CN104212833A - Novel genetically modified plasmid vector, construction method and cultivation method of silkworms containing novel recombination gene in vivo - Google Patents

Abstract

The invention relates to a new type of transgenic carrier and its construction, and a method for cultivating silkworm containing a new recombinant gene in the body. The new type of transgenic silkworm has a recombined silk fibroin heavy chain protein gene containing an MMP enzyme cleavage site. This invention belongs to agriculture The field of breeding and utilization of fine insect varieties; the flow chart of the construction of new transgenic vectors is shown in the attached figure; using the characteristics that the MMPs restriction site can be digested by MMPs, the MMPs restriction site is introduced into the silkworm silk fibroin, so that the silkworm silk fibroin has a new performance, to achieve the purpose of modifying silkworm silk fibroin through biological methods; this patent clones the recombinant silk fibroin gene containing MMPs restriction sites into the PiggyBac transposon carrier and injects the carrier by microinjection Inside the silkworm eggs of the silkworm, after the silkworm eggs are hatched, the newly bred transgenic silkworms are determined by selecting individuals with green fluorescent eyes.

Description

A Novel Transgenic Plasmid Vector and Its Construction Method, and a Method for Cultivating Bombyx mori Containing Novel Recombinant Genes in Its Body

technical field

The invention belongs to the field of cultivating and utilizing fine varieties of agricultural insects, and in particular relates to the field of a method for cultivating novel recombinant gene silkworms.

Background technique

As a natural material, silk has strong advantages in the field of biomedicine, such as biocompatibility, safety, degradability, good mechanical properties, etc. As a medical material, silk has many requirements and also has some limitations. For example, as a tissue engineering scaffold, its degradation rate is required to be consistent with the growth rate of tissue cells, but the existing silk cannot be better controlled in a suitable time. degraded; as a drug delivery system, the identification of the drug action site on silk itself is very low, if silk has a better guiding effect, it will play a greater role in the drug delivery process; when silk participates in the treatment of tumors and other diseases , as a sensor, the fast response time needs to be further explored. Most of the silk currently used is natural silk, and the modification of silk protein by transgenic technology at the genetic level will make the modified silk protein have better biocompatibility, degradability and unique drug delivery targeting properties. Has good potential value.

Matrix metalloproteinases (MMPs) are the most important enzyme system for degrading the extracellular matrix (ECM), accounting for more than 70% of the total activity of ECM degrading enzymes, and the activation of MMPs is considered to be the rate-limiting rate of ECM degradation. link. Utilizing the feature that MMPs cleavage sites can be cleaved by MMPs, relevant technological innovations have appeared, such as the invention patent of Huang Dinan et al.: matrix metalloproteinase-directed recombinant human tumor necrosis factor fusion protein and its preparation (patent application number: 2007100280375). But there is no report about introducing MMPs restriction sites into recombinant silk fibroin heavy chain protein to construct a new type of silk fibroin. At present, there are physical methods for modifying silk fibroin, such as making silk into microspheres/nanoparticles, etc., but physical modification cannot change the biochemical properties of silk fibroin. There are also chemical methods, such as graft modification of silk fibroin, etc. The processing and modification of silkworm silk fibroin by chemical methods will consume more energy and even cause environmental pollution.

Contents of the invention

The purpose of the present invention is to breed a new type of transgenic silkworm, which has a recombined silk fibroin heavy chain protein gene, and this recombined silk fibroin heavy chain protein contains an MMP enzyme cleavage site, which solves the problem of energy consumption of chemical methods. Many, environmental pollution problems, physical methods can not change the biochemical properties of silk fibroin

Technical solution of the invention: This patent introduces the MMP enzyme cleavage site into the recombinant silk fibroin heavy chain protein gene, constructs a new type of silkworm transgenic plasmid vector, and injects this new type of vector into the production within a certain period of time by microinjection. In the silkworm eggs, by observing the fluorescence phenomenon of the moth's eyes to identify whether the transgene is successful, a new type of transgenic silkworm is bred. The basic technology of MMP enzyme cleavage site achieves the purpose of silk fibroin modification.

The first purpose of the present invention is to use gene cloning technology to construct a novel silkworm transgenic plasmid vector, which has a recombined silk fibroin heavy chain protein gene, and this recombined silk fibroin heavy chain protein contains an MMP enzyme cleavage site point, achieved through the following schemes:

First, the silkworm (Jingsong) genome was extracted by ethanol precipitation, and then the N-terminal and C-terminal gene sequence fragments of the heavy chain of the silkworm were amplified by PCR with the designed primers, and then the pMD-18T simple vector ligation technology, PCR technology, and enzyme digestion technology were used , T4 DNA ligase technology to construct a new silkworm transgenic plasmid vector (see Figure 1 for the construction flow chart).

The second purpose of the present invention is to inject the constructed novel silkworm transgenic plasmid carrier into the silkworm eggs laid within a certain period of time by microinjection, and to identify whether the transgene is successful by observing the fluorescence phenomenon in the eyes of the silkworm moth, and to cultivate a new type of silkworm. The transgenic silkworm, the silkworm genome has a recombinant silk fibroin heavy chain protein gene, and this recombinant silk fibroin heavy chain protein contains an MMP enzyme cleavage site, which is achieved by the following scheme:

Firstly, the newly constructed silkworm transgenic plasmid vector pBac[3×P3-EGFPafm]-FibH-GS-site and Piggybac Help Plasmid were mixed in equal amounts, and the mixed plasmid was injected into silkworm eggs laid within a certain period of time by microinjection. middle. The success of the transgene was identified by observing the fluorescence phenomenon of the eyes of the offspring silkworm moths, and a new type of transgenic silkworm was obtained through genomic PCR and sequencing identification.

The beneficial effects of the present invention are:

1. In the technology provided by the present invention, the N-terminal and C-terminal genes of the silk fibroin heavy chain in the silkworm (Pine pine) genome and the restriction site gene sequence of matrix metalloproteinase are cloned into the piggyBac vector, and the method of microinjection is adopted The transgenic silkworm eggs have achieved the effect of cultivating a new type of transgenic silkworm. The novel transgenic silkworm has a recombined silk fibroin heavy chain protein gene, and the recombined silk fibroin heavy chain protein contains an MMP restriction site.

2. In the technology provided by the present invention, the green fluorescent protein gene transcribed under the control of the eye-specific promoter 3×P3 promoter is placed in the piggyBac vector and transferred into silkworm eggs together with the recombinant silk fibroin heavy chain protein gene. The effect of intuitively separating and cultivating the successful novel transgenic silkworm has been achieved.

3. Modification of silkworm silk fibroin by biological methods can reduce the steps of silk fibroin product processing, thereby reducing energy consumption.

Description of drawings

Fig. 1 is used to construct the flowchart of a kind of novel silkworm transgenic plasmid vector

Figure 2 PCR verification of the new silkworm transgenic plasmid vector

Figure 3 Observation of silk moths in the control group and the experimental group under a stereo microscope

Figure 4PCR amplification detection transgenic silk moth genome

Fig. 5 Sequencing results of the fragment Fib H-GS-site in the genome of the transgenic silkworm moth.

Detailed ways

The present invention will be further described in conjunction with specific embodiments. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the invention.

Example 1

The invention adopts gene cloning technology to construct a novel silkworm transgenic plasmid carrier, the plasmid carrier has a recombined silk fibroin heavy chain protein gene, and the recombined silk fibroin heavy chain protein contains an MMP restriction site. Specific steps are as follows:

1. Design a new silkworm transgenic plasmid vector construction process

The flow chart designed to construct a novel silkworm transgenic plasmid vector is shown in Figure 1. Among them, fibroin H refers to silk fibroin heavy chain gene, PCR refers to polymerase chain reaction technology, FibH-N refers to silk fibroin heavy chain protein N-terminal gene sequence, A, B, F refer to Asc I, BamH I, Fse I enzyme digestion site, pMD-FibH-N refers to the pMD-18T simple vector with FibH-N cloned (purchased from Takara Company), and FibH-GS-site-C refers to the C-terminus of silk fibroin heavy chain protein with MMPs restriction site gene sequence.

2. Genome Extraction of Bombyx mori (Jingsong Song)

Take the midgut of the 5th instar three-day-old silkworm (Jingsong), put it into a pre-cooled mortar, add liquid nitrogen and quickly grind it into powder; scrape the powder into a 1.5ml EP tube, add extraction buffer (10Mm Tris -Cl (PH8.0), 0.1M EDTA (PH8.0), 0.5% SDS) 700μl, place at room temperature for 1h, then add proteinase K to a final concentration of 500μg/mL, and add RNase to a final concentration of 50μg/ml, Place in a water bath at 56°C for 5 hours (shake every 1 hour); take out the digested sample, add an equal volume of phenol, shake well, centrifuge at 12,000 rpm for 10 minutes, take 650 μl of the supernatant into a new 1.5ml EP tube; add Equal volume of phenol/chloroform/isoamyl alcohol (25:24:1), shake well, centrifuge at 12000rpm for 10min, take 600μl of the supernatant into a new 1.5ml EP tube; add an equal volume of phenol/chloroform (1:1 ), shake well, centrifuge at 12000rpm for 10min, take 550μl of the supernatant into a new 1.5ml EP tube; add an equal volume of chloroform, shake well, centrifuge at 12000rpm for 10min, take 500μl of the supernatant into a new 1.5ml EP tube; Add 900 μl of pre-cooled absolute ethanol, then add 100 μl of 3M NaAC (PH5.2), freeze in a -20°C refrigerator for 15 min; centrifuge at 12,000 rpm for 10 min, precipitate DNA, and discard the supernatant; wash the precipitate twice with 1 ml of 70% ice ethanol, Then discard the supernatant as much as possible, dry it naturally to evaporate the ethanol; add 40 μl dd H2O to sterilize dd H2O, dissolve the precipitate for 2 hours or overnight at 4°C, and store it at -20°C for later use.

3. Polymerase chain reaction (PCR) amplification of the target sequence of silk fibroin heavy chain protein gene

(1) Primer design

Using the silkworm (Jingsong) genome as a template, use Primer Premier5.0 to design the following primers:

Wherein Fib-H-p1 and Fib-H-p2 are primer pairs used to amplify silk fibroin heavy chain N-terminal gene, and Fib-H-p1 and Fib-H-p2 are upstream primers and downstream primers respectively. Big-o-p3 and Fib-H-p4 are primer pairs used to amplify the C-terminal gene of silk fibroin heavy chain, Big-o-p3 and Fib-H-p4 are upstream primers and downstream primers respectively, Big-o -The p3 primer contains the MMPs restriction site sequence.

(2) PCR amplification: PCR amplification was carried out on the extracted silkworm genome using the designed primers to obtain the N-terminal and C-terminal gene sequence fragments of silk fibroin heavy chain. The PCR amplification system is as follows:

PCR conditions: 94°C for 5min; 94°C for 30s; 56°C for 30s; 72°C for 1min/1kb; 72°C for 10min; 12°C forever.

4. Construction of a new silkworm transgenic plasmid vector

(1) The PCR products of primers Fib-H-p1 and Fib-H-p2 were directly connected to the pMD-18T simple vector to construct the plasmid pMD-FibH-N, and the reaction system was: system: solution I5μl;

Target fragment 4.5μl;

simpleT-180.5μl;

Conditions: 16°C connection for 1-2h

(2) Preparation of competent cells (calcium chloride method)

Pick a single colony point from the plate of the cultured bacteria, transfer it to two test tubes containing 3-5ml LB medium, and shake the bacteria at 210rpm on a 37°C shaker for 12-16h; prepare 15% glycerol bacteria from the two tubes of cultured bacteria ( 425 μl of bacterial solution + 75 μl of glycerol (glycerol needs to be pre-sterilized and stored at 4 degrees), stored at -70°C; take 200 μl from No. 1 test tube and add it to 200ml of LB medium, shake the bacteria at 210rpm at 37°C for about 6 hours , shake until the OD value is 0.498, divide the culture into 6 tubes of sterile pre-cooled 40ml centrifuge tubes on a sterile ultra-clean bench, 30ml per tube, place on ice for 10min; 4°C, 4000rpm, 10min centrifugation, Pour off the culture solution on the ultra-clean platform, and invert it on the filter paper, remove the culture solution, cover it and put it on ice; add 1ml0.1MMgCl2 to each tube, mix in one tube, ice bath for 20min, 4000rpm, and centrifuge for 10min; Pour off the culture medium on the clean platform, and invert it on the filter paper, remove the culture medium, cover it and put it on ice; add 6ml of 0.05M ice-cold calcium chloride (after high-temperature sterilization and store at -20°C), and shake the precipitate gently Gently place on ice for 30min; 4°C, 4000rpm, centrifuge for 10min, pour off the culture medium on the ultra-clean platform, and invert it on the filter paper for 1min, remove the culture medium, cover it and put it on ice; add 2ml0. 1M ice-cold calcium chloride, then add 1ml 50% glycerol (final concentration 15%), mix the precipitate gently, and put it in an ice bath; divide the competent bacterial solution into EP tubes as soon as possible, quickly seal, 200μl per tube, -70℃ save.

(3) Transformation and storage of bacteria

Take out the competent cells from the refrigerator at -70°C, bathe in ice until they melt, transform one competent cell with one ligation product, and keep one competent cell as a negative control, add 10ul of the ligation product to the competent cells, and place on ice for 5 -10min, heat shock at 42°C for 90s (do not move), quickly transfer to ice and place for 1-2min, add 800μl of LB medium on a sterile ultra-clean table; , Centrifuge for 4min; operate the following steps in the ultra-clean platform: Discard 800μl of the supernatant respectively, mix the remaining precipitates evenly, and spread them on the plates respectively; incubate in a constant temperature incubator at 37°C for 12-18h, and observe the colonies; use a test tube (4ml LB Culture medium, with antibiotics), 37°C, 210rpm, shaking for 14 hours, after PCR verification, glycerol bacteria were stored (15-30% glycerol, 85-70% bacterial liquid), labeled with glycerol bacteria, and stored at -70°C.

(4) Plasmid extraction

Operate according to the instructions of the plasmid extraction kit, take 1-2ml of overnight cultured bacterial solution, centrifuge at 12000rpm for 1min, discard the supernatant, add 200μl Solution I, fully suspend the bacteria with a pipette tip or a shaker; add 200μl Solution II, immediately gently and Fully turn up and down and mix 4-6 times to fully lyse the bacteria until a clear egg white-like solution is formed. This step should not exceed 5 minutes. Add 350μl Solution III, gently and thoroughly mix up and down 8-10 times, place at room temperature for 2-5min, centrifuge at 12000rpm for 10min. Transfer the supernatant to a GenClean Column set in a 2ml collection tube, centrifuge at 6000rpm for 1min at room temperature, take out the GenClean Column, and discard the waste liquid in the collection tube. Put the GenClean Column back into the collection tube, add 500 μl Solution IV, centrifuge at 12000 rpm for 1 min at room temperature, and discard the waste liquid in the collection tube. Put the GenClean Cloumn back into the collection tube, add 500μl Wash Solution, centrifuge at 12000rpm, room temperature for 1min, and discard the waste liquid in the collection tube. repeat. Pour off the waste liquid in the collection tube, centrifuge at 12000rpm at room temperature for 1min, and completely remove the WashSolution. Put the GenClean Column into a clean 1.5ml centrifuge tube, add 50μl-70μl of Elution Buffer to the center of the GenClean Cloumn membrane, and place it at 37°C for 2min. Centrifuge at 12000rpm at room temperature for 1min. The liquid in the centrifuge tube is the solution containing the target plasmid. Take 2-5μl for agarose gel electrophoresis detection. The purified DNA can be used immediately for subsequent experiments or frozen at -20°C.

(5) DNA agarose gel electrophoresis and gel recovery

Electrophoresis conditions:

Prepare 1% agarose gel, 250bp DNA Marker, voltage 120V, constant voltage electrophoresis.

Glue recovery steps:

Operate according to the instructions of the agarose gel recovery kit, and estimate the concentration of the recovered sample according to the electrophoresis conditions.

(6) Perform double enzyme digestion on the extracted plasmid pMD-FibH-N and the PCR product FibH-GS-site-C recovered by agarose gel electrophoresis and gel recovery. The enzyme digestion system is as follows:

Digestion conditions: 37°C, 3-5h.

After enzyme digestion, the FibH-N sequence and FibH-GS-site-C sequence were recovered from the gel. And ligated with T4 DNA ligase.

The connection system is as follows:

System: The molar ratio of fragments is 1:1; FibH-N sequence and FibH-GS-site-C sequence are each 4 μl.

T4 DNA ligase 1μl;

T4 DNA ligase buffer 1μl

Condition: 16°C connection 14-20h

(7) After the gel recovery of the ligation product in step 6, use the primer pair (Fib-H-p1 and Fib-H-p4) to PCR amplify the recombinant silk fibroin heavy chain protein gene containing the MMPs restriction site.

(8) Perform double enzyme digestion on the gel recovery product of the PCR product in step 7 and the transgenic vector plasmid pBac[3×P3-EGFPafm]. The enzyme digestion system is as follows:

Digestion conditions: 37°C, 3-5h.

(9) Retrieve the target sequence by gel after enzyme digestion. T4 DNA ligase was used to connect to construct a new silkworm transgenic plasmid vector, and the ligated product was transformed into competent cells.

The connection system is as follows:

System: The molar ratio of fragment and carrier is 3-8:1;

T4 DNA ligase 1μl;

T4 DNA ligase buffer 1μl

Condition: 16°C connection 14-20h

5. PCR verification of the new silkworm transgenic plasmid vector

The plasmid pBac[3×P3-EGFPafm ]-Fib H-GS-site was verified by PCR using primers Fib-H-p1 and Fib-H-p4, and the electrophoresis results are shown in Figure 2. Among them, M refers to the 250bp DNA standard molecular weight Marker, and GS refers to the fragment Fib H-GS-site; the number is the molecular weight, and the unit is bp.

Example 2

In the present invention, the constructed novel silkworm transgenic plasmid vector pBac[3×P3-EGFPafm]-FibH-GS-site is mixed with Piggybac Help Plasmid in equal amounts, and the mixed plasmid is injected into silkworms that have been born for a certain period of time by microinjection. in the egg. Since the 3×P3 promoter is used in the transgenic vector, the success of the transgene is identified by observing the fluorescence phenomenon in the eyes of the silkworm moth, and the obtained transgenic silkworm is identified by genome PCR and sequencing. Specific steps are as follows:

1 plasmid extraction

The plasmid was extracted using the kit QIAprep Spin Miniprep Kit (Cat. No.: 27104), and the operation was strictly in accordance with the instructions of the plasmid extraction kit. (Note: Add LyseBlue reagent to Buffer P1. Add the RNase A solution included in the kit to Buffer P1, mix, and store P1 at 2-8°C. When using Buffer PE, add absolute ethanol to PE .All centrifugation conditions are 13000rpm.)

Collect 1-5ml of overnight cultured bacterial solution, centrifuge at 10000rpm for 3min at room temperature; discard the supernatant, suspend the pellet with 250μl Buffer P1 and transfer to a microcentrifuge tube; add 250μl Buffer P2, and rotate the centrifuge tube 4-6 times until The solution becomes clear, and if Buffer P1 is added with a blue reagent, the solution becomes clear blue. The reaction in this step should not exceed 5 minutes; add 350 μl Buffer N3, and immediately rotate the centrifuge tube 4-6 times, if Buffer P1 is added with a blue reagent, the solution becomes colorless; centrifuge at 13000 rpm for 10 minutes at room temperature. Take out the supernatant and transfer it to the QIAprep spin column, centrifuge at room temperature for 1 min, discard the waste liquid; wash the QIAprep spin column with 500 μl Buffer PB, centrifuge at room temperature for 1 min, discard the waste liquid; add 750 μl Buffer PE to wash the QIAprep spin column, centrifuge at room temperature for 1 min, discard Remove the waste liquid; centrifuge at room temperature for 1 min to remove the remaining washbuffer; put the QIAprep column on a clean centrifuge tube, add 50 μl Buffer EB, place at room temperature for 1 min, and centrifuge for 1 min.

2 Microinjection

(1) Inoculate the Escherichia coli strains containing the vector pBac[3×P3-EGFPafm]-FibH-GS-site and Piggybac Help plasmids of the target gene into 5ml of fresh LB medium, culture for 14h, and follow the QIAGEN Plasimd MiniKit plasmid Extract the ultrapure plasmid through the operation steps described in the manual of the extraction kit, measure the concentration of the plasmid with One-Drop, and store it at -20°C for later use;

(2) Prepare raw powder into 60% water by weight, steam it into a paste, apply it evenly on the silkworm paper, and let it dry naturally;

(3) Put the mated female moths on the starched silkworm paper, lay eggs in a dark environment, collect silkworm eggs every 40 minutes, and move the female moths to another silkworm paper to continue laying eggs, until spawning is complete;

(4) Soak the collected silkworm eggs and silkworm paper in single distilled water at about 25°C for 1 min. After the paste on the silkworm paper is infiltrated, use a fine brush to transfer the silkworm eggs to a glass slide sterilized with 75% alcohol. On the slide, neatly arrange the silkworm eggs one by one on the glass slide with a fine brush, fix the silkworm eggs on the slide, with the hole of the silkworm egg facing upwards and the egg fossa facing the right, and let it dry naturally;

(5) Adjust the concentration of the recombinant plasmid and Helper plasmid mixture to about 0.4 μg/μl with injection buffer, and inject it into non-diapausing early embryos at a volume ratio of 1:1, and the time is 1 to 3 hours after oviposition , the injection volume is 15-20ng (10-15nl) DNA;

(6) After being sterilized with formaldehyde vapor, it is placed under the conditions of 25°C and relative humidity of 95%-100% for greening.

3 silkworm breeding

The silkworm eggs in the greening period are disinfected with formaldehyde steam every 2-3 days, and the relative humidity of the environment where the silkworm eggs are located is 95%-100% every day. Collect silkworm ants, silkworm ants use the tenderest mulberry leaves to crush and feed, feed 4 times a day, and sleep after 2-3 days; after molting, they will be the second instar, and the corresponding mulberry leaves should be the tenderest leaves, and they can start netting every day. Silkworm excrement, feed 3-4 times a day, and then sleep for 3-4 days; after molting, it is the third instar, and the corresponding mulberry leaves are tender leaves, 1-2 times the size of the silkworm body. Feed 3 times a day, except Sand 1 time. The temperature of the silkworm room before this is preferably 26-27°C, and the relative humidity is 85-90%. , sleep after 4-5 days; it is the fourth instar after molting, and the mulberry leaves fed accordingly are large leaves, twice the size of the silkworm body, fed 3 times a day, and cleaned silkworm excrement 2 times. To prevent bacterial infection, you can start to spray norfloxacin (50μg/ml) on the mulberry leaves, the temperature is about 25°C, the relative humidity is about 85%, sleep after 5-6 days; after molting is the fifth instar, feed accordingly It can slowly change into old leaves, twice the size of the silkworm body, the temperature is about 25°C, and the relative humidity is 80-85%. It is fed for 3-4 days a day. Before the cocooning, it needs to be fed a lot of mulberry leaves to provide silkworms Next, prepare a large amount of silk spinning. On the seventh day, the body of the silkworm becomes transparent, the head turns yellow, and the silkworm is about to start spinning. At this time, the silkworm needs to be placed in a net that can cocoon, and when the silkworm starts to cocoon. The cocoon is made into a pupa. At this time, the temperature is about 25°C and the humidity is about 85%. There is no need to feed any more. Clean up the silkworm room and wait for the silkworm to break out of the cocoon and become a moth. On the left and right, the cocoons are artificially broken, and the male and female are selected and separated to help silkworms safely change from the pupa stage to moths. After becoming moths, naturally mate the required male and female moths to lay eggs. You can also disassemble the pair after 4 hours of mating, and place the female moths in the egg storage circle of silkworm paper. After about 24 hours, take out the female moths and place them on the Eggs are preserved, and after a new round of greening, they can be raised periodically.

4. Fluorescence Screening

The silkworm eggs injected with the plasmid are the G0 generation silkworm eggs. A total of 251 silkworm eggs were injected, and a total of 48 silkworm moths in the experimental group were obtained from breeding to the silkworm moth stage, including 20 female moths and 28 male moths. The male and female silkworm moths in the experimental group were paired for seed production , the remaining male moths were mated with female moths that had not been injected with the plasmid, and the G1 generation of silkworm eggs (27 egg circles) were obtained, and continued to be green; each egg circle was raised separately until the G1 generation became moths, and the G1 generation The silkworm moths were observed one by one under a stereomicroscope, and after fluorescence screening, the living experimental group silkworms with green fluorescent eyes were screened out, and 5 female silkworms and 12 male silkworms were obtained. The male and female silkworms in the experimental group were paired with each other for seed production to obtain silkworms. Eggs of the G2 generation (a total of 4 moth circles were obtained), continued to be greened, and the 4 moth circles of the G2 generation were raised separately until the G2 generation became adult moths, and the G2 generation silkworm moths were observed under a stereo microscope one by one, and after fluorescence After screening, only one female silkworm moth with green fluorescent eyes was obtained.

The G1 and G2 generation silkworm moths transferred with the plasmid and the original silkworm moths without the plasmid were observed with a stereo microscope, and the results are shown in Figure 3. Silkworm moths with green fluorescent eyes can be preliminarily screened as transgenic silkworm moths.

5 PCR amplification and sequencing of transgenic silk moth

The transgenic silkworm moths preliminarily determined by fluorescence screening, after laying eggs, took part of the moth body, ground it with liquid nitrogen, extracted the genome according to the method of extracting the silkworm genome, and used the primer pair Fib-H-P1, Fib-H- P2, using the extracted silk moth genome as a template for PCR amplification to obtain a band of about 3kb in size, as shown in Figure 4, where M refers to the DNA standard molecular weight marker (250bp), and B2 refers to the amplified fragment Fib H-GS-site . This band was cut and sent directly for sequencing. The sequencing primer used was the reverse PCR primer Fib-H-P2. The sequencing results are shown in the sequence table, see also Figure 5, where the part marked in gray is the base sequence of GS-site. It shows that the Fib H-GS-site sequence has been integrated into the genome of silkworm.

Claims (10)

1. a novel silkworm transgenosis plasmid vector, is characterized in that, plasmid vector contains restructuring fibroin heavy chain protein gene, and restructuring fibroin heavy chain protein contains matrix metalloproteinase (MMPs) restriction enzyme site.

2. a kind of novel silkworm transgenosis plasmid vector according to claim 1, is characterized in that, plasmid vector is PiggyBac transposon carrier.

3. a kind of novel silkworm transgenosis plasmid vector according to claim 1, is characterized in that, PiggyBac transposon contains the green fluorescence protein gene that a specific promoter 3 * P3 starts.

4. a novel silkworm transgenosis plamid vector construction method, is characterized in that, step is as follows:

A: adopt the precipitator method to extract domestic silkworm gene group;

B:PCR amplification fibroin heavy chain protein gene aim sequence;

C, build novel silkworm transgenosis plasmid vector.

5. novel silkworm transgenosis plamid vector construction method according to claim 4, it is characterized in that, pcr amplification fibroin heavy chain protein gene aim sequence process, first PCR design of primers, silkworm (cyanines pine) genome of take is template, utilize Primer Premier5.0 design primer, in primer, contain MMPs restriction enzyme site sequence.

6. novel silkworm transgenosis plamid vector construction method according to claim 4, is characterized in that, build novel silkworm transgenosis plasmid vector, its carrier construction method is as follows:

(1) build plasmid: the PCR product of primers F ib-H-p1 and Fib-H-p2 is connected directly in pMD-18T simple carrier, builds plasmid pMD-FibH-N;

(2) prepare competent cell;

(3) transform, deposit bacterium;

(4) plasmid extraction;

(5) agarose gel electrophoresis of DNA and glue reclaim;

(6) to the plasmid pMD-FibH-N extracting with through the PCR product FibH-GS-site-C of agarose gel electrophoresis and glue recovery, carry out double digestion; Enzyme is cut rear glue and is reclaimed FibH-N sequence and FibH-GS-site-C sequence, and connects with T4DNAligase;

(7) the 6th steps increase containing the restructuring fibroin heavy chain protein gene of MMPs restriction enzyme site with primer pair Fib-H-p1 and Fib-H-p4PCR after connecting the recovery of product glue;

(8) glue of the 7th step PCR product is reclaimed to product and transgene carrier plasmid pBac[3 * P3-EGFPafm] carry out double digestion;

(9) enzyme is cut rear glue and is reclaimed aim sequence, and connects with T4DNA ligase, builds novel silkworm transgenosis plasmid vector, and connection product is converted in competent cell.

7. the method for cultivation of a novel transgenic bombyx mori, it is characterized in that, adopt microinjection to be injected in the silkworm seed of giving birth in certain hour the novel silkworm transgenosis plasmid vector having built, by observing the fluorescence phenomenon of silkworm moth eye, identify and successfully carry out genetically modified silkworm, and cultivate by the transgenic bombyx mori of Genomic PCR method and order-checking evaluation acquisition, its step is as follows:

(1) plasmid extraction: the PiggyBac transposon vector having built is extracted stand-by;

(2) microinjection: the method that PiggyBac transposon vector is injected in silkworm seed is after PiggyBac transposon carrier mixes with Help plasmid, is expelled in the body early embryo of non-diapause with 0.4 μ g/ μ l;

(3) mulberry silkworm rearing;

(4) fluorescent screening: screening has the silkworm of PiggyBac transposon vector;

(5) pcr amplification of transgenosis silkworm moth order-checking: further detect the silkworm filtering out and whether there is transgenosis.

(6) silkworm is cultivated: to having genetically modified silkworm, carry out routine cultivation.

8. the method for cultivation of novel transgenic bombyx mori according to claim 7, is characterized in that, PiggyBac transposon carrier and Help plasmid volume ratio are 1:1.

9. the method for cultivation of novel transgenic bombyx mori according to claim 7, is characterized in that, injection volume is 15-20ng(10-15nl) DNA.

10. the method for cultivation of novel transgenic bombyx mori according to claim 7, is characterized in that, body early embryo is 1～3h after laying eggs.