WO2023273420A1 - 大豆基因启动子pEIF1和pEIF1-I在大豆、拟南芥及烟草中的应用 - Google Patents

大豆基因启动子pEIF1和pEIF1-I在大豆、拟南芥及烟草中的应用 Download PDF

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WO2023273420A1
WO2023273420A1 PCT/CN2022/081297 CN2022081297W WO2023273420A1 WO 2023273420 A1 WO2023273420 A1 WO 2023273420A1 CN 2022081297 W CN2022081297 W CN 2022081297W WO 2023273420 A1 WO2023273420 A1 WO 2023273420A1
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peif1
gus
promoter
soybean
expression
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PCT/CN2022/081297
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French (fr)
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王学路
王海娇
彭亚齐
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河南大学
河南大学三亚研究院
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Priority to BR112023010403A priority Critical patent/BR112023010403A2/pt
Publication of WO2023273420A1 publication Critical patent/WO2023273420A1/zh
Priority to US18/167,878 priority patent/US20230250442A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation
    • C12N15/8223—Vegetative tissue-specific promoters

Definitions

  • the invention belongs to the technical field of plant genetic engineering, and specifically relates to the application of soybean gene promoters pEIF1 and pEIF1-I in soybean, Arabidopsis and tobacco.
  • Promoters are an important part of regulating gene expression in plants.
  • promoters can be divided into exogenous promoters and endogenous promoters. Exogenous promoters can regulate gene expression heterologously, but transgenic safety issues often arise when preparing transgenic materials, especially commonly used promoters derived from viruses, such as the tobacco mosaic virus 35S promoter. In addition, there is the potential for transgene silencing to occur when the same promoter is used in the same transgenic event. Therefore, the endogenous promoter is a more favored promoter for the preparation of transgenic materials.
  • promoters are divided into constitutive promoters, tissue-specific promoters, inducible promoters, etc. The expression regulated by the constitutive promoter is not affected by tissue specificity, time and space, etc. When preparing some transgenic materials, gene expression driven by constitutive promoters will be more stable.
  • Soybean is an important oil crop and protein crop and is able to live in symbiosis with rhizobia, which form root nodules that convert nitrogen in the air into ammonia that can be used by plants.
  • rhizobia form root nodules that convert nitrogen in the air into ammonia that can be used by plants.
  • soybean can not only provide nitrogen source for itself, but also provide nitrogen source for other crops through intercropping and crop rotation, thereby reducing the use of chemical fertilizers and establishing a green ecological agricultural system.
  • the yield of soybean has been very low, and obtaining soybean varieties with excellent traits and high yield through transgenic means is one of the important directions of soybean research in the future.
  • endogenous promoters In addition to exogenous viral promoters, such as 35S promoter and CMV promoter, which are used for soybean transformation, some endogenous promoters have also been reported successively. These endogenous promoters include constitutive promoters with high expression and ubiquitous expression. , inducible promoters, tissue-specific promoters, etc., wherein constitutive promoters refer to promoters that can drive stable expression of genes at different stages of growth and development, in different tissues, and in different growth conditions.
  • the Gmubi promoter is currently the most widely used promoter due to its high expression level.
  • the screening conditions of the reported constitutive promoters are mostly incomplete. Most of the promoters are obtained from tissue expression in a single stage of soybean growth and development, while the constitutive expression in soybean root nodule development promoters have hardly been reported. In terms of screening technology, most of the reported promoters are detected by the traditional qRT-PCR method, and there are not many reports on the detection of expression levels by high-throughput RNA sequencing methods.
  • soybean transgenic engineering it is very important for the application of soybean transgenic engineering to develop promoters that are ubiquitously expressed in different developmental stages of soybean and during the development of tissues and root nodules.
  • the purpose of the present invention is to obtain internal reference genes RPS28 and EIF1 for analyzing soybean growth and development in different stages, expression in different tissues, and root nodule development, and simultaneously obtain their promoters pRPS28 and pEIF1.
  • Another object of the present invention is to provide the application of promoter pRPS28 and promoter pEIF1 in regulating the constitutive or non-tissue-specific expression of soybean genes or other plant (such as tobacco, Arabidopsis) genes, specifically to achieve full-length
  • the pRPS28-type promoter and the intron-existing promoter pRPS28-I, the full-length promoter pEIF1 and the intron-existing promoter pEIF1-I respectively drive the high expression and Broad expression.
  • the present invention firstly provides the application of soybean gene promoter in exogenous gene expression.
  • the promoters include the full-length promoter pRPS28 or the intron-existing promoter pRPS28-I, the full-length promoter pEIF1 or the intron-existing promoter pEIF1-I, and the promoter is used as a general expression promoter , used to drive exogenous gene expression.
  • the exogenous gene is GUS gene.
  • soybean gene promoter When the above-mentioned soybean gene promoter is used for soybean gene expression, it can drive the expression of foreign genes in soybean cotyledons, radicles, germs, true leaves, compound leaves, buds, petioles, internodes, roots and root nodules.
  • soybean gene promoter When the above-mentioned soybean gene promoter is used for Arabidopsis gene expression, it can drive the expression of foreign genes in the whole plant, flower and pod of Arabidopsis.
  • soybean gene promoter When the above-mentioned soybean gene promoter is used for tobacco gene expression, it can drive the expression of foreign genes in tobacco leaves.
  • the present invention also provides a recombinant vector containing soybean gene promoter.
  • the soybean gene promoter is recombined into the vector pCAMBIA1391Z-BAR to construct and obtain, and the specific steps are as follows: use the PCR amplified fragments of pRPS28, pRPS28-I, pEIF1, pEIF1-I as templates, and PCR The amplified fragment was cloned into soybean stable transformation vector pCAMBIA1391Z-BAR to obtain soybean stable transformation vectors pRPS28-GUS-BAR, pRPS28-I-GUS-BAR, pEIF1-GUS-BAR, pEIF1-I-GUS-BAR.
  • the present invention also provides a primer pair for PCR amplification of soybean RPS28 gene and EIF1 gene.
  • primers for amplifying RPS28 are identical to primers for amplifying RPS28.
  • RPS28-F ATGGAGTCTCAGGTGAAGCAC
  • RPS28-R CTAGCGCAATCTTCTTGCTTC
  • EIF1-F ATGTCTGAATTAGACGATCAAATTCC
  • EIF1-R TCAGAAACCATGAATCTTGATATGATC.
  • the present invention also provides a primer pair for PCR amplifying soybean gene promoter.
  • the primers used to construct the pRPS28-GUS expression vector are: pRPS28-GUS-bar-F:
  • the primers used to construct the pRPS28-I-GUS expression vector are:
  • the primers used to construct the pEIF1-GUS expression vector are: pEIF1-GUS-bar-F:
  • the primers used to construct the pEIF1-I-GUS expression vector are:
  • the present invention obtains the nucleotide sequence of the promoter pRPS28 as shown in SEQ ID NO.2 through the above method, and the nucleotide sequence of the promoter pRPS28-I is shown in SEQ ID NO.3.
  • the present invention obtains the nucleotide sequence of the promoter pEIF1, as shown in SEQ ID NO.5, and the nucleotide sequence of the promoter pEIF1-I, as shown in SEQ ID NO.6, through the above method.
  • the present invention further provides the application of the full-length promoter pRPS28 and the intron-existing promoter pRPS28-I in improving the expression activity of exogenous genes in soybean.
  • the present invention further provides the application of the full-length promoter pEIF1 and the intron-existing promoter pEIF1-I in improving the expression activity of exogenous genes in soybean.
  • RNA sequencing analysis of RPS28 gene and EIF1 gene in different stages of soybean growth and development, and in different tissues through the RNA sequencing analysis of different stages and different tissues corresponding to before and after inoculation of rhizobia, it is found that there are higher, and constitutive expression.
  • the development period involved in the present invention includes: 8 days after the germination of the aboveground part or 8 days after the germination of the underground part, and the development of inoculation with Bradyrhizobium USDA110. Tissue development was studied while comparing the effects of rhizobia treatment on the various tissues.
  • the pRPS28 promoter and the pEIF1 promoter of the present invention can drive the target gene to achieve constitutive expression in various tissue parts (including root nodules) in soybean stably transformed plants.
  • the activity of GUS driven by the pRPS28 promoter of the present invention in soybean stably transformed plants is equivalent to that of the existing GmUbi promoter.
  • the intron of the pEIF1 promoter contributes a lot to the promoter driving performance, and the activity of the pEIF-I promoter of the present invention to drive GUS in soybean stably transformed plants is equivalent to that of the existing GmUbi promoter .
  • Fig. 1 is the expression analysis of RPS28 gene in soybean growth and development and root nodule development process
  • Fig. 2 is the expression analysis of EIF1 gene during soybean growth and development and root nodule development
  • Fig. 3 is the structural representation of the expression vector of promoter pRPS28 and promoter pEIF1;
  • Figure 4 is the detection of the activity of GUS driven by promoter pRPS28 and promoter pEIF1 during the stable transformation of soybean;
  • Figure 5 shows the expression of GUS driven by the promoter pRPS28 in different parts of transgenic soybean T2 generation plants
  • Figure 6 shows the expression of GUS driven by the promoter pEIF1 in different parts of transgenic soybean T2 generation plants
  • Figure 7 shows the detection results of the GUS activity of the GUS protein driven by the pRPS28 promoter, pRPS28-I promoter, pEIF1 promoter, pEIF1-I promoter and pGmUbi promoter respectively in transgenic soybeans;
  • Figure 8 shows the expression of GUS protein driven by the pRPS28 promoter, pRPS28-I promoter and pGmUbi promoter respectively in different parts of transgenic Arabidopsis T3 generation plants;
  • Figure 9 shows the expression of GUS protein driven by the pEIF1 promoter, pEIF1-I promoter and pGmUbi promoter respectively in different parts of transgenic Arabidopsis T3 generation plants;
  • Figure 10 shows the transient expression of GUS protein driven by pRPS28 promoter and pEIF1 promoter respectively in transgenic tobacco leaves.
  • the present invention clones gene RPS28 and gene EIF1 from soybean; obtains promoter pRPS28 and promoter pEIF1 by CTAB method, then constructs pRPS28-GUS, pRPS28-I-GUS, pEIF1-GUS, pEIF1-I-GUS promoter soybean stable
  • the vector was transformed, and the expression of GUS protein driven by pRPS28 and pEIF1 was realized at each stage of soybean genetic transformation. They were expressed in buds, petioles, internodes, roots and root nodules, indicating that pRPS28 and pEIF1 are ubiquitously expressed promoters, and the activity of GUS protein expressed in soybean was determined.
  • pRPS28 and pEIF1 drive the expression of GUS protein in Arabidopsis and tobacco.
  • the soybean variety--Jidou 17 was selected as the material. After sowing, the bradyrhizobium USDA110 was inoculated when the true leaves were unfolded (8 days after sowing). , 25 and 30 days, samples such as root, root nodule, hypocotyl, cotyledon, epicotyl, true leaf, true leaf node, compound leaf, internode, petiole and terminal bud were selected respectively, and RNA- Seq data is screened, and RNA is extracted for RNA sequencing.
  • the RNA extraction and sequencing methods can use existing technologies, and are not the invention point of the present invention, so they will not be described in detail. and filtered according to the following criteria:
  • a gene encoding ribosomal 40S small subunit protein S28 was screened, and it was named RPS28, and the cDNA sequence was obtained as shown in SEQ ID NO.1; a gene encoding eukaryotic translation initiation factor SUI1 was screened, and the It was named as EIF1, and the cDNA sequence was obtained as shown in SEQ ID NO.4.
  • Fig. 1 is the expression analysis of RPS28 gene during soybean growth and development and root nodule development.
  • Figure 1a-b shows that the RPS28 gene was sampled 1, 4, 6, 8, 10 and 20 days after soybean true leaves were inoculated with rhizobia and not inoculated with rhizobia.
  • the expression levels of hypocotyls, true leaves, true leaf nodes, compound leaves, internodes, petioles, terminal buds and other tissue parts were found to be stable and highly expressed in all tissue parts tested, and the effect of inoculation with rhizobia on its expression level not big.
  • Figure 1c shows that the RPS28 gene was sampled at 1, 2, 4, 6, 8, 10, 15, 20, 25 and 30 days after soybean true leaves were inoculated with and without rhizobia, and the RPS28 gene was detected by RNA-Seq. and the expression in root nodule development, it was found that the RPS28 gene was stably and highly expressed in roots and nodules.
  • FIG. 2 is the expression analysis of EIF1 gene during soybean growth and root nodule development.
  • Figure 2a-b shows that the EIF1 gene was sampled 1, 4, 6, 8, 10 and 20 days after soybean true leaves were inoculated with rhizobia and not inoculated with rhizobia.
  • the expression levels of epicotyls, true leaves, true leaf nodes, compound leaves, internodes, petioles, terminal buds and other tissue parts were found to be stable and highly expressed in all tissue parts tested, and the expression level of inoculated rhizobia Has little effect.
  • FIG. 2c shows that the EIF1 gene was sampled at 1, 2, 4, 6, 8, 10, 15, 20, 25 and 30 days after soybean true leaves were inoculated with and without rhizobia, and the RPS28 gene was detected by RNA-Seq. and the expression in root nodule development, it was found that EIF1 gene was stably and highly expressed in roots and nodules.
  • EIF1 has an intron between the translation initiation codon ATG and the 5'UTR. Therefore, we designed two promoter forms, one full-length promoter and one intron-existing promoter, named pRPS28, pRPS28-I, and pEIF1, pEIF1-I, respectively.
  • the soybean DNA extracted by the CTAB method is used as a template to amplify the RPS28 fragment and the RPS28-I fragment; amplify the pEIF1 fragment and the pEIF1-I fragment.
  • the specific amplification system and primer design are as follows:
  • the total volume of the reaction system is 50 ⁇ l
  • the template is 1 ⁇ l (about 100 ng) of genomic DNA of soybean line Williams82 (WS82), 25 ⁇ l of 2 ⁇ phanta MAX fertility enzyme reaction buffer, 1 ⁇ l of 10 mM dNTP, 5 ⁇ l of 4 ⁇ M primers (each primer concentration is 10 mM , both 2 ⁇ l), 1 ⁇ l phanta MAX fertility enzyme, add ddH 2 O (sterile deionized water) to 50 ⁇ l.
  • ddH 2 O sterile deionized water
  • the reaction program was: denaturation at 94°C for 2 minutes, 10s at 94°C, 30s at 58°C, 30s at 72°C, 30 cycles, and extension at 72°C for 5 minutes.
  • the primers are:
  • GUS proteins driven by pRPS28 and pEIF1 are expressed in all stages of soybean stable genetic transformation, including co-cultivation, bud induction, shoot elongation and other processes.
  • pRPS28-GUS, pRPS28-I-GUS, pEIF1-GUS, pEIF1- The I-GUS soybean stable transformation vector was constructed, and the pGmUbi-GUS soybean stable transformation vector was constructed as a control group.
  • the structure of the constructed expression vector is shown in FIG. 3 . The specific test steps are:
  • the total volume of the reaction system is 50 ⁇ l, template (the amplified DNA fragment above) 1 ⁇ l (about 100 ng), 2 ⁇ phanta MAX fertility enzyme reaction buffer 25 ⁇ l, 10 mM dNTP 1 ⁇ l, 4 ⁇ M primer 5 ⁇ l (each primer concentration is 10 mM, average 2 ⁇ l), 1 ⁇ l phanta MAX fertilizer enzyme, add ddH 2 O (sterile deionized water) to 50 ⁇ l.
  • the reaction program was: denaturation at 94°C for 2 minutes, 10s at 94°C, 30s at 58°C, 30s at 72°C, 30 cycles, and extension at 72°C for 5 minutes.
  • the primers used to construct the pRPS28-GUS expression vector are:
  • the primers used to construct the pRPS28-I-GUS expression vector are:
  • the primers used to construct the pEIF1-GUS expression vector are:
  • the primers used to construct the pEIF1-I-GUS expression vector are:
  • the primers used to construct the pGmUbi-GUS expression vector are:
  • the transformation vector pCAMBIA1391Z-BAR used in the present invention is the commercially available pCAMBIA1391Z vector.
  • the vector is linearized with the restriction endonuclease XhoI, and then the screening marker HygR gene is replaced by the seamless cloning method.
  • the primer sequence used when replacing the screening marker gene is pCAMBIA1391Z-BAR-F: TACAATCTATCTCTCTCTCGAGatgagcccagaacgacgcccg, pCAMBIA1391Z-BAR-R: CATTATTATGGAGAAACTCGAGTCAGATCTCGGTGACGGGCAGGAC.
  • the method of seamless cloning used in the present invention can be implemented by conventional means in the field, and is not the invention point of the present invention, so it will not be described again.
  • the methods for transferring the obtained promoter-driven GUS soybean stable transformation vector into the soybean line WS82 all adopt the soybean cotyledon node transformation method mediated by Agrobacterium EHA105.
  • references Lith D, Warnberg K, Wang K .Soybean[Glycine max(L.)Merr].Methods Mol Biol.2015; 1223:275-84.doi:10.1007/978-1-4939-1695-5_22.PMID:25300848. Transformation method, at the same time
  • the test has made adaptive improvements, and the specific steps are as follows:
  • Sterilization and germination of soybean seeds Pick soybean seeds without damage and disease spots and sterilize them with chlorine gas (measure 100ml of sodium hypochlorite into a 250ml beaker, slowly add 5ml of concentrated hydrochloric acid along the wall of the cup to generate chlorine gas, seal and sterilize 16 hours). Place the sterilized soybean seeds in a germination medium, place them in an incubator at 22° C. and culture them in dark for 16-24 hours to germinate.
  • Activation of Agrobacterium and preparation of infection solution the expression vectors of pRPS28-GUS-BAR, pRPS28-I-GUS-BAR, pEIF1-GUS-BAR, pEIF1-I-GUS-BAR, and pGmUbi-GUS-BAR were electroporated Transformed into Agrobacterium EHA105 and grown on LB solid medium containing kanamycin resistance. Take a single positive clone and put it into 6 mL of LB liquid medium containing kanamycin, and culture overnight at 28° C. with a shaker speed of 220 rpm.
  • Explant preparation and infection keep the 3-5 mm long hypocotyl of the germinated seeds, separate the two cotyledons, remove the seed coat, cut off the primary buds, and use a razor blade to make several cuts at the cotyledon nodes to obtain the explants for transformation.
  • Cotyledonary node explants Put it in the infection solution, and shake it on a horizontal rotator (50-80r/min) to infect for 30 minutes.
  • Co-cultivation Pour off the bacterial liquid, transfer the explants to a solid co-culture medium covered with a layer of sterile filter paper, 15-20 per dish, culture in the dark for 3-5 days in a 22°C incubator, and take the explants Perform GUS staining.
  • Bud induction culture After 3 to 5 days of co-cultivation, the explants were transferred to the bud induction medium, and 5 explants were placed in each dish. The photoperiod was 16/8 hours (light/dark), cultured at 25°C, subcultured once every 2 weeks, and subcultured twice. GUS staining was performed on sprouted explants.
  • Plant elongation culture remove the dead buds, cut off the cotyledons, transfer the explants to the shoot elongation medium, put 5 explants in each dish, photoperiod 16/8 hours (light/dark), 25°C Cultivate under conditions, subculture once every 3 weeks, and subculture 2 to 4 times. Explants were taken for GUS staining. Rooting induction: when the elongated shoots grow to 3 cm in length, they are cut off and transferred to root induction medium, cultured at 25°C with a photoperiod of 16/8 hours (light/dark).
  • the promoter pRPS28 and the promoter pEIF1 respectively drive the expression of GUS in the soybean genetic transformation tissue culture stage.
  • the figure is divided into co-cultivation (a), bud induction for 2 weeks (b), bud induction for 4 weeks (c), the results of the shoot elongation (d) stage, the results in Figure 4 indicate that the GUS protein driven by the promoter pRPS28 and the promoter pEIF1 respectively has expression in the soybean genetic transformation stage.
  • Seedling hardening and transplanting culture When the regenerated plant takes root and grows more than two compound leaves, take out the plant, wash the medium of the root, plant it in a small flowerpot filled with sterilized vermiculite, and place it in the artificial intelligence incubator (Temperature 25° C., photoperiod 16/8 hours, relative humidity 85% RH, light intensity 90 ⁇ M/m 2 /s) harden the seedlings for 5 to 7 days.
  • the artificial intelligence incubator Tempoture 25° C., photoperiod 16/8 hours, relative humidity 85% RH, light intensity 90 ⁇ M/m 2 /s
  • Plants are identified by transgenic T1 generation regenerated plants: extract DNA from the leaves of T1 transgenic soybean plants, use BAR gene to perform PCR amplification on all plants of T1 generation, and select a single plant that can amplify the specific band of BAR. Successful transgenic plants.
  • the total volume of the reaction system is 10 ⁇ l
  • the template the DNA fragment of the above-mentioned T1 generation plant
  • the 2 ⁇ taq mix reaction solution 5 ⁇ l
  • the primer 0.5 ⁇ l (0.25 ⁇ l for both BAR-F and BAR-R )
  • ddH 2 O sterile deionized water
  • the reaction program was denaturation at 94°C for 2 minutes, 10s at 94°C, 30s at 58°C, 30s at 72°C, 30 cycles, and extension at 72°C for 5 minutes.
  • the primers used are as follows:
  • samples were taken at 5 days and 15 days after growth, and analyzed by GUS staining.
  • the specific staining method is as follows: fix with acetone for 30-60 minutes, then discard the acetone, add GUS stain buffer to wash off the residual acetone on the surface of the sample, add an appropriate amount of GUS working solution (GUS buffer+1mg/L X-GLuc), and shading treatment Vacuum down for 30-60 minutes, shading treatment at 37°C for 4-8 hours, add 75% alcohol for decolorization, and take pictures to observe the dyeing results.
  • a is the soybean that germinated in roll paper for 5 days
  • b is the embryo
  • c is the soybean plant that has grown for 15 days
  • d is the soybean plant that has grown for 15 days
  • Figure 6 shows the expression of GUS driven by the promoter pEIF1 in different parts of transgenic soybean T2 generation plants
  • a is the soybean that germinated 5 days after roll paper
  • b is the embryo
  • c is the soybean plant that has grown for 15 days
  • d is the true leaf of the plant that has grown for 15 days , compound leaves, buds, petioles, internodes, e for roots and nodules, f for immature embryos, and g for pods.
  • GUS driven by pRPS28, pRPS28-I, pEIF1, and pEIF1-I was found to be expressed in cotyledons, radicles, and germs; 15 days later, pRPS28, pRPS28-I, pEIF1 , pEIF1-I-driven GUS were expressed in true leaves, compound leaves, buds, petioles, internodes, roots and root nodules, and pRPS28, pRPS28-I, pEIF1, pEIF1-I-driven GUS were found in immature embryos , pods and seeds at the bulging stage were expressed. The above results indicated that pRPS28 and pEIF1 are ubiquitous expression promoters.
  • the T3 generation homozygous soybean transgenic plants containing pRPS28-GUS-BAR, pRPS28-I-GUS-BAR, pEIF1-GUS-BAR, pEIF1-I-GUS-BAR, pGmUbi-GUS-BAR recombinant vectors were cultured for 15 days, and the Roots, three compound leaves, true leaves, cotyledons and other tissues were stored in liquid nitrogen quick-frozen.
  • 4-MU is used as a standard
  • 4-MUG is used as a substrate, and the value generated is measured by a microplate reader under the conditions of excitation light of 365nm and emission light of 455nm. Then according to the standard curve, calculate the value of 4-MU produced, and calculate the GUS activity pmol 4-MU/ ⁇ g total protein/min.
  • the pRPS28 promoter, pRPS28-I promoter, pEIF1 promoter, pEIF1-I promoter and pGmUbi promoter respectively drive the activity of GUS protein in soybean roots, compound leaves, true leaves, cotyledon and other tissues Test results.
  • the activity of the GUS protein driven by the pRPS28 promoter in the cotyledon of soybean #20 is 25.93 pmolMU/min/ ⁇ g
  • the activity of the GUS protein driven by the pRPS28 promoter in the cotyledon of soybean #35 is 38 pmolMU/min/ ⁇ g
  • pEIF1 The activity of -I promoter-driven GUS protein in soybean #13 cotyledons was 42.77pmolMU/min/ ⁇ g
  • the activity of pEIF1 promoter-driven GUS protein in soybean #77 cotyledons was 26.92pmolMU/min/ ⁇ g, indicating that the pRPS28 promoter
  • the pRPS28-I promoter, pEIF1 promoter, and pEIF1-I promoter can drive foreign proteins to have higher expression and activity in specific parts of soybean, and at the same time, it shows that the promoter of the present invention is more suitable for transformation events.
  • Reaction system a total volume of 50 ⁇ l, template (the amplified DNA fragment) 1 ⁇ l (about 100 ng), 2 ⁇ phanta MAX fertility enzyme reaction buffer 25 ⁇ l, 10 mM dNTP 1 ⁇ l, 4 ⁇ M primers 5 ⁇ l (each primer concentration is 10 mM, average 2 ⁇ l), 1 ⁇ l phanta MAX fertilizer enzyme, add ddH 2 O (sterile deionized water) to 50 ⁇ l.
  • template the amplified DNA fragment
  • 2 ⁇ phanta MAX fertility enzyme reaction buffer 25 ⁇ l
  • 10 mM dNTP 1 ⁇ l 4 ⁇ M primers 5 ⁇ l (each primer concentration is 10 mM, average 2 ⁇ l)
  • 1 ⁇ l phanta MAX fertilizer enzyme add ddH 2 O (sterile deionized water) to 50 ⁇ l.
  • the reaction program was: denaturation at 94°C for 2 minutes, 10s at 94°C, 30s at 58°C, 30s at 72°C, 30 cycles, and extension at 72°C for 5 minutes.
  • the primers used to construct the pRPS28-GUS-HYG expression vector are:
  • the primers used to construct the pRPS28-I-GUS-HYG expression vector are:
  • the primers used to construct the pEIF1-GUS-HYG expression vector are:
  • the primers used to construct the pEIF1-I-GUS-HYG expression vector are:
  • transgenes with consistent expression of GUS protein were screened in pEIF1-GUS-HYG line#3, #7, #12, transgenes with consistent expression of GUS protein were screened in pEIF1-I-GUS-HYG For line#10, #11, and #12, the T3 seedlings of the above transgenic Arabidopsis were sown in the Arabidopsis hygromycin selection medium, and placed in a refrigerator at 4°C for vernalization for 2 days.
  • the pRPS28 promoter, pRPS28-I promoter and pGmUbi promoter respectively drive the expression of GUS protein in different parts of the transgenic Arabidopsis T3 generation plants
  • a is the GUS protein driven by the pRPS28 promoter in the Arabidopsis
  • b is the expression situation of the GUS protein driven by the pRPS28-I promoter in the whole plant, flower and pod of Arabidopsis thaliana, indicating that pRPS28 can drive the target gene (GUS gene) in Heterologous ubiquitous expression in Arabidopsis.
  • Figure 9 shows the expression of GUS protein driven by pEIF1 promoter, pEIF1-I promoter and pGmUbi promoter respectively in different parts of transgenic Arabidopsis T3 generation plants
  • a is the expression of GUS protein driven by pEIF1 promoter in Arabidopsis
  • b is the expression of GUS protein driven by the pEIF1-I promoter in the whole plant, flower and pod of Arabidopsis, indicating that pEIF1 can drive the target gene (GUS gene) in Arabidopsis Heterologous ubiquitous expression in A. thaliana.
  • the pRPS28 promoter and pEIF1 promoter respectively drive the transient expression of GUS protein in transgenic tobacco leaves.
  • the leaves of the mock group injected with the empty vector pCAMBIA1391Z had no expression of GUS, while the leaves of the pRPS28 and pEIF1 promoter groups There is GUS expression, and the test results show that GUS driven by pRPS28 and pEIF1 promoters can be transiently expressed in tobacco leaves, indicating that pRPS28 and pEIF1 promoters can heterologously drive gene expression in tobacco.

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Abstract

提供了大豆基因启动子pEIF1和pEIF1-I，含有这些启动子的重组载体，扩增这些启动子的引物对，以及上述启动子在大豆、拟南芥及烟草中的应用。

Description

大豆基因启动子pEIF1和pEIF1-I在大豆、拟南芥及烟草中的应用 技术领域
本发明属于植物基因工程技术领域，具体涉及大豆基因启动子pEIF1和pEIF1-I在大豆、拟南芥及烟草中的应用。
背景技术
启动子是调控植物基因表达的重要组成部分。按照来源，启动子可以分为外源启动子和内源启动子。外源启动子可以异源调控基因的表达，但是在制备转基因材料时往往会出现转基因安全问题，尤其是常用的来源于病毒的启动子，如烟草花叶病毒35S启动子。此外，在同一个转基因事件中使用同一个启动子还有可能发生转基因沉默现象。因此，内源启动子是制备转基因材料更加受到青睐的启动子。按照表达特征，启动子分为组成型启动子、组织特异性启动子、诱导型启动子等。其中组成型启动子调控的表达不受组织特异性、时空等的影响。在制备一些转基因材料时，组成型启动子驱动的基因表达会更加稳定。
大豆是重要的油料作物和蛋白质作物，并且能够与根瘤菌共生，形成根瘤，将空气中的氮气转化为可供植物利用的氨。通过根瘤固氮，大豆不但可以为自己提供氮源，还可以通过间作、轮作的方式为其他作物提供氮源，进而减少化肥施用，建立绿色生态型农业系统。但是大豆的产量一直很低，通过转基因手段获得性状优良、产量高的大豆品种是未来大豆研究的重要方向之一。除了外源的病毒启动子，如35S启动子、CMV启动子等用于大豆的转化外，一些内源启动子也被陆续报道，这些内源启动子包括高表达和泛表达的组成型启动子、诱导型启动子、组织特异性启动子等，其中组成型启动子是指在生长发育的不同阶段、不同组织、不同生长条件都能够驱动基因稳定表达的启动子。
已经报道的大豆组成型启动子中，Gmubi启动子由于较高的表达量使其成为目前应用比较广泛的启动子。此外，在筛选手段方面，已经报道的组成型启动子的筛选条件大都不完善，大部分启动子都是根据大豆生长发育的单一时期的组织表达得到的，而在大豆根瘤发育过程中组成型表达的启动子几乎没有报道。在筛选技术上，大部分报道的启动子是通过传统的qRT-PCR方法检测的，通过高通量的RNA测序方法检测表达水平的报道并不多。文献(Ning Zhang,Leah K.McHale,John J.Finer.Isolation and characterization of“GmScream”promoters that regulate highly expressing soybean(Glycine max Merr.)genes[J].Plant Science,2015,241.)根据来源于大豆品系Williams82(WS82)的31个文库的RNA测序数据 筛选到了10个可能的组成型启动子的表达基因，但是其中并没有涉及一个大豆生长发育的连续过程及根瘤发育中的表达情况。同时，在同一株植物中转入多个基因时，如果用同一个启动子驱动容易造成基因沉默。
因此，开发在大豆不同发育时期和组织及根瘤发育过程中泛表达的启动子对大豆转基因工程的应用非常重要。
发明内容
本发明的目的在于获得用于分析大豆生长发育不同时期、不同组织中表达情况，及根瘤发育情况的内参基因RPS28和EIF1，并同时获得其启动子pRPS28和pEIF1。
本发明的另一个目的是提供启动子pRPS28、启动子pEIF1在调控大豆基因或其他植物(例如烟草、拟南芥)基因的组成型、或者非组织特异性表达中的应用，具体为实现全长型启动子pRPS28及内含子存在型启动子pRPS28-I，全长型启动子pEIF1及内含子存在型启动子pEIF1-I分别驱动目的基因在大豆、拟南芥、烟草中的高表达和泛表达。
为了实现上述目的，本发明首先提供了大豆基因启动子在外源基因表达中的应用。
所述启动子包括全长型启动子pRPS28或内含子存在型启动子pRPS28-I，全长型启动子pEIF1或内含子存在型启动子pEIF1-I，所述启动子作为泛表达启动子，用于驱动外源基因表达。
所述外源基因为GUS基因。
上述大豆基因启动子在用于大豆基因表达时，能够驱动外源基因在大豆子叶、胚根、胚芽、真叶、复叶、芽、叶柄、节间、根和根瘤中表达。
上述大豆基因启动子在用于拟南芥基因表达时，能够驱动外源基因在拟南芥全株、花、荚果中表达。
上述大豆基因启动子在用于烟草基因表达时，能够驱动外源基因在烟草叶片中表达。
进一步的，本发明还提供了一种含有大豆基因启动子的重组载体。
所述重组载体在构建时，将大豆基因启动子重组进入载体pCAMBIA1391Z-BAR中构建获得，具体步骤如下：以pRPS28、pRPS28-I、pEIF1、pEIF1-I的PCR扩增片段为模板，并将PCR扩增片段克隆进大豆稳定转化载体pCAMBIA1391Z-BAR中，获得大豆稳定转化载体pRPS28-GUS-BAR、pRPS28-I-GUS-BAR、pEIF1-GUS-BAR、pEIF1-I-GUS-BAR。
进一步的，本发明还提供了PCR扩增大豆RPS28基因和EIF1基因用引物对。
具体为，扩增RPS28的引物：
RPS28-F：ATGGAGTCTCAGGTGAAGCAC
RPS28-R：CTAGCGCAATCTTCTTGCTTC
扩增EIF1的引物：
EIF1-F：ATGTCTGAATTAGACGATCAAATTCC
EIF1-R：TCAGAAACCATGAATCTTGATATGATC。
进一步的，本发明还提供了PCR扩增大豆基因启动子的引物对。
构建pRPS28-GUS表达载体所用引物为：pRPS28-GUS-bar-F：
Figure PCTCN2022081297-appb-000001
pRPS28-GUS-bar-R：
Figure PCTCN2022081297-appb-000002
构建pRPS28-I-GUS表达载体所用引物为：
pRPS28-I-GUS-bar-F：
Figure PCTCN2022081297-appb-000003
pRPS28-I-GUS-bar-R：
Figure PCTCN2022081297-appb-000004
构建pEIF1-GUS表达载体所用引物为：pEIF1-GUS-bar-F：
Figure PCTCN2022081297-appb-000005
pEIF1-GUS-bar-R：
Figure PCTCN2022081297-appb-000006
构建pEIF1-I-GUS表达载体所用引物为：
pEIF1-I-GUS-bar-F：
Figure PCTCN2022081297-appb-000007
pEIF-I-GUS-bar-R：
Figure PCTCN2022081297-appb-000008
本发明通过上述方法获得了启动子pRPS28的核苷酸序列如SEQ ID NO.2所示，启动子pRPS28-I的核苷酸序列如SEQ ID NO.3所示。
本发明通过上述方法获得了启动子pEIF1的核苷酸序列，如SEQ ID NO.5所示，以及启动子pEIF1-I的核苷酸序列，如SEQ ID NO.6所示。
本发明进一步提供了全长型启动子pRPS28及内含子存在型启动子pRPS28-I在提高外源基因在大豆中表达活性的应用。
本发明进一步提供了全长型启动子pEIF1及内含子存在型启动子pEIF1-I在提高外源 基因在大豆中表达活性的应用。
与现有技术相比，本发明的有益效果为：
本发明通过RPS28基因、EIF1基因在大豆生长发育的不同时期、不同组织的RNA测序分析，以及在接种根瘤菌前后相对应的不同时期、不同组织的RNA测序分析，发现都有较高的、及组成型的表达。本发明涉及的发育时期包括：地上部分萌发后8天或地下部分萌发后8天，并接种慢生型根瘤菌USDA110的发育情况，在现有报道的大豆启动子中，本发明首次对多种组织的发育情况进行了研究，同时比较了根瘤菌处理对各个组织的影响。
本发明pRPS28启动子和pEIF1启动子在大豆稳定转化植株中各个组织部位(包括根瘤中)能够驱动目的基因实现组成型表达。
本发明的pRPS28启动子驱动GUS在大豆稳定转化植株中的活性与现有的GmUbi启动子相当。与现有的GmUbi启动子相比，pEIF1启动子的intron对启动子驱动性能贡献很大，本发明的pEIF-I启动子驱动GUS在大豆稳定转化植株中的活性与现有的GmUbi启动子相当。
附图说明
图1为RPS28基因在大豆生长发育和根瘤发育过程中的表达分析；
图2为EIF1基因在大豆生长发育和根瘤发育过程中的表达分析；
图3为启动子pRPS28和启动子pEIF1的表达载体的结构示意图；
图4为启动子pRPS28和启动子pEIF1驱动GUS在大豆稳定转化过程中的活性检测；
图5为启动子pRPS28驱动GUS在转基因大豆T2代植株不同部位中的表达情况，；
图6为启动子pEIF1驱动GUS在转基因大豆T2代植株不同部位中的表达情况；
图7为pRPS28启动子、pRPS28-I启动子、pEIF1启动子、pEIF1-I启动子与pGmUbi启动子分别驱动GUS蛋白在转基因大豆中GUS的活性检测结果；
图8为pRPS28启动子、pRPS28-I启动子与pGmUbi启动子分别驱动GUS蛋白在转基因拟南芥T3代植株不同部位中的表达情况；
图9为pEIF1启动子、pEIF1-I启动子与pGmUbi启动子分别驱动GUS蛋白在转基因拟南芥T3代植株不同部位中的表达情况；
图10为pRPS28启动子、pEIF1启动子分别驱动GUS蛋白在转基因烟草叶片的瞬时表达情况。
具体实施方式
本发明从大豆中克隆了基因RPS28及基因EIF1；通过CTAB法获取启动子pRPS28 和启动子pEIF1，然后构建pRPS28-GUS、pRPS28-I-GUS、pEIF1-GUS、pEIF1-I-GUS启动子大豆稳定转化载体，并实现pRPS28和pEIF1驱动的GUS蛋白在大豆遗传转化各阶段的表达，发现pRPS28、pRPS28-I、pEIF1、pEIF1-I驱动的GUS在子叶、胚根、胚芽、真叶、复叶、芽、叶柄、节间、根和根瘤中均有表达，表明pRPS28和pEIF1是泛表达启动子，并对GUS蛋白在大豆中表达产物的活性进行了测定。另外，还实现了pRPS28和pEIF1驱动GUS蛋白在拟南芥和烟草中表达。
本发明所述技术方案，如未特别说明，均为本领域的常规方案，所述试剂或生物材料，如未特别说明，均已公开。
实施例
一、RPS28基因和EIF1基因的筛选
选取大豆品种--冀豆17作为材料，播种后，待真叶展开(播种后8d)时接种慢生型根瘤菌USDA110，接菌后1、2、4、6、8、10、15、20、25和30d时分别选取根、根瘤、下胚轴、子叶、上胚轴、真叶、真叶节、复叶、节间、叶柄和顶芽等样品，同时在所选大豆品种的RNA-seq数据中进行筛选，提取RNA做RNA测序，RNA提取和测序方法采用现有技术即可，且不是本发明的发明点所在，故不再赘述。并根据以下条件进行了筛选：
1、根据本实验大豆RNA-seq数据，分析在各个时期、各个组织部位均有表达的基因，根据其FPKM值从大到小排序，挑选排名靠前的20个大豆泛表达基因为候选基因。
2、将这些候选基因的RNA测序数据作图，挑选各个时期、组织部位表达量波动不大，且不受接种根瘤菌影响(变异系数CV≤0.3)的10个基因作为进一步分析的候选基因。
3、然后用这10个大豆泛表达候选基因，以本实验大豆整合转录组数据中的表达量作图，挑选不易受环境影响的两个基因，并分析它们的启动子序列，以及进一步的表达分析相关研究。
筛选到一个编码核糖体40S小亚基蛋白S28的基因，将其命名为RPS28，并获得cDNA序列如SEQ ID NO.1所示；筛选到一个编码真核生物翻译起始因子SUI1的基因，将其命名为EIF1，并获得cDNA序列如SEQ ID NO.4所示。
根据基因RPS28和EIF1的表达量的结果显示，这两个基因在大豆真叶展开后1d～30d内各个组织部位均有高表达，且接种根瘤菌前后表达量影响不大(图1和图2所示)。
图1为RPS28基因在大豆生长发育和根瘤发育过程中的表达分析。其中图1a-b是RPS28基因在大豆真叶展开后接种根瘤菌和不接种根瘤菌1、4、6、8、10和20天取样，通 过RNA-Seq检测RPS28基因在下胚轴、子叶、上胚轴、真叶、真叶节、复叶、节间、叶柄和顶芽等组织部位的表达量，发现RPS28基因在检测的各个组织部位均稳定且高表达，接种根瘤菌对其表达量影响不大。1c图是RPS28基因在大豆真叶展开后接种根瘤菌和不接种根瘤菌1、2、4、6、8、10、15、20、25和30天取样，通过RNA-Seq检测RPS28基因在根和根瘤发育中的表达，发现RPS28基因在根和根瘤中稳定且高表达。
图2为EIF1基因在大豆生长发育和根瘤发育过程中的表达分析。其中图2a-b图是EIF1基因在大豆真叶展开后接种根瘤菌和不接种根瘤菌1、4、6、8、10和20天取样，通过RNA-Seq检测EIF1基因在下胚轴、子叶、上胚轴、真叶、真叶节、复叶、节间、叶柄和顶芽等组织部位的表达量，发现EIF1基因在检测的各个组织部位均稳定且高表达，接种根瘤菌对其表达量影响不大。2c图是EIF1基因在大豆真叶展开后接种根瘤菌和不接种根瘤菌1、2、4、6、8、10、15、20、25和30天取样，通过RNA-Seq检测RPS28基因在根和根瘤发育中的表达，发现EIF1基因在根和根瘤中稳定且高表达。
二、启动子pRPS28和启动子pEIF1的获得
根据试验一、的筛选结果，发现RPS28和EIF1这两个基因的基因组序列及转录起始位点上游分别2357bp(RPS28)和1640bp(EIF1)的启动子序列，分析发现这两个基因(RPS28和EIF1)的翻译起始密码子ATG与5’UTR之间具有内含子。因此我们设计两种启动子形式，一种全长型启动子，一种内含子存在型启动子，分别命名为pRPS28、pRPS28-I，以及pEIF1、pEIF1-I。
采用CTAB法提取的大豆DNA为模板，扩增RPS28片段和RPS28-I片段；扩增pEIF1片段和pEIF1-I片段。具体的扩增体系及引物设计如下：
反应体系的总体积为50μl，模板为大豆品系Williams82(WS82)的基因组DNA 1μl(约100ng)、2×phanta MAX fertility酶反应缓冲液25μl、10mM dNTP 1μl、4μM引物5μl(每条引物浓度为10mM，均为2μl)、1μl phanta MAX fertility酶，加ddH 2O(无菌去离子水)至50μl。
反应程序为：94℃变性2min，94℃10s、58℃30s、72℃30s，30cycles，72℃延伸5min。
所述引物为：
扩增RPS28基因片段的引物：
F：ATGGAGTCTCAGGTGAAGCAC
R：CTAGCGCAATCTTCTTGCTTC
扩增EIF1基因片段的引物：
F：ATGTCTGAATTAGACGATCAAATTCC
R：TCAGAAACCATGAATCTTGATATGATC
扩增pRPS28的引物：
pRPS28-F：CACCACCCAATCCATAACCACCAC
pRPS28-R：CTGATGCAAAACACGAACAAAGAAAG
最终获得的DNA序列见SEQ ID NO.2
扩增pRPS28-I的引物：
pRPS28-I-F：CACCACCCAATCCATAACCACCAC
pRPS28-I-R：CCTGCTCAAACACAATCAACAG
最终获得的DNA序列见SEQ ID NO.3
扩增pEIF1的引物：
pEIF1-F:GGAGAGAAGTTGAACTCTGAGTTGTG
pEIF1-R:CTGATCGTAAATTTAAGGTTTCG
最终获得的DNA序列见SEQ ID NO.5
扩增pEIF1-I的引物：
pEIF1-I-F:GGAGAGAAGTTGAACTCTGAGTTGTG
pEIF1-I-R:AAAACTTGACTCACTAAGACCAAAGG
最终获得的DNA序列见SEQ ID NO.6
三、启动子pRPS28和启动子pEIF1在大豆中的稳定遗传转化
pRPS28和pEIF1驱动的GUS蛋白在大豆稳定遗传转化各阶段都有表达，包括共培养、芽诱导、芽伸长等过程，本试验构建pRPS28-GUS、pRPS28-I-GUS、pEIF1-GUS、pEIF1-I-GUS大豆稳定转化载体，同时构建pGmUbi-GUS大豆稳定转化载体作为对照组，所构建的表达载体的结构如图3所示。具体的试验步骤为：
1、大豆稳定转化载体的构建
以试验二、中得到的pRPS28、pRPS28-I、pEIF1、pEIF1-I的PCR扩增片段为模板，并将上述PCR产物片段通过无缝克隆法克隆进大豆稳定转化载体pCAMBIA1391Z-BAR中，获得大豆稳定转化载体pRPS28-GUS-BAR、pRPS28-I-GUS-BAR、pEIF1-GUS-BAR、pEIF1-I-GUS-BAR、pGmUbi-GUS-BAR，并转入大豆品系WS82中。
具体的扩增体系及引物设计如下：
反应体系的总体积为50μl，模板(上述扩增的DNA片段)1μl(约100ng)、2×phanta  MAX fertility酶反应缓冲液25μl、10mM dNTP 1μl、4μM引物5μl(每条引物浓度为10mM，均为2μl)、1μl phanta MAX fertility酶，加ddH 2O(无菌去离子水)至50μl。
反应程序为：94℃变性2min，94℃10s、58℃30s、72℃30s，30cycles，72℃延伸5min。
构建pRPS28-GUS表达载体所用引物为：
pRPS28-GUS-bar-F：
Figure PCTCN2022081297-appb-000009
pRPS28-GUS-bar-R：
Figure PCTCN2022081297-appb-000010
构建pRPS28-I-GUS表达载体所用引物为：
pRPS28-I-GUS-bar-F：
Figure PCTCN2022081297-appb-000011
pRPS28-I-GUS-bar-R：
Figure PCTCN2022081297-appb-000012
构建pEIF1-GUS表达载体所用引物为：
pEIF1-GUS-bar-F：
Figure PCTCN2022081297-appb-000013
pEIF1-GUS-bar-R：
Figure PCTCN2022081297-appb-000014
构建pEIF1-I-GUS表达载体所用引物为：
pEIF1-I-GUS-bar-F：
Figure PCTCN2022081297-appb-000015
pEIF-I-GUS-bar-R：
Figure PCTCN2022081297-appb-000016
构建pGmUbi-GUS表达载体所用引物为：
pGmUbi-GUS-bar-F：
Figure PCTCN2022081297-appb-000017
pGmUbi-GUS-bar-R：
Figure PCTCN2022081297-appb-000018
本发明中所使用的转化载体pCAMBIA1391Z-BAR是将商业化市售的pCAMBIA1391Z载体，首先采用限制性内切酶XhoI将载体线性化，然后再通过无缝克隆的方法将其中的筛选 标记HygR基因替换为BAR基因实现的，具体的，替换筛选标记基因时采用的引物序列为pCAMBIA1391Z-BAR-F：TACAAATCTATCTCTCTCGAGatgagcccagaacgacgcccg，pCAMBIA1391Z-BAR-R：CATTATTATGGAGAAACTCGAGTCAGATCTCGGTGACGGGCAGGAC。
本发明所使用的无缝克隆的方法采用本领域的常规手段实现即可，且不是本发明的发明点所在，故不再赘述。
2、大豆遗传转化及遗传转化中的GUS染色
具体的，将得到的启动子驱动GUS的大豆稳定转化载体转入大豆品系WS82中的方法均采用农杆菌EHA105介导的大豆子叶节转化法，具体方法参考文献(Luth D,Warnberg K,Wang K.Soybean[Glycine max(L.)Merr].Methods Mol Biol.2015；1223:275-84.doi:10.1007/978-1-4939-1695-5_22.PMID:25300848.)中的转化方法，同时本试验在此基础上作了适应性改进，具体步骤如下：
大豆种子灭菌及萌发：挑取无破损、无病斑的大豆种子用氯气灭菌(量取100ml次氯酸钠置入250ml烧杯中，沿杯壁缓缓加入5ml浓盐酸，即产生氯气，密封灭菌16小时)。将灭过菌的大豆种子放置于萌发培养基中，放置于22℃培养箱中暗培养16～24小时使其萌发。
农杆菌的活化和侵染液的制备：将pRPS28-GUS-BAR、pRPS28-I-GUS-BAR、pEIF1-GUS-BAR、pEIF1-I-GUS-BAR、pGmUbi-GUS-BAR表达载体通过电激法转化到农杆菌EHA105中，并在含有卡那霉素抗性的LB固体培养基上生长。取单个阳性克隆到6mL含有卡那霉素的LB液体培养基中，于28℃，摇床转速220rpm条件下过夜培养。取250μl的菌液涂布于含有卡那霉素抗性的LB固体平板上，28℃培养过夜。用一次性接种环刮取菌膜，垂悬于液体共培养基里，用分光光度计测定菌液浓度，使终浓度OD 600＝0.5～0.6。
外植体准备和侵染：保留萌发后种子3～5mm长的下胚轴，分离两片子叶，去除种皮，切除初生芽后，用刀片在子叶节部位划几刀，得到用于转化的子叶节外植体。将其置于侵染液中，于水平旋转仪上振荡(转速50～80r/min)侵染30min。
共培养：倒掉菌液，将外植体转移至铺有一层无菌滤纸的固体共培养基上，每皿15～20个，22℃培养箱中暗培养3～5天，取外植体进行GUS染色。
筛选培养和植株再生(具体的培养基以及培养条件参见文献Luth D,Warnberg K,Wang K.Soybean[Glycine max(L.)Merr].Methods Mol Biol.2015；1223:275-84.doi:10.1007/978-1-4939-1695-5_22.PMID:25300848.)：芽诱导培养：共培养3～5天之后，将外植体转移至芽诱导培养基中，每皿放5个外植体，光周期16/8小时(光照/黑暗)、25℃条件下培养，每2周继代一次，继代2次。对长出芽的外植体进行GUS染色。芽伸长培养：除 去死芽，切掉子叶部分，将外植体转移至芽伸长培养基中，每皿放5个外植体，光周期16/8小时(光照/黑暗)、25℃条件下培养，每3周继代一次，继代2～4次。取外植体进行GUS染色。生根诱导：当伸长苗长至3cm长之后，切下转移至根诱导培养基中，光周期16/8小时(光照/黑暗)、25℃条件下培养。
如图4所示，为启动子pRPS28和启动子pEIF1分别驱动GUS在大豆遗传转化组织培养阶段的表达情况，图中分为共培养(a)、芽诱导2周(b)、芽诱导4周(c)、芽伸长(d)阶段的结果，图4的结果说明启动子pRPS28和启动子pEIF1分别驱动的GUS蛋白在大豆遗传转化阶段有表达。
炼苗及移栽培养：当再生植株生根并长出两片以上复叶后，取出植株，洗净根部的培养基，栽入盛有灭菌蛭石的小花盆中，在人工智能培养箱(温度25℃，光周期16/8小时，相对湿度85％RH，光照强度90μM/m 2/s)炼苗5～7天。待壮苗之后将其移栽到大花盆(营养土：蛭石＝1:1)中，移至培养室(温度28±2℃，光周期13.5/10.5h，相对湿度40％～60％RH，光照强度90μM/m 2/s)中生长至成熟。
利用转基因T1代再生植株对植株进行鉴定：取T1代转基因大豆植株的叶片提取DNA，利用BAR基因对T1代的全部植株进行PCR扩增，挑选能扩增出BAR特异条带的单株即为转基因成功植株。
PCR体系和反应条件：反应体系的总体积为10μl，模板(上述T1代植株DNA片段)0.5-1μl、2×taq mix反应液5μl、引物0.5μl(BAR-F和BAR-R均为0.25μl)，加ddH 2O(无菌去离子水)至10μl。反应程序为94℃变性2min，94℃10s、58℃30s、72℃30s，30cycles，72℃延伸5min。
所用引物如下：
BAR-F:ATGAGCCCAGAACGACGCCCGGCC
BAR-R:TTAGATCTCGGTGACGGGCAGGAC
最终获得的BAR基因序列如SEQ ID NO.7所示
3、GUS染色结果测定
上述方法获得的稳定转化植株，得到纯合植株后，在生长后5天和15天取样，进行GUS染色分析。具体的染色方法为：丙酮固定30～60min，后弃去丙酮，加入GUS染液缓冲液洗去样品表面残留的丙酮，加适量GUS工作液(GUS buffer﹢1mg/L X-GLuc)，遮光处理下抽真空30～60min，37℃遮光处理4～8h，加入75％酒精脱色，对染色结果进行拍照观察。
如图5所示，为启动子pRPS28驱动GUS在转基因大豆T2代植株不同部位中的表达情况，a为卷纸萌发5d的大豆，b为胚芽，c为生长15d的大豆植株，d为生长15d植株的 真叶、复叶、芽、叶柄、节间，e为根和根瘤，f为未成熟的胚，g为荚果。
图6为启动子pEIF1驱动GUS在转基因大豆T2代植株不同部位中的表达情况，a为卷纸萌发5d的大豆，b为胚芽，c为生长15d的大豆植株，d为生长15d植株的真叶、复叶、芽、叶柄、节间，e为根和根瘤，f为未成熟的胚，g为荚果。
上述转基因方法获得的植株在萌发后5天，发现pRPS28、pRPS28-I、pEIF1、pEIF1-I驱动的GUS在子叶、胚根、胚芽处均有表达；15天后，发现pRPS28、pRPS28-I、pEIF1、pEIF1-I驱动的GUS在真叶、复叶、芽、叶柄、节间、根和根瘤中均有表达，并发现pRPS28、pRPS28-I、pEIF1、pEIF1-I驱动的GUS在未成熟的胚、鼓粒期荚果和种子中均有表达。以上结果表明pRPS28和pEIF1是泛表达启动子。
4、pRPS28启动子、pEIF1启动子与pGmUbi启动子分别驱动GUS蛋白在大豆中表达产物的活性比较。
将含pRPS28-GUS-BAR、pRPS28-I-GUS-BAR、pEIF1-GUS-BAR、pEIF1-I-GUS-BAR、pGmUbi-GUS-BAR重组载体的大豆转基因植株T3代纯合体培养15天，取根、三出复叶、真叶、子叶等组织，液氮速冻保存。对GUS活性进行定量测定，以4-MU为标准品，以4-MUG为底物，通过酶标仪，在激发光为365nm，发射光为455nm条件下测定产生的值。然后根据标准曲线，计算产生的4-MU的值，并计算GUS活性pmol 4-MU/μg总蛋白/min。
如图7所示，为pRPS28启动子、pRPS28-I启动子、pEIF1启动子、pEIF1-I启动子与pGmUbi启动子分别驱动GUS蛋白在大豆根、复叶、真叶、子叶等组织部位的活力检测结果。从图中可以看出pRPS28、pRPS28-I、pEIF1、pEIF1-I驱动的GUS在上述组织部位泛表达，结果表明pRPS28启动子驱动GUS蛋白的活性强于pRPS28-I启动子，pEIF1-I启动子驱动GUS蛋白的活性强于pEIF1启动子。从图中可以看出pRPS28启动子驱动GUS蛋白在大豆#20子叶中的活力为25.93pmolMU/min/μg，pRPS28启动子驱动GUS蛋白在大豆#35子叶中的活力为38pmolMU/min/μg，pEIF1-I启动子驱动GUS蛋白在大豆#13子叶中的活力为42.77pmolMU/min/μg，pEIF1启动子驱动GUS蛋白在大豆#77子叶中的活力为26.92pmolMU/min/μg，说明pRPS28启动子、pRPS28-I启动子、pEIF1启动子、pEIF1-I启动子能够驱动外源蛋白在大豆特定部位中具有较高的表达量以及活性，同时说明本发明的启动子更加适用于转化事件。
四、pRPS28和pEIF1驱动GUS蛋白在拟南芥和烟草中的转化
1、pRPS28-GUS、pRPS28-I-GUS、pEIF1-GUS、pEIF1-I-GUS启动子拟南芥转化载体、以及 烟草转化载体的构建。
以试验二、中得到的pRPS28、pRPS28-I、pEIF1、pEIF1-I的PCR扩增片段为模板，并将上述PCR产物通过无缝克隆克隆进pCAMBIA1391Z-GUS-HYG(在转基因植物中为潮霉素抗性)的载体中，获得能够在拟南芥和烟草中转化的载体pRPS28-GUS-HYG、pRPS28-I-GUS-HYG、pEIF1-GUS-HYG、pEIF1-I-GUS-HYG载体，并分别转入拟南芥和烟草中，具体的扩增体系及引物设计如下：
反应体系：总体积为50μl，模板(上述扩增的DNA片段)1μl(约100ng)、2×phanta MAX fertility酶反应缓冲液25μl、10mM dNTP 1μl、4μM引物5μl(每条引物浓度为10mM，均为2μl)、1μl phanta MAX fertility酶，加ddH 2O(无菌去离子水)至50μl。
反应程序为：94℃变性2min，94℃10s、58℃30s、72℃30s，30cycles，72℃延伸5min。
构建pRPS28-GUS-HYG表达载体所用引物为：
pRPS28-GUS-HYG-F：
Figure PCTCN2022081297-appb-000019
pRPS28-GUS-HYG-R：
Figure PCTCN2022081297-appb-000020
构建pRPS28-I-GUS-HYG表达载体所用引物为：
pRPS28-I-GUS-HYG-F：
Figure PCTCN2022081297-appb-000021
pRPS28-I-GUS-HYG-R：
Figure PCTCN2022081297-appb-000022
构建pEIF1-GUS-HYG表达载体所用引物为：
pEIF1-GUS-HYG-F：
Figure PCTCN2022081297-appb-000023
pEIF1-GUS-HYG-R：
Figure PCTCN2022081297-appb-000024
构建pEIF1-I-GUS-HYG表达载体所用引物为：
pEIF1-I-GUS-HYG-F：
Figure PCTCN2022081297-appb-000025
pEIF-I-GUS-HYG-R：
Figure PCTCN2022081297-appb-000026
2、pRPS28和pEIF1驱动GUS在拟南芥中的表达分析。
1)将含pRPS28-GUS-HYG、pRPS28-I-GUS-HYG、pEIF1-GUS-HYG、pEIF1-I-GUS-HYG重组载体的农杆菌GV3101重悬于拟南芥转化缓冲液中，花序浸染法转化拟南芥Col-0，筛选阳性苗，得到转基因拟南芥。
2)在pRPS28-GUS-HYG中筛选到GUS蛋白表达量一致的转基因line#5、#25、#38，在pRPS28-I-GUS-HYG中筛选到GUS蛋白表达量一致的转基因line#2、#5、#13，在pEIF1-GUS-HYG中筛选到GUS蛋白表达量一致的转基因line#3、#7、#12，在pEIF1-I-GUS-HYG中筛选到GUS蛋白表达量一致的转基因line#10、#11、#12，将以上转基因拟南芥的T3代苗撒种于拟南芥潮霉素筛选培养基中，置于4℃冰箱春化2d。
3)然后移至光照培养箱(22±2℃，16/8h)中培养7d，每个line挑选5株T3代阳性植株幼苗进行GUS染色。然后将T3代阳性幼苗种入盆中(每盆5株)，22±2℃，16/8h条件下生长。待开花结荚期，选取拟南芥的花、荚果进行GUS染色，观察GUS的表达情况。
如图8所示，为pRPS28启动子、pRPS28-I启动子与pGmUbi启动子分别驱动GUS蛋白在转基因拟南芥T3代植株不同部位中的表达情况，a为pRPS28启动子驱动的GUS蛋白在拟南芥全株、花、荚果中的表达情况，b为pRPS28-I启动子驱动的GUS蛋白在拟南芥全株、花、荚果中的表达情况，说明pRPS28可以驱动目的基因(GUS基因)在拟南芥中异源泛表达。
图9所示，为pEIF1启动子、pEIF1-I启动子与pGmUbi启动子分别驱动GUS蛋白在转基因拟南芥T3代植株不同部位中的表达情况，a为pEIF1启动子驱动的GUS蛋白在拟南芥全株、花、荚果中的表达情况，b为pEIF1-I启动子驱动的GUS蛋白在拟南芥全株、花、荚果中的表达情况，说明pEIF1可以驱动目的基因(GUS基因)在拟南芥中异源泛表达。
3、pRPS28和PEIF1驱动GUS在烟草叶片中的表达分析。
将含pRPS28-GUS-HYG、pRPS28-I-GUS-HYG、pEIF1-GUS-HYG、pEIF1-I-GUS-HYG重组载体的农杆菌GV3101重悬于烟草叶片侵染液中，将浸染液用注射器注射到烟草Nicotiana benthamiana的叶片中，共培养2d之后剪取转化的烟草叶片，GUS染色。
如图10所示，为pRPS28启动子、pEIF1启动子分别驱动GUS蛋白在转基因烟草叶片的瞬时表达情况，注射了空载体pCAMBIA1391Z的mock组的叶片无GUS表达，而pRPS28和pEIF1启动子组的叶片有GUS表达，试验结果表明pRPS28和pEIF1启动子驱动的GUS可以在烟草叶片中瞬时表达，表明pRPS28和pEIF1启动子可以异源驱动基因在烟 草中表达。

Claims (9)

  1. 大豆基因启动子在外源基因表达中的应用，其特征在于，所述启动子为全长型启动子pEIF1或内含子存在型启动子pEIF1-I，作为泛表达启动子，用于驱动外源基因表达。
  2. 如权利要求1所述的应用，其特征在于，所述外源基因为GUS基因。
  3. 如权利要求1所述的应用，其特征在于，用于大豆时，能够驱动外源基因在大豆子叶、胚根、胚芽、真叶、复叶、芽、叶柄、节间、根和根瘤中表达。
  4. 如权利要求1所述的应用，其特征在于，用于拟南芥时，能够驱动外源基因在拟南芥全株、花、荚果中表达。
  5. 如权利要求1所述的应用，其特征在于，用于烟草时，能够驱动外源基因在烟草叶片中表达。
  6. 含有大豆基因启动子的重组载体，其特征在于，将大豆基因启动子重组进入载体pCAMBIA1391Z-BAR中构建获得，具体步骤如下：以pEIF1、pEIF1-I的PCR扩增片段为模板，并将PCR扩增片段通过无缝克隆法克隆进大豆稳定转化载体pCAMBIA1391Z-BAR中，获得大豆稳定转化载体pEIF1-GUS-BAR、pEIF1-I-GUS-BAR。
  7. PCR扩增大豆基因启动子的引物对，其特征在于，构建pEIF1-GUS表达载体所用引物为：pEIF1-GUS-bar-F：
    GACCATGATTACGCCAAGCTTGGAGAGAAGTTGAACTCTGAGTTGTG
    pEIF1-GUS-bar-R：
    CCAGTGAATTCCCGGGGATCCCTGATCGTAAATTTAAGGTTTCG
    构建pEIF1-I-GUS表达载体所用引物为：
    pEIF1-I-GUS-bar-F：
    GACCATGATTACGCCAAGCTTGGAGAGAAGTTGAACTCTGAGTTGTG
    pEIF-I-GUS-bar-R：
    CCAGTGAATTCCCGGGGATCCAAAACTTGACTCACTAAGACCAAAGG。
  8. PCR扩增大豆EIF1基因用引物对，其特征在于，所述引物对为：
    EIF1-F：ATGTCTGAATTAGACGATCAAATTCC
    EIF1-R：TCAGAAACCATGAATCTTGATATGATC。
  9. 如权利要求1-5任一所述应用，其特征在于，启动子pEIF1的核苷酸序列如SEQ ID NO.5所示，启动子pEIF1-I的核苷酸序列如SEQ ID NO.6所示。
PCT/CN2022/081297 2021-07-02 2022-03-17 大豆基因启动子pEIF1和pEIF1-I在大豆、拟南芥及烟草中的应用 WO2023273420A1 (zh)

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