KR20130006151A - Heat shock protein 70 promoter for silkworm transformation, primers for amplification therof, expression vector comprising promoter, silkworm transformant transformed with expression vector and production method therof - Google Patents

Abstract

PURPOSE: A HSP70 promoter for transforming silkworm, a primer for amplifying the same, and an expression vector containing the promoter are provided to express green fluorescent genes in silkworm eggs and larvae, and to early determine transformation. CONSTITUTION: A HSP70 promoter for transforming silkworm is denoted by sequence number 1, and is isolated in a silkworm. A primer for amplifying the promoter has sequences of sequence numbers 2 and 3. An expression vector for transforming silkworm contains the HSP70 promoter. A method for preparing a silkworm transformant comprises: a step of treating a dormant silkworm egg with hydrochloric acid at 25 deg. C for 1 hour; a step of transducing green fluorescent gene at downstream of the HSP70 promoter(sequence number 1) and cloning fused vector to prepare the expression vector; and a step of microinjecting the expression vector to the silkworm egg.

Description

Heat shock protein 70 promoter for silkworm transformation, primer for amplification thereof, expression vector comprising the promoter, silkworm transformant transformed with the expression vector and a method for producing the same {HEAT SHOCK PROTEIN 70 PROMOTER FOR SILKWORM TRANSFORMATION, PRIMERS FOR AMPLIFICATION THEROF, EXPRESSION VECTOR COMPRISING PROMOTER, SILKWORM TRANSFORMANT TRANSFORMED WITH EXPRESSION VECTOR AND PRODUCTION METHOD THEROF}

The present invention relates to a silkworm transformed heat shock protein 70 promoter, amplification primers thereof, an expression vector comprising the promoter, a silkworm transformant transformed with the expression vector, and more specifically, a silkworm egg It is possible to select transformants from both larvae and larvae so that they can be identified early in their transformation. The silkworm transforming heat shock protein 70 promoter, its amplification primer, the expression vector including the promoter, and the expression vector are transformed with the expression vector. It relates to a silkworm transformant and a method for producing the same.

Silkworm (silkworm, Bombyx) mori ) is a larva of the silkworm moth belonging to the genus Moth, and the larvae hatched from eggs develop into pupae and become adult (moths) to lay eggs and finish their lives.

Silkworm egg (silkworm egg) is a silkworm egg used for the production of cocoons. It is a trait that is determined by genotyping, which occurs once a year in voltinism. ), Two occurrences of Mars, and multiple occurrences of Mars.

The polymorphic silkworms are adapted to tropical or subtropical areas, and all of the silkworms raised in Korea are bisexual. 2 Martian varieties are large, strong eggs, silkworms, pupa, and moths compared to polymorphism.

The first silkworm transformation study was attempted by Nawa et al. Of Japan in 1971 before Drosophila. The silkworm transformation at that time did not have the concept of transposon, so that the whole genomic DNA of the black egg silkworm was selected. An attempt was made to induce black eggs by injecting them into eggs. In this generation, black eggs were induced, but failed because the trait did not transfer to the next generation.

Afterwards, the silkworm was continuously transformed, but continued to fail. In 2000, a donor vector and a helper plasmid were prepared using a piggyBac transposon derived from T. ni cells. Then, by mixing 1: 1 and microinjection to silkworm eggs within 4 hours after spawning before nuclear fission (microinjection) has been successful in silkworm transformation by selecting a silkworm expressing the marker gene in the next generation of silkworm larvae.

Until now, the promoters used for silkworm transformant selection were the actin 3 promoter (BmA3) of silkworms and the 3xP3 promoter specifically expressed in the single eye and nervous system of Drosophila.

When the BmA3 promoter is used for the expression of a marker gene (EGFP) for silkworm transformant selection, EGFP expression can be confirmed in larval, pupal and adult stages by expression control of the BmA3 promoter. Can't.

Therefore, when using the BmA3 promoter, it is disadvantageous that it takes a lot of cost and time because all the subtypes of the G1 stage induced transformation by microinjection must be raised for at least 2 days and 1 day.

In addition, when the 3xP3 promoter of Drosophila is used for EGFP expression, EGFP can be identified from silkworm eggs about 5 days after spawning, which can significantly reduce the cost and time required to select a transformant compared to the BmA3 promoter. Selection of transformants is difficult at the larval stage. Therefore, when the BmA3 promoter is used, normal silkworms and transformed silkworms are mixed in the subgroup of the G1 stage, which is still a considerable cost and time for selection of transformants.

Therefore, there is an urgent need for the development of promoters capable of transformant selection in both silkworm eggs and larvae.

An object of the present invention is to express a green fluorescence gene in both silkworm eggs and larvae can be determined early transformation or transformation of the silkworm heat shock protein 70 promoter, primers for amplification thereof, expression vector comprising the promoter, this expression There is provided a silkworm transformant transformed with a vector and a method for producing the same.

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory and are not intended to limit the invention to the particular embodiments that are described. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory and are not restrictive of the invention, There will be.

Silkworm transformed heat shock protein 70 promoter of the present invention is characterized by consisting of the nucleotide sequence of SEQ ID NO: 1.

The silkworm transformed heat shock protein 70 promoter amplification primers are characterized by consisting of SEQ ID NO: 2 and SEQ ID NO: 3.

Silkworm transformation expression vector of the present invention is characterized in that it comprises a silkworm transformation heat shock protein 70 promoter represented by SEQ ID NO: 1 as described above.

Method for producing a silkworm transformant of the present invention is to prepare a silkworm egg for transforming the dormant egg obtained by mating of female and male moths into a dormant state, the promoter of heat shock protein 70 gene (SEQ ID NO: 1 (C) introducing a green fluorescent gene downstream to clone a fusion-expressed vector to produce an expression vector for silkworm transformation, and micro-injecting the silkworm transgenic expression vector into the prepared silkworm eggs for transfection. Preparing a sieve.

In the silkworm egg preparation step for transformation, the low temperature acid treatment is characterized in that the hydrochloric acid treatment for 1 hour at 25 ℃.

Silkworm transformant of the present invention is transformed through the manufacturing method as described above, characterized in that the green fluorescent gene is expressed in silkworm eggs or larvae.

According to the present invention, there is provided a heat shock protein 70 promoter for silkworm transformation, which expresses a green fluorescent gene in silkworm eggs or larvae, and an expression vector comprising the promoter.

By generating a silkworm transformant using the expression vector, it is possible to select a transformant in both silkworm eggs and larvae, thereby reducing the cost and time required for silkworm transformant selection.

In addition, the development of useful material production technology by the source silkworm transformation technology as described above will reduce the production cost of useful materials such as fluorescent silk, biopharmaceuticals.

1 shows randomly selected 950 cDNAs from a silkworm cDNA gene bank and a dot blot ybridization experiment using the actin 3 cDNA of silkworms as a control.
Source: Actin 3
Arrow: Similar or High Expressing cDNA Clone Compared to Actin3
Figure 2 is a diagram showing the results of the transcript expression analysis by the time of the early embryo development for heat shock protein 70 (bHsp70) isolated from silkworms.
2h: 2 hours after spawning
1d-8d: date passed after spawning
Figure 3a and Figure 3b is a view showing the results of the silkworm tissue and time-specific transcript expression analysis by real-time PCR.
Fa of Figure 3a: Fatty body
Ma of FIG. 3A: Malvertebral Organs
Mi of Figure 3a: Lieutenant General
MS of FIG. 3A: Central Landline
PS of FIG. 3A: Posterior oblique line
Ep of FIG. 3A: Epidermis
Te of Fig. 3A: Testis
Ov of FIG. 3A: Ovarian Tissue
1-8 of FIG. 3B: Development stage of 5-year-old silkworm
FIG. 4 shows the results of comparative analysis of luciferase activity against four deletion constructs of the silkworm heat shock protein 70 promoter.
Figure 5 shows the optimal promoter sequence of the heat shock protein 70 gene for the production of expression vectors for silkworm transformation.
FIG. 6 shows luciferase activity of silkworm heat shock protein 70 promoter versus actin 3 promoter of silkworm and heat shock protein 70 promoter of Drosophila.
White bars: steady state
Black bar: Luciferase activity after treatment for 4 hours at 42 ° C.
7 is a view showing a transition vector for producing a transgenic silkworm.
piggyBacR: Right arm of the piggyBac transfer factor
piggyBacL: Left arm of the piggyBac transfer factor
bHSP70: Silkworm heat shock protein 70 promoter
SV40pA: simian virus 40 polyadenylation signal
dHSP70: Drosophila heat shock protein 70 promoter
dHSP70pA: Heat shock protein 70 polyadenylation signal in Drosophila
Basic vector: pUC18
8 shows EGFP labeling gene expression in silkworm eggs and larvae.
Number: days since spawning

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will now be described in detail with reference to the accompanying drawings.

Silkworm transformation technology is currently developed by Lepidoptera insect Trichoplusia A helper plasmid expressing a transgenic plasmid containing a marker gene GFP (green fluorescent protein) and a transfer factor piggyBac transposase downstream of the silkworm Actin3 promoter using a nitric-derived piggyBac transfer factor plasmids were mixed 1: 1, and then microinjected into silkworm early embryos (1-2 hours after spawning), and transformants were selected with hatching larvae and the presence or absence of GFP expression in the next generation. .

As a result, 695 eggs hatched from 1,058 silkworm eggs that were microinjected, and 425 of them were allied to moths. As a result of the GFP expression assay of the G1 generation obtained by crossing them, silkworms expressing GFP from 3 subpopulations were obtained. Confirmed.

Thus, in order to select a G1 generation transgenic silkworm by the presence or absence of GFP expression, which is a marker gene, it is necessary to raise about 50,000-100,000 silkworms.

The reason why it takes a lot of time and money for the selection of silkworm transformants is that in the silkworm egg state (early embryo), GFP expression by actin 3 promoter regulation is not sufficiently expressed by fluorescence microscopy. It is believed that it is possible to select a transformant only after hatching the silkworm eggs and raising them for a certain period of time.

Therefore, not only the selection of silkworm transformants by direct marker gene expression in silkworm eggs, but also easier selection of silkworm transformants by overexpression of strong marker genes in silkworm larvae and overexpression of useful foreign genes are required. Development of a strong promoter is urgently needed in the larval stage as well as the actin 3 promoter. However, until now, silkworms have not developed strong promoters such as Drosophila heat shock protein 70 promoter.

Thus, the "thermal shock protein 70 gene promoter" of the present invention refers to a novel promoter represented by SEQ ID NO: 1 separated from silkworms, and begins to express from the early embryonic development stage, and operates in all tissues and whole time of silkworms. In particular, it is a promoter specifically expressing silkworm eggs or larvae.

The present invention is not only a heat shock protein 70 gene promoter (hereinafter, referred to as "bHsp70") which is a promoter of SEQ ID NO: 1, but also a heat shock protein 70 promoter activity as a modified sequence in which some bases of SEQ ID NO: 1 are substituted, deleted or added. It may include all of the base sequence that represents.

The present invention also provides a pair of primers consisting of SEQ ID NO: 2 and SEQ ID NO: 3 as a primer for amplifying the heat shock protein 70 gene promoter of SEQ ID NO: 1.

The primer is composed of a forward primer (5'-ATTTAAATTATATAGGTATTGACCCAAATTACTATA-3 ') consisting of 36 bp to specifically amplify bHsp70, a heat shock protein 70 gene promoter, and a reverse primer (5'-GCTAGCCTATAGATTAAAAGACTCCGAAATTAAC-3') consisting of 34 bp.

The primer may include a modification sequence in which a mutation occurs in a part of the individual nucleotide sequences constituting the primer. The modified sequence is a result of performing a PCR using a primer having a mutation and a PCR using a primer (SEQ ID NO: 2 and SEQ ID NO: 3) consisting of an unmodified sequence, a deletion within substantially the same range, It means a substituted or added sequence.

In addition, the present invention provides an expression vector pPIGbHsp70-EGFP (hereinafter referred to as "pPIGbHsp70-EGFP") comprising the heat shock protein 70 gene promoter of SEQ ID NO: 1.

The present invention also provides a transformant transformed with the expression vector pPIGbHsp70-EGFP comprising a promoter of SEQ ID NO: 1.

The present invention introduces a transformation technology using the expression vector pPIGbHsp70-EGFP in the silkworm practical varieties such as Geumokjam, which is currently distributed to the latent farmers, and produces various useful substances such as fluorescent silk and biopharmaceuticals based on this technology. In addition, the following process is useful for producing transformants that can be used to select transformants from both silkworm eggs and larvae, thereby reducing the cost and time required for silkworm transformant selection.

1) Preparation of transformed silkworm eggs

A dormant egg obtained by mating female and male moths was acid-treated to prepare a silkworm egg prepared in a dormant state.

At this time, after breeding females and females of the dormant system, the laid eggs are hydrochloric acid treated at low temperature (25 ° C.) for 1 hour to wake up dormant to produce silkworm eggs in a dormant state.

At this time, when the hydrochloric acid treatment is less than 1 hour, the hydrochloric acid treatment is not performed well, so it is difficult to awaken the dormant state, and when the treatment is performed for more than 1 hour, the hatching rate is lowered.

It is preferable to use Geumjam (home silk moth) as the silkworm practical varieties, but other practical article types can also be used.

2) Preparation of expression vector (carrier) for silkworm transformation

As a silkworm transformation vector for silkworm transformation, a green fluorescence gene is introduced downstream of the promoter of the heat shock protein 70 gene (SEQ ID NO: 1) to produce a cloned vector.

3) Silkworm transformant preparation

A silkworm transformant is prepared by microinjecting the silkworm transformed expression vector into the prepared silkworm eggs.

A silkworm transformant is prepared by microinjecting the silkworm transgenic expression vector into the silkworm egg for transformation of 1) to induce transformation, thereby expressing a green fluorescent gene in the eyes or larvae of the silkworm.

In this case, when inducing the transformation, first attach the silkworm eggs to the slide glass in a directional manner, and then first drill a hole in the hard eggshell of the silkworm eggs using a fine tungsten needle, and then a glass capillary in the hole. By inserting the silkworm transformation vector and the helped plasmid mixed solution in the silkworm egg is good to induce.

Hereinafter, the present invention will be described in detail with reference to Examples, but these are not intended to limit the scope of the present invention.

Example 1 Selection of High Expressing Gene Compared to Silkworm Actin 3 Gene

In order to select a high-expression gene compared to the conventional silkworm actin 3 gene, first, total RNA was isolated from the larvae of silkworm 5 days and 3 days, and then purely poly (A) + RNA was isolated.

Single-chain cDNA and double-chain cDNA were synthesized sequentially using purely isolated poly (A) + RNA, and EcoR I and Xho I restriction enzyme recognition sequences were assigned to the 5'-end and 3'-end, respectively.

The cDNA thus prepared was directionally inserted into a Lambda ZAP II carrier containing pBluescript SK (-) Plasmid, followed by in vitro packaging to prepare cDNA gene bank. The pfu (plaque forming unit) of the constructed cDNA gene bank was 1.2 × 10 ⁶ .

950 cDNA phage clones randomly selected from the prepared cDNA gene bank were converted into plasmid pBluescript SK (-) using in vivo excision, and then hybrid-dot manifold system (hybri). -dot manifold system), the control actin 3 gene was blotted at the A1 position, and randomly selected clones were blotted at the remaining positions.

Hybridization was performed using single-stranded cDNA synthesized using poly (A) + RNA isolated from silkworms at 5 days and 3 days of the bled 10 nylon membranes as a probe.

As a result, as shown in FIG. 1, cDNA clones showing similar or stronger signals than actin 3 of silkworms were selected.

<Example 2> Partial sequencing and transcriptome expression analysis of high-expressing clones compared to silkworm actin 3 gene

Partial sequencing of the similar or high expression clones selected from the silkworm actin 3 gene was performed using an automatic base sequence analysis device (Perkin Elmer), and then analyzed the nucleotide sequence of the NCBI gene bank database (Blast). -X) was performed to carry out homology analysis with the genes previously found.

As a result of homology analysis, the selected clone showed high homology with the heat shock 70 kDa protein (hereinafter, referred to as 'bHsp70') gene.

As shown in FIG. 2, the bHsp70 gene began to be expressed from the early embryonic development stage and was compared to actin 3 (BmA3) of silkworms during the entire embryonic development as shown in FIG. 2. We could confirm that we expressed strongly

In addition, as shown in Figures 3a and 3b, as a result of performing transcript expression analysis by real-time PCR for each of the stages of development of silkworm tissue and silkworm silkworms for the clones, the expression of each tissue is somewhat different expression by tissue However, the expression was confirmed in all the tissues tested, and the expression of transcripts in each developmental stage was gradually increased as time elapsed.

Therefore, the clone could be estimated to be expressed in all tissues and developmental time of silkworm similarly to actin3.

<Example 3> Preparation of eight deletion construct carriers to determine the active site of the thermal shock protein 70 promoter

In order to secure the entire genome gene of heat shock protein 70 clone, the silkworm genomic gene bank was screened using this cDNA probe to select phage clones containing the genomic gene of heat shock protein 70.

Phage DNA was isolated from the selected phage clones, co-treated with restriction enzymes EcoR I and Xho I and sub-cloned to the EcoR I and Xho I positions of the plasmid carrier pBluescript SK (-).

The sequencing of each subclone was performed using an automatic sequencing device (Perkin Elmer) to determine the total sequencing.

In order to determine the active site of the isolated thermal shock protein 70 promoter, 8 forward primers and 1 reverse primer were prepared as shown in Table 1 below.

Table 1 is a primer sequence for the production of eight deletion construct carriers for determining the active site of the heat shock protein 70 promoter, the underline of ACGCGT represents the recognition sequence of the restriction enzyme Mlu I, CTCGAG recognition of restriction enzyme XhoI The sequence is shown.

Eight forward primers prepared as described above were synthesized by adding ACGCGT, which is the recognition sequence of restriction enzyme MluI, and one reverse primer, which was added to the recognition sequence of restriction enzyme XhoI, which added CTCGAG. ) To synthesize.

Each of the synthesized forward and reverse primers was used for PCR reactions based on Fiji DNA.

Eight PCR products amplified in the PCR reaction were cloned into pGEM-T easy carriers, respectively, to identify eight deletion constructs: pGEMT-bHsp70-D1, pGEMT-bHsp70-D2, pGEMT-bHsp70-D3, and pGEMT. -bHsp70-D4, pGEMT-bHsp70-D5, pGEMT-bHsp70-D6, pGEMT-bHsp70-D7 and pGEMT-bHsp70-D8 were prepared.

The eight deletion constructs were treated with restriction enzymes Mlu I and Xho I at the same time to obtain eight insertion genes, and then pGL3-, a promoter-free carrier containing a firefly luciferase gene as a marker gene. It was introduced between Mlu I and Xho I, the restriction enzyme positions of the basic carrier, and the eight types of promoter activity assays, pGL3-bHsp70-D1, pGL3-bHsp70-D2, pGL3-bHsp70-D3, pGL3-bHsp70-D4, and pGL3- bHsp70-D5, pGL3-bHsp70-D6, pGL3-bHsp70-D7 and pGL3-bHsp70-D8 were prepared.

On the other hand, the preparation of a control vehicle for normalization of eight deletion constructs for the thermal shock protein 70 promoter was performed by the SV40 promoter in a pRL-SV40 carrier containing a Renilla luciferase gene as a marker gene. Was removed by treatment with restriction enzymes Kpn I and Hind III, and then the heat shock protein 70 promoter of Drosophila was introduced to prepare pRL-dHsp70-Rluc, a control carrier that can standardize firefly luciferase activity.

PGL3-bHsp70-D1, pGL3-bHsp70-D2, pGL3-bHsp70-D3, pGL3-bHsp70-D4, pGL3-bHsp70-D5, pGL3-bHsp70-D6, pGL3-bHsp70-D7 Luciferase activity assays for pGL3-bHsp70-D8 were performed using the Dual-Luciferase Reporter Assay System.

PGL3-bHsp70-D1, pGL3-bHsp70-D2, pGL3-bHsp70-D3 and pGL3-bHsp70-D4, pGL3-bHsp70-D5, pGL3-bHsp70-D6, pGL3-bHsp70-D7, pGL3-bHsp70-D8 and 400 ng for each pGL3-Basic carrier and 40 ng of the control carrier, pRL-dHsp70-Rluc, were mixed in 25 μl of sterile distilled water, and then 2 μl of intracellular nucleic acid transduction reagent (FuGENE HD Transfection Reagent) was mixed again. The Bm5 silkworm cultured cell line was grown to.

After 48 hours of treatment, the culture medium was removed, washed with 1 × PBS solution, and 100 µl of 1 × passive Lysis Buffer was added thereto, followed by rotation stirring for 15 minutes to decompose the cultured cells.

Dispense 100 μl of 1 × Firefly Luciferase Assay Reagent II into 6 wells in a 96 well white plate, and then prepare each cell lysate prepared above in each well. After mixing 20 μl, firefly luciferase activity was measured for each deletion construct using a luminometer.

In order to normalize Firefly luciferase activity, immediately after firefly luciferase activity measurement, 100 μl of firefly luciferase activity inhibitor and Renilla luciferase activity measurement solution (1 × Stop & Glo Subtrate) were added, followed by using a luminometer. Renilla luciferase activity was measured for each deletion construct. Luciferase activity measurements were performed in three replicates.

The firefly luciferase measurements for each deletion construct measured by the luminometer were normalized by dividing by the Renilla luciferase measurements measured for each deletion construct.

At this time, the standardization value of pGL3-bHsp70-D1, the deletion construct, was set to 100, and then the relative deletion construct was calculated as a relative value.

As a result, as shown in FIG. 4, the relative value of pGL3-bHsp70-D2 was 129.9%, and the relative values of pGL3-bHsp70-D3 and pGL3-bHsp70-D4 were 102.2% and 92.2%, respectively. After D5, promoter activity was rapidly decreased. And when only pGL3-Basic carrier was introduced, the relative value was very low (0.3%).

Therefore, as shown in Figure 5, the appropriate promoter region of the heat shock protein 70 gene introduced into the carrier for the production of silkworm transformant was determined to be up to the D4 (-503 ~ +147) base position.

That is, the size of the silkworm transformant carrier has a great influence on the transformation efficiency. In particular, as the size of the carrier increases, the transformation efficiency decreases. Therefore, although D4 exhibited somewhat lower promoter activity than D1, D2, and D3, it was determined that D4 is an optimal sequence that can be used for silkworm transformation carrier production in consideration of the silkworm transformation efficiency.

In addition, using the same method as shown in Figure 4, the activity intensity of the silkworm heat shock protein 70 promoter (pGL3-bHsp70-D4) compared to the silkworm actin 3 promoter (pGL3-BmA3) and Drosophila heat shock protein 70 promoter (pGL3-dHsp70) Was measured. Luciferase activity measurements were performed in three replicates.

As a result, as shown in Figure 6, the luciferase activity of the thermal shock protein 70 promoter (pGL3-bHsp70-D4) was 187, respectively, compared to the silkworm actin 3 promoter and the Drosophila heat shock protein 70 promoter under normal conditions (25 ° C) without thermal shock. It was found to be 3.3 times higher and 3 times higher than the silkworm actin 3 promoter and 2.4 times higher than Drosophila heat shock protein 70 promoter under heat shock at 42 ° C. for 4 hours.

<Example 4> Construction of a carrier capable of selecting a transformant in both transformed silkworm eggs and larvae

In the silkworm heat shock protein 70 promoter, a transfection vector for silkworm transformation was constructed in which the sequence of FIG. 5 was introduced.

The base vector was a pBac3xP3EGFP vector that was supplied. In order to introduce the FIG. 5 sequence from the silkworm heat shock protein 70 promoter instead of the 3xP3 promoter specifically expressed in the insect eye and nervous system of the base vector, the front of the 3xP3 promoter using a Muta-Direct ™ Site Directed Mutagenesis Kit from iNtRON The ATCTAATT sequence in 5'-) was replaced with ATTTAAAT, the recognition sequence of restriction enzyme SwaI, and the GGTCGC sequence in the rear (3'-), GCTAGC, recognition sequence of restriction enzyme NheI.

In order to amplify the bHsp70-D4 promoter sequence (FIG. 5) by PCR, a forward primer was synthesized with 5'-ATTTAAATTATATAGGTATTGACCCAAATTACTATA-3 'by adding ATTTAAAT, a recognition sequence of restriction enzyme SwaI, and a reverse primer, a recognition sequence of restriction enzyme NheI. GCTAGC was added to synthesize 5'-GCTAGCCTATAGATTAAAAGACTCCGAAATTAAC-3 '. Each of the synthesized forward and reverse primers was used for PCR reactions using the pGL3-bHsp70-D4 vector as a template. PCR products amplified in the PCR reaction were cloned into pGEM-T easy carrier (Promega, USA) to prepare pGEMT-SwaI-bHsp70-D8-NheI. To replace the 3xP3 promoter in the pBac3xP3EGFP vector with the bHsp70-D4 promoter, pBac3xP3EGFP and pGEMT-SwaI-bHsp70-D8-NheI were treated with restriction enzymes SwaI and NheI, respectively, and the insertion gene bHsp70-D4 was restricted to the pBac3xP3 enzyme of pBac3xP3. The pPIGbHsp70-EGFP of the present invention was finally prepared by introducing between positions SwaI and NheI (FIG. 7).

Example 5 Preparation of Silkworm Transformant by Microinjection

Microinjection for silkworm transformation was performed as follows.

Male moths of the 140 varieties of the silkworms (Pam 140 × Jam 125) and female moths of the 125 varieties of silkworm were bred in the dark at room temperature for 4 hours and then stored in the dark at 4 ° C. for 12 hours.

The mated female moths were laid on a 0.2 mm polyvinyl chloride (PVC) plastic cover for 60 hours in a dark room at 25 ° C, and the eggs were collected using a separable Buchner Funnels PP.

The collected silkworm eggs were washed twice with running water and then 3M paper was used to remove excess water.The silkworm eggs were soaked in hydrochloric acid solution with specific gravity of 1.0955 at 25 ℃, and then kept in a 25 ℃ thermostat for 1 hour. Pickled.

Pickled silkworms were washed in running water for 30 minutes and then dried at room temperature. The dried eggs were attached to the slide glass in four rows of 15 pieces with a vinyl acetate resin in a directional manner, followed by microinjection of a 1: 1 mixture of a pPIGbHsp70-EGFP transition vector and a helper plasmid in silkworm eggs to induce transformation.

After micro-injection in a total of 3,060 silkworm eggs, put the silkworm eggs in a moisturized petri dish at 25 ℃ and examined the hatching rate, as shown in Table 2 below.

Carrier Micro injection
to know hatching
Days imago
Number Mating
Imago Transformation
Aggus pPIGbHsp70-EGFP 3,060 772 280 122 38

As shown in Table 2, the hatching rate after microinjection in silkworm eggs of the transforming carrier was 25%, the transformation rate was analyzed by fluorescence microscopy. Transformation rate compared to mating appendix was 31%.

Example 6 Expression of Marker Gene (EGFP) in Both Silkworm Eggs and Larvae

Silkworm transformation was analyzed by expression microscopy of EGFP expression by fluorescence microscopy.

As a result, as shown in Figure 8, when using the bHsp70 promoter for the expression of a marker gene (EGFP) for the selection of silkworm transformants, it was possible to confirm the expression of EGFP from the 3rd day after spawning, the number of days increased until 8 days after spawning EGFP expression increased. However, on day 9, just before hatching, the expression of EGFP decreased dramatically. This could be due to epidermal formation. Also, hatching larvae (ant worms) were able to clearly identify EGFP expression.

The silkworm transformation carrier was microinjected into the silkworm eggs of the cultivars Geumokjam, to induce transformation, and then confirmed the expression of green fluorescent gene (EGFP) in both silkworm eggs and larvae of the next generation. The cost and time required for screening were greatly reduced. In addition, when using this technique, you can observe the transformation in silkworm eggs two days before the 3xP3 promoter of Drosophila. There is an economic advantage that you do not have to raise silkworms by 1 day. The selection of transformants in both silkworm eggs and larvae is our own original source technology.

The present invention has been described with reference to the preferred embodiments, and those skilled in the art to which the present invention pertains to the detailed description of the present invention and other forms of embodiments within the essential technical scope of the present invention. Could be. Here, the essential technical scope of the present invention is shown in the claims, and all differences within the equivalent range will be construed as being included in the present invention.

<110> REPUBLIC OF KOREA (MANAGEMENT: RERAL DEVELOPMENT ADMINISTRATION) <120> HEAT SHOCK PROTEIN 70 PROMOTER FOR SILKWORM TRANSFORMATION,          PRIMERS FOR AMPLIFICATION THEROF, EXPRESSION VECTOR COMPRISING          PROMOTER, SILKWORM TRANSFORMANT TRANSFORMED WITH EXPRESSION          VECTOR AND PRODUCTION METHOD THEROF <130> P2011-2079 <160> 3 <170> Kopatentin 2.0 <210> 1 <211> 651 <212> DNA <213> Bombyx mori <400> 1 tatataggta ttgacccaaa ttactatact tttattatca cagtaattat gtacacattt 60 taactatagt ttccaaaaat ttacaaattt ggtggcagat ggaaagaaac aaacaaaaat 120 aaggtatcaa aatgtaatta ttacattctg ttttttgagc taggaaagac ataactgtct 180 gaatggtaca tatataggaa tgtatttgaa aacatttttt gtattttaat aatatttttt 240 ttagtttttt tttaatgttg cacaattttg catgtaaaac tttcataatt catcatactc 300 ttatccaaga atttgattat gccgcccttt aactttccac tcaaaggcca cttagaaacg 360 tgggccaatt ggaatgatcc agatcgactt acttgtacca attatacaca ctgtacagct 420 acgtcatcac aaagtggacc aatagtaatc gcccacgatg aacactagcg ctaaataata 480 gcgacggcag cgtgtcaaac ttcagttttt cttgaacaat tgaacgaaaa gtattggtgt 540 actgtttgaa tacgcaagtg tttgataaaa gcattttaaa gtgatcttat cgtggctgtg 600 aatataattt tatttagatt tttgttaatt tcggagtctt ttaatctata g 651 <210> 2 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 atttaaatta tataggtatt gacccaaatt actata 36 <210> 3 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 gctagcctat agattaaaag actccgaaat taac 34

Claims (6)

Represented by SEQ ID NO: 1, isolated from silkworms,
Silkworm transformed heat shock protein 70 promoter.
A primer for amplifying a heat shock protein 70 promoter for silkworm transformation, comprising SEQ ID NO: 2 and SEQ ID NO: 3.
Silkworm transformed heat shock protein represented by SEQ ID NO: 1 comprising a 70 promoter,
Expression vector for silkworm transformation.
Preparing a transgenic silkworm egg prepared in a non-dormant state by acid treatment of the dormant egg obtained by mating female and male moths;
Preparing a silkworm transformation expression vector by cloning the fusion-expressed vector by introducing a green fluorescent gene downstream of the promoter of the heat shock protein 70 gene (SEQ ID NO: 1);
And producing a silkworm transformant by micro-injecting the silkworm transformed expression vector into the prepared silkworm eggs.
Method for producing silkworm transformants.
5. The method of claim 4,
In the silkworm egg preparation step for transformation, the low temperature acid treatment is made of hydrochloric acid treatment at 25 ℃ for 1 hour,
Method for producing silkworm transformants.
Transformed through the method of any one of claims 4 or 5,
In which green fluorescent genes are expressed in silkworm eggs or larvae,
Silkworm transformant.

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