Studies On The Genes Involving In Histidine Biosynthetic Pathway Of Nilaparvata Lugens St(?)l

Nilaparvata lugens,is a serious phloem-feeding pest of rice in China.Rice phloem sap can not meet its nutrition requirement for essential amino acid.The endosymbionts harboured by N.lugens are considered relative to the biosynthetic pathway of essential amino acid.Based on genome and transcriptome data of N.lugens and yeast-like symbiont(YLS,also named Entomomyces delphacidicola str.NLU),the major biosynthesis pathways for amino acids in N.lugens were constructed.In this study,we focused on the histidine biosynthetic pathway,cloned the genes involving this pathway,analysised their origin,and revealed the negative effects of double-stranded RNA(dsRNA)targeting the coresspsonding genes on the growth,development and survival rate of N.lugens.These results are shown below.1.Cloning and origin analysis of genes involving in histidine biosynthetic pathwayIn the current study,7 putative histidine biosynthetic pathway genes were cloned,and named EdeHis1,EdeHis2,EdeHis3,EdeHis4,EdeHis5,EdeHis6 and EdeHis7,respectively.Multiple alignment and phylogenetic analysis showed that all these genes origin from YLS genome,share high similarities with that of Metarhizium acridum and formed a monophyletic group in the phylogenetic tree.However,there is no sequence that is homologous to these genes in N.lugens.Temporal expression profiles showed that all genes were ubiquitously but unevenly expressed among the different life stages,and the spatial expression patterns showed they have higher expression levels in the fat body rather than head,leg,integument and midgut.Furthermore,no target products were amplified in head and wing genomic DNA of N.lugens,rather than that in abdomen genomic DNA.In conclusion,the results suggest all the 7 genes are from YLSs rather than BPH or ARS.2.Studies on histidine biosynthetic key gene EdeHis1One critical gene,EdeHis1,was chosed for futher study.Reare d on rice or histidine-free diet,BPH individuals that injected with 0.05,0.5 and 1.5 μg/μL dsEdeHis1 were used to detect the m RNA expressiom level on day 3,respectively.The result showed that dsRNA-aided knockdown of EdeHis1 significant suppressed the EdeHis1 expression level,compared to the blank control.Furthermore,this two diet,rice and histidine-free diet,have no influence on EdeHis1 m RNA level(p=0.22).We found that free histidine level and body weight of adult were significantly reduced,respectively.These results indicated that EdeHis1 was involved in the histidine biosynthetic pathway and contributed to the development of N.lugens.Furthermore,high levels of histidine exposure(through injection and feeding)significantly inhibited the EdeHis1 m RNA levels,whereas low levels of histidine significantly increased EdeHis1 expression levels.In addition,the result of gel filtration showed that the presence of excessive histine may change the secondary structure of EdeHis1,forming hexamer rather than dimer.3.Studies on histidine biosynthetic pathway genes,EdeHis2 and EdeHis6EdeHis2 and EdeHis6,which are also catalyzed important steps in histidine biosynthesis pathway.At day two,four and six of dsEdeHis2 or dsEdeHis6 injection,the m RNA abundance of target genes were decreased by 45%~60%(EdeHis2)or 27%~55%(EdeHis6),comparing with blank control.Down-regulation of EdeHis2 or dsEdeHis6 slightly increased the mortality by 8.3% or 9.2%,and delayed the nymphal duration of male and female by 0.43 and 0.65 days and 0.33 and 0.36 days,respe ctively.Moreover,both male(11 %)and female adult(13%)showed wing deformation after injection of dsEdeHis6,higher than that in the blank control.In conclusion,EdeHis2 and EdeHis6 that origin from YLS involved in histidine biosynthesis pathway,contr ibuted to the survivor,development and wing formation of N.lugens.