CN110551733A

CN110551733A - sedum lineare salt-tolerant gene SlR2R3-MYB and application thereof

Info

Publication number: CN110551733A
Application number: CN201910820432.XA
Authority: CN
Inventors: 王洁华; 岳靖; 杨合宇; 白婧平
Original assignee: Tianjin University
Current assignee: Tianjin University
Priority date: 2019-09-01
Filing date: 2019-09-01
Publication date: 2019-12-10

Abstract

The invention discloses a sedum lineare salt-tolerant gene SlR2R3-MYB and application thereof, wherein the nucleotide sequence of the sedum lineare salt-tolerant gene SlR2R3-MYB is shown in SEQ ID NO.1, and experiments prove that salt tolerance is enhanced by adopting Arabidopsis thaliana and poplar transfected by the SlR2R3-MYB gene, so that the sedum lineare salt-tolerant gene SlR 3-MYB provided by the invention plays an important role in improving the salt resistance of crops.

Description

Sedum lineare salt-tolerant gene SlR2R3-MYB and application thereof

Technical Field

The invention relates to a Sedum lineare salt-tolerant gene SlR2R3-MYB and application thereof, belonging to the field of molecular biology and biotechnology.

Background

During the growth and development of plants, salt stress has become one of the most serious adverse factors in abiotic stress, and many breeding workers take the improvement of salt tolerance of crops as an important breeding target. According to incomplete statistics, about 10% of the land area in the world is salinized, and the salinized soil area in China is close to l × 108hm2 and is increasingly serious. How to improve the salt tolerance of crops is a problem to be solved urgently. Traditional breeding programs that seek to enhance abiotic stress tolerance in crops have achieved some success, but the extent of improvement is still limited based on the polygenic nature of the tolerance trait. Therefore, the plant salt-tolerant promoter can be transferred into plants, enhances the salt tolerance of the plants, and has important strategic significance for restoring ecological environment and improving land utilization rate.

Disclosure of Invention

the invention aims to overcome the defects of the prior art and provides a sedum lineare salt-tolerant gene SlR2R 3-MYB.

the second purpose of the invention is to provide a cloning vector pJET1.2Jr2R 3-MYB containing a sedum lineare salt-tolerant gene SlR2R 3-MYB.

The third purpose of the invention is to provide an expression vector pBI121_ SlR2R3-MYB containing a sedum lineare salt-tolerant gene SlR2R 3-MYB.

A fourth object of the present invention is to provide a host cell containing the expression vector pBI121_ SlR2R 3-MYB.

The fifth purpose of the invention is to provide the application of the sedum lineare salt-tolerant gene SlR2R3-MYB in enhancing the salt tolerance of plants.

The technical scheme of the invention is summarized as follows:

The nucleotide sequence of the sedum lineare salt-tolerant gene SlR2R3-MYB is shown in SEQ ID NO. 1.

The cloning vector pJET1.2_ SlR2R3-MYB containing the sedum lineare salt-tolerant gene SlR2R 3-MYB.

An expression vector pBI121_ SlR2R3-MYB containing the sedum lineare salt-tolerant gene SlR2R 3-MYB.

A host cell comprising the expression vector pBI121_ SlR2R 3-MYB.

The sedum lineare salt-tolerant gene SlR2R3-MYB is used for enhancing the salt tolerance of plants.

The plant is preferably Arabidopsis thaliana or poplar.

The invention has the advantages that:

Experiments prove that the arabidopsis and poplar transfected by the SlR2R3-MYB gene show salt tolerance, and the salt tolerance gene SlR2R3-MYB provided by the invention plays an important role in improving the salt tolerance of crops. Thereby providing an effective method for cultivating salt-tolerant transgenic species,

Drawings

FIG. 1 is a schematic diagram of electrophoresis of a linear stonecrop salt-tolerant gene SlR2R3-MYB clone.

FIG. 2 is a schematic diagram of the inserting of a sedum lineare salt-tolerant gene SlR2R3-MYB into an expression vector.

FIG. 3 shows the results of PCR screening of transformant genomes after Arabidopsis thaliana transformation with pBI121_ SlR2R3-MYB (Nos. 1-9 represent single colony solutions of pBI121_ SlR2R3-MYB, respectively).

FIG. 4 shows the results of expression level measurement by semi-quantitative PCR of T3 homozygote after transformation of Arabidopsis thaliana into pBI121_ SlR2R3-MYB (Arabidopsis thaliana expressed in 2 and 3; Arabidopsis thaliana with high expression level No. 1; Arabidopsis thaliana with low expression level No.4 and 5).

FIG. 5 is a photograph of the experiment effect of salt tolerance of a homozygote of a stonecrop salt-tolerant gene SlR2R3-MYB transgenic Arabidopsis T3.

FIG. 6 is a photograph of the experiment effect of salt tolerance of sedum lineare salt-tolerant gene SlR2R3-MYB transgenic poplar.

Detailed Description

The present invention will be further described with reference to the following examples.

The experimental methods in the examples, in which the specific conditions are not specified, are generally performed under the conditions described in the manual and the conventional conditions, or under the conditions recommended by the manufacturer.

Vector pJET1.2 Thermo, Clone JET PCR Cloning Kit # K1231

The vector pBIl21 is purchased from China plasmid vector strain cell strain gene collection center, http:// biovector

Example 1

1. Cloning of Sedum lineare (Sl for short) SlR2R3-MYB gene

From Sedum lineare (obtained from the coastal new area of Tianjin city) subjected to salt-tolerant treatment with 100uMABA (abscisic acid C ₁₅ H ₂₀ O ₄; relative molecular mass: 264.32) aqueous solution, total RNA was extracted using a Plant RNeasy Plant Mini Kit (Transgene Code # E101-0150rxns), and cDNA was reverse-transcribed using EasyScript Frist-Strand cDNA SynSgesis Supermix (Transgene Code # AE301-03100 rxns). high-throughput sequencing of cDNA gave 78407 transcripts (high-throughput sequencing by Nozao Source company), the 3' -end sequence of SlR2R3-MYB gene was obtained by comparison analysis with GO database, the full-length cDNA sequence of SlR2R3-MYB gene was amplified using RACE technology (Takara-RACE Kit), the full-length SLR2R3-MYB gene sequence was amplified using the SLR2R3-MYB gene, the full-length primer was constructed as a late-stage expression vector (SEQ ID 5, 5. downstream PCR amplification primer was constructed, and the upstream primer was constructed as a late-stage expression vector (SEQ ID-5, pBR 2R 5, and the expression vector was constructed as a late stage.

The method comprises the following specific steps:

1) First Strand cDNA Synthesis

Synthesizing a first cDNA chain under the action of AMV reverse transcriptase by using a reverse transcription Kit TaKaRaRNAPCR Kit (AMV) Ver.3.0, taking total RNA as a template and oligo (dT) as a primer, wherein a reverse transcription system comprises the following steps:

Reaction conditions are as follows: 60min at 42 ℃ and 5min at 99 ℃.

2) Detection of reverse transcription quality PCR amplification of sedum lineare SlR2R3-MYB gene

the specific primer of the sedum lineare Actin gene SEQ ID No.5:5'-GAACTTACTAGCCGACTG-3', SEQ ID No. 6: 5'-CCTCAAGCCTTATACGCAA-3', PCR to verify reverse transcription and RNA quality.

The PCR reaction system is as follows:

reaction conditions are as follows: 3min at 94 ℃; 94 ℃ for 30s, 40 ℃ for 30s, 72 ℃ for 50s, 35 cycles; 5min at 72 ℃.

3) PCR amplification of sedum lineare SlR2R3-MYB gene fragment

The SlR2R3-MYB gene obtained by Takara RACE kit amplification is used for sequencing analysis, and the whole length of the obtained whole sedum lineare SlR2R3-MYB gene is 798bp (SEQ ID No. 1). The protein coded by the sedum lineare SlR2R3-MYB gene is an amino acid sequence shown in SEQ ID No. 2. Designing an upstream primer and a downstream primer of the SlR2R3-MYB gene by using primer software according to a known cDNA sequence:

SEQ ID No.7:5'-CTGATAAGTCTCCGCCATCG-3'

The PCR reaction program of SEQ ID No.8:5'-CTCCCACAACTCATTTTCCAAA-3' was as follows:

After the PCR reaction, 1. mu.L of the PCR product was subjected to 1.0% agarose gel electrophoresis to examine the quality of the PCR product (see FIG. 1), and the remainder was used for purification and recovery of the product.

4) Constructing a cloning vector containing the sedum lineare SlR2R3-MYB gene

Construction of a vector pJET1.2JSlR 2R3-MYB containing a Sedum lineare SlR2R3-MYB gene

The gel recovered and purified target fragment of the sedum lineare SlR2R3-MYB gene is recombined on a vector pJET1.2 by using Clone JET PCR Cloning Kit (pJET1.2: Thermo, Clone JET PCR Cloning Kit # K1231) to obtain the vector pJET1.2_ SlR2R 3-MYB.

The reaction procedure is as follows:

The reaction conditions are 24 ℃,10min of standing on ice for 30min, 42 ℃ of heat shock for 1min of 30s and 2min of standing on ice for 30s, the cells are transferred into competent cells DH5 alpha, 37 ℃, 180rpm and 45min, after the procedure is finished, the bacterial liquid is coated into LB (antibiotic added Amp100uM) solid culture medium (10 g of peptone, 5g of yeast extract, 5g of sodium chloride and 15g of agar, the volume is constant to 1L, and the pH value is 7) and cultured at 37 ℃ overnight.

and respectively carrying out colony PCR verification on different colonies by using upstream and downstream primers (SEQ ID No.7 and SEQ ID No.8) of the target fragment, screening positive colonies and sequencing to obtain a host cell containing the cloning vector pJET1.2_ SlR2R 3-MYB.

Note: pJET1.2 Thermo, Clone JET PCR Cloning Kit # K1231 vector from invitrogen; the Escherichia coli used was DH 5. alpha. competent cells, TIANGEN, CB 101-2.

5) Constructing an expression vector containing the sedum lineare SlR2R3-MYB gene

constructing an expression vector pBI121_ SlR2R3-MYB containing sedum lineare SlR2R3-MYB genes,

when constructing the super expression vector, pBI121 recombination sites are respectively added at the 5 'end and the 3' end of the specific primer,

SEQ ID No.3:5'–ACGGGGGACTCTAGAGGATCC-3',

SEQ ID No.4:5'-CGATCGGGGAAATTCGAGCTC-3'

Obtaining:

SEQ ID No.9:5'-ACGGGGGACTCTAGAGGATCCCTGATAAGTCTCCGCCATCG-3'

SEQ ID No.10:5'-CGATCGGGGAAATTCGAGCTCCTCCCACAACTCATTTTCCAAA-3'

Extracting pJET 1.2-gene plasmid with correct sequencing as template, and PCR amplification with primers SEQ ID No.9 and SEQ ID No.10 as recombination sites

After the PCR reaction, 1. mu.L PCR product was subjected to 1.0% agarose gel electrophoresis to detect the quality of the PCR product, and the remainder was used for purification and recovery of the product.

The pBI121 plasmid, its vector map (see FIG. 2), was extracted and subjected to double-restriction linearization, as follows:

Reaction conditions are as follows: inactivating at 37 deg.C for 12 hr and 80 deg.C for 20 min.

The genes and linearized pBI121 plasmid were recombinantly constructed using the Clone Express Entry One Step Cloning Kit, the reaction program was as follows:

(50-200 ng for linearized pBI121 plasmid; 20-200ng for gel recovery gene fragment; 100 ng/. mu.L for linearized pBI121 plasmid; 100 ng/. mu.L for gel recovery gene fragment)

Reaction procedure: the culture medium is incubated at 37 deg.C for 30min on ice for 5min, and at 42 deg.C for 1min and 30s on ice for 2min and 30s, and then transferred into competent cell DH5 alpha, at 37 deg.C for 180r and 45min, after the procedure is finished, the bacterial liquid is smeared into LB (antibiotic kan 50uM) solid culture medium, and cultured overnight at 37 deg.C.

And performing colony PCR double verification on the same colony by using the vector and upstream and downstream primers (shown as SEQ ID No.9 and SEQ ID No.10) of the target fragment respectively, and screening positive colony sequencing (shown as SEQ ID No. 11).

Note: this Step was performed using the clone Express One Step Cloning Kit available from vazyme,

6) Transforming agrobacterium-competent cells by using recombinant vector containing sedum lineare SlR2R3-MYB gene

The Agrobacterium strain used in the experiment was C58 (purchased from China plasmid vector strain Gene Collection, http:// bioselector. blog.163.com /), C58 had rifampicin resistance (Rif), and the helper plasmid had gentamicin resistance (Gen).

An E.coli expression vector pBI121_ SlR2R3-MYB containing sedum lineare SlR2R3-MYB genes is transformed into agrobacterium tumefaciens strain C58(pMP90) competent cells by an agrobacterium tumefaciens electric shock transformation method, the competent cells are cultured for 36h at 28 ℃, and positive clone colonies are selected by colony PCR.

Example 2

1. Transformation of Arabidopsis thaliana

(1) Arabidopsis thaliana was transformed.

The specific operation steps for transforming Arabidopsis are as follows:

Activation and expanded culture of the colonies of the positive clones obtained in example 1

And (3) activation: the positive clone colonies thus selected were cultured in 3ml of LYEB liquid medium (peptone 5g, yeast extract 1g, beef extract 5g, sucrose 5g, constant volume to 1L, pH 7) (with Gen, Rift, kana antibiotics added to make the concentrations 30mg/L, 25mg/L, and 50mg/L, respectively) for about 15 hours (to OD600 about 0.8), 180rpm, 28 ℃.

And (3) amplification culture of positive clone bacteria: a proper amount of antibiotics (Gen, Rift, kana antibiotics, concentration of 30mg/L, 25mg/L, 50mg/L, respectively) was added to a fresh 10ml YEB liquid medium, and then a proper amount of positive clone bacteria liquid was inoculated to the YEB liquid medium for culture at 180rpm until OD600 became 0.6 at 28 ℃.

② transformation

The bacterial solution was centrifuged (3000rpm, 15 ℃,10min), the supernatant was discarded, and the cells were resuspended in an aqueous sucrose solution having a volume of 5% twice the mass concentration of the taken bacterial solution (slow operation was performed to ensure cell viability), so that the cells were dispersed, and the OD600 was adjusted to 0.8.

Selecting wild type arabidopsis thaliana (commodity) which is cultured for 3-4 weeks and has 5-7cm of bolting, inversely placing the wild type arabidopsis thaliana in a container filled with a transformation liquid, soaking the whole inflorescence in a bacterial liquid for 15 seconds, taking out the arabidopsis thaliana, transversely laying the arabidopsis thaliana in a tray, covering the arabidopsis thaliana with a plastic film for moisturizing, and performing dark treatment for 12 hours to ensure that the arabidopsis thaliana grows vertically under the culture conditions of 25 ℃, 16 hours of light cycle, 8 hours of dark and 70% of relative humidity until seeds are mature. The seeds were collected and dried in a 37 ℃ oven for two weeks for subsequent testing.

(2) Screening of transgenic Arabidopsis Positive transformant homozygote

The collected T1 generation seeds are disinfected and placed in a refrigerator for three days at 4 ℃, then the transgenic arabidopsis seeds are uniformly sown on a 1/2MS solid screening culture medium (2.2 g of MS salt, 10g of cane sugar, constant volume to 1L, pH value of 5.7 and agar 7.2g) containing 50 mu g/mL kanamycin on a super clean bench, and the transgenic arabidopsis seeds grow for 8-10 days under 1800Lux light/8 h dark in a light cycle of 16h, and leaves are dark green, namely the T1 generation transformants of the transgenic arabidopsis are positive. When the T1 generation positive transformant plants grow to 3-4 true leaves, the plants are transplanted to soil (purchased from EPAGMA, the Netherlands, http:// www.epagma.eu /), and continue to grow for 14 days under the culture conditions of 25 ℃, 1800Lux, 16h light/8 h dark light in a light cycle and 70% relative humidity, the positive transformants are identified (see figure 3; No. 1-9 respectively represent single colony bacterial liquid of pBI121_ SlR2R 3-MYB), the expression level of transgenes is identified by semi-quantitative PCR (see figure 4; wherein No.2 and 3 represent expressed arabidopsis thaliana, No.1 represents high expression amount arabidopsis thaliana, and No.4 and No.5 represent low expression amount arabidopsis thaliana), and an independent transformation strain No.1 with high expression level and an independent transformation strain No.4 with low expression level are selected. The growth is continued under the conditions, and the seeds are collected after about one and a half months, namely T2 transformed seeds. Repeating the above steps to obtain T3 generation homozygote seeds of No.1 and No. 4.

(3) Salt-tolerant treatment of transgenic arabidopsis

Respectively planting seeds of generation 1T 3 homozygote, seeds of generation 4T 3 homozygote and seeds of wild Arabidopsis thaliana in soil, growing for 21 days under the culture conditions of 25 ℃ of temperature, 1800Lux, 16H light/8H dark photoperiod and 70% of relative humidity, randomly dividing each plant into three groups of parallel experiments, carrying out salt tolerance treatment (irrigation) on 7 plants of each group with different types, treating once every 3 days by using 100uMABA (abscisic acid C ₁₅ H ₂₀ O ₄; relative molecular mass: 264.32) aqueous solution, and photographing the plants after 15 days of total treatment (see figure 5).

Example 3

1. Transformation of poplar

the poplar for transforming the positive clone bacteria is a tissue culture seedling of Populus tremula x Populus alba (Popululus tremula x P. albainra clone N7171-B4, hereinafter referred to as 717 poplar). (commercial products)

(1) Placing the 717 poplar axillary buds or terminal buds on a basic culture medium (2.2 g of MS salt, 30g of cane sugar, constant volume to 1L, pH 5.7 and 7.2g of agar) for subculture propagation, and culturing for 6 weeks to obtain tissue culture seedlings; cutting 1cm stem without axillary buds of the tissue culture seedling, scratching the mouth, and pre-culturing for 3 days at 24 ℃ in the dark;

(2) Centrifuging the selected positive clone bacterium liquid (OD600 is 0.8) obtained in example 1 at room temperature and 4000rpm for 10min, discarding the supernatant, resuspending the precipitate with an equal volume of M liquid (M liquid: MS salt 4.4g, sucrose 30g, auxin NAA1.86mg, cytokinin 2ip1.02mg, acetosyringone As19.86mg, constant volume to 1L, pH is 5.7), activating at 24 ℃ and 100rpm for 1h to obtain an infection liquid; the stem segments pre-cultured in the step (1) are placed into the infection solution according to the proportion of 40, 25mL, and infected for 1h at 100rpm under the condition of 24 ℃. Co-culturing (M1 solid culture medium: MS salt 4.4g, sucrose 30g, agar 7.2g, auxin NAA1.86mg, cytokinin 2ip1.02mg, acetosyringone As19.86mg, constant volume to 1L, pH 5.7; culture condition: 26 ℃, co-culturing for 36 hours under dark condition), delayed selection (CIM delayed selection culture medium: MS salt 4.4g, sucrose 30g, auxin NAA1.86mg, cytokinin 2ip1.02mg, cefamycin 500mg, agar 7.2g, constant volume to 1L, pH 5.7; culture condition: 800Lux culturing for 8 days under weak light, light/dark constant volume to 16h/8h), inducing adventitious bud (in SIM screening culture medium: MS salt 4.4g, sucrose 30g, cytokinin TDZ0.05mg, cefamycin 500mg, kanamycin, 7.2g, constant volume to 1 h/8h, light/dark constant volume to 26.8 h, Lux culturing for 26.8 h/2000 ℃, Lux culturing for 16 h/2000 h, Lux culture condition: 8 h/8h, light/8 h, After elongation culture (SEM screening culture medium: 4.4g MS salt, 30g sucrose, 2ip1.02mg cytokinin, 500mg cefamycin, 500mg kanamycin and 7.2g agar, constant volume is 1L, pH is 5.7; culture conditions: 26 ℃, 2000Lux illumination, illumination/darkness is 16h/8h, culture is 4 weeks), induced rooting (RM culture medium comprises 2.2g MS salt, 30g sucrose, 500mg cefamycin, 500mg kanamycin, 7.2g agar, constant volume is 1L, pH is 5.7; culture conditions: 26 ℃, 2000Lux illumination, illumination/darkness is 16h/8h), after the regeneration poplar grows normally, each transformant is independently identified as positive.

And selecting an independent transformation strain with high expression level and an independent transformation strain with low expression level through semi-quantitative PCR to perform a salt resistance experiment.

(3) Subjecting 717 poplar to salt treatment

Transplanting transgenic high-expression poplar, low-expression transgenic poplar and wild 717 poplar which grow uniformly for 2 months into a soil pot, after the seedlings of the soil pot grow for 30 days, keeping 21 seedlings with consistent growth vigor for each plant, randomly dividing each plant into three groups, carrying out irrigation treatment on 7 plants in each group of different types by using a salinity treatment solution (100uMABA (abscisic acid C ₁₅ H ₂₀ O ₄; relative molecular mass: 264.32) aqueous solution), irrigating once every 3 days, wherein the irrigation amount is 0.5 times of the soil mass each time so as to keep the concentration of the treatment solution in the pot constant, and observing and photographing the plants after 30 days of total treatment (see figure 6).

Sequence listing

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Claims

1. The sedum lineare salt-tolerant gene SlR2R3-MYB is characterized in that the nucleotide sequence of the gene is shown in SEQ ID NO. 1.

2. A cloning vector pJET1.2_ SlR2R3-MYB containing the sedum lineare salt-tolerant gene SlR2R3-MYB of claim 1.

3. An expression vector pBI121_ SlR2R3-MYB containing the sedum lineare salt-tolerant gene SlR2R3-MYB of claim 1.

4. A host cell comprising the expression vector pBI121_ SlR2R3-MYB of claim 3.

5. The use of the salt-tolerant gene SlR2R3-MYB of linear stonecrop as claimed in claim 1 to enhance the tolerance of plants to salt.

6. The use according to claim 5, wherein the plant is Arabidopsis or poplar.