Functional Research Of The Mutant Gene Of Quail-Like Mutant Q-l In Silkwrom,Bombyx Mori

The purple quail-like（q-l^p）mutant and brown quail-like（q-l^b）mutant,a type of pigmentation mutant,were all found from the plain silkworm.The phynotype of quail-like（q-l）mutant was similar to another pigmentation mutant quail（q）.But the controlled gene of q-l was different of that of q mutant.Classical genetic analysis indicated that the mutant genes of two q-l mutants were alleles and that q-l^b was dominant to q-l^p.The mutant genes of two q-l mutants were located on the 8^thchromosome by positional cloning.Furthermore,orcokinin（OK）gene was responsible to q-l^p mutant.One base deletion resulted in early termination of translation of two orcokinin transcripts,which led to completely fuctional loss of orcokinin in q-l^pmutant.Orcokinin gene is a neuropeptide gene unique to Arthropoda,which is first found in Orconectes limosus.This new neuropeptide is named as orcokinin（OK）because of its myotropic property.Orcokinin is first found in insect（Blattella germanica）in 2004.Resently,orcokinin is thought to work in biorhythm,ecdysone signaling pathway etc..But the mechanism was unknown,and even no one receptor of orcokinin has been identified at present.In this study,we firstly confirmed that orcokinin was also the target gene of q-l^bmutant.Then the structure and expression profile of BmOK gene were analyzed.After fuctional analysis,transcriptome and proteome were used to analyze the differentially expressed genes and proteins as well as their roles in q-l mutant.Our study could provide the experimental evidences of formation mechanism of q-l mutant characterestics.The main results were listed below:First,we had testified that the mutant gene of q-l^b was also orcokinin.Two transcipts BmOKA and BmOKB were all downregulated in q-l^bmutant.The m RNA structure of BmOKB was normal in q-l^b,but there were two types of abnormal mRNAs of BmOKA in q-l^b mutant.The exon5 was deleted in q-l^b_type1（80%proportion）,and both exon4 and exon5 were deleted in q-l^b_type2（20%proportion）.In genome,we found a 4.3 kb fragment insertion in exon5.There was a 11 bp forward repeat in both ends of inserted fragment,because of which we guess the inserted fragment was a transposon.Second,the structure and expression analysis of BmOK were performed.There was alternative splicing for BmOK gene,which could form two transcripts BmOKA and BmOKB.BmOKA itself also had different splicings.A 30 bp fragment in 5’end of exon4 and a 36 bp fragment in 3’end of exon6 could be randomly spliced into mRNA,which led to 4hypotypes of BmOKA include BmOKA1,BmOKA2,BmOKA3 and BmOKA4 and the proportion was 22:13:5:1.The RACE results showed that BmOKA and BmOKB had the same 5’UTR（62 bp）.The ORF length of 4 BmOKA hypotypes were 534 bp,564 bp,570 bp and 600 bp respectively.BmOKA had a unique 3’UTR（188 bp）.Bm OKB had only one type,and the ORF length and 3’UTR of BmOKB were 864 bp and 168 bp respectively.BmOKA was mainly expressed in ventral nerve cord.In ventral nerve cord,BmOKA had the lowest expression level at 18 hours after molting.BmOKB relatively had high expression level in head and midgut,and had higher expression level in molting than in other periods.BmOKA had the highest homology with Spodoptera litura orcokinin.And the homology of C terminal mature peptide region was 80.57%.Third,the fuctional research of BmOK gene.The mutant gene of q-l^b mutant was also orcokinin gene.Two mutants could prove each other,which indicated that BmOK was major gene of forming mutant characterestics of q-l mutant.BmOK is a neuropeptide gene and logically mature peptides play functional roles.Thus,based on prediction of mature peptides in references and the results of peptidomics identification,six mature peptides of BmOK were injected under epidermis for the newly exuviated silkworm of 4^thh instar of q-l mutant.Of all mature peptides,OKA_type2 could rescue the pigmentation characterestics of both q-l^pmutant and q-l^bmutant.We found the pigmentation phenomenon disappeared after newly exuviated of 5^thh instar,which seemed no differences to wildtype.The results on the one hand approved that BmOK was the key gene of q-l mutant,and on the other hand firstly indicated that OKA_type2 played roles in the regulation of pigmentation.Besides,two mature peptides OKA_type5 and OKB_type1 could significantly improve developmental abnormalities phynotype of q-l^p mutant,of which the effect of OKB_type1 was to an extremely significant level.These facts preliminarily proved that BmOK gene could affect the grouth and development of silkworm.Fourth,transcriptome and qRT-PCR were used to analyze the expression of pigmentation-related genes of two q-l mutant.From the results,wo found three types of pigments all increased in q-l^p and the same in q-l^b except ommochrome.The cuticular proteins and chitin as well as several pigmentation-related nuclear receptors were all increased in q-l^pand q-l^b.Apt-like always regulate the synthesis of melanin,was downregulated in new epidermis and old epidermis both in q-l^p and q-l^b.We suspected that apt-like played important roles in the pigmentation of q-l mutant.Fifth,fat bodys of q-l^pand 932VR in the third day of 5^thinstar were dissected for two-dimensional electrophoresis analysis.Among all identified proteins,Bombyx aldose reductase（BmAR）was significantly downregulated in q-l^p mutant,which was related to the developmental abnormalities phynotype of q-l^pmutant.In different period in fat body,BmAR was always downregulated in q-l^pmutant verified by qRT-PCR.RNAi proved that BmAR could affect the development of silkworm,and downregulation of BmAR decreased the weight of silkworm.In mammals,AR always catalyzes the conversion of glucose to sorbitol.Thus,determination of sorbitol levels was performed.The results showed that sorbitol level of q-l^p was significantly lower than that of 932VR,which indicated that Bm AR also had the activity of aldose reductase that catalyzes the conversion of glucose to sorbitol.These results indicated that downregulation of BmAR might be one of reasons for the abnormal development of q-l^p.Through the analysis above,we not only clearly testified that BmOK was the major control gene of q-l mutant,but also firstly proved that the BmOKA mature peptide OKA_type2 maily regulated the pigmentation of silkworm.Meanwhile,expression of pigmentation-related genes were analyzed.These results provided direct experimental evidences for further researches of pigmentation mechanism of q-l mutant.