A primer set and method for analyzing the nanos1 gene of Macrobrachium rosenbergii

Abstract

The present invention provides a primer set and method for analyzing the nanos1 gene of Macrobrachium rosenbergii, relating to the biotechnology field of reproductive-related genes in Macrobrachium rosenbergii. The Nanos1 gene obtained by the present invention has a cDNA sequence of 2811 base pairs (bp) long, encoding 243 amino acids. Nanos1 is specifically expressed in the ovary, and the expression level of nanso1 mRNA is highest in unfertilized eggs, significantly higher than after fertilization and at all stages of embryonic development. During embryonic development, expression is highest during fertilization, significantly higher than during the cleavage stage and extremely significantly higher than during the late embryonic development stage. The gene's expression level during the cleavage stage is significantly higher than from the blastocyst stage to the larvae stage; however, the expression level is lower and does not differ between the blastocyst stage and the larvae stage. Nanos1 mRNA is expressed in the cytoplasm of oogonia and primary oocytes (Oc1, Oc2, Oc3, and Oc4). Nanos1 is closely related to the development of female germ cells in Macrobrachium rosenbergii and plays an important role in the reproductive development of Macrobrachium rosenbergii, providing a theoretical basis and ideas for research on reproductive development and sex identification in Macrobrachium rosenbergii.

Description

Technical Field

[0001] The present invention relates to the biotechnology field of Macrobrachium rosenbergii reproduction-related genes, and in particular to a primer set and a method for analyzing Macrobrachium rosenbergii nanos1 gene. Background Art

[0002] Macrobrachium rosenbergii belongs to the family Palaemonidae and the genus Macrobrachium. Native to Southeast Asia, it was introduced to China in the late 1970s. Due to its fast growth, large size, and delicious flavor, it has gradually become a staple of freshwater aquaculture. Like other crustaceans, Macrobrachium rosenbergii exhibits sexual dimorphism: the size and growth rate of male and female individuals of the same age vary significantly. Under the same culture conditions, males grow faster than females, and the average weight of sexually mature individuals is approximately twice that of females. Furthermore, studies have found that some females weighing only 6 grams and males weighing only 2 grams reach sexual maturity. These precocious individuals produce gametes and mate prematurely, resulting in significant energy loss, stunted development, and size variation. Therefore, research on the reproductive development and regulatory mechanisms of Macrobrachium rosenbergii is of great significance to the development of the Macrobrachium rosenbergii industry. Nanos is an RNA-binding protein with two highly conserved zinc-finger domains (CCHCs), which play a crucial role in reproductive development. As a gene with maternal effects, nanos has different homologs and regulatory functions in different species. For example, in Drosophila, nanos has one homolog that plays an important role in germ cell migration, somatic cell inhibition, and stem cell self-repair. In the nematode Caenorhabditis elegans, three nanos homologs (nanos1, nanos2, and nanos3) have been found. Nanos1 and nanos2 maintain germ cell viability, while nanos3 primarily controls sex change in the hermaphroditic nematode. In the vertebrate zebrafish (Danio rerio), the nanos1 gene is responsible for the survival of primordial germ cells during embryonic development and for maintaining the survival of oocytes in adult zebrafish. In mice (Mus musculus), nanos2 and nanos3 play a role in the growth and development of germ cells. Knocking down the nanos2 gene leads to the loss of spermatogonia and a decrease in sperm quality in male mice, while blocking nanos3 completely leads to the stagnation of germ cell development in both sexes. Currently, research on nanos family genes in crustaceans is relatively weak. Only nanos1 and nanos2 genes have been found in the Henan Chinese river crab (Sinopotamon henanense), and it is speculated that they play an important regulatory role in ovarian development. No research has been found on the nanos gene in Macrobrachium rosenbergii. Summary of the Invention

[0003] In view of this, the purpose of the present invention is to provide the nanos1 gene of Macrobrachium rosenbergii, an amplification primer set and its application. The present invention clarifies the important role of the nanos1 gene in the reproductive development of Macrobrachium rosenbergii, and provides a theoretical basis and ideas for the research of reproductive development and sex identification of Macrobrachium rosenbergii.

[0004] In order to achieve the above object, the present invention provides the following technical solutions:

[0005] A primer set for analyzing the nanos1 gene of Macrobrachium rosenbergii, comprising a semi-quantitative PCR amplification primer set and a fluorescent quantitative PCR amplification primer set;

[0006] The semi-quantitative PCR amplification primer set consists of two pairs of primers, namely nanos1 F, nanos1 R, β-actin F, and β-actin R. The nucleotide sequences of nanos1 F, nanos1 R, β-actin F, and β-actin R are shown in SED ID NOs. 1 to 4.

[0007] The fluorescent quantitative PCR amplification primer set consists of two pairs of primers, namely nanos1 QF, nanos1 QR, β-actin QF, and β-actin QR. The nucleotide sequences of nanos1 QF, nanos1 QR, β-actin QF, and β-actin QR are shown in SED ID NOs. 5 to 8.

[0008] The nanos1 F and nanos1 R can also be used for PCR amplification and in situ hybridization of the nanos1 gene.

[0009] The present invention also provides a method for obtaining the primer set for analyzing the Macrobrachium rosenbergii nanos1 gene according to claim 1, comprising the following steps:

[0010] (1) Sequencing was used to obtain transcriptome data of Macrobrachium rosenbergii and screen out the predicted sequence of the nanos1 gene.

[0011] (2) Randomly select the gonads of healthy individuals of Macrobrachium rosenbergii, extract total RNA, obtain cDNA after reverse transcription, and verify the screened Macrobrachium rosenbergii nanos1 gene;

[0012] (3) Design of a semi-quantitative PCR amplification primer set: Based on the nanos1 gene obtained in step (2), upstream and downstream specific primers and a β-actin internal reference primer were designed to obtain a semi-quantitative PCR amplification primer set for the nanos1 gene of Macrobrachium rosenbergii;

[0013] (4) Design of fluorescent quantitative PCR amplification primer set: Based on the nanos1 gene obtained in step (2), a fluorescent quantitative PCR amplification primer set was designed to obtain a fluorescent quantitative PCR amplification primer set for Macrobrachium rosenbergii.

[0014] The present invention also provides a method for analyzing the Macrobrachium rosenbergii nanos1 gene, which uses the primer set for analyzing the Macrobrachium rosenbergii nanos1 gene according to claim 1.

[0015] Preferably, the gene analysis method includes semi-quantitative PCR, fluorescent quantitative PCR, PCR and in situ hybridization.

[0016] Preferably, the PCR is used to amplify the Macrobrachium rosenbergii nanos1 gene; the PCR amplification uses the gonad cDNA of Macrobrachium rosenbergii as a template;

[0017] The reaction conditions of the PCR amplification are: 94° C. for 2 min; 94° C. for 30 s, 57° C. for 30 s, and 72° C. for 90 s for 38 cycles; and 72° C. for 10 min.

[0018] Preferably, the semi-quantitative PCR is used to evaluate the expression of nanos1 gene in different tissues of Macrobrachium rosenbergii; the semi-quantitative PCR uses cDNA from different tissues of Macrobrachium rosenbergii as template;

[0019] The reaction conditions of the semi-quantitative PCR amplification were: 94°C for 2 min; 38 cycles of 94°C for 30 s, 57°C for 30 s, and 72°C for 40 s; 72°C for 10 min;

[0020] The different tissues include the brain, hepatopancreas, heart, muscle, gonad, gill, intestine and eye of Macrobrachium rosenbergii.

[0021] Preferably, the fluorescent quantitative PCR is used to evaluate the expression of the nanos1 gene during the early developmental period of Macrobrachium rosenbergii;

[0022] The fluorescent quantitative PCR uses cDNA from Macrobrachium rosenbergii eggs, embryos, and early developmental stages as templates; the eggs include unfertilized eggs, fertilized eggs, and cleavage stages; the embryos include blastocyst stages and gastrula stages; and the early developmental stages include zoa larvae and larvae stages.

[0023] The reaction conditions for the fluorescent quantitative PCR amplification are: 50°C for 2 min, 95°C for 2 min; 95°C for 15 s, 60°C for 30 s, 72°C for 30 s, 40 cycles; melting curve detection: 95°C for 15 s, 60°C for 30 s, 95°C for 15 s.

[0024] Preferably, the in situ hybridization is used to evaluate the expression position of the nanos1 gene in the gonadal tissue of Macrobrachium rosenbergii; the in situ hybridization is performed on the gonadal tissue of Macrobrachium rosenbergii.

[0025] The present invention also provides a use of the primer set for analyzing the Macrobrachium rosenbergii nanos1 gene according to claim 1 in regulating the development of female germ cells of Macrobrachium rosenbergii.

[0026] The present invention also provides a use of the primer set for analyzing the Macrobrachium rosenbergii nanos1 gene according to claim 1 in sex identification during the early developmental period of Macrobrachium rosenbergii.

[0027] Beneficial technical effects: The nanos1 gene obtained by the present invention has a cDNA sequence of 2811bp long, encoding 243 amino acids, with the highest homology to the Chinese mitten crab, a relatively high homology to the nanos1 of fish, and a relatively low homology to mammals. Nanos1 is specifically expressed in the ovary, and the expression level of nanso1 mRNA is highest in unfertilized eggs, significantly higher than after fertilization and in each stage of embryonic development; during embryonic development, the expression level is highest during the fertilization period, significantly higher than the cleavage period and extremely significantly higher than the late embryonic development period; the expression level of this gene in the cleavage period is significantly higher than that from the blastocyst stage to the larvae stage; while the expression level between the blastocyst stage and the larvae is low and there is no difference. In situ hybridization technology detection shows that nanos1 mRNA is expressed in the cytoplasm of oogonia and primary oocytes (Oc1, Oc2, Oc3, Oc4). Nanos1 is closely related to the development of female germ cells of Macrobrachium rosenbergii and plays an important role in the reproductive development of Macrobrachium rosenbergii, providing a theoretical basis and ideas for research on reproductive development and sex identification of Macrobrachium rosenbergii. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The cDNA sequence and amino acid sequence of Macrobrachium rosenbergii nanos1 are shown; the start and stop codons are highlighted in bold and underlined; the gray shaded area is the conserved -CCHC-zinc finger domain;

[0029] Figure 2The phylogenetic tree of Nanos1 amino acid sequence was compared with that of Nanos family proteins from other species. The sequences were obtained from the following gene databases: carp (GenBank ID: BAJ76659); zebrafish (GenBank ID: AAL15474); medaka (GenBank ID: NP_001116300, NP_001153919, BAG84599 and NP_001153941); Atlantic cod (GenBank ID: ADV36251); house mouse (GenBank ID: NP_918948, NP_918953 and NP_848508); human (GenBank ID: NP_001092092, NP_001025032 and NP_955631); sea bass (GenBank ID: CBN81978); African clawed frog (GenBank ID: NP_001116300); ID:NP_001081503); Chinese sturgeon (GenBank ID:JQ410472); Smelt (GenBank ID:ACO09328); Atlantic salmon (GenBank ID:NP_001135057); Drosophila (GenBank ID:NP_476658); Nematode (GenBank ID:NP_496358); Chinese mitten crab (GenBank ID:AMP 42752.1);

[0030] Figure 3The phylogenetic tree of the nanos1 amino acid sequence was compared with that of other species of Nanos family proteins. Among them, the sequences were obtained from the gene database: common carp (GenBank ID: BAJ76659); zebrafish (GenBank ID: AAL15474); medaka (GenBank ID: NP_001116300, NP_001153919, BAG84599 and NP_001153941); Atlantic cod (GenBank ID: ADV36251); house mouse (GenBank ID: NP_918948, NP_918953 and NP_848508); human (GenBank ID: NP_001092092, NP_001025032 and NP_955631); sea bass (GenBank ID: CBN81978); African clawed frog (GenBank ID: NP_001116300, NP_001153919, BAG84599 and NP_001153941). ID:NP_001081503); Chinese sturgeon (GenBank ID:JQ410472); Smelt (GenBank ID:ACO09328); Atlantic salmon (GenBank ID:NP_001135057); Drosophila (GenBank ID:NP_476658); Nematode (GenBank ID:NP_496358); Chinese mitten crab (GenBank ID:AMP 42752.1);

[0031] Figure 4 This is the expression analysis of nanos1 gene in different tissues of Macrobrachium rosenbergii; 1 is brain; 2 is hepatopancreas; 3 is heart; 4 is muscle; 5 is gonad; 6 is gill; 7 is intestine; 8 is eye; M is standard 2000;

[0032] Figure 5 The relative expression levels of nanos1 in different embryonic developmental stages and early larvae of Macrobrachium rosenbergii are shown in Figure 1, where 1 represents the unfertilized egg stage; 2 represents the fertilized egg stage; 3 represents the cleavage stage; 4 represents the blastula stage; 5 represents the gastrula stage; 6 represents the pre-zoa stage; 7 represents the zoa stage; and 8 represents the larval stage. Different letters indicate significant differences among the experimental groups at the same time (P<0.05).

[0033] Figure 6The subcellular distribution of Nanos1 mRNA in the ovary of Macrobrachium rosenbergii; (A) Macrobrachium rosenbergii ovary section (HE staining); (B) Macrobrachium rosenbergii ovary section (antisense probe result); (C) Macrobrachium rosenbergii ovary section (sense probe result); (D, E, F) show the enlarged fields of view in the boxes of Figures A, B, and C, respectively; Og: oogonia; Oc1: primary oocyte stage I cell; Oc2: primary oocyte stage II cell; Oc3: primary oocyte stage III cell; Oc4: primary oocyte stage IV cell. DETAILED DESCRIPTION

[0034] The present invention provides a primer set for analyzing the nanos1 gene of Macrobrachium rosenbergii, wherein the primer set comprises a semi-quantitative PCR amplification primer set and a fluorescent quantitative PCR amplification primer set;

[0035] In the present invention, the semi-quantitative PCR amplification primer set consists of two pairs of primers, namely nanos1 F, nanos1 R, β-actin F, and β-actin R. The nucleotide sequences of nanos1 F, nanos1 R, β-actin F, and β-actin R are shown in SED ID NOs. 1 to 4; among them, nanos1 F and nanos1 R are upstream and downstream specific primers, and β-actin F and β-actin R are internal reference primers.

[0036] In the present invention, the fluorescent quantitative PCR amplification primer set consists of two pairs of primers, namely nanos1 QF, nanos1 QR, β-actin QF, and β-actin QR. The nucleotide sequences of nanos1 QF, nanos1 QR, β-actin QF, and β-actin QR are shown in SED ID NOs. 5 to 8; among them, nanos1 QF and nanos1 QR are upstream and downstream specific primers, and β-actin QF and β-actin QR are internal reference primers.

[0037] In the present invention, the nucleotide sequences of the semi-quantitative PCR amplification primer set and the fluorescent quantitative PCR amplification primer set are shown in Table 1.

[0038] Table 1 Primer sets for analysis of the nanos1 gene in Macrobrachium rosenbergii

[0039]

[0040]

[0041] The nanos1 F and nanos1 R can also be used for PCR amplification and in situ hybridization of the nanos1 gene.

[0042] In the present invention, the primer set for analyzing the Macrobrachium rosenbergii nanos1 gene obtains a single band and has high amplification efficiency, which can well meet the needs of subsequent research on the Macrobrachium rosenbergii nanos1 gene.

[0043] The present invention also provides a method for obtaining the primer set for analyzing the Macrobrachium rosenbergii nanos1 gene according to claim 1, comprising the following steps:

[0044] (1) Sequencing was used to obtain transcriptome data of Macrobrachium rosenbergii and screen out the predicted sequence of the nanos1 gene.

[0045] (2) Randomly select the gonads of healthy individuals of Macrobrachium rosenbergii, extract total RNA, obtain cDNA after reverse transcription, and verify the screened Macrobrachium rosenbergii nanos1 gene;

[0046] (3) Design of a semi-quantitative PCR amplification primer set: Based on the nanos1 gene obtained in step (2), upstream and downstream specific primers and a β-actin internal reference primer were designed to obtain a semi-quantitative PCR amplification primer set for the nanos1 gene of Macrobrachium rosenbergii;

[0047] (4) Design of fluorescent quantitative PCR amplification primer set: Based on the nanos1 gene obtained in step (2), a fluorescent quantitative PCR amplification primer set was designed to obtain a fluorescent quantitative PCR amplification primer set for Macrobrachium rosenbergii.

[0048] The present invention also provides a method for analyzing the Macrobrachium rosenbergii nanos1 gene, which uses the primer set for analyzing the Macrobrachium rosenbergii nanos1 gene according to claim 1.

[0049] In the present invention, the gene analysis methods include semi-quantitative PCR, fluorescent quantitative PCR, PCR and in situ hybridization.

[0050] In the present invention, the PCR is used to amplify the nanos1 gene of Macrobrachium rosenbergii; the PCR amplification uses the gonad cDNA of Macrobrachium rosenbergii as a template; the reaction conditions of the PCR amplification are: 94°C for 2 min; 94°C for 30 s, 57°C for 30 s, and 72°C for 90 s for 38 cycles; 72°C for 10 min.

[0051] In the present invention, the PCR amplification product is recovered using a Gel Extraction Kit (Omega, China) to obtain the target fragment, which is then ligated to a pMD19-T (Takara, China) vector and transformed into DH5α competent cells (Takara, China). Positive clone strains are screened and sent to Guangzhou Tianyi Huiyuan Gene Technology Co., Ltd. for sequencing. After sequencing, alignment is performed, and the positive strains with consistent alignment results are expanded and cultured, the plasmids are extracted, and the strains are stored in a -20°C refrigerator.

[0052] In the present invention, the sequencing results were analyzed using Sequence Manipulation Suite (SMS) (http: / / www.bio-soft.net / sms / ) to analyze the nucleotide and amino acid sequences of the nanos1 gene, and the Simple Modular Architecture Research Tool (SMART) (http: / / smart.embl-heidelberg.de / ) was used to predict the domain structure of its protein. The amino acid sequence of Nanos1 was homologously aligned with the amino acid sequences of the Nanos protein family of other species using BLAST (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) in NCBI (http: / / www.ncbi.nlm.nih.gov / ), and multiple sequence alignment analysis was performed using DNAMAN and BioEdit. The maximum likelihood estimate (MLE) method in MEGA7 software was used to construct a phylogenetic tree, and homology analysis of the nanos1 sequence was performed.

[0053] The nanos1 gene was cloned and its nucleotide sequence was uploaded to NCBI (GenBank: ON155838). The cDNA sequence is 2811 bp long, with a 686 bp 5' untranslated region (UTR), a 1396 bp 3' untranslated region (UTR), and a 729 bp open reading frame (ORF), encoding 243 amino acids. This gene plays an important role in the reproductive development of Macrobrachium rosenbergii.

[0054] In the present invention, the semi-quantitative PCR is used to evaluate the expression of nanos1 gene in different tissues of Macrobrachium rosenbergii; the semi-quantitative PCR uses cDNA from different tissues of Macrobrachium rosenbergii as template;

[0055] In the present invention, the reaction conditions for semi-quantitative PCR amplification are: 94°C for 2 min; 94°C for 30 s, 57°C for 30 s, and 72°C for 40 s for 38 cycles; 72°C for 10 min; the different tissues include the brain, hepatopancreas, heart, muscle, gonads, gills, intestines, and eyes of Macrobrachium rosenbergii.

[0056] The present invention detects and analyzes the expression levels of the nanos1 gene in different tissues, and obtains the expression distribution of nanos1 in different tissues such as the liver, spleen, heart, female gonads, muscles, gills, intestines, brain and eyes of normal Macrobrachium rosenbergii individuals, providing a theoretical basis and ideas for the study of reproductive development of Macrobrachium rosenbergii.

[0057] In the present invention, the products amplified by the semi-quantitative PCR were separated using 1% agarose gel and photographed using a gel imaging analysis system (GenoSens 2200, CN).

[0058] In the present invention, the fluorescent quantitative PCR is used to evaluate the expression of the nanos1 gene in the early developmental period of Macrobrachium rosenbergii; the fluorescent quantitative PCR uses cDNA of Macrobrachium rosenbergii eggs, embryos and early developmental period as templates; the eggs include unfertilized eggs, fertilized eggs, and cleavage stage; the embryos include blastocyst stage and gastrula stage; the early developmental period includes zoa larvae stage and larval stage;

[0059] The reaction conditions for the fluorescent quantitative PCR amplification are: 50°C for 2 min, 95°C for 2 min; 95°C for 15 s, 60°C for 30 s, 72°C for 30 s, 40 cycles; melting curve detection: 95°C for 15 s, 60°C for 30 s, 95°C for 15 s.

[0060] In the present invention, the in situ hybridization is used to evaluate the expression position of the nanos1 gene in the gonadal tissue of Macrobrachium rosenbergii; the in situ hybridization is performed on the gonadal tissue of Macrobrachium rosenbergii.

[0061] In the present invention, the specific operation steps of the in situ hybridization refer to the literature method (YU L, XU D, YE H, et al. Gonadal Transcriptome Analysis of Pacific Abalone Haliotis discus discus: Identification of Genes Involved in Germ Cell Development [J]. Marine Biotechnology, 2018, 20 (5): 1-14.).

[0062] The present invention also provides a use of the primer set for analyzing the nanos1 gene of Macrobrachium rosenbergii according to claim 1 in regulating the development of female germ cells of Macrobrachium rosenbergii. The present invention is not particularly limited to the type of application, and conventional application methods in the art can be used.

[0063] The present invention also provides a use of the primer set for analyzing the nanos1 gene of Macrobrachium rosenbergii according to claim 1 for sex identification during the early developmental period of Macrobrachium rosenbergii. The present invention is not particularly limited to the type of application, and conventional application methods in the art can be used.

[0064] In order to better understand the present invention, the content of the present invention is further illustrated below in conjunction with the examples, but the content of the present invention is not limited to the following examples.

[0065] Example 1

[0066] 1 Methods and steps

[0067] 1.1 Collection and processing of experimental materials

[0068] Twenty healthy male and female 3-month-old Macrobrachium rosenbergii were purchased from Foshan Sanshui Platinum Co., Ltd. The prawns were dissected, and tissues including the brain, hepatopancreas, heart, muscle, gonads, gills, intestine, and eyes were collected and quickly frozen in liquid nitrogen before being stored in a -80°C freezer for RNA extraction. Some gonadal tissue was fixed in Bonn's solution for 8 hours, then transferred to 75% ethanol and stored in a 4°C refrigerator for paraffin sectioning. Eggs, embryos, and samples from different stages of early development were collected, quickly frozen in liquid nitrogen, and stored in a -80°C freezer for subsequent experiments.

[0069] 1.2 Total RNA extraction from different tissues and cDNA synthesis

[0070] The collected tissues were lysed and Total RNA was extracted using a Super kit (Promega, China). RNA concentration and purity were determined using a Nano Q™ spectrophotometer (Thermo Fisher Scientific, USA). RNA integrity was verified by 1% agarose gel electrophoresis. Qualified RNA was stored at −80°C. cDNA was subsequently transcribed and synthesized using the M-MLV reverse transcription kit (Invitrogen, USA).

[0071] 1.3 Cloning of nanos1 cDNA from gonadal tissue

[0072] Based on the nanos1 gene (Nanos1) cDNA sequence (unpublished) obtained from gonadal transcriptome sequencing in our laboratory, upstream and downstream specific primers nanos1 F and nanos1 R were designed (Table 1). PCR amplification was performed using the gonadal cDNA as a template. The reaction conditions were: 94°C for 2 min; 94°C for 30 s, 57°C for 30 s, and 72°C for 90 s (38 cycles in total); and 72°C for 10 min. The PCR product was recovered using a Gel Extraction Kit (Omega, China). The target fragment was then ligated into the pMD19-T vector (Takara, China) and transformed into DH5α competent cells (Takara, China). Clones were screened and positive clones were sent to Guangzhou Tianyi Huiyuan Gene Technology Co., Ltd. for sequencing. After sequencing, alignment was performed. Positive strains that matched the alignment were expanded, and plasmids were extracted and stored at -20°C.

[0073] 1.4 nanos1 cDNA sequence analysis

[0074] The nucleotide and amino acid sequences of nanos1 were analyzed using the Sequence Manipulation Suite (SMS) (http: / / www.bio-soft.net / sms / ). The protein domain structure was predicted using the Simple Modular Architecture Research Tool (SMART) (http: / / smart.embl-heidelberg.de / ). The amino acid sequence of Nanos1 was compared with the amino acid sequences of the Nanos protein family of other species using BLAST (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) in NCBI (http: / / www.ncbi.nlm.nih.gov / ). Multiple sequence alignments were performed using DNAMAN and BioEdit. A phylogenetic tree was constructed using the Maximum Likelihood Estimate (MLE) method in MEGA7 software.

[0075] 1.5 Expression of nanos1 in different tissues

[0076] Based on the nanos1 sequence, a pair of primers, nanos1 F and nanos1 R, were designed, using β-actin as an internal reference gene (GenBank NO: AY626840) (Table 1). RT-PCR amplification (Applied Biosystems, BRD) was performed using cDNA from brain, hepatopancreas, heart, muscle, gonads (male and female), gills, intestine, and eye as templates to determine the expression distribution of Nanos1 in different tissues. The reaction protocol was as follows: 94°C for 2 min; 94°C for 30 s, 57°C for 30 s, and 72°C for 40 s (38 cycles total); and extension at 72°C for 10 min. RT-PCR products were separated on 1% agarose gels and photographed using a gel imaging system (GenoSens 2200, CN).

[0077] 2 Results

[0078] 2.1 nanos1 cDNA sequence characteristics

[0079] Figure 1 Figure 2 shows the cDNA sequence and amino acid sequence of Macrobrachium rosenbergii nanos1, where the start and stop codons are highlighted in bold and underlined; the grey shaded area is the conserved -CCHC-zinc finger domain. Figure 2 Comparison of the zinc finger region structure of Macrobrachium rosenbergii nanos1 with that of other species. Figure 1 It can be seen that the cDNA sequence of the cloned nanos1 gene (GenBank: ON155838) is 2811 bp long, with a 5' non-coding region (UTR) of 686 bp, a 3'-UTR of 1396 bp, and an open reading frame (ORF) of 729 bp, encoding 243 amino acids. Figure 1 、 2 It can be seen that the prediction of the Nanos1 amino acid sequence revealed that the protein has two conserved -CCHC-(-Cys-Cys-His-Cys-) zinc finger domains, as well as structural and functional domains unique to the nanos family.

[0080] 2.2 Nanos1 phylogeny

[0081] The phylogenetic tree of nanos1 was constructed using MEGA7.0 with the maximum likelihood method, and the homology analysis of nanos1 sequences was performed. Figure 3 Phylogenetic tree analysis of nanos1 amino acid sequence and Nanos family proteins of other species. Figure 3The nanos gene family is divided into two major clades: one branch encompassing the nanos1 protein cluster and the other containing the nanos2 and nanos3 proteins. A phylogenetic tree shows that nanos1 clusters with nanos1 proteins from other species and with the Chinese mitten crab (Eriocheir sinensis). The Macrobrachium rosenbergii nanos gene obtained in this study belongs to the nanos1 gene family. To further understand the homology of the nanos1 gene, the amino acid sequence of nanos1 was compared and identified with amino acid sequences from fish, amphibians, and mammals. The zinc finger base sequence of the nanos1 gene was found to be highly homologous to those of other animals. In the nanos1 cluster, the nanos1 sequence has the highest homology with the Chinese mitten crab nanos, with a similarity of 51%. It also has higher homology with fish, with 40.7% and 46% similarity with the medaka (Oryzias latipes) nanos1a and Atlantic salmon (Salmo salar) nanos1, respectively, and 40%, 22% and 23% similarity with the African clawed frog (Xenopus laevis), mouse and human (Homo sapiens) nanos1, respectively; while in the Nanos2 and Nanos3 clusters, it shows lower similarity.

[0082] 2.3 Analysis of nanos1 mRNA tissue expression

[0083] To understand the expression pattern of nanos1 in different tissues, RT-PCR technology was used to detect and analyze the expression distribution of Nanos1 in different tissues such as liver, spleen, heart, female gonads, muscles, gills, intestines, brain and eyes of normal male and female individuals using β-actin as the internal reference gene. Figure 4 This is the expression analysis of nanos1 gene in different tissues of Macrobrachium rosenbergii. Figure 4 It can be seen that nanos1 expression was only detected in the ovary, and no expression was found in other tissues of females or all tissues of males.

[0084] 2.4 Expression patterns of nanos1 in eggs, embryos, and early development

[0085] To understand the expression pattern of nanos1 in eggs, embryos and early development, qPCR detection was performed on eggs, embryos and other samples. Figure 5 is the relative expression level of nanos1 in different embryonic development stages and early larvae of Macrobrachium rosenbergii, Figure 5It can be seen that the expression level of nanso1 mRNA is highest in the unfertilized (egg) stage, which is significantly higher than that in the fertilized (egg) stage, and extremely significantly higher than that in the cleavage stage and other late stages of embryonic development (blastocyst stage, gastrula stage, daphnia stage, daphnia stage) and juvenile stage; during embryonic development, the expression level is highest in the fertilized (egg) stage, which is significantly higher than that in the cleavage stage and extremely significantly higher than that in the late stages of embryonic development (blastocyst stage, gastrula stage, daphnia stage, daphnia stage); and the expression level of this gene in the cleavage stage is significantly higher than that in the blastocyst stage, gastrula stage, daphnia stage and daphnia stage; the expression level of this gene is low and there is no significant difference between the blastocyst stage, gastrula stage, daphnia stage, daphnia stage and juvenile stage.

[0086] 2.5 Subcellular localization of nanos1 in ovarian tissue

[0087] The subcellular localization of nanos1 in the ovary of Macrobrachium rosenbergii was studied using in situ hybridization. Figure 6 The subcellular distribution of nanos1 mRNA in the ovary of Macrobrachium rosenbergii is shown in Figure 2. Figure 6 It can be seen that in the ovaries of Macrobrachium rosenbergii, nanos1 mRNA positive signals were detected in oocytes, and the positive signals were strongest in oocytes at stages Oc1, Oc2, Oc3, and Oc4. The positive signals in primary oocytes at stages Oc1, Oc2, Oc3, and Oc4 were mainly concentrated in the cytoplasm of oocytes.

[0088] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A primer set for analyzing the nanos1 gene of Macrobrachium rosenbergii, characterized in that: The primer set includes a semi-quantitative PCR amplification primer set and a fluorescent quantitative PCR amplification primer set; The semi-quantitative PCR amplification primer set consists of two pairs of primers, namely nanos1 F, nanos1 R, β-actin F, and β-actin R. The nucleotide sequences of nanos1 F, nanos1 R, β-actin F, and β-actin R are shown in SED ID NOs. 1 to 4. The fluorescent quantitative PCR amplification primer set consists of two pairs of primers, namely nanos1 QF, nanos1 QR, β-actin QF, and β-actin QR. The nucleotide sequences of nanos1 QF, nanos1 QR, β-actin QF, and β-actin QR are shown in SEDID NOs. 5 to 8. The nanos1 F and nanos1 R are also used for PCR amplification and in situ hybridization of the Macrobrachium rosenbergii nanos1 gene.

2. A method for obtaining the primer set for analyzing the Macrobrachium rosenbergii nanos1 gene according to claim 1, characterized in that: The following steps are involved: (1) Sequencing was used to obtain transcriptome data of Macrobrachium rosenbergii and screen out the predicted sequence of the nanos1 gene; (2) Randomly select the gonads of healthy individuals of Macrobrachium rosenbergii, extract total RNA, obtain cDNA after reverse transcription, and verify the screened Macrobrachium rosenbergii nanos1 gene; (3) Design of a semi-quantitative PCR amplification primer set: Based on the nanos1 gene obtained in step (2), upstream and downstream specific primers and a β-actin internal reference primer were designed to obtain a semi-quantitative PCR amplification primer set for the nanos1 gene of Macrobrachium rosenbergii; (4) Design of fluorescent quantitative PCR amplification primer set: Based on the nanos1 gene obtained in step (2), a fluorescent quantitative PCR amplification primer set was designed to obtain a fluorescent quantitative PCR amplification primer set for Macrobrachium rosenbergii.

3. A method for analyzing the nanos1 gene of Macrobrachium rosenbergii, characterized in that: The primer set for analyzing the Macrobrachium rosenbergii nanos1 gene according to claim 1 is used.

4. The method for analyzing the nanos1 gene of Macrobrachium rosenbergii according to claim 3, characterized in that: The gene analysis methods include semi-quantitative PCR, fluorescent quantitative PCR, PCR and in situ hybridization.

5. The method for analyzing the nanos1 gene of Macrobrachium rosenbergii according to claim 4, characterized in that: The PCR is used to amplify the nanos1 gene of Macrobrachium rosenbergii; the PCR amplification uses the gonad cDNA of Macrobrachium rosenbergii as a template; The reaction conditions of the PCR amplification were: 94° C. for 2 min; 38 cycles of 94° C. for 30 s, 57° C. for 30 s, and 72° C. for 90 s; and 72° C. for 10 min.

6. The method for analyzing the nanos1 gene of Macrobrachium rosenbergii according to claim 4, characterized in that: The semi-quantitative PCR is used to evaluate the expression of nanos1 gene in different tissues of Macrobrachium rosenbergii; the semi-quantitative PCR uses cDNA from different tissues of Macrobrachium rosenbergii as template; The reaction conditions of the semi-quantitative PCR amplification were: 94°C for 2 min; 38 cycles of 94°C for 30 s, 57°C for 30 s, and 72°C for 40 s; 72°C for 10 min; The different tissues include the brain, hepatopancreas, heart, muscle, gonad, gill, intestine and eye of Macrobrachium rosenbergii.

7. The method for analyzing the nanos1 gene of Macrobrachium rosenbergii according to claim 4, characterized in that: The fluorescent quantitative PCR is used to evaluate the expression of nanos1 gene in the early development period of Macrobrachium rosenbergii; The fluorescent quantitative PCR uses cDNA from Macrobrachium rosenbergii eggs, embryos, and early developmental stages as templates; the eggs include unfertilized eggs, fertilized eggs, and cleavage stages; the embryos include blastocyst stages and gastrula stages; and the early developmental stages include zoa larvae and larvae stages. The reaction conditions for the fluorescent quantitative PCR amplification are: 50°C for 2 min, 95°C for 2 min; 95°C for 15 s, 60°C for 30 s, 72°C for 30 s, 40 cycles; melting curve detection: 95°C for 15 s, 60°C for 30 s, 95°C for 15 s.

8. The method for analyzing the nanos1 gene of Macrobrachium rosenbergii according to claim 4, characterized in that: The in situ hybridization is used to evaluate the expression position of the nanos1 gene in the gonadal tissue of Macrobrachium rosenbergii; the in situ hybridization is performed on the gonadal tissue of Macrobrachium rosenbergii.

9. Use of the primer set for analyzing the nanos1 gene of Macrobrachium rosenbergii according to claim 1 in identifying whether Macrobrachium rosenbergii is fertilized or in the embryonic development stage.

10. Use of the primer set for analyzing the nanos1 gene of Macrobrachium rosenbergii according to claim 1 in sex identification during the early developmental period of Macrobrachium rosenbergii.

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