Cloning And MRNA Expression Analysis Of Four Development-related Genes In The Brown Plant Hopper,Nilaparvata Lugens (Stal)

The brown planthopper （BPH）, Nilaparvata lugens （Stal）（Homoptera:Delphacidae） is an important rice pest in Asian countries. At present, chemical control is still the primary means of BPH management. However, irrational use of large amounts of chemical insecticides has lead to the development of BPH resistance to insecicides including organophosphates, carbamates, pyrethroids, neonicotinoids and phenylpyrazoles insecticides as well as some insect growth regulators. On the other hand, some chemical insecticdes cause BPH resurgence by exhibiting high toxicity against BPH natural enemies or stimulating BPH reproduction under sublethal dosages, which has been a serious problem in BPH management. Histone acetyltransferases （HATs） and deacetylases （HDACs） as well as Vacuolar H+-ATPase （V-ATPase） play an important role in insect growth and development. In this study, cDNA sequences of HATs （ELP3and Mof）, HDACs （HDAC3） and V-ATPase d subunit （NIVHA-d） gene in BPH were cloned by RT-PCR and RACE. The mRNA expression of these genes in different developmental stages of BPH and the effects of sublethal dose of triazophos on the expression level of these genes were analyzed by real-time qPCR.Full-length cDNA sequences of ELP3, Mof, HDAC3and V-ATPase d subunit gene were cloned by RT-PCR and RACE. Sequence analysis showed that the1656bp ORF of ELP3encoded551amino acid residues, and predicted a protein with molecular mass of63.06251kDa. The1440bp cDNA sequence of Mof contains a1353bp ORF encoding450amino acid residues, and predicted a protein with molecular mass of53.44258kDa. The1299bp ORF of HDAC3encoded349amino acid residues, and predicted a protein with molecular mass of49.07634kDa. The1247bp cDNA sequence of V-ATPase d subunit gene contains a1050bp ORF encoding349amino acid residues, and predicted a protein with molecular mass of39.52216kDa. Online SMART analysis showed that ELP3contains elongation factor Elp3domain and HATs domain （Pfam:Acetyltransf₁）, Mof contains CHROMO domain, HATs domain （Pfam:MOZ_SAS） and Zinc finger domain （ZnF_C2H2）, HDAC3contains HDACs domain （Pfam:Hist_deacetyl） and Blast:STYKc domain, V-ATPase d subunit contains Pfam:vATP-synt_AC39domain. Multiple sequence alignment showed that these genes have high sequence identity with homologous genes in other insects.The real-time qPCR analysis revealed that the mRNA expression of ELP3gene was the highest in macropterous female adults, while lowest expression was observed in fourth instar nymphs. The mRNA expression of ELP3in female adults was significantly higher than that in male adults, and the expression level in first instar nymphs was higher than other nymphs. In contrast to ELP3gene, there was no significant difference between the expressions of Mof gene in BPH nymphs at each stage. However, the expression level of Mof gene in female adults was also significantly higher than that in male adults, and the expression level was the highest in macropterous female adults and the lowest in macropterous male adults. As for HDAC3gene, its mRNA expression level in female adults was also significantly higher than that in male adults, and the highest expression level was observed in macropterous female adults. In case of NIVHA-d gene, its mRNA expression was developmentally regulated and the female adults showed higher levels of expression than the male adults.The effects of sublethal dose of triazophos on the expression levels of these genes were analyzed by real-time qPCR. The results showed that the relative expressions of ELP3in brachypterous female adults treated with LD30of triazophos at first and third day after eclosion were1.60-and2.03-fold higher than that in control treatment with acetone, respectively. However, the relative expressions of ELP3in macropterous female adults treated with LD30of triazophos at first and third day after eclosion were0.31-and0.57-fold lower than that in control treatment with acetone, respectively. At first day after eclosion, both of the relative expressions of Mof gene in brachypterous male adults treated with LD10and LD30of triazophos as well as macropterous female and male adults treated with LD30of triazophos significantly decreased to0.43,0.61,0.52and0.70times of control treatment, respectively. At third day after eclosion, the relative expressions of Mof gene in male adults treated with LD10of triazophos significantly reduced to0.52and0.54times of control treatment, respectively. As for HDAC3gene, its relative expressions in brachypterous and macropterous male adults treated with LD30of triazophos at first day after eclosion significantly reduced to0.67and0.65times of control treatment, respectively. At third day after eclosion, its relative expressions in macropterous male adults significantly reduced to0.37and0.69times of control treatment, respectively. However, its relative expressions in brachypterous female adults treated with LD10and LD30of triazophos significantly increased to1.94and2.00times of control treatment, respectively. Furthermore, at third day after eclosion, the relative expressions of NIVHA-d in brachypterous male adults treated with LD10and LD30of triazophos were2.15and2.46times higher than that in control treatment with acetone, respectively.