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Sex reversal siRNA of Chinese mitten crab and application of siRNA

A Chinese mitten crab, nucleotide sequence technology, applied in the direction of DNA / RNA fragments, recombinant DNA technology, and other methods of inserting foreign genetic materials, can solve the problems of low survival rate of androgenic glands, difficult surgery, etc. Achieve the effect of reducing infection mortality, avoiding immune stress response, and improving success rate

Inactive Publication Date: 2019-07-09
WEIFANG UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Implantation of androgenic glands in female Japanese mitten crabs prompts females to develop male-like appendages [5,6] , but the transplantation operation is difficult, and the survival rate of the androgenic gland is low; injecting the androgenic gland extract into the female mitten crab can also make the male crab produce male appendages [7]

Method used

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  • Sex reversal siRNA of Chinese mitten crab and application of siRNA
  • Sex reversal siRNA of Chinese mitten crab and application of siRNA
  • Sex reversal siRNA of Chinese mitten crab and application of siRNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] The IAG gene fragment of Chinese mitten crab was obtained by transcriptome technology, and its full-length cDNA sequence was cloned, and siRNA was designed and synthesized according to the IAG gene sequence of Chinese mitten crab (accession number: MF098773); the online siRNA design software ( http: / / sidirect2.rnai.jp / ) Design alternative siRNA sequences, exclude SNP-containing and non-open reading frame fragments from candidate siRNAs, remove degenerate bases, toxic structural domains, repetitive sequences, high GC / low GC sequences, from the perspective of thermodynamics , high-level structure, etc., and list the sequences from high to low. The siRNA fragments were subjected to Blast analysis to remove non-specific binding sequences. The design was entrusted to Shanghai Gemma Gene Co., Ltd. to synthesize three siRNAs and one negative control dsRNA ( See Table 1); in order to enhance the sequence stability, the two bases at the 3' end were replaced with dTdT; at the...

Embodiment 2

[0026] The siRNA obtained in the above examples was dissolved with EntransterTM-in vivo (Engreen Biosystem Co, Lid.) transfection reagent, and the amount of nucleic acid was in accordance with the standard of 2.5 mg / kg body weight. The complex of siRNA and transfection reagent was obtained from Eriocheir sinensis The joint membrane of the base of the fifth step of the male crab’s stage III juvenile crab was injected into the vicinity of the ejaculatory duct by a capillary tube. Before the injection and 12 hours after the injection, the tissue around the base of the penis of the male crab was taken for fluorescent quantitative PCR verification. The qRT- The PCR primers are listed in Table 2.

[0027] Table 2 Primer sequences used in qRT-PCR

[0028]

[0029] The reaction system is as follows:

[0030] 25μL qRT-PCR reaction system includes:

[0031]

[0032] The PCR reaction procedure is:

[0033]

[0034] A negative control was set up, with 3 biological replicates i...

Embodiment 3

[0042] Take the above-mentioned siRNA-391 and use EntransterTM-in vivo (Engreen Biosystem Co, Lid.) transfection reagent to dissolve it in proportion, and set aside;

[0043] Experiment: Take 90 young male crabs of Eriocheir sinensis stage III, and inject the complex of siRNA and transfection reagent into the ejaculatory duct from the joint membrane at the base of the foot in the fifth step according to the standard of 2.5mg / kg body weight for nucleic acid, and wait until The juvenile crabs at stage III molted into the second day after juvenile crabs at stage IV and stage V were reintroduced by the existing transfection means respectively. After the juvenile crabs moulted at stage V, the changes in the abdomen shape and male appendages were observed. The observation results can be seen: after the molting of young crabs at the V stage, the shape of the abdomen of 9 male crabs of 90 experimental mitten crabs changed from a narrow and long shape to a triangle, indicating that 9 ma...

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Abstract

The invention relates to a sex development regulation and control technology for the male Chinese mitten crab, in particular to a sex reversal siRNA of the Chinese mitten crab and application of the siRNA. A nucleotide sequence of a positive-sense strand of the sex reversal siRNA of the male Chinese mitten crab is shown in SEQ ID NO.1, and a nucleotide sequence of an antisense strand is shown in SEQ ID NO.2. according to the sex reversal siRNA, an IAG gene segment of the Chinese mitten crab is screened from an androgenic gland transcriptome of the Chinese mitten crab, the total-length cDNA sequence of the segment is cloned, according to an IAG gene sequence of the Chinese mitten crab, the siRNA is designed, synthesized and guided into a body of the Chinese mitten crab in the sex differentiation period, expression of an IAG gene in an androgenic gland cell is disturbed, and sex reversal of the male crab is achieved. Compared with androgenic gland removal through surgery, a micro-injection method is adopted and has the advantages that the trauma to the body of the Chinese mitten crab is small, and the situation is avoided that due to wound infection after surgery, the Chinese mittencrab dies; the injection dose is extremely low, the immunological stress reaction of a tested animal is not easily triggered, and accordingly the success rate of sex reversal is increased.

Description

technical field [0001] The invention relates to a sex development regulation technology of Chinese mitten crab male crab, in particular to a sex-reversal siRNA of Chinese mitten crab and its application. Background technique [0002] The androgenic gland (insulin-like androgenic gland, IAG) is a unique organ of male crustaceans, and the androgenic hormone secreted by it plays an important role in regulating the sex differentiation of crustaceans [1-4] . Implantation of androgenic glands in female Japanese mitten crabs prompts females to develop male-like appendages [5,6] , but the transplantation operation is difficult, and the survival rate of the androgenic gland is low; injecting the androgenic gland extract into the female mitten crab can also make the male crab produce male appendages [7] . [0003] RNAi technology is also vividly called gene knockout (Knock-down) or gene silencing (Gene silencing), which is a phenomenon of efficient and specific degradation of homol...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/87A01K67/033
CPCA01K67/0333A01K2217/075A01K2227/70A01K2267/02C07K14/43509C12N15/113C12N15/87C12N2310/14
Inventor 付春鹏李法君王丽芳
Owner WEIFANG UNIV OF SCI & TECH
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