Macrobrachium Nipponense Cathepsin L Gene, dsRNA Thereof, and Use Thereof

Abstract

The present disclosure relates to the technical field of biology, and in particular to a Macrobrachium nipponense Cathepsin L gene and use of dsRNA thereof, including the Macrobrachium nipponense Cathepsin L gene, a gene fragment and the dsRNA thereof, and use of the dsRNA in inhibiting ovary development of Macrobrachium nipponense. The Macrobrachium nipponense Cathepsin L gene is obtained at first with the full-length nucleotide sequence as shown in SEQ ID No. 1 and the amino acid sequence as shown in SEQ ID No. 2. Gene fragments with sequences of SEQ ID No. 3 and SEQ ID No. 8 are obtained by using technologies such as RNA interference, and dsRNA1 and dsRNA2 are synthesized from the two gene fragments. The synthesized dsRNA1 and dsRNA2 are injected into a pericardial cavity of a female Macrobrachium nipponense and the result shows that the dsRNA1 can effectively slow down the ovary development speed of the female Macrobrachium nipponense.

Claims

1. Use of a Macrobrachium nipponense Cathepsin L gene or a protein encoded by the Macrobrachium nipponense Cathepsin L gene as a target in preparing an inhibitor for ovary development of Macrobrachium nipponense; wherein the nucleotide sequence of the Macrobrachium nipponense Cathepsin L gene is set forth in SEQ ID NO. 1; and wherein the amino acid sequence of the protein encoded by the Macrobrachium nipponense Cathepsin L gene is set forth in SEQ ID NO. 2.

2. Use of a Macrobrachium nipponense Cathepsin L gene fragment in preparing an inhibitor for ovary development of Macrobrachium nipponense, wherein the nucleotide sequence of the Macrobrachium nipponense Cathepsin L gene fragment is set forth in SEQ ID No. 3.

3. A primer set for amplifying a Macrobrachium nipponense Cathepsin L gene fragment, wherein the nucleotide sequence of an upstream primer of the primer set is set forth in SEQ ID NO. 4 and the nucleotide sequence of a downstream primer is set forth in SEQ ID NO. 5; and the nucleotide sequence of the Macrobrachium nipponense Cathepsin L gene fragment is set forth in SEQ ID NO. 3.

4. A dsRNA, obtained by transcription using a Macrobrachium nipponense Cathepsin L gene fragment as a template; and the nucleotide sequence of the Macrobrachium nipponense Cathepsin L gene fragment is set forth in SEQ ID NO. 3.

5. Use of the dsRNA according to claim 4 in inhibiting mRNA expression of a Macrobrachium nipponense Cathepsin L gene.

6. Use of the dsRNA according to claim 4 in inhibiting ovary development of Macrobrachium nipponense.

Description

BRIEF DESCRIPTION OF FIGURES

[0032] In order to more clearly illustrate the examples of the present disclosure or the technical solutions in the prior art, the following will briefly introduce the drawings that need to be used in the description of the examples or the prior art.

[0033] FIG. 1 shows the mRNA expression of a Cathepsin L gene in ovary and hepatopancreas at different sampling time points after injection of dsGFP, dsRNA1 and dsRNA2 into female Macrobrachium nipponense at an ovary development stage I, where a Macrobrachium nipponense β-Actin gene is used as an internal reference gene; 1, 4 and 7 are the 1st, 4th, and 7th days after injection respectively (n=9); and different letters represent p<0.05. A green fluorescent protein (GFP) gene is used as a reporter gene in a control group and the Macrobrachium nipponense in the control group is injected with the same dose of dsGFP.

[0034] FIG. 2 is a graph showing changes of an ovary gonadosomatic index of the female Macrobrachium nipponense at the ovary development stage I after injection with dsGFP, dsRNA1 and dsRNA2; 1 and 30 are the 1st and 30th days after injection respectively (n=9); and different letters a and b represent p<0.05.

DETAILED DESCRIPTION

[0035] The present disclosure discloses a Macrobrachium nipponense Cathepsin L gene, a gene fragment and a dsRNA thereof, and use of the dsRNA in inhibiting ovary development of Macrobrachium nipponense. Those skilled in the art can learn from the content of this document and appropriately improve process parameters. Of particular note, all similar substitutions and alterations will be apparent to those skilled in the art, and they are all deemed to be included in the present disclosure. The method and use of the present disclosure have been described through preferred embodiments. It is obvious that relevant persons can make modifications or appropriate alterations and combinations to the method and use described herein without departing from the content, spirit and scope of the present disclosure to achieve and use the technology of the present disclosure.

[0036] The present disclosure provides a Macrobrachium nipponense Cathepsin L gene whose nucleotide sequence is as shown in SEQ ID No. 1 and amino acid sequence is as shown in SEQ ID No. 2.

[0037] The present disclosure provides a high-efficiency Macrobrachium nipponense Cathepsin L interference gene fragment whose nucleotide sequence is as shown in SEQ ID No. 3.

[0038] The present disclosure provides a method for obtaining a Macrobrachium nipponense Cathepsin L interference gene fragment: after the amino acid sequence of Macrobrachium nipponense Cathepsin L is analyzed, a specific region of Cathepsin L is selected and two pairs of interference primers are designed: one interference primer pair has an upstream primer as shown in SEQ ID No. 4 and a downstream primer as shown in SEQ ID No. 5; and the other interference primer pair has an upstream primer as shown in SEQ ID No. 6 and a downstream primer as shown in SEQ ID No. 7; and then PCR amplification is conducted to obtain DNA fragments SEQ ID No. 3 and SEQ ID No. 8 by using total cDNA of Macrobrachium nipponense as a template.

[0039] Further, the DNA fragments SEQ ID No. 3 and SEQ ID No. 8 are used as templates, and dsRNA1 and dsRNA2 are synthesized by using kits.

[0040] Use of dsRNA1 and dsRNA2 synthesized by using the DNA fragments SEQ ID No. 3 and SEQ ID No. 8 as templates in inhibiting ovary development of female Macrobrachium nipponense: the dsRNA1 and the dsRNA2 are injected into the pericardial cavity of the Macrobrachium nipponense at the injection concentration of 4 μg/g. Results show that injection of the dsRNA1 synthesized with SEQ ID No. 3 as the template can effectively reduce the mRNA expression of the Macrobrachium nipponense Cathepsin L gene and inhibit the ovary development speed of Macrobrachium nipponense. But the injection of the dsRNA2 synthesized with SEQ ID No. 8 as the template does not effectively reduce the mRNA expression of the Macrobrachium nipponense Cathepsin L gene and does not inhibit the ovary development speed of the Macrobrachium nipponense.

[0041] Where:

TABLE-US-00001 SEQ ID No. 1 ttgaaatctcccgttcaataaacattttctccacaactacgattctcta atccgagactttccgcagatgaaattcctgctcttcctctgtggtttgg ccatcgctgccgccagtcagtcatgggaaagctttaagctgacccatgg caaggcctactccaacgccaaggaggagctctacaggaagaccattttc gagagcaaccttaaattcgtagcagaacacaatgaacgcttccgaaagg gcctagtcaccttcaacgtcgccatgaacagatttggtgacttgaccac agaggagtttgtagcccagatgactggtctgcagaaactggagagcacc gagggaatggaattcgctcacttccctgaggcccccagagctgccgatg ttgactggagaaacaagggagctgtcactcctgtcaaggaccagggaca gtgtggatcctgctggtccttctctactactggagctctagaaggcgca catttcatcaaaaccggaagtctgccaagcctctccgaacagcagctgg ttgattgctcaaaggaaaacagcggttgcaacggaggagttgtgcaatg ggcctacgattacctcaagtcctgcggaggaagccagactgagtcttcc tatccttacgaggctattgacaacatatgccgcttcgattcatctcagg tggctgccactgtgaggggatacacgaacatcccctatggcgatgaggt gactcaggcctctgctgtccacgacgaaggtccagtcagtgtctgcgtc gatgctggacacttgtccttccagttgtacagctcaggtgtctactacg aaccaaactgcaaccctcagggcatcaaccacgccgtgttggctgttgg ctacggaaccgaaggcggctccgactactggatcatcaagaactcgtgg ggcagcagctggggtgagtctggatacatgaagctcaccaggaacaaga acaaccactgcggtgttgccacccagtcttgctacccaaccgtctaagg attccaagaaagtctggttgctttattccatgaagagttatgagtatac atcgacaccttaactcataagaccatagcttgataatcatgtctggctt tatatcttgtttatgaaaaataaagtggaatcgattaaaaaaaaaa SEQ ID No. 2 MKFLLFLCGLAIAAASQSWESFKLTHGKAYSNAKEELYRKTIFESNLKF VAEHNERFRKGLVTFNVAMNRFGDLTTEEFVAQMTGLQKLESTEGMEFA HFPEAPRAADVDWRNKGAVTPVKDQGQCGSCWSFSTTGALEGAHFIKTG SLPSLSEQQLVDCSKENSGCNGGVVQWAYDYLKSCGGSQTESSYPYEAI DNICRFDSSQVAATVRGYTNIPYGDEVTQASAVHDEGPVSVCVDAGHLS FQLYSSGVYYEPNCNPQGINHAVLAVGYGTEGGSDYWIIKNSWGSSWGE SGYMKLTRNKNNHCGVATQSCYPTV SEQ ID No. 3 gctctacaggaagaccattttcgagagcaaccttaaattcgtagcagaa cacaatgaacgcttccgaaagggcctagtcaccttcaacgtcgccatga acagatttggtgacttgaccacagaggagtttgtagcccagatgactgg tctgcagaaactggagagcaccgagggaatggaattcgctcacttc SEQ ID No. 4 gctctacaggaagaccattttc SEQ ID No. 5 gaagtgagcgaattccattcc SEQ ID No. 6 gcctacgattacctcaagtcc SEQ ID No. 7 gtggacagcagaggcctgagtc SEQ ID No. 8 gcctacgattacctcaagtcctgcggaggaagccagactgagtcttcct atccttacgaggctattgacaacatatgccgcttcgattcatctcaggt ggctgccactgtgaggggatacacgaacatcccctatggcgatgaggtg actcaggcctctgctgtccac

[0042] In the Macrobrachium nipponense Cathepsin L gene, the gene fragment and the dsRNA thereof, and the use of the dsRNA in inhibiting ovary development of Macrobrachium nipponense provided by the present disclosure, all of the raw materials and reagents used can be purchased from the market.

[0043] The present disclosure will be described in detail below in connection with specific examples:

Example 1 Obtaining of full-length sequence of Macrobrachium nipponense Cathepsin L gene

[0044] Transcriptomes at each ovary development stage of female Macrobrachium nipponense were compared and analyzed, thus a full-length differential Cathepsin L gene was obtained with the full-length nucleotide sequence of SEQ ID No. 1 and the amino acid sequence of SEQ ID No. 2.

Example 2 Obtaining of Macrobrachium nipponense Cathepsin L gene fragment and dsRNA thereof

[0045] Based on the nucleotide sequence SEQ ID No. 1 of a Macrobrachium nipponense Cathepsin L gene, an NCBI online dsRNA primer design software (https://www.flyrnai.org/cgi-bin/RNAi_find_primers.pl) was used in its open reading frame to design specific primers for RNA interference (SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6 and SEQ ID No. 7), and a T7 promoter sequence of TAATACGACTCACTATAGGG was added before the primers. An upstream primer SEQ ID No. 4 and a downstream primer SEQ ID No. 5 as well as an interference upstream primer SEQ ID No. 6 and a downstream primer SEQ ID No. 7 containing the T7 promoter were used to obtain PCR products (sequences as shown in SEQ ID No. 3 and SEQ ID No. 8) by PCR amplification, after purification by a PCR product purification kit, according to the manufacturer's instructions, a Transcript AidTM T7 High Yield Transcription kit (Fermentas, Inc., USA) was used for in vitro transcription to synthesize dsRNA1 and dsRNA2; 1.2% agarose gel electrophoresis was used to detect purity and integrity of dsRNA1 and dsRNA2; the concentration of the dsRNA1 and the dsRNA2 was measured with a UV spectrophotometer (Eppendorf, Hamburg, Germany) at 260 nm; and the dsRNAs were stored at −80° C. for later use.

Example 3 Experiment of injecting dsRNA1 and dsRNA2 synthesized by Cathepsin L gene fragment to inhibit ovary development of female Macrobrachium nipponense

[0046] (1) Injection of dsRNA1 and dsRNA2 synthesized by cathepsin L gene fragment

[0047] According to appearance characteristics of ovary development of Macrobrachium nipponense, 180 healthy Macrobrachium nipponense (0.61±0.14 g) at an ovary development stage I (white and transparent at an oogonia proliferation stage) were selected to ensure consistency of an initial ovary development period; and the Macrobrachium nipponense injected with dsGFP was set as a control group, while the Macrobrachium nipponense injected with dsRNA1 and dsRNA2 were set as experimental groups. The dsRNA synthesized in vitro was injected into the pericardial cavity of the Macrobrachium nipponense with a microsyringe at a dose of 4 μg/g, and 60 Macrobrachium nipponense were set in each group, where three parallel replicates (n=20) were set. Before injection, the Macrobrachium nipponense were temporarily cultured in a glass tank for three days to adapt to the laboratory culture environment (at the water temperature of 25±1° C.) and fed with fresh margarya melanoide every day in the morning and evening.

[0048] (2) Detection of Cathepsin L gene silencing efficiency

[0049] On the 1st, 4th, and 7th days after the injection, 3 Macrobrachium nipponense were randomly collected from each group and the ovary and hepatopancreas were dissected. Total

[0050] RNA was extracted using an RNAiso Plus reagent (TaKaRa, Japan) and a template cDNA was obtained by reverse transcription using a Primer ScriptII1st Strand cDNA Synthesis reverse transcription kit (Bio-Rad) and an M-MLV kit (TaKaRa, Japan). The relative expression of the Cathepsin L was detected by Real Time PCR using the template, and β-Actin was used as an internal reference gene to calculate the silencing efficiency of the target gene.

TABLE-US-00002 TABLE 1 Original data of hepatopancreas Day/Group 1 4 7 Control group 2435.50 2929.96 3455.85 2336.28 2991.53 3774.80 2241.11 3228.54 3639.40 dsRNA1 2215.37 1.05 17.39 1969.12 0.95 16.22 2154.79 1.00 14.93 dsRNA2 2345.78 2987.32 3512.34 2412.45 2887.98 3487.05 2278.21 3098.32 3698.07

TABLE-US-00003 TABLE 2 Mean value and standard deviation Day/Group 1 4 7 Control group 2337.63 ± 97.20a 3050.01 ± 157.65b 3623.35 ± 160.08d dsRNA1  2113.09 ± 128.31a  1.00 ± 0.05c 16.18 ± 1.23c dsRNA2 2345.48 ± 67.12a 2991.21 ± 105.22b 3565.82 ± 115.23d

TABLE-US-00004 TABLE 3 Original data of ovary Day/Group 1 4 7 Control group 74.54 391.63 528.83 68.12 388.92 384.45 88.03 443.67 392.53 dsRNA1 82.33 20.30 1.10 94.57 18.29 1.10 80.63 15.71 0.84 dsRNA2 75.32 389.25 387.56 69.89 398.32 378.98 86.56 376.21 369.54

TABLE-US-00005 TABLE 4 Mean value and standard deviation Day/Group 1 4 7 Control group  76.90 ± 10.16.sup.a 408.07 ± 30.86.sup.b 435.27 ± 81.13.sup.b dsRNA1 85.85 ± 7.61.sup.a 18.10 ± 2.30.sup.c  1.01 ± 0.15.sup.c dsRNA2 77.26 ± 8.50.sup.a 387.93 ± 11.11.sup.b 378.69 ± 9.01.sup.b 

[0051] FIG. 1 and Tables 1-4 show the mRNA expression of the Cathepsin L gene in the ovary and the hepatopancreas at different sampling time points after injection of the dsGFP, the dsRNA1 and the dsRNA2 in female Macrobrachium nipponense at the ovary development stage I. 1, 4 and 7 were the 1st, 4th, and 7th days after the injection respectively (n=9). Different letters represented p<0.05. A green fluorescent protein (GFP) gene was used as a reporter gene in the control group and the Macrobrachium nipponense in the control group was injected with the same dose of dsGFP.

[0052] The results showed that compared with the dsGFP control group, the silencing efficiencies were 99.97% and 99.55% respectively in the hepatopancreas and 95.56% and 99.79% respectively in the ovary in the dsRNA1 experimental group on the 4th and 7th days after injection, while the silencing efficiencies showed no significant differences between the dsRNA2 experimental group and the control group (p>0.05).

[0053] (3) Changes of gonadosomatic index (GSI) of Macrobrachium nipponense after injection of dsGFP, dsRNA1 and dsRNA2

TABLE-US-00006 TABLE 5 Original date of gonadosomatic index (GSI) Day/Group 1 30 Control group 1.60% 4.01% 2.38% 3.74% 1.50% 3.90% dsRNA1 1.45% 1.94% 2.38% 1.59% 2.08% 1.74% dsRNA2 2.20% 4.11% 1.45% 3.97% 2.10% 3.91%

TABLE-US-00007 TABLE 6 Mean value and standard deviation Day/Group 1 30 Control group 1.83% ± 0.48%.sup.a 3.89% ± 0.13%.sup.b dsRNA1 1.97% ± 0.48%.sup.a 1.76% ± 0.17%.sup.a dsRNA2 1.90% ± 0.41%.sup.a 4.00% ± 0.10%.sup.b

[0054] FIG. 2 and Tables 5-6 show a graph showing changes of the ovary gonadosomatic index of the female Macrobrachium nipponense at the ovary development stage I after injection with the dsGFP, the dsRNA1 and the dsRNA2. 1 and 30 were the 1st and 30th days after injection respectively (n=9). Different letters a and b represented p<0.05.

[0055] According to the formula of gonadosomatic index (GSI)=wet weight of gonad/wet weight of body weight×100%, the GSI of the Macrobrachium nipponense in the dsGFP, dsRNA1 and dsRNA2 three groups was calculated respectively. The GSI of the control group was compared with that of the dsRNA1 injection experimental group; it was found that after the Cathepsin L is silenced, the ovary development speed of the Macrobrachium nipponense was significantly inhibited; after cultured for 30 days, the Macrobrachium nipponense in the control group developed from the ovary development stage I (GSI=1.83%, white and transparent at an oogonia proliferation stage) to an ovary development stage III (GSI=3.89%, light green at a secondary vitellogenesis phase), while the Macrobrachium nipponense in the dsRNA1 experimental group had the GSI of 1.76% and still remained at the stage I (white and transparent at the oogonia proliferation stage); and after cultured for 30 days, the Macrobrachium nipponense in the dsRNA2 experimental group had the GSI of 4.00% and developed to the stage III (light green at the secondary vitellogenesis phase), which was not significantly different compared with the control group (p>0.05). The results of the GSI showed that the exogenous injection of the dsRNA1 of the Cathepsin L could effectively inhibit the ovary development speed of the Macrobrachium nipponense, while the dsRNA2 did not inhibit the ovary development speed.

[0056] The above descriptions are merely preferred implementations of the present disclosure. It should be noted that a person of ordinary skill in the art may further make several improvements and modifications without departing from the principle of the present disclosure, and such improvements and modifications should be deemed as falling within the protection scope of the present disclosure.