A method for regulating the formation of epidermal melanin in silkworms by estrogen-related receptors

Abstract

The present invention belongs to the field of biotechnology and relates to a method for regulating the formation of epidermal melanin in silkworms by using estrogen-related receptors. On the one hand, the present invention changes the expression of the BmERR gene in silkworms through genetic modification means based on transgenic technology, thereby affecting the formation of epidermal patterns. Its efficiency is higher and the cycle is shorter. Generally, stable inheritance traits of patterns can be obtained in half a year. On the other hand, the present invention has high adaptability and can change epidermal patterns by regulating the expression level of the BmERR gene in any silkworm variety without variety restrictions.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to a method for regulating the formation of silkworm cuticular melanin by estrogen-related receptors. Background Art

[0002] The diversity of insect body colors and patterns is beneficial for insects to avoid predators, regulate body temperature, seek mates, adapt to geography, and resist ultraviolet rays, etc. It is the result of biological evolution and natural selection and has important biological significance. The silkworm is a model insect of Lepidoptera and has high economic value. There are many silkworm varieties, and the patterns on the body surfaces of different varieties are not the same. Some researchers have used genetic operations such as cross-breeding to breed silkworms with limited pattern varieties (Yao Lusong, Du Xin, Chen Jin'e, etc. Breeding of a new multi-cocoon silk silkworm variety Nongke No. 2 with full limited pattern in a four-way cross [J]. Acta Sericologica Sinica, 2019, 45(04): 594-597. DOI: 10.13441 / j.cnki.cykx.2019.04.018.; Gong Dagang, Xian Yuerong, Feng Guangqiang. Breeding of a new spring-autumn dual-purpose four-way cross silkworm variety Jin·Yuan × Ling·Zhou with full limited pattern [J]. Acta Sericologica Sinica, 2017, 43(06): 1039-1044. DOI: 10.13441 / j.cnki.cykx.2017.06.021.; Zhang Youhong, Xiao Jinshu, Xiao Wenfu, etc. Breeding of a spring-autumn dual-purpose silkworm variety Shu 63 × Xian 16 with dual limited pattern [J]. Acta Sericologica Sinica, 2015, 41(06): 1017-1022. DOI: 10.13441 / j.cnki.cykx.2015.06.007.). Silkworms with limited pattern varieties can distinguish between males and females depending on the different patterns on their body surfaces during the larval stage, enabling separate rearing of male and female silkworms or exclusive rearing of male silkworms, thus having advantages such as reducing the production cost of silkworm eggs, improving the quality of silkworm eggs, increasing the breeding coefficient of silkworm eggs, and broadening the application range of cocoon silk.

[0003] Traditional cross-breeding is to cross a limited pattern variety with a normal variety multiple times to transfer the trait of limited pattern to the normal variety, thereby obtaining a new limited pattern variety. It also has obvious deficiencies: (1) Long cycle; it takes about two years to breed a new pattern variety, consuming a high amount of manpower and material resources; (2) Variety restriction. It is difficult to conduct genetic improvement on these mainstream varieties through cross-breeding.

[0004] The formation of silkworm patterns is mainly controlled by genes. The melanin metabolic pathway is the main signaling pathway affecting the generation of silkworm body surface patterns. For example, yellow-y genes and tyrosine hydroxylase ( tyrosine, TH ) genes are the main reasons for the chocolate-colored body color of ch mutant larvae; the dark mutant of silkworms ( mln) Due to AA-NAT1 a base deletion in the gene, it shows melanism. This indicates that there is a certain theoretical basis for controlling the formation of epidermal patterns by changing the gene expression in silkworms.

[0005] Estrogen-related receptor ( ， ERR), an important nuclear receptor, plays an important regulatory role in the energy metabolism and growth and development of insects. Neither in mammals nor in invertebrates has it been previously reported that increasing the BmERR expression in silkworms would lead to an increase in epidermal patterns. SUMMARY OF THE INVENTION

[0006] In view of this, on the one hand, the present invention changes the BmERR gene expression in silkworms through genetic modification means based on transgenic technology, thereby affecting the formation of epidermal patterns, with higher efficiency and shorter cycle (stable inheritance of patterns can generally be obtained in half a year); on the other hand, the present invention has high adaptability and can change epidermal patterns by regulating the BmERR gene expression level in any silkworm variety, without variety restrictions.

[0007] To achieve the above object, the present invention provides the following technical solutions:

[0008] The present invention provides the application of the silkworm BmERR gene in regulating the formation of epidermal melanin in silkworms. The present invention changes the black epidermal patterns of silkworms by regulating the BmERR gene expression level in silkworms. The CDS of the silkworm BmERR gene is shown as SEQ IDNO:1.

[0009] The present invention also provides a method for regulating the formation of epidermal melanin in silkworms using the silkworm BmERR gene, comprising the following steps:

[0010] 1. Cloning of the silkworm BmERR gene:

[0011] Using the cDNA of the fat body of the fifth instar third day silkworm variety dazao as a template for PCR amplification, designing primers, the upstream primer is: 5’-ATGATGTCCGCAGTCAGTGG-3’, the downstream primer is: 5’-TTACCGCAGACAGGCCTC GA-3’, the amplification conditions are pre-denaturation at 95°C for 5 minutes; denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, extension at 72°C for 90 seconds, a total of 30 cycles; finally extension at 72°C for 10 minutes and preservation at 4°C to obtain the sequence shown as SEQ ID NO:1 ( BmERR CDS sequence).

[0012] 2. Construction of silkworm-containing BmERR Recombinant gene vector

[0013] A. Silkworm BmERR TA cloning of gene CDS

[0014] The silkworm Bombyx mori sequence shown in SEQ ID NO: 1 BmERR The gene CDS was TA cloned with the vector pMD-19T simple to obtain the recombinant vector pMD-19T simple-BmERR;

[0015] B. Containing enhancer-systemic expression promoter-Bombyx mori BmERR Construction of recombinant vector of gene CDS

[0016] Homologous recombination primers were designed, the upstream primer was: 5'-AGGATTGGTGGATCCATGATGTCCGCAGTCAGTG G-3', the downstream primer was: 5'-AGTTGTAGCGGCCGCTTACCGCAGACAGGCCTCGA-3', and pMD-19T simple-BmERR was used as a template for amplification. The amplification conditions were the same as those of "Bombyx mori" BmERR The amplification conditions in "Gene Cloning" are consistent, and the amplified BmERR Gene CDS fragment. Not I and BamH I Double enzyme digestion of pSL1180[HR3-A4-DsRed-SV40] plasmid, recovery of pSL1180[HR3-A4-SV40] vector fragment, and the recovered BmERR The gene CDS was ligated with the pSL1180[HR3-A4-SV40] vector fragment using homologous recombination to construct the pSL1180[HR3-A4-BmERR-SV40] recombinant plasmid;

[0017] C. Preparation of microinjection vectors

[0018] Design homologous recombination primers. The upstream primer is: 5’-TTATCGATACGCGTACGGCGCAGCGTCGTGAAAAGAGGCAATGAC-3’, and the downstream primer is: 5’-GAGATCGGCCGGCCTAGGCGTTCGTCAATGTATCAG TTTTGGTGC-3’. Using the recombinant plasmid pSL1180[HR3-A4-BmERR-SV40] as a template for PCR amplification, the amplification conditions are: pre-denaturation at 95°C for 5 minutes; denaturation at 95°C for 30 seconds, annealing at 56°C for 30 seconds, extension at 72°C for 210 seconds, for a total of 30 cycles; finally, extension at 72°C for 10 minutes to obtain the HR3-A4-BmERR-SV40 fragment. Using the restriction endonuclease AscI Digest the pBac[3×P3-Red-SV40] basic vector with the enzyme, and ligate the recovered HR3-A4-BmERR-SV40 fragment to the digested pBac[3×P3-Red-SV40] basic vector to construct the pBac[3×P3-Red-SV40, HR3-A4-BmERR-SV40] microinjection vector;

[0019] 3. Overexpression in silkworms BmERR The gene promotes the formation of epidermal markings in silkworms

[0020] Mix the plasmid pBac[3×P3-RsRed-SV40, HR3-A4-BmERR-SV40] with the helper plasmid at a molar ratio of 1:1 and microinject silkworm eggs.

[0021] After the newly hatched silkworm larvae emerge, raise the G0 generation of silkworms with conventional mulberry leaves. After they turn into moths, perform in-cage mating to obtain G1 generation individuals; conventionally incubate the G1 generation of silkworm eggs. After 6 days of incubation, screen the silkworm eggs that emit red fluorescence in the eyes, which are the positive G1 generation transgenic overexpressing BmERR Silkworm individuals; after the silkworm eggs hatch into larvae, raise the positive G1 generation transgenic silkworm individuals in a single-moth cage with mulberry leaves. After they turn into moths, further confirm by fluorescence screening and perform in-cage mating within a single-moth cage to obtain the G2 generation positive transgenic overexpressing BmERR Silkworm individuals.

[0022] The beneficial effects of the present invention are as follows:

[0023] The present invention discloses the function of the silkworm BmERR gene affecting the formation of epidermal markings in silkworms, which is a gene target controlling the formation of epidermal markings in silkworms.

[0024] The present invention has high adaptability and can change the epidermal markings by regulating the expression level of the BmERR gene in any silkworm variety, without variety restrictions.

[0025] Other advantages, objects and features of the present invention will be set forth in part in the following description, and in part will be obvious to those skilled in the art upon examination of the following, or may be learned by practice of the present invention. The objects and other advantages of the present invention may be realized and obtained by the following description. Brief Description of the Drawings

[0026] In order to make the objects, technical solutions and advantages of the present invention clearer, the present invention will be described in detail preferably with reference to the accompanying drawings, where:

[0027] Figure 1 : Transgenic overexpression BmERR Schematic diagram of the vector and transgenic overexpression BmERR Results of fluorescence observation of individual silkworms. A, Transgenic overexpression BmERR Schematic diagram of the structure of the vector. 3×p3, eye-specific expression promoter; Red, red fluorescent protein; SV40, terminator; HR3, enhancer; A4, systemic expression promoter; The arrow indicates the transcription start site and the transcription direction. B, Results of fluorescence observation of transgenic positive silkworm eggs and moths. B1 / B2 are photos observed under white light, and B3 / B4 are photos observed under fluorescence.

[0028] Figure 2 : Transgenic overexpression BmERR In the silkworm epidermis BmERR Expression level detection. (A) qRT-PCR was used to detect the expression of BmERR in the epidermis of transgenic overexpression silkworms; (B) Western blot was used to detect the expression of BmERR protein in the epidermis of transgenic overexpression silkworms; L2D2: the second day of the second instar; L2M: the second instar molting stage; L3D2: the second day of the third instar; L3M: the third instar molting stage; L4D1, L4D3: the first and third days of the fourth instar; L5D1, L5D3, L5D5: the first, third and fifth days of the fifth instar; W0: the spinning stage; WT: wild-type silkworms; OE: transgenic overexpression BmERR silkworms;

[0029] Figure 3 : Transgenic overexpression BmERR Observation of the silkworm epidermis. L3M: the third instar molting stage; L4D3: the third day of the fourth instar; L4M: the fourth instar molting stage; L5D5: the fifth day of the fifth instar; WT: wild-type silkworms; OE: transgenic overexpression BmERR silkworms. Detailed Description of the Preferred Embodiments

[0030] The following uses specific specific examples to illustrate the implementation manners of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific implementation manners, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0031] The silkworm varieties used in the present invention dazao and the silkworm strain D9L are both from the Center for Biological Research of the School of Frontier and Interdisciplinary Sciences, Southwest University.

[0032] Example 1 Cloning of silkworm BmERR gene

[0033] Take the fat body of the third instar larvae of the fifth instar of the silkworm variety dazao Extract the total RNA of the tissue with TRIzol reagent (Invitrogen, Carlsbad, CA), and synthesize cDNA with M-MLV reverse transcriptase (Promega, Madison, WI). Using the fat body cDNA as a template, use upstream and downstream primers (upstream primer: 5’-ATGATGTCCGCAGTCAGTGG-3’, downstream primer: 5’-TTACCGCAGACAGGCCTCGA-3’) for PCR amplification. The amplification conditions are pre-denaturation at 95°C for 5 minutes; denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, extension at 72°C for 90 seconds, for a total of 35 cycles; finally, extension at 72°C for 10 minutes and preservation at 4°C. The sequence of the obtained PCR product is shown in SEQ ID NO:1, that is, the CDS sequence of the silkworm BmERR gene.

[0034] Example 2 Obtaining a recombinant vector containing the silkworm BmERR gene

[0035] Clone the PCR product obtained in Example 1 into the pMD19-T simple vector (TAKARA, Japan) through TA cloning to obtain pMD-19T simple-BmERR, and perform sequencing verification.

[0036] Design homologous recombination primers. The upstream primer is: 5’-AGGATTGGTGGATCCATGATGTCCGCAGTCAGTG G-3’, and the downstream primer is: 5’-AGTTGTAGCGGCCGCTTACCGCAGACAGGCCTCGA-3’. Use the pMD19-T simple-BmERR vector with correct sequencing verification in Example 1 as a template for amplification. The amplification conditions are the same as those in Example 1, and recover the amplified BmERR gene fragment. Use Not Iand BamH I Digest the plasmid pSL1180[HR3-A4-DsRed-SV40] (preserved and provided by the laboratory where the inventors are located, Deng, D., Xu, H., Wang, F. et al. The promoter of Bmlp3 genecan direct fat body-specific expression in the transgenic silkworm, Bombyx mori . Transgenic Res 22, 1055–1063 (2013). The sequence of HR3-A4 is shown in SEQ ID NO:2), and recover the vector fragment of pSL1180[HR3-A4-SV40]. The BmERR gene and the vector fragment of pSL1180[HR3-A4-SV40] are ligated by homologous recombination to construct the recombinant plasmid pSL1180[HR3-A4-BmERR-SV40];

[0037] Design homologous recombination primers. The upstream primer is: 5’-TTATCGATACGCGTACGGCGCAGCGTCGTGAAAAAGGCAATGAC-3’, and the downstream primer is: 5’-GAGATCGGCCGGCCTAGGCGTTCGTCAATGTATCGTT GC-3’. Use the recombinant plasmid pSL1180[HR3-A4-BmERR-SV40] as a template for PCR amplification. The amplification conditions are: pre-denaturation at 95°C for 5 minutes; denaturation at 95°C for 30 seconds, annealing at 56°C for 30 seconds, extension at 72°C for 210 seconds, for a total of 30 cycles; finally, extension at 72°C for 10 minutes to obtain the HR3-A4-BmERR-SV40 fragment. Use restriction endonuclease AscI to digest pBac[3×P3-Red-SV40] (preserved and provided by the laboratory where the inventors are located, Deng, D., Xu, H., Wang, F. et al. The promoterof Bmlp3 gene can direct fat body-specific expression in the transgenicsilkworm, Bombyx mori . Transgenic Res22, 1055–1063 (2013).) The basic vector, the recovered HR3-A4-BmERR-SV40 fragment was ligated to the digested pBac[3×P3-Red-SV40] basic vector to construct the pBac[3×P3-Red-SV40, HR3-A4-BmERR-SV40] microinjection vector, as Figure 1 shown in A.

[0038] Example 3 BmERR Regulation of the epidermal markings of silkworms

[0039] The pBac[3×P3-Red-SV40, HR3-A4-BmERR-SV40] microinjection vector and the helper plasmid were mixed at a molar ratio of 1:1, and then the mixed plasmid was injected into the D9L silkworm eggs within 2 h after they were laid using a microinjector; after the newly hatched silkworms emerged, the G0 generation of silkworms was reared with conventional mulberry leaves. After they pupated, they were mated within the same circle to obtain G1 generation individuals; the G1 generation of silkworm eggs was conventionally incubated. After 6 days of incubation, the silkworm eggs were observed under a fluorescence microscope for the fluorescence emission situation. The silkworm eggs emitting red fluorescence in the eyes were screened, which were the positive G1 generation transgenic overexpression [[ID= silkworm individuals ( ​ B); after the silkworm eggs hatched, the positive G1 generation transgenic silkworm individuals in a single-moth circle were reared with mulberry leaves. After they pupated, they were further confirmed by fluorescence screening and mated within the single-moth circle to obtain G2 generation positive transgenic overexpression ​ silkworm individuals.

[0040] The expression levels were detected at the transcriptional level and protein level respectively for ​ to further confirm the overexpression of the transgene ​ in the epidermis of silkworms ​ incremental expression. Specifically, the total RNA and proteins of the epidermis of the transgenic overexpression ​ silkworms were extracted on the second day of the second instar, during the second instar dormancy, the second day of the third instar, during the third instar dormancy, the first day of the fourth instar, the third day of the fourth instar, the first day of the fifth instar, the third day of the fifth instar, the fifth day of the fifth instar, and the spinning stage.

[0041] The extracted total RNA was reverse transcribed into cDNA and detected by fluorescence quantitative PCR (qRT-PCR). The primer sequences for detection were as follows: ​ The upstream primer for gene fluorescence quantitative detection was 5’-CGCCGACCTGTACGACC-3’, and the downstream primer was 5’-CACGCCCGACACCTGTAGAAA-3’; the internal reference gene ​Forward primer 5’-TTCGTACTGGCTTTCT -3’, reverse primer 5’-CAAAGTTGATAGCAATTCCCT-3’. The detection conditions for fluorescence quantitative PCR were: pre-denaturation at 95°C for 30 s; denaturation at 95°C for 5 s, annealing at 60°C for 30 s, extension at 95°C for 15 s, for a total of 40 cycles.

[0042] The overexpression of the transgene was detected by Western blot ​ in the epidermis of silkworms ​ for the expression level of the protein. The extracted protein was mixed evenly with 5× loading buffer and denatured at 100°C for 10 min, then loaded onto a 10% SDS-polyacrylamide gel for Western blot analysis. The antibody used for Western blot was ​ a polyclonal antibody (preserved and provided by the laboratory where the inventors are located, G. Shen, J. Wu, C. Han, H. Liu, Y. Xu, H. Zhang, Y. Lin and Q.Xia. Oestrogen-related receptor reduces vitellogenin expression by crosstalkwith the ecdysone receptor pathway in female silkworm, Bombyx mori. InsectMol Biol, 2018.). The detection results showed that, compared with the control, during the larval stage of silkworms, in the epidermis of transgenic overexpressing silkworms ​ gene ( ​ A) and the expression level of BmERR protein ( ​ B) were significantly higher than those of the wild type.

[0043] Comparing the changes in the body color of transgenic silkworm larvae with overexpression ​ , it was found that starting from the late larval stage (the fourth and fifth instars), compared with the control group of silkworms, in the overexpressing ​ silkworms, obvious differences began to occur in the body surface markings. The colors of the ocellar spots, semilunar spots and star spots became significantly darker, and obvious black markings also appeared in the intersegmental membrane regions of each body segment, especially obvious during the peak feeding period of the late larvae ( ​ ). This indicates that the high-level expression of ​ in the epidermis of transgenic overexpressing silkworms will lead to an increase in the black markings on the epidermis.

[0044] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the purpose and scope of the present technical solution, and they should all be covered within the scope of the claims of the present invention.

Claims

1. A method using silkworm BmERR A method for genetically regulating the formation of melanin in the silkworm epidermis, characterized in that: The following steps are involved: S1: Cloning of silkworms BmERR Gene; The silkworm BmERR The gene CDS is shown in SEQ ID NO: 1; S2: Constructing silkworm-containing BmERR Recombinant vectors of genes; Including the preparation of microinjection vectors: Homologous recombination primers were used to construct a gene containing enhancer-systemic expression promoter-Bombyx mori BmERR The gene CDS-terminator recombinant vector was used as a template for PCR amplification to obtain the enhancer-systemic expression promoter-silkworm BmERR gene CDS-terminator fragment; The vector containing eye-specific expression promoter-fluorescent protein-terminator was digested and the recovered vector fragment was combined with enhancer-systemic expression promoter-silkworm BmERR The gene CDS-terminator fragments were connected by homologous recombination to construct an eye-specific expression promoter-fluorescent protein-terminator and enhancer-systemic expression promoter-silkworm BmERR gene CDS-terminator microinjection vector; S3: Injecting the recombinant vector plasmid into silkworm embryos, specifically, mixing the microinjection vector with the helper plasmid and then microinjecting it into silkworm eggs; Hatching, raising, mating, and screening to obtain silkworms with black stripes on their skin; The colors of the eye-shaped patterns, half-moon patterns and star-shaped patterns of the obtained silkworms became darker, and black spots appeared in the intersegmental membrane area of each segment.

2. The method according to claim 1, characterized in that Step S1 is specifically as follows: PCR amplification was performed using upstream and downstream primers with cDNA of the silkworm fat body as a template to obtain the sequence shown in SEQ ID NO: 1; The upstream primer is: 5'-ATGATGTCCGCAGTCAGTGG-3', The downstream primer is: 5'-TTACCGCAGACAGGCCTCGA-3'.

3. The method according to claim 1, characterized in that Step S2 is specifically as follows: A. Silkworm BmERR TA cloning of gene CDS The silkworm Bombyx mori shown in SEQ ID NO: 1 was cloned using the TA cloning vector. BmERR Gene CDS was TA cloned to obtain TA cloning recombinant vector; B. Containing enhancer-systemic expression promoter-Bombyx mori BmERR Construction of gene CDS-terminator recombinant vector Homologous recombination primers were used to amplify the TA clone recombinant vector obtained in step A as a template to obtain BmERR Gene CDS fragment; The plasmid containing enhancer-systemic expression promoter and terminator was recovered by enzyme digestion and then compared with the recovered BmERR Gene CDS fragments were connected by homologous recombination to form a construct containing enhancer-systemic expression promoter-Bombyx mori BmERR Gene CDS-terminator recombinant vector; C. Preparation of microinjection vectors.

4. The method according to claim 3, characterized in that The sequence of the enhancer-systemic expression promoter is shown in SEQ ID NO:

2.

5. The method according to claim 3, characterized in that The terminator is SV40, the fluorescent protein is red fluorescent protein, and the eye-specific expression promoter is 3×p3.

6. The method according to claim 3, characterized in that Step 3 is as follows: The microinjection vector is mixed with the helper plasmid and then microinjected into silkworm eggs; After the larvae hatch, the G0 generation of silkworms are raised on mulberry leaves; After the moths emerge, they mate in the same circle to obtain G1 generation individuals; Accelerate G1 silkworm eggs; after 6 days of incubation, screen for eggs with fluorescent eyes, which are positive G1 transgenic overexpression eggs. BmERR Silkworm individuals; After silkworm eggs hatched, single moth circle positive G1 generation transgenic silkworm individuals were raised on mulberry leaves; After the moths have emerged, they are confirmed by fluorescence screening and mating is performed in a single moth circle to obtain G2 generation positive transgene overexpression. BmERR Silkworm individuals.

7. The method according to claim 6, characterized in that The microinjection vector and helper plasmid were mixed at a 1:1 molar ratio.

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