,which was fed with T.cinnabarinus increased significantly(P<0.05),and after feeding BRA900,the enhancements were greater,with the increase multiples ranging from 2.51 to 3.46 times.In this study,the full-length sequence of TcNrf2 of Tetranychus cinnabar was cloned,and the effect of TcNrf2 inhibitor and activator on the expression of downstream protective enzymes gene was further confirmed at the transcriptional level.Which can provide theoretical basis on using TcNrfl as control target amd genetic resources that applied to molecular design breeding of crop resistance to insect(mite).