CN113621650A - Establishment and application of efficient silk fibroin heavy chain promoter secretion expression system - Google Patents

Abstract

The invention relates to establishment and application of a high-efficiency silk fibroin heavy chain secretory recombinant expression system, wherein functional elements for enhancing gene expression, such as an enhancer hr3, an untranslated region sequence 3' UTR and the like, are utilized to optimize the efficiency of a silk fibroin heavy chain promoter (Fib-H promoter) of a silkworm, and on the basis, the high-efficiency and stable secretory silk fibroin heavy chain (Fib-H) recombinant expression system is integrated and established to realize the high-efficiency secretory recombinant expression of foreign protein in silk glands at the back of transgenic silkworms. The invention establishes an efficient silk fibroin heavy chain secretion expression system, realizes the secretion of foreign proteins in silk with the natural size and the large molecular weight and has activity, and has more prospect in application compared with the prior silk fibroin expression system.

Description

Establishment and application of efficient silk fibroin heavy chain promoter secretion expression system

Technical Field

The invention is applied to the technical fields of biotechnology and genetic engineering, and particularly relates to establishment and application of a high-efficiency silk fibroin heavy chain promoter secretion expression system.

Background

With the rapid development of bioengineering and genetic engineering in the 21 st century, people have increasingly increased demands for functional proteins for various purposes such as medical use, medicine, food, beauty, health care and the like, and the rapidly increasing market demands cannot be met by virtue of protein extraction and production from natural sources. Establishing and perfecting various high-efficiency prokaryotic and eukaryotic expression systems, and utilizing bacterial strains, cells, insects and the like as host bioreactors, is an effective and sustainable method for realizing low-cost large-scale production of recombinant foreign proteins with biological activity, and becomes a hotspot of current world research. The method for producing the foreign protein by using the Chinese hamster ovary cells as the bioreactor is the most standard expression mode at present, but the operation cost and the requirement on the environment are extremely strict, and the large-scale popularization and application of the foreign protein are severely limited. In order to establish a low-cost, large-scale, safe, sustainable and efficient bio-factory for producing foreign proteins, since 2000, researchers have tried to produce recombinant foreign proteins using organs of transgenic organisms such as mammals, birds, insects and plants as bioreactors.

Silkworm is known as serials and is one of the first economic animals (insects) completely domesticated and utilized by human beings. The silk gland is the only organ for synthesizing and secreting fibroin, and is the biological basis of the whole silk industry. After thousands of years of artificial domestication, the silk gland of the silkworm has super strong protein synthesis and secretion capacity, and the silkworm with the weight of about 5g can synthesize and secrete about 0.5g of fibroin, which is the best of the known insects at present. Fibroin is mainly composed of silk fibroin (fibrin) and sericin (sericin) coated on the outer layer thereof: the silk fibroin is the main body of silk, accounts for about 75 percent, is synthesized by the posterior silk gland of the silkworm, comprises three main components of fib-H chain, fib-L chain and P25, and is insoluble in water; the balance is Sericin, accounting for about 25 percent, is synthesized by the middle silk gland of the silkworm and comprises three main components of Sericin 1(Sericin1), Sericin 2(Sericin2) and Sericin 3(Sericin3), wherein the Sericin1 has the highest protein content and is soluble in water. With the development of modern molecular biology and transgenic technology, the characteristics of high-efficiency synthesis and silk protein secretion of silkworm spun silk glands and the protein post-translational modification processing capacity of glycosylation, methylation and the like which are extremely important for maintaining the activity of foreign proteins are achieved, the feeding cost is low, the silkworm silk gland protein can be produced in an industrial manner, and the silkworm silk gland protein is safe to people and livestock, so that the silkworm silk gland protein becomes an ideal bioreactor model and is concerned by researchers in various countries and competitively developed and utilized.

In 2000, rural et al used piggyBac transposon mediated microinjection of silkworm eggs and obtained stably inherited transgenic silkworms; in 2003, Xiuqingyou et al completed the silkworm genome project, and important coding genes involved in silk protein synthesis in silkworm silk glands, such as promoter regulatory elements of fibroin heavy chain (FibH chain) gene, fibroin light chain (FibL chain) gene, Sericin 1(Sericin1) gene, Sericin 2(Sericin2) gene, Sericin 3(Sericin3) gene, P25 gene and the like, were identified and cloned, and these basic research results made it possible to produce recombinant foreign proteins in silk glands on a large scale by using a transgenic silkworm tissue specific expression system. In recent years, a plurality of foreign proteins are tried to be expressed in silk glands at home and abroad by utilizing a piggyBac transposon mediated transgenic technology and a silkworm tissue specific promoter element, and the foreign proteins comprise: EGFP fused with fibroin heavy chain (Zhao et al 2010), feline interferon (Kurihara et al 2007), enhanced green fluorescent protein (Kojima et al 2007), spider silk traction protein (Zhu et al 2010), a partial peptide segment of human type III collagen fused with fibroin light chain (Tomita et al 2003), enhanced green fluorescent protein (Tomita et al 2003), hydroxyproline collagen partial peptide segment (Adachi et al 2006), fibroblast growth factor (Hino et al 2006), partial collagen peptide segment (Yanagisawa et al 2007), red fluorescent protein fused with P25 (Royer et al 2005) and the like are expressed in posterior silk gland; human serum albumin (Ogawa et al 2007), enhanced green fluorescent protein (Tomita et al 2007), murine monoclonal antibody (Iizuka et al 2009), human collagen alpha chain gene (Adachi et al 2010), soluble GM-Csf receptor alpha (Urano et al 2010), and the like are expressed in the middle silk gland, but in general, the industrialization of the above-described research results is considerably difficult, mainly because such fibroin heavy chain expression systems require a single regulatory element and fusion of exogenous proteins with endogenous silk proteins to achieve secretory expression, adversely affect the expression level and biological activity of exogenous proteins, and researchers try over a protein renaturation strategy, but the process is complicated and has little effect, and thus greatly restrict the application of the fibroin heavy chain expression system, so that it is necessary to modify the existing fibroin heavy chain expression system, so as to improve the expression efficiency and the biological activity of the foreign protein and promote the application of the silkworm silk gland bioreactor technical system in the fields of biological pharmacy and functional silk genetic improvement.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides a high-efficiency silk fibroin heavy chain promoter secretory expression system, and solves the problems that the prior silk fibroin heavy chain expression system has a single regulation and control element, and the secretory expression can be realized only by fusing exogenous protein with endogenous silk protein, so that the expression quantity and the biological activity of the exogenous protein are adversely affected.

The technical problem to be solved by the invention is realized by adopting the following technical scheme:

a construction method of a high-efficiency fibroin heavy chain pBac vector is characterized in that the pBac vector comprises a pBac-FRF vector, a pBac-HFRF vector, a pBac-HFRS vector and a pBac-HFlRS vector; the pBac-HFRF vector, the pBac-HFRS vector and the pBac-HFlRS vector are insertion type vectors formed by the pBac-FRF vector; the construction of the pBac vector comprises the following steps:

1) carrying out PCR amplification by adopting a specific primer and taking a phSRSV vector as a template to obtain red fluorescent protein DsRed1, an hr3 CQ enhancer and a Ser1pA terminator, respectively cloning the obtained fragments into a pMD19T simple vector to respectively obtain a pDsRed1 vector, a phr3 vector and a pSer1pA vector;

2) carrying out PCR amplification by adopting specific primers and silkworm genome DNA as a template to obtain F (1967), cloning the obtained F (1967) fragment into a pMD19T simple vector to obtain a pF1967 vector, wherein the sequence of the F (1967) is shown as SEQ ID No. 1;

performing PCR amplification by using a specific primer and a FibHpolyA (FibHpA) terminator as a template, and cloning the obtained DNA fragment into a pMD19T simple vector to obtain a pFibHpA vector;

3) connecting a DsRed1 fragment obtained by enzyme digestion of a pDsRed1 vector by using BamH I and Not I to a pF1967 vector obtained by enzyme digestion of the BamH I and Not I to obtain a pFR vector;

connecting a FibHpA fragment obtained by enzyme digestion of a p-FibHpA vector by Not I and Kpn I to an pFR vector obtained by enzyme digestion of Not I and Kpn I to obtain a pFRF vector, wherein the sequence of the pFRF is shown as SEQ ID No. 2;

4) the pFRF vector is digested by AscI to obtain an FRF fragment, and the FRF fragment is inserted into a pBac [3xP3DsRedaf ] transgenic vector to obtain a pBac-FRF vector.

Further, the construction method of the efficient fibroin heavy chain pBac-HFRF vector comprises the following steps: hr3 was inserted into pFRF vector to obtain pHFRF vector whose sequence is shown in SEQ ID No.2, pHFRF vector was digested with AscI to obtain HFRF fragment, which was inserted into pBac [3xP3DsRedaf ] transgenic vector to generate pBac-HFRF.

Further, a construction method of a high-efficiency fibroin heavy chain pBac-HFRS vector comprises the following steps: replacing the FibHpA in the pHFRF vector with Ser1pA to generate pHFRS; the sequence of pHFRS is shown in SEQ ID No.2, the pHFRS vector is cut by AscI enzyme to obtain an HFRS fragment, and the HFRS fragment is inserted into a pBac [3xP3DsRedaf ] transgenic vector to generate pBac-HFRS.

Further, a construction method of a high-efficiency fibroin heavy chain pBac-HFIRS vector comprises the following steps: (ii) replacing Exon2 NTD 2AA with Exon2(GenBank accession No. AF226688, nt.63451-63846) in pHFRS to generate vector pHFlRS; the pHFIRS vector is digested by AscI to obtain an HFIRF fragment, and the fragment is inserted into a pBac [3xP3DsRedaf ] transgenic vector to generate pBac-HFIRS.

An application of the efficient fibroin heavy chain pBac vector in preparation of an efficient fibroin heavy chain promoter secretion expression system.

A high-efficiency silk fibroin heavy chain promoter secretion expression system is characterized in that the preparation method comprises the following steps:

1) injecting the pBac vector and the helper plasmid pHA3PIG into G0 generation eggs at a mass ratio of 1:1, feeding to obtain G0 moth, and mating to obtain G1 generation;

2) and screening positive individuals of eyes or nerves of G1 generation eggs by using a fluorescence microscope to obtain transgenic silkworms, feeding and passaging the obtained transgenic silkworms, and reserving the positive transgenic individuals of pBac-FRF vector, pBac-HFRF vector and pBac-HFRS vector single copy as FRF strains, HFRF strains and HFRS strains respectively.

Further, the insertion sites of the pBac-FRF vector, the pBac-HFRF vector and the pBac-HFRS vector in the pBac vector on the chromosome are chromosome 1, chromosome 25 and chromosome 20, respectively.

An application of the high-efficiency fibroin heavy chain promoter secretion expression system in synthesizing and secreting mature recombinant protein in a silkworm bioreactor.

Compared with the prior art, the invention has the beneficial effects that:

1) an efficient fibroin heavy chain secretion expression system is established, exogenous protein is secreted in silk with a large molecular weight of the natural size and has activity, and the silk fibroin heavy chain secretion expression system has a bright prospect in application compared with the prior fibroin expression system.

2) The efficient silk fibroin heavy chain secretion expression system can be used for expressing and secreting mature recombinant protein in a bioreactor, and the size of the protein is not influenced without any additional silk fibroin heavy chain amino acid residues at the N-terminal.

Drawings

FIG. 1 is a schematic diagram of construction of a silk fibroin heavy chain promoter pBac expression vector in establishment and application of a high-efficiency silk fibroin heavy chain promoter secretion expression system.

FIG. 2 is a schematic diagram of screening transgenic silkworm positive individuals in the establishment and application of a high-efficiency silk fibroin heavy chain promoter secretion expression system.

In FIG. 2, a, b, g and h are FRF strains, c, d, i and j are HFRF strains, and e, f, k and l are HFRS strains. a. c, e, g, i and k were observed under white light, respectively, and b, d, f, h, j and l were observed under DsRed excitation light, respectively.

FIG. 3 is a map of the location on the chromosome of DsRed1 gene in the establishment and application of a high-efficiency silk fibroin heavy chain promoter secretion expression system of the present invention.

FIG. 4 is a schematic diagram of expression of DsRed1 in transgenic silkworms in the establishment and application of a high-efficiency silk fibroin heavy chain promoter secretion expression system of the present invention.

In FIG. 4, a is WT, b is FRF, c is HFRF, and d is HFRS. a. b, c, d and e were observed under white light, and f was observed under DsRed excitation light, respectively.

FIG. 5 is a schematic diagram of the distribution of DsRed1 in the posterior and middle silk glands in the establishment and application of a high-efficiency silk fibroin heavy chain promoter secretory expression system of the present invention.

FIG. 6 is a schematic diagram of different transgenic silkworm cocoons in the establishment and application of a high-efficiency silk fibroin heavy chain promoter secretory expression system.

FIG. 7 is a schematic diagram showing the comparison of the silkworm cocoon layer rate of transgenes in the establishment and application of a high-efficiency silk fibroin heavy chain promoter secretion expression system of the present invention.

FIG. 8 is a schematic view of the observation of DsRed1 on the inner and outer layers of transgenic silkworm cocoons in the establishment and application of the efficient silk fibroin heavy chain promoter secretory expression system of the present invention.

FIG. 9 is a schematic diagram of the distribution of DsRed1 in transgenic silkworm cocoons in the establishment and application of a high-efficiency silk fibroin heavy chain promoter secretory expression system of the present invention.

FIG. 10 is a relative expression diagram of DsRed1 mRNA in posterior silk gland in the establishment and application of a high-efficiency silk fibroin heavy chain promoter secretion expression system of the present invention.

FIG. 11 is a relative expression diagram of DsRed1 mRNA in the middle silk gland in the establishment and application of a high-efficiency silk fibroin heavy chain promoter secretion expression system of the present invention.

FIG. 12 is a relative expression diagram of FibH mRNA in posterior and middle silk glands in the establishment and application of a high-efficiency silk fibroin heavy chain promoter secretion expression system of the present invention.

FIG. 13 is a protein electrophoresis analysis diagram of DsRed1 in the establishment and application of a high-efficiency silk fibroin heavy chain promoter secretion expression system of the present invention.

FIG. 14 is an immunoblot analysis of DsRed1 in the establishment and application of a high efficiency silk fibroin heavy chain promoter secretory expression system of the present invention.

In FIG. 14, the dsRed-6XHis sample is a standard control.

Detailed Description

The technical scheme of the invention is further described in detail in the following by combining the drawings and specific examples, and the experimental method without specific conditions noted in the examples is generally carried out according to conventional conditions. It is to be understood that the following examples are only illustrative and explanatory of the present invention and should not be construed as limiting the scope of the present invention. All the technologies realized based on the above-mentioned contents of the present invention are covered in the protection scope of the present invention.

Example 1: construction of efficient fibroin heavy chain pBac vector

The invention uses specific primer to amplify a DNA segment with length of 1967bp from silkworm genome DNA by PCR, which is called F (1967) vector, the silk fibroin heavy chain promoter basic element sequence on the F (1967) vector is consistent with silkworm FibH gene Accession No. AF226688 (nt.61490-63456) registered on GenBank, including 5' UTR (628bp), promoter region (320bp), Exon1(42bp), Intron1(971bp), Exon2-NTD 2AA (6 bp). The hr3 CQ enhancer, Ser1pA terminator and FibHpA terminator sequences are known sequences (Wang et al 2013).

The pBac vector comprises a pBac-FRF vector, a pBac-HFRF vector, a pBac-HFRS vector and a pBac-HFlRS vector; the pBac-HFRF vector, the pBac-HFRS vector and the pBac-HFlRS vector are insertion type vectors formed by a pBac-FRF vector; the construction of the pBac vector comprises the following steps:

1) carrying out PCR amplification by adopting a specific primer and taking a phSRSV vector as a template to obtain red fluorescent protein DsRed1, an hr3 CQ enhancer and a Ser1polyA (Ser1pA) terminator, and cloning the obtained fragments into a pMD19T simple vector (Takara) respectively to obtain a pDsRed1 vector, a phr3 vector and a pSer1pA vector respectively;

2) carrying out PCR amplification by using a silkworm genome DNA as a template by adopting a specific primer to obtain F (1967) (the sequence is shown as SEQ ID No. 1), and cloning the obtained F (1967) fragment (GenBank accession number AF226688, nt.61490-63456) into a pMD19T single vector (Takara) to obtain a pF1967 vector;

carrying out PCR amplification by using a specific primer and a FibHpolyA (FibHpA) terminator as a template, and cloning the obtained DNA fragment into a pMD19T simple vector (Takara) to obtain a pFibHpA vector;

3) connecting a DsRed1 fragment obtained by enzyme digestion of a pDsRed1 vector by using BamH I and Not I to a pF1967 vector obtained by enzyme digestion of the BamH I and Not I to obtain a pFR vector;

connecting a FibHpA fragment obtained by cutting the p-FibHpA vector with Not I and Kpn I to an pFR vector obtained by cutting the p-FibHpA vector with Not I and Kpn I to obtain a pFRF vector (the sequence is shown as SEQ ID No. 2);

4) the pFRF vector is digested by AscI to obtain an FRF fragment, and the FRF fragment is inserted into a pBac [3xP3DsRedaf ] transgenic vector to obtain a pBac-FRF vector.

Inserting hr3 into pFRF vector to obtain pHFRF vector (shown in SEQ ID No. 3), digesting pHFRF vector with AscI to obtain HFRF fragment, inserting into pBac [3xP3DsRedaf ] transgenic vector to generate pBac-HFRF vector;

replacing the FibHpA in the pHFRF vector with Ser1pA to generate pHFRS (the sequence is shown as SEQ ID No. 4); the pHFRS vector is digested by AscI to obtain an HFRS fragment, and the HFRS fragment is inserted into a pBac [3xP3DsRedaf ] transgenic vector to generate a pBac-HFRS vector.

(ii) replacing Exon2 NTD 2AA with Exon2(GenBank accession No. AF226688, nt.63451-63846) in pHFRS to generate vector pHFlRS; the pHFIRS vector is digested by AscI to obtain an HFIRF fragment, and the fragment is inserted into a pBac [3xP3DsRedaf ] transgenic vector to generate a pBac-HFIRS vector.

The nucleotide sequences of the specific primers required for PCR amplification in the pBac vector construction are shown in Table 1.

TABLE 1 nucleotide sequence Listing

Note: the sequences of the restriction sites of the respective restriction enzymes are underlined.

Example 2: preparation of efficient silk fibroin heavy chain promoter secretion expression system

A high-efficiency silk fibroin heavy chain promoter secretion expression system is prepared by the following steps:

1) injecting the pBac vector and the helper plasmid pHA3PIG into G0 generation eggs at a mass ratio of 1:1, feeding to obtain G0 moth, and mating to obtain G1 generation;

2) and screening positive individuals of eyes or nerves of G1 generation eggs by using a fluorescence microscope to obtain transgenic silkworms, feeding and passaging the obtained transgenic silkworms, and reserving the positive transgenic individuals of pBac-FRF vector, pBac-HFRF vector and pBac-HFRS vector single copy as FRF strains, HFRF strains and HFRS strains respectively.

Example 3: detection of insertion site of pBac vector on chromosome by inverse PCR method

Genomic DNA was digested with HaeIII overnight at 37 ℃ and the digestion products were recovered and ligated with T4DNA ligase overnight at 16 ℃ to circularize the DNA fragment. The ligation products were PCR amplified using transposon specific primers pBacL-F/pBacL-R (for piggyBac left arm) and pBacR-F/pBacR-R (for piggyBac right arm). PCR fragments were cloned and sequenced. The insertion site of the pBac expression vector on the silkworm genome was determined using SilkDB 3.0(https:// Silkbb. bioinfotoolkits. net).

Through detection, the insertion sites of the pBac-FRF vector, the pBac-HFRF vector and the pBac-HFRS vector in the pBac vector on the chromosome are chromosome 1, chromosome 25 and chromosome 20 respectively.

Example 4: distribution of DsRed1 recombinant protein in silk gland tissue at upper cluster stage

The transgenic silkworm in the upper cluster stage is observed for the three strains of FRF strain, HFRF strain and HFRS strain under natural illumination, and the transgenic silkworm is shot by a digital camera. Then, silkworms were dissected to take out silk glands, and red fluorescent protein was observed with a body type fluorescence microscope equipped with an RFP filter. Finally, the middle and posterior silk glands (MSG and PSG) were embedded with the embedding medium Tissue-Tek o.c.t.compound (sakurafeinetechnical), respectively, and frozen with liquid nitrogen. Sections were sliced with a cryostat (typically-22 ℃) 10 μm thick and the sections were mounted on pre-treated slides. Dyeing with DAPI, then dripping an anti-fluorescence quenching agent to complete the mounting, and finally placing the section in a fluorescence microscope for observation and shooting.

Through the accompanying drawings of fig. 4 and fig. 5, the transgenic silkworms in the upper cluster stage are observed to find that the whole bodies of the HFRS (fig. 4d) and HFRF (fig. 4c) transgenic lines are orange red, and are dissected to find that the middle parts and the back parts of the HFRS and HFRF silk glands are pink, and the FRF is basically consistent with the WT group (as shown in fig. 4 e); the mid and posterior silk glands of FRF, HFRF and HFRS were observed under a fluorescence microscope to fluoresce red and the fluorescence was secondary enhanced (as shown in fig. 4 f). Dissecting silkworm, taking out silk gland, and freezing slice observing rear and middle silk glands (as shown in FIG. 5), wherein the expression of DsRed1 is in FRF, HFRF and HFRS rear silk gland cells, the distribution of DsRed1 is in rear silk gland cavity, the silk glue layer is slightly red fluorescence, and the red fluorescence is enhanced by the red fluorescence; a large number of spherical red fluorescent dots are distributed in the lumen of the middle silk gland, and the HFRS strain is strongest.

Example 5: distribution of DsRed1 recombinant protein in silkworm cocoons

The transgenic silkworm in the upper cluster stage is observed for the three strains of FRF strain, HFRF strain and HFRS strain under natural illumination, and the transgenic silkworm is shot by a digital camera. 30 transgenic cocoons are selected respectively for counting cocoon layer rate, the transgenic cocoons are punched by a puncher to obtain round pieces with the diameter of 5mm, the outer layer and the inner layer of the cocoons are further observed by fluorescence, the cocoons are observed by freezing and slicing, and shooting is carried out by a fluorescence microscope with an RFP filter.

Referring to FIGS. 6-9, the cocoons collected from positive individuals were observed to have an orange red color with HFRS observed in the inner layer, the HFRF and FRF groups were reduced in the outer layer, the WT group showed no red fluorescence, and the HFRS group showed minimal cocoons. Statistical analysis of cocoon layer rate is carried out, the cocoon layer rate of the FRF group is not obviously different from that of the WT group, and both the HFRF and HFRS groups are obviously lower than that of the WT group. The silk is frozen and sliced, and the silk fibroin and sericin layers of the positive individuals are both distributed with red fluorescent protein, and the HFRS groups are distributed most.

Example 6: relative expression of mRNA for DsRed1 in silk glands

Extracting total RNA in MSG and PSG from 3 days to upper cluster stage of 5-year-old transgenic silkworms. Then cDNA is obtained through reverse transcription, qPCR detection is carried out by taking the cDNA as a template, and silkworm ribosomal protein 49(rp49) is taken as an internal reference. qPCR reactions were performed using ABI 7500 fast real-time system (Applied Biosystems) under the following conditions: at 95 ℃ for 30s, 40 cycles at 95 ℃, 3s and 60 ℃ for 30 s. Table 1 describes the FibH, DsRed1 and rp49 primers used for qPCR.

Referring to the attached figures 10-14, RNA extraction is respectively carried out on silkworm larvae L5D3, L5D4, L5D5, L5D6, L5D7 and silk glands in the upper cluster stage, fluorescent quantitative PCR detection is respectively carried out on the DsRed1 gene and the FibH gene of the middle silk gland and the rear silk gland, the relative expression amounts of the HFRF and the rear silk gland DsRed1 of the HFRS strain in the upper cluster stage are respectively 5.7 times and 6.9 times of that of FRF and are obviously higher than those of the FRF group, and the HFRF and the HFRS strain DsRed1 also have small expression in the middle silk gland. Furthermore, HFRS and HFRF strains affect FibH relative expression.

Example 7: analysis of DsRed1 content in silkworm cocoon

80mg of cut transgenic silkworm cocoons were dissolved with 1ml of 9.3ml of LiBr under vortex shaking at 40 ℃ for 10min, and then 1ml of 8M urea was added thereto and mixed with a mixing vortex mixer (Eppendorf). The mixture was diluted with 9-fold PBS. Finally, protein electrophoresis, Coomassie blue R-250 staining and Western blotting analysis were performed on 4-20% gradient acrylamide gel (Genscript). The content of recombinant RFP in the transgenic silkworm cocoon is subjected to gray level analysis by using Image J software.

The nucleotide sequences of the primers used in the inverse PCR/qPCR reactions of examples 3 and 6 are shown in Table 2.

TABLE 2 nucleotide sequence Listing

Referring to FIGS. 13 and 14, the protein extraction of cocoons from FRF, HFRF and HFRS lines was performed, and the DsRed1 content was compared, and the DsRed1 content in cocoons from HFRS and HFRF groups was 7.2 times and 4.1 times that of cocoons from FRF groups.

The above description is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above-described embodiments. It will be understood by those skilled in the art that various changes, substitutions of equivalents, and alterations can be made without departing from the spirit and scope of the invention.

Sequence listing

<110> university of southwest

<120> establishment and application of efficient fibroin heavy chain promoter secretion expression system

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1967

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

aaaatgctct agtgttatac ctttccaaaa tcaccattaa ttaggtagtg tttaagcttg 60

ttgtacaaaa ctgccacacg catttttttc tccactgtag gttgtagtta cgcgaaaaca 120

aaatcgttct gtgaaaattc aaacaaaaat attttttcgt aaaaacactt atcaatgagt 180

aaagtaacaa ttcatgaata atttcatgta aaaaaaaaat actagaaaag gaatttttca 240

ttacgagatg cttaaaaatc tgtttcaagg tagagatttt tcgatatttc ggaaaatttt 300

gtaaaactgt aaatccgtaa aattttgcta aacatatatt gtgttgtttt ggtaagtatt 360

gacccaagct atcacctcct gcagtatgtc gtgctaatta ctggacacat tgtataacag 420

ttccactgta ttgacaataa taaaacctct tcattgactt gagaatgtct ggacagattt 480

ggctttgtat ttttgattta caaatgtttt tttggtgatt tacccatcca aggcattctc 540

caggatggtt gtggcatcac gccgattggc aaacaaaaac taaaatgaaa ctaaaaagaa 600

acagtttccg ctgtcccgtt cctctagtgg gagaaagcat gaagtaagtt ctttaaatat 660

tacaaaaaaa ttgaacgata ttataaaatt ctttaaaata ttaaaagtaa gaacaataag 720

atcaattaaa tcataattaa tcacattgtt catgatcaca atttaattta cttcatacgt 780

tgtattgtta tgttaaataa aaagattaat ttctatgtaa ttgtatctgt acaatacaat 840

gtgtagatgt ttattctatc gaaagtaaat acgtcaaaac tcgaaaattt tcagtataaa 900

aaggttcaac tttttcaaat cagcatcagt tcggttccaa ctctcaagat gagagtcaaa 960

acctttgtga tcttgtgctg cgctctgcag gtgagttaat tattttacta ttatttcaga 1020

aggtggccag acgatatcac gggccacctg ataataagtg gtcgccaaaa cgcacagata 1080

tcgtaaattg tgccatttga tttgtcacgc ccgggggggc tacggaataa actacattta 1140

tttatttaaa aaatgaacct tagattatgt aacttgtgat ttatttgcgt caaaagtagg 1200

caagatgaat ctatgtaaat acctgggcag acttgcaata tcctatttca ccggtaaatc 1260

agcattgcaa tatgcaatgc atattcaaca atatgtaaaa caattcgtaa agcatcatta 1320

gaaaatagac gaaagaaatt gcataaaatt ataaccgcat tattaattta ttatgatatc 1380

tattaacaat tgctattgcc tttttttcgc aaattataat cattttcata acctcgaggt 1440

agcattctgt tacattttaa tacattggta tgtgattata acacgagctg cccactgagt 1500

ttctcgccag atcttctcag tgggtcgcgt taccgatcac gtgatagatt ctatgaagca 1560

ctgctcttgt tagggctagt gttagcaaat tctttcaggt tgagtctgag agctcaccta 1620

cccatcggag cgtagctgga ataggctacc agctaatagg tagggaaaac aaagctcgaa 1680

acaagctcaa gtaataacaa cataatgtga ccataaaatc tcgtggtgta tgagatacaa 1740

ttatgtactt tcccacaaat gtttacataa ttagaatgtt gttcaacttg cctaacgccc 1800

cagctagaac attcaattat tactattacc actactaagg cagtatgtcc taactcgttc 1860

cagatcagcg ctaacttcga ttgaatgtgc gaaatttata gctcaatatt ttagcactta 1920

tcgtattgat ttaagaaaaa attgttaaca ttttgtttca gtatgtc 1967

<210> 2

<211> 9324

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60

cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120

ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180

accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240

attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300

tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360

tttcccagtc acgacgttgt aaaacgacgg ccagtgccaa gctttgttta aaatataaca 420

aaattgtgat cccacaaaat gaagtggggc aaaatcaaat aattaactag tgtccgtaaa 480

cttgttggtc ttcaactttt tgaggaacac gttggacggc aaatcgtgac tataacacaa 540

gttgatttaa taattttagc caacacgtcg ggctgcgtgt tttttgcgct ctgtgtacac 600

gttgattaac tggtcgagat taaataattt aatttttggt tcttctttaa atctgtgatg 660

aaatttttta aaataacttt aaattcttca ttggtaaaaa atgccacgtt ttgcaacttg 720

tgagggtcta atatgaggtc aaactcagta ggagttttat ccaaaaaaga aaacatgatt 780

acgtctgtac acgaacgcgt attaacgcag agtgcaaagt ataagagggt taaaaaatat 840

attttacgca ccatatacgc atcgggttga tatcgttaat atggatcaat ttgaacagtt 900

gattaacgtg tctctgctca agtctttgat caaaacgcaa atcgacgaaa atgtgtcgga 960

caatatcaag tcgatgagcg aaaaactaaa aaggctagaa tacgacaatc tcacagacag 1020

cgttgagata tacggtattc acgacagcag gctgaataat aaaaaaatta gaaactatta 1080

tttaacccta gaaagataat catattgtga cgtacgttaa agataatcat gcgtaaaatt 1140

gacgcatgtg ttttatcggt ctgtatatcg aggtttattt attaatttga atagatatta 1200

agttttatta tatttacact tacatactaa taataaattc aacaaacaat ttatttatgt 1260

ttatttattt attaaaaaaa aacaaaaact caaaatttct tctataaagt aacaaaactt 1320

ttaaacattc tctcttttac aaaaataaac ttattttgta ctttaaaaac agtcatgttg 1380

tattataaaa taagtaatta gcttaactta tacataatag aaacaaatta tacttattag 1440

tcagtcagaa acaactttgg cacatatcaa tattatgctc tcgacaaata acttttttgc 1500

attttttgca cgatgcattt gcctttcgcc ttattttaga ggggcagtaa gtacagtaag 1560

tacgtttttt cattactggc tcttcagtac tgtcatctga tgtaccaggc acttcatttg 1620

gcaaaatatt agagatatta tcgcgcaaat atctcttcaa agtaggagct tctaaacgct 1680

tacgcataaa cgatgacgtc aggctcatgt aaaggtttct cataaatttt ttgcgacttt 1740

gaaccttttc tcccttgcta ctgacattat ggctgtatat aataaaagaa tttatgcagg 1800

caatgtttat cattccgtac aataatgcca taggccacct attcgtcttc ctactgcagg 1860

tcatcacaga acacatttgg tctagcgtgt ccactccgcc tttagtttga ttataataca 1920

taaccatttg cggtttaccg gtactttcgt tgatagaagc atcctcatca caagatgata 1980

ataagtatac catcttagct ggcttcggtt tatatgagac gagagtaagg ggtccgtcaa 2040

aacaaaacat cgatgttccc actggcctgg agcgactgtt tttcagtact tccggtatct 2100

cgcgtttgtt tgatcgcacg gttcccacaa tggttaattc gagctcgccc ggggatctaa 2160

ttcaattaga gactaattca attagagcta attcaattag gatccaagct tatcgatttc 2220

gaaccctcga ccgccggagt ataaatagag gcgcttcgtc tacggagcga caattcaatt 2280

caaacaagca aagtgaacac gtcgctaagc gaaagctaag caaataaaca agcgcagctg 2340

aacaagctaa acaatcgggg taccgctaga gtcgacggta ccgcgggccc gggatccacc 2400

ggtcgccacc atggtgcgct cctccaagaa cgtcatcaag gagttcatgc gcttcaaggt 2460

gcgcatggag ggcaccgtga acggccacga gttcgagatc gagggcgagg gcgagggccg 2520

cccctacgag ggccacaaca ccgtgaagct gaaggtgacc aagggcggcc ccctgccctt 2580

cgcctgggac atcctgtccc cccagttcca gtacggctcc aaggtgtacg tgaagcaccc 2640

cgccgacatc cccgactaca agaagctgtc cttccccgag ggcttcaagt gggagcgcgt 2700

gatgaacttc gaggacggcg gcgtggtgac cgtgacccag gactcctccc tgcaggacgg 2760

ctgcttcatc tacaaggtga agttcatcgg cgtgaacttc ccctccgacg gccccgtaat 2820

gcagaagaag accatgggct gggaggcctc caccgagcgc ctgtaccccc gcgacggcgt 2880

gctgaagggc gagatccaca aggccctgaa gctgaaggac ggcggccact acctggtgga 2940

gttcaagtcc atctacatgg ccaagaagcc cgtgcagctg cccggctact actacgtgga 3000

ctccaagctg gacatcacct cccacaacga ggactacacc atcgtggagc agtacgagcg 3060

caccgagggc cgccaccacc tgttcctgta gcggccgcga ctctagatca taatcagcca 3120

tgcggccgcg actctagacc acatttgtag aggttttact tgctttaaaa aacctcccac 3180

acctccccct gaacctgaaa cataaaatga atgcaattgt tgttgttaac ttgtttattg 3240

cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt 3300

tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttaa agcttatcga 3360

tacgcgtacg gcgcgccagt cgtcgacaaa atgctctagt gttatacctt tccaaaatca 3420

ccattaatta ggtagtgttt aagcttgttg tacaaaactg ccacacgcat ttttttctcc 3480

actgtaggtt gtagttacgc gaaaacaaaa tcgttctgtg aaaattcaaa caaaaatatt 3540

ttttcgtaaa aacacttatc aatgagtaaa gtaacaattc atgaataatt tcatgtaaaa 3600

aaaaaatact agaaaaggaa tttttcatta cgagatgctt aaaaatctgt ttcaaggtag 3660

agatttttcg atatttcgga aaattttgta aaactgtaaa tccgtaaaat tttgctaaac 3720

atatattgtg ttgttttggt aagtattgac ccaagctatc acctcctgca gtatgtcgtg 3780

ctaattactg gacacattgt ataacagttc cactgtattg acaataataa aacctcttca 3840

ttgacttgag aatgtctgga cagatttggc tttgtatttt tgatttacaa atgttttttt 3900

ggtgatttac ccatccaagg cattctccag gatggttgtg gcatcacgcc gattggcaaa 3960

caaaaactaa aatgaaacta aaaagaaaca gtttccgctg tcccgttcct ctagtgggag 4020

aaagcatgaa gtaagttctt taaatattac aaaaaaattg aacgatatta taaaattctt 4080

taaaatatta aaagtaagaa caataagatc aattaaatca taattaatca cattgttcat 4140

gatcacaatt taatttactt catacgttgt attgttatgt taaataaaaa gattaatttc 4200

tatgtaattg tatctgtaca atacaatgtg tagatgttta ttctatcgaa agtaaatacg 4260

tcaaaactcg aaaattttca gtataaaaag gttcaacttt ttcaaatcag catcagttcg 4320

gttccaactc tcaagatgag agtcaaaacc tttgtgatct tgtgctgcgc tctgcaggtg 4380

agttaattat tttactatta tttcagaagg tggccagacg atatcacggg ccacctgata 4440

ataagtggtc gccaaaacgc acagatatcg taaattgtgc catttgattt gtcacgcccg 4500

ggggggctac ggaataaact acatttattt atttaaaaaa tgaaccttag attatgtaac 4560

ttgtgattta tttgcgtcaa aagtaggcaa gatgaatcta tgtaaatacc tgggcagact 4620

tgcaatatcc tatttcaccg gtaaatcagc attgcaatat gcaatgcata ttcaacaata 4680

tgtaaaacaa ttcgtaaagc atcattagaa aatagacgaa agaaattgca taaaattata 4740

accgcattat taatttatta tgatatctat taacaattgc tattgccttt ttttcgcaaa 4800

ttataatcat tttcataacc tcgaggtagc attctgttac attttaatac attggtatgt 4860

gattataaca cgagctgccc actgagtttc tcgccagatc ttctcagtgg gtcgcgttac 4920

cgatcacgtg atagattcta tgaagcactg ctcttgttag ggctagtgtt agcaaattct 4980

ttcaggttga gtctgagagc tcacctaccc atcggagcgt agctggaata ggctaccagc 5040

taataggtag ggaaaacaaa gctcgaaaca agctcaagta ataacaacat aatgtgacca 5100

taaaatctcg tggtgtatga gatacaatta tgtactttcc cacaaatgtt tacataatta 5160

gaatgttgtt caacttgcct aacgccccag ctagaacatt caattattac tattaccact 5220

actaaggcag tatgtcctaa ctcgttccag atcagcgcta acttcgattg aatgtgcgaa 5280

atttatagct caatatttta gcacttatcg tattgattta agaaaaaatt gttaacattt 5340

tgtttcagta tgtcggatcc atggtgcgct cctccaagaa cgtcatcaag gagttcatgc 5400

gcttcaaggt gcgcatggag ggcaccgtga acggccacga gttcgagatc gagggcgagg 5460

gcgagggccg cccctacgag ggccacaaca ccgtgaagct gaaggtgacc aagggcggcc 5520

ccctgccctt cgcctgggac atcctgtccc cccagttcca gtacggctcc aaggtgtacg 5580

tgaagcaccc cgccgacatc cccgactaca agaagctgtc ctttcccgag ggcttcaagt 5640

gggagcgcgt gatgaacttc gaggacggcg gcgtggtgac cgtgacccag gactcctccc 5700

tgcaggacgg ctgcttcatc tacaaggtga agttcatcgg cgtgaacttc ccctccgacg 5760

gccccgtaat gcagaagaag accatgggct gggaggcctc caccgagcgc ctgtaccccc 5820

gcgacggcgt gctgaagggc gagatccaca aggccctgaa gctgaaggac ggcggccact 5880

acctggtgga gttcaagtcc atctacatgg ccaagaagcc cgtgcagctg cccggctact 5940

actacgtgga ctccaagctg gacatcacct cccacaacga ggactacacc atcgtggagc 6000

agtacgagcg caccgagggc cgccaccacc tgttcctgta ggcggccgct ttttaatata 6060

aaataaccct tgtttcttac ttcgtcctgg atacatctat gttttttttt tcgttaataa 6120

atgagagcat ttaagttatt gtttttaatt actttttttt agaaaacaga tttcggattt 6180

tttgtatgca ttttatttga atgtactagg taccgtcagg cgcgcctagg ccggccgatc 6240

tcggatctga caatgttcag tgcagagact cggctacgcc tcgtggactt tgaagttgac 6300

caacaatgtt tattcttacc tctaatagtc ctctgtggca aggtcaagat tctgttagaa 6360

gccaatgaag aacctggttg ttcaataaca ttttgttcgt ctaatatttc actaccgctt 6420

gacgttggct gcacttcatg tacctcatct ataaacgctt cttctgtatc gctctggacg 6480

tcatcttcac ttacgtgatc tgatatttca ctgtcagaat cctcaccaac aagctcgtca 6540

tcgctttgca gaagagcaga gaggatatgc tcatcgtcta aagaactacc cattttatta 6600

tatattagtc acgatatcta taacaagaaa atatatatat aataagttat cacgtaagta 6660

gaacatgaaa taacaatata attatcgtat gagttaaatc ttaaaagtca cgtaaaagat 6720

aatcatgcgt cattttgact cacgcggtcg ttatagttca aaatcagtga cacttaccgc 6780

attgacaagc acgcctcacg ggagctccaa gcggcgactg agatgtccta aatgcacagc 6840

gacggattcg cgctatttag aaagagagag caatatttca agaatgcatg cgtcaatttt 6900

acgcagacta tctttctagg gttaaaaaag atttgcgctt tactcgacct aaactttaaa 6960

cacgtcatag aatcttcgtt tgacaaaaac cacattgtgg ccaagctgtg tgacgcgacg 7020

cgcgctaaag aatggcaaac caagtcgcgc gagcgtcgac tctagaggat ccccgggtac 7080

cgagctcgaa ttcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca 7140

caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag 7200

tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt 7260

cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc 7320

gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg 7380

tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa 7440

agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg 7500

cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga 7560

ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg 7620

tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg 7680

gaagcgtggc gctttctcaa tgctcacgct gtaggtatct cagttcggtg taggtcgttc 7740

gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg 7800

gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca 7860

ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt 7920

ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag 7980

ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg 8040

gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc 8100

ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt 8160

tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt 8220

ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca 8280

gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg 8340

tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac 8400

cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg 8460

ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc 8520

gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta 8580

caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac 8640

gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc 8700

ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac 8760

tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact 8820

caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa 8880

tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt 8940

cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca 9000

ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa 9060

aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac 9120

tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg 9180

gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc 9240

gaaaagtgcc acctgacgtc taagaaacca ttattatcat gacattaacc tataaaaata 9300

ggcgtatcac gaggcccttt cgtc 9324

<210> 3

<211> 10277

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60

cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120

ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180

accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240

attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300

tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360

tttcccagtc acgacgttgt aaaacgacgg ccagtgccaa gctttgttta aaatataaca 420

aaattgtgat cccacaaaat gaagtggggc aaaatcaaat aattaactag tgtccgtaaa 480

cttgttggtc ttcaactttt tgaggaacac gttggacggc aaatcgtgac tataacacaa 540

gttgatttaa taattttagc caacacgtcg ggctgcgtgt tttttgcgct ctgtgtacac 600

gttgattaac tggtcgagat taaataattt aatttttggt tcttctttaa atctgtgatg 660

aaatttttta aaataacttt aaattcttca ttggtaaaaa atgccacgtt ttgcaacttg 720

tgagggtcta atatgaggtc aaactcagta ggagttttat ccaaaaaaga aaacatgatt 780

acgtctgtac acgaacgcgt attaacgcag agtgcaaagt ataagagggt taaaaaatat 840

attttacgca ccatatacgc atcgggttga tatcgttaat atggatcaat ttgaacagtt 900

gattaacgtg tctctgctca agtctttgat caaaacgcaa atcgacgaaa atgtgtcgga 960

caatatcaag tcgatgagcg aaaaactaaa aaggctagaa tacgacaatc tcacagacag 1020

cgttgagata tacggtattc acgacagcag gctgaataat aaaaaaatta gaaactatta 1080

tttaacccta gaaagataat catattgtga cgtacgttaa agataatcat gcgtaaaatt 1140

gacgcatgtg ttttatcggt ctgtatatcg aggtttattt attaatttga atagatatta 1200

agttttatta tatttacact tacatactaa taataaattc aacaaacaat ttatttatgt 1260

ttatttattt attaaaaaaa aacaaaaact caaaatttct tctataaagt aacaaaactt 1320

ttaaacattc tctcttttac aaaaataaac ttattttgta ctttaaaaac agtcatgttg 1380

tattataaaa taagtaatta gcttaactta tacataatag aaacaaatta tacttattag 1440

tcagtcagaa acaactttgg cacatatcaa tattatgctc tcgacaaata acttttttgc 1500

attttttgca cgatgcattt gcctttcgcc ttattttaga ggggcagtaa gtacagtaag 1560

tacgtttttt cattactggc tcttcagtac tgtcatctga tgtaccaggc acttcatttg 1620

gcaaaatatt agagatatta tcgcgcaaat atctcttcaa agtaggagct tctaaacgct 1680

tacgcataaa cgatgacgtc aggctcatgt aaaggtttct cataaatttt ttgcgacttt 1740

gaaccttttc tcccttgcta ctgacattat ggctgtatat aataaaagaa tttatgcagg 1800

caatgtttat cattccgtac aataatgcca taggccacct attcgtcttc ctactgcagg 1860

tcatcacaga acacatttgg tctagcgtgt ccactccgcc tttagtttga ttataataca 1920

taaccatttg cggtttaccg gtactttcgt tgatagaagc atcctcatca caagatgata 1980

ataagtatac catcttagct ggcttcggtt tatatgagac gagagtaagg ggtccgtcaa 2040

aacaaaacat cgatgttccc actggcctgg agcgactgtt tttcagtact tccggtatct 2100

cgcgtttgtt tgatcgcacg gttcccacaa tggttaattc gagctcgccc ggggatctaa 2160

ttcaattaga gactaattca attagagcta attcaattag gatccaagct tatcgatttc 2220

gaaccctcga ccgccggagt ataaatagag gcgcttcgtc tacggagcga caattcaatt 2280

caaacaagca aagtgaacac gtcgctaagc gaaagctaag caaataaaca agcgcagctg 2340

aacaagctaa acaatcgggg taccgctaga gtcgacggta ccgcgggccc gggatccacc 2400

ggtcgccacc atggtgcgct cctccaagaa cgtcatcaag gagttcatgc gcttcaaggt 2460

gcgcatggag ggcaccgtga acggccacga gttcgagatc gagggcgagg gcgagggccg 2520

cccctacgag ggccacaaca ccgtgaagct gaaggtgacc aagggcggcc ccctgccctt 2580

cgcctgggac atcctgtccc cccagttcca gtacggctcc aaggtgtacg tgaagcaccc 2640

cgccgacatc cccgactaca agaagctgtc cttccccgag ggcttcaagt gggagcgcgt 2700

gatgaacttc gaggacggcg gcgtggtgac cgtgacccag gactcctccc tgcaggacgg 2760

ctgcttcatc tacaaggtga agttcatcgg cgtgaacttc ccctccgacg gccccgtaat 2820

gcagaagaag accatgggct gggaggcctc caccgagcgc ctgtaccccc gcgacggcgt 2880

gctgaagggc gagatccaca aggccctgaa gctgaaggac ggcggccact acctggtgga 2940

gttcaagtcc atctacatgg ccaagaagcc cgtgcagctg cccggctact actacgtgga 3000

ctccaagctg gacatcacct cccacaacga ggactacacc atcgtggagc agtacgagcg 3060

caccgagggc cgccaccacc tgttcctgta gcggccgcga ctctagatca taatcagcca 3120

tgcggccgcg actctagacc acatttgtag aggttttact tgctttaaaa aacctcccac 3180

acctccccct gaacctgaaa cataaaatga atgcaattgt tgttgttaac ttgtttattg 3240

cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt 3300

tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttaa agcttatcga 3360

tacgcgtacg gcgcgccagt cgtcgaccag cgtcgtgaaa agaggcaatg acaaatacaa 3420

aacgacgtat gagcagaccc gtcgccaaga cgggtctacc tctaagatga tgtcatttgt 3480

tttttaaaac taactcgctt tacgagtaga attctacgtg taaaacataa tcaagagatg 3540

atgtcatttg tttttcaaaa ccaaactcgc tttacgagta gaattctacg tgtaaaacac 3600

aatcaaaaga tgatgtcatt cgtttttcaa aaccgaattt aagaaatgat gtcatttgtt 3660

tttcaaaacc aaactcgctt tacgagcaga attctacgtg taaaacacaa tcaagagatg 3720

atgtcatttg tttttcaaaa ctgaatgatg tcatttgttt ttcaaaacta aacttgcttt 3780

gcgagtagaa ttctacgtgt aaaacacagt caagagatga tgtcatttgt ttttcaaaac 3840

tgaaccggct ttacgagtag aattctactt gtaaaacata atcaagagat gatgtcattt 3900

gtttttcaaa actgaactgg ctttacgagt agaattctac gtgtaaaaca taatcaagag 3960

atgatgtcat cattaaactg atgtcatttt atacacgatt gttaacatgt ttaataatga 4020

ctaatttgtt tttccaaatt aaactcgctt tacgagtaga attctacttg taacgcacga 4080

ttaagtatga atcataagct gatgtcattt gttttcgaca taaaatgttt atacaatgga 4140

atcttcttgt aaattatcca aataatataa tttatccgat tctacgttac atttaaattc 4200

gttgttatcg tacaattctt caggacacgc catgtattgg tcatttttag cgtgcaacca 4260

acgattgtat ttgacgccgt cgttggattg cgtgttcagg ttggcgtaca cgtgactggg 4320

cacggcttct ttttgtcgac aaaatgctct agtgttatac ctttccaaaa tcaccattaa 4380

ttaggtagtg tttaagcttg ttgtacaaaa ctgccacacg catttttttc tccactgtag 4440

gttgtagtta cgcgaaaaca aaatcgttct gtgaaaattc aaacaaaaat attttttcgt 4500

aaaaacactt atcaatgagt aaagtaacaa ttcatgaata atttcatgta aaaaaaaaat 4560

actagaaaag gaatttttca ttacgagatg cttaaaaatc tgtttcaagg tagagatttt 4620

tcgatatttc ggaaaatttt gtaaaactgt aaatccgtaa aattttgcta aacatatatt 4680

gtgttgtttt ggtaagtatt gacccaagct atcacctcct gcagtatgtc gtgctaatta 4740

ctggacacat tgtataacag ttccactgta ttgacaataa taaaacctct tcattgactt 4800

gagaatgtct ggacagattt ggctttgtat ttttgattta caaatgtttt tttggtgatt 4860

tacccatcca aggcattctc caggatggtt gtggcatcac gccgattggc aaacaaaaac 4920

taaaatgaaa ctaaaaagaa acagtttccg ctgtcccgtt cctctagtgg gagaaagcat 4980

gaagtaagtt ctttaaatat tacaaaaaaa ttgaacgata ttataaaatt ctttaaaata 5040

ttaaaagtaa gaacaataag atcaattaaa tcataattaa tcacattgtt catgatcaca 5100

atttaattta cttcatacgt tgtattgtta tgttaaataa aaagattaat ttctatgtaa 5160

ttgtatctgt acaatacaat gtgtagatgt ttattctatc gaaagtaaat acgtcaaaac 5220

tcgaaaattt tcagtataaa aaggttcaac tttttcaaat cagcatcagt tcggttccaa 5280

ctctcaagat gagagtcaaa acctttgtga tcttgtgctg cgctctgcag gtgagttaat 5340

tattttacta ttatttcaga aggtggccag acgatatcac gggccacctg ataataagtg 5400

gtcgccaaaa cgcacagata tcgtaaattg tgccatttga tttgtcacgc ccgggggggc 5460

tacggaataa actacattta tttatttaaa aaatgaacct tagattatgt aacttgtgat 5520

ttatttgcgt caaaagtagg caagatgaat ctatgtaaat acctgggcag acttgcaata 5580

tcctatttca ccggtaaatc agcattgcaa tatgcaatgc atattcaaca atatgtaaaa 5640

caattcgtaa agcatcatta gaaaatagac gaaagaaatt gcataaaatt ataaccgcat 5700

tattaattta ttatgatatc tattaacaat tgctattgcc tttttttcgc aaattataat 5760

cattttcata acctcgaggt agcattctgt tacattttaa tacattggta tgtgattata 5820

acacgagctg cccactgagt ttctcgccag atcttctcag tgggtcgcgt taccgatcac 5880

gtgatagatt ctatgaagca ctgctcttgt tagggctagt gttagcaaat tctttcaggt 5940

tgagtctgag agctcaccta cccatcggag cgtagctgga ataggctacc agctaatagg 6000

tagggaaaac aaagctcgaa acaagctcaa gtaataacaa cataatgtga ccataaaatc 6060

tcgtggtgta tgagatacaa ttatgtactt tcccacaaat gtttacataa ttagaatgtt 6120

gttcaacttg cctaacgccc cagctagaac attcaattat tactattacc actactaagg 6180

cagtatgtcc taactcgttc cagatcagcg ctaacttcga ttgaatgtgc gaaatttata 6240

gctcaatatt ttagcactta tcgtattgat ttaagaaaaa attgttaaca ttttgtttca 6300

gtatgtcgga tccatggtgc gctcctccaa gaacgtcatc aaggagttca tgcgcttcaa 6360

ggtgcgcatg gagggcaccg tgaacggcca cgagttcgag atcgagggcg agggcgaggg 6420

ccgcccctac gagggccaca acaccgtgaa gctgaaggtg accaagggcg gccccctgcc 6480

cttcgcctgg gacatcctgt ccccccagtt ccagtacggc tccaaggtgt acgtgaagca 6540

ccccgccgac atccccgact acaagaagct gtcctttccc gagggcttca agtgggagcg 6600

cgtgatgaac ttcgaggacg gcggcgtggt gaccgtgacc caggactcct ccctgcagga 6660

cggctgcttc atctacaagg tgaagttcat cggcgtgaac ttcccctccg acggccccgt 6720

aatgcagaag aagaccatgg gctgggaggc ctccaccgag cgcctgtacc cccgcgacgg 6780

cgtgctgaag ggcgagatcc acaaggccct gaagctgaag gacggcggcc actacctggt 6840

ggagttcaag tccatctaca tggccaagaa gcccgtgcag ctgcccggct actactacgt 6900

ggactccaag ctggacatca cctcccacaa cgaggactac accatcgtgg agcagtacga 6960

gcgcaccgag ggccgccacc acctgttcct gtaggcggcc gctttttaat ataaaataac 7020

ccttgtttct tacttcgtcc tggatacatc tatgtttttt ttttcgttaa taaatgagag 7080

catttaagtt attgttttta attacttttt tttagaaaac agatttcgga ttttttgtat 7140

gcattttatt tgaatgtact aggtaccgtc aggcgcgcct aggccggccg atctcggatc 7200

tgacaatgtt cagtgcagag actcggctac gcctcgtgga ctttgaagtt gaccaacaat 7260

gtttattctt acctctaata gtcctctgtg gcaaggtcaa gattctgtta gaagccaatg 7320

aagaacctgg ttgttcaata acattttgtt cgtctaatat ttcactaccg cttgacgttg 7380

gctgcacttc atgtacctca tctataaacg cttcttctgt atcgctctgg acgtcatctt 7440

cacttacgtg atctgatatt tcactgtcag aatcctcacc aacaagctcg tcatcgcttt 7500

gcagaagagc agagaggata tgctcatcgt ctaaagaact acccatttta ttatatatta 7560

gtcacgatat ctataacaag aaaatatata tataataagt tatcacgtaa gtagaacatg 7620

aaataacaat ataattatcg tatgagttaa atcttaaaag tcacgtaaaa gataatcatg 7680

cgtcattttg actcacgcgg tcgttatagt tcaaaatcag tgacacttac cgcattgaca 7740

agcacgcctc acgggagctc caagcggcga ctgagatgtc ctaaatgcac agcgacggat 7800

tcgcgctatt tagaaagaga gagcaatatt tcaagaatgc atgcgtcaat tttacgcaga 7860

ctatctttct agggttaaaa aagatttgcg ctttactcga cctaaacttt aaacacgtca 7920

tagaatcttc gtttgacaaa aaccacattg tggccaagct gtgtgacgcg acgcgcgcta 7980

aagaatggca aaccaagtcg cgcgagcgtc gactctagag gatccccggg taccgagctc 8040

gaattcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc 8100

acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta 8160

actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca 8220

gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc 8280

cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc 8340

tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat 8400

gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt 8460

ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg 8520

aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc 8580

tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt 8640

ggcgctttct caatgctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa 8700

gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta 8760

tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa 8820

caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa 8880

ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt 8940

cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt 9000

ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat 9060

cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat 9120

gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc 9180

aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc 9240

acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta 9300

gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga 9360

cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg 9420

cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc 9480

tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat 9540

cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag 9600

gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat 9660

cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa 9720

ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa 9780

gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga 9840

taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg 9900

gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc 9960

acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg 10020

aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact 10080

cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat 10140

atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt 10200

gccacctgac gtctaagaaa ccattattat catgacatta acctataaaa ataggcgtat 10260

cacgaggccc tttcgtc 10277

<210> 4

<211> 10497

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60

cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120

ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180

accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240

attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300

tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360

tttcccagtc acgacgttgt aaaacgacgg ccagtgccaa gctttgttta aaatataaca 420

aaattgtgat cccacaaaat gaagtggggc aaaatcaaat aattaactag tgtccgtaaa 480

cttgttggtc ttcaactttt tgaggaacac gttggacggc aaatcgtgac tataacacaa 540

gttgatttaa taattttagc caacacgtcg ggctgcgtgt tttttgcgct ctgtgtacac 600

gttgattaac tggtcgagat taaataattt aatttttggt tcttctttaa atctgtgatg 660

aaatttttta aaataacttt aaattcttca ttggtaaaaa atgccacgtt ttgcaacttg 720

tgagggtcta atatgaggtc aaactcagta ggagttttat ccaaaaaaga aaacatgatt 780

acgtctgtac acgaacgcgt attaacgcag agtgcaaagt ataagagggt taaaaaatat 840

attttacgca ccatatacgc atcgggttga tatcgttaat atggatcaat ttgaacagtt 900

gattaacgtg tctctgctca agtctttgat caaaacgcaa atcgacgaaa atgtgtcgga 960

caatatcaag tcgatgagcg aaaaactaaa aaggctagaa tacgacaatc tcacagacag 1020

cgttgagata tacggtattc acgacagcag gctgaataat aaaaaaatta gaaactatta 1080

tttaacccta gaaagataat catattgtga cgtacgttaa agataatcat gcgtaaaatt 1140

gacgcatgtg ttttatcggt ctgtatatcg aggtttattt attaatttga atagatatta 1200

agttttatta tatttacact tacatactaa taataaattc aacaaacaat ttatttatgt 1260

ttatttattt attaaaaaaa aacaaaaact caaaatttct tctataaagt aacaaaactt 1320

ttaaacattc tctcttttac aaaaataaac ttattttgta ctttaaaaac agtcatgttg 1380

tattataaaa taagtaatta gcttaactta tacataatag aaacaaatta tacttattag 1440

tcagtcagaa acaactttgg cacatatcaa tattatgctc tcgacaaata acttttttgc 1500

attttttgca cgatgcattt gcctttcgcc ttattttaga ggggcagtaa gtacagtaag 1560

tacgtttttt cattactggc tcttcagtac tgtcatctga tgtaccaggc acttcatttg 1620

gcaaaatatt agagatatta tcgcgcaaat atctcttcaa agtaggagct tctaaacgct 1680

tacgcataaa cgatgacgtc aggctcatgt aaaggtttct cataaatttt ttgcgacttt 1740

gaaccttttc tcccttgcta ctgacattat ggctgtatat aataaaagaa tttatgcagg 1800

caatgtttat cattccgtac aataatgcca taggccacct attcgtcttc ctactgcagg 1860

tcatcacaga acacatttgg tctagcgtgt ccactccgcc tttagtttga ttataataca 1920

taaccatttg cggtttaccg gtactttcgt tgatagaagc atcctcatca caagatgata 1980

ataagtatac catcttagct ggcttcggtt tatatgagac gagagtaagg ggtccgtcaa 2040

aacaaaacat cgatgttccc actggcctgg agcgactgtt tttcagtact tccggtatct 2100

cgcgtttgtt tgatcgcacg gttcccacaa tggttaattc gagctcgccc ggggatctaa 2160

ttcaattaga gactaattca attagagcta attcaattag gatccaagct tatcgatttc 2220

gaaccctcga ccgccggagt ataaatagag gcgcttcgtc tacggagcga caattcaatt 2280

caaacaagca aagtgaacac gtcgctaagc gaaagctaag caaataaaca agcgcagctg 2340

aacaagctaa acaatcgggg taccgctaga gtcgacggta ccgcgggccc gggatccacc 2400

ggtcgccacc atggtgcgct cctccaagaa cgtcatcaag gagttcatgc gcttcaaggt 2460

gcgcatggag ggcaccgtga acggccacga gttcgagatc gagggcgagg gcgagggccg 2520

cccctacgag ggccacaaca ccgtgaagct gaaggtgacc aagggcggcc ccctgccctt 2580

cgcctgggac atcctgtccc cccagttcca gtacggctcc aaggtgtacg tgaagcaccc 2640

cgccgacatc cccgactaca agaagctgtc cttccccgag ggcttcaagt gggagcgcgt 2700

gatgaacttc gaggacggcg gcgtggtgac cgtgacccag gactcctccc tgcaggacgg 2760

ctgcttcatc tacaaggtga agttcatcgg cgtgaacttc ccctccgacg gccccgtaat 2820

gcagaagaag accatgggct gggaggcctc caccgagcgc ctgtaccccc gcgacggcgt 2880

gctgaagggc gagatccaca aggccctgaa gctgaaggac ggcggccact acctggtgga 2940

gttcaagtcc atctacatgg ccaagaagcc cgtgcagctg cccggctact actacgtgga 3000

ctccaagctg gacatcacct cccacaacga ggactacacc atcgtggagc agtacgagcg 3060

caccgagggc cgccaccacc tgttcctgta gcggccgcga ctctagatca taatcagcca 3120

tgcggccgcg actctagacc acatttgtag aggttttact tgctttaaaa aacctcccac 3180

acctccccct gaacctgaaa cataaaatga atgcaattgt tgttgttaac ttgtttattg 3240

cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt 3300

tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttaa agcttatcga 3360

tacgcgtacg gcgcgccagt cgtcgaccag cgtcgtgaaa agaggcaatg acaaatacaa 3420

aacgacgtat gagcagaccc gtcgccaaga cgggtctacc tctaagatga tgtcatttgt 3480

tttttaaaac taactcgctt tacgagtaga attctacgtg taaaacataa tcaagagatg 3540

atgtcatttg tttttcaaaa ccaaactcgc tttacgagta gaattctacg tgtaaaacac 3600

aatcaaaaga tgatgtcatt cgtttttcaa aaccgaattt aagaaatgat gtcatttgtt 3660

tttcaaaacc aaactcgctt tacgagcaga attctacgtg taaaacacaa tcaagagatg 3720

atgtcatttg tttttcaaaa ctgaatgatg tcatttgttt ttcaaaacta aacttgcttt 3780

gcgagtagaa ttctacgtgt aaaacacagt caagagatga tgtcatttgt ttttcaaaac 3840

tgaaccggct ttacgagtag aattctactt gtaaaacata atcaagagat gatgtcattt 3900

gtttttcaaa actgaactgg ctttacgagt agaattctac gtgtaaaaca taatcaagag 3960

atgatgtcat cattaaactg atgtcatttt atacacgatt gttaacatgt ttaataatga 4020

ctaatttgtt tttccaaatt aaactcgctt tacgagtaga attctacttg taacgcacga 4080

ttaagtatga atcataagct gatgtcattt gttttcgaca taaaatgttt atacaatgga 4140

atcttcttgt aaattatcca aataatataa tttatccgat tctacgttac atttaaattc 4200

gttgttatcg tacaattctt caggacacgc catgtattgg tcatttttag cgtgcaacca 4260

acgattgtat ttgacgccgt cgttggattg cgtgttcagg ttggcgtaca cgtgactggg 4320

cacggcttct ttttgtcgac aaaatgctct agtgttatac ctttccaaaa tcaccattaa 4380

ttaggtagtg tttaagcttg ttgtacaaaa ctgccacacg catttttttc tccactgtag 4440

gttgtagtta cgcgaaaaca aaatcgttct gtgaaaattc aaacaaaaat attttttcgt 4500

aaaaacactt atcaatgagt aaagtaacaa ttcatgaata atttcatgta aaaaaaaaat 4560

actagaaaag gaatttttca ttacgagatg cttaaaaatc tgtttcaagg tagagatttt 4620

tcgatatttc ggaaaatttt gtaaaactgt aaatccgtaa aattttgcta aacatatatt 4680

gtgttgtttt ggtaagtatt gacccaagct atcacctcct gcagtatgtc gtgctaatta 4740

ctggacacat tgtataacag ttccactgta ttgacaataa taaaacctct tcattgactt 4800

gagaatgtct ggacagattt ggctttgtat ttttgattta caaatgtttt tttggtgatt 4860

tacccatcca aggcattctc caggatggtt gtggcatcac gccgattggc aaacaaaaac 4920

taaaatgaaa ctaaaaagaa acagtttccg ctgtcccgtt cctctagtgg gagaaagcat 4980

gaagtaagtt ctttaaatat tacaaaaaaa ttgaacgata ttataaaatt ctttaaaata 5040

ttaaaagtaa gaacaataag atcaattaaa tcataattaa tcacattgtt catgatcaca 5100

atttaattta cttcatacgt tgtattgtta tgttaaataa aaagattaat ttctatgtaa 5160

ttgtatctgt acaatacaat gtgtagatgt ttattctatc gaaagtaaat acgtcaaaac 5220

tcgaaaattt tcagtataaa aaggttcaac tttttcaaat cagcatcagt tcggttccaa 5280

ctctcaagat gagagtcaaa acctttgtga tcttgtgctg cgctctgcag gtgagttaat 5340

tattttacta ttatttcaga aggtggccag acgatatcac gggccacctg ataataagtg 5400

gtcgccaaaa cgcacagata tcgtaaattg tgccatttga tttgtcacgc ccgggggggc 5460

tacggaataa actacattta tttatttaaa aaatgaacct tagattatgt aacttgtgat 5520

ttatttgcgt caaaagtagg caagatgaat ctatgtaaat acctgggcag acttgcaata 5580

tcctatttca ccggtaaatc agcattgcaa tatgcaatgc atattcaaca atatgtaaaa 5640

caattcgtaa agcatcatta gaaaatagac gaaagaaatt gcataaaatt ataaccgcat 5700

tattaattta ttatgatatc tattaacaat tgctattgcc tttttttcgc aaattataat 5760

cattttcata acctcgaggt agcattctgt tacattttaa tacattggta tgtgattata 5820

acacgagctg cccactgagt ttctcgccag atcttctcag tgggtcgcgt taccgatcac 5880

gtgatagatt ctatgaagca ctgctcttgt tagggctagt gttagcaaat tctttcaggt 5940

tgagtctgag agctcaccta cccatcggag cgtagctgga ataggctacc agctaatagg 6000

tagggaaaac aaagctcgaa acaagctcaa gtaataacaa cataatgtga ccataaaatc 6060

tcgtggtgta tgagatacaa ttatgtactt tcccacaaat gtttacataa ttagaatgtt 6120

gttcaacttg cctaacgccc cagctagaac attcaattat tactattacc actactaagg 6180

cagtatgtcc taactcgttc cagatcagcg ctaacttcga ttgaatgtgc gaaatttata 6240

gctcaatatt ttagcactta tcgtattgat ttaagaaaaa attgttaaca ttttgtttca 6300

gtatgtcgga tccatggtgc gctcctccaa gaacgtcatc aaggagttca tgcgcttcaa 6360

ggtgcgcatg gagggcaccg tgaacggcca cgagttcgag atcgagggcg agggcgaggg 6420

ccgcccctac gagggccaca acaccgtgaa gctgaaggtg accaagggcg gccccctgcc 6480

cttcgcctgg gacatcctgt ccccccagtt ccagtacggc tccaaggtgt acgtgaagca 6540

ccccgccgac atccccgact acaagaagct gtcctttccc gagggcttca agtgggagcg 6600

cgtgatgaac ttcgaggacg gcggcgtggt gaccgtgacc caggactcct ccctgcagga 6660

cggctgcttc atctacaagg tgaagttcat cggcgtgaac ttcccctccg acggccccgt 6720

aatgcagaag aagaccatgg gctgggaggc ctccaccgag cgcctgtacc cccgcgacgg 6780

cgtgctgaag ggcgagatcc acaaggccct gaagctgaag gacggcggcc actacctggt 6840

ggagttcaag tccatctaca tggccaagaa gcccgtgcag ctgcccggct actactacgt 6900

ggactccaag ctggacatca cctcccacaa cgaggactac accatcgtgg agcagtacga 6960

gcgcaccgag ggccgccacc acctgttcct gtaggcggcc gctacaacta aacacgactt 7020

ggagtattcc ttgtagtgtt taagatttta aatcttactt aatgacttcg aacgatttta 7080

acgataactt tctctttgtt taactttaat cagcatacat aaaaagcccc ggttttgtat 7140

cgggaagaaa aaaaatgtaa ttgtgttgcc tagataataa acgtattatc aaagtgtgtg 7200

gttttccttt accaaagacc cctttaagat gggcctaatg ggcttaagtc gagtcctttc 7260

cgatgtgtta aatacacatt tattacactg atgcgtcgaa tgtacacttt taataggata 7320

gctccactaa aaattatttt atttatttaa tttgttgcac caaaactgat acattgacga 7380

aggtaccgtc aggcgcgcct aggccggccg atctcggatc tgacaatgtt cagtgcagag 7440

actcggctac gcctcgtgga ctttgaagtt gaccaacaat gtttattctt acctctaata 7500

gtcctctgtg gcaaggtcaa gattctgtta gaagccaatg aagaacctgg ttgttcaata 7560

acattttgtt cgtctaatat ttcactaccg cttgacgttg gctgcacttc atgtacctca 7620

tctataaacg cttcttctgt atcgctctgg acgtcatctt cacttacgtg atctgatatt 7680

tcactgtcag aatcctcacc aacaagctcg tcatcgcttt gcagaagagc agagaggata 7740

tgctcatcgt ctaaagaact acccatttta ttatatatta gtcacgatat ctataacaag 7800

aaaatatata tataataagt tatcacgtaa gtagaacatg aaataacaat ataattatcg 7860

tatgagttaa atcttaaaag tcacgtaaaa gataatcatg cgtcattttg actcacgcgg 7920

tcgttatagt tcaaaatcag tgacacttac cgcattgaca agcacgcctc acgggagctc 7980

caagcggcga ctgagatgtc ctaaatgcac agcgacggat tcgcgctatt tagaaagaga 8040

gagcaatatt tcaagaatgc atgcgtcaat tttacgcaga ctatctttct agggttaaaa 8100

aagatttgcg ctttactcga cctaaacttt aaacacgtca tagaatcttc gtttgacaaa 8160

aaccacattg tggccaagct gtgtgacgcg acgcgcgcta aagaatggca aaccaagtcg 8220

cgcgagcgtc gactctagag gatccccggg taccgagctc gaattcgtaa tcatggtcat 8280

agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa 8340

gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc 8400

gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc 8460

aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact 8520

cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac 8580

ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa 8640

aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg 8700

acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa 8760

gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc 8820

ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct caatgctcac 8880

gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac 8940

cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg 9000

taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt 9060

atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagga 9120

cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct 9180

cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga 9240

ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg 9300

ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct 9360

tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt 9420

aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc 9480

tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg 9540

gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag 9600

atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt 9660

tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag 9720

ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt 9780

ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca 9840

tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg 9900

ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat 9960

ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta 10020

tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca 10080

gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct 10140

taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat 10200

cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa 10260

agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt 10320

gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa 10380

ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtctaagaaa 10440

ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc tttcgtc 10497

Claims (8)

1. A construction method of a high-efficiency fibroin heavy chain pBac vector is characterized in that the pBac vector comprises a pBac-FRF vector, a pBac-HFRF vector, a pBac-HFRS vector and a pBac-HFlRS vector; the pBac-HFRF vector, the pBac-HFRS vector and the pBac-HFlRS vector are insertion type vectors formed by the pBac-FRF vector; the construction of the pBac vector comprises the following steps:

1) carrying out PCR amplification by adopting a specific primer and taking a phSRSV vector as a template to obtain red fluorescent protein DsRed1, an hr3 CQ enhancer and a Ser1pA terminator, respectively cloning the obtained fragments into a pMD19T simple vector to respectively obtain a pDsRed1 vector, a phr3 vector and a pSer1pA vector;

2) carrying out PCR amplification by adopting specific primers and silkworm genome DNA as a template to obtain F (1967), and cloning the obtained F (1967) fragment into a pMD19T simple vector to obtain a pF1967 vector;

carrying out PCR amplification by using a specific primer and a FibHpA terminator as a template, and cloning the obtained DNA fragment into a pMD19T simple vector to obtain a pFibHpA vector;

3) connecting a DsRed1 fragment obtained by enzyme digestion of a pDsRed1 vector by using BamH I and Not I to a pF1967 vector obtained by enzyme digestion of the BamH I and Not I to obtain a pFR vector;

connecting a FibHpA fragment obtained by enzyme digestion of a p-FibHpA vector by Not I and Kpn I to an pFR vector obtained by enzyme digestion of Not I and Kpn I to obtain a pFRF vector;

4) the pFRF vector is digested by AscI to obtain an FRF fragment, and the FRF fragment is inserted into a pBac [3xP3DsRedaf ] transgenic vector to obtain a pBac-FRF vector.

2. The method for constructing the high-efficiency fibroin heavy chain pBac vector as claimed in claim 1, wherein the pBac-HFRF vector comprises the following steps: hr3 was inserted into pFRF vector to obtain pHFRF vector, pHFRF vector was digested with AscI to obtain HFRF fragment, which was inserted into pBac [3xP3DsRedaf ] transgenic vector to generate pBac-HFRF.

3. The method for constructing the high-efficiency fibroin heavy chain pBac vector as claimed in claim 1, wherein the pBac-HFRS vector comprises the following steps: replacing the FibHpA in the pHFRF vector with Ser1pA to generate pHFRS; the pHFRS vector is digested by AscI to obtain an HFRS fragment, and the HFRS fragment is inserted into a pBac [3xP3DsRedaf ] transgenic vector to generate pBac-HFRS.

4. The method for constructing the high-efficiency fibroin heavy chain pBac vector as claimed in claim 1, wherein the pBac-HFIRS vector comprises the following steps: in pHFRS, Exon2 NTD 2AA was replaced with Exon2 to generate vector pHFlRS; the pHFIRS vector is digested by AscI to obtain an HFIRF fragment, and the fragment is inserted into a pBac [3xP3DsRedaf ] transgenic vector to generate pBac-HFIRS.

5. Use of the proficient silk fibroin heavy chain pBac vector of any of claims 1-4 in the preparation of a proficient silk fibroin heavy chain promoter secretion expression system.

6. A high-efficiency silk fibroin heavy chain promoter secretion expression system obtained by using the construction method of the high-efficiency silk fibroin heavy chain pBac vector in any one of claims 1-4, which is characterized in that the preparation method comprises the following steps:

1) injecting the pBac vector and the helper plasmid pHA3PIG into G0 generation eggs at a mass ratio of 1:1, feeding to obtain G0 moth, and mating to obtain G1 generation;

2) and screening positive individuals of eyes or nerves of G1 generation eggs by using a fluorescence microscope to obtain transgenic silkworms, feeding and passaging the obtained transgenic silkworms, and reserving the positive transgenic individuals of pBac-FRF vector, pBac-HFRF vector and pBac-HFRS vector single copy as FRF strains, HFRF strains and HFRS strains respectively.

7. The efficient silk fibroin heavy chain promoter secretory expression system of claim 6, wherein: the insertion sites of the pBac-FRF vector, the pBac-HFRF vector and the pBac-HFRS vector in the pBac vector on the chromosome are chromosome 1, chromosome 25 and chromosome 20 respectively.

8. The use of the high-efficiency silk fibroin heavy chain promoter secretory expression system of claim 6 in the synthesis and secretion of mature recombinant proteins in a silkworm bioreactor.

Images