Genes Cloning And Expression Of Wing Development Genes In Rice Planthoppers And Role Of Wg Gene In Regulating Wing Forms Of Laodelphax Striatellus

The brown planthopper(BPH)Nilaparvata lugens(Stal),small brown planthopper(SBPH)Laodelphax striatellus(Fallen)and white back planthopper(WBPH)Sogatella furcifera(Horvath)are three important pests of rice in China,and their adult wings are either macropterous or brachypterous.Determination factors and molecular mechanism of wing dimorphism in rice planthopper are interesting topics.However,the interaction between environmental and inherent factors to regulate the wing dimorphism is still not entirely clear.Therefore,the influences of photoperiod on wing forms of the pure macropterous and brachypterous lineages,cloning and expression of three wing development genes of SBPH and WBPH,and wg gene role in regulating wing forms of SBPH were studied in this study,using the pure or near pure macropterous and brachypterous lineages of three rice planthoppers whose wing forms had been selected for several generations in laboratory.The main results were summarized as follows:(1)The results showed that the effect of photoperiod on wing forms of rice planthoppers was strongly dependent on rice planthopper’s genetic backgrounds,and the differentiation of wing forms in pure macropterous and brachypterous lineages was less subject to photoperiod.(2)The full cDNA sequence of fng and srf in SBPH and WBPH,and the partial fragment of dll gene were obtained by RACE method.The full-length cDNA of fng in SBPH is 865 bp,and its ORF is 645 bp,which encodes 214 amino acid residues.The full-length cDNA of fng in WBPH is 876 bp,and its ORF is 645 bp,which also encodes 214 amino acid residues.The estimated molecular mass is 24.9 kDa and an estimated isoelectric point(PI)is 7.61 in SBPH and WBPH.The full-length cDNA of srf in SBPH is 1297 bp,and its ORF is 1023 bp,which encodes 340 amino acid residues with an estimated molecular mass of 36.4 kDa and an estimated PI of 9.01.The full-length cDNA of srf in WBPH is 1265 bp,and its ORF is 1005 bp,which encodes 334 amino acid residues with an estimated molecular mass of 35.9 kDa and an estimated PI of 8.76.The partial fragment of dll gene is 381 bp,which encodes 127 amino acid residues in SBPH and WBPH.There is a high similarity between the SBPH and WBPH in fng,srf and dll protein sequences and the similarity by pairwise comparison of them is more than 96%.(3)Using the macropterous and brachypterous lineages of SBPH to check the relative expression levels of wg,fng,srf,dll gnes in nymphs and adults by qRT-PCR technology,the results indicated that the expression levels of the four genes was different between the macropterous and brachypterous lineages.The wg gene was highly expressed in nymphs and low expressed in adults,and the expression of wg in the macropterous lineage was significantly higher than in the brachypterous lineage,although there were no significant difference in adult between the two lineages.Relative expression level of fng gene increased significantly with the nymphal instars increased and the expression level was the highest in the 5th instar nymphs of the macropterous lineage of SBPH.The expression levels of fng gene were not significantly different among different instars of brachypterous lineage,but the expression level in female adults was significantly higher than in nymphs and male adults.The expression level of fng in the 3rd and 5th instar nymphs of macropterous lineage was significantly higher than of the brachypterous lineage,but the expression level in female adults of macropterous lineage was significantly lower than that of the brachypterous lineage.Relative expression levels of srf gene in the macropterous lineage were higher than that in the brachypterous lineage,and these differences were significant in the 3rd,4th,5th instar nymphs,female and male adults of SBPH.In the macropterous lineage,the expression level was the highest in the 5th nymph stage,and significantly higher than the other instar nymphs and adults.However,in the brachypterous lineage there were not significantly different in nymph and adult stages.Relative expression levels of dll gene decreased first and then increased as the nymphal instars increased and the expression level was the lowest in the 3rd instar nymphs and the highest in the 5th instar nymphs in the macropterous lineage of SBPH.The relative expression level decreased with the nymphal instars increased and the expression levels was the lowest in female adults.The expression level of dll in brachypterous lineage was significantly higher than macropterous lineage in the 2nd and 3rd instar nymph stages,but the expression levels in the 5th instar and female adults of the macropterous lineage were significantly higher than that of the brachypterous lineage.(4)Using the microinjection of double-stranded wg(dswg)with different concentrations to interfere the wg gene of the macropterous and brachypterous lineages from the 2nd instar nymphs of SBPH,the results showed that the relative expression levels of wg was significantly decreased in the macropterous and brachypterous lineages after RNA interference.The macropterous rate in the macropterous lineages was significantly decreased,and the length of fore-and hind-wing of the macropterous and brachypterous lineages became shorter when the wg was interfered.Moreover,the brachypterous adults were found in the macropterous lineages after wg interference.The wg interference caused a decrease of the eclosion rate and abnormal wings which were unfolded or curling in the macropterous lineages,but the abnormal wings in the brachypterous lineage were not found.After injection of dswg,the relative expression levels of ap,fng,dll,sd,srf were not significantly different,but led to a significant decline of the relative expression level of vg in the macropterous and brachypterous lineages.Wg gene plays a role in wing development of SBPH.