Construction and application of recombinant lentivirus of giant salamander NR5A2 gene

Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to construction and application of a recombinant lentivirus of a giant salamander NR5A2 gene. The invention provides siRNA for targeted inhibition of giant salamander gonad Nr5a2 gene expression, and a positive-sense strand and an antisense strand of shRNA are also designed and synthesized based on the sequence of the siRNA, so as to prepare a recombinant vector and a virus interference vector for targeted inhibition of giant salamander gonad Nr5a2 gene expression. Test results prove that the siRNA, shRNA designed based on the siRNA, a recombinant vector, a virus interference vector, an engineering cell and the like can inhibit expression inhibition of the Nr5a2 gene in the giant salamander gonad, and can be used for silence and functional verification of the Nr5a2 gene of the giant salamander gonad and related research of sex differentiation of the giant salamander.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and particularly relates to the construction and application of a recombinant lentivirus of the Andrias davidianus NR5A2 gene. Background Art

[0002] The fibroblast nuclear receptor NR5A2 belongs to the NR5A or FTZ-F1 subfamily of nuclear receptors, also known as LRH-1 (liver receptor homolog) and fetal protein transcription factor (FTF). It is an orphan nuclear receptor that mainly participates in regulating transcription, morphogenesis, and homeostasis. The Nr5a2 gene is expressed in the endodermal tissues of mammals and certain vertebrates such as the gonads, liver, pancreas, and intestine, and is involved in the processes of promoting embryonic development, cell proliferation, and inhibiting tumorigenesis. The Nr5a2 gene is highly expressed in mouse follicular granulosa cells and luteal cells, and plays an important role in the growth of follicular cells and the proliferation of granulosa cells. The deletion of the Nr5a2 gene can cause abnormal expression of the progesterone receptor (PR), leading to certain breeding disorders.

[0003] Andrias davidianus is the largest existing amphibian in the world and is a national second-class protected wild animal. Currently, the artificial breeding of Andrias davidianus can be legally utilized. With the breakthrough of artificial breeding technology, the yield of Andrias davidianus has been increasing. However, there is a large growth difference between male and female Andrias davidianus, with males growing fast and females growing slow, which seriously hinders the development of the industry. In addition, Andrias davidianus is an amphibian and a key group in the transition from aquatic animals to terrestrial animals. The research on its sex differentiation mechanism not only has important significance for the development of the industry, but also can enrich the sex differentiation mechanism of amphibians. Previous studies have found that high temperature can induce sex reversal from female to male in Andrias davidianus. However, at present, due to the lack of technical means for analyzing gene functions in Andrias davidianus, the molecular mechanism of sex differentiation in Andrias davidianus still cannot be analyzed. Summary of the Invention

[0004] The purpose of the present invention is to provide the construction and application of a recombinant lentivirus of the Andrias davidianus NR5A2 gene, design siRNAs targeting and inhibiting the expression of the Nr5a2 gene in the gonads of Andrias davidianus, design shRNAs based on the siRNAs, construct recombinant vectors, viral interference vectors, engineering cells, etc., inhibit the expression of the Nr5a2 gene in the gonads of Andrias davidianus, and be used for related research on sex differentiation in Andrias davidianus.

[0005] The present invention provides an siRNA targeting and inhibiting the expression of the Nr5a2 gene in the gonads of Andrias davidianus, including Nr5a2-1F293 and / or Nr5a2-3M, and the nucleotide sequences of Nr5a2-3M and Nr5a2-1F293 are shown as SEQ ID NO:1 and SEQ ID NO:2 respectively.

[0006] The present invention also provides an shRNA for targeted inhibition of the Nr5a2 gene in the gonads of Andrias davidianus, including Nr5a2-3M shRNA and Nr5a2-1F293 shRNA. The Nr5a2-3M shRNA is designed based on Nr5a2-3M described in the above technical solution, and the Nr5a2-1F293 shRNA is designed based on Nr5a2-1F293 described in the above technical solution.

[0007] Preferably, the nucleotide sequences of the sense strand and the antisense strand of the Nr5a2-3M shRNA are shown in SEQ ID NO:5 and SEQ ID NO:6 respectively; the nucleotide sequences of the sense strand and the antisense strand of the Nr5a2-1F293 shRNA are shown in SEQ ID NO:7 and SEQ ID NO:8 respectively.

[0008] The present invention also provides a recombinant vector for targeted inhibition of the Nr5a2 gene in the gonads of Andrias davidianus. The recombinant vector includes a plasmid vector and the shRNA inserted into the plasmid vector; the shRNA is the shRNA for targeted inhibition of the Nr5a2 gene in the gonads of Andrias davidianus described in the above technical solution.

[0009] Preferably, the plasmid vector includes a lentiviral plasmid vector.

[0010] Preferably, the lentiviral plasmid vector includes the LV3(H1 / GFP&Puro) vector, and the shRNA is inserted between BamHI and EcoR I of the LV3(H1 / GFP&Puro) vector.

[0011] The present invention also provides a viral interference vector for targeted inhibition of the expression of the Nr5a2 gene in the gonads of Andrias davidianus. The interference vector includes the recombinant vector described in the above technical solution.

[0012] Preferably, the viral interference vector includes a lentiviral interference vector.

[0013] The present invention also provides a genetically engineered cell, which includes the viral interference vector described in the above technical solution.

[0014] The present invention also provides the application of the siRNA for targeted inhibition of the expression of the Nr5a2 gene in the gonads of Andrias davidianus, the shRNA for targeted inhibition of the expression of the Nr5a2 gene in the gonads of Andrias davidianus, the recombinant vector, the viral interference vector, or the genetically engineered cell described in the above technical solution in one or more of gene silencing of the Nr5a2 gene in the gonads of Andrias davidianus, verification of the function of the Nr5a2 gene in the gonads of Andrias davidianus, and regulation of sex differentiation of Andrias davidianus.

[0015] Beneficial effects:

[0016] The present invention provides an siRNA for targeted inhibition of the expression of the Nr5a2 gene in the gonads of Andrias davidianus, including Nr5a2-1F293 and / or Nr5a2-3M, and the nucleotide sequences of Nr5a2-3M and Nr5a2-1F293 are shown in SEQ ID NO:1 and SEQ ID NO:2 respectively. Based on the sequence of the siRNA, the present invention also designs and synthesizes the sense strand and antisense strand of shRNA for preparing a recombinant vector and a viral interference vector for targeted inhibition of the expression of the Nr5a2 gene in the gonads of Andrias davidianus. The test results prove that the siRNA of the present invention and the shRNA designed based on the siRNA, the constructed recombinant vector, viral interference vector, engineering cells, etc. can all inhibit the expression of the Nr5a2 gene in the gonads of Andrias davidianus, and can be used for the silencing of the Nr5a2 gene in the gonads of Andrias davidianus, function verification, and related research on the sex differentiation of Andrias davidianus. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments.

[0018] Figure 1 It is the structural map of the lentiviral expression vector LV3 (H1 / GFP&Puro) in Example 2;

[0019] Figure 2 It is the white light and fluorescence images before and after dissection of the Andrias davidianus injected with the lentiviral packaging control particles LV3 (H1 / GFP&Puro) in Example 4;

[0020] Figure 3 It is the fluorescence inspection results of the gonads of the Andrias davidianus 72h after injection with the target lentiviral packaging particles Nr5a2-3M-siRNA and Nr5a2-1F293-siRNA in Example 4;

[0021] Figure 4 It is the expression level of the Nr5a2 gene in the gonads after the lentivirus carrying the Nr5a2-3M and Nr5a2-1F293 interference fragments and the siRNA control lentivirus infect the juvenile Andrias davidianus for 72h; among them, "****" indicates a highly significant difference P < 0.01.

[0022] Figure 5 It is the expression level of the Nr5a2 gene in the gonads after the lentivirus carrying the Nr5a2-1F42, Nr5a2-1F139, and Nr5a2-1F293 interference fragments and the siRNA control lentivirus infect the juvenile Andrias davidianus for 72h; among them, "***" indicates a highly significant difference P < 0.01; "ns" indicates no significant difference P > 0.05. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0023] The present invention provides an siRNA for targeted inhibition of the expression of the Nr5a2 gene in the gonads of Andrias davidianus, including Nr5a2-1F293 and / or Nr5a2-3M, and the nucleotide sequences of Nr5a2-3M and Nr5a2-1F293 are shown in SEQ ID NO:1 and SEQ ID NO:2 respectively.

[0024] The present invention also provides an shRNA for targeted inhibition of the Nr5a2 gene in the gonads of Andrias davidianus, including Nr5a2-3M shRNA and Nr5a2-1F293 shRNA. The Nr5a2-3M shRNA is designed based on Nr5a2-3M described in the above technical solution, and the Nr5a2-1F293 shRNA is designed based on Nr5a2-1F293 described in the above technical solution.

[0025] As an implementation manner, the loop structure of the shRNA can select "TTCAAGAGA" to avoid forming a termination signal. As an implementation manner, sticky ends complementary to the restriction enzyme sites are added to the 5' end and 3' end of the sense strand of the shRNA respectively for subsequent recombinant vector construction; as another implementation manner, "GATCC" and "AATTC" are added to the 5' end and 3' end of the sense strand of the shRNA respectively, which are complementary to the sticky ends after digestion with BamHI enzyme and EcoR I enzyme. As another implementation manner, the nucleotide sequences of the sense strand and antisense strand of the Nr5a2-3M shRNA are shown in SEQ ID NO:3 and SEQ ID NO:4 respectively; the nucleotide sequences of the sense strand and antisense strand of the Nr5a2-1F293 shRNA are shown in SEQ ID NO:5 and SEQ ID NO:6 respectively.

[0026] The present invention also provides a recombinant vector for targeted inhibition of the Nr5a2 gene in the gonads of Andrias davidianus. The recombinant vector includes a plasmid vector and the shRNA inserted into the plasmid vector; the shRNA is the shRNA for targeted inhibition of the Nr5a2 gene in the gonads of Andrias davidianus described in the above technical solution. As an implementation manner, the plasmid vector can be a lentiviral plasmid vector; as another implementation manner, the lentiviral plasmid vector includes an LV3 (H1 / GFP&Puro) vector, and the shRNA is inserted between BamHI and EcoR I of the LV3 (H1 / GFP&Puro) vector. As an implementation manner, in the present invention, the sense strand and antisense strand of the shRNA are annealed to synthesize a double-stranded DNA fragment and then inserted into the plasmid vector.

[0027] The present invention also provides a viral interference vector for targeted inhibition of the expression of the Nr5a2 gene in the gonads of Andrias davidianus, and the interference vector comprises the recombinant vector described in the above technical solution. As an implementation manner, the viral interference vector comprises a lentiviral interference vector; as another implementation manner, the lentiviral interference vector is obtained by mixing and packaging a recombinant lentiviral vector and a packaging plasmid; the packaging process is not particularly limited, and a conventional packaging process in the art can be adopted.

[0028] The present invention also provides a genetically engineered cell, and the genetically engineered cell comprises the viral interference vector described in the above technical solution. As an implementation manner, the initial cell of the genetically engineered cell can be 293T cells. By infecting the initial cells with the viral interference vector, a packaged viral interference vector can be obtained.

[0029] Lentiviral vectors encompass the genetic information required for packaging, transfection, and integration. Recombinant lentiviral vectors carrying foreign genes need the assistance of lentiviral packaging plasmids and transfected cells to obtain infectious viral plasmids. By infecting cells or living tissues, long-term expression of foreign genes in cells or individual organs can be achieved. Compared with other retroviruses, lentiviral vectors can achieve continuous and stable expression after integrating foreign genes into host cells, can accommodate larger gene fragments, and have the ability to infect both dividing and non-dividing cells, etc., and can be widely used in the research of expressing RNAi.

[0030] The present invention also provides the application of the siRNA for targeted inhibition of the expression of the Nr5a2 gene in the gonads of Andrias davidianus, or the shRNA for targeted inhibition of the expression of the Nr5a2 gene in the gonads of Andrias davidianus, or the recombinant vector, or the viral interference vector, or the genetically engineered cell described in the above technical solution in one or more of gene silencing of the Nr5a2 gene in the gonads of Andrias davidianus, verification of the function of the Nr5a2 gene in the gonads of Andrias davidianus, and regulation of sex differentiation of Andrias davidianus.

[0031] The viral interference vector constructed by the present invention can inhibit the expression of the Nr5a2 gene in the gonads of Andrias davidianus, change its expression pattern, and further indicate that the viral interference vector constructed based on the siRNA has an impact on the sex differentiation of Andrias davidianus.

[0032] In order to further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0033] Example 1

[0034] Design interfering siRNA sequences targeting and inhibiting the expression of two splice variants of the giant salamander Nr5a2 gene. According to the mRNA sequences of the two splice variants Nr5a2-F and Nr5a2-M of the giant salamander Nr5a2 gene, 5 groups of siRNA sequences targeting the expression of the Nr5a2 gene and siRNA control sequences were designed. The specific sequence information is as follows:

[0035]

[0036]

[0037] Nr5a2 - 3M: 5'-GAAGTGCCTGAGCGTGGGCAT-3' (SEQ ID NO:1);

[0038] Nr5a2 - 1F293: 5'-GGCTACCCCTCTTCTGAAT-3' (SEQ ID NO:2);

[0039] si - NC: 5'-TTCTCCGAACGTGTCACGT-3' (SEQ ID NO:13);

[0040] Nr5a2 - 1F42: 5'-AGCACGCCTACGCTGATTT-3' (SEQ ID NO:3);

[0041] Nr5a2 - 1F139: 5'-GAGCCTGAACTTTATGGTA-3' (SEQ ID NO:4);

[0042] Among them, Nr5a2 - 3M, Nr5a2 - 1F293, Nr5a2 - 1F42, and Nr5a2 - 1F139 are siRNAs targeting the expression of the Nr5a2 gene, and si - NC is the negative control group.

[0043] Example 2

[0044] Construct siRNA plasmids and siRNA control plasmids targeting and inhibiting the expression of two splice variants Nr5a2 - F and Nr5a2 - M of the Andrias davidianus Nr5a2 gene

[0045] Design and synthesize the sense and antisense strands of shRNA with the siRNA sequences in Example 1, and their structures are as follows:

[0046] Nr5a2 - 3M shRNA:

[0047] Sense strand: 5'-GATCCGAAGTGCCTGAGCGTGGGCATTTCAAGAGAATGCCCACGCTCAGGCACTTCTTTTTTG-3' (SEQ ID NO:5);

[0048] Antisense strand: 5'-AATTCAAAAAAGAAGTGCCTGAGCGTGGGCATTCTCTTGAAATGCCCACGCTCAGGCACTTCG-3' (SEQ ID NO:6);

[0049] Nr5a2 - 1F293 shRNA:

[0050] Sense strand: 5'-GATCCGGCTACCCCTCTTCTGAATTTCAAGAGAATTCAGAAGAGGGGTAGCCTTTTTTG-3' (SEQ ID NO:7);

[0051] Antisense strand: 5'-AATTCAAAAAAGGCTACCCCTCTTCTGAATTCTCTTGAAATTCAGAAGAGGGGTAGCCG-3' (SEQ ID NO:8);

[0052] Nr5a2-1F42 shRNA:

[0053] Sense strand: 5'-GATCCAGCACGCCTACGCTGATTTTTCAAGAGAAAATCAGCGTAGGCGTGCTTTTTTTG-3' (SEQ ID NO:9);

[0054] Antisense strand: 5'-AATTCAAAAAAAGCACGCCTACGCTGATTTTCTCTTGAAAAATCAGCGTAGGCGTGCTG-3' (SEQ ID NO:10);

[0055] Nr5a2-1F139 shRNA:

[0056] Sense strand: 5'-GATCCGAGCCTGAACTTTATGGTATTCAAGAGATACCATAAAGTTCAGGCTCTTTTTTG-3' (SEQ ID NO:11);

[0057] Antisense strand: 5'-AATTCAAAAAAGAGCCTGAACTTTATGGTATCTCTTGAATACCATAAAGTTCAGGCTCG-3' (SEQ ID NO:12);

[0058] Dissolve the synthesized shRNA dry powder described above in annealing buffer, incubate in a water bath at 95°C for 5 minutes, and after cooling to room temperature, form double-stranded oligo fragments containing the interference target, with sticky ends containing BamHI and EcoR I restriction enzyme cleavage sites at both ends.

[0059] Use BamH I and EcoR I restriction endonucleases to treat the LV3 (H1 / GFP&Puro) vector (vector map as Figure 1As shown, it was purchased from GenePharma and linearized. After identifying the digested fragments by agarose gel electrophoresis, they were purified and recovered. The linearized LV3 (H1 / GFP&Puro) plasmid was ligated with the annealed product in Example 2 at 16°C overnight using T4 ligase. After transforming competent cells, recombinant positive clones were picked and sent for sequencing. If the homology of the sequencing result with the target sequence is above 96%, the clone is the successfully constructed interfering lentiviral vector for the expression of the Andrias davidianus Nr5a2 gene, named LV3-Nr5a2-3M-siRNA plasmid, LV3-Nr5a2-1F293-siRNA plasmid, LV3-Nr5a2-3M-si-NC-siRNA plasmid (siRNA control plasmid), LV3-Nr5a2-3M-Nr5a2-1F42-siRNA, and LV3-Nr5a2-3M-Nr5a2-1F139-siRNA plasmid respectively.

[0060] Example 3

[0061] Construction of an interfering lentiviral vector for targeted inhibition of the expression of the Andrias davidianus Nr5a2 gene

[0062] The LV3-Nr5a2-3M-siRNA plasmid, siRNA control plasmid, and three lentiviral packaging plasmids pGag / Pol, pRev, pVSV-G (purchased from GenePharma) were extracted using the plasmid extraction kit from GenePharma. The LV3-Nr5a2-1F293-siRNA plasmid was extracted in the same way.

[0063] One day before transfection, 293T cells in the logarithmic growth phase were digested with trypsin. After sucking off the trypsin solution, 2 mL of DMEM culture medium containing 10% FBS was added, and the cells were pipetted to form a single-cell suspension.

[0064] The cell suspension was inoculated into a 15-cm culture dish, and 18 mL of DMEM culture medium containing 10% FBS was added. After mixing, it was cultured at 37°C in 5% CO 2 overnight.

[0065] Add 1.5 mL of serum-free DMEM to a sterile 5 mL centrifuge tube, add the LV3-Nr5a2-3M-siRNA plasmid and packaging plasmids (20 μg of pGag / Pol, 15 μg of pRev, 10 μg of pVSV-G), mix well. Take another 5 mL centrifuge tube containing 1.5 mL of serum-free DMEM, add 300 μL of RNAi-Mate, mix well. After standing at room temperature for 5 minutes, mix the two tubes and then stand at room temperature for 20 - 25 minutes. The LV3-Nr5a2-1F293-siRNA plasmid, LV3-Nr5a2-3M-Nr5a2-1F42-siRNA plasmid, LV3-Nr5a2-3M-Nr5a2-1F139-siRNA plasmid and siRNA control plasmid are operated in the same way.

[0066] Remove the culture medium from a 15 cm culture dish and add 8 mL of serum-free DMEM culture medium.

[0067] Dropwise add the transfection mixture into the 15 cm culture dish, gently shake to mix well, and incubate in an incubator at 37 °C and 5% CO 2 for 4 - 6 hours.

[0068] Aspirate and discard the transfection solution, add 18 mL of DMEM culture medium containing 10% FBS, and continue to culture at 37 °C and 5% CO 2 After culturing for 72 hours, collect the supernatant of 293T cells into a 50 mL centrifuge tube and centrifuge at 4000 rpm for 4 minutes at 4 °C.

[0069] After low-speed centrifugation, pour the supernatant of the centrifuge tube into a 50 mL syringe and filter through a 0.45 μm filter.

[0070] Centrifuge the filtrate at 20000 rpm for 2 hours at 4 °C in a centrifuge, collect the supernatant to obtain the lentivirus concentrate. After aliquoting, store it at -80 °C, and take one tube for virus biological titer determination. The interfering lentivirus containing the LV3-Nr5a2-3M-siRNA plasmid and the interfering lentivirus containing the LV3-Nr5a2-1F293-siRNA plasmid are named Nr5a2-3M-siRNA and Nr5a2-1F293-siRNA respectively. The lentivirus containing the siRNA control plasmid is named lentivirus packaging control particle LV3(H1 / GFP&Puro). The interfering lentivirus containing the LV3-Nr5a2-1F42-siRNA plasmid and the interfering lentivirus containing the LV3-Nr5a2-1F139-siRNA plasmid are named Nr5a2-1F42-siRNA and Nr5a2-1F139-siRNA respectively.

[0071] Example 4

[0072] In vivo infection test of the lentivirus prepared in Example 3

[0073] 1. In vivo injection infection test of lentivirus

[0074] Before injection, thaw the lentivirus concentrate prepared in Example 3 on ice for later use. Select healthy juvenile Chinese giant salamanders with a body length of about 10 cm, anesthetize them with MS222 solution or place them on ice to enter a semi-dormant state. Use a 25 μL syringe to aspirate 20 μL of the lentivirus concentrate and inject it into the abdominal cavity of Chinese giant salamanders before gonadal differentiation.

[0075] 2. First, inject lentivirus particles without carrying the target siRNA. After 72 hours, dissect and observe the transfection of the gonads of juvenile Chinese giant salamanders under a fluorescence microscope. Figure 2 The fluorescence of the gonads of juvenile Chinese giant salamanders injected with lentivirus packaging particles carrying the target Nr5a2-3M-siRNA and Nr5a2-1F293-siRNA is shown in Figure 3 .

[0076] 3. Mark the gonads of Chinese giant salamanders showing green fluorescence under the fluorescence microscope as successfully transfected with lentivirus, and collect the gonad-mesonephros complex (i.e., Figure 3 as shown by the fluorescence position) together and put them into a cryotube. After rapid freezing in liquid nitrogen, immediately store them at -80 °C.

[0077] Example 5

[0078] Analysis of the inhibitory effect of lentivirus on the expression of Nr5a2 gene in Chinese giant salamanders

[0079] 1. RNA extraction from the gonads of Chinese giant salamanders

[0080] 1) Grind the gonads into powder in liquid nitrogen, take 0.1 g of tissue powder and transfer it into a sterile and enzyme-free centrifuge tube containing 1 mL of Trizol reagent, mix well and let it stand for 5 minutes.

[0081] 2) Add 200 μL of chloroform, shake vigorously for 1 minute and then let it stand for 5 minutes. Centrifuge at 12000 rpm at 4 °C for 10 minutes, aspirate the supernatant into a new 1.5 mL centrifuge tube, and repeat this step once.

[0082] 3) Aspirate the supernatant into a new 1.5 mL centrifuge tube, add isopropanol with the same volume as the supernatant, mix well and let it stand for 10 minutes, then put it into a centrifuge at 12000 rpm at 4 °C for 10 minutes.

[0083] 4) Discard the supernatant, remove all isopropanol, add 1 mL of ethanol solution with a volume percentage of 75%, gently pipette the precipitate, and then put it into a centrifuge at 12000 rpm at 4 °C for 5 minutes.

[0084] 5) Discard the supernatant. After the RNA precipitate is dried, add DEPC-treated water and store it at -80 °C.

[0085] 2. Reverse transcription of RNA into cDNA

[0086] The reverse transcription kit was purchased from Yeasen. The specific steps were carried out according to the operation manual of Ⅲ 1stStrand cDNA Synthesis SuperMix for qPCR (gDNA digester plus) from Yeasen

[0087] 1) Removal of residual genomic DNA: Prepare the mixture in Table 1 in an RNase-free centrifuge tube, gently pipette and mix well, and incubate at 42 °C for 2 minutes to obtain the reaction solution.

[0088] Table 1 Mixture for removing residual genomic DNA

[0089] Component Dosage 5 × gDNA digester Mix Make up to 15 μL Total RNA 1 μg <![CDATA[Rnase-freeH 2 O]]> 3 μL

[0090] 2) Preparation of the reverse transcription reaction system (20 μL system)

[0091] Directly add Ⅲ SuperMix plus to the reaction tube from step 1), and gently pipette and mix well. The reaction conditions are 25 °C for 5 min; 55 °C for 15 min; 85 °C for 5 min.

[0092] Table 2 Reverse transcription reaction system

[0093]

[0094] 3. Real-time fluorescence quantitative detection

[0095] Using the ACTB gene of Andrias davidianus as an internal reference, the expression level of the Nr5a2 gene in Andrias davidianus was detected by fluorescence quantitative PCR. The primer sequences are shown in Table 3, the reaction system is shown in Table 4, and the reaction program is shown in Table 5.

[0096] Table 3 Primers for real-time fluorescence quantitative detection

[0097]

[0098] Table 4 Reaction system for real-time fluorescence quantitative detection

[0099]

[0100] Table 5 Reaction program for real-time fluorescence quantitative detection

[0101]

[0102] The results of fluorescence quantitative PCR are as Figure 4 , Figure 5As shown in Figure 4 In Figure 4 , NC-siRNA represents the change in the expression level of the Nr5a2 gene in the gonads of juvenile giant salamanders after being infected with siRNA control lentivirus for 72 h, serving as the control group for the change in the expression level of the Nr5a2 gene in the gonads of juvenile giant salamanders after being infected with Nr5a2-1F42-siRNA, Nr5a2-1F139-siRNA, and Nr5a2-1F293-siRNA lentiviruses for 72 h. In Figure 5 In Figure 5 , Control1 represents the change in the expression level of the Nr5a2-M gene in the gonads of juvenile giant salamanders after being infected with siRNA control lentivirus for 72 h, serving as the control group for the change in the expression level of the Nr5a2-M gene in the gonads of juvenile giant salamanders after being infected with Nr5a2-3M-siRNA lentivirus for 72 h; control2 represents the change in the expression level of the Nr5a2-F gene in the gonads of juvenile giant salamanders after being infected with siRNA control lentivirus for 72 h, serving as the control group for the change in the expression level of the Nr5a2-F gene in the gonads of juvenile giant salamanders after being infected with Nr5a2-1F293-siRNA lentivirus for 72 h.

[0103] According to Figure 4 and Figure 5 It can be seen that the virus interference vectors designed based on Nr5a2-1F293 and Nr5a2-3M of the present invention can effectively inhibit the expression of the Nr5a2 gene in the gonads of giant salamanders, and have significant differences from the virus interference vectors designed based on Nr5a2-1F42 and Nr5a2-1F139 and the siRNA control lentivirus; there is no significant difference in the inhibitory effect between the virus interference vectors designed based on Nr5a2-1F42 and Nr5a2-1F139 and the siRNA control lentivirus. Nr5a2-1F293 and Nr5a2-3M are highly efficient interference fragments, while Nr5a2-1F42 and Nr5a2-1F139 are low-efficiency interference fragments.

[0104] It can be concluded from the above examples that the siRNA and the shRNA designed based on the siRNA, as well as the recombinant vectors, virus interference vectors, and engineered cells constructed, can all inhibit the expression of the Nr5a2 gene in the gonads of giant salamanders and are used for related research on the sex differentiation of giant salamanders.

[0105] Although the above examples have described the present invention in detail, they are only a part of the embodiments of the present invention, not all embodiments. People can also obtain other embodiments based on these embodiments without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A siRNA targeting and inhibiting the expression of Nr5a2 gene in the gonad of giant salamander, characterized in that: It includes Nr5a2-1F293 and / or Nr5a2-3M, and the nucleotide sequences of Nr5a2-3M and Nr5a2-1F293 are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

2. A shRNA targeting and inhibiting the Nr5a2 gene of the giant salamander gonad, characterized in that: It comprises Nr5a2-3M shRNA and Nr5a2-1F293 shRNA, wherein the Nr5a2-3M shRNA is designed based on the Nr5a2-3M described in claim 1, and the Nr5a2-1F293 shRNA is designed based on the Nr5a2-1F293 described in claim 1.

3. The shRNA targeting and inhibiting the Nr5a2 gene of the giant salamander gonad according to claim 2, characterized in that: The nucleotide sequences of the sense strand and antisense strand of the Nr5a2-3M shRNA are shown in SEQ ID NO:5 and SEQ ID NO:6, respectively; the nucleotide sequences of the sense strand and antisense strand of the Nr5a2-1F293 shRNA are shown in SEQ ID NO:7 and SEQ ID NO:8, respectively.

4. A recombinant vector for targeted inhibition of the Nr5a2 gene in the gonads of giant salamanders, characterized in that: The recombinant vector comprises a plasmid vector and a shRNA inserted into the plasmid vector; the shRNA is the shRNA targeted at inhibiting the giant salamander gonad Nr5a2 gene as claimed in claim 2 or 3.

5. The recombinant vector according to claim 4, characterized in that The plasmid vector comprises a lentiviral plasmid vector.

6. The recombinant vector according to claim 5, characterized in that The lentiviral plasmid vector includes a LV3 (H1 / GFP&Puro) vector, and the shRNA is inserted between BamHI and EcoR I of the LV3 (H1 / GFP&Puro) vector.

7. A viral interference vector for targeted inhibition of Nr5a2 gene expression in the gonads of giant salamanders, characterized in that: The interference vector comprises the recombinant vector according to any one of claims 4 to 6.

8. The viral interference vector according to claim 7, characterized in that The viral interference vector includes a lentiviral interference vector.

9. An engineered cell, characterized in that: The engineered cell comprises the viral interference vector according to claim 7 or 8.

10. Use of the siRNA targeting the inhibition of Nr5a2 gene expression in the gonads of giant salamanders as described in claim 1, or the shRNA targeting the inhibition of Nr5a2 gene expression in the gonads of giant salamanders as described in claim 2 or 3, or the recombinant vector as described in any one of claims 4 to 6, or the viral interference vector as described in claim 7 or 8, or the engineered cell as described in claim 9 in one or more of silencing the Nr5a2 gene in the gonads of giant salamanders, verifying the function of the Nr5a2 gene in the gonads of giant salamanders, and regulating the sex differentiation of giant salamanders.

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