Application and method for knocking down bombyx mori BmGDAP2 gene in prolonging growth cycle of bombyx mori

Abstract

The invention discloses an application and a method for knocking down a bombyx mori BmGDAP2 gene in prolonging the growth cycle of bombyx mori, the service life of bombyx mori is prolonged by using Cas9 mediated target BmGDAP2 gene knockout, the economic benefit can be remarkably improved, and deep development of scientific research can be promoted. And by prolonging the service life, the silkworms have more time to grow and spin, so that the yield of the silk is increased. Meanwhile, the silkworms with the prolonged service life generally have higher adaptability and stress resistance and can better adapt to the environment, frequent breeding caused by diseases and death is reduced, and the breeding cost is reduced. In addition, by deeply researching a silkworm life prolonging mechanism, a life regulation and control mechanism can be further disclosed, and scientific development is promoted.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to an application of knocking down the silkworm BmGDAP2 gene in extending the growth cycle of the silkworm, and also to a method for extending the growth cycle of the silkworm. Background Art

[0002] Lifespan refers to the time it takes for a living organism to develop, grow, mature, age and die from birth. Lifespan is regulated by many factors, including genetics and the environment. With the aging of the population, aging has become an important issue facing human society, and delaying aging and prolonging lifespan has become very urgent. Enhancing the body's ability to resist adversity and prolonging its lifespan will provide more possibilities for solving the aging problem. At present, a variety of mechanisms have been found to be involved in the regulation of lifespan, such as the dietary restriction R pathway, the insulin / IGF-1 signaling pathway, the AMPK signaling pathway, the TOR signaling pathway, mitochondrial respiration, the JNK pathway, etc. The discovery of these lifespan regulation signaling mechanisms provides a direction for further exploring the lifespan mechanism.

[0003] Invertebrate models have many advantages, such as short life cycle, complete physiological structure, small size and easy research. Silkworm is a holometabolous insect, which goes through four developmental stages with completely different morphology and functions in its life: egg, larva, pupa and adult. Generally speaking, the growth period of silkworm larvae is about 30 days, during which they undergo five moltings. During the growth period, they continuously eat mulberry leaves to absorb and store energy. As a new type of invertebrate experimental animal, silkworm has been widely used in the field of lifespan research. Ganglioside-directed differentiation-associated protein 2 (GDAP2) is a member of the family. GDAP2 may play an important role in cell differentiation and development, especially in the development and function maintenance of the nervous system. Through gene editing technology and targeting GDAP2, we prepared a silkworm lifespan mechanism research strain with longer larval stage, which can provide theoretical basis and technical support for further research on new life extension mechanisms. Summary of the invention

[0004] In view of this, one of the objects of the present invention is to provide an application of knocking down the silkworm BmGDAP2 gene in extending the growth cycle of silkworm; the second object of the present invention is to provide a method for extending the growth cycle of silkworm.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] 1. Application of knocking down the silkworm BmGDAP2 gene in extending the growth cycle of silkworms, wherein the nucleic acid sequence of the silkworm BmGDAP2 gene is shown in SEQ ID NO.1.

[0007] Preferably, the knockdown of the silkworm BmGDAP2 gene uses Cas9 to mediate target BmGDAP2 gene knockout.

[0008] Preferably, the target BmGDAP2 gene is constructed on an sgRNA expression vector, and SEQ ID NO.2 and SEQ ID NO.3 are annealed to form a double strand that is connected to the AarΙ restriction site of the piggyBac-{3xp3-EGFP-sv40-TTTTT-gRNA-U6} vector.

[0009] 2. A method for extending the growth cycle of silkworms, by knocking out the expression of the BmGDAP2 gene in silkworms, screening mutant individuals, and obtaining a silkworm whose growth cycle is extended.

[0010] Preferably, the method for knocking out the expression of the BmGDAP2 gene is to knock out the target BmGDAP2 gene by ZFNs, TALENs and CRISPR / Cas9.

[0011] Preferably, the knockout of BmGDAP2 gene expression uses Cas9 to mediate a sequence of sgRNA containing the target BmGDAP2 gene.

[0012] Preferably, the sequence of the sgRNA is formed by annealing SEQ ID NO.2 and SEQ ID NO.3.

[0013] Preferably, the sgRNA expression vector of the target BmGDAP2 gene is transformed into silkworm eggs to obtain sgRNA transgenic silkworms, which are then hybridized with Cas9 transgenic silkworms to obtain the F1 generation, thereby obtaining silkworms with extended growth cycles.

[0014] The beneficial effect of the present invention is that the present invention can extend the life span of silkworms by knocking down the silkworm BmGDAP2 gene, which can not only significantly improve economic benefits, but also promote the in-depth development of scientific research. By extending the life span, silkworms will have more time to grow and spin silk, thereby increasing the yield of silk. At the same time, silkworms with extended life spans usually have stronger adaptability and stress resistance. They can better adapt to the environment, reduce frequent breeding caused by disease and death, and reduce breeding costs. In addition, by in-depth research on the mechanism of extending the life span of silkworms, we can further reveal the life span regulation mechanism and promote the development of science. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to make the purpose, technical solution and beneficial effects of the present invention clearer, the present invention provides the following drawings for illustration:

[0016] Figure 1Schematic diagram of the sgRNA transgenic vector structure (3×P3 is the eye-specific promoter; EGFP is the green fluorescent protein; SV40 is the stop codon; U6 is the promoter; gRNA is the designed primer);

[0017] Figure 2 Screening for positive BmGDAP2-sgRNA (the compound eyes of positive individuals emit green light under a fluorescence microscope);

[0018] Figure 3 Screening for BmGDAP2-KO strain (the silkworm strain N4-Cas9 that expresses Cas9 systemically can stimulate green fluorescence, which is hybridized with BmGDAP2-sgRNA to prepare F1 generation silkworm eggs. The F1 double-light individuals hybridized with BmGDAP2-sgRNA and N4-Cas9 are named BmGDAP2-KO);

[0019] Figure 4 BmGDAP2-KO knockout detection (A: nucleotide sequence of target site changes; B: amino acid sequence of target site changes; C: relative expression of GDAP2. The blue font sequence (GCCCTTTTCGTGGCT CA) in Figure A is the designed sg sequence, and the red font sequence (AGG) is PAM (Protospacer adjacent motif));

[0020] Figure 5 Statistical diagram of the growth cycle of BmGDAP2-KO (A: Statistics of the time of WT and BmGDAP2-KO mutants to the fifth instar; B: Proportion of each instar of WT and BmGDAP2-KO in the total growth cycle). DETAILED DESCRIPTION

[0021] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0022] Example 1: Construction of sgRNA transgenic vector of BmGDAP2

[0023] (1) The gene sequence of BmGDAP2 (SEQ ID NO. 1) was obtained from the silkworm gene database Silk DB3.0, and then the gRNA sequence was designed through the CC-TOP online analysis website, and the primers KO-BmGDAP2-sgF: 5'-AAGTGCCCTTTTCGTGGCTCAAGG-3' (SEQ ID NO. 2); KO-BmGDAP2-sgR: 5'-AAACCCTTGAGCCACGAAAAGGGC-3' (SEQ ID NO. 3) were designed.

[0024] (2) Use restriction endonuclease Aar Ι to digest the plasmid piggyBac-{3xp3-EGFP-sv40-TTTTT-gRNA-U6}

[0025] (U6-gRNA-TTTTT was used to replace the pBac [U6-gRNAgLMN, 3xp3-EGFP] vector, see Sanyuan Ma. CRISP R / Cas9 mediated multiplex genome editing and heritable mutagenesis of BmKu70 in Bomby x mor. 2014), and Yuanyuan Liu. Tissue-specific genome editing of laminA / C in the posterior silk glands of Bombyx mor. 2017)), and the vector after enzyme digestion was recovered.

[0026] (3) 10 μL of primers SEQ ID NO.2 and SEQ ID NO.3 were taken and mixed, denatured at 95°C for 10 min, and then cooled from 95°C to 25°C, decreasing by 0.4°C every 8 seconds, for a total of 175 cycles. The double-stranded annealed primers were connected to the restriction vector, and the ligation reaction was carried out under the action of T4 DNA ligase at 25°C for 10 min, and then transformed into Trans1-T1 competent cells. The positive clones were obtained and sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing. The sequencing results showed that we successfully connected the gRNA primers to the restriction vector and obtained piggyBac [3×p3-EGFP-SV40-hr3-U6-BmGDAP2gRNA-TTTTTT] bacterial solution ( Figure 1 ) to extract ultrapure plasmid.

[0027] Example 2: Screening of BmGDAP2-sgRNA strains

[0028] (1) The recombinant plasmid piggyBac [3×p3-EGFP-SV40-hr3-U6-BmGDAP2gRNA-TTTTTT] was mixed with the auxiliary plasmid A3H at a ratio of 1:1 (concentration of 600 ng / μL), and the embryos were microinjected into D9L silkworm eggs, sealed with non-toxic glue, and incubated in an incubator at 25°C;

[0029] (2) G0 generation silkworms were reared to adults and self-pollinated to obtain G1 generation silkworm eggs, which were incubated at 25°C. Six days later, positive individuals with green eyes were screened under a fluorescence microscope and named BmGDAP2-sgRNA.

[0030] (3) The BmGDAP2-sgRNA genome was extracted and amplified using primers U6-R: 5'-AGCTGTCCAAGGAATGCG-3' (SEQ ID NO. 4) and gRNA-F: 5'-CGACTCGGTGCCACTTT-3' (SEQ ID NO. 5). The PCR reaction conditions were: pre-denaturation at 98°C for 5 minutes, followed by denaturation at 98°C for 5 seconds, annealing at 53°C for 10 seconds, and extension at 72°C for 30 seconds, for a total of 30 cycles, and finally extension at 72°C for 10 minutes. The PCR product was identified and recovered by agarose gel electrophoresis, and then PCR products were purified by pMD1.

[0031] The 9-T vector was connected and then transformed into Trans1-T1 competent cells. After obtaining positive clones, they were sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing. The sequencing results showed that the sgRNA expression fragment was successfully inserted into the transgenic silkworm.

[0032] Example 3, BmGDAP2-KO strain screening

[0033] (1) The silkworm strain N4-Cas9 that expresses Cas9 systemically carries a green fluorescent reporter gene ( Figure 2 ), BmRPA2-sgRNA moths were hybridized with them respectively to obtain three kinds of F1 silkworm eggs, and then incubated at 25℃ in the dark ( Figure 3 ).

[0034] (2) Screening for double individuals that can emit green light in both the eyes and body under a fluorescence microscope. The F1 double-light individual hybridized by BmGDAP2-sgRNA and N4-Cas9 was named BmGDAP2-KO.

[0035] Example 4 BmGDAP2-KO knockout form detection

[0036] (1) BmGDAP2-KO larvae were fed with mulberry leaves and their developmental cycles were counted;

[0037] (2) The whole genome of BmGDAP2-KO silkworm was extracted and amplified using primers BmGDAP2-KO-F: 5'-ACCAGCCGCAGAAGAAACA-3' (SEQ ID NO.6), BmGDAP2-KO-R: 5'-CCTCCTTGAGCCACGAAAA-3' (SEQ ID NO.7). The PCR reaction conditions were: pre-denaturation at 98°C for 5 minutes, followed by denaturation at 98°C for 10 seconds, annealing at 58°C for 15 seconds, and extension at 72°C for 30 seconds, for a total of 30 cycles, and finally extension at 72°C for 10 minutes. The PCR product was identified and recovered by agarose gel electrophoresis, then ligated with the pMD19-T vector and transformed into Trans1-T1 competent cells. After obtaining positive clones, they were sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing. The sequencing results were compared with the sequences in the silkworm database. The results showed that BmGDAP2-KO had a base deletion ( Figure 4 , A). The amino acids also changed after the base deletion in BmGDAP2-KO ( Figure 4 , B).

[0038] The relative expression of GDAP2 was detected. Figure 4 As shown in middle C. The results showed that the expression of GDAP2 gene was reduced in BmGDAP2-KO mutant individuals.

[0039] Example 5 Statistics of growth and development cycle of BmGDAP2-KO

[0040] BmGDAP2-KO larvae were fed with mulberry leaves, and their growth cycles were recorded and recorded ( Figure 5 , A and Figure 5 , B). 15 days after the silkworms emerged, the wild-type silkworms had entered the fourth instar dormancy period, while the BmGDAP2-KO mutants were still in the fourth instar. 17 days after the silkworms emerged, the wild-type silkworms had entered the fifth instar, and were in the fifth instar two days period, while the F1 generation mutants began to enter the fourth instar dormancy period, which extended the mutants' development time in the fourth instar as a whole. 22 days after the silkworms emerged, the wild-type silkworms entered the wandering stage, while the BmGDAP2-KO mutants were in the fifth instar five days, and were still in the feeding stage and had not entered the wandering stage. 26 days after the silkworms emerged, at this time, all the wild-type silkworms were in the pupal stage, and about 25% of the BmGDAP2-KO began to cluster. Until 28 days after the silkworms emerged, all the F1 generation mutants entered the wandering stage. The development time of the pupal stage and the survival time of the moth stage were further observed, and no obvious difference was found between the F1 generation mutants and the wild type. Overall, the BmGDAP2-KO mutant delayed the growth and development of silkworm larvae at the fourth and fifth instars and increased the lifespan of silkworms.

[0041] In the embodiment of the present invention, Cas9 is used to mediate the knockout of the target BmGDAP2 gene. It is well known to those skilled in the art that any means capable of knocking out the BmGDAP2 gene can achieve the purpose of the present invention, such as: ZFNs, TALENs and CRISPR-Cas9 or its variants mediating the knockout of the target BmGDAP2 gene.

[0042] The above-described embodiments are only preferred embodiments for fully illustrating the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or changes made by those skilled in the art based on the present invention are within the protection scope of the present invention. The protection scope of the present invention shall be subject to the claims.

Claims

1. Application of knocking down the silkworm BmGDAP2 gene in extending the growth cycle of silkworm, characterized in that: The nucleic acid sequence of the silkworm BmGDAP2 gene is shown in SEQ ID NO.

1.

2. The use of knocking down the silkworm BmGDAP2 gene in extending the growth cycle of silkworm according to claim 1, characterized in that: The method for knocking down the silkworm BmGDAP2 gene uses Cas9 to mediate target BmGDAP2 gene knockout.

3. The use of knocking down the silkworm BmGDAP2 gene in extending the growth cycle of silkworm according to claim 1, characterized in that: The target BmGDAP2 gene was constructed on the sgRNA expression vector, and SEQ ID NO.2 and SEQ ID NO.3 were annealed to form a double strand and connected to the AarΙ restriction site of the piggyBac-{3xp3-EGFP-sv40-TTTTT-gRNA-U6} vector.

4. A method for extending the growth period of silkworms, characterized in that: By knocking out the expression of the BmGDAP2 gene in silkworms and screening mutant individuals, the growth cycle of the silkworms was extended.

5. The method according to claim 4, characterized in that: The method for knocking out the expression of the BmGDAP2 gene is to knock out the target BmGDAP2 gene by mediating ZFNs, TALENs and CRISPR / Cas9.

6. The method according to claim 4, characterized in that: The knockout of BmGDAP2 gene expression uses Cas9 to mediate the sequence of sgRNA containing the target BmGDAP2 gene.

7. The method according to claim 4, characterized in that: The sequence of the sgRNA is formed by annealing SEQ ID NO.2 and SEQ ID NO.

3.

8. The method according to claim 4, characterized in that: The sgRNA expression vector of the target BmGDAP2 gene was transformed into silkworm eggs to obtain sgRNA transgenic silkworms, which were then hybridized with Cas9 transgenic silkworms to obtain the F1 generation and silkworms with extended growth cycles.

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