Cloning And Functional Verification Of Silk Fibrion Light Chain In Sylepta Derogata Fabricius

Sylepta derogata(Lepidoptera:Pyralidae)is a main pest for cotton and other cash crops,the larvae of which damage cotton by-spinning silk and rolling leaves.In recent years,increasing studies have reported the damage of this pest to cotton and other cash crops.In this study,in order to explore the molecular mechanism of silk-spinning of this pest,the fibroin light-chain-encoding gene(SdFib)was interfered using RNAi technology after obtaining the cDNA fragments and full-length cDNA by cloning,and the effects of SdFib interference on the morphological structure,mechanical properties and amino acid types and contents of larval silk of S.derogata and its growth and development were analyzed.The results are as follows.Primers were designed based on the conserved regions in fibroin light-chain-encoding genes of other insects that had been sequenced and registered.The 636-bp fragment of SdFib was cloned by RT-PCR.Multiple sequence alignment analysis showed that the amino acid sequence similarity between SdFib and homologous genes of Corcyra cephalonica was the highest,reaching 71%.The amino acid sequence similarity between SdFib and homologous genes of Galleria mellonella and Bombyx mori was 70%and 67%,respectively.The dsSdFib vector was constructed by in vitro dsSdFib synthesis and fermentation of Escherichia coli.The effects of dsSdFib at different concentrations and different feeding time on SdFib expression in Sylepta derogata were analyzed by feeding method.On the basis of fully verifying that the obtained strain could produce dsRNA and interfere SdFib expression in the larvae of S.derogata,analysis was carried out and demonstrated that the concentration of dsSdFib could affect SdFib expression in the larvae.With the increase of the concentration of dsSdFib,the relative expression of SdFib decreased.The expression of SdFib in S.derogata at different stages and instars decreased with the increase of feeding days.The 3rd and 4th instar S.derogata was fed with dsSdFib and after 7 d,there was a significant difference of SdFib relative expression between the control and the treatment which demonstrates that the interference effect on larvae at the 3rd instar was the most obvious,with the decrease of SdFib expression by 59.7%.After treated with dsSdFib at different concentrations,the difference in SdFib expression between different stages(instars)was consistent with the field-feeding damage characteristics of the pest at different stages(instars).To further verify the effects of dsFib liquid constructed by fermentation of Escherichia coli on the morphological characteristics and elastic properties of the silk spun by S.derogata larvae at the 5th instar,observation was performed using a VANOX AHB-1 microscope.The results showed that the degree of winding and curling of the silk spun by S.derogata fed with water and dsGFP(green fluorescent protein)liquid was lower,and the smoothness of silk was better compared with that by S.derogata fed with dsFib liquid.The silk spun by S.derogata fed with dsFib liquid showed obviously uneven surface,greatly varied thickness,and swelling and rupture in many parts.In addition,the silk spun by S.derogata fed with water and dsGFP liquid presented larger cross-sectional area and rounder shape compared with tha by S.derogata fed with dsFib liquid.The cross-sectional area of the silk spun by S.derogata fed with dsFib liquid was smaller by 84.7%,and the roundness was lower by 52.8%than the water control,suggesting that the quality of larval silk of S.derogata was seriously affected by gene interference.On the basis of feeding larvae of S.derogata,graph matching was carried out with the help of relevant mechanical indicators,and the relationship between gene interference and the mechanical properties of the silk spun by S.derogata larvae at the 5th instar was investigated in this study.The results showed that the strength and elongation of silk spun by S.derogata fed with dsFib liquid decreased significantly compared with that fed with water and dsGFP liquid.The results of mechanical analysis further proves that gene interference has a great effect on the elasticity of larval silk of S.derogata.Moreover,the effects of feeding with water,dsGFP liquid and dsFib liquid on the composition of protein amino acids in silk and the growth and development of S.derogata were compared in this study.The results presented that the ratio of basic/acidic amino acids and hydrophilic/hydrophobic amino acids in silk of S.derogata treated with dsFib liquid significantly reduced compared with that treated with water and dsGFP liquid.The ratio of amino acids with small-side groups/amino acids with bulk-side groups in silk of S.derogata treated with dsFib liquid was only 1.07.The results suggested that the water absorption of silk of S.derogata treated with dsFib liquid was weakened,and the aggregation structure of the silk was not intensive,which increased the difficulty in crystallization of the silk fibers.On the other hand,this indicates that gene interference affects the strength of the silk,which was consistent with the results of above mechanical property test.The analysis on indicators related to the growth and development of S.derogata demonstrated that the larval duration,pupal duration and pupal weight of S.derogata fed with dsFib liquid showed significant differences from S.derogata fed with water and dsGFP liquid,with larval duration increased by 3.0 d and pupal weight reduced by 23.73%,which suggests that SdFib interference might affect the growth and development of S.derogata.