Seed-specific expression vector and its construction methods and applications
09745362 · 2017-08-29
Assignee
Inventors
- Shengjun An (Shijiazhuang, CN)
- Xiqing Chai (Shijiazhuang, CN)
- Kunsheng Wang (Beijing, CN)
- Tiemei Shao (Shijiazhuang, CN)
- Zhan Jiao (Shijiazhuang, CN)
- Xin Wen (Shijiazhuang, CN)
- Xue Li (Shijlazhuang, CN)
- Pei Liu (Shijiazhuang, CN)
- Haigang Lu (Shijiazhuang, CN)
- Yunyu Chen (Shijiazhuang, CN)
- Liangyuan Hu (Beijing, CN)
- Haimin Xu (Beijing, CN)
- Chenggang Yu (Beijing, CN)
Cpc classification
International classification
Abstract
A seed-specific expression vector and its construction methods and applications are disclosed. A fusion protein expression cassette consisting of Arachis hypogaea oleosin gene-apolipopoprotein A-I.sub.Milano (A-IM) gene driven by Brassica napus oleosin gene promoter is inserted between the HindIII and SacI sites of a plant binary expression vector pBI121, obtaining the plant expression vector pBINOA of the invention. In addition, a method for producing apolipoprotein A-I.sub.Milano is provided, in which the expression vector is used to transform oil sunflower which is used as a plant bioreactor. The method can not only improve the yield of apolipoprotein A-I.sub.Milano, but also greatly reduce production costs, and is suitable for industrial production.
Claims
1. An expression vector comprising a Brassica napus oleosin gene promoter operably linked to a DNA sequence encoding: an apolipoprotein A-I.sub.Milano fused with an Arachis hypogaea oleosin, wherein the vector comprises the sequence as shown in SEQ ID NO: 15.
2. A method for the construction of the expression vector as described in claim 1, including the following procedures: 1) isolate and clone the Brassica napus oleosin gene promoter and the Arachis hypogaea oleosin gene; 2) design and synthesis the apolipoprotein A-I.sub.Milano gene according to Helianthus annuus codon usage; and 3) construct the expression vector in which the nucleic acid encoding the fusion protein of Arachis hypogaea oleosin with apolipoprotein A-I.sub.Milano is driven by the Brassica napus oleosin gene promoter; wherein the expression vector comprises SEQ ID NO: 15.
3. A method according to claim 1, wherein in step (3), Arachis hypogaea oleosin gene and apolipoprotein A-I.sub.Milano gene are constructed as a fusion gene by overlapping PCR.
4. A method for producing apolipoprotein A-I.sub.Milano, including the following steps: 1) introducing the expression vector as claimed in claim 1 into an explant of an oil sunflower; 2) cultivating the oil sunflower material into a mature plant, the plant including seeds, and 3) collecting the seeds thereof; and; 4) purifying apolipoprotein A-I.sub.Milano from the seeds.
5. The method according to claim 4, wherein the introduction method in Step 1) is Agrobacterium-mediated transformation.
6. The method according to claim 4, wherein the oil sunflower seed has a kanamycin resistance phenotype.
7. The method according to claim 4, wherein the purification method is high-performance liquid chromatography (HPLC).
8. The method according to claim 4, wherein the purification includes grinding the seed containing apolipoprotein A-I.sub.Milano in a buffer solution, separating an oil body from other components by centrifugation, washing the oil body, releasing the apolipoprotein A-I.sub.Milano from the surface of the oil body through digestion, and obtaining apolipoprotein A-I.sub.Milano through HPLC purification.
Description
DESCRIPTION OF FIGURES
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DETAILED DESCRIPTION OF THE INVENTION
(12) The following embodiments are provided for further description of this invention, and are not construed as limiting to the scope of the invention. Given the present disclosure, alterations may be made to this invention without departing from the spirit of this invention. All these alterations are within the scope of the present invention.
(13) Unless otherwise specified, the methods referred to in the following embodiments are practiced according to general practice in this field.
Example 1: Seed-Specific Plant Expression Vector
(14) Brassica napus oleosin gene promoter (NOP) was amplified by PCR, inserted into pUC19 between the HindIII and BamHI sites, obtaining pUCN. Apolipoprotein gene was designed according to apolipoprotein A-I-.sub.Milano (AIM) gene sequence and the codon usage of Helianthus annuus, synthetically produced, and inserted at the 3′ end of the Arachis hypogaea oleosin gene (Ole), obtaining the fusion gene of Arachis hypogaea oleosin and apolipoprotein A-I-.sub.Milano. Thrombin cleavage site was added between the Arachis hypogaea oleosin gene and the apolipoprotein A-I-.sub.Milano gene. The fusion gene was inserted into pUCN between the BamHI and SacI sites to obtain pUCNOA. pUCNOA was double digested with HindIII and SacI. The 2202 bp exogenous fragment was collected on agarose gel, and inserted between the and SacI sites of plant binary expression vector pBI121, obtaining the plant expression vector pBINOA of the invention. The expression cassette of pBINOA is the Ole/apoA-I.sub.M fusion gene driven by Brassica napus oleosin promoter. The structure of pBINOA is shown in
Example 2: Construction of Seed-Specific Plant Expression Vector pBINOA
(15) The construction of the plant expression vector pBINOA is shown in
(16) Cloning of Brassica napus oleosin gene promoter: Brassica napus is an important oil crop. The oil content is up to 42˜45%. The 20 kD oleosin in Brassica napus oil body is 10 times the amount of 24 kD oleosin. Forward primer pBINOA-1: CCC AAG CTT TTC AAC GTG GTC GGA TCA TGA CG (SEQ ID NO:1) and reverse primer pBINOA-2: CGC GGA TCC GAA TTG AGA GAG ATC GAA GAG (SEQ ID NO:2) for the PCR amplification of Brassica napus 20 kD oleosin gene promoter were designed according to the nucleotide sequence of Brassica napus oleosin gene promoter (Genbank No. AF134411) in which HindIII and BamHI cleavage sites were introduced (the underlined section). Using the genome DNA of Brassica napus Qingyou 14 variety as the template and pBINOA-1 and pBINOA-2 as primers, Brassica napus oleosin gene promoter was amplified by PCR with the following conditions: 94° C. 1 min, 63-73° C. 1 min, and 68° C. 1 min, and 10 min of extension at 68° C. after 30 cycles. The amplification product was recovered by agarose gel electrophoresis, double digested with HindIII and BamHI, and connected to pUC19 digested with HindIII and BamHI. The ligation product was mixed with 2004 of DH5α competent cell (purchased from Tiangen Biotech (Beijing) Co., Ltd.), and then subjected to ice bath for 30 min, heat shock for 1.5 min at 42° C., and ice bath for 3 min. 8004 LB culture medium was added and cultured for 45 min at 37° C. Aliquots of the transformation reaction was plated on LB agar containing 50 μg/mL ampicillin and incubated overnight at 37° C. The transformants were screened by PCR using pBINOA-1 and pBINOA-2 as primers. PCR conditions were 94° C. 1 min, 60-73° C. 1 min, 72° C. 1 min, and 10 min of extension at 72° C. after 30 cycles. The PCR product was subjected to electrophoresis with agarose gel for verification. The positive transformant was named as pUCN. The positive transformant was shaken in liquid culture medium. Plasmid was extracted through alkaline lysis. The plasmid was subjected to single enzyme digestion identification with HindIII and double enzyme digestion identification with HindIII and BamHI. The results displayed by agarose gel electrophoresis are shown in
(17) Artificial synthesis of apolipoprotein A-I-.sub.Milano gene: Based on apolipoprotein A-I gene sequence (SEQ ID NO:4, NM000039) (amino acid sequence shown in SEQ ID NO:5) and the codon usage of Helianthus annuus (http://www.kazusa.or.jp/codon/cgi-bin/showcodon.cgi?species=4232), as well as the GC content in Helianthus annuus (genome, apolipoprotein A-I-.sub.Milano gene is redesigned and synthesized. Residue C at position 517 was mutated into T, and at the 5′ end of the gene a thrombin cleavage site was added, with the nucleotide sequence shown in SEQ ID NO:6 (CTGGTCCCAA GGGGTAGC) and the amino acid sequence shown in SEQ ID NO:7 (L V P R G S). The molecular weight of the synthesized apolipoprotein A-I-.sub.Milano gene was 462.4 kDa, and the sequence is shown in SEQ ID NO:8. The encoded protein is composed of 249 amino acid residues and the molecular weight is 28.585 kDa.
(18) Amplification of Ole/apoA-I.sub.M fusion protein gene: Two pairs of specific primers (pBINOA-3/pBINOA-4 and pBINOA-5/pBINOA-6, wherein the sequences are SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12 respectively) were designed according to the sequence of Arachis hypogaea oleosin gene (Genbank No. AF325917) and the sequence of apolipoprotein A-I-.sub.Milano gene (SEQ ID NO:8). pBINOA-3 and pBINOA-6 were provided with BamHI and SacI restriction sites (the underlined section) respectively. Moreover, Kozak sequence (the bolded part in the sequence, to improve the transcription and expression efficiencies) is positioned near the initiator codon of oleosin gene in pBINOA-3 primer. pBINOA-4 and pBINOA-5 were reverse complementary sequences.
(19) TABLE-US-00001 SEQ ID NO.: 9 pBINOA-3: CGC GGA TCC AGC AAA GCC GCC ACC ATG GCT ACT GCT ACT GAT CG SEQ ID NO.: 10 pBIN0A-4: GCT ACC CCT TGG GAC CAG TGA TGA TGA CCT CTT AAC SEQ ID NO.: 11 pBINOA-5: GTT AAG AGG TCA TCA TCA CTG GTC CCA AGG GGT AGC SEQ ID NO.: 12 pBINOA-6: C GAG CTC TTA TTG TGT GTT AAG TTT CTT TG
(20) Using pBINOA-3/pBINOA-4 as the primer, Arachis hypogaea (variety Jihua 4) genome DNA as template, the Arachis hypogaea oleosin gene lacking the terminate codon was amplified. PCR conditions are 94° C. 1 min, 50-55° C. 1 min, 68° C. 1 min, and 10 min of extending at 68° C. after 30 cycles. Using pBINOA-5/pBINOA-6 as the primer, the optimized apolipoprotein A-I-.sub.Milano as template, the apolipoprotein A-I-.sub.Milano gene was amplified. PCR conditions are 94° C. 1 min, 63-73° C. 1 min, 68° C. 1 min, and 10 min of extending at 68° C. after 30 cycles. The two PCR amplification products were recovered by agarose gel electrophoresis, and mixed at the molar ratio of 1:1 to serve as template. pBINOA-3/pBINOA-6 were used as primer for overlapping PCR. PCR conditions are 94° C. 1 min, 50-55° C. 1 min, 68° C. 2 min, and 10 min of extending at 68° C. after 30 cycles. Ole/apoA-I.sub.M fusion gene was obtained through agarose gel electrophoresis of the amplification product. The construction of Ole/apoA-I.sub.M fusion gene is shown in
(21) Construction of intermediate vector pUCNOA: The ole/apoA-I.sub.M fusion gene was BamHI and SacI double digested and ligated with pUCN which was double digested in the same way. The ligation product was mixed with 200 μL DH5α competent cell (purchased from Tiangen Biotech (Beijing) Co., Ltd.), and subjected to ice bath for 30 min, heat shock for 1.5 min at 42° C., and ice batch for 3 min. 8004 LB culture medium was added and grown at 37° C. for 45 min. LB agar plate containing 100 μg/mL ampicillin was innoculated and incubated at 37° C. overnight. The transformants were selected by PCR using pBINOA-3 and pBINOA-6 as primers. PCR conditions were 94° C. 1 min, 60-73° C. 1 min, 72° C. 1.5 min, and extension of 10 min at 72° C. after 30 cycles. The PCR product was run on agarose gel. The positive transformant was named as pUCNOA and was shaken in liquid medium. Plasmid was extracted by alkaline lysis. The plasmid was identified by HindIII single digestion identification, HindIII and BamHI double digestion identification, and BamHI and SacI double digestion identification. The identification results of agarose gel electrophoresis are shown in
(22) Construction of seed-specific plant expression vector pBINOA: DNA of pUCNOA plasmid was extracted by alkaline lysis, and cleaved with HindIII and SacI. The exogenous fragment of 2202 bp was recovered by agarose gel electrophoresis and ligated to pBI121 cleaved with HindIII and SacI. The ligation product was mixed with 200 μL DH5α competent cell (purchased from Tiangen Biotech (Beijing) Co., Ltd.), and subjected to ice bath for 30 min, heat shock at 42° C. for 1.5 min, and ice bath for 3 min. 800 μL LB culture medium was added and cultivated for 45 min. LB plate containing 100 μg/mL kanamycin was plated and cultivated at 37° C. overnight. Transformants are screened by PCR using pBINOA-1 and pBINOA-6 as primers. PCR conditions were 94° C. 1 min, 60-73° C. 1 min, 72° C. 2 min, and extension or 10 min at 72° C. after 30 cycles. The PCR product were screened through agarouse gel electrophoresis. The positive transformant was designated as pBINOA. The positive transformant was cultured in liquid while shaking. Plasmid was extracted with alkaline lysis, and subjected to HindIII single digestion identification and HindIII and SacI double digestion. The identification results of agarose gel electrophoresis are shown in
Example 3: Production of Apolipoprotein A-I-Milano (AIM) with the Vector
(23) 3.1 Introduce the Seed-Specific Expression Vector Constructed Above into the Explants of the Receptor Plant;
(24) 3.1.1 Preparation of the Competent Agrobacterium Cells
(25) (1) Transfer Agrobacterium tumefacien LBA4404 single bacterium into 3 mL YEB medium (containing streptomycin Sm 125 μg/mL), and grow the cells at 28° C. overnight;
(26) (2) Transfer 5004 overnight culture into 50 mL YEB (Sm 125 μg/mL) medium, and grow the cells at 28° C. until OD.sub.600 is 0.5;
(27) (3) 5,000 rpm, centrifuge for 5 min;
(28) (4) Resuspend Agrobacterium cells in 10 mL 0.15M NaCl solution, 5,000 rpm, and centrifuge for 5 min;
(29) (5) Resuspend Agrobacterium cells in 1 mL precooled 20 mM CaCl.sub.2 for ice bath and use within 24 h, or dispense aliquots (200 μl) of the suspensions into tube and quick freeze for 1 min in liquid nitrogen, and preserve at −70° C. for later use.
(30) 3.1.2. Transformation of Agrobacterium Competent Cells with Seed-Specific Plant Expression Vector
(31) 1 μg thus constructed plasmid DNA was added to 2004 competent cells, and stored in liquid nitrogen for 1 min, in water bath at 37° C. for 5 min. Then 1 mL YEB medium was added, cultivated in liquid medium at 28° C. while slowly shaking for 4 h; and centrifuged at 1,000 rpm for 30 sec. The supernatant was discarded and 0.1 mL YEB medium was added for resuspension. Aliquots of the transformation reaction were plated on YEB agar plate containing 100 μg/mL Kan and 124 μg/mL Sm, and incubated at 28° C. for approximately 48 h.
(32) Identification of Positive Clone
(33) Single colony was picked into YEB medium (containing 100 μg/mL Kan and 125 μg/mL Sm), and cultivated in liquid medium at 28° C. overnight. Small amount of plasmid DNA was extracted with alkaline lysis. Using the plasmid DNA as template and pBINOA-1 and pBINOA-6 as primers, PCR amplification identification was carried out under the following conditions: 94° C. 1 min, 60-73° C. 1 min, 72° C. 2 min, and extension of 10 min at 72° C. after 30 cycles. Positive transformants were obtained after agar gel electrophoresis of PCR product.
(34) Preparation of Agrobacterium Suspension Used for Oil Sunflower Transformation
(35) 5 mL YEB medium containing 100 μg/mL Kan and 125 μg/mL Sm was inoculated with a single colony of transformed Agrobacterium. The culture was grown overnight with shaking. 100-200 mL YEB liquid medium containing 100 μg/mL Kan and 125 μg/mL Sm was inoculated with 1 mL culture. The culture was grown at 28° C. with vigorous shaking until OD.sub.600 is 0.4˜0.8, and centrifuged at 3500 rpm for 10 min to recover cells. The pellet was resuspended with MS (free of plant growth regulators or antibiotics) to make OD.sub.600 at approximately 0.6 for transformation.
(36) 3.1.3 Genetic Transformation of Oil Sunflower Explants Mediated by Agrobacterium
(37) The explants, in the forms of shoot apexes excised from sterile seedlings, cotyledon, cotyledonary node or seedlings with one cotyledon detached, of the seedling of oil sunflower seeds sprouting for 3˜4 d were immersed in said Agrobacterium suspension for 6˜8 min and transferred to MS solid medium for culture for 3 d (at 25° C., in dark). The seedlings with one cotyledon detached is preferred.
(38) 3.2 Cultivation of the Above Receptor Plant Materials into Complete Plant to Obtain Seeds for the Detection of Target Gene and Protein
(39) 3.2.1 Cultivate the Receptor Plant Materials into Complete Plant and Obtain Seeds
(40) The transformed explants were transferred to MS agar medium containing 300 mg/L cephalosporin for approximately 7 d, then transferred to MS resistance screening medium (containing 300 mg/L cephalosporin and 70 mg/L kanamycin) for selective culture. The medium was exchanged every 15˜20 d. Resistance buds were obtained after three rounds of screening. 2˜3 cm resistance buds were transferred to rooting medium MS2 (MS+IBA0.1 mg/L+Kan 70 mg/L+cef 300 mg/L) and transplanted after rootage of resistance seedling into greenhouse for vermiculite and Nutritional soil mixture culture until maturity, seeds harvested.
(41) 3.2.2 Target Gene and Protein Detection
(42) PCR detection was performed on apolipoprotein A-I-.sub.Milano gene during the Seedling Stage. Western blotting detection was performed on Arachis hypogaea oleosin and apolipoprotein A-I-.sub.Milano fusion protein after harvesting kernels.
(43) PCR Detection and PCR-Southern Blotting Detection of Transgenic Oil Sunflower Seedling
(44) SDS method was adopted to extract the genome DNA of the young leaves of resistant oil sunflower seedling as the template. PCR amplification was carried out with two pairs of primers nptIIF/nptIIR and pBINOA-5/pBINOA-6. The sequences of the premiers are nptIIF: ATG AAC TGC AGG ACG AGG (SEQ ID NO:17) and GCG ATA CCG TAA AGC ACG (SEQ ID NO:18) respectively. The PCR condition of nptIIF/nptIIR and pBINOA-5/pBINOA-6 includes 94° C. for 1 min, 60° C. for 1 mm, 72° C. for 1 min, and final extension for 10 min at 72° C. after 30 cycles. As anticipated, fragments of 567 bp (partial nptII gene) and apoA-I.sub.M gene fragment of 750 bp were amplified respectively. The results are shown in
(45) PCR-Southern Blotting Detection
(46) 1) Genomic DNA of the young leave of the transgenic oil sunflowers, in which both nptII and apoA-I.sub.M are positive, was extracted with SDS method. PCR amplification was performed on the genome DNA with pBINOA-1/pBINOA-6 as the primer. The PCR reaction condition includes 30 cycles of 94° C. for 1 min, 60° C. for 1 min, and 72° C. for 2.5 min; and final extension at 72° C. for 10 min. 2) The DNA was transferred from agarose gel to a nylon membrane, denatured and neutralized after electrophoresis, and subjected to semi-dry blotting. The membrane was dried and baked for 1.2 hr at 80° C. in a vacuum oven. 3) DNA probe marking
(47) The pBINOA plasmid DNA digested with BamH□ and Sac□ was recovered. 3 μg DNA was used for labeling. 4) Hybridization
(48) The membrane was pre-hybridized at 63° C. for 30 min and hybridized at 63° C. overnight, washed twice with 2×SSC, 0.1% SDS, and then washed twice with 0.5×SSC, 0.1% SDS preheated to 65° C. at 63° C. 5) Detection
(49) The hybridized and washed membrane was briefly rinsed once with washing buffer, incubated in 100 ml Blocking solution for 30 min, incubated for 30 min in 20 ml Antibody solution, Washed 2×15 min in 100 ml Washing buffer, and equilibrated for 2-5 min in 20 ml Detection buffer. The membrane was placed in a hybridization bag (with DNA side facing up) and 1 ml CSPD added. The membrane was incubated for 10 min at 37° C. to enhance the luminescent reaction, and exposed to X-ray film at room temperature. The results are shown in
(50) Western Blotting Detection of Arachis hypogaea Oleosin and Apolipoprotein A-I-.sub.Milano Fusion Protein in Transgenic Oil Sunflower Seeds
(51) Transgenic oil sunflower seeds were ground in five volumes of grinding buffer (50 mM Tris-HCl pH 7.5, 0.4 M sucrose, 0.5M NaCl), centrifugated 10×g for 30 min, and separated into three parts. The oil phase was collected and resuspend in one volume of grinding buffer and mixed even. Five volumes of precooled 50 mM Tris-HCl pH 7.5 buffer was added, centrifugated 10×g for 30 min, and the oil phase collected. The above processes were repeated for two times to further remove the remaining water-soluble ingredients and insoluble ingredients, obtaining pure oil body (the ingredients of oil body include: neutral lipids, phosphatides, and oleosin). To the oil body was added 2V of diethyl ether and centrifugated. The neutral lipids were in the upper diethyl ether layer and phosphatides were left in the lower water phase. The intermediate protein layer was collected and suspended in 0.1M sucrose buffer. Chloroform methanol (2:1) mixture was added and extracted twice. The intermediate protein layer was collected, extracted with diethyl ether once and dissolved in sterile water. SDS polyacrylamide gel electrophoresis was performed, and then Western blotting analysis was performed using polyclonal goat anti-rabbit apolipoprotein A-I after transmembrane. The results are shown in
(52) 3.3 Obtain Apolipoprotein A-I-.sub.Milano from the Seeds by Separation and Purification.
(53) Step 1: Separate Oil Body from Other Components in Seeds
(54) The kernel was ground in five volumes of grinding buffer (50 mM Tris-HCl pH 7.5, 0.4M sucrose, 0.5 M NaCl), centrifuged at 10×g for 30 min, and divided into three parts. The bottom part is insoluble precipitation (hull, fiber materials, insoluble sugar, protein and other insoluble dirt); the middle layer is aqueous phase, containing soluble cellular constituents (storage protein); the upper layer is the oil body and the associated oil body protein.
(55) Step 2: Wash the Oil Body
(56) The oil phase obtained from Step 1 was resuspended in the same volume of grinding butter and mixed even. Five volumes of precooled 50 m MTris-HCl pH 7.5 buffer are added and centrifuged at 10×g for 30 min. The oil phase was collected. The above processes were repeated twice to further remove the residual water-soluble ingredients and insoluble ingredients. The washed oil body was resuspended in precooled 50 mM Tris-HCl pH 7.5 of equivalent volume. The resulting oil body was substantially pure oil body, and he only protein left was oil body protein.
(57) Step 3: Release Apolipoprotein A-I-.sub.Milano Protein by Restrictive Digestion
(58) The oil body was washed with thrombin digestion buffer (20 m M Tris-HCl pH8.4, 150 m M NaCl, and 2.5 m M CaCl.sub.2) for two times. Appropriate amount of thrombin was added, stored at 37° C. overnight, and centrifuged. Apolipoprotein protein exists in the aqueous phase.
(59) Step 4: Purify Apolipoprotein A-I-.sub.Milano Protein with High Performance Liquid Chromatography (HPLC)
(60) Reversed-phase chromatography C4 column (5μ, 0.24*25 cm) was used, at the ultraviolet wavelength of 214 nm. The column was equilibrated with 2 mL/min buffer A (10% acetonitrile, 0.1% trifluoroacetic acid), loaded with the aqueous phase obtained in the last step, and applied linear gradient elution of 0-60% buffer B (95% acetonitrile, 0.1% trifluoroacetic acid). Pure apolipoprotein A-I-.sub.Milano protein was obtained with the purity above 99.5%.
Example 4: Comparison Between Oil Sunflower and Carthamus Tinctorius as Bioreactor for the Production of Apolipoprotein A-I-Milano (AIM)
(61) The same amount (280 mg) of trans-apolipoprotein A-I-.sub.Milano gene oil sunflower seed and carthamus tinctorius seed were used to obtain apolipoprotein A-I-.sub.Milano protein by separation and purification according to Example 3. The loading quantity was one-tenth of the total quantity obtained. Western blotting detection was performed, and the results are shown in