Application and method of silencing bombyx mori bmgdap2 gene in prolonging the growth cycle of bombyx mori

Abstract

The application discloses application and method of knocking down domestic silkworm BmGDAP2 gene in prolonging growth cycle of domestic silkworm, and life span of the domestic silkworm is prolonged by using Cas9 mediated target BmGDAP2 gene knockout, which can not only significantly improve economic benefits, but also promote in-depth development of scientific research. Through prolonging the life span, the domestic silkworm has more time for growth and silk spinning, thereby increasing the yield of silk. Meanwhile, the domestic silkworm with prolonged life span usually has stronger adaptability and stress resistance, and can better adapt to the environment, reduce frequent breeding caused by diseases and death, and reduce breeding cost. In addition, through in-depth research on the mechanism of prolonging the life span of the domestic silkworm, the life span regulation mechanism can be further revealed, and the development of science is promoted.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to the application of knocking down the BmGDAP2 gene in silkworms to prolong their growth cycle, and also to methods for prolonging the silkworm growth cycle. Background Technology

[0002] Lifespan refers to the time from birth, through development, growth, maturity, aging, and death of a living organism. Lifespan is regulated by numerous factors, including genetics and environment. With the increasing aging of the population, aging has become a significant problem facing human society, making the slowing of aging and the extension of lifespan extremely urgent. Enhancing the body's ability to resist adversity and extending its lifespan will provide more possibilities for solving the aging problem. Currently, multiple mechanisms have been discovered 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, and the JNK pathway; the discovery of these lifespan-regulating signaling mechanisms provides direction for further research into the mechanisms of lifespan.

[0003] Invertebrate models offer numerous advantages, including short lifespans, intact physiological structures, small size, and ease of research. The silkworm, a holometabolous insect, undergoes four distinct developmental stages throughout its life cycle: egg, larva, pupa, and adult. Generally, the silkworm larva's life cycle is approximately 30 days, during which it molts five times, continuously consuming mulberry leaves to obtain and store energy. As a novel invertebrate experimental animal, the silkworm has been widely used in lifespan research. Ganglioside-guided differentiation-associated protein 2 (GDAP2) is a member of this family and may play a crucial role in cell differentiation and development, particularly in the development and maintenance of the nervous system. By targeting GDAP2 using gene editing technology, we have prepared silkworm strains with longer larval stages for studying lifespan mechanisms, providing a theoretical basis and technical support for further research into novel lifespan extension mechanisms. Summary of the Invention

[0004] In view of this, one objective of the present invention is to provide an application of knocking down the BmGDAP2 gene in silkworms to prolong the silkworm growth cycle; another objective of the present invention is to provide a method for prolonging the silkworm growth cycle.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] 1. Application of knockdown of the silkworm BmGDAP2 gene in prolonging the silkworm growth cycle, 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 is achieved using Cas9-mediated knockout of the target BmGDAP2 gene.

[0008] Preferably, the target BmGDAP2 gene is constructed on an sgRNA expression vector, and the double strands formed by annealing SEQ ID NO.2 and SEQ ID NO.3 are linked to the AarI restriction site of the piggyBac-{3xp3-EGFP-sv40-TTTTT-gRNA-U6} vector.

[0009] 2. A method for extending the growth cycle of silkworms, which involves knocking out the expression of the BmGDAP2 gene in silkworms, screening for mutant individuals, and obtaining silkworms with extended growth cycles.

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

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

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

[0013] In a preferred embodiment of the present invention, 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 effects of this invention are as follows: By knocking down the BmGDAP2 gene in silkworms, the lifespan of silkworms is extended, which not only significantly improves economic benefits but also promotes in-depth scientific research. Extended lifespan allows silkworms more time to grow and spin silk, thus increasing silk production. Simultaneously, silkworms with extended lifespans typically exhibit stronger adaptability and resilience; they are better able to adapt to their environment, reducing frequent rearing due to disease and mortality, and lowering rearing costs. Furthermore, by studying the mechanisms of silkworm lifespan extension, we can further elucidate lifespan regulation mechanisms, advancing scientific progress. Attached Figure Description

[0015] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the following figures are provided for illustration:

[0016] Figure 1This is a schematic diagram of the sgRNA transgenic vector structure (3×P3 is the eye-specific promoter; EGFP is 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 the BmGDAP2-KO strain (the N4-Cas9 silkworm strain, which expresses Cas9 systemically, can emit green fluorescence. It was hybridized with BmGDAP2-sgRNA to prepare F1 generation silkworm eggs. The F1 dual-fluorescent individuals resulting from the hybridization of BmGDAP2-sgRNA and N4-Cas9 were named BmGDAP2-KO);

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

[0020] Figure 5 The growth cycle statistics of BmGDAP2-KO are shown in the figure (A: statistics of the time to fifth age of WT and BmGDAP2-KO mutants; B: the proportion of each age of WT and BmGDAP2-KO in the total growth cycle). Detailed Implementation

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

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

[0023] (1) The gene sequence of BmGDAP2 (SEQ ID NO.1) was obtained from the silkworm gene database Silk DB3.0. Then, gRNA sequences were designed using the CC-TOP online analysis website, and 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) The plasmid piggyBac-{3xp3-EGFP-sv40-TTTTT-gRNA-U6} was digested with the restriction endonuclease AarI.

[0025] (Replace the pBac[U6-gRNAgLMN,3xp3-EGFP] vector with U6-gRNA-TTTTT, see Sanyuan Ma. CRISP R / Cas9 mediated multiplex genome editing and heritable mutagenesis of BmKu70 in Bombyx mor. 2014) and Yuanyuan Liu. Tissue-specific genome editing of laminA / C in the posterior silk glands of Bombyx mor. 2017)), and recover the digested vector.

[0026] (3) Mix 10 μL each of primers SEQ ID NO.2 and SEQ ID NO.3, denature at 95℃ for 10 min, then gradually decrease the temperature from 95℃ to 25℃, decreasing by 0.4℃ every 8 seconds, for a total of 175 cycles. The double-stranded annealing primers were ligated to the restriction enzyme vector. The ligation reaction was carried out at 25℃ for 10 min using T4 DNA ligase. The mixture was then transformed into Trans1-T1 competent cells. Positive clones were obtained and sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. The sequencing results showed that we successfully ligated the gRNA primers to the restriction enzyme vector, obtaining piggyBac[3×p3-EGFP-SV40-hr3-U6-BmGDAP2gRNA-TTTTTT] bacterial culture. Figure 1 ), and extract ultrapure plasmids.

[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 helper plasmid A3H at a ratio of 1:1 (concentration of 600ng / μL), and D9L silkworm eggs were microinjected into the embryos. The eggs were sealed with non-toxic glue and incubated in a 25℃ incubator.

[0029] (2) The G0 generation silkworms were raised to adulthood and then self-crossed to obtain G1 generation silkworm eggs. They were then incubated at 25°C. After six days, positive individuals with green eyes were selected under a fluorescence microscope and named BmGDAP2-sgRNA.

[0030] (3) BmGDAP2-sgRNA genomic DNA 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: 98℃ pre-denaturation for 5 minutes, followed by 98℃ denaturation for 5 seconds, 53℃ annealing for 10 seconds, and 72℃ extension for 30 seconds, for a total of 30 cycles, with a final extension at 72℃ for 10 minutes. The PCR products were identified and recovered by agarose gel electrophoresis and then compared with pMD1.

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

[0032] Example 3: Screening of the BmGDAP2-KO strain

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

[0034] (2) Under a fluorescence microscope, dual individuals that can emit green light simultaneously in the eyes and body were screened. The F1 dual-light individuals of BmGDAP2-sgRNA and N4-Cas9 were named BmGDAP2-KO.

[0035] Example 4: Detection of BmGDAP2-KO knockout

[0036] (1) The BmGDAP2-KO larvae were fed with mulberry leaves and their development cycle was recorded.

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

[0038] The relative expression level of GDAP2 was detected, and the results are as follows: Figure 4 As shown in Figure C. The results showed that the expression level of the GDAP2 gene was reduced in individuals with the BmGDAP2-KO mutation.

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

[0040] BmGDAP2-KO larvae were fed mulberry leaves, and their growth cycle was recorded. Figure 5 A and Figure 5 (B) Fifteen days after emergence, wild-type silkworms had entered the fourth instar molting stage, while the BmGDAP2-KO mutant remained in the fourth instar. Seventeen days after emergence, wild-type silkworms had entered the fifth instar, at stage two, while the F1 mutants were only beginning to enter the fourth instar molting stage, thus prolonging the development time of the mutants in the fourth instar. Twenty-two days after emergence, wild-type silkworms entered the migratory stage, while the BmGDAP2-KO mutants were at stage five in the fifth instar, still in the feeding stage and not yet in the migratory stage. Twenty-six days after emergence, all wild-type silkworms were in the pupal stage, and approximately 25% of the BmGDAP2-KO mutants began to ascend to the pupa. It wasn't until 28 days after emergence that all F1 mutants entered the migratory stage. Continued observation of pupal development time and moth survival time revealed no significant differences between the F1 mutants and wild-types. Overall, the BmGDAP2-KO mutant delayed the growth and development of silkworm larvae during the fourth and fifth instars, thus increasing the lifespan of the silkworms.

[0041] In this embodiment of the invention, Cas9 is used to mediate the knockout of the target BmGDAP2 gene. As is known to those skilled in the art, any means that can knock out the BmGDAP2 gene can achieve the purpose of this invention, such as ZFNs, TALENs and CRISPR-Cas9 or their variants mediating the knockout of the target BmGDAP2 gene.

[0042] The above-described embodiments are merely preferred embodiments provided to fully illustrate the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are all within the scope of protection of the present invention. The scope of protection of the present invention is defined by the claims.

Claims

1. The application of knocking down the BmGDAP2 gene in silkworms to prolong the silkworm growth cycle, characterized by: The nucleic acid sequence of the silkworm BmGDAP2 gene is shown in SEQ ID NO.1; the knockdown of the silkworm BmGDAP2 gene was performed using Cas9-mediated knockout of the target BmGDAP2 gene; the sgRNA targeting the BmGDAP2 gene was constructed in an sgRNA expression vector: a double-stranded RNA formed by annealing SEQ ID NO.2 and SEQ ID NO.3 was inserted into the piggyBac-{3xp3-EGFP-sv40-TTTTT-gRNA-U6} vector. Obtained at the enzyme cleavage site.

2. A method for extending the growth cycle of silkworms, characterized in that: By knocking out the BmGDAP2 gene in silkworms and screening for mutant individuals, the growth cycle of silkworms was extended. The knockout of the BmGDAP2 gene was achieved using Cas9-mediated targeting of the BmGDAP2 gene's sgRNA. The sgRNA was obtained by annealing SEQ ID NO.2 and SEQ ID NO.

3.

3. The method according to claim 2, characterized in that: The sgRNA expression vector targeting the BmGDAP2 gene was transformed into silkworm eggs to obtain sgRNA transgenic silkworms. These silkworms were then hybridized with Cas9 transgenic silkworms to obtain the F1 generation, which produced silkworms with extended growth cycles.

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