| Salivary protein Nlsalivap-7 is a class of proteins in the saliva of Nilaparvata lugens which are presumed to be involved in regulating rice tricin metabolism.The purpose of this study was to elucidate the expression pattern of the salivary protein Nlsalivap-7 gene in N.lugens and the effect of tricin metabolism in host rice during N.lugens feeding on rice.In this study,through the screening of salivary proteins of N.lugens in response to tricin treatment,a salivary protein Nlsalivap-7 gene with high response to tricin was found by RTq PCR.Two spliceosomes 12896 and 19287 of the salivary protein Nlsalivap-7 gene were cloned using RT-PCR and RACE technology.The real-time quantitative PCR(RT-q PCR)was used to detect the temporal and spatial expression pattern of the salivary protein Nlsalivap-7 gene of N.lugens and the expression of this gene when feeding on different insect-resistant rice variety TN1,Rathu Heenati and IR36.The results are as follows:(1)The salivary proteomics response of N.lugens to tricin stress was clarified from the proteomic level.we analyzed the salivary proteomic response of N.lugens to tricin stimulation by using differential proteomic methods.The results showed that the differential proteins in N.lugens salivary treated with tricin for 6 h reaches 90(including 62 up-regulated proteins and 28 down-regulated proteins).The differential proteins inside the N.lugens sample treated with tricin for 24 h significantly less than treated for 6 h,and the number of different proteins just was 27(including 10 up-regulated proteins and 17 down-regulated proteins).Tricin stimulation can make serine protease(SP)and its homologous(SPH)inside the N.lugens salivary gland changing obviously.Through by GO function analysis and KEGG pathway analysis towards the differential proteins,we found that the differential proteins in N.lugens salivary gland mainly belongs to stress proteins and metabolism proteins,and total of their ratio more than 50 percent.The N.lugens salivary gland proteins responses to tricin stimulation participating the following pathways,such as amino metabolism,salivary secretion system,energy metabolism,carbohydrate metabolism,and so on.By RTq PCR analysis of the N.lugens proteome,it was found that the expression of the salivary protein gene Nlsalivap-7 was up-regulated by 11.5 times compared with the blank control for tricin stimulation.(2)Nlsalivap-7 spliceosomes clone and expression pattern analysis of N.lugens.Results by comparing the Brown Planthopper Genome Database of the State Key Laboratory of Pest Control and Resource Utilization,Sun Yat-sen University(Unpublished),it was shown that the salivary protein Nlsalivap-7 gene has two similar sequences,using RACE technology to amplify the full length of salivary protein Nlsalivap-7 gene,sequencing results obtained two transcripts of salivary protein Nlsalivap-7 gene(19287 and 12896).19287 is hardly expressed in 1-5th instar nymph stages,and it is expressed to varying degrees in adult stages.The expression level of adults is higher than other adult stages at 9,11,13,and 15 d.The expression of 19287 in N.lugens feeded on Rathu Heenati rice varieties was lower than that of N.lugens feeded on TN1 rice varieties by 83.09%.The difference was significant.The population of TN1 N.lugens was not significantly different from the N.lugens of IR36 population.19287 was the highest expressed in the salivary glands of N.lugens,followed by the midgut,head,and it is almost no expression in fat bodies,feet and ovaries.12896 is hardly expressed in the 2-4th instar nymph stages,and it is slightly expressed in the 1st instar nymphs and adults at 9,13,and 15 d.The expression is highest at 11 d than in other adult developmental ages.There is no significant difference in expression of 12896 in N.lugens populations feeding on three different insect-resistant rice varieties.12896 had higher expression levels in the midgut and salivary glands,and it is also expressed in the feet,and the expression level in the fat bodies,head,and ovaries is low.(3)Study on the function of Nlsalivap-7 gene of salivary protein of N.lugens.The RNA interferes with the 12896 and 19287 spliceosome of female adults that have just emerged in1 d,The RNA interferes can significantly reduced the expression levels of the two spliceosomes.The expression of 19287 spliceosome decreased by 81.36% compared with the control group at 48 h.It reached a significant level after 72 h,which was only 1.13% of the control group,while the expression of 12896 decreased by 91.27% at 48 h,and the expression level was only 1.58% at 72 h,which was hardly expressed.The amount of honeydew produced by the silenced 19287 N.lugens was reduced by 7.8% compared with the control group,and the amount of honeydew produced by the silenced 12896 spliceosome was reduced by 52.5%;Silencing N.lugens of 19287 and 12896 spliceosomes could stimulate feeding frequency of N.lugens on host plants.The frequency of feeding is specifically manifested by the fact that the feeding holes caused by RNA interference on rice stalks are significantly higher than those of the control group.Feeding holes left on rice stalks by N.lugens that silenced 19287 spliceosome doubled,After silencing 12896 spliceosome,the feeding holes left by N.lugens on rice stalks doubled;the weight of silenced 19287 spliceosome N.lugens decreased by 31.79%,and the weight of silenced 12896 spliceosome N.lugens decreased by 24.16%,which were significantly lower than the control group.It has been clarified that the salivary protein Nlsalivap-7 gene is involved in regulating tricin metabolism in host plant rice.Different spliceosomes play different roles in regulating tricin metabolism and N.lugens metabolism in TN1 rice.After TN1 rice was damaged by the RNA interference 19287 spliceosome N.lugens,the tricin content increased by 2.3,which was higher than TN1 rice damaged by N.lugens of the control group.There was no significant difference between the RNA interference 12896 spliceosome N.lugens and the control group feeded rice plant.It can be seen that the 19287 and 12896 spirosomes of N.lugens salivary protein Nlsalivap-7 gene both inhibit rice flavonoid metabolism,and the 12896 spliceosome may also play an important role in regulating the induction of flavonoids and metabolism in N.lugens. |
