| The insulin/IGF-like growth factor signaling pathway plays an important role in growth, development, reproduction, stress resistance, metabolism and lifespan in most multi-cellular organisms. Insect insulin-like peptides (ILPs) are a structurally diverse group encoded by a large multi-gene family and expressed in brains and other tissues,which are analogical in structure to that of vertebrate but serve different functions.As a model insect of Lepidoptera, Bombyxin (BBX) of silkworm is the first discovered insect insulin-like peptide and contains 7 gene families(A, B, C, D, E, F, G)consisting of 38 members. Accordingly, silkworm is a good material as the Lepidopteron model organism to study the function of insect ILPs. The B8 (GenBank Accession No. D00784) gene of BBX-B family encodes 88 amino acid residuces, is highly expressed in the neurosecretory cells of silkworm brain. It has been certified that silkworm BBX can promote cell proliferation of hematogenic organs, participates in saccharometabolism, regulates the secreting activity of eri silkworm pupae prothoracic gland. To further study the function of the BBX-B8 gene, the effect of up-regulation and down-regulation of BBX-B8 gene on the development, stress resistance, voltinism and fibroin synthesis were investigated by using RNAi and transgenic technology. After cloning the BBX-B8 cDNA, the expression profile was analyzed with RT-PCR, the results showed that the BBX-B8 gene was expressed in the brain, ovary, testis, midgut, anterior silk gland, malpighian tubules, fat body and embryonic, etc. in distinct expression levels, whereas the mRNA of BBX-B8 was not detected in the wing disc and the yolk, suggesting that BBX-B8 was expressed specifically in distinct tissues. The results of Western blotting showed that the expression level of BBX-B8 was lower in the brain of the third day larvae of fifth-instar, however, increased obviously from the fifth days of the fifth-instar. After injecting the synthesized BBX-B8 dsRNA in vitro into the blood of the 3-day larvae of fifth-instar, sqRT-PCR and western blotting were carried out at 3 days post injection, the results displayed that the level of mRNA declined in a dose-dependent manner, and that the level of BBX was declined strikingly by 30% in the brain after injection with BBX-B8 dsRNA (10μg per larva), indicating that the BBX expression was inhibited by injecting BBX-B8 dsRNA into silkworm. Using this BBX-B8 RNAi-silkworm model, the functions of BBX-B8 were investigated, the results indicated that the development of pupal wing disc was repressed, the proportion of deformed wing was increased and the morphological development of adult brain was significantly delayed after injecting BBX-B8 dsRNA into the 3-day larvae of fifth-instar. Compared to the control group, the pupation rate of the larvae which injected with BBX-B8 dsRNA was increased by 12.76% after injection followed by high-low temperature shock (40℃3 h, 4℃3 h) while the pupation rate of the injected larvae was decreased by 9.29% under starvation condition. Three days after the injection of BBX-B8 dsRNA into the 3-day larvae of fifth-instar (10μg per larva), the activity of SOD and T-AOC in hemolymph elevated by 17.81 % and 41.86 % respectively. The activity of CAT exhibited no discernable difference compared with control group, however, the levels of trehalose and trehalase in hemolymph increased by 12.68% and 68.24% respectively. It was also noted that the spawning number of the pupa injected with BBX-B8 dsRNA in 10 and 15μg per moth (10μL) one day after pupation increased by 7.86% and 12.62% respectively.Wild type BBX-B8 (BBX-B8w) and modified BBX-B8(BBX-B8m ) were cloned into insect non-transposon vector pIZT/V5-His to generate respectively recombination vector pIZT/V5-B8w and pIZT/V5-B8m. The BmN cells were transfected with the two respectively, after screening with Zeocin and molecular biological identification, the transformed BmN cells named B8w and B8m respectively, were obtained. Compared with normal BmN cells, the growth of transformed cells B8w and B8m was slower slightly and their infectivity to Bombyx mori nucleopolyhedrovirus was analogous to that of normal BmN cells. the vector pIZT/V5-B8w was transferred into silkworm eggs by sperm-mediated gene transfer. And after being screened for gfp gene and verified by molecular biological identification, the transgenic silkworms was obtained. The investigation demonstrated that extracts from the head of transgenic silkworms had higher activity of facilitating eclosion of brain-removed eri silkworm pupa compared with that of normal silkworm, and that the development of transgenic silkworms were delayed from 2 to 8 days, additionally, the weight of transgenic pupa were decreased, the ability of synthesizing silk protein was declined compared with normal silkworms, furthermore, in all cases influence on the male was more obvious than that on the female. It is also observed that about 5.56 % to 14.29 % transgenic moths laid non-diapausing eggs. All results mentioned above clearly indicated that BBX-B8 is an important developmental regulatory factor, serves to multiple specific biological functions and play an important role on the development of tissue and organ, voltinism, stress resistance, the amount of laying eggs, body size, synthesizing activity of silk protein.
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