CN115806986B - Milk goat Zfy gene interference fragment, expression vector and application thereof - Google Patents
Milk goat Zfy gene interference fragment, expression vector and application thereof Download PDFInfo
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Abstract
The invention relates to a Zfy gene interference fragment of a milk goat, an expression vector and application thereof, and belongs to the technical field of animal breeding. The invention provides a Zfy gene interference fragment of a milk goat, and the sequence of the Zfy gene interference fragment of the milk goat is shown as SEQ ID NO. 1-2. The Zfy gene interference fragment of the invention is utilized to artificially interfere the expression of the Zfy gene of the milk goat, so that the generation and development of Y sperms are blocked, the activity is reduced, but the development and the activity of X sperms are not influenced, thereby creating more fertilization opportunities for the X sperms and increasing the proportion of the mother lambs of the child generation. And has low cost and convenient use. Provides a theoretical basis for further application of the milk goats.
Description
Technical Field
The invention relates to the technical field of animal breeding, in particular to a Zfy gene interference fragment of a milk goat, an expression vector and application thereof.
Background
Sex control (sex control) of animals refers to a biological technique that allows adult females to produce offspring of a desired sex through human intervention in the normal reproductive process of the animal. The significance of sex control of animals is mainly that: (1) improving the animal husbandry production benefit; (2) Producing an animal having a sex of utility value (typically female animals are of higher utility value than male animals); (3) The twin sterility caused by the foreign embryo in the embryo transfer process is avoided; (4) improving seed selection intensity; (5) preventing the occurrence of concomitant genetic diseases, etc.
Related studies on sex control of animals have begun as early as many years ago. At present, domestic and foreign livestock sex control methods mainly comprise three methods (1) separating X sperms from Y sperms. Separating sperm containing two different chromosomes by the weak biological difference of X, Y sperm, wherein the accuracy of the separation of the sperm can reach 90% by adopting a flow cytometry at present, but the separation speed is too low (18 mil-lion/h), the semen is expensive, the separated semen is not resistant to freezing, and the sperm mortality is higher, so that the application of the method is limited; (2) sex identification of embryos. Embryo sex determination is mainly by identifying the sex of the embryo, thereby manually intervening in the sex ratio of birth. The early embryo sex identification method mainly comprises a cytology method, an immunology method, a molecular biology method, a PCR amplification method, a male specificity DNA probe method and the like, but partial cells are cut from the embryo and then frozen and thawed to cause certain damage to the embryo so as to reduce the success rate of embryo transfer, and meanwhile, the sex identification success rate of the inferior embryo is lower, so that the commercialization process of providing the embryo of livestock with predicted sex is restricted; (3) controlling the external environment of livestock fertilization. Some factors in the livestock external environment are also important conditions for sex determination mechanisms, mainly including nutrition, ph of body fluids, temperature, insemination time, age, birth times, hormone levels, etc. However, the factors determining the sex of animals are mainly internal factors of animals, and the sex of the animals is controlled by adopting a method for controlling the external environment, so that the sex is highly random and unstable and is not easy to control. Nowadays, research on sex control from the genetic level has become a new direction with the development of the scientific and technological level, and the cost of genetic technology has also been greatly reduced with the increasing maturity of genetic technology, so that the genetic technology is possible to be popularized in a large scale in production and application.
RNA interference (RNAInterface) is a post-transcriptional gene silencing phenomenon, and is characterized in that double-stranded RNA is artificially synthesized in vitro or homologous mRNA is specifically degraded in cells by double-stranded RNA in vivo, so that the corresponding gene is silenced, the purpose of preventing gene expression is achieved, and the RNA interference is not transgenic, and has the advantages of specificity, high efficiency, easiness in operation and the like.
The Zfy gene is located on the Y chromosome of mammals and is responsible for the specific genes encoding 12-13 transcription factors on a large acid chain zinc finger protein. The Zfy gene has specific nuclear localization signal region and DNA binding site, can be specifically combined with target gene, can directionally guide the target gene to pass through nuclear membrane and locate in sperm cell nucleus, and its coded protein is used as transcription regulating factor, and is related to development of Y sperm. Silencing the Zfy gene by RNA interference techniques is a viable approach to achieving sex control. However, animals capable of sex control by silencing Zfy gene of the prior art do not include dairy goats. Therefore, in order to improve the utilization efficiency of the milk goats, a method for controlling the gender of the milk goats by using the silent Zfy gene is imperative to be studied.
Disclosure of Invention
The invention aims to provide a Zfy gene interference fragment of a milk goat, an expression vector and application thereof.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a Zfy gene interference fragment of a milk goat, and the sequence of the Zfy gene interference fragment of the milk goat is shown as SEQ ID NO. 1-2.
The invention also provides application of the Zfy gene interference fragment of the milk goat in preparation of a preparation for silencing the Zfy gene in sperm cells of the milk goat.
The invention also provides application of the Zfy gene interference fragment of the milk goat in preparing a medicament for controlling the sex of the milk goat.
The invention also provides an expression vector which comprises the milk goat Zfy gene interference fragment and an empty vector.
Preferably, the empty vector is pGPU6/GFP/Neo.
The invention also provides application of the expression vector in preparation of a preparation for silencing Zfy gene in sperm cells of milk goats.
The invention also provides application of the expression vector in preparation of a medicament for controlling sex of milk goats.
The invention provides a Zfy gene interference fragment of a milk goat, an expression vector and application thereof. The RNA interference fragment provided by the invention can be used for carrying out artificial interference on the expression of the Zfy gene of the dairy goat, so that the generation and development of Y sperms are blocked, the vitality is reduced, but the development and vitality of X sperms are not influenced, thereby creating more fertilization opportunities for the X sperms and increasing the mother lamb proportion of the child generation. The invention effectively controls the sex of offspring before fertilization, achieves the purpose of controlling the sex of offspring milk goats, and has low cost and convenient use. Provides a theoretical basis for further application of the milk goats.
Drawings
FIG. 1 shows pGPU6/GFP/Neo empty vector map.
FIG. 2 shows the expression level of Zfy/Zfx gene mRNA in sperm cells.
Detailed Description
The invention provides a Zfy gene interference fragment of a milk goat, and the sequence of the Zfy gene interference fragment of the milk goat is shown as SEQ ID NO. 1-2.
In the invention, the sequence of the sense strand of the Zfy gene interference fragment of the milk goat is shown as SEQ ID NO.1, specifically 5'-CACCGTGCTCAGATATCTTAGAATTCAAGAGATTCTAAGATATCTGAGCACTTTTTTG-3'; the sequence of the antisense strand is shown in SEQ ID NO.2, specifically 5'-GATCCAAAAAAGTGCTCAGATATCTTAGAATCTCTTGAATTCTAAGATATCTGAGCAC-3'.
In the invention, the Zfy gene interference fragment of the milk goat is a short hairpin RNA (shRNA), the nucleotide sequence of the loop structure in the DNA template of the shRNA is TTCAAGAGA so as to avoid forming a termination signal, and the transcription termination sequence of the shRNA adopts a T6 structure. The nucleotide sequence of the 5' end of the sense strand template is CACC and is complementary with the sticky end formed after BbsI enzyme digestion; the 5' -end of the antisense strand template had a nucleotide sequence of GATC, which was complementary to the sticky end formed after BamHI cleavage.
In the invention, the shRNA is constructed on the basis of siRNA fragments. If the first base of the siRNA is not G, then a G is added after CACC. Enzyme cutting sites, loop loops and termination sequences are added to the 5 'and 3' of each siRNA segment.
The invention also provides application of the Zfy gene interference fragment of the milk goat in preparation of a preparation for silencing the Zfy gene in sperm cells of the milk goat.
The invention also provides application of the Zfy gene interference fragment of the milk goat in preparing a medicament for controlling the sex of the milk goat.
The invention also provides an expression vector which comprises the milk goat Zfy gene interference fragment and an empty vector.
In the present invention, the empty vector is pGPU6/GFP/Neo.
The invention also provides application of the expression vector in preparation of a preparation for silencing Zfy gene in sperm cells of milk goats.
The invention also provides application of the expression vector in preparation of a medicament for controlling sex of milk goats.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
Construction of interference fragment and expression vector of Zfy Gene of milk goat
The sequence of 1165bp is obtained by designing a primer by taking the Zfy gene (NCBI accession number: XM_ 018044894.1) of the milk goat as a template in the early stage and amplifying. The sequence of 1165bp is designed by siRNA design software, 2 pairs of siRNA fragments are screened out by culturing the milk goat sperm cells, carrying out cell transfection and real-time fluorescence quantification, and cDNA templates of the siRNA fragments are shown as SEQ ID NO. 5-6.
The 1165bp Zfy gene sequence is shown in SEQ ID NO.13, and SEQ ID NO.13 is TTTTGATGGAATAGGAACTGATGCTACACACATGGATGGTGATCAAATTGTTGTGGAAGTACAAGAAACTGTTTTTGTTTCAGATGTTGTGGATTCAGATATAACTGTGCATAATTTTGTTCCTGATGATCCAGACTCAGTTGTAATCCAAGATGTTATTGAGAATGTTGTTATTGAGGATGTTCAGTGCTCAGATATCTTAGAA GAAGCAGATGTGTCTGAAAATGTCATCATTCCTGAGCAAATGCTTTCCTCAGATGTAACAGAAGAAGTGTCTTTAGCACATTGCACAGTCCCAGATGACGTCTTAGCTTCTGATATTACTTCAGCCTCAATGTCTATGCCAGAACATGTCTTGACCAGTGAGTCTGTACATGTGTCTGATGTTGGACATGTTGAACACATTGTTCGTGGTAGTGTAGTAGAGGCAGAAATTGTCACTGATCCTCTGACAGACGACGTAGTGTCAGAAGAAGTATTGGTAGCAGATTGTGCCTCAGAAGCAGTCATAGATGCCAACGGGATCCCTGTGGACCAGCAGGATGATGACAAAGGCAACTGTGAGGACTACCTTATGATTTCCTTGGATGATGATGGCAAAATTGAACAGGACTGTTCTGCTGGTATGACCATAGACAGAGAGTCGGAAATTGATCCTTGTAAGGTGGATGGCACTTGCCCTGAAGTCATCAAGGTGTATATTTTTAAAGCTGACCCTGGAGAGGATGACTTAGGTGGGACTGTTGACATTGTGGAGAGTGAGCCTGAGAATGATCATGGAGTTGAACTCCTTGATCAGAGTAACAGTATTCGTATGCCAAGGGAAAAGATGGTTTATATGACTGTCAGTGACTCTCAACAAGAAGATGAAGATTTAAATGTTGCTGAAATTGCAGATGAAGTTTATATGGAAGTGATTGTAGGAGAGGAAGATGCTGCTGTTGCTGCAGCAGCTGCTACCACTGTTCATGAACAGGAAATGGATGACAGTGAAATCAAAACCTTCATGCCAATAGCATGGGCAGCAGCTTATGGTAATAATTCTGATGGAATTGAAAATCGGAATGGCACTGCAAGTGCTCTCTTGCACATAGATGAGTCTGCTGGACTTGGCAGACTGGCTAAACAAAAGCCAAAGAAAAGGAGAAGACCTGATTCCAGGCAG.
SEQ ID No.5 is 5 'GTGCTCAGACTATTTAGAAA3';
SEQ ID No.6 is 5 'ATGCCAGAACATGTCTTTGA3'.
Enzyme cutting sites, loop loops and termination sequences are added to the 5 'and 3' of the siRNA fragments. TTCAAGAGA is selected as Loop structure, and T6 structure is adopted as termination sequence. The finally obtained shRNA oligonucleotide sequence is the interference fragment of the Zfy gene of the milk goat, and the shRNA oligonucleotide sequence is the T+sense strand 19nt target sequence+stem-loop structure (CTTGAAAGA) +target sequence reverse complementary sequence+RNA PolyIII polymerase transcription termination site (TTTTTT) +XhoI cleavage site (GAGCT). CACC is added to the 5' end of a specific shRNA sense strand template and complements the sticky end formed after BbsI enzyme digestion; the 5' end of the antisense strand template was added with GATC, complementary to the sticky end formed after BamHI cleavage. The shRNA oligonucleotide sequence was synthesized by Shanghai Ji Ma pharmaceutical technologies Co. The synthesized shRNA fragment is annealed to be double-stranded, and the name of shRNA oligonucleotide synthesized according to the fragment shown in SEQ ID NO.5 is pGPU6-2, and the specific sequence is shown in SEQ ID NO. 1-2. The shRNA oligonucleotide synthesized according to the fragment shown in SEQ ID NO.6 is named pGPU6-1, and the specific sequence is shown in SEQ ID NO. 3-4.
SEQ ID NO.1 is 5'-CACCGTGCTCAGATATCTTAGAATTCAAGAGATTCTAAGATATCTGAGCACTTTTTTG-3';
SEQ ID NO.2 is 5'-GATCCAAAAAAGTGCTCAGATATCTTAGAATCTCTTGAATTCTAAGATATCTGAGCAC-3';
SEQ ID NO.3 is 5'-CACCATGCCAGAACATGTCTTGATTCAAGAGAAGACATTTCTTTACTATGCTTTTTTG-3';
SEQ ID NO.4 is 5'-GATCCAAAAAAATGCCAGAACATGTCTTGATCTCTTGAAAGACATTTCTTTACTATGC-3'.
pGPU6-1 and pGPU6-2 obtained above were ligated to pGPU6/GFP/Neo vector, respectively, to obtain expression vector, which pGPU6/GFP/Neo vector is shown in FIG. 1. Then transferring into E.coli DH5 alpha competent cells for amplification, extracting plasmids to obtain pGPU6-1 and pGPU6-2 plasmids, and preserving at-20 ℃ for standby.
Example 2
In vitro interference test on Zfy gene of milk goat
Collecting testis tissue of mature milk goat, and separating testis supporting cells and spermatogenic cells by two-step enzyme digestion. Placing the separated support cells and sperm cells into a complete culture solution (containing FBS, diabody, insulin, transferrin, retinoic acid, vitamin A, vitamin E, vitamin C, pyruvic acid, testosterone) at 35deg.C, 5% CO 2 Culturing for 24h under the condition of 95% humidity, and using liposome 2000 as a transfection reagent, the two plasmids, namely pGPU6-1 plasmid and pGPU6-2 plasmid, stored in example 1 are respectively transfected into spermatogenic cells, and each group is repeated three times. And (3) extracting total RNA of the spermatogenic cells after 60h transfection, detecting mRNA expression levels of Zfy and Zfx genes by qRT-PCR, and judging the interference effect of 2 pairs of interference fragments by taking an empty vector as a control group. The primers used in the test are shown in Table 1 and SEQ ID NO. 7-12, and the mRNA expression of genes Zfy and Zfx of spermatogenic cells is detected by qRT-PCR as shown in FIG. 2. The primers for detecting the Zfy gene were set using the series of genes with accession number xm_018044894.1 in NCBI as templates. The primer for detecting Zfx gene was set using the gene with accession number XM_018043810.1 in NCBI as a template.
TABLE 1qRT-PCR detection of primer sequences of Zfy and Zfx genes and internal reference genes
As shown in fig. 2, the expression level of Zfy gene mRNA in pGPU6-2 plasmid in the experimental group was lower than that in the control group (empty vector group), and the difference was very significant (P < 0.01) compared with the experimental group; preliminary determination of pGPU6-2 interference fragment has silencing effect on Zfy gene of milk goat. The expression level of Zfx gene mRNA in the experimental group pGPU6-2 interference fragment is not significantly different from that of the control group (P is less than 0.05), which shows that the expression of Zfx gene is not significantly influenced. The expression level of Zfy gene mRNA in the pGPU6-1 plasmid of the experimental group is also lower than that of the control group (empty vector group), and the difference is very obvious compared with the control group, but the expression level of Zfx gene mRNA in the pGPU6-1 interference fragment of the experimental group is also obvious compared with the control group, which indicates that the pGPU6-1 interference fragment also influences the expression of Zfx genes.
Example 3
Milk goat in vivo interference test of Zfy gene
A10 mL centrifuge tube was charged with plasmid (pGPU 6-2 plasmid stored in example 1) and 500. Mu.L of the double antibody in a total amount of 3.0mg, and the volume was made up to 6mL with PBS. 3 mature healthy goats are selected for testing in a Shaanxi Long county milk goat breeding field and a Qianyang county breeding sheep field respectively, the testis skin is punctured by 1.0 multiplied by 18 10-size puncture, and then plasmids are extracted by 0.6 multiplied by 122-size 6-size needle and enter the testis through the puncture needle to be injected, wherein the injection method avoids the damage in the testis caused by over-thick needle, each goats is injected with 3.0mg each side testis, the injection is carried out once every 10 days, the total injection is carried out for 3 times, and the breeding can be carried out 3 days after the 3 rd injection is finished (the Shang county milk goat breeding field adopts fresh sperm artificial insemination and the Qianyang county breeding sheep field adopts natural mating), and the injection can last for 30 days. And counting the number of male and female offspring of the lambs. The final statistics are shown in table 2.
TABLE 2 offspring milk goat gender statistics
| Male male | Female | Female ratio |
| 34 | 128 | 79.01％ ** |
As shown in Table 2, the female ratio in the offspring of the milk goats after being interfered by pGPU6-2 interference fragment is 79.01%, and the difference is very remarkable (P < 0.01) compared with the sex ratio of 1:1.
As can be seen from the above examples, the present invention provides a Zfy gene interference fragment of a milk goat, an expression vector and application thereof. The sequence of the Zfy gene interference fragment of the milk goat is shown as SEQ ID NO. 1-2. The interference fragment can silence Zfy gene of the milk goat, does not influence the expression of Zfx gene, and can improve the female lamb rate in offspring lambs to 79.01%.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (7)
1. The Zfy gene interference fragment of the milk goat is characterized in that the sense strand sequence of the Zfy gene interference fragment of the milk goat is shown as SEQ ID NO.1, and the antisense strand sequence is shown as SEQ ID NO. 2.
2. Use of the Zfy gene interference fragment of a dairy goat of claim 1 in the preparation of a formulation for silencing Zfy gene in sperm cells of a dairy goat.
3. Use of the Zfy gene interference fragment of the dairy goat of claim 1 in the preparation of a formulation for controlling the sex of the dairy goat.
4. An expression vector comprising the Zfy gene interference fragment of the dairy goat of claim 1 and an empty vector.
5. The expression vector of claim 4, wherein the empty vector is pGPU6/GFP/Neo.
6. Use of the expression vector of claim 4 or 5 in the preparation of a formulation for silencing Zfy gene in sperm cells of a dairy goat.
7. Use of the expression vector of claim 4 or 5 in the preparation of a formulation for controlling sex in a dairy goat.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6580017B1 (en) * | 1998-11-02 | 2003-06-17 | Genzyme Transgenics Corporation | Methods of reconstructed goat embryo transfer |
| CN104232643A (en) * | 2014-08-25 | 2014-12-24 | 石河子大学 | RNAi (ribonucleic acid interference) interference segment, interference vector, and preparation method and application thereof |
| CN105255889A (en) * | 2015-09-30 | 2016-01-20 | 石河子大学 | RNAi fragment, RNAi vector, preparation method and application of RNAi vector |
| CN113122539A (en) * | 2021-04-15 | 2021-07-16 | 石河子大学 | RNA interference fragment of donkey Zfy gene, expression vector and application thereof |
| CN115786343A (en) * | 2022-11-17 | 2023-03-14 | 石河子大学 | RNA interference fragment of swine Zfy gene, expression vector and application thereof |
-
2022
- 2022-10-28 CN CN202211335178.2A patent/CN115806986B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6580017B1 (en) * | 1998-11-02 | 2003-06-17 | Genzyme Transgenics Corporation | Methods of reconstructed goat embryo transfer |
| CN104232643A (en) * | 2014-08-25 | 2014-12-24 | 石河子大学 | RNAi (ribonucleic acid interference) interference segment, interference vector, and preparation method and application thereof |
| CN105255889A (en) * | 2015-09-30 | 2016-01-20 | 石河子大学 | RNAi fragment, RNAi vector, preparation method and application of RNAi vector |
| CN113122539A (en) * | 2021-04-15 | 2021-07-16 | 石河子大学 | RNA interference fragment of donkey Zfy gene, expression vector and application thereof |
| CN115786343A (en) * | 2022-11-17 | 2023-03-14 | 石河子大学 | RNA interference fragment of swine Zfy gene, expression vector and application thereof |
Non-Patent Citations (4)
| Title |
|---|
| Sex control by Zfy siRNA in the dairy cattle;Ji-feng Xia等;Animal Reproduction Science;20180517;1-6 * |
| Zfy基因与哺乳动物性别控制研究进展;张永生等;畜牧与兽医;20161210;105-108 * |
| 山羊Zfy基因克隆分析及其敲除载体构建;黄敏等;畜牧兽医学报;20220323;711-721 * |
| 绵羊Zfy基因干扰载体筛选及性控效果的验证;刘帅兵;中国优秀硕士学位论文全文数据库 农业科技辑;20150315;D050-70 * |
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