Regulation Of Homeodomain Transcription Factors On The Biosynthesis Of Juvenile Hormone And 20-hydroxyecdysone In The Silkworm

Juvenile hormone（JH） and 20-hydroxyecdysone（20E） are the most two important endocrine hormones which strictly orchestrate insect growth, molting and metamorphosis. JH is responsible for maintaining insect growth, while 20 E plays important roles in controlling larval molting and the process of matemorphosis from larvae to pupa. Studying on the regulation of biosynthesis of JH and 20 E is helpful to improve and perfect the molecular mechenism of insect endocrine system.The silkworm, Bombyx mori, is not only an important insect with economical value, but also a best-characterized model for studying the lepidopterous insects. Although there are much research about the biosynthesis pathway and the signaling transduction of JH and 20 E in silkworm, the molecular mechenism of the regulation of their biosynthesis remains to be further studied. Our study took the genes involved in JH and 20 E biosynthesis as the targets and explored the regulatory mechenism of JH and 20 E biosynthesis by homeodomain transcription factors in cells, tissues, and individuals with a serious methods such as bioinformatics analysis, genetic operation, biochemical and molecular biological experiments. The main results are presented as follows:1. The homeodomain transcription factor Scr regulates JH biosynthesis in silkworm1)The JH titer is increased during early larval periods in the M3 mutationCompared with the wild-type（WT） silkworm that has five larval instars, the M3 mutation only has four larval instars. However, the duration of its 2nd and 3rd instars are obviously prolonged. The body length and body weight of M3 are also greater than thoseof WT begining from the 2nd instar. Its body size in the 4th instar is almost equal to that of WT in the 5th instar, and the M3 larvae start wandering at the end of the 4th instar to initiate the process of metamorphosis from larvae to pupa.We determined the developmental changes of JH titers during larval periods both in M3 and WT using GS-MS. The result showed that the JH titers gradually decrease along with larvae growing and developing both in M3 and WT, and the highest JH levels are present at the beginning of each instar. Then the JH titers quickly decrease to the lowest levels at molting periods. It is worth noting that the JH titer in M3 is obviously higher than that in WT during the 2nd instar. q RT-PCR analysis showed that the temporal expression profiles of the JH reponsive gene Kr-h1 which also transduces JH signaling are consistent with the developmental changes of JH titers. In addition, we also determined the developmental changes of 20 E titers during larval periods both in M3 and WT using RIA. The result showed that the 20 E titers are lowest at the beginning of each instar, then gradually increase, and reach the highest levels at molting and wandering periods in both M3 and WT. q RT-PCR analysis showed that the temporal expression profiles of the 20 E responsive receptor gene Ec R exhibited no difference between M3 and WT. These results suggest that the higher JH titer during the 2nd instar in M3 is closely linked to its mutant phenotype.We estimated the effects of JH on silkworm growth and development by treating the newly molted 2nd instar WT larvae with the JHA, methoprene. The result showed that compared with control, the duration of each larval instar of most treated larvae are prolonged, and their body size are also increased. Furthermore, some treated larvae start wandering and metamorphosis at the end of the 4th instar previously, which is similar to the mutant phenotype of M3. These results indicate that JHA application on the newly molted 2nd instar larvae can not only prolong the duration of larval periods, but also induce the conversion of the silkworm from tetramolter to trimolter, which further suggest that the higher JH titer during the 2nd instar is the main reason to cause the M3 mutation.2)Scr gene is tightly linked to the M3 mutationGiven that the locus of the M3 mutation has been located at the position around the 24.1 c M on the silkworm chromosome₀6, we identified the potential dominating gene on the base of the sequences of the fine map of silkworm genome with the method of positional cloning. The result showed that the M3 locus is located in the region that isabout 117.5 kb in length on Bm_nscaf2853 on the silkworm chromosome₀6. This region contains only one predicted protein coding gene, the homeodomain transcription factor Scr（Sex combs reduced） coding gene. Further analysis showed that the coding sequences of Scr have no difference between M3 and WT. However, compared with WT, there are some insertion or deletion of transposon in intron 2 of Scr in M3, which is supposed to be related to its mutant phenotype.3)Scr regulates the transcription of genes involved in JH biosynthesisGiven that Scr gene is linked to the M3 mutation, and the M3 mutation is related to abnormal changes of JH titer, we supposed that Scr gene probably participates the regulation of JH biosynthesis in silkworm. To confirm this hypothesis, we first analyzed the temporal expression profiles of Scr in Br-CC-CA and PG where JH and the precursor of 20 E is respectively synthesized in silkworm larvae. The results showed that the temporal expression profile of Scr in Br-CC-CA is consistent to the developmental changes of JH titer, but its temporal expression profile in PG is not closely linked to the developmental changes of 20 E titer. In addition, we overexpressed Scr in the silkworm embryo-derived（Bm E） cells, and q RT-PCR analysis showed that Scr overexpression can up-regulate the expression of genes involved in JH biosynthesis, but has no effect on the expression of genes involved in 20 E biosynthesis. These results suggest that Scr probably affects the biosynthesis of JH by regulating the transcription of genes involved in JH biosynthesis.Online prediction showed that there are conserved cis-regulatory elements（CREs） of homeodomain transcription factors on the promoter regions of the genes involved in JH biosynthesis such as Allototropin, FPPS2 and JHAMT. We further performed a serious experiments including promoter truncating, luciferase activity analysis, and electrophoretic mobility shift assay（EMSA）. The results confirmed that Scr can directly bind to the related CREs in the promoter regions of Allototropin, FPPS2 and JHAMT. These indicate that Scr can regulate the biosynthesis of JH by directly regulating the transcription of genes involved in JH biosynthesis in silkworm.2.The regulation of 20 E biosynthesis by the homeodomain transcription factors Antp and POU-M2 in silkworm1)Antp and POU-M2 co-regulate the transcription of 20 E biosynthetic enzyme genes via a protein interactionOnline prediction showed that there are many CREs of homeodomain transcriptionfactors Antp（Antennapedia） and POU-M2 on the promoter regions of the 20 E biosynthetic enzyme genes Spook, Shroud, Phantom, Disembodied, and Shadow. q RT-PCR results showed that the temporal expression profiles of Antp and POU-M2 in PG of silkworm larvae are similar to those of the 20 E biosynthetic enzyme genes. These clues suggest that Antp and POU-M2 probably play roles in regulating the transcription of the 20 E biosynthetic enzyme genes.We mainly analyzed the roles of Antp and POU-M2 in regulating the transcription of the 20 E biosynthetic enzyme gene Phantom. A serious experiments including promoter truncating, luciferase activity analysis, EMSA, and chromatin immunoprecipitation（Ch IP） proved that Antp and POU-M2 can regulate the transcription of Phantom by binding to the adjacent core motif of the CRE in its promoter region directly. Furthermore, using the experiments of pull-down and Co-immunoprecipitation（Co-IP）, we found that Antp can interact with POU-M2 to co-regulate the transcription of Phantom.2) RNAi of Antp and POU-M2 affect 20 E biosynthesis and interrupt the metamorphosis in silkwormWe performed the experements of RNAi of Antp and POU-M2 in silkworm. First, we synthetised the ds RNA of Antp and POU-M2, and injected them into the silkworm at wandering stage. Following analyses showed that the expression of Antp, POU-M2, and most of the 20 E biosynthetic enzyme genes in PG are obviously decreased at 48 h after the injection of ds RNA, and the 20 E titer in hemolymph as well as the expression of 20 E responsive genes（Ec R, E74 A, βftz-f1, and Br-C） are also obviously decreased. At 60 h after the injection of ds RNA, the control larvae had accomplished pupation, but the metamorphosis of the RNAi larvae were interrupted and they finally died. These results further confirmed at individual level that Antp and POU-M2 can regulate 20 E biosynthesis by directly regulating the transcription of 20 E biosynthetic enzyme genes in silkworm.