Positional Cloning And Function Research Of Nm2(non-molting In The 2^nd Instar) Gene In Silkwrom, Bombyxmori

As a kind of important economical lepidoptera insect, silkworm(Bombyx mori) has become an important experiment materials because of its special research value. For a large number of researchs about the gene function and basic study, silkworm has become the model organism for lepidoptera research.Silkworm is completely metamorphosisinsect, the moulting is an important physiological processes and is essential for its growth, development and metamorphosis.The silkworm mutant that non-molting in the 2nd instar(nm2) is a recently discoverednew mutantfrom the silkworm variety C603.The mutant cannot moult in the 2nd instar and exuviate and finally dies.In the present study,based on the genetic analysis of the mutants, we mapped the candidate gene involved in the nm2 mutant by positional cloning.The transcriptional expression analysis and functional identification of the candidate gene were conducted by qRT-PCR, 2-DE, RNAi, and feeding expriment of 20 E and cycloheximide.The main results of the study are as follows. 1. Genetic analysis of nm2 mutantThe new mutant of non-moulting in the 2nd instar(nm2) is discovered from the silkworm variety C603. The mutant develops normally in the 1st instar and moults on time, but in the beginning of the pre-moulting stage of the 2nd instar, the mutant larvae becomes lustrous, lasts for 6-8 days with hardly any development, cannot moult and exuviate and finally dies. Genetic analysis revealed that nm2 was controlled by a single recessive gene, and has homogeneous lethality. 2. Positional cloning of nm2 mutant geneThe population of genetic analysis including P1,P2,F1,BC1 F and BC1 M were conducted. Polymorphic markers on each linkage group of silkworm were found.These polymorphic markers as well as 10 normal and 10 nm2 BC1 F progenies that from the same parents were used to determine the linkage group on which the nm2 gene was located. The results showed that the nm2 gene was located on the 5th linkage group. 594 nm2 BC1 M individuals were used for fine mapping, and the results indicated that nm2 was located in a region of approximately 275 kb between two polymorphic markers S2529-27 and S2529-32. There were thirteen genes within the particular region. 3. Identification of nm2 genewe analysed the expression profiles of the thirteen initial candidate genes of 2nd instar pre-moulting silkworm between nm2 and the wild-type by semi-quantitative RT-PCR. There were two genes that had different mRNA expression level between the wild-type and nm2. One was BMgn002601 and the other was BMgn002602. And the key gene responsible for the nm2 was further determined by cloning of their ORF and RNAi of BMgn002602. The ORF of BMgn002601 wasidentical between wild type and nm2, but the BMgn002602 ORF in nm2 involved a deletion of 217 bp in its functional domain, but the 3’UTRand 5’UTRof the BMgn002602 were normal. In addition, the mRNA expression of BMgn002601 could be influenced by the expression ofBMgn002602. The moulting time of the individuals injected with BMgn002602 dsRNA in the 2nd instar could be delayed 48 h to 72 h. Based on these results, we concluded that the mutation of BMgn002602 was the most likely key gene responsible for thenon-moulting in the 2nd instar silkworm mutant.BMgn002602 encodes a cuticle protein, BmCPG10, rich in glycine. The sequence of BmCPG10 3’ UTR, 5’UTR and promoter were obtained by RACE and cloning. The 5’UTR and promoter of BmCPG10 wasidentical between wild-type and nm2, but 3’UTR ofBmCPG10 in nm2 was influeced by the 217 bp deletion, and was different with that of the wild-type. 4. The expression and function validation of nm2 mutant geneThe temporal and spatial expression profiles of BmCPG10 were examined using the entire body except the midgut of wild-type larvae by qRT-PCR. The results indicated that temporal BmCPG10 mRNA was detectable throughout the development, from the egg to the moth, and was low during the pre-moulting and moulting while high in the newly molted silkworm. The spatial expression profiles of BmCPG10 gene transcription was high in the epidermis, head and trachea, while low in the midgut, malpighian tubule, prothoracic gland and ventral nerve cord, but hardly detected in haemocytes. The titre of ecdysone in nm2 and wild-type were determined by ELISA. The result indicated that ecdysone titre in nm2 was significantly lower than that of the wild-type. Cholesterol, 7dC and 20 E dissolved or suspended in water and were fed to each group, including 50 nm2; water was used as control. As a result, some mutants moulted in the 2nd instar and grew into the 3rd instar stage, and the partly rescued nm2 then moulted and grew into the 4th instar stage but could not moult in the 4th instar and finally died. Based on the results of the feeding expriment of 20 E and cycloheximide to wild-type glutonous stage larva and the over expression, it is indicated that the expression of BmCPG10 mRNA was negative correlation with the ecdysone titer.Based on the aboving results, we concluded that the mutation of BmCPG10 had an influence on sterol utilization and resulted in a deficiency of cholesterol, which was the raw material needed for the synthesis of ecdysone that were synthesized from dietary sterols. The low ecdysone titerin nm2 led to non-molting in 2nd instar. 5.Differential expression of the epidermis proteins between wild-type and nm2 mutant2-DE was used to investigate the differential expression of the epidermis proteins between wild-type and nm2 mutant, and the result showed that one of the protein spot was higher expressed than that of the wild-type. The spot protein was BmCP-like protein identified by MS, and encoded by BmCP-like gene.And the expression of BmCP-like mRNA of nm2 was higher than that of the wild-type by qRT-PCR, which in accordance with the result of 2-DE. Thedevelopmental transcription pattern of BmCP-like showed that BmCP-like mRNA was high in the molting and newly exuviated larvae and low in the premolting larvae of the 1st to 3rd stage, but in the 4th instar its expression was high in the feeding stage and low in the molting and pre-molting stage. The tissue-specific transcription pattern of BmCP-like showed that BmCP-like gene mRNA was high in the epidermis, head and trachea, and low in the other tissues.Furthermore the ORF of BmCP-like gene in nm2 was identical to the wild-type. The loss of BmCP-like function resulted in the high mortality of 2nd wild-type larvae by BmCP-like RNAi, and the body of survived twolarvaeformed a dark spots around the injection place.These studies give us a hint that the BmCP-like gene maybe take part in the immune systemof silkworm, and its transcription upregulationin the nm2 might be affected or regulated by BmCPG10 in order to survive.These study can provide theoretical basis for elucidation of molecular mechanisms of the nm2 mutant, and help to better understanding the regulation mechanism of ecdysone on silkworm growth,moreover,possess important practical significance for using the functional genes, such asprevention and control of lepidoptera pests.