RNA interference fragment of donkey Eif2s3y gene, expression vector and application thereof

Abstract

The invention provides an RNA interference fragment of a donkey Eif2s3y gene, an expression vector and application thereof, and relates to the technical field of molecular genetics, the nucleotide sequence of the RNA interference fragment of the donkey Eif2s3y gene is shown as SEQ ID NO: 1 or SEQ ID NO: 2; the RNA interference fragment of the donkey Eif2s3y gene can artificially interfere expression of the donkey Eif2s3y gene, so that generation and development of Y sperms are blocked, and more fertilization opportunities are created for X sperms, so that the proportion of female foals of a first filial generation is increased, the purpose of controlling the gender of offspring foals is achieved, and X sperm cells are not damaged and the activity of the X sperm cells is not reduced. According to the invention, the gender of the offspring is effectively controlled before fertilization, the purpose of controlling the gender of the offspring donkey foals is achieved, the cost is low, and the RNA interference fragment provided by the invention has a relatively good silencing effect on the donkey Eif2s3y gene.

Description

Technical Field

[0001] The invention relates to the technical field of molecular genetics technology, and in particular to an RNA interference fragment of donkey Eif2s3y gene, an expression vector and applications thereof. Background Art

[0002] At present, it is of great significance to control the sex of livestock. The significance of animal sex control is: (1) improving the production efficiency of animal husbandry; (2) producing animals of the same sex with utilization value (generally, the utilization value of female animals is higher than that of male animals); (3) avoiding twin infertility caused by opposite-sex embryos during embryo transplantation; (4) improving the intensity of seed selection; (5) preventing the occurrence of sex-linked genetic diseases, etc. Relevant research on animal sex control has begun many years ago. Animal sex control (sex control) refers to a biotechnology that artificially intervenes in the normal reproductive process of animals to make adult female animals produce offspring of the desired sex. At present, there are three main methods for livestock sex control at home and abroad: (1) separation of X sperm and Y sperm. Through the slight biological differences between X and Y sperm, sperm containing two different chromosomes are separated. Currently, physical separation methods are mainly used. For example, the accuracy of sperm separation using flow cytometry can reach 90%, but its separation speed is too slow (18mil-lion / h), and the semen is expensive. After separation, the semen is not resistant to freezing, and the sperm deformity rate is high, which is limited in production application; (2) Embryo sex identification. Embryo sex identification is mainly through identifying the sex of the embryo, so as to artificially select the embryo and intervene in the sex ratio at birth. The main methods for early embryo sex identification include cytological methods, immunological methods, molecular biological methods, PCR amplification methods, male-specific DNA probe methods, etc. However, cutting some cells from the embryo and then freezing and thawing them will cause certain damage to the embryo, reducing the embryo survival rate and the success rate of embryo transplantation. At the same time, the success rate of sex identification of inferior embryos is even lower, which restricts the commercialization process of providing livestock embryos with predicted sex; (3) Controlling the external environment of livestock fertilization. Certain factors in the external environment of livestock are also important conditions for the sex determination mechanism, including nutrition, body fluid pH, temperature, insemination time, age and parity, hormone levels, etc. However, the factors that determine the sex of animals are mainly internal factors of the animal body. The method of controlling the external environment to control the sex of newborn animals is highly random, unstable, and difficult to control. Nowadays, with the development of science and technology, studying sex control at the genetic level has become a new direction. At the same time, with the increasing maturity of genetic technology, the cost of genetic technology has also dropped significantly, making it possible to promote it on a large scale in production applications.

[0003] RNA interference (RNA Interference) is a gene silencing phenomenon at the post-transcriptional level. It is achieved by synthesizing double-stranded RNA in vitro or double-stranded RNA in vivo to specifically degrade its homologous mRNA in cells, silencing the corresponding gene and preventing gene expression. It has the advantages of specificity, high efficiency, and easy operation. RNA interference is not a transgenic or gene editing technology.

[0004] The Eif2s3y gene is a sequence of the third subunit of the second initiation factor of protein translation encoded on the short arm of the Y chromosome. It is located in the non-recombination region of the short arm of the Y chromosome, and the genes in this region are only expressed in male animals. Eif2s3y encodes eIF2γ, which forms a ternary complex with the translation initiation codon Met-tRNAiMet, and is closely related to the initiation of translation of specific proteins and the development of Y sperm. Silencing the Eif2s3y gene through RNA interference technology affects the development of Y sperm, but has no effect on X sperm, which is a feasible way to achieve sex control. At present, the invention patents that have been authorized for interfering with the Zfx / Zfy gene to control sex in animals include mouse (female) (CN ZL 201110072013.6), mouse (male) (CN ZL 201310053235.2), deer (CN ZL 201710506560.8), donkey (CN ZL 201410421472.4), pig (CN ZL202211458361.1; CNZL201410421472.4), and dairy goat (CN ZL 202211335178.2). The Eif2s3y gene is located on the short arm of the Y chromosome with the Zfy gene, and is the upstream gene of the Zfy gene. It has the same effect as the Zfy gene on the development of Y sperm.

[0005] Through the above analysis, the problems and defects of the prior art are as follows:

[0006] (1) Among the current methods for sex control of livestock, physical separation is too slow, and the semen is expensive. The semen after separation is not resistant to freezing, and the sperm deformity rate is high, which limits its application in production. The amount of semen ejaculated by a donkey is large and the semen is thin, which increases the difficulty of physical separation.

[0007] (2) In early embryo sex identification, cutting out some cells from the embryo and then freezing and thawing them will cause certain damage to the embryo, reducing the embryo survival rate and the success rate of embryo transplantation. The success rate of sex identification of poor quality embryos is even lower.

[0008] (3) Among the current methods for controlling the sex of livestock, the method of controlling the external environment to control the sex of newborn animals is highly random, unstable, has poor repeatability, and is difficult to control. Therefore, the present invention proposes an RNA interference fragment of the donkey Eif2s3y gene, an expression vector, and its application to solve the problems existing in the prior art. Summary of the invention

[0009] In view of the above problems, the purpose of the present invention is to propose an RNA interference fragment, an expression vector and an application thereof of the donkey Eif2s3y gene, which effectively controls the sex of offspring before fertilization, thereby achieving the purpose of controlling the sex of offspring donkey foals. Moreover, the method is simple, low-cost, easy to promote, and can solve the problems existing in the prior art.

[0010] To achieve the purpose of the present invention, the present invention is implemented by the following technical scheme: an RNA interference fragment of the donkey Eif2s3y gene, the nucleotide sequence of the RNA interference fragment of the donkey Eif2s3y gene is shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0011] An expression vector constructed by RNA interference fragment of donkey Eif2s3y gene.

[0012] A further improvement is that the expression vector comprises the sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4.

[0013] A further improvement is that the expression vector comprises a pPLK GFP+Puro vector, and the pPLK GFP+Puro vector is connected to the RNA interference fragment of the donkey Eif2s3y gene.

[0014] The invention discloses an application of an expression vector constructed by RNA interference fragment of donkey Eif2s3y gene in preparing a reagent for sex control of animals before fertilization.

[0015] A further improvement is that the reagent for sex control of the animal before fertilization is injected directly into the animal's testicles to interfere with the Eif2s3y gene on the animal's Y chromosome.

[0016] The invention discloses an expression vector constructed by RNA interference fragment of donkey Eif2s3y gene and its application in preparing a preparation for controlling the sex of offspring donkey foals. The expression vector is used for silencing Eif2s3y gene in donkey spermatogenic cells.

[0017] The invention discloses an application of an RNA interference fragment of a donkey Eif2s3y gene in preparing a preparation for controlling the sex of offspring donkey foals. The RNA interference fragment of the donkey Eif2s3y gene is used for silencing the Eif2s3y gene in donkey spermatogenic cells.

[0018] The invention discloses an application of an RNA interference fragment of donkey Eif2s3y gene in preparing a kit for controlling the sex of offspring donkey foals.

[0019] The invention discloses an application of an expression vector constructed by RNA interference fragment of donkey Eif2s3y gene in preparing a kit for controlling the sex of offspring donkey foals.

[0020] The beneficial effects of the present invention are as follows: (1) The Eif2s3y gene provided by the present invention is responsible for encoding eIF2γ, which forms a ternary complex with the translation initiation codon Met-tRNAiMet, is closely related to the translation initiation of specific proteins, and is associated with the development of Y sperm.

[0021] (2) The RNA interference fragment provided by the present invention artificially interferes with the expression of the donkey Eif2s3y gene, thereby hindering the generation and development of Y sperm and not affecting X sperm, thereby creating more fertilization opportunities for X sperm, thereby increasing the proportion of female foals in the next generation. The present invention effectively controls the sex of offspring before fertilization, achieving the purpose of controlling the sex of offspring donkey foals, and the method is simple, low-cost, and easy to promote.

[0022] (3) The RNA interference fragment provided by the present invention artificially interferes with the expression of the donkey Eif2s3y gene, thereby hindering the generation and development of Y sperm and reducing the motility of Y sperm, but having no effect on X sperm, creating more fertilization opportunities for X sperm, thereby increasing the proportion of female foals in the first generation and achieving the purpose of controlling the gender of offspring foals. The RNA interference fragment provided by the present invention has a good silencing effect on the donkey Eif2s3y gene. After interfering with the Eif2s3y gene, the female foal rate in the offspring foals is 73.68%.

[0023] This technology uses direct testicular injection, does not require special expensive instruments and equipment, is simple, easy to operate, and low-cost. In particular, unlike the semen obtained by the separation method, which can only be used for artificial insemination and has high technical requirements, this technology can be used for natural mating, artificial insemination, and the production of frozen sperm tubes, which can be transported over long distances for application. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is a schematic diagram of the interference carrier spectrum provided by the present invention.

[0025] Figure 2 It is a schematic diagram of the mRNA expression level of the Eif2s3y gene in donkey spermatogenic cells provided by the present invention. DETAILED DESCRIPTION

[0026] In order to deepen the understanding of the present invention, the present invention will be further described in detail below in conjunction with examples. The examples are only used to explain the present invention and do not constitute a limitation on the protection scope of the present invention.

[0027] according to Figure 1-Figure 2 As shown, this embodiment proposes an RNA interference fragment (RNA Interference fragment) of the donkey Eif2s3y gene, and the nucleotide sequence of the RNA interference fragment of the donkey Eif2s3y gene is shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0028] The Eif2s3y gene provided in this example is responsible for encoding eIF2γ, which forms a ternary complex with the translation initiation codon Met-tRNAiMet, is closely related to the initiation of translation of specific proteins, and is associated with the development of Y sperm.

[0029] An expression vector constructed by RNA interference fragment of donkey Eif2s3y gene.

[0030] The expression vector comprises the sequence shown in SEQ ID NO:3 or SEQ ID NO:4.

[0031] The expression vector includes a pPLK GFP+Puro vector, and the pPLK GFP+Puro vector is connected with an RNA interference fragment of donkey Eif2s3y gene.

[0032] The invention discloses an application of an expression vector constructed by RNA interference fragment of donkey Eif2s3y gene in preparing a reagent for sex control of animals before fertilization.

[0033] The reagent for sex control before animal fertilization is injected directly into the animal's testicles to interfere with the Eif2s3y gene on the animal's Y chromosome.

[0034] The invention discloses an expression vector constructed by RNA interference fragment of donkey Eif2s3y gene and its application in preparing a preparation for controlling the sex of offspring donkey foals. The expression vector is used for silencing Eif2s3y gene in donkey spermatogenic cells.

[0035] The invention discloses an application of an RNA interference fragment of donkey Eif2s3y gene in preparing a preparation for controlling the sex of offspring donkey foals. The RNA interference fragment of donkey Eif2s3y gene is used for silencing the Eif2s3y gene in donkey spermatogenic cells.

[0036] The invention discloses an application of an RNA interference fragment of donkey Eif2s3y gene in preparing a kit for controlling the sex of offspring donkey foals.

[0037] The invention discloses an application of an expression vector constructed by RNA interference fragment of donkey Eif2s3y gene in preparing a kit for controlling the sex of offspring donkey foals.

[0038] The embodiments of the present invention have achieved some positive effects during the development or use process, and indeed have great advantages over the prior art. The following content is described in conjunction with data, charts, etc. of the test process.

[0039] 1. Construction of RNA interference fragment and expression vector of donkey Eif2s3y gene

[0040] The present invention screened out 2 pairs of siRNA fragments through preliminary work, and then added restriction sites, loops and termination sequences to the 5' and 3' of each siRNA fragment according to the requirements of the vector and the promoter it carried. The loop structure in the template selected TCAAGAG, and the transcription termination sequence of shRNA adopted the T6 structure. GATCC was added to the 5' end of the positive chain template. If the first base of the siRNA is not G, a G is added after GATCC, and there are two Ts at the end. (Nanjing Jikai Gene Biotechnology Co., Ltd. was commissioned to synthesize the shRNA oligonucleotide sequence), as shown in Table 1.

[0041] Table 1 2 pairs of shRNA oligonucleotide sequences

[0042]

[0043]

[0044] The synthesized shRNA fragments were annealed into double strands and connected to the vector pPLK to obtain an expression vector. The vector map is shown in Figure 1 As shown; then transformed into E.coli DH5α competent cells for amplification, the plasmid was extracted and stored at -20°C for future use.

[0045] 2. In vitro interference test of donkey Eif2s3y gene

[0046] Testicular tissue of sexually mature donkeys was collected, and the testicular supporting cells and spermatogenic cells were separated by a two-step enzymatic digestion method. The separated supporting cells and spermatogenic cells were placed in complete culture medium (containing FBS, double antibody, insulin, transferrin, retinoic acid, vitamin A, vitamin E, vitamin C, pyruvate, testosterone) and cultured at 35°C, 5% CO2, and 95% humidity for 24 hours. Lipofectamine 2000 was used as a transfection reagent to transfect the two plasmids stored in Table 1 of step 1 into the spermatogenic cells. Each group was repeated three times. 58 hours after transfection, the total RNA of the spermatogenic cells was extracted, and the mRNA expression level of the Eif2s3y gene was detected by qRT-PCR. The empty vector was used as the control group to determine the interference effect of the two pairs of interference fragments. The primers used in the experiment are shown in Table 2. The mRNA expression of the Eif2s3y gene in spermatogenic cells detected by qRT-PCR is shown in Table 2. Figure 2 shown.

[0047] Table 2 Primer sequences for qRT-PCR detection of Eif2s3y gene and internal reference gene

[0048]

[0049] like Figure 2 The results showed that the expression of Eif2s3y gene mRNA in the Y-217 interference vector in the experimental group was significantly different from that in the control group (P < 0.01); it was preliminarily determined that the Y-217 interference fragment had a silencing effect on the donkey Eif2s3y gene. Figure 2 The results showed that there was no significant difference in the expression level of Eif2s3y gene mRNA in the interference vector between the experimental group Y-323 and the control group (P>0.05).

[0050] 3. Donkey in vivo interference test of Eif2s3y gene

[0051] Add 1.5 mg of Y-217 plasmid and 500 μL of double antibody to a 10 mL centrifuge tube, and add PBS to the volume to 6 mL. Nine sexually mature and healthy male donkeys were selected for the experiment at the donkey farm of the Black Donkey Research Center of Dong'e Ejiao Co., Ltd. The plasmid was extracted with a syringe and punctured with a 0.3 mm*60 mm puncture needle to the testicular skin and injected into the testicle. This injection method avoids damage to the testicle due to the needle being too thick. Each male donkey was injected with 1.5 mg per testicle, once every 10 days, for a total of 3 injections. Fresh semen artificial insemination can be performed 3 days after the third injection, and the ear number of the injected male donkey, the ear number and pen number of the female donkey, and the number of male and female offspring foals were counted.

[0052] Table 3 Sex statistics of offspring donkeys

[0053] Plasmid name Number of foals born female Female ratio Y-217 57 42 73.68％

[0054] As shown in Table 3, after the Y-217 plasmid interference, the female proportion of the donkey offspring was 73.68%, which was significantly different from the 1:1 sex ratio (P < 0.01).

[0055] It can be seen from the above examples that the RNA interference fragment provided by the present invention has a good silencing effect on the donkey Eif2s3y gene. After interfering with the Eif2s3y gene, the female foal rate in the offspring donkey foals is 73.68%.

[0056] Then the technical solution of the present invention is transformed, and the expected economic and social benefits after the transformation are:

[0057] Since the gestation period of donkeys is 13 months, which is one of the longest gestation periods among all mammals, the reproductive efficiency of donkeys is relatively low. In addition, the investment in donkey farming is large and the capital recovery time is long, which further causes the current situation of survival difficulties for donkey farmers. If dairy donkeys are raised, the proportion of milk-producing donkeys in donkey farms can reach 80% to 85% through gender control. On the one hand, the base of the female donkey group can be continuously expanded, and the donkey milk produced can continuously increase income, maintain the survival of the donkey farm and even make profits. In particular, it has a good curbing effect on the trend of a sharp decline in the number of donkeys in the donkey farming industry year by year. It plays a very important role in promoting the vigorous development of my country's donkey farming industry and the development of extended industries.

[0058] The present invention can also solve the technical problem of achieving sex control for small animals such as dairy goats and animals with low individual economic benefits, because the present invention has low cost, is easy to use, and has various breeding methods.

[0059] The above shows and describes the basic principles, main features and advantages of the present invention. It should be understood by those skilled in the art that the present invention is not limited by the above embodiments. The above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the framework and scope of application of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention to be protected. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. An RNA interference fragment of donkey Eif2s3y gene, characterized in that: The nucleotide sequence of the RNA interference fragment of the donkey Eif2s3y gene is shown in SEQ ID NO: 1 or SEQ ID NO:

2.

2. An expression vector constructed by using an RNA interference fragment of the donkey Eif2s3y gene described in claim 1.

3. The expression vector constructed by the RNA interference fragment of the donkey Eif2s3y gene according to claim 2, characterized in that: The expression vector comprises the sequence shown in SEQ ID NO:3 or SEQ ID NO:

4.

4. The expression vector constructed by the RNA interference fragment of the donkey Eif2s3y gene according to claim 2, characterized in that: The expression vector comprises a pPLK GFP+Puro vector, and the pPLK GFP+Puro vector is connected with the RNA interference fragment of the donkey Eif2s3y gene.

5. Use of an expression vector constructed by using an RNA interference fragment of the donkey Eif2s3y gene as claimed in any one of claims 2 to 4 in the preparation of a reagent for sex control of animals before fertilization.

6. The RNA interference fragment, expression vector and application of the donkey Eif2s3y gene according to claim 5, characterized in that: The reagent for sex control of the animal before fertilization is injected into the animal by direct testicular injection, so as to interfere with the Eif2s3y gene on the Y chromosome of the animal.

7. An application of an expression vector constructed by using an RNA interference fragment of a donkey Eif2s3y gene as claimed in any one of claims 2 to 4 in the preparation of a preparation for controlling the sex of offspring donkey foals, characterized in that: The expression vector is used to silence the Eif2s3y gene in donkey spermatogenic cells.

8. An application of an RNA interference fragment of a donkey Eif2s3y gene according to claim 1 in the preparation of a preparation for controlling the sex of offspring donkey foals, characterized in that: The RNA interference fragment of the donkey Eif2s3y gene is used to silence the Eif2s3y gene in donkey spermatogenic cells.

9. Use of an RNA interference fragment of the donkey Eif2s3y gene according to claim 1 in preparing a kit for controlling the sex of offspring donkey foals.

10. Use of an expression vector constructed using an RNA interference fragment of the donkey Eif2s3y gene as claimed in any one of claims 2 to 4 in preparing a kit for controlling the sex of offspring donkey foals.

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