Effect Of RALDH Gene On Triglyceride Content In Mythimna Separata Larva

Mythimna separata（Lepidoptera:Noctuidae）is a worldwide agricultural migratory pest.The population density affects the synthesis of triglycerides,which are the energy sources for migratory of M.separata.The triglyceride content of gregarious moths is higher than that in the solitary ones,and the gregarious moths generally tends to flight for long-distance resulted in a more wider range pest.It has been reported that RALDH gene may regulate the triglyceride synthesis in Mus musculus.There are few reports that RALDH gene regulates triglyceride synthesis in M.separata in response to population density change.Therefore,according to the candidate gene principle,the role of RALDH gene on the triglyceride synthesis in M.separata larvae was investigated,which may provide basic data for clarifying insect migration mechanisms and a theoretical basis for pest control.In this paper,the GPO-PAP enzyme method was used to determine the triglyceride contents in various instars larvae of two lifestyles（solitary phase:1larva/jar;gregarious phase:10 larvae/jar）.Using the RACE and RT-qPCR techniques,the full-length cDNA sequence of RALDH gene in M.separata was cloned and the expression pattern of RALDH mRNA was established.The role of the RALDH gene on the triglyceride synthesis of the larvae was investigated by using plant-mediated insect RNAi technology.1.Triglyceride synthesis pathway was analyzed based on transcriptome data.The cDNA library of 5th instar larvae（L5）in two lifestyles was constructed by IIllumina Hi Seq^TM2000 high through put sequencing platform.The result showed that about the data size of each sample is 8.76 Gigabyte（Gb）,and the Q20 content of all samples is above 98%.In addition,the G+C content is between 51.29%and 53.04%.A total of58 094 unigenes were assembled by Trinity software,30 184（51.96%）unigenes were annotated in the Nr,Swissport,KOG,and KEGG databases.The result of gene differential expression analysis revealed that RALDH gene in the gregarious larvae was up-regulated expression in the triglyceride metabolism pathway.It was speculated that this gene affected triglyceride synthesis in response to population density.2.Determination of triglyceride content in larvae of two lifestyles.The triglyceride（TG）GPO-PAP enzyme method was used to determine the triglyceride content in 4th,5th and 6th instars larvae of the two lifestyles.The result showed that the triglyceride content of gregarious larvae was higher than those of solitary larvae,and the triglyceride content increased with the growth of larvae.The triglyceride content were not significantly different between solitary larvae（0.774±0.01 mmol/L）and gregarious larvae（0.8165±0.03 mmol/L）at 4th instar（P>0.05）;The triglyceride contents of the gregarious larvae at 5th instar（1.508±0.05 mmol/L）and6th instar（2.015±0.03 mmol/L）were significantly higher than those of the solitary larvae at 5th instar（1.232±0.01 mmol/L）and 6th instar（1.596±0.04 mmol/L）（P<0.05）.3.Cloning and bioinformatics analysis of RALDH gene.Based on RALDH unigene sequence,the primers for cloning RALDH full-length cDNA sequence were designed.The sequences of 5’-RACE and 3’-RACE of RALDH gene cDNA were amplified by using RACE and RT-qPCR technology.The 5’-RACE,3’-RACE and RALDH unigene sequences were spliced to obtain the full-length 1 901 base-pair（bp）cDNA sequence of RALDH gene.This sequence contains a 5’untranslated region（5’-UTR）of 98 bp,a 3’untranslated region（3’-UTR）of 342 bp,and an open reading frame（ORF）of 1 461 bp encoding a 486-amino acid protein.The initiating codon ATG is located positions 99-101 and the stop codon TAA is located positions 1 557-1559.The deduced protein has a molecular weight of 52.382 KDa and a theoretical isoelectric point of 5.98.The function domain assay revealed that it belongs to the ALDH super family.The deduced RALDH separately shares 83.74%and 82.72%identity with those from Helicoverpa armigera and Spodoptera litura.These results indicate that the newly isolated cDNA is RALDH homologue.Construction of RALDH phylogenetic tree.Based on RALDH amino acid sequence,the phylogenetic tree was constructed by comparing RALDH amino acid sequences of M.separata and other 12 insects using MEGA7.0 Neighbor-joining method.The result showed that M.separata and Spodoptera Litura clustered in the same branch and the bootstrap value is 87.The result indicated that their genetic distance is the closest.4.Expression pattern of RALDH gene in M.separata larvae.（1）Expression pattern of RALDH gene in larval tissues.RT-qPCR was used to determine RALDH mRNA levels in the head,epidermis,foregut,midgut,hindgut,fat body and malpighian tubules of L5 larvae.The results showed that RALDH gene was expressed in various tissues with the highest level in fat body,which was significantly higher than that of other tissues（P<0.01）,followed by malpighian tubules,foregut and midgut,and the head expression level was the lowest.（2）The expression pattern of RALDH gene in M.separata larvae of two lifestyles.The expression levels of RALDH gene mRNA in 4th,5th and 6th instars larvae of two lifestyles was investigated by RT-qPCR.The results showed that the mRNA levels of RALDH gene in the gregarious larvae were higher than those in the solitary larvae,and RALDH mRNA levels increased with the growth of larvae.There was no significant difference in between two lifestyles larvae in 4th instar（P>0.05）.The mRNA levels in the gregarious larvae of 5th and 6th instars were significantly higher than those in the solitary larvae（P<0.05）.5.The role of RALDH gene on the triglyceride synthesis of M.separata.（1）Plant（maize）-mediated RNAi system（PMRi）in M.separata was established.The 400 bp target region for RNAi was cloned based on ORF sequence of RALDH using specific primers adding the restriction enzyme sites of Bam HI（CGGGGATCC）and Kpn I（CGGGGTACC）at the 5′end.This fragment was ligated into p TRV2 after digestion of both vector and PCR product with Bam HI and Kpn I,generating the p TRV2-RALDH plasmid.Agroinfiltration liquid was made in a flask by mixing p TRV1 Agrobacterium tumefaciens strain GV3101,respectively,with various A.tumefaciens strain GV3101 carrying different p TRV2 recombinant plasmids（p TRV2-RALDH and p TRV2-GFP（Labs produce））in 1:1 ration.The germinated maize seeds was infiltrated by vacuum in above agroinfiltration liquid.The RALDH mRNA levels and triglyceride contents of the larvae after feeding with maize seedlings expressing RALDH-ds RNA or GFP-ds RNA were measured.The results showed that there was no significant difference in the RALDH gene mRNA between two control groups（P>0.05）.The RALDH gene mRNA in the experimental group（RALDH-ds RNA）was significantly reduced compared with the control groups（P<0.05）.These findings clearly indicated that RALDH was specifically silenced by maize-mediated RNAi aproach.（2）Determination of triglyceride contents in M.separate larvae.The triglyceride contents in the control groups and the experimental group were tested by the triglyceride GPO-PAP enzyme method.The findings showed that there was no significant difference in triglyceride contents between the black control group and the negative control group（P>0.05）.The triglyceride content in 4th-instar larvae of the experimental group was not significantly different from that of the control group（P>0.05）,however the triglyceride contents in the 5th-instar and 6th-instar experimental group larvae was significantly lower than that in the control groups（P<0.05）.These findings showed that feeding maize expressing RALDH-ds RNA to larvae has effectively reduced triglyceride contents.Therefore,we speculated that the RALDH gene may play a key role in regulating triglyceride synthesis of M.separata.In summary,the full-length cDNA sequence of RALDH and its temporal and spatial expression patterns were obtained by RACE and RT-qPCR technologies.Using the host plant maize-mediated RNAi approach,the RALDH expression of larval was inhibited so that larval triglyceride contents decreased.So,we speculated that RALDH gene may take part in regulating the synthesis of triglyceride content in M.separata.