Dissertation > Agricultural Sciences > Aquaculture, fisheries > Aquatic basic science > Aquatic Biology > Aquatic Zoology

Identification of Sex-Specific Markers and EST Fuctional Analysis by Constructing Gonadal cDNA Differential Libraries in Hypophthalmichthys Molitrix

Author ChenJianJun
Tutor ChangZhongJie
School Henan Normal
Course Zoology
Keywords Yellow River carp Gender specific markers SSH EST Gonadal development
CLC S917.4
Type PhD thesis
Year 2011
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Yellow River carp, experimental materials, select 220 10bp of random primer material 10 pairs of SSR primers material and 100 ISSR cited material, the use of RAPD-PCR SSR-PCR and ISSR-PCR three molecular marker technology on the Yellow River carp gene group carried scan , trying to find some linked markers associated with the Yellow River carp gender. SSR results show that in the process of scanning its DNA pools, was found on a pair of primers amplified bands a possible gender chain suspected bands, but when using this pair of primers in more individuals verify, found no gender-specific; the ISSR results show, screened 10 primers amplified bands were not found with the the gender chain of suspected bands. RAPD technology to scan the Yellow River carp, the male and female gene pool the results show that there are the suspected gender specific bands exist in one of the five random primers amplified bands, five random primers individuals repeatedly verified , only random primers object S2107 amplification product in the presence of a male-specific bands. After recovery, cloning, sequencing the to a size 909bp candidate male-specific bar band, named Ccmf1. BLAST searches have certain similarities to this fragment with linkage groups in zebrafish of a repetitive sequence of 1 (DKEY-24 H 22). In addition, this study also used the suppression subtractive hybridization successfully constructed a subtractive library of the Yellow River carp genomic DNA by nested primer PCR validation of the randomly selected 150 clones, 88 positive clones with a forward, reverse secondary PCR product as a probe, Southern dot blotting hybridization with 88 positive clones plasmid DNA from the selected 22 candidate gender-specific positive clones were sequenced after specific primers PCR validation, two length 387bp and 183bp, respectively. The male-specific DNA fragments, BLASTN and BLASTX retrieve not found their nucleotide and amino acid sequence homology, named Ccmf2 and Ccmf3. Specific primers were designed according to the sequence of the three male-specific fragment as the template for specific PCR amplification of the housekeeping gene GAPDH as internal control, male and female individuals genome. The results show that in all males amplified steady purpose specific bands, while the female individuals without the emergence of the strip. Due to the separation into three fragments with male gender chain, so should speculated Yellow River carp chromosome type XX / XY type. To learn more about the three sex-specific fragments using specific PCR technology 100 Yellow River carp, the male and female genome validation results, the the gender of Ccmf1 and Ccmf2 specificity 99% and 98%; Ccmf3 gender specificity of 100%, the reorganization of the three male-specific fragments between two chromosomes (sex chromosomes and autosomes) values ??are very low, can be used for the identification of the Yellow River carp genetic gender. Probe, and carp were Ccmf1, Ccmf2 and Ccmf3 other three varieties: wild Yellow River carp, Jian carp abundance of carp genome dot blot hybridization (male and female of each 5), in order to judge the three gender specific The fragment molecular gender effectiveness (i.e. sexing efficiency). The results showed that the molecular gender effectiveness in female individuals, Ccmf1 Ccmf2 and Ccmf3 respectively 67%, 80% and 93%; in males, the effectiveness of its molecule gender were 67%, 100%, and 73 %. Fully illustrated the diversity of molecular types of carp between the different kinds of sex chromosomes. Three male linkage discovery and identification of the mark, can be used as identification of the Yellow River carp genetic gender and to identify the sex chromosomes of the species molecular entry point, so as to provide a basis to explore the species, sex determination and differentiation mechanisms. To a more comprehensive understanding of the Yellow River carp gonad development mechanism, the mRNA level, respectively, to the testis and ovary each other authenticator successfully build the Yellow River carp mature female, the male gonads bidirectional subtractive cDNA library, broth PCR preliminary screening of the library, respectively 280 (from males Library) and 240 (from females library) containing the inserted fragment positive clones; then take advantage of the Southern dot bltting randomly selected 180 clones (90 female and male identified) in the positive and negative blots, to find the difference of the 64 candidates (including from 21 male library 43 females library) expression clones, after sequencing, fragment differences of these candidate genes by BLASTN and BLASTX functional classification, 50 ESTS sequence. 18 male library differences ESTS, including 14 known functions and fragments of four genes of unknown function. In accordance with its function, 14 ESTS belong to the structural protein gene, protein translation mechanism related genes, ion channels and protein transporter gene, tumor-related factors and other genes and tumor suppressor; 32 difference from the female library the ESTS represents 27 known functional genes and five genes of unknown function, genes of known function, the higher frequency is related gene fragments from the respiratory chain, and protein synthesis gene fragment; Apart from some unknown proteins, other 1 times. The reference to the zebrafish and other vertebrate gene function analysis the female library of known functions EST belong to adjust factors, structural proteins, enzymes, transcription factors, signal regulation. Part of homologous genes in the Yellow River carp sperm, ovarian semi-quantitative expression of 10 differentially expressed genes: 5 testis-specific expressed genes found in male differences in library; 5 testes highly expressed genes; including 2 first discovered genes of unknown function. 8 differentially expressed genes found in female differences in library: 2 ovary-specific expression of the gene; 6 ovarian highly expressed genes, including an unknown functional genes. According to the nature of the gene fragment encoding function, select the three gene fragments may be involved in gonad development, its total length was amplified using RACE technology, respectively, to obtain the size of 2173bp, 1763bp and 884bp of full-length gene, they are Hmwt1a the gene, HmSetd6 gene HmPsmb2 gene. Finally, found three genes are gonad differentially expressed genes in different tissues of adult semi-quantitative fluorescence quantitative expression pattern analysis Hmwt1a gene expression in the testis, HmSetd6 gene expressed mainly in the ovary, and the expression level of much higher than the testis, and HmPsmb2 genes ovarian specific expressed genes; contrast with vertebrate gonadal development related gene function, suggesting Hmwt1a genes may be important regulatory genes of the Yellow River carp male sexual development; Hmsetd6 genes as epigenetic regulation The genes involved in the development of the Yellow River carp female traits; HmPsmb2 gene maintain physiological or behavioral gender bias in the genes of the Yellow River carp female sex; the discovery of these genes and gonadal differentially expressed EST library construction, will understand the Yellow River carp gonads lay the foundation for development and the molecular mechanisms of sex differentiation, as well as the study of the control mechanism of the Yellow River carp gender gene identified resources.

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