CN117016497A - Method for regulating and controlling formation of melanin on silkworm epidermis by utilizing estrogen related receptor - Google Patents

Abstract

The invention belongs to the technical field of biology, and relates to a method for regulating and controlling formation of silkworm epidermis melanin by utilizing estrogen related receptors. On one hand, the expression of BmERR genes in silkworms is changed by genetic modification means based on a transgenic technology, so that the formation of epidermis patches is influenced, the efficiency is higher, the period is shorter, and the stable inheritance of the patches can be obtained in half a year; on the other hand, the invention has higher adaptability, can change the epidermis speckle by regulating and controlling the expression quantity of BmERR genes in any silkworm variety, and has no variety restriction.

Description

Method for regulating and controlling formation of melanin on silkworm epidermis by utilizing estrogen related receptor

Technical Field

The invention belongs to the technical field of biology, and relates to a method for regulating and controlling formation of silkworm epidermis melanin by utilizing estrogen related receptors.

Background

The diversity of the body color and the speckle of the insects is beneficial to the insects to avoid enemy, regulate the body temperature, seek partners, adapt to geography, resist ultraviolet rays and the like, is the result of biological evolution and natural selection, and has important biological significance. The silkworms are model insects of lepidoptera insects and have higher economic value. The varieties of silkworms are numerous, and the stripes on the surfaces of the silkworms among different varieties are different. Researchers have used genetic operations such as hybridization breeding to cultivate a zebra limited variety of silkworms (Yao Liusong, du Xin, chen Jine, etc. zebra full-limited quaternary hybridization multi-silk amount silkworms of new variety of agronomic 2 # breeding [ J ]. Silkworm industry science, 2019,45 (04): 594-597.DOI:10.13441/j.cnki.cykx.2019.04.018.; dagang, xiansheng, feng Guangjiang. Spring and autumn dual-purpose quaternary hybridization zebra full-limited silkworms of new variety of brocade @ @ silk-screen @ silkworm industry science, 2017,43 (06): 1039-1044.DOI:10.13441/j.cnki.cykx.2017.06.021, [ J ]; zhang Youhong, chinese golds, sho, wen Fu, etc. dual-limited variety of hollyx.2019.04.018, [ J ]. Silkworm industry science, 2015,41 (06): 1017-1347.13441/j.cnki @.2015); the female silkworms and the male silkworms of the zebra limiting variety can be separated into male and female silkworms according to different body surface zebra during the larval stage, so that the male and female silkworms or the male silkworms can be bred separately, and the method has the advantages of reducing the production cost of silkworm seeds, improving the quality of silkworm seeds, increasing the breeding coefficient of silkworm seeds, widening the application range of cocoon silkworms and the like.

In the traditional cross breeding, the zebra limited variety and the normal variety are hybridized for a plurality of times, so that the characters of the zebra limited variety are transferred into the normal variety, and a new zebra limited variety is obtained. It also has obvious disadvantages: (1) long period; the breeding of a new speckle variety takes about two years, and high manpower and material resources are consumed; (2) has variety restriction. At present, different main varieties exist in each silkworm area in China, for example, the 'two Guangdong No. 2' has wider coverage and the most popularization in the two Guangdong (Guangxi) silkworm areas; the southwest silkworm area is mainly used for pushing the silkworm variety Chuan shan XShushui. Hybrid breeding is difficult to genetically improve on these mainstream varieties.

The formation of silkworm patches is mainly controlled by genes. The melanin metabolic pathway is a main signal pathway affecting the generation of body surface patches of silkworms. For example, the yellow-y gene and the Tyrosine Hydroxylase (TH) gene are the main reasons for controlling the color of ch mutant larvae to appear chocolate color; the silkworm darkening type mutant (mln) is darkened in vivo due to the base deletion of the AA-NAT1 gene. This suggests that it is a theoretical basis to control the formation of epidermal patches by altering the expression of genes in silkworms.

Estrogen-related receptor (ERR) is an important nuclear receptor that plays an important regulatory role in the energy metabolism and growth and development of insects. In both mammals and invertebrates, it has not been previously reported that increasing BmERR expression in silkworms results in increased epidermis speckle.

Disclosure of Invention

Therefore, on one hand, the expression of BmERR genes in silkworms is changed by a genetic modification means based on a transgenic technology, so that the formation of epidermis patches is influenced, the efficiency is higher, and the period is shorter (the stable inheritance character of the patches can be obtained in half a year); on the other hand, the invention has higher adaptability, can change the epidermis speckle by regulating and controlling the expression quantity of BmERR genes in any silkworm variety, and has no variety restriction.

In order to achieve the above purpose, the present invention provides the following technical solutions:

the invention provides an application of a silkworm BmERR gene in regulating and controlling the formation of melanin on the skin of a silkworm. The invention changes the black spots and lines of the silkworm epidermis by regulating and controlling the expression quantity of BmERR genes in the silkworm body. The CDS of the silkworm BmERR gene is shown as SEQ ID NO. 1.

The invention also provides a method for regulating and controlling the formation of the melanin on the skin of the silkworm by utilizing the silkworm BmERR gene, which comprises the following steps:

1. cloning of silkworm BmERR gene:

PCR amplification is carried out by taking cDNA of the dazao fat body of the third day five-year silkworm variety as a template, and primers are designed, wherein the upstream primers are as follows: 5'-ATGATGTCCGCAGTCAGTGG-3', the downstream primer is: 5'-TTACCGCAGACAGGCCTC GA-3', the amplification conditions were 95℃for 5 minutes of pre-denaturation; denaturation at 95℃for 30 seconds, annealing at 55℃for 30 seconds, elongation at 72℃for 90 seconds, 30 cycles total; finally, the sequence shown in SEQ ID NO. 1 (BmERR CDS sequence) is obtained after extension at 72 ℃ for 10 minutes and preservation at 4 ℃.

2. Construction of recombinant vector containing silkworm BmERR Gene

A. TA cloning of silkworm BmERR gene CDS

TA cloning is carried out on the silkworm BmERR gene CDS with the sequence shown in SEQ ID NO. 1 and a vector pMD-19T simple to obtain a recombinant vector pMD-19T simple-BmERR;

B. construction of recombinant vector containing enhancer-systemic expression promoter-silkworm BmERR Gene CDS

Designing homologous recombination primers, wherein the upstream primers are as follows: 5'-AGGATTGGTGGATCCATGATGTCCGCAGTCAGTG G-3', the downstream primer is: 5'-AGTTGTAGCGGCCGCTTACCGCAGACAGGCCTCGA-3' and amplifying by using pMD-19T simple-BmERR as a template, wherein the amplification conditions are consistent with those in the silkworm BmERR gene cloning, and recovering the amplified BmERR gene CDS fragment. The pSL1180[ HR3-A4-DsRed-SV40] plasmid is digested by Not I and BamHI, pSL1180[ HR3-A4-SV40] vector fragment is recovered, and the recovered BmERR gene CDS and pSL1180[ HR3-A4-SV40] vector fragment are connected by utilizing a homologous recombination method to form pSL1180[ HR3-A4-BmERR-SV40] recombinant plasmid;

C. preparation of microinjection vector

Designing homologous recombination primers, wherein the upstream primers are as follows: 5'-TTATCGATACGCGTACGGCGCAGCGTCGTGAAAA GAGGCAATGAC-3', the downstream primer is: 5'-GAGATCGGCCGGCCTAGGCGTTCGTCAATGTATCAG TTTTGGTGC-3' PCR amplification is carried out by taking pSL1180[ HR3-A4-BmERR-SV40] recombinant plasmid as a template, and the amplification conditions are as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 95℃for 30 seconds, annealing at 56℃for 30 seconds, extension at 72℃for 210 seconds, 30 cycles total; finally, the fragment HR3-A4-BmERR-SV40 was obtained by extension at 72℃for 10 minutes. Cutting a pBac [3 XP 3-Red-SV40] basal vector by using restriction enzyme AscI, connecting the recovered HR3-A4-BmERR-SV40 fragment to the cut pBac [3 XP 3-Red-SV40] basal vector, and constructing a pBac [3 XP 3-Red-SV40, HR3-A4-BmERR-SV40] microinjection vector;

3. over-expression of BmERR gene in silkworm to promote formation of silkworm epidermis speckle

The pBac [3 XP 3-RsRed-SV40, HR3-A4-BmERR-SV40] plasmid and the helper plasmid were mixed in a molar ratio of 1:1 and injected into silkworm eggs.

After the newly hatched silkworms, raising the G0-generation silkworms by using conventional mulberry leaves. After the moth is transformed, carrying out copulation in the same circle to obtain G1 generation individuals; conventional silkworm eggs with G1 generation for hastening the hatching. After 6 days of hastening the blue, screening silkworm eggs with eyes emitting red fluorescence, namely, positive G1 generation transgenic over-expressed BmERR silkworm individuals; after the silkworm eggs come out of the ants, mulberry leaves are raised to single-moth-circle positive G1 generation transgenic silkworm individuals. After the moth is converted, the silkworm is further confirmed by fluorescent screening, and single moth in-loop mating is carried out, so that the G2 generation positive transgene over-expression BmERR silkworm individual is obtained.

The invention has the beneficial effects that:

the invention discloses a function of silkworm BmERR genes affecting formation of silkworm epidermis patches, which is a gene target for controlling the formation of silkworm epidermis patches.

The invention has higher adaptability, can change the epidermis speckle by regulating and controlling the expression quantity of BmERR genes in any silkworm variety, and has no variety restriction.

Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention. The objects and other advantages of the invention may be realized and obtained by means of the instrumentalities and combinations particularly pointed out in the specification.

Drawings

For the purpose of making the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in the following preferred detail with reference to the accompanying drawings, in which:

fig. 1: schematic diagram of transgenic over-expression BmERR vector and fluorescent observation result of transgenic over-expression BmERR silkworm individual. A, structural schematic diagram of a transgenic over-expression BmERR vector. 3×p3, an eye-specific expression promoter; red, red fluorescent protein; SV40, terminator; HR3, enhancer; a4, a systemic expression promoter; the arrow indicates the transcription initiation site and the transcription direction. B, the results of transgenic positive silkworm eggs and moths under fluorescent observation. B1/B2 is a photograph observed under white light, and B3/B4 is a photograph observed under fluorescence.

Fig. 2: detecting the expression quantity of BmERR in the epidermis of the transgenic over-expressed BmERR silkworm. (A) qRT-PCR detection of the expression of BmERR in the epidermis of transgenic overexpressed silkworm; (B) Detecting the expression of BmERR protein in the epidermis of the transgenic overexpressed silkworm by Western blot; L2D2: the second day of age two; L2M: sleep stage of second age; L3D2: the next day of three ages; L3M: sleep period of three ages; L4D1, L4D3: four days at first and third; L5D1, L5D3, L5D5: five days of age one, three and five; w0: a cluster loading period; WT: wild silkworm; OE: transgenic overexpressing BmERR silkworms;

fig. 3: and (5) observing the epidermis of the transgenic over-expressed BmERR silkworm. L3M: sleep period of three ages; L4D3: third day of four ages; L4M: sleep stage of four ages; L5D5: fifth day of fifth age; WT: wild silkworm; OE: transgenic overexpressing BmERR silkworms.

Detailed Description

Other advantages and effects of the present invention will become apparent to those skilled in the art from the following disclosure, which describes the embodiments of the present invention with reference to specific examples. The invention may be practiced or carried out in other embodiments that depart from the specific details, and the details of the present description may be modified or varied from the spirit and scope of the present invention.

Both the silkworm variety dazao and the silkworm strain D9L used in the invention are from the biological research center of the front intersection academy of sciences of the university of southwest.

EXAMPLE 1 cloning of silkworm BmERR Gene

Fat bodies of the third day larvae of the silkworm species dazao five years old were taken, total RNA of the tissues was extracted with TRIzol reagent (Invitrogen, carlsbad, calif.), and cDNA was synthesized with M-MLV reverse transcriptase (Promega, madison, wis.). Performing PCR amplification by using the fat body cDNA as a template and using an upstream primer and a downstream primer (the upstream primer is 5'-ATGATGTCCGCAGTCAGTGG-3' and the downstream primer is 5'-TTACCGCAGACAGGCCTCGA-3'), wherein the amplification condition is 95 ℃ for 5 minutes; denaturation at 95℃for 30 seconds, annealing at 55℃for 30 seconds, elongation at 72℃for 90 seconds, 35 cycles total; finally, the mixture is extended at 72 ℃ for 10 minutes and stored at 4 ℃. The sequence of the obtained PCR product is shown as SEQ ID NO. 1, namely the CDS sequence of the silkworm BmERR gene.

EXAMPLE 2 obtaining of recombinant vector containing silkworm BmERR Gene

The PCR product obtained in example 1 was TA cloned into pMD19-T simple vector (TAKARA, japan), pMD-19T simple-BmERR was obtained, and sequencing verified.

Designing homologous recombination primers, wherein the upstream primers are as follows: 5'-AGGATTGGTGGATCCATGATGTCCGCAGTCAGTG G-3', the downstream primer is: 5'-AGTTGTAGCGGCCGCTTACCGCAGACAGGCCTCGA-3' the amplification was carried out using the pMD19-T simple-BmERR vector as a template, which was confirmed to be correct by sequencing in example 1, under the same conditions as in example 1, and the amplified BmERR gene fragment was recovered. pSL1180[ HR3-A4-DsRed-SV40] vector fragment was recovered by double cleavage of pSL1180[ HR3-A4-DsRed-SV40] plasmid (stored and supplied by the laboratory of the inventors, deng, D., xu, H., wang, F.et al. The pro of Bmlp3 gene can direct fat body-specific expression in the transgenic silkworm, bombyx mori. Transgenic Res 22,1055-1063 (2013), wherein the sequence of HR3-A4 is shown in SEQ ID NO: 2). Connecting the recovered BmERR gene with pSL1180[ HR3-A4-SV40] vector fragment by utilizing a homologous recombination method to form pSL1180[ HR3-A4-BmERR-SV40] recombinant plasmid;

designing homologous recombination primers, wherein the upstream primers are as follows: 5'-TTATCGATACGCGTACGGCGCAGCGTCGTGAAAA AGGCAATGAC-3', the downstream primer is: 5'-GAGATCGGCCGGCCTAGGCGTTCGTCAATGTATCGTT GC-3' PCR amplification is carried out by taking pSL1180[ HR3-A4-BmERR-SV40] recombinant plasmid as a template, and the amplification conditions are as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 95℃for 30 seconds, annealing at 56℃for 30 seconds, extension at 72℃for 210 seconds, 30 cycles total; finally, the fragment HR3-A4-BmERR-SV40 was obtained by extension at 72℃for 10 minutes. The recovered HR3-A4-BmERR-SV40 fragment was ligated to the digested pBac [3 XP 3-Red-SV40] base vector using the restriction enzyme AscI (saved and supplied by the laboratory of the inventors, deng, D., xu, H., wang, F.et al. The promoter of Bmlp3 gene can direct fat body-specific expression in the transgenic silkworm, bombyx mori. Transgenic Res 22,1055-1063 (2013)), and the pBac [3 XP 3-Red-SV40, HR3-A4-BmERR-SV40] microinjection vector was constructed as shown in FIG. 1A.

EXAMPLE 3BmERR modulation of silkworm epidermis speckle

Mixing pBac [3×P3-Red-SV40, HR3-A4-BmERR-SV40] microinjection vector and auxiliary helper plasmid according to the mass ratio of 1:1, and then injecting the mixed plasmid into D9L silkworm eggs within 2h immediately after production by using a microinjection instrument; after the newly hatched silkworms, raising the G0-generation silkworms by using conventional mulberry leaves. After the moth is transformed, carrying out copulation in the same circle to obtain G1 generation individuals; conventional silkworm eggs with G1 generation for hastening the hatching. After 6 days of hastening the silkworm eggs are placed under a fluorescence microscope to observe the fluorescence of the silkworm eggs. Screening silkworm eggs emitting red fluorescence from eyes, namely positive G1 generation transgenic over-expression BmERR silkworm individuals (figure 1B); after the silkworm eggs come out of the ants, mulberry leaves are raised to single-moth-circle positive G1 generation transgenic silkworm individuals. After the moth is converted, the silkworm is further confirmed by fluorescent screening, and single moth in-loop mating is carried out, so that the G2 generation positive transgene over-expression BmERR silkworm individual is obtained.

The expression quantity of BmERR is detected through the transcription level and the protein level respectively, and the incremental expression condition of BmERR in the epidermis of the transgenic over-expressed BmERR silkworm is further confirmed. Specifically, the total RNA and protein of the epidermis of the BmERR silkworm are respectively extracted from the transgenic over-expressed in the second-age day, the second-age sleep period, the third-age sleep period, the fourth-age day, the fifth-age day and the upper cluster period.

The extracted total RNA is reversely transcribed into cDNA, and fluorescence quantitative PCR (qRT-PCR) detection is carried out, wherein the detection primer sequence is as follows: the BmERR gene is used for quantitatively detecting an upstream primer 5'-CGCCGACCTGTACGACC-3' and a downstream primer 5'-CACGCCCGACACCTGTAGAAA-3' by fluorescence; the upstream primer 5'-TTCGTACTGGCTTTCT-3' and the downstream primer 5'-CAAAGTTGATAGCAATTCCCT-3' of the internal reference gene BmTIF 4A. The detection conditions of the fluorescent quantitative PCR are as follows: pre-denaturation at 95 ℃ for 30 seconds; denaturation at 95℃for 5 seconds, annealing at 60℃for 30 seconds, extension at 95℃for 15 seconds, 40 cycles total.

The expression quantity of BmERR protein in the epidermis of the transgenic over-expressed BmERR silkworm is detected by Western blot. The extracted protein was mixed with 5 Xloading buffer, denatured at 100℃for 10 min, loaded with 10% SDS-polyacrylamide gel and Western blot analysis was performed. Antibodies used in Western blot were BmERR polyclonal antibodies (saved and supplied by the laboratory of the inventors, G.Shen, J.Wu, C.Han, H.Liu, Y.Xu, H.Zhang, Y.Lin and Q.Xia. Oestrogen-related receptor reduces vitellogenin expression by crosstalk with the ecdysone receptor pathway in female silkworm, bombyx mori. Instrument Mol Biol, 2018.). The test results showed that, compared with the control, the expression levels of BmERR gene (FIG. 2A) and BmERR protein (FIG. 2B) in the epidermis of the transgenic overexpressed silkworm were significantly higher than those of the wild-type silkworm during the larva period.

Comparing the change condition of the body color of the transgenic silkworm larva over-expressing BmERR, the phenomenon that the body surface stripes of the silkworm over-expressing BmERR are obviously different from those of the silkworm in the control group from the beginning of the period of the large silkworm (four ages and five ages), the colors of the eye stripes, the half moon stripes and the star stripes are obviously deepened, obvious black stripes are also generated in the internode film area of each body segment, and the full feeding period of the large silkworm is particularly obvious (figure 3). This suggests that overexpression of BmERR in the epidermis of silkworms by transgenic overexpression leads to an increase in the black spots of the epidermis.

Finally, it is noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications and equivalents may be made thereto without departing from the spirit and scope of the present invention, which is intended to be covered by the claims of the present invention.

Claims (8)

1. The application of the silkworm BmERR gene in regulating and controlling the formation of melanin on the silkworm epidermis is characterized in that the black spots on the silkworm epidermis are changed by regulating and controlling the expression level of the BmERR gene in the silkworm, and the CDS of the silkworm BmERR gene is shown as SEQ ID NO. 1.

2. A method for regulating and controlling the formation of melanin on the epidermis of a silkworm by utilizing a silkworm BmERR gene, which is characterized by comprising the following steps:

s1: cloning silkworm BmERR gene; the CDS of the silkworm BmERR gene is shown as SEQ ID NO. 1;

s2: constructing a recombinant vector containing a silkworm BmERR gene;

s3: injecting the recombinant vector plasmid into silkworm embryo, incubating, raising, mating and screening to obtain silkworm with black skin and color changed.

3. The method according to claim 2, wherein step S1 is specifically:

performing PCR amplification by using cDNA of silkworm fat body as a template by adopting an upstream primer and a downstream primer to obtain a sequence shown in SEQ ID NO. 1;

the upstream primer is as follows: 5'-ATGATGTCCGCAGTCAGTGG-3' the number of the individual pieces of the plastic,

the downstream primer is: 5'-TTACCGCAGACAGGCCTCGA-3'.

4. The method according to claim 2, wherein step S2 is specifically:

A. TA cloning of silkworm BmERR gene CDS

Performing TA cloning on the silkworm BmERR gene CDS shown in SEQ ID NO. 1 by adopting a TA cloning vector to obtain a TA cloning recombinant vector;

B. construction of recombinant vector containing enhancer-systemic expression promoter-silkworm BmERR Gene CDS

Amplifying by adopting a homologous recombination primer and taking the TA cloning recombination vector obtained in the step A as a template to obtain a BmERR gene CDS fragment;

the plasmid containing enhancer-systemic expression promoter and terminator is recovered by enzyme digestion, and is connected with the CDS fragment of the BmERR gene obtained by recovery by utilizing a homologous recombination method to form a recombinant vector containing enhancer-systemic expression promoter-silkworm BmERR gene CDS-terminator;

C. preparation of microinjection vector

Carrying out PCR amplification by using a homologous recombination primer and a recombination vector containing an enhancer-systemic expression promoter-silkworm BmERR gene CDS-terminator as a template, thereby obtaining an enhancer-systemic expression promoter-silkworm BmERR gene CDS-terminator fragment;

the vector containing the eye specific expression promoter-fluorescent protein-terminator is cut, the recovered vector fragment is connected with the enhancer-systemic expression promoter-silkworm BmERR gene CDS-terminator fragment by utilizing a homologous recombination method, and a microinjection vector containing the eye specific expression promoter-fluorescent protein-terminator and the enhancer-systemic expression promoter-silkworm BmERR gene CDS-terminator is constructed.

5. The method of claim 4, wherein the sequence of the enhancer-systemic expression promoter is set forth in SEQ ID NO. 2.

6. The method of claim 4, wherein the terminator is SV40, the fluorescent protein is red fluorescent protein, and the eye-specific expression promoter is 3 x p3.

7. The method according to claim 4, wherein step 3 is specifically:

microinjection of silkworm eggs after mixing microinjection vector with auxiliary helper plasmid;

after the newly hatched silkworms are hatched, feeding the mulberry leaves with G0 generation silkworms;

after the moth is transformed, carrying out copulation in the same circle to obtain G1 generation individuals;

accelerating the egg production of the G1 generation silkworm; after 6 days of hastening the hatching, screening silkworm eggs with fluorescence emitted from eyes, namely, positive G1 generation transgenic over-expressed BmERR silkworm individuals;

after the silkworm eggs come out of the ants, the mulberry leaves are raised to single moth circle positive G1 generation transgenic silkworm individuals;

after the moth is converted, the silkworm is confirmed by fluorescent screening, and the single moth is mated in a circle, so that the G2 generation positive transgene over-expression BmERR silkworm individual is obtained.

8. The method of claim 7, wherein the microinjection vector and the helper plasmid are mixed in a molar ratio of 1:1.