Gene Cloning, Expression And Function Identification Of Cadherin-like Protein Of The Oriental Armyworm, Mythimna Separata (Walker)

Mythimna separata (Walker) is a typical polyphagous and migratory pest in China. It is also one ofthe most important pests of corn and other food crops. With the development new varieties of Bt corn,its potential risk analysis and evaluation of ecological safety is especially important. Bt toxins bind toreceptors in the insect midgut, causing a series of changes in cells that ultimately lead to cell death. Thisinteraction between Bt toxins and receptors is the key factor affecting toxicity. The cadherin-likeprotein plays a key role in insecticidal process as it is the first receptor of Cry toxins. It is reportedGM maize expressing Cry1Ab proteins has good efficacy against M. separata, and the orientalarmyworm has lower sensitivity to the Cry1Ac toxin than H. armigera. However, there is no publishedinformation about the Bt receptors in M. separata, as well as the molecular mechanism of Bt toxins toM. separata. This study is to examine the expression of cadehrin-like protein gene in M. separata in thedifferent developmental larvae and different tissues on the basis of cloning, sequencing of this gene, andthen to examine the function of cadehrin-like protein by using RNA interference, in order to reveal themechanism of Bt toxins to M. separata from molecular level. The results are as follows:Cloning the full-length cDNA of the cadherin-like protein gene from M. separata. Thecomplete cDNA sequence of cadherin-like protein gene was amplified from the oriental armyworm,M.separata, using RT-PCR and RACE techniques. The full cDNA sequence (named as Ms-CAD,GenBank accession no. JF951432) was5642bp in length, encoding1757amino acid residues, and thededuced amino acid sequence showed typical characteristics of the known insect cadherin proteins,including a signal peptide, a proprotein region, twelve cadherin repeats, a membrane-proximal region, atransmembrane region and a cytoplasmic region. The predicted molecular mass and isoelectric pointwere196.786kD and4.5, respectively. The amino acid sequence of the cadherin-like protein from M.separata was highly similar with some other lepidopteran insect species and showed the closerelationship to cadherin-like proteins from Sesamia inferens, Sesamia nonagrioides, Helicoverpaassulta, Heliothis virescens and Helicoverpa armigera, with the amino acid identities of more than60%.Among them, it showed the closest relationship to cadherin-like proteins from S. inferens and S.nonagrioides, with the amino acid identities of70.55%and70.51%, respectively. This suggested thecadherin-like proteins share evolutionarily conserved structures.The expression levels of Ms-CAD mRNA in different developmental larvae and differenttissues. β-actin of M. separata was used as a reference gene and the plasmid with Ms-CAD specialfragment was used as a template. A real-time fluorescence quantitative PCR was constructed to examinethe transcriptional expression levels of Ms-CAD from different larval instars and different tissues. Theresult showed that relative expression levels of cadherin-like gene were significantly different indifferent instars of larvae (P<0.05), which was the highest in the4thinstar larvae and the lowest in thenewly-hatched larvae. However, there were no significant differences among instars3-6(P>0.05).Ms-CAD expression was much higher in the midgut than in the rest of the larva cadaver (P<0.01). Relative expression levels of Ms-CAD in different tissues of4th,5thand6thinstar larvae of M. separatawere detected and it showed that Ms-CAD expression was much higher in the midgut than in the rest ofthe larva cadaver (P<0.01), suggesting that Ms-CAD was majorly expressed in midgut of M. separata.The function of Ms-CAD was verified by using RNA interference technology. Three specialdouble-stranded RNA were designed and synthesized according to Ms-CAD, named ds1, ds2and ds3,respectively. The mRNA expression of Ms-CAD was detected at24,48,72and96hours after feedingon dsRNA by using semi-quantitative PCR. The results showed that Ms-CAD mRNA was substantiallyreduced in the larvae24hours after ingesting a relatively high dose of ds1, and was reduced24and48hours after ingesting a relatively high dose of ds2, compared to control larvae of the same age. Larvaefed relatively low doses of ds1and ds2showed only a slight reduction in Ms-CAD transcript levels.Ms-CAD transcript levels were not affected in larvae fed on either high or low doses of ds3. Thus, onlya sufficient concentration and a specific target site of dsRNA can induce target gene knockdown. Afterobserving specific gene silencing following ingestion of dsRNA, we examined whether inhibition ofMs-CAD expression changes Cry1Ab susceptibility of M. separata larvae. After Ms-CAD gene silencing,developmental duration was significantly reduced in3rd instars fed a continuous diet containing45μg/gCry1Ab toxin (P <0.05). After7days, the weight of larvae fed diet containing0.25μg/μl ds1and ds2was significantly greater than ds3-fed and control larvae. In addition, mortality of Ms-CADgene-silenced larvae was reduced significantly relative to that of control larvae and larvae withoutMs-CAD gene silencing. These results showed that Cry1Ab susceptibility of M. separata was decreasedafter knock-down of Ms-CAD expression, suggesting that Ms-CAD is associated with the Cry1Abtoxicity and cadherin-like protein of M. separata serves as a receptor of the Cry1Ab toxin.