CN111172170A - Sedum lineare drought-resistant gene SlAP2 and application thereof - Google Patents
Sedum lineare drought-resistant gene SlAP2 and application thereof Download PDFInfo
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- CN111172170A CN111172170A CN201910820409.0A CN201910820409A CN111172170A CN 111172170 A CN111172170 A CN 111172170A CN 201910820409 A CN201910820409 A CN 201910820409A CN 111172170 A CN111172170 A CN 111172170A
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Abstract
The invention discloses a sedum lineare drought-resistant gene SlAP2 and application thereof, wherein the nucleotide sequence of the sedum lineare drought-resistant gene SlAP2 is shown as SEQ ID NO.1, and experiments prove that the arabidopsis thaliana and populus nicotiana through SlAP2 gene transfection enhance drought tolerance, so that the drought-resistant gene SlAP2 provided by the invention plays an important role in improving the drought resistance of crops.
Description
Technical Field
The invention relates to a Sedum lineare drought-resistant gene SlAP2 and application thereof, belonging to the field of molecular biology and biotechnology.
Background
The growth of plants is greatly influenced by environmental abiotic factors, such as drought, high salinity, low temperature, and the like. Plants respond to and adapt to these stress conditions to survive in stress situations, where drought is the most severe environmental factor limiting plant growth and crop yield. At present, the temperature is continuously raised due to global climate change, and the regional distribution and survival of crops are limited due to unbalanced distribution of Chinese water resources. Drought has become one of the stresses of adversity that severely affect crop yield worldwide. Therefore, the plant drought tolerance can be enhanced by transferring the plant drought tolerance into the plant, and the plant drought tolerance enhancing agent 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 drought-resistant gene SlAP 2.
The second purpose of the invention is to provide a cloning vector pJET1.2_ SlAP2 containing a sedum lineare drought-resistant gene SlAP 2.
The third purpose of the invention is to provide an expression vector pBI121_ SlAP2 containing a sedum lineare drought-resistant gene SlAP 2.
It is a fourth object of the invention to provide a host cell containing the expression vector pBI121_ SlAP 2.
The fifth purpose of the invention is to provide the application of the sedum lineare drought-resistant gene SlAP2 in enhancing the drought tolerance of plants.
The technical scheme of the invention is summarized as follows:
the nucleotide sequence of the sedum lineare drought-resistant gene SlAP2 is shown in SEQ ID No. 1.
A cloning vector pJET1.2_ SlAP2 containing a sedum lineare drought-resistant gene SlAP 2.
An expression vector pBI121_ SlAP2 containing a sedum lineare drought-resistant gene SlAP 2.
A host cell comprising the expression vector pBI121_ SlAP 2.
The application of the sedum lineare drought-resistant gene SlAP2 in enhancing the drought tolerance of plants.
The plant is preferably Arabidopsis thaliana or tobacco.
The invention has the advantages that:
experiments prove that arabidopsis and tobacco transfected by the SlAP2 gene show drought tolerance, and the drought-resistant gene SlAP2 provided by the invention plays an important role in improving the drought resistance of crops.
Drawings
FIG. 1 is the electrophoresis diagram of the cloning of sedum lineare drought-resistant gene SlAP 2.
FIG. 2 is a schematic diagram of a sedum lineare drought-resistant gene SlAP2 after being inserted into an expression vector.
FIG. 3 shows the results of PCR screening of transformant genomes after Arabidopsis thaliana transformation with pBI121_ SlAP2 (Nos. 1-8 represent the single colony bacterial solution of pBI121_ SlAP2, respectively).
FIG. 4 shows the results of expression level measurement by semi-quantitative PCR of T3 homozygote after transformation of Arabidopsis thaliana into pBI121_ SlAP2 (Arabidopsis thaliana expressed in3, 5; Arabidopsis thaliana with high expression level in No.1, 4, 7; Arabidopsis thaliana with low expression level in No.2, 6).
FIG. 5 is a photograph of the effect of drought resistance experiment of a T3 homozygote of the Sedum lineare drought-resistant gene SlAP2 transgenic Arabidopsis thaliana.
FIG. 6 is a photograph of the effect of the test of the P.lineare drought-resistant gene SlAP2 transgenic tobacco drought resistance.
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) SlAP2 Gene
From 0.3mol/L mannitol (C)6H1406Relative molecular mass: 182.17) water solution simulation drought stress treatment of Sedum lineare (obtained from New coastal region of Tianjin), total RNA was extracted using the 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). Carrying out high-throughput sequencing on cDNA to obtain 78407 transcripts (high-throughput sequencing carried out by Nozaoyuan company), carrying out comparative analysis on the transcripts and a GO database to obtain a 3' end sequence of the SlAP2 gene, obtaining a full-length cDNA sequence of the SlAP2 gene by using RACE technology (Takara-RACE kit), carrying out sequencing analysis on the amplified SlAP2 gene to obtain a complete SlAP2 geneThe total length is 513 bp. When constructing the super expression vector, a pBI121 recombination site, an upstream 5'-ACGGGGGACTCTAGAGGATCC-3' (SEQ ID No.3) and a downstream 5'-CGATCGGGGAAATTCGAGCTC-3' (SEQ ID No.4) are respectively added at the 5' end of the specific primer, so as to be beneficial to the construction of the later expression vector.
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 TaKaRa RNA PCR 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 SlAP2 gene
The specific primer of the stonecrop Actin gene SEQ ID No.5:5'-GAACTTACTAGCCGACTG-3', SEQ IDNo. 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 SlAP2 gene fragment
The SlAP2 gene obtained by Takara RACE kit amplification is used for sequencing analysis, and the whole linear length of the linear stonecrop SlAP2 gene is 513bp (SEQ ID No. 1). The protein coded by the sedum lineare SlAP2 gene is an amino acid sequence shown in SEQ ID No. 2. Designing upstream and downstream primers of the SlAP2 gene by using primer software according to the known cDNA sequence:
SEQ ID No.7:5'-ACAGCAATCACTTCCAACAACC-3'
the PCR reaction program of SEQ ID No.8:5'-TCATAAATCTGAGCACCCCAAC-3' was as follows:
reaction conditions are as follows: 3min at 94 ℃; 94 ℃ for 30s, 40 ℃ for 30s, 72 ℃ for 50s, 35 cycles; 5min at 72 ℃.
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 SlAP2 gene
Construction of a vector pJET1.2_ SlAP2 containing Sedum lineare SlAP2 Gene
The gel recovered and purified Sedum lineare SlAP2 gene target fragment was recombined onto the vector pJET1.2 by using Clone JET PCR Cloning Kit (pJET1.2: Thermo, Clone JET PCR Cloning Kit # K1231), and the vector pJET1.2_ SlAP2 was obtained.
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 α, 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_ SlAP 2.
Note that pJET1.2, Thermo, Clone JET PCR Cloning Kit # K1231 vector was purchased from invitrogen, and the E.coli used was DH5 α competent cells, TIANGEN, CB 101-2.
5) Constructing an expression vector containing the sedum lineare SlAP2 gene
Constructing an expression vector pBI121_ SlAP2 containing sedum lineare SlAP2 gene,
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'-ACGGGGGACTCTAGAGGATCCACAGCAATCACTTCCAACAACC-3'
SEQ ID No.10:5'-CGATCGGGGAAATTCGAGCTCTCATAAATCTGAGCACCCCAAC-3'
extracting pJET 1.2-gene plasmid with correct sequencing as template, and PCR amplification with primers of SEQ ID No.9 and SEQ ID No.10, and the reaction program is as follows
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)
the reaction program is that the bacteria liquid is coated in LB (adding antibiotic kan 50uM) solid culture medium and cultured overnight at 37 ℃ after the reaction program is finished, wherein the reaction program is that the bacteria liquid is coated on ice for 5min, heat shock is carried out at 42 ℃ for 1min and 30s, the reaction program is carried out on ice for 2min and 30s, and the cells are transferred into competent cells DH5 alpha, the temperature is 37 ℃ and 180r and 45 min.
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-infected competent cell with recombinant vector containing sedum lineare SlAP2 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_ SlAP2 containing sedum lineare SlAP2 gene is transformed into competent cells of an agrobacterium strain C58(pMP90) by an agrobacterium tumefaciens transformation method, cultured for 36h at 28 ℃, and colony PCR is carried out to select positive clone colonies.
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 (commercial) with 5-7cm of bolts and bolts cultured for 3-4 weeks, inversely placing the wild type Arabidopsis in a container filled with transformation liquid, soaking the whole inflorescence in the bacterial liquid for 15 seconds, taking out the Arabidopsis, transversely laying the Arabidopsis in a tray, covering the Arabidopsis with a plastic film for moisturizing, and performing dark treatment for 12 hours to ensure that the Arabidopsis grows vertically under the culture conditions of 25 ℃ of temperature, 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 (MS salt is 2.2g, cane sugar is 10g, constant volume is 1L, pH is 5.7, agar is 7.2g) containing 50 mu g/mL kanamycin on a super clean bench, the seeds grow for 8-10 days under 1800Lux light with 16h light/8 h darkness of a photoperiod, and leaves are dark green, namely the T1 generation positive transformants of the transgenic arabidopsis. When the T1 generation positive transformant plants grow to 3-4 true leaves, the positive transformant 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. 1-8 respectively represent single colony bacterial liquid of pBI121_ SlAP 2), the expression level of the transgenes is identified by semi-quantitative PCR (see figure 4; 3, 5 represent expressed Arabidopsis thaliana; No.1, 4,7 represent high expression arabidopsis thaliana; and No.2, 6 represent low expression arabidopsis), and the independent transformation line No.4 with high expression level and the independent transformation line No.6 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.4 and No. 6.
(3) Drought-resistant treatment of transgenic arabidopsis
Respectively planting No. 4T 3 generation homozygote seed, No. 6T 3 generation homozygote seed and wild Arabidopsis seed in soil, growing for 21 days under the culture conditions of 25 deg.C, 1800Lux, 16h light/8 h dark photoperiod and 70% relative humidity, keeping 21 seedlings with consistent growth vigor for each plant, randomly dividing into three groups of parallel experiments, respectively planting 7 plants in each group, and adding 0.3mol/L mannitol (C)6H1406Relative molecular mass: 182.17) aqueous solution simulating drought stress. The plants were treated every 5 days and photographed after 20 days of total treatment (see FIG. 5).
Example 3
1. Transformed tobacco
The tobacco transformed with the positive clone was NC89 (6855-2X 6772) tissue culture (commercial).
(1) Selecting tobacco tissue culture seedlings with good growth condition for 30 days, selecting thick leaves, reducing leaf edges, and cutting the leaves into 1cm multiplied by 1cm explants. The selected positive clone bacterial liquid (OD600 ═ 0.8) obtained in example 1 was put on LB liquid medium (peptone 10g, yeast extract 5g, sodium chloride 5g, constant volume to 1L, pH 7) of Agrobacterium, and shake-cultured at 24 ℃ for 10 min. After infection, the explants were blotted dry and placed on MS medium (4.4 g MS salt, 30g sucrose, constant volume to 1L pH 5.7 agar 7.2g) and cultured in the dark for 3 days. Washing the dark cultured explant with sterilized distilled water for 2-3 times, drying by filter paper, and placing the explant on a selective medium (MS salt 4.4g, sucrose 30g, auxin NAA1.86mg, cytokinin 2ip1.02mg, kanamycin 500mg, cefamycin 500mg, agar 7.2g, constant volume to 1L, pH 5.7); the culture conditions are as follows: culturing under 2000Lux light for 14 days, and the light/dark is 16h/8h).
(2) After a culture medium is selected, when the explant grows out of a small seedling, the whole small seedling is cut off, and a root primordium is vertically inserted onto an MS culture medium to induce rooting. After rooting is finished, each independent transformant is subjected to positive identification.
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 tobacco to drought resisting treatment
Transplanting 7-day-old transgenic high-expression tobacco, low-expression transgenic tobacco and wild tobacco into a soil pot, after the seedlings in the soil pot grow for 5 days, keeping 21 seedlings with consistent growth vigor for each plant, randomly dividing the seedlings into three groups, adding 0.3mol/L mannitol (C) into 7 seedlings of each group of different types of plants, and culturing with a culture medium6H1406Relative molecular mass: 182.17) aqueous solution simulating drought stress. The plants were treated every 5 days and photographed 50 days after treatment (see FIG. 6).
Sequence listing
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<120> sedum lineare drought-resistant gene SlAP2 and application thereof
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Claims (6)
1. The sedum lineare drought-resistant gene SlAP2 is characterized in that the nucleotide sequence of the gene is shown in SEQ ID No. 1.
2. A cloning vector pJET1.2_ SlAP2 containing the sedum lineare drought-resistant gene SlAP2 of claim 1.
3. An expression vector pBI121_ SlAP2 containing the sedum lineare drought-resistant gene SlAP2 of claim 1.
4. A host cell comprising the expression vector pBI121_ SlAP2 of claim 3.
5. Use of the sedum lineare drought-resistant gene SlAP2 to enhance the tolerance of plants to drought according to claim 1.
6. The use according to claim 5, wherein the plant is Arabidopsis thaliana or tobacco.
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