Molecular Mechanism Of Leg Specialization And Phenomenon Of Leg Regeneration In Locust Migratoria

Locusta migratoria is a common agricultural pest which has strong reproductive and adaptive ability. It always feed on a large amount of crop and has also the habit of migration. The plague of lo-custs always resulted in huge economic losses in agricultural crops. As we know, Locusta migratoria is one member of Orthoptera insect which has two advantages:one is the specialization of the hind leg which become jumping legs, the other is the ability to regenerate amputated appendages. These two characters play an important role in migration, feeding and mating for L. migratoria. In this study, we studied the mechanism of specialization of the hind leg and the leg regeneration ability of L.migratoria, which was beneficial to reveal the mechanism of morphological differentiation of homologs and to pro-vide theoretical basis for the control of L.migratoria. The aim of the research is improving the integrat-ed management of L.migratoria. There were six sections in this doctoral dissertation.(1) This work provided systematic selection and assessment of reference genes in L.migratoria for gene profile analyses covering biotic and abiotic experimental contexts:EF1a and RPL32 were found to be optimal for embryo and larvae stage; SDH and RP49 were reliable for adult stage; RPL32, Hsp70 and RP49 would be appropriate for study in different tissues; Act and SDH should be recommended for lar-vae treated with insecticide treatment; Act and Ach should be selected for larvae suffering starvation; RPL32 and RP49 were appropriated for larvae subjected to different temperature treatment.(2) For L.migratoria, the specialization of hind leg was from 40% to 55% of embryogenesis. The legs at these developmental stages were mixed to extract total RNA which were provided for DGE li-brary preparation and sequencing. The fore leg, middle leg and hind leg was performed separately. There were 33,484 unigenes which were annotated using 7 databases including GenBank NR, GenBank NT, KOG, PFAM, SwissProt, GO and KEGG.(3) Digital Gene Expression Profiling (DGE) was used to identify differentially expressed genes (DEGs) among the fore leg, middle leg and hind leg. We found the number of DEGs in LM-FL vs LM-ML, LM-HL vs LM-FL and LM-HL vs LM-ML were 16,446,404 respectively. The expression lev-el of genes involved in four signaling pathways which play an essential role in leg development was similar among different legs. The expression level of Homeotic slector gene was significantly different among different legs, especially for the gene ubx.(4) Dpp gene was cloned by RT-PCR to get the partial sequence. The relative expression level of dpp was analyzed by real-time PCR.The result showed there was no difference for the expression of dpp among three pairs of legs during the specialization process of hind leg. RNAi was operated by injecting dsRNA into 40% of embryogenesis. Dpp gene expression was successfully inhibited. After that, it was delayed for the development of femur and tibia of metathoracic leg at the beginning time. In later stage of embryogenesis, the leg was malformed. But the femur of hind leg was still wider than that of precor-dial and mesothoracic legs. The tibia of metathoracic leg was longer than that of the other two legs. This study confirmed the dpp gene palyed an important role in pattern of leg development. However, it did not participate in the specialization of hind leg.(5) Ubx gene was also cloned by RT-PCR to get the partial sequence. The amino acids shared sig-nificant homology with other insects. This study showed ubx gene was only expressed in metathoracic leg. RNAi was operated by injecting siRNA into 40% of embryogenesis to inhibit ubx gene. ubx gene expression was successfully inhibited. After that, the femur of hind leg was no longer wider than that of fore and middle legs. The tibia of metathoracic leg was not longer than that of the other two legs any more. The size of three pairs of legs has no significant difference. This study confirmed the ubx gene participated in the specialization of hind leg.(6) This report also studied the regeneration potency of L. migratoria. The results showed the legs of L. migratoria have partial regeneration ability when amputation was conducted at the joint of two seg-ments, although the regeneration capacity was weak. The regeneration potency might be stronger the more proximal the operation. The time interval before emergence was delayed if regeneration occured for L. migratoria.