CN106434659B

CN106434659B - Soybean low-temperature inducible promoter, recombinant expression vector containing promoter and application

Info

Publication number: CN106434659B
Application number: CN201610847705.6A
Authority: CN
Inventors: 翟莹; 邵淑丽; 张军; 赵艳; 任巍巍; 张闯
Original assignee: Qiqihar University
Current assignee: Qiqihar University
Priority date: 2016-09-23
Filing date: 2016-09-23
Publication date: 2020-01-10
Anticipated expiration: 2036-09-23
Also published as: CN106434659A

Abstract

The invention discloses a soybean low-temperature inducible promoter, a recombinant expression vector containing the promoter and application, relates to the field of plant genetic engineering, and particularly relates to expression of a soybean gene promoter sequence which can be used as a promoter regulatory gene under low-temperature stress. The invention aims to solve the problem that an effective soybean low-temperature inducible promoter is not developed. The promoter is derived from the promoter sequence of soybean ethylene response factor gene GmERF9, is named GmERF9P, and has a base sequence shown as SEQ ID NO: 1 is shown. The sequence contains various stress-related cis-acting elements. The cis-acting elements are GT-1, BIHD10S, WRKY, MYB, MYC and G-box, respectively. The soybean low-temperature inducible promoter is used for inducing cold-resistant gene expression at low temperature.

Description

Soybean low-temperature inducible promoter, recombinant expression vector containing promoter and application

Technical Field

The invention relates to the field of plant genetic engineering, in particular to a soybean gene promoter sequence which can be used as a promoter to regulate the expression of genes under low temperature stress.

Background

Soybeans are important economic crops, are generally planted worldwide, are not only main sources of human proteins and lipids, but also important livestock feed and industrial raw materials, and have great medical value. However, adverse environmental conditions such as low temperature, drought, high salt, etc. can affect the growth and development of soybeans, thereby causing the yield of the soybeans to be reduced and causing serious economic loss. In addition to conventional breeding techniques, in recent years, with the development of molecular biology and the maturation of plant genetic engineering breeding techniques, it has become possible to improve soybean stress resistance using genetic engineering methods.

A promoter is a DNA sequence located upstream of a structural gene on a DNA molecule that binds RNA polymerase to initiate transcription of the structural gene. The efficiency of gene expression depends mainly on the activity of the promoter, and high-level expression of endogenous or exogenous genes requires efficient promoters. In genetic engineering, some constitutive promoters such as CaMV35S are commonly used at present to control the expression of a target gene. Although these promoters allow overexpression of the gene of interest, they are constitutively expressed without tissue, environment, and temporal specificity. Although the use of a constitutive promoter to control the expression of a stress-resistance-associated gene can improve the stress resistance of a plant under stress conditions, the associated stress-resistant protein is overexpressed even under normal plant growth conditions, which is wasteful for plant metabolism. And the long-time excessive accumulation of the stress-resistant protein in the plant body under the normal environment can also generate negative influence on the normal growth of the plant, cause abnormal morphology of the transgenic plant, and delay or even death of the growth. The stress-induced promoter can greatly start the expression of downstream genes only when the plant is stressed by stress, so that the plant growth defect caused by the continuous expression of exogenous genes in the transgenic plant can be avoided. Therefore, the cloning and application of the stress-inducible promoter can realize the expression of the high-efficiency, controllable and specific (fixed point, timing and quantitative) regulation and control stress-resistant gene, has great superiority in the aspect of improving the stress resistance of plants, and becomes a hotspot of the current research.

At present, some reports about low-temperature inducible promoters exist at home and abroad. For example, the arabidopsis thaliana rd29A promoter can be induced by abiotic stresses such as low temperature, drought and high salt, and is the most widely applied inducible promoter in stress-resistant genetic engineering at present. The blt101.1 promoter induced by low temperature was obtained from winter wheat. The cold inducible promoter p-LTT1 cloned from wild rice and having low temperature inducible expression properties. A low-temperature strong inducible promoter POscold6 is obtained from a rice variety Nipponbare, and the promoter can specifically drive an exogenous gene to express in a plant under the condition of low temperature/cold stress. However, in general, the number of low-temperature inducible promoters that can be used in genetic engineering is still small, and therefore cloning and research of effective novel low-temperature inducible promoters are still important for future research.

Disclosure of Invention

The invention aims to solve the problem that an effective soybean low-temperature inducible promoter is not developed, and provides a soybean low-temperature inducible promoter, a recombinant expression vector containing the promoter and application. .

The promoter is derived from the promoter sequence of soybean ethylene response factor gene GmERF9, is named GmERF9P, and has a base sequence shown as SEQ ID NO: 1 is shown.

The sequence contains various stress-related cis-acting elements.

The recombinant expression vector comprises a soybean low-temperature inducible promoter, and the original vector of the recombinant expression vector is pCAMBIA 1301.

The soybean low-temperature inducible promoter disclosed by the invention is applied to low-temperature induction of cold-resistant gene expression.

The expression quantity of the GmERF9 gene in the soybean leaf is detected by using a real-time fluorescent quantitative PCR technology when the soybean leaf is treated at 4 ℃ for 2 hours, and the expression quantity of the GmERF9 is obviously increased, so that the GmERF9 gene is proved to have the low-temperature induced expression characteristic. Therefore, a soybean genome database is searched for a sequence of about 2000bp upstream of the GmERF9 gene, a primer is designed, and a 1885bp GmERF9 promoter sequence is cloned from a soybean leaf genome and named as GmERF 9P. GmERF9P was constructed on a plant expression vector pCAMBIA1301, and tobacco was transformed by the leaf disc method. Obtaining T through hygromycin screening and PCR identification₁Generation positive transgenic tobacco. And carrying out low-temperature treatment on the transgenic tobacco for 2 h. GUS histochemical staining and real-time fluorescent quantitative PCR detection of GUS gene expression quantity are carried out on untreated tobacco and low-temperature treated tobacco, which shows that GmERF9P has low-temperature induction starting characteristic under low-temperature stress and is a low-temperature induction type promoter.

Compared with the prior art, the invention has the advantages that:

although the use of a constitutive promoter to control the expression of a stress-resistance-associated gene can improve the stress resistance of a plant under stress conditions, the associated stress-resistant protein is overexpressed even under normal plant growth conditions, which is wasteful for plant metabolism. And the long-time excessive accumulation of the stress-resistant protein in the plant body under the normal environment can also generate negative influence on the normal growth of the plant, cause abnormal morphology of the transgenic plant, and delay or even death of the growth. The stress-induced promoter can greatly start the expression of downstream genes only when the plant is stressed by stress, so that the plant growth defect caused by the continuous expression of exogenous genes in the transgenic plant can be avoided. The invention clones a soybean low-temperature inducible promoter, the promoter provides a favorable tool for the research of plant cold-resistant gene engineering, and the high-efficiency expression of cold-resistant related genes can be started under the low-temperature condition, so that practical and effective cold-resistant plant varieties can be cultivated.

Drawings

FIG. 1 shows the real-time fluorescent quantitative PCR detection results of the expression level of GmERF9 gene in 0h and 2h of low-temperature treatment;

FIG. 2 shows the PCR amplification result of the promoter sequence of GmERF9 gene, wherein M is DL2000Marker, and 1 is amplification band;

FIG. 3 prediction analysis of cis-acting elements of the promoter sequence of GmERF 9P;

FIG. 4 shows the plasmid double digestion results of the recombinant vector pCAMBIA1301-GmERF9P, wherein M is DL2000Marker, and 1-3 are double digestion bands;

FIG. 5 GmERF9P T₁The PCR identification result of the generation-positive transgenic tobacco, wherein M is DL2000Marker, and 1-6 are T₁Generating positive transgenic tobacco amplification strips, wherein 7 is a wild tobacco negative control, and 8 is a pCAMBIA1301-GmERF9P plasmid positive control;

FIG. 6 GUS histochemical staining of untreated wild type tobacco leaves;

FIG. 7 GUS histochemical staining results of 2h of cold-treated wild-type tobacco leaves;

FIG. 8 untreated T₁GUS organization of transgenic tobacco leavesThe result of the chemical dyeing is obtained;

FIG. 9 cryogenic treatment 2h T₁GUS histochemical staining results of transgenic tobacco leaves;

FIG. 10 is the real-time fluorescent quantitative PCR detection result of GUS gene expression level in transgenic tobacco after low temperature treatment for 2h, wherein 1 is untreated T₁The expression level of GUS gene in transgenic tobacco is 2, T is treated at low temperature for 2h₁The expression quantity of GUS gene in transgenic tobacco is 3, which is positive control pCAMBIA1301 transgenic tobacco.

Detailed Description

The technical scheme of the invention is not limited to the specific embodiments listed below, and any reasonable combination of the specific embodiments is included.

The first embodiment is as follows: the sequence of the soybean low-temperature inducible promoter of the embodiment is shown as SEQ ID NO: 1 is shown.

The second embodiment is as follows: this embodiment differs from the specific embodiment in that the sequence contains a stress-related cis-acting element. Other steps and parameters are the same as those in the first embodiment.

The third concrete implementation mode: this embodiment differs from the second embodiment in that the cis-acting elements are GT-1, BIHD10S, WRKY, MYB, MYC, and G-box, respectively. Other steps and parameters are the same as those in the first embodiment.

The fourth concrete implementation mode: the recombinant expression vector of the present embodiment includes a soybean low-temperature inducible promoter.

The fifth concrete implementation mode: the fourth difference between this embodiment and the embodiment is that the original vector of the recombinant expression vector is pCAMBIA 1301. Other steps and parameters are the same as those in the first embodiment.

The sixth specific implementation mode: the soybean low-temperature inducible promoter is applied to the induction of cold-resistant gene expression at low temperature.

Example 1: this example will verify the sequence of the soybean low temperature inducible promoter of the present invention from the following experiment:

real-time fluorescent quantitative PCR detection of GmERF9 gene expression amount under low-temperature treatment

The method comprises the following steps of placing potted four-leaf-stage soybean seedlings in an incubator at 4 ℃ under normal conditions for low-temperature treatment, cutting 0.1g of leaves respectively and rapidly placing the cut leaves in liquid nitrogen when the treatment is carried out for 0h and 2h, and storing the cut leaves at-80 ℃ for later use. Total RNA of leaves of soybeans treated at low temperature and untreated was extracted using Plant RNAzol reagent (available from Biotech, Beijing ancient China), and first strand cDNA was synthesized according to the instructions of cDNA first strand synthesis kit (available from Biotech, Beijing ancient China).

Real-time fluorescent quantitative PCR primers (F: 5'-CATACCAACCTTCAAATGCCTC-3'; R: 5'-TTTCTATTAGGGTCACGGATTTC-3') were designed based on the cDNA sequence of GmERF9 gene (GenBank accession No.: AK 24092). On a BIO-RADCFX96Real-Time PCR instrument, a soybean constitutive expression gene beta-tubulin (GenBank accession number: GMU12286) is used as an internal reference gene (F: 5'-GGAAGGCTTTCTTGCATTGGTA-3'; R: 5'-AGTGGCATCCTGGTACTGC-3'), and soybean leaf cDNA is used as a template. The reaction system was 2 XSSYBR Premix Ex Taq (available from TaKaRa) 10. mu.L, ROXReference Dye II 0.2. mu. L, cDNA 2. mu.L, Primer F0.4. mu.L, Primer R0.4. mu.L, supplemented with water to a total volume of 20. mu.L. Reaction procedure: pre-denaturation at 95 ℃ for 10 s; denaturation at 95 ℃ for 20s, annealing at 58 ℃ for 20s, and extension at 72 ℃ for 30s, for 40 cycles. The relative expression level of the gene was calculated in 3 replicates for each treatment. The test results were analyzed by BIO-RAD CFX Manager software. As shown in table 1 and fig. 1, the results showed that the expression level of GmERF9 gene was significantly increased when treated at low temperature for 2 h.

TABLE 1 relative expression level of GmERF9 gene under low temperature treatment (value is the average of triplicates)

(II) cloning of promoter sequence of GmERF9 gene and analysis of cis-acting element

The soybean genome database GmGDB (http:// www.plantgdb.org/GmGDB /) was searched based on the soybean GmERF9 gene cDNA sequence to obtain a promoter sequence of about 2000bp upstream thereof. Primers were designed using Primer design software Primer5, the sequence was as follows,F：5'-ACGCGTCGACCCGTGCAACTTGATATTCGT-3' (underlined represents SalI cleavage site); r: 5' -GGCCATGGTTTTTGGTTGTGAAATTGAGG-3' (underlined for NcoI cleavage site). Extracting soybean leaf genome DNA by using a plant genome DNA extraction kit (purchased from Beijing ancient Biotechnology company), and performing PCR amplification by using the genome DNA as a template. PCR procedure: 8min at 94 ℃; 30 cycles of 94 ℃ for 40s, 56 ℃ for 40s and 72 ℃ for 1 min; extension at 72 ℃ for 8 min. The resulting amplified bands are shown in FIG. 2. The amplified fragment is recovered and then is connected with a cloning vector pMD18-T (purchased from TaKaRa company), a positive plasmid obtained by screening is named pMD18-T-GmERF9P, and the positive plasmid is sent to Shanghai's company for sequencing to verify that the sequence is correct. PLACE (http:// www.dna.affrc.go.jp/PLACE /) database was used for predictive analysis of promoter cis-acting elements.

The promoter sequence GmERF9P of the GmERF9 gene is obtained by PCR amplification, and the length is 1885bp (shown as SEQ ID NO: 1 in a sequence table). The sequence of GmERF9P is predicted to contain a plurality of cis-acting elements related to stress, and as can be seen from FIG. 3, the cis-acting elements related to stress comprise 3 GT1 elements, 1G-box element, 2 BIHD10S binding sites, 5 WRKY binding sites, 3 MYB binding sites and 1 MYC binding site. The sequence of GmERF9P was subjected to homologous Blast in NCBI database, and no promoter sequence having homology with the sequence was obtained, indicating that GmERF9P is a novel promoter sequence.

(III) tobacco transformation and transgenic tobacco identification of GmERF9P

The construction method of the plant expression vector comprises the following steps:

the plasmid pMD18-T-GmERF9 was digested with both SalI and Nco I restriction enzymes (each restriction enzyme was purchased from TaKaRa), the digested products were purified with an agarose gel DNA purification recovery kit (purchased from Beijing Ding Biotech), and ligated with pCAMBIA1301 vector (in order to excise the CaMV35S promoter) similarly digested with SalI and Nco I, to obtain pCAMBIA1301-GmERF 9P. The ligation product was transformed into E.coli DH5 alpha (purchased from Biovector), and the plasmid was verified by double digestion (the result is shown in FIG. 4), and transformed into Agrobacterium EHA105 (purchased from Biovector).

The method of infecting tobacco leaf discs with agrobacterium is used for transforming pCAMBIA1301-GmERF9P into tobacco NC89 (purchased from Zhongyan seed Co., Ltd.), and meanwhile, transforming pCAMBIA1301 empty vector as a positive control. The specific method comprises the following steps:

1, putting tobacco seeds into a 1.5mL centrifuge tube, soaking and disinfecting for 3min by using 10% sodium hypochlorite, and washing for 4-5 times by using sterile water;

2, spreading tobacco seeds on an MS culture medium, wherein the culture conditions are as follows: 16h (28 ℃) light/8 h (22 ℃) dark;

3 taking sterile tobacco leaf which grows for 1-2 months after germination, and shearing the sterile tobacco leaf into 0.5cm²The small pieces (main veins are removed) are inoculated on an MS differentiation medium (3 mg/L6-BA and 0.2mg/L NAA are added into MS), and preculture is carried out for 2 days;

4 picking pCAMBIA1301-GmERF9P monoclonal in 5mL YEP (peptone 10g/L, yeast powder 10g/L, NaCl 5g/L) with 50 μ g/mL rifampicin and 50mg/L kanamycin, culturing at 28 deg.C for 24h with 120rpm shaking;

5, according to the proportion of 1: 100 ratio the above culture was transferred to 50mL YEP supplemented with 50. mu.g/mL rifampicin and 50. mu.g/mL kanamycin, and cultured at 28 ℃ with shaking at 120rpm to OD₆₀₀＝0.4-0.5；

65000 rpm, centrifuging at room temperature for 15min, removing supernatant, and suspending the thallus in MS liquid culture medium to OD₆₀₀About 0.5;

7 infecting the pre-cultured tobacco leaves in the heavy suspension for 20min, sucking off bacterial liquid on the surfaces of the leaves, placing the leaves on a co-culture medium (the pH is adjusted to about 5.4), and culturing for 3 days;

8, cleaning the co-cultured tobacco leaves by using an MS liquid culture medium containing 500mg/L carbenicillin, air-drying, transferring to a screening culture medium (adding 3 mg/L6-BA, 0.2mg/L NAA, 500mg/L carbenicillin and 8mg/L hygromycin to MS), and subculturing once every 15 days;

9 when the resistant bud grows to 1cm, transferring into a rooting culture set (200 mg/L carbenicillin and 5mg/L hygromycin are added into MS) to promote the growth of the root;

and 10, after the root system of the tobacco seedling grows well, transplanting the tobacco seedling into soil and performing conventional management.

The screening and identification method of the transgenic tobacco comprises the following steps:

1 get T₀Extracting total DNA of tobacco leaf according to the method of Universal Genomic DNA Extraction Kit (from TaKaRa) instruction;

2 diluting the extracted DNA by 50 times, taking 1 uL as a template, and carrying out conventional PCR verification by using primers (F: 5'-CCGTGCAACTTGATATTCGT-3'; R: 5'-TTTTTGGTTGTGAAATTGAGG-3'). Wherein, untransformed wild tobacco is used as a negative control, and plasmid pCAMBIA1301-GmERF9P is used as a positive control;

3 harvesting the seeds of the positive tobacco plants, i.e. T₁Seed generation;

4 will T₁Planting seeds in the soil to obtain T₁Replacing tobacco seedling, extracting DNA continuously for PCR detection, and taking T₁And carrying out subsequent resistance identification test on the generation positive tobacco plants.

FIG. 5 shows GmERF9P at portion T₁The PCR identification result in the generation of transgenic tobacco shows that the GmERF9P promoter is successfully integrated into the tobacco genome.

(IV) GUS histochemical staining of leaves under transgenic tobacco low-temperature treatment

6 weeks old T from Normal potting culture₁The transgenic tobacco is put into an incubator at 4 ℃ for low-temperature treatment. Clipping unprocessed T₁Transgenic tobacco leaf generation and T low-temperature treatment for 2h₁And (3) replacing transgenic tobacco leaves, simultaneously shearing untreated and low-temperature treated wild tobacco leaves for 2h as negative control, and carrying out GUS histochemical staining: adding GUS staining solution (clone of soybean stearic acid-ACP desaturase gene promoter and its expression activity analysis, Zhang Qinglin, etc., 2011), incubating overnight at 37 deg.C, and decolorizing with 75% ethanol until the background color completely disappears.

GUS histochemical staining results As shown in FIGS. 6-9, the untreated and cryopreserved 2h leaves of wild type tobacco were not stained blue; untreated T₁The transgenic tobacco leaves are dyed light blue, but are lighter in color, which indicates that GmERF9P has the promoter activity of a promoter; t of low-temperature treatment for 2h₁The transgenic tobacco leaves were stained blue and darker, indicating that the priming activity of GmERF9P was observed at 2h of low temperature treatmentInducing to increase the expression level of downstream reporter gene GUS.

(V) real-time fluorescence quantitative PCR detection of GUS gene expression quantity under transgenic tobacco low-temperature treatment

6 weeks old T from Normal potting culture₁And (3) placing the transgenic tobacco in an incubator at 4 ℃ for low-temperature treatment for 2 h. Clipping of cryogenically treated 2h and untreated T₁0.1g of transgenic tobacco leaf is replaced, and pCAMBIA1301T is cut₁The transgenic tobacco leaf is used as positive control and is quickly placed in liquid nitrogen, and is preserved at minus 80 ℃ for standby. Total RNA of tobacco leaves was extracted using Plant RNAzol reagent (available from Biotech, Beijing ancient China) and first strand cDNA was synthesized according to the instruction of cDNA first strand synthesis kit (available from Biotech, Beijing ancient China).

On a BIO-RAD CFX96Real-Time PCR instrument, a tobacco constitutive expression gene alpha-tubulin (GenBank accession number: AB052822) is used as an internal reference gene (F: 5'-ATGAGAGAGTGCATATCGAT-3'; R: 5'-TTCACTGAAGAAGGTGTTGAA-3'), and GUS gene Real-Time fluorescence quantitative PCR primers are as follows, F: 5'-GATCGCGAAAACTGTGGAAT-3', respectively; r: 5'-TAATGAGTGACCGCATCGAA-3' are provided. Taking tobacco leaf cDNA as a template. The real-time fluorescent quantitative PCR reaction system and the reaction procedure are the same as above, and the annealing temperature is changed to 55 ℃. As shown in FIG. 10 and Table 2, the real-time fluorescent quantitative PCR result shows that the expression level of downstream reporter GUS can be obviously improved by GmERF9P when the GmERF9 is treated at low temperature for 2 h.

TABLE 2 relative expression level of GUS gene in transgenic tobacco under low temperature treatment (value is mean value of triplicates)

Claims

1. The application of the soybean low-temperature inducible promoter in inducing cold-resistant gene expression at low temperature is characterized in that: the nucleotide sequence of the promoter is shown as SEQ ID NO: 1 is shown.

2. The use of the soybean cold-inducible promoter for inducing cold-resistant gene expression at low temperature according to claim 1, wherein: the sequence contains a stress-related cis-acting element.

3. The use of the soybean cold-inducible promoter for inducing cold-resistant gene expression at low temperature according to claim 2, wherein: the cis-acting elements are GT-1, BIHD10S, WRKY, MYB, MYC, and G-box.