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Reproduction marker gene vasa of Sinopotamon henanense and application thereof

A technology for marking genes and genes, applied in the application, genetic engineering, plant genetic improvement and other directions, can solve the problem of gene vasa separation that still needs to be further studied

Inactive Publication Date: 2017-10-20
SHANXI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, so far, the isolation of the gene vasa in river crabs with special reproductive development methods still needs further study

Method used

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  • Reproduction marker gene vasa of Sinopotamon henanense and application thereof
  • Reproduction marker gene vasa of Sinopotamon henanense and application thereof
  • Reproduction marker gene vasa of Sinopotamon henanense and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1. Cloning and isolation of Henan Huaxi crab gene vasa.

[0027] 1.1 Cloning of the partially conserved sequence of Huaxi crab vasa in Henan.

[0028] 1.1.1 Extraction of total RNA from gonad tissue and synthesis of cDNA first strand.

[0029] According to the operating instructions of the RNeasy Mini Kit (Qiagen) kit, total RNA was extracted from the ovaries or testes of early-stage Huaxi crabs in Henan. The integrity of the total RNA was detected by 1% agarose gel electrophoresis, and the concentration and purity of the total RNA were detected by a BioSpectrometer micro-spectrophotometer. According to the instructions of the TransScript One-Step gDNA Removal and cDNASynthesis SuperMix (full gold) kit, the first strand of cDNA was synthesized.

[0030] 1.1.2 Cloning of partially conserved sequences of vasa.

[0031] According to the conserved sequence of VASA protein in crustaceans reported in the public database, search for its corresponding nucleotide sequ...

Embodiment 2

[0058] Example 2. Structural analysis of the vasa gene sequence and VASA protein sequence of Henan Huaxi crab.

[0059] Analyze and compare the obtained vasa nucleotide sequences, and use NCBI / BLASTn (http: / / www.ncbi.nlm.nih.gov / BLAST) for homologous alignment; use NCBI / ORF Finder (http: / / www.ncbi.nlm.nih.gov / projects / gorf / orfig.cgi) and Expasy-Translate tool (http: / / web.expasy.org / translate / ) online tools to analyze and predict gene coding regions and their encoded proteins Sequence (Sequence Listing SEQ ID NO. 2).

[0060] Analyze predicted protein sequences and structures. The amino acid composition and basic physicochemical properties of SHVASA protein were analyzed using Expasy (http: / / web.expasy.org / protparam / ) online software. The results showed that the molecular weight of the SHVASA protein was 75247.8KD; the theoretical isoelectric point was 5.00, and it contained 101 negatively charged amino acid residues (aspartic acid and glutamic acid), and 76 positively charg...

Embodiment 3

[0061] Example 3. RT-PCR detection of the specific expression of the gene vasa in the gonad tissue of Henan Huaxi crab.

[0062] According to the Henan Huaxi crab vasa full-length sequence obtained in 1.3 of Example 1, referring to the results of sequence analysis in Example 2, the species-specific segment in the Henan Huaxi crab vasa sequence was selected, and the gene-specific gene used for quantitative PCR was designed. With primers, rpl38 gene (60S ribosomal protein L38) was used as an internal reference gene, and the expression of gene vasa in different tissues of Henan Huaxi crab was detected by quantitative PCR.

[0063] Using the method described in 1.1.1 of Example 1, extract total RNA from tissues such as Henan Huaxi crab ovary, testis, accessory gonads, gills, heart, hepatopancreas, intestine, stomach, muscle, etc.; synthesize the first strand by reverse transcription cDNA; use Vq and R38 as primers respectively, as shown in the sequence table SEQ ID NO.9-12; use th...

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Abstract

The present invention discloses Henan Huaxi crab (Sinopotamon henanense) reproductive marker gene vasa and its application, the nucleotide sequence of the gene is shown in the sequence table SEQ ID NO.1; also discloses the protein encoded by Henan Huaxi crab gene vasa Sequence, as shown in the sequence table SEQ ID NO.2, and the physical and chemical properties of the protein, the characteristics of amino acid residues, and the characteristics of the protein structure and functional domains. The coding region of the gene starts at the 142nd nucleotide at the 5' end and ends at the 2214th nucleotide, encoding 690 amino acid residues. The reproductive marker gene of Henan Huaxi crab of the present invention is specifically expressed in the ovary and testis tissue of Henan Huaxi crab through identification, and can be used for the development and application of reproductive marker molecules of Henan Huaxi crab and its similar species, and can be used to guide river crab Artificial breeding has practical significance.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a reproductive marker gene of Sinopotamon henanense, in particular to a full-length cDNA clone of the germ cell-specific expression gene vasa and its application. Background technique [0002] Freshwater crabs, a special branch of crustacean decapoda (decapoda) brachyura, live in freshwater environment for life. River crabs are freshwater crabs widely distributed in wild water resources, and their reproductive and developmental patterns are significantly different from those of other decapods. The eggs of the river crab are particularly large, the egg production is small, the egg shell is thick, the embryo develops directly, and the egg metamorphosis. However, most sea crabs and migratory mitten crabs have very small eggs, a large amount of eggs, thin egg shells, and indirect embryo development. [0003] However, so far, the reproductive and developmental regulation of r...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/55C12N9/14C12N15/10C07K14/435
CPCC12N9/14C07K14/43509C12Q1/6888C12Q2600/158
Inventor 孙敏王兰杜晓琳李怡婷
Owner SHANXI UNIV
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