CN103555761B - Silkworm calcium ion-ATP (adenosine triphosphate) enzyme gene recombination expression vector and application thereof - Google Patents
Silkworm calcium ion-ATP (adenosine triphosphate) enzyme gene recombination expression vector and application thereof Download PDFInfo
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Abstract
The invention discloses a silkworm calcium ion-ATP (adenosine triphosphate) enzyme gene recombination expression vector, wherein the specific expression of silkworm calcium ion-ATP enzyme on a silkworm anterior silkgland is regulated by a silkworm anterior silkgland specific promoter, and the recombination expression vector is micro-injected in silkworm eggs to obtain transgenic silkworm positive individuals; researches on the transgenic silkworm positive individuals find that the calcium ion-ATP enzyme is subjected to overexpression in both mRNA (messenger ribonucleic acid) and protein level; a mechanical tensile test on transgenic silkworm fibres finds that the mechanical properties of the transgenic silkworm fibres are remarkably improved; an infrared spectroscopic analysis finds that the structures of the transgenic silkworm fibres are changed. Therefore, the mechanical properties of the silkworm fibres can be changed by applying the silkworm calcium ion-ATP enzyme gene recombination expression vector constructed by the method disclosed by the invention, thus contributing to obtain more excellent silkworm fibres.
Description
Technical field
The invention belongs to biological technical field, relate to a kind of domestic silkworm gene recombinant expression vector, and it is improving the application in silk fiber mechanical property.
Background technology
Silk fiber is protein fibre, is a kind of filamentary material in the field such as textile industry, biomedicine with widespread use.But due to the structural defect of fibroin, it is poor that the mechanical property of silk fiber compares synthon, thus constrain its application in fields such as novel high polymer fiber, special materials.
Summary of the invention
In view of this, an object of the present invention is to provide a kind of domestic silkworm gene recombinant expression vector, and two of object is to provide this recombinant expression vector improving the application in silk fiber mechanical property; Three of object is to provide the method utilizing this recombinant expression vector to improve silk fiber mechanical property.
For achieving the above object, after deliberation, the invention provides following technical scheme:
1. silkworm calcium ion-ATP enzyme gene recombinant vectors, be by by silkworm anterior division of silkgland specificity promoter, silkworm calcium ion-ATP enzyme gene C DS sequence and stop molecular expression cassette insert containing fluorescent reporter gene transposon vector in and obtain; Described silkworm calcium ion-ATP enzyme gene C DS sequence is as shown in SEQ ID No.1.
Further, described silkworm anterior division of silkgland specificity promoter is silkworm epidermal protein BmCP231 promotor or BmCP274 promotor, described BmCP231 promoter sequence as shown in the 1st to the 1457th Nucleotide in SEQ ID No.4, or as shown in the 1st to the 1511st Nucleotide in SEQ ID No.4; Described BmCP274 promoter sequence is as shown in SEQ ID No.9.
Further, described recombinant expression vector is obtained by the Asc I restriction enzyme site being stopped molecular expression cassette insertion pBac [3xP3-EGFPafm] carrier by silkworm epidermal protein BmCP231 promotor, silkworm calcium ion-ATP enzyme gene C DS sequence and SV40; Described BmCP231 promoter sequence is as shown in the 1st to the 1511st Nucleotide in SEQ ID No.4.
2. silkworm calcium ion-ATP enzyme gene recombinant vectors is improving the application in silk fiber mechanical property.
3. utilize silkworm calcium ion-ATP enzyme gene recombinant vectors to improve the method for silk fiber mechanical property, comprise the following steps: silkworm calcium ion-ATP enzyme gene recombinant vectors is mixed with the transgenosis assistant carrier equimolar ratio of encoding transposase, microinjection enters in the silkworm seed of after laying eggs of termination of diapause 2 ~ 5 hours, the nontoxic glue sealing of silkworm seed after injection, 25 DEG C are hastened the hatching of silkworms to hatching, the larva hatched adopts artificial diet to raise, the selfing production of hybrid seeds is carried out to adult, get the silkworm seed of the 7th day ovum phase again, carry out fluoroscopic examination under the microscope, filter out the trans genie individual at eyes or neural specific emitting fluorescence, obtain transgenic bombyx mori, the silk fiber that the mechanical property that is its silk fiber produced improves.
Beneficial effect of the present invention is: the present invention constructs silkworm calcium ion-ATP enzyme gene recombinant vectors, by silkworm anterior division of silkgland specificity promoter regulation and control silkworm calcium ion-ATP enzyme at silkworm anterior division of silkgland specifically expressing, this recombinant expression vector microinjection is entered in Eggs of Silkworm to obtain transgenic bombyx mori positive individuals, the research of transgenic bombyx mori positive individuals is found, calcium ion-ATP enzyme is obtained for overexpression on mRNA and protein level, mechanical stretch test is carried out to transgenic silkworm silky fibre and finds that its mechanical property is significantly improved, Infrared spectroscopy finds that the structure of transgenic silkworm silky fibre there occurs change.Thus the silkworm calcium ion-ATP enzyme gene recombinant vectors using the present invention to build can change the mechanical property of silk fiber, contributes to obtaining more excellent silk fiber.
Accompanying drawing explanation
In order to make object of the present invention, technical scheme and beneficial effect clearly, the invention provides following accompanying drawing and being described:
Fig. 1 is the Southern Blot detected result of transgenic bombyx mori, and wherein 1 is transgenic bombyx mori genome; 2 is non-transgenic domestic silkworm gene group.
Fig. 2 is the fluorescence quantitative PCR detection result of transgenic bombyx mori, and wherein 1 is transgenic bombyx mori anterior division of silkgland genome, and 2 is non-transgenic silkworm anterior division of silkgland genome.
Fig. 3 is the Western Blot detected result of transgenic bombyx mori, and wherein 1 is non-transgenic silkworm anterior division of silkgland protein sample, and 2 is transgenic bombyx mori anterior division of silkgland protein sample.
Fig. 4 is the Infrared spectrum scanning result of transgenic silkworm silky fibre, wherein A is non-transgenic silk fiber, and B is transgenic silkworm silky fibre, and curve a is infrared absorption curve, curve b is the curve of infrared absorbance values by being formed after deconvoluting of Fourier, and arrow place is 1660cm
-1peak.
Embodiment
Below in conjunction with accompanying drawing, the preferred embodiments of the present invention are described in detail.The experimental technique of unreceipted actual conditions in preferred embodiment, usually conveniently condition, the such as condition described in Molecular Cloning: A Laboratory guide (third edition, the work such as J. Pehanorm Brooker), or according to the condition that manufacturer advises.
The cultivated silkworm breed variety used in preferred embodiment, for make greatly, is provided by Southwestern University's domestic silkworm gene resources bank.
One, the clone of silkworm calcium ion-ATP enzyme gene
According to the CDS sequence (SEQ IDNo.1) of calcium ion in domestic silkworm gene group database-ATP enzyme gene (BGIBMGA006603), design 1 pair of upstream and downstream primer, entrust Sangon Biotech (Shanghai) Co., Ltd. to synthesize.Primer sequence is as follows: 6603-F:5'-cg
acgcgtatggaggacgctcacacga-3'(SEQ ID No.2, underscore part is Mlu I restriction enzyme site); 6603-R:5'-attt
gcggccgcttatagtggtccgtagatgatgatg-3'(SEQ ID No.3, underscore part is Not I restriction enzyme site).
Make greatly Malpighian tube cDNA for template with the 3rd day five ages, adopt 6603-F and 6603-R primer PCR amplification BGIBMGA006603 gene C DS fragment.PCR reaction conditions is: 94 DEG C of denaturations 4 minutes; Then 94 DEG C of sex change 40 seconds, 65 DEG C of annealing 40 seconds, 72 DEG C extend 3 minutes, totally 24 circulations; Last 72 DEG C extend 10 minutes.PCR primer carries out agarose gel electrophoresis separation, cuts glue and reclaims purifying object fragment, then be cloned into carrier pMD19-T Simple(TaKaRa), obtain recombinant vectors pMD19-BGIBMGA006603.
Two, the structure of silkworm calcium ion-ATP enzyme gene recombinant vectors
By recombinant vectors pMD19-BGIBMGA006603 Mlu I and Not I double digestion, reclaim BGIBMGA006603 gene C DS fragment, connect with the same carrier pSL [BmCP231-DsRed-SV40] through Mlu I and Not I double digestion again, obtain recombinant vectors pSL [BmCP231-BGIBMGA006603-SV40].The construction process of described carrier pSL [BmCP231-DsRed-SV40] is see Chinese patent CN102604954B, and silkworm epidermal protein BmCP231 promoter sequence is as shown in the 1st to the 1511st Nucleotide in SEQ IDNo.4.
Recombinant vectors pSL [BmCP231-BGIBMGA006603-SV40] Asc I enzyme is cut, reclaim BmCP231-BGIBMGA006603-SV40 fragment, connect with the same transposon vector pBac [3xP3-EGFPafm] cut through Asc I enzyme again, obtain recombinant vectors pBac [BmCP231-BGIBMGA006603-SV40,3xP3EGFP].The construction process of described carrier pBac [3xP3-EGFPafm] is see document (Horn and Wimmer, 2000), and it carries enhanced green fluorescence protein (EGFP) gene as reporter gene.
Three, the acquisition of transgenic bombyx mori
By recombinant vectors pBac [BmCP231-BGIBMGA006603-SV40, 3xP3EGFP] mix with the transgenosis assistant carrier pHA3PIG equimolar ratio of coding piggyBac transposase, what microinjection entered termination of diapause makes greatly body early embryo (after laying eggs 2 ~ 5 hours, G0 generation) in, the nontoxic glue sealing of silkworm seed after injection, 25 DEG C are hastened the hatching of silkworms to hatching, the larva hatched adopts artificial diet to raise, the selfing production of hybrid seeds is carried out to adult, get the silkworm seed (G1 generation) of the 7th day ovum phase again, detect with the exciting light of 460 ~ 490nm wavelength under macroscopical Stereo fluorescence microscope (Olympus MVX10), filter out the trans genie individual at eyes or neural specific transmitting green fluorescence, namely transgenic bombyx mori is obtained.
Random selecting transgenic bombyx mori G1 is for silkworm moth, extract genomic dna and carry out Southern blot detection, result as shown in Figure 1, the insertion copy number of EGFP is 2, because BGIBMGA006603 gene C DS fragment and EGFP are in same transposon region, then it inserts copy number is also 2, shows that the piggyBac transposable element carrying BGIBMGA006603 gene C DS fragment there occurs 2 transposition event in the genome of transgenic bombyx mori.
Four, in transgenic bombyx mori, the expression amount of calcium ion-ATP enzyme and encoding gene thereof detects
1, in transgenic bombyx mori, the gene expression amount of calcium ion-ATP enzyme detects
Get the transgenic bombyx mori in the 3rd day five ages and the anterior division of silkgland of non-transgenic silkworm respectively, grind and extract total serum IgE fast in liquid nitrogen, be cDNA by total serum IgE reverse transcription, employing special primer 6603qPCR-F [5'-aatcccttcaatgttcctaa-3'(SEQID No.5)] and 6603qPCR-R [5'-tgctcccttgacaaatagtt-3'(SEQ ID No.6)], the relative expression quantity of fluorescence quantitative PCR detection BGIBMGA006603 gene C DS fragment, take sw as internal reference, internal reference primer sw-F:5'-ttcgtactg-gctcttctcgt-3'(SEQ ID No.7), sw-R:5'-caaagttgatagcaattccct-3'(SEQ ID No.8).PCR reaction conditions is: 95 DEG C of denaturations 30 seconds, then 95 DEG C of sex change 3 seconds, 60 DEG C of annealing 30 seconds, totally 40 circulations.As shown in Figure 2, BGIBMGA006603 gene C DS fragment obtains overexpression to result in the anterior division of silkgland of transgenic bombyx mori.
2, in transgenic bombyx mori, the expressing quantity of calcium ion-ATP enzyme detects
Get the transgenic bombyx mori in the 3rd day five ages and the anterior division of silkgland of non-transgenic silkworm respectively, in liquid nitrogen, grinding makes to be dissolved in RIPA lysate fast, place 1 hour for 4 DEG C, centrifuging and taking supernatant, after measuring total protein concentration, mixes with 5 × sample loading buffer, hatch 30 minutes for 37 DEG C, carry out 8%SDS-PAGE, after electrophoresis, with Anti-BGIBMGA006603 antibody be primary antibodie, Tubulin antibody carries out Western blot analysis for internal reference antibody.As shown in Figure 3, calcium ion-ATP enzyme obtains overexpression to result in the anterior division of silkgland of transgenic bombyx mori.
Five, the mechanical property of transgenic silkworm silky fibre and structure detection
1, the mechanics properties testing of transgenic silkworm silky fibre
Respectively the cephalothorax of transgenic bombyx mori and non-transgenic silkworm is fixed, utilize machine motor at the uniform velocity to be pulled out from silkworm orifice of spinneret by silk according to the speed of 60r/min.Get a part of silk fiber, utilize universl tester to carry out tension test, test and carry out under temperature 26 DEG C, atmospheric moisture 54% condition, use 50N sensor, draw speed is 3mm/min, the crushing load of record silk and Fault displacement; Separately get a part of silk fiber and carry out electron-microscope scanning, measure the diameter of silk fiber; Calculate maximum stress and maximum strain as follows again: maximum stress=crushing load/cross-sectional area, length × 100% of maximum strain=Fault displacement/silky fibre.The results are shown in Table 1, compare non-transgenic silk fiber, rigidity and the toughness of transgenic silkworm silky fibre are obtained for raising.
The mechanical performance data of table 1 transgenosis and non-transgenic silk
* represent to there is significant difference, p<0.05 between transgenosis silk and non-transgenic silk.
2, the structure detection of transgenic silkworm silky fibre
Get transgenic silkworm silky fibre respectively and non-transgenic silk fiber carries out infrared spectra detection, test and carry out under temperature 22 DEG C, atmospheric moisture 46% condition, adopt infrared transmission scanning, acquisition time is 51s, and times of collection is 256 times.As shown in Figure 4, transgenic silkworm silky fibre is at 1660cm for result
-1the peak area at place is large compared with non-transgenic silk fiber, due to the content of β-pleated sheet structure in this peak-to-peak area representative sample, comparatively non-transgenic silk fiber is high for the β-pleated sheet structure content of therefore transgenic silkworm silky fibre, illustrate that the structure of transgenic silkworm silky fibre there occurs change, consequently leads to the raising of its mechanical property.
The silkworm anterior division of silkgland specificity promoter used in above preferred embodiment is sequence silkworm epidermal protein BmCP231 promotor of (containing signal peptide) as shown in the 1st to the 1511st Nucleotide in SEQ ID No.4, except this promotor, other silkworm anterior division of silkgland specificity promoter is all applicable to the present invention, such as, silkworm epidermal protein BmCP231 promotor (CN102604954B) that sequence (does not contain signal peptide) as shown in the 1st to the 1457th Nucleotide in SEQ ID No.4, or silkworm epidermal protein BmCP274 promotor (CN102604953B) of sequence as shown in SEQ ID No.9.These specificity promoters can regulate and control the specifically expressing of calcium ion-ATP enzyme at silkworm anterior division of silkgland, improve the mechanical property of transgenic silkworm silky fibre, just the amount of calcium ion-ATP enzyme overexpression in transgenic bombyx mori may be different, thus cause transgenic silkworm silky fibre slightly to be distinguished on the increase rate of mechanical property.
What finally illustrate is, above preferred embodiment is only in order to illustrate technical scheme of the present invention and unrestricted, although by above preferred embodiment to invention has been detailed description, but those skilled in the art are to be understood that, various change can be made to it in the form and details, and not depart from claims of the present invention limited range.
Claims (6)
1. silkworm calcium ion-ATP enzyme gene recombinant vectors is improving the application in silk fiber mechanical property, and described silkworm calcium ion-ATP enzyme gene recombinant vectors obtains by silkworm anterior division of silkgland specificity promoter, silkworm calcium ion-ATP enzyme gene C DS sequence with in stopping transposon vector that molecular expression cassette inserts containing fluorescent reporter gene; Described silkworm calcium ion-ATP enzyme gene C DS sequence is as shown in SEQ ID No.1.
2. application according to claim 1, it is characterized in that: described silkworm anterior division of silkgland specificity promoter is silkworm epidermal protein BmCP231 promotor or BmCP274 promotor, described BmCP231 promoter sequence as shown in the 1st to the 1457th Nucleotide in SEQ ID No.4, or as shown in the 1st to the 1511st Nucleotide in SEQ ID No.4; Described BmCP274 promoter sequence is as shown in SEQ ID No.9.
3. application according to claim 1, is characterized in that: described recombinant expression vector is obtained by the Asc I restriction enzyme site being stopped molecular expression cassette insertion pBac [3xP3-EGFPafm] carrier by silkworm epidermal protein BmCP231 promotor, silkworm calcium ion-ATP enzyme gene C DS sequence and SV40; Described BmCP231 promoter sequence is as shown in the 1st to the 1511st Nucleotide in SEQ ID No.4.
4. utilize silkworm calcium ion-ATP enzyme gene recombinant vectors to improve the method for silk fiber mechanical property, it is characterized in that, comprise the following steps: silkworm calcium ion-ATP enzyme gene recombinant vectors is mixed with the transgenosis assistant carrier equimolar ratio of encoding transposase, microinjection enters in the silkworm seed of after laying eggs of termination of diapause 2 ~ 5 hours, the nontoxic glue sealing of silkworm seed after injection, 25 DEG C are hastened the hatching of silkworms to hatching, the larva hatched adopts artificial diet to raise, the selfing production of hybrid seeds is carried out to adult, get the silkworm seed of the 7th day ovum phase again, carry out fluoroscopic examination under the microscope, filter out the trans genie individual at eyes or neural specific emitting fluorescence, obtain transgenic bombyx mori, the silk fiber that the mechanical property that is its silk fiber produced improves, described silkworm calcium ion-ATP enzyme gene recombinant vectors is by silkworm anterior division of silkgland specificity promoter, silkworm calcium ion-ATP enzyme gene C DS sequence and the molecular expression cassette of termination insert in the transposon vector containing fluorescent reporter gene and obtain, described silkworm calcium ion-ATP enzyme gene C DS sequence is as shown in SEQ ID No.1.
5. method according to claim 4, it is characterized in that: described silkworm anterior division of silkgland specificity promoter is silkworm epidermal protein BmCP231 promotor or BmCP274 promotor, described BmCP231 promoter sequence as shown in the 1st to the 1457th Nucleotide in SEQ ID No.4, or as shown in the 1st to the 1511st Nucleotide in SEQ ID No.4; Described BmCP274 promoter sequence is as shown in SEQ ID No.9.
6. method according to claim 4, is characterized in that: described recombinant expression vector is obtained by the Asc I restriction enzyme site being stopped molecular expression cassette insertion pBac [3xP3-EGFPafm] carrier by silkworm epidermal protein BmCP231 promotor, silkworm calcium ion-ATP enzyme gene C DS sequence and SV40; Described BmCP231 promoter sequence is as shown in the 1st to the 1511st Nucleotide in SEQ ID No.4.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102604953A (en) * | 2012-03-28 | 2012-07-25 | 西南大学 | BmCP274 promoter of bombyx mori cuticular protein, recombinant expression vector and application of the promoter |
| CN102604954A (en) * | 2012-03-28 | 2012-07-25 | 西南大学 | Domestic silkworm cuticle protein BmCP231 promoter as well as recombinant expression vector and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102604953A (en) * | 2012-03-28 | 2012-07-25 | 西南大学 | BmCP274 promoter of bombyx mori cuticular protein, recombinant expression vector and application of the promoter |
| CN102604954A (en) * | 2012-03-28 | 2012-07-25 | 西南大学 | Domestic silkworm cuticle protein BmCP231 promoter as well as recombinant expression vector and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| Bombyx mori sarco/endoplasmic reticulum calcium ATPase (LOC100302733), mRNA;NM_001164476.1;《GenBank》;20130824;全文 * |
| 家蚕性信息素腺体肌质网膜Ca2+-ATP酶基因的分子鉴定;张松斗等;《河南农业大学学报》;20121031;第46卷(第5期);577-581 * |
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