| Bactrocera dorsails(Hendel)is a kind of pests that do harm to more than 250 kinds of fruits and vegetables, it has high reproduction, widely range of host plants,seriously damaging etc., and has brought severe losses to agricultural production especially in southern China. B. dorsails pupae albino mutant was found by accident during breeding wild type in the laboratory, the color of pupa is white, albino trait is stable, doesn't have separation change or back mutation, become a kind of stable pupae of B. dorsails albino mutant strains. In this research, we compared the biological indicators of albino mutant strains, revealed the albino regularity of genetic mutations. In addition, we also did transcriptome sequencing of 5 different strains pupa that breeding in the laboratory, studied the different expression of melanin synthesis related genes in two strains during pupa stage and the effect of tyrosine hydroxylase on B. dorsails pupate pupation, provides the basis for the further study of researching B. dorsails albino mutation mechanism. Our main results as follows:1. Illustrated the genetic structure of B. dorsails albino mutant strains In order to get B. dorsails albino pupa isolation transgenerational breeding, from now on, we have raised more than 20 generations, from quantity of population from four(2males and 2 females)initially to quite a lot of quantity. Through to the wild strains and normal strain group 20 consecutive generation of hybrid, the offspring had no character separation phenomenon. The results indicated that mutant strains of body color control is likely to be homozygous genes. Through single hybridization experiment results showed that the albino mutant strains of genetic law follows the laws of mendelian inheritance, was controlled by a single gene autosomal, showed albino character relative to the wild properties was recessive. In addition, through cross experiment showed that the wild strains and the mutant strains can mate with each other and produced fertile offsprings, illustrated that two kinds of strains are still belong to a kind.2 Determined and compared biological indicators of two strains of B.dorsails The experimental results indicated that the same indoor conditions in winter, relative to the wild strains, albino mutant strains of larvae and pupae of development period,hatching rate and eclosion rate had no differences between males and females. The eggs of albino mutant strains development period reached 52.61 h, significantly higher than that the normal strains, 48.34 h. We weighed the weight of 10 pupae of two strains respectively,albino strain of pupae's weight was 0.1387 g, significantly higher than those of wild strains,0.1223 g. Finally, two strains of male and female body length and head width were measurement and analysis, the results showed that body length of two strains of females were longer than males, mutant strain females head width was significant longer than males.3 Pupa transcriptome sequencing of five B. dorsails strains Using illumine high-throughput sequencing technology. transcriptome data construction of five B. dorsails pupae,CK?CB?CZ?LB and LX. they were respectively get3.16G?2.56G?3.35G?3.35 G and 3.15 G raw reads,the Q20 and Q30 up to 96% and93%?By sequence assembly,we get a total of 136941 Transcripts and 97809 unigenes. The average of unigene was 729 bp. A total of 26527 unigenes was successfully annotation using the databases of NR,Nt?Pfam?KOG/COG?Swiss-prot?KEGG and GO, accounting for27.12% of the total unigene. there are 3173 genes annotated in all Databases. through the GO function classification, biological process, most of the genes gathered on the process of cells; Molecular function category, combination has gathered the most gene; In cells, gene is mainly gathered in the cells and part.Analysis of different expressed gene showed that 400 genes differentially expressed in wild strains compared with albino mutant strains. There are 72 genes up-regulated and 328down-regulated in albino mutant strains. GO function of enrichment revealed that a majority of these genes were significantly clustered into structural constituent of cuticle?structural molecule activity and metalloexopeptidase activity function entry. KEGG Pathway Analysis of metabolic pathways(KEGG),these genes were significantly clustered into Fructose and mannose metabolism ? Fatty acid degradation and Glycolysis /Gluconeogenesis.4 Quantitative expression of related genes of melanin synthesis pathways and feeding with tyrosine hydroxylase inhibitor(3- IT)In the transcriptome annotation screening Tyrosine hydroxylase gene(Tyrosine hydroxylase, TH), dopamine decarboxylase(Dopa decarboxylase, DDC) gene, N- beta-propionyl dopamine synthetase(NBAD synthetase, ebony) gene and two phenol oxidase(phenoloxidase, PO1 & PO2), these five black melanin synthesis pathways related gene segments, and compared the relative expression of five genes of wild strain and mutant strains in the process of pupation by rt-pcr method, it was found that in two strains B.dorsails pupae on the same day, the amount expression of tyrosine hydroxylase genes in whole worm and cuticle of wild strains were 4.31 times and 4.31 times than those of in albino strains respectively. Feeding wild strains instar larvae with tyrosine hydroxylase inhibitor(3- IT) until pupating, all pupa bodies were soft and atrophy, and with the increasing of the concentration of the 3 – IT, pupa color shallow rate increased, 1 mg/g3-IT could make 86.7% of wild strains pupa color become shallow.The results showed that the tyrosine hydroxylase genes involved in the orange small fruit fly pupae shell in the process of coloring and ossification.Quantitative expression of the melanin synthesis pathways related genes and tyrosine hydroxylase inhibitor(3- IT) feeding In the transcriptome annotation screening Tyrosine hydroxylase gene(Tyrosine hydroxylase, TH), dopamine decarboxylase(Dopa decarboxylase, DDC) gene,N-beta-propionyl dopamine synthetase(NBAD synthetase, ebony) gene and two phenol oxidase(phenoloxidase, PO1 & PO2), and other five black melanin on the way to related genes, and by rt-pcr method compared the five genes in the process of wild strain and the mutant strains of pupation relative expression, it was found that in two strains orange small fruit fly pupae on the same day, Tyrosine hydroxylase genes in wild strains whole worm and amount of expression in the epidermis, respectively 4.31 times and 4.31 times that of albino strain.With tyrosine hydroxylase inhibitor(3- IT) feeding wild strains instar larvae pupate, all pupa body soft atrophy, and with the increase of the concentration of the 3- IT pupa color becomes shallow rate increased, 1 mg/g3- IT can make 86.7% of wild strains pupa color becomes shallow.The results showed that the tyrosine hydroxylase genes involved in the process of coloring puparium and ossification of B. dorsails. |
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