Molecular Cloning And Characterization Of Genes For Biosynthesis Of Branch Chain Amino Acid And Aromatic Amino Acids In Nilaparvata Lugens

Branched chain amino acids includes valine,leucine and isoleucine.Aromatic amino acids have three members,i.e.,phenylalanine,tyrosine and tryptophan.Both of the two groups belong to the essential amino acids that can not be biosynthesized in the brown planthopper,Nilaparvata lugens(Stal)(Hemiptera:Delphacidae).In the present paper,based on N.lugens-symbiont transcriptome and genome data,we focused on the identification of synthesis-related genes of the six amino acids.Morevoer,using RT-PCR and RACE,we obtained main genes encoding enzymes catalyzing the biosynthesis of the six amino acids.Finally,using RNA interference,we successfully knocked down two genes,NIBCAT and NlPDH,and observed their negative effects on nymphal survival.The results are summarized as follows.1.Molecular cloning and characterization of genes involved in biosynthesis of branched chain and aromatic amino acidsBased on N.lugens-symbiont transcriptome and genome data,49 fragments involved in biosynthesis of branched chain and aromatic amino acids were found.After combination,36 fragments were obtained.Using RT-PCR,19 fragments were substantiated.By RACE,8 full-length cDNAs were obtained,along with five cDNAs lacking 5’-ends and three cDNAs lacking 3’-ends.Comparison with genome data revealed that those genes from N.lugens had more and longer introns,whereas those genes from symbiont had less and shorter introns.According to these enzymes,N.lugens and symbiont enzymes may work collaboratively to catalyze the biosynthesis of valine,leucine,isoleucine,phenylalanine and tyrosine.Symbionts function in the biosynthesis of tryptophan.2.Origins of NIBCAT and NlPDH genesFrom genome data,genes encoding branched-chain amino acid aminotransferase(EC 2.6.1.42,BCAT)and prephenate dehydratase(EC 4.2.1.51,PDH)were present in both N.lugens and symbiont genome.In order to determine the origins of NIBCAT and NlPDH cloned in this study,phylogenetic trees were constructed.Unrooted phylogenetic trees revealed that either NIBCAT or NlPDH was grouped together with fungi proteins,supporting by high bootstrap values.Moreover,the tissue-biased expression of NIBCAT and NlPDH were tested using RT-PCR.All the two genes were highly expressed in abdomen,but were hardly detectable in thorax.Since symbionts have been reported to be distributed in abdominal fat body,ovary and eggs,rather than in head,leg,thorax and wing,our results suggested that NlBCAT and NlPDH originate from YLSs.3.Function analysis of NIBCAT and NlPDH by RNA inteferenceBranched-chain amino acid aminotransferase(EC 2.6.1.42,BCAT)and prephenate dehydratase(EC 4.2.1.51,PDH)were the key enzymes for biosynthesis of branched chain amino acids(valine,leucine and isoleucine)and aromatic amino acids(phenylalanine,tyrosine and tryptophan).The genes encoding the two enzymes(NlBCAT and NlPDH)showed similar expression patterns.Both genes were lowly expressed in the second and fourth instars,whereas they were highly expressed in the third and fifth instars.Moreover,the expression levels of the two genes were higher in newly-eclosed female adults than those in newly-eclosed males.Continuous ingestion of either dsNIPDH or dsNIPDH for 4 days by the newly-ecdysed second-instar nymphs significantly knocked down the expression levels of NlPDH or NlPDH respectively.Continuous ingestion for 6 days had similar silencing effects.However,ingestion of dsNIPDH or dsNIPDH for 6 days did not affected nymphal survival.