The Role Of Transferrin Family Genes In The Survival And Fecundity Of Brown Planthopper

The brown planthopper（BPH）,Nilaparvata lugens St（?）l,is a serious hemiptera insect pest of rice.Insect transferrin is a protein with multiple functions,which plays a critical role in insects.Different transferrin family members may play different roles in iron homeostasis and innate immunity.However,roles of different transferrins in survival and fecundity of BPH have been rarely demonstrated,and their potentials in controlling BPH are still unknown.Therefore,it is of great necessary to study the influence of transferrin family genes interference on the BPH and analyze the potential of transferrin family genes as a control target for the BPH.In this study,three transferrin genes NlTsf1,NlTsf2 and NlTsf3 were screened out based on the previous transcriptome information of the interaction between BPH and different rice varieties.The full-length cDNA of the three genes was obtained by PCR amplification.The expression patterns at different developmental stages and in different tissues of BPH were studied by real-time fluorescence quantitative PCR（RT-PCR）.The expression levels of these three genes were knocked down by RNA interference（RNAi）technology to study their effects on growth,development and fecundity of the BPH.Results are as follows:1.Cloning and expression of transferrin genes NlTsf1,NlTsf2 and NlTsf3 in BPH1.1 Cloning and sequence analysis of full-length cDNAThe full-length cDNA sequences of NlTsf1,NlTsf2 and NlTsf3 were obtained by PCR.The full-length cDNA sequences of NlTsf1,NlTsf2 and NlTsf3 were 2085 bp,2451 bp and 2241 bp,respectively.The proteins encoded by NlTsf1,NlTsf2 and NlTsf3contained 694,800 and 744 amino acids,respectively.SMART online tools analysis showed that NlTsf1,NlTsf2 and NlTsf3 all had a signal peptide and two transferrin domains,but only NlTsf2 had a transmembrane domain.In addition,NlTsf3 contained a phosphonate-bd domain（391-605）that overlaping with a second transferrin domain（378-708）.1.2 The expression pattern revealed by RT-qPCRRT-qPCR results showed that three transferrin genes,NlTsf1,NlTsf2 and NlTsf3,were expressed in different tissues and at different developmental stages of the brown planthopper.The expression patterns were similar in all tissues,but they showed different expression patterns at different developmental stages.The expression of NlTsf1 gene was higher in nymphs and males,but lower in eggs and females.NlTsf2gene was highly expressed in the middle stage of male and egg development,but rarely expressed in nymphs and females.The expression level of NlTsf3 gene was low in eggs at day 1 and high at all other developmental stages.NlTsf1,NlTsf2 and NlTsf3genes showed similar expression patterns in different tissues or body parts of adults.In terms of tissue expression level,the expression levels of NlTsf2 and NlTsf3 genes were higher in the thorax,epidermis and head,while the expression levels of NlTsf2and NlTsf3 genes were the lowest in the ovary.The expression levels of NlTsf2 and NlTsf3 genes were similar in other tissues except the head.In addition,RT-qPCR results showed that the relative expression levels of three transferrin genes,NlTsf1,NlTsf2 and NlTsf3,were all suppressed to varying degrees in the BPH reared on resistant rice R1.Among the transferrin genes NlTsf1,NlTsf2 and NlTsf3,NlTsf3 gene was most severely inhibited by resistant rice R1,and its relative expression was only0.1,while that of BPH reared on susceptible rice S9 was 0.9.The expression of NlTsf1gene was also significantly inhibited by resistant rice R1,and the inhibition of NlTsf2gene expression to resistant rice R1 showed no significant difference compared with the control group.2.Functional studies of transferrin gene family NlTsf1,NlTsf2 and NlTsf3 in BPH2.1 Detection of RNA interference effectRNAi of NlTsf1,NlTsf2 and NlTsf3 genes by microinjection showed good interference effect on the 3^rd instar nymphs,females and males.RT-qPCR results showed that the gene expression levels of the three transferrin family genes in the dsNlTsfs-treated group were significantly lower than those in the GFP control group within 5 days after the nymphs were interfered,and the efficiency of RNAi was the best on the day 5,with the expression levels reduced by 99.4%,96.00%and 97.3%,respectively.The expression levels of three transferrin family genes were significantly decreased by 93.8%,96.3%and 55.1%,respectively,on day 4 after interference on females.Similarly,compared with the control group,the expression level was significantly decreased by 80.1%,93.3%and 65.1%,respectively,on day 4 after interference on males.Therefore,RNAi of NlTsf1,NlTsf2 and NlTsf3 genes can effectively inhibit the expression of target genes.2.2 The effect of RNA interference on the survival of BPHRNAi of three transferrin genes had different effects on the survival of BPH.compared with the control group treated with dsGFP,the survival rate of nymphs treated with dsNlTsf3 decreased sharply from day 2 to day 4,and then gradually slowed down to 3.3%at day 11,while the survival rate of nymphs treated with dsNlTsf1 and dsNlTsf2 decreased to 58.9%and 56.7%,respectively.However,the survival rate in the dsGFP control group was 78.9%.The male survival rate showed similar trendsafter dsNlTsf1,dsNlTsf2 and dsNlTsf3 injection.The male survival rate on day 11 was 11.1%,0%and 0%,respectively,while the male survival rate in dsGFP control group was 73.3%.RNAi of three transferrin genes showed different lethal effectson female.The lethal effect of dsNlTsf3 was the strongest,followed by dsNlTsf1.Compared with dsGFP control,dsNlTsf2 showed no significant lethal effect except for a slight decrease on days 11 and 12.The survival rates of dsNlTsf1 and dsNlTsf3 on the 15th day were 36.9%and 5.6%,respectively,while the survival rates of dsNlTsf2group and dsGFP control group were still higher,68.9%and 72.2%,respectively.2.3 Effects of RNA interference on fecundity and reproductive organs of BPHRNAi of three transferrin genes also had different effects on the fecundity and reproductive organs of BPH.Compared with dsGFP control group,RNAi of NlTsf3gene significantly affected the shape of ovary.Oocytes in ovary were abnormally enlarged,and there were almost no banana-shaped eggs,which were spherical.There was no significant difference in the shape of ovary in dsNlTsf1/dsNlTsf2-treated females compared with dsGFP control group.However,some eggs in the dsNlTsf1treatment group were L-shaped.There was no significant difference in morphology of testis between dsRNA treatment group and dsGFP control group.After interfering with NlTsf3 gene by dsNlTsf3,the number of eggs of the female was seriously reduced by 99.4%,and after interfering with NlTsf1 gene,the number of eggs of the female was decreased by 48.6%.However,after interfering with NlTsf2 gene,the number of eggs of the female was not significantly affected.Interference with NlTsf2 and NlTsf3significantly reduced the egg hatching rate,and dsNlTsf3 significantly reduced the egg hatching rate from 86.5%to 10.8%,while interference with NlTsf1 had no significant effect on the egg hatching rate.In conclusion,three transferrin family genes of NlTsf1,NlTsf2 and NlTsf3 were cloned and identified in this study.The spatio-temporal expression patterns of NlTsf1,NlTsf2 and NlTsf3 genes were revealed,and their expression patternsin BPH on resistant rice and sensibility were explored.The roles of three genes in survival and fecundity of BPH were studied using RNAi tecknique.Therefore,our work shed light on more general aspects of the functional properties of different transferrin members and revealed that transferrin family genes NlTsf1,NlTsf2 and NlTsf3 were differentially involved in the interaction between the rice and the BPH.Most importantly,our work suggested potential targets NlTsf1 and NlTsf3 for BPH control.Our work provided a novel clue for further study on the mechanism of how rice resists against BPH.