Bactrocera cucurbitae egg-entering protein LP and application thereof in gene editing

Abstract

The invention discloses bactrocera cucurbitae egg-entering protein LP and application thereof in gene editing. The amino acid sequence is shown in a sequence table SEQ ID NO: 1. The invention proves that the bactrocera cucurbitae egg-entering protein LP can be successfully absorbed by the ovary of bactrocera cucurbitae, and can guide a Cas9 / gRNA system to enter eggs and perform gene editing of offspring. The LP sequence and the LP-Cas9 fusion protein have very high application value in gene editing of bactrocera cucurbitae and related species.

Description

Technical Field

[0001] The invention belongs to the technical field of gene editing, and specifically relates to cucurbit fruit fly egg protein LP and its application in gene editing. Background Art

[0002] The CRISPR / Cas9 system is currently the most commonly used gene editing technology. The system includes clustered regulatory interspaced short palindromic repeats (CRISPR) and Cas9 nuclease (CRISPR associated system 9). Under the guidance of a specific RNA (single guide RNA, sgRNA), Cas9 can cut the target DNA by recognizing a specific sequence, and after the genomic DNA is broken, it will activate the cell's DNA repair mechanism; by utilizing the repair mechanism, the genome can be precisely edited, such as: conditional gene knockout, gene knock-in, gene replacement, point mutation, etc.

[0003] At present, the main method for introducing the CRISPR / Cas9 system into animals is fertilized egg microinjection: such as co-injecting the ribonucleoprotein complex formed by Cas9 and sgRNA into fertilized eggs or early embryos to obtain corresponding mutants, or transcribing the expression box of Cas9 and sgRNA in vitro to form the corresponding mRNA, and co-injecting it to achieve the purpose of editing the target gene. However, fertilized egg microinjection has great limitations. First of all, this injection technology requires expensive equipment and professional training, which limits the general application of this technology, especially for non-professional laboratories. During the injection process, it is easy to cause damage to the fertilized eggs or it is difficult to collect a large number of fertilized eggs for injection. In addition, it is difficult to control the timing of injection in the early stage of cleavage, especially for some ovoviviparous species, which produce larvae instead of eggs. This greatly limits the use of CRISPR-Cas9 technology in different systems. Due to the application limitations of fertilized egg microinjection to introduce CRISPR / Cas9 system, researchers have tried to develop other introduction methods.

[0004] In recent years, researchers have developed a new CRISPR / Cas9 introduction method, namely, a method of vertically transferring CRISPR / Cas9 for gene editing mediated by ovarian surface receptors, and first applied it in Aedes aegypti. The researchers took advantage of the fact that the egg development of Aedes mosquitoes requires receptor-mediated endocytosis to absorb yolk protein, screened a peptide sequence P2C from the yolk protein of Drosophila as a leader protein, and fused it with Cas9 for expression. The fusion protein can be recognized and actively absorbed by the receptors on the surface of the egg, so that it can successfully enter the egg and edit the genes in the nucleus, realizing the idea of ​​introducing Cas9 / sgRNA into the mother and editing the genes of its offspring, which is also called receptor-mediated ovarian transfer of cargo (ReMOT Control). After that, this ovarian transfer of germline gene mutation technology has been successfully applied to species such as whiteflies, red castanea, silkworms, and Ixodes scapularis. Compared with the classic fertilized egg injection method, the biggest advantage of this germline gene editing technology is that it gets rid of the dependence on microinjection, greatly reducing the difficulty of operation and technical labor costs. And because insects lay a large number of eggs, a single injection of the parent can edit a large number of offspring, improving the editing efficiency. Therefore, this germline gene editing method has great application potential in scientific research and molecular breeding.

[0005] The key to the successful implementation of this receptor-mediated ovarian delivery gene editing technology is to obtain the key sequence that guides the fusion protein into the egg. Due to the differences in egg surface receptors of different species, the identified and used leading protein sequences are different, and the ability of different leading proteins identified in the same species to guide entry into the egg also varies. The egg entry sequences identified in other species are not suitable for the ovarian delivery gene editing technology of the melon fruit fly. Therefore, the identification of protein sequences that can efficiently guide entry into the melon fruit fly eggs is of great significance for the gene editing of the melon fruit fly and the study of egg developmental biology. Summary of the invention

[0006] The purpose of the present invention is to provide the egg protein LP of Bactrocera melonensis and its application in gene editing.

[0007] The invention discloses a Bactrocera melonii egg protein LP, the amino acid sequence of which is shown in SEQ ID NO: 1 in the sequence listing.

[0008] The gene sequence of the egg protein LP of Bactrocera melonensis is shown in SEQ ID NO: 2 in the sequence listing.

[0009] Application of Bactrocera melonii egg protein LP in gene editing.

[0010] Preferably, the LP-Cas9 / sgRNA complex is injected into the eggs of the melon fruit fly, which are then hatched and continued to be raised to adulthood.

[0011] Beneficial effects of the invention: The invention first identified a leading protein sequence for Bactrocera cucurbita (named as leading protein, abbreviated as LP), and confirmed that the fusion protein produced by the sequence can be successfully absorbed by the ovary of Bactrocera cucurbita, and can guide the Cas9 / gRNA system into the egg and perform gene editing of the offspring. The LP sequence and LP-Cas9 fusion protein have high application value in gene editing of Bactrocera cucurbita and related species. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 To test the ability of LP to guide egg entry;

[0013] In the figure, Blank: untreated cucurbit fruit fly ovary; eGFP: cucurbit fruit fly ovary 24 hours after injection of eGFP protein; LP-eGFP: cucurbit fruit fly ovary 24 hours after injection of LP-eGFP protein; the upper half is the field of view under green fluorescence, and the lower half is the field of view under white light.

[0014] Figure 2 For the expression and purification of LP-Cas9 protein;

[0015] In the figure, M-protein molecular weight marker; Lane 1-total protein of Escherichia coli Rosseta strain before IPTG induction; Lane 2-total protein of Escherichia coli Rosseta strain after IPTG induction; Lane 3-purified LP-Cas9 protein; the arrow indicates the target protein LP-Cas9.

[0016] Figure 3 The changes in eye phenotype of Bactrocera melonensis after knocking out the eye pigment gene white;

[0017] In the figure, Control is the uninjected control, and sgRNA1 and sgRNA2 are gudie RNAs designed and synthesized for different sites of the white gene. DETAILED DESCRIPTION

[0018] In order to facilitate the understanding of the present invention, the present invention will be described more fully below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0019] Example 1 Vector Construction

[0020] The amino acid sequence of the egg protein LP of Bactrocera cucurbitae is shown in SEQ ID NO: 1 in the sequence listing; the gene sequence of the egg protein LP of Bactrocera cucurbitae is shown in SEQ ID NO: 2 in the sequence listing.

[0021] Construction of LP-Cas9 expression vector: The DNA sequence encoding LP protein and the connecting linker with restriction sites was obtained by chemical synthesis (Shanghai Sangon Biotechnology) as shown in the sequence table SEQ ID NO: 5. The fragment and the pET28a-Cas9-Cys vector were double-digested (Nde I and EcoR I), and the vector and fragment that completed the restriction digestion were detected and recovered. The recovered fragment was connected to the vector to construct an expression vector. The connection product of the above step was transformed into competent Escherichia coli DH5α, positive strains were screened, inoculated into LB medium containing kanamycin, and cultured at 37°C 180rpm for 10 hours, and then the expression plasmid was extracted with a kit and transformed into Escherichia coli Rosseta, and positive strains were screened.

[0022] The construction process of LP-eGFP expression vector is similar to the above method, except that the vector is replaced with pET30a-eGFP and the restriction sites are replaced with Bgl II and EcoR I.

[0023] Example 2 Protein Expression

[0024] The proteins expressed in large quantities in this embodiment are: secretory LP-eGFP protein and secretory LP-Cas9 protein. The positive strain was grown in LB medium at 37°C and 180rpm. According to the ratio of 1:100, 3ml of bacterial solution was transferred to 300ml of LB medium, and cultured at 37°C and 180rpm for 3h, and then IPTG inducer with a final concentration of 0.5mM was added to continue induction culture for 4h, followed by centrifugation at 4°C, 6000rpm, 10min, and the supernatant and precipitate were collected respectively. LP-eGFP protein is expressed in the supernatant and appears green, and the supernatant is retained and the precipitate is discarded. The LP-Cas9 protein is expressed in the precipitate, so the precipitate is suspended with 20ml 1×PBS, and 1% Tween is added to the precipitate in an ice bath with ultrasonic crushing, power 35%, 3s on 3s off, 45min, and then centrifuged at 4°C, 12000rpm, 10min, and the supernatant and precipitate are collected respectively. The collected precipitate was suspended and mixed with 20 ml of Buffer A (5 mM EDTA, 50 mM Tris-HCl; pH 8.0), and centrifuged at 4°C, 10,000 rpm for 20 min; the supernatant was discarded, and the precipitate was washed and mixed with 20 ml of Buffer B (5 mM EDTA, 50 mM Tris-HCl, 2 M urea; pH 8.0), and centrifuged at 4°C, 10,000 rpm for 20 min (Buffer B was repeated once); the supernatant was discarded, and 20 ml of denaturing solution (0.1 M Tris-HCl, 10 mM DTT, 8M urea; pH 8.0) to dissolve the precipitate, shake on a shaker at 37°C, 220rpm, oscillate for 1h; centrifuge at 4°C, 10000rpm for 10min, collect the supernatant and precipitate respectively, dialyze the supernatant (dialysis fluid is 50 times the volume of the supernatant), dialyze at 4°C for more than 16h, change the dialysate the next day and dialyze for another day, finally centrifuge at 4°C, 10000rpm, 10min, keep the supernatant containing the target protein and store it at -80°C until use. Figure 2 ).

[0025] Example 3 Female worm injection

[0026] Select female melon flies that have just emerged (less than 6 hours old) and inject LP-eGFP protein (1 μL, 0.5 μg) or an equal amount of control eGFP protein into the female insect's chest using a syringe and a microinjection needle. 24 hours after the injection, dissect the female flies and remove the ovaries. Rinse the ovaries repeatedly with PBS buffer and then press them into slices. Observe and take pictures under a fluorescence microscope ( Figure 1 ).

[0027] like Figure 1 As shown, 24 hours after the injection of LP-eGFP protein, the ovaries of the melon fly appeared green under fluorescence, proving that LP has the ability to guide into the eggs.

[0028] Example 4 Gene Editing

[0029] The purified LP-Cas9 protein was mixed with a guide RNA (gRNA) designed and synthesized for the eye pigment gene white of the melon fruit fly to form an RNP complex and injected into the fertilized eggs of the melon fruit fly. The primer sequence of the gRNA is shown in the sequence table SEQ ID NO: 3 or SEQ ID NO: 4. TM sgRNA was synthesized using the In Vitro Transcription and Screening System (Takara, Dalian) kit.

[0030] First, collect the melon fruit fly eggs. The egg collection method is as follows: slice the mature pumpkin and put it in a culture dish. Cover the mouth of the dish with plastic wrap and poke small holes in the plastic wrap to facilitate the melon fruit fly to lay eggs. Continue to observe the egg-laying situation and continue to collect newly laid eggs (within 30 minutes). Use an injection device to inject the LP-Cas9 / sgRNA complex (LP-Cas9 protein concentration 200ng / μl, sgRNA concentration 150ng / μl) into the eggs, and the injection volume is 0.05μl. The injected eggs are placed in an environment of 20°C and 60% relative humidity to wait for them to hatch and continue to be raised to adults. Observe the changes in its eye color to determine whether the gene editing is successful ( Figure 3 ).

[0031] like Figure 3 As shown, melon flies with mutated eye color were observed in the hatched eggs, confirming the gene editing activity of the LP-Cas9 protein.

[0032] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A melon fruit fly egg protein LP, characterized in that: Its amino acid sequence is shown in SEQ ID NO: 1 in the sequence listing.

2. The egg protein LP of melon fly according to claim 1, characterized in that: The gene sequence of the egg protein LP of Bactrocera melonensis is shown in SEQ ID NO: 2 in the sequence listing.

3. Application of Bactrocera melonii egg protein LP in gene editing.

4. The use of the egg protein LP of melon fly in gene editing according to claim 3, characterized in that: The LP-Cas9 / sgRNA complex was injected into the eggs of the melon fruit fly, which then hatched and continued to be raised to adulthood.

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