阅读说明：本技术 小疣刺参促***短肽及其编码基因与应用 (Ovulation-promoting short peptide of stichopus parviflora as well as coding gene and application thereof ) 是由 陈廷 任春华 吴菲菲 称楚航 江晓 于 2019-11-15 设计创作，主要内容包括：本发明公开了小疣刺参促排卵短肽及其编码基因与应用。本发明首次从小疣刺参卵巢克隆到小疣刺参cubifrin基因的开放读码框,其核苷酸序列如SEQ ID NO.1所示,其编码cubifrin前体蛋白的氨基酸序列如SEQ ID NO.2所示。其编码的3种衍生NG小肽NGIWY、NGFWY和NGLWY的氨基酸序列如SEQ ID NO.3、SEQ ID NO.4和SEQ ID NO.5所示。NGIWY、NGFWY和NGLWY可作为刺激小疣刺参雌参排卵的短肽应用于小疣刺参人工育苗。(The invention discloses an ovulation-promoting short peptide of stichopus parviflora and a coding gene and application thereof. The invention clones the open reading frame of the cubifrin gene from the ovary of the stichopus parvitus for the first time, the nucleotide sequence of the open reading frame is shown as SEQ ID NO.1, and the amino acid sequence of the encoded cubifrin precursor protein is shown as SEQ ID NO. 2. The amino acid sequences of the 3 kinds of derived NG small peptides NGIWY, NGFWY and NGLWY coded by the polypeptide are shown in SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO. 5. NGIWY, NGFWY and NGLWY can be used as short peptide for stimulating ovulation of Stichopus japonicus selenka for artificial breeding of Stichopus japonicus selenka.)

1. The precursor protein of Cubifrin of Acanthopanax parviflorus is characterized in that the amino acid sequence is shown as SEQ ID NO. 2.

2. A gene encoding the precursor protein of stichopus parviflora cubifrin according to claim 1.

3. The gene of claim 2, wherein the nucleotide sequence is represented by SEQ ID No. 1.

4. A recombinant expression vector comprising the gene of claim 2 or 3.

5. A host cell comprising the recombinant expression vector of claim 4.

6. A derivative NG small peptide is characterized in that the amino acid sequence of the derivative NG small peptide is shown as SEQ ID NO.3, SEQ ID NO.4 or SEQ ID NO. 5.

7. The use of the derivative NG small peptide as a short peptide for stimulating ovulation of stichopus japonicus selenka in the artificial breeding of stichopus japonicus selenka.

8. An agent for stimulating ovulation in female apostichopus japonicus selenka comprising the derivative NG small peptide according to claim 6 as an active ingredient.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to an ovulation-promoting short peptide of stichopus parviflora and a coding gene and application thereof.

Background

Stichopus japonicus selenka (Stichopus japonicus selenka) is commonly called as yellow meat ginseng, is a representative high-economic-value variety of tropical sea cucumbers in China, can be eaten, and can also be used as a raw material of cosmetics and medicines.

The artificial breeding of the stichopus japonicus selenka has been primarily successful in recent years, the induced spawning is mainly carried out by adopting a shade-drying running water method, the induced spawning effect of the method on male stichopus japonicus selenka is good, but the ovulation proportion of female stichopus japonicus selenka is low by adopting the method, and the ovulation and spermiation of the female stichopus japonicus selenka exist in a monthly rule, namely, the induced spawning can be successfully carried out only in one or two days of about 5-8 months in the lunar calendar every year, and the industrialized artificial breeding of the stichopus japonicus selenka is hindered. Therefore, an efficient oxytocic method for stichopus parviflora is urgently needed.

Echinoderm NGIWYamide (NG small peptide), originally isolated from the radial nerve cord and nerve loop of apostichopus japonicus, is a neuropeptide structurally similar to vertebrate NG peptide, which induces contraction of the body wall and intestinal muscles of apostichopus japonicus, stimulates the tubular-foot activity of starfish and the tubular-foot and esophageal contractions of echinocandis. The head of the apostichopus japonicus is swung after the NG small peptide is injected, so the precursor protein of the apostichopus japonicus is named cubifrin. The research shows that the injection of the NG small peptide can stimulate the generation and the maturation of the apostichopus japonicus oocyte. However, no related research on the gene sequence of the Cubifrin of the small wart stichopus japonicus and the derived small peptide exists at present.

Disclosure of Invention

The invention aims to provide an ovulation-promoting short peptide of stichopus parviflora and a coding gene and application thereof.

In order to realize the purpose, the invention obtains the Open Reading Frame (ORF) of the cubifrin gene by RT-PCR amplification on the basis of the transcript sequence of the cubifrin gene in the ovary transcriptome library of stichopus parviensis constructed by the subject group, wherein the nucleotide sequence of the Open Reading Frame (ORF) is shown as SEQ ID No.1, and the amino acid sequence of the coding cubifrin precursor protein is shown as SEQ ID No. 2. The precursor protein of the gene comprises 5NG small peptides (NGIWY, NGFWY, NGIWY and NGLWY) (figure 1), and can be processed after translation to form 3 derived NG small peptides: the amino acid sequences of NGIWY, NGFWY and NGLWY are respectively shown as SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO. 5. Because the NG small peptide of the cubifrin gene can stimulate the generation and maturation of sea cucumber oocytes, the NGIWY, the NGFWY and the NGLWY can be used as short peptides for stimulating the ovulation of the stichopus parviflora juveniles and applied to the artificial breeding of the stichopus parviflora.

The invention also provides a recombinant expression vector which contains the Cubifrin gene of the stichopus parviflorus, and the nucleotide sequence of the Cubifrin gene of the stichopus parviflorus is shown in SEQ ID No. 1.

The invention also provides a host cell containing the recombinant expression vector.

The invention also provides an agent for stimulating ovulation of stichopus japonicus selenka, which comprises NGIWY, NGFWY and/or NGLWY in the derivative NG small peptide.

Compared with the prior art, the invention has the advantages that:

the invention discloses a Cubifrin gene sequence of small stichopus japonicas for the first time, and related researches on a coding protein and derivative NG small peptides of the Cubifrin gene, and finds that a precursor protein of the Cubifrin gene of the small stichopus japonicas contains 5NG small peptides (NGIWY, NGFWY, NGIWY and NGLWY), and can be processed into 3 derivative NG small peptides after translation: NGIWY, NGFWY, and NGLWY. Compared with NG small peptides of cubifrin genes of apostichopus japonicus, holothuria rugosa and holothuria leucospilota, NGFWY and NGLWY are unique to apostichopus japonicus suffering from verruca minor. The NGIWY, NGFWY and NGLWY can be used as short peptides for stimulating ovulation of the stichopus japonicus selenka and can be applied to artificial breeding of the stichopus japonicus selenka.

Drawings

FIG. 1 is the nucleotide sequence of Cubifrin gene of lesser wart stichopus japonicus and its coding protein.

FIG. 2 is an alignment of derived NG small peptide sequences formed by post-translational processing of four sea cucumber cubifrin precursor proteins.

FIG. 3 shows the effect of 3 derived NG small peptides of Cubifrin precursor protein on ovulation of Culex parviflora.

Detailed Description

In order to make the objects, technical solutions and advantageous technical effects of the present invention clearer, the present invention is further described in detail with reference to the following embodiments. It should be understood that the embodiments described in this specification are only for the purpose of illustrating the invention and are not to be construed as limiting the invention, and the parameters, proportions and the like of the embodiments may be suitably selected without materially affecting the results. The examples are all reagents and process steps conventional in the art, except where specifically indicated. Unless otherwise indicated, reagents and materials used in the following examples are commercially available.