WO2014026310A1 - Cotton ion channel protein and coding genes and use thereof - Google Patents
Cotton ion channel protein and coding genes and use thereof Download PDFInfo
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- WO2014026310A1 WO2014026310A1 PCT/CN2012/079996 CN2012079996W WO2014026310A1 WO 2014026310 A1 WO2014026310 A1 WO 2014026310A1 CN 2012079996 W CN2012079996 W CN 2012079996W WO 2014026310 A1 WO2014026310 A1 WO 2014026310A1
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- C—CHEMISTRY; METALLURGY
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- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
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- 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)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
- C12N15/8273—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for drought, cold, salt resistance
Definitions
- the present invention relates to ion channel proteins and their encoding genes and applications, and more particularly to a cotton-derived ion channel protein NHX1-1 and its encoding gene, and its application in breeding transgenic plants with improved salt tolerance .
- Salt stress is one of the most important abiotic stress hazards in agricultural production in the world. Salted soil is usually dominated by sodium, calcium or magnesium salts, and is a major factor affecting plant growth and causing food and economic crop yield reduction. The world's saline-alkali soil covers an area of about 400 million hectares, accounting for one-third of the irrigated farmland.
- Saline-alkali land is widely distributed in China, and the existing saline-alkali land area is about 0.4 million hectares. With the increase of population in China and the reduction of cultivated land, the development and utilization of saline-alkali resources has extremely important practical significance.
- the improvement of plant resistance to salt and alkali, drought-tolerant ability and the selection of plant species or strains suitable for growth on saline-alkali land with high economic and ecological value is an economical and effective measure to utilize saline-alkali land.
- most plants are poorly tolerant to saline and alkali, and can only grow on soils with a sodium chloride content of less than 0.3%. Excess Na + in the soil will be plant Normal growth metabolism produces toxic effects. Therefore, how to increase crop yield in a salted environment has become a very important issue in agricultural production worldwide.
- the salt tolerance of plants is a very complex quantitative trait, and its salt tolerance mechanism involves various levels from plants to organs, tissues, physiology and biochemistry to molecules.
- scientists from various countries have also done a lot of work for this purpose, and have made a lot of new progress, especially in the use of the higher model plant Arabidopsis to study the salt-tolerant molecular mechanism of plants, making breakthroughs in research in this field ( Zhu JK. 2002. Sal t and drought stress s ingal transduct ion in plants. Annu. Rev. Plant Biol. 53: 1247-1273; Zhang ZL. 2011.
- Arabidops is Floral Init iator SKBl Confers High Salt Tolerance by Regulat ing Transcript ion And Pre-mRNA Spl icing through Altering Hi stone H4R3 and Smal l Nuclear Ribonucleoprotein LSM4 Methylat ion. Plant Cel l, 23: 396-411). Higher plant cells can have multiple ways to sense changes in the physical and chemical parameters in the external environment, thereby transforming extracellular signals into intracellular signals, and finally transmitting stress signals to the nucleus through a series of signal transductions, activating transcription factors, and activating transcription. The factor acts on the functional gene and initiates the expression of the stress response gene to increase the tolerance of the plant.
- the first aspect of the present invention provides a gene encoding an ion channel-like protein NHX1-1 of cotton having the sequence of SEQ ID NO: 2.
- a second aspect of the invention provides a recombinant expression vector comprising the gene of the first aspect of the invention, and the nucleotide sequence of the gene is operably linked to an expression control sequence of the expression vector; preferably, The vector is the rd29A-GhNHX1-1-2300 carrier shown in Fig. 2.
- a third aspect of the invention provides a recombinant cell comprising the gene of the first aspect of the invention or the recombinant expression vector of the second aspect of the invention; preferably, the recombinant cell is a recombinant Agrobacterium cell.
- a fourth aspect of the present invention provides a method for improving salt tolerance of a plant, comprising: introducing the gene of the first aspect of the invention or the recombinant expression vector of the second aspect of the invention into a plant or plant tissue and expressing the gene
- the plant is tobacco.
- a fifth aspect of the invention provides a method for producing a transgenic plant, comprising: cultivating a plant or plant tissue comprising the gene of the first aspect of the invention, the recombinant expression vector of the second aspect of the invention under conditions effective to produce a plant;
- the plant is tobacco.
- a sixth aspect of the present invention provides the gene according to the first aspect of the present invention, the recombinant expression vector of the second aspect of the present invention or the recombinant cell of the third aspect of the present invention for improving salt tolerance of a plant and for use in plant breeding Use;
- the plant is tobacco.
- a seventh aspect of the invention provides the amino acid sequence encoded by the gene of the first aspect of the invention, as shown in SEQ ID NO: 1.
- Brief Description of the Drawings Fig. 1 is a construction flow of a plant expression vector (rd29A-GhNHX1-1-2300) of GhNHX1-1.
- Figure 2 is a plasmid map of the plant expression vector (rd29A-GhNHX1-1-2300) of GhNHXl-1.
- FIG 3 shows the growth results of GhNHXl-1 transgenic tobacco seeds and non-transgenic tobacco seeds (control) on conventional MS medium.
- the left side of the culture dish (GM) is the growth result of the transgenic tobacco seed; the right side (CK) is non- Growth results of transgenic tobacco seeds (control).
- Figure 4 shows the growth results of GhNHXl-1 transgenic tobacco seeds and non-transgenic tobacco seeds (control) on MS medium supplemented with 100 mM NaCl.
- the left side of the culture dish (GM) is the growth result of the transgenic tobacco seed (T Q H2); the right side (CK) is the growth result of the non-transgenic tobacco seed (control).
- Figure 5 shows the results of protein expression verification at the transcriptional level of transgenic T1 tobacco plants and non-transgenic control plants.
- M is Marker
- 1_5 is the control tobacco
- 6_15 is the salt-tolerant transgenic tobacco T1 plant
- 16_21 is the salt-tolerant transgenic tobacco T1 plant.
- BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be further described below in conjunction with non-limiting examples.
- Example 1 Cotton SSH library construction under salt stress:
- a subtractive library was constructed by inhibition subtractive hybridization using the method shown in Clontech's PCR-selectTM cDNA Subtraction Kit.
- the mRNA of the roots of the cotton seedlings treated with NaCl for 6 hours was used as a tester during the experiment, and the mRNA of the roots of the untreated cotton seedlings was used as a driver.
- the test seedlings were divided into 2 groups of 4 plants each.
- the first group was a control group, cultured at 25 ° C under light, and placed in 1/2 MS liquid medium.
- the second group was treated with 25 ° C, light culture, placed in 1/2 MS liquid medium supplemented with a final concentration of 200 mM NaCl, and treated for 6 hours. After the treatment, the roots of the two groups were cut out in time. After rapid freezing with liquid nitrogen, it was stored in a -70 ° C refrigerator.
- RNA of cotton was extracted with 0.5 g of control and NaCl-treated cotton roots, respectively, using a plant RNA extraction kit (invitrogen). Determination of total RNA at 260 nm using HITACHI's UV spectrophotometer U-2001 And the absorbance value at 280 nm, the ratio of 0D260/0D280 is 1. 8-2 ⁇ 0, indicating that the total RNA purity is high, and the integrity of total A and the brightness of the 28S strip are detected by 1.0% agarose gel electrophoresis. It is about twice as large as the 18S band, indicating good RNA integrity.
- mRNA was isolated using the Qiagen Oligotex mRNA Purification Kit (purification of polyA+ RNA from total RNA).
- the gene In order to increase the availability of the Expressed Sequence Tag (EST) (unigene), the gene is not cleavable and the obtained sequence is in the untranslated region.
- the laboratory uses Rsal, Haelll to digest the double-stranded cDNA (according to the protocol described in the subtractive kit, obtained by reverse transcription of mRNA), and performs two sets of suppression subtraction. Other steps and methods are based on PCR_select TM cDNA Subtraction of Clontech The method shown in the Kit was used for suppression subtractive hybridization, and finally the second PCR product of the two groups of forward subtracted hybrid cDNA fragments was combined.
- the second PCR product of the forward subtracted hybrid cDNA fragment (QIAquick PCR Purification Kit purified from Qiagen) was ligated with the pGEM_T Easy (purchased from Promega kit) vector.
- the specific steps are as follows: l
- the PCR tube is sequentially added with the following components: Purified positive subtractive hybridization cDNA fragment second PCR product 3 ⁇ 1, ⁇ 4 ligase buffer 5 ⁇ 1, pGEM-T Easy vector 1 ⁇ 1, T4 DNA ligase 1 ⁇ ⁇ , connected overnight at 4 ° C. 10 ⁇ L of the ligation reaction product was added to 100 ⁇ L of competent E.
- coli JMI09 (purchased from TAKARA), ice bath for 30 min, heat shock for 60 s, ice bath for 2 min, and 250 L LB medium (1%). Tryptone was purchased from 0X0ID, 0. 5% Yeast Extract was purchased from 0X0ID, 1% NaCl was purchased from Sinopharm. It was placed in a 37°C water bath, shaken at 225 r/min for 30 min, and 200 ⁇ L of bacterial solution was planted in 50 ⁇ m.
- PCR primers Primer l and Primer 2R (Clontech's PCR-selectTM cDNA Subtraction Kit kit) were used to carry out PCR amplification of the bacterial cells, and 211 positive clones were obtained, and all positive clones were sent to the British ⁇ jieji ( Shanghai) Trading Co., Ltd. sequencing (6) cDNA sequencing analysis of differential clones:
- sequence of the cloned Gh-S037 is SEQ ID No: 3. Sequence analysis indicated that the encoded amino acid sequence of the sequence belongs to the ion channel-like protein NHX1.
- the full-length gene encoded by the clone Gh-S037 was named GhNHXl-1.
- the corresponding protein was named NHX1-1.
- GhNHXl GSP1 SEQ ID NO: 4:
- GhNHXl GSP2 SEQ ID NO: 5:
- the experimental procedure was performed according to the kit instructions (3' RACE System for Rapid Amplification of cDNA Ends kit purchased from Invitrogen).
- the first round of PCR amplification was carried out using SEQ ID NO: 4 and the 3' primer AUAP (provided by the kit), using mRNA reverse transcribed cDNA as a template.
- the specific steps are as follows: ExTaq is purchased from TAKARA, 50 l PCR reaction system: 5 ⁇ 1 ⁇ ⁇ ⁇ Buffer, 3 ⁇ 1 2. 5 mM dNTP, 2. 0 ⁇ 1 mRNA reverse transcribed cDNA, 1. 0 ⁇ 1 Ex Taq 10 ⁇ ⁇ primers SEQ ID NO: 4 and AUAP 2. 0 ⁇ 1, and 35 ⁇ 1 of double distilled water.
- PCR reaction conditions pre-denaturation at 94 °C for 5 min, denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 2 min, after 33 cycles, extension at 72 °C for 10 min e
- the obtained PCR product was diluted 50-fold with double-distilled water, and then 2.0 ⁇ l was used as a template, and the second round of PCR amplification was carried out using SEQ ID NO: 5 and the 3' primer AUAP, and the specific steps were as follows: 50 ⁇ 1 PCR reaction System: 5 ⁇ 1 ⁇ ⁇ ⁇ Buffer, 3 ⁇ 1 2. 5 mM dNTP, 2. 0 ⁇ 1 diluted first round PCR product, 1. 0 ⁇ 1 Ex Taq 10 ⁇ ⁇ primer SEQ ID NO : 5 And AUAP each of 2.0 ⁇ l, and 35 ⁇ l of double distilled water.
- PCR reaction conditions pre-denaturation at 94 °C for 5 min, denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 2 min, after 33 cycles, extension at 72 °C for 10 min.
- the second PCR product was recovered from a fragment of approximately 1200 bp (Gel Extraction Kit from OMEGA) linked to pGEM_T Easy Vector, transformed into E.
- coli JM109 (specifically as above), and randomly picked 10 white colonies containing 50 ⁇ g/ The mL ampicillin was cultured in LB liquid medium, cultured at 37 ° C overnight, and then glycerin was added to a final concentration of 20%, and stored at -80 ° C until use.
- SEQ ID NO: 5 and 3' primers AUAP were used for PCR amplification of the bacterial cells, and 5 positive clones were obtained, which were sent to Yingjie Jieji (Shanghai) Trading Co., Ltd. for sequencing and sequencing, and the 3' end of the cDNA of the gene was obtained.
- GhNHXl-1 GSP3 SEQ ID NO: 6:
- GhNHXl-1 GSP4 SEQ ID NO: 7:
- GhNHXl-1 GSP5 SEQ ID NO: 8:
- the experimental procedure was performed according to the kit instructions (5' RACE System for Rapid Amplification of cDNA Ends kit purchased from Invitrogen).
- the first round of PCR amplification was carried out using SEQ ID NO: 7 and 5' universal primer AAP (provided with the kit), and cDNA reverse transcription cDNA (reverse transcription primer SEQ ID NO: 6) was used as a template for the first round of PCR amplification.
- the specific steps are as follows: Ex Taq was purchased from TAKARA, 50 ⁇ PCR reaction system: 5 ⁇ ⁇ Buffer, 3 ⁇ 2.5 mM dNTP, 2.0 ⁇ mRNA reverse transcribed cDNA, 1.0 ⁇ Ex Taq, 10 ⁇ primers SEQ ID NO: 7 and AAP 2.0 ⁇ l, and 35 ⁇ double distilled water.
- PCR reaction conditions predenaturation at 94 °C for 5 min, denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 2 min, after 33 cycles, extension at 72 °C for 10 min.
- the obtained PCR product was diluted 50-fold with double distilled water, and 2.0 ⁇ L was used as a template, and the second round of PCR was carried out using SEQ ID NO: 8 and the 3' primer AUAP.
- Amplification the specific steps are as follows: 50 ⁇ 1 ⁇ Reaction system: 5 ⁇ lOxEx Buffer, 3 ⁇ 2.5 mM dNTP, 2.0 ⁇ l diluted first round PCR product, 1.0 l Ex Taq, 10 ⁇ primer SEQ ID NO: 8 and AUAP each 2.0 ⁇ l, and 35 ⁇ double distilled water.
- PCR reaction conditions predenaturation at 94 °C for 5 min, denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 2 min, after 33 cycles, extension at 72 °C for 10 min.
- the second PCR recovery fragment was approximately 1200 bp band (Gel Extraction Kit was purchased from OMEGA).
- GEM-T Easy Vector was ligated to JM109 (the same method as above), and 10 white colonies were randomly picked up to contain 50 g/mL ampicillin.
- the penicillin was cultured in LB liquid medium, cultured at 37 ° C overnight, and then glycerin was added to a final concentration of 20%, and stored at -80 ° C until use.
- SEQ ID NO: 8 and the 3' primer AUAP were used for PCR amplification (reaction system and reaction conditions as above), and 4 positive clones were obtained and sent to Yingji Jieji (Shanghai) Trading Co., Ltd. for sequencing and sequencing, and the gene was obtained. The 5' end of the cDNA.
- the obtained 5' RACE product was cloned and sequenced, and spliced with the 3' RACE product sequencing result.
- the full-length cDNA sequence of GhNHXl_l was obtained as SEQ ID NO: 19.
- GhNHXl-lR SEQ ID NO: 10:
- the PCR reaction was carried out using TaKaRa's PrimeSTAR HS DNA polymerase and cotton cDNA as a template.
- 50 ⁇ 1 PCR reaction system 10 ⁇ 1 5 X PS Buffer, 3 ⁇ 1 2 ⁇ 5 mM dNTP, 2. 0 ⁇ 1 cDNA, 1. 0 ⁇ 1 PrimeSTAR, 10 ⁇ ⁇ primer SEQ ID NO: 9 and SEQ ID NO: 10 each of 2 ⁇ 0 ⁇ 1, and 30 ⁇ of double distilled water.
- PCR reaction conditions pre-denaturation at 94 °C for 5 min, denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 2 min, after 33 cycles, extension at 72 °C for 10 min.
- PCR amplification product plus A tail PCR product plus 2.5 times absolute ethanol, _20 ° C for 10 minutes, centrifuge, remove the supernatant, air dry, dissolved in 21 ⁇ ⁇ double distilled water.
- Reaction conditions The reaction was carried out at 70 ° C for 30 minutes.
- a DNA fragment of about 1600 bp was recovered (Omega Recovery Kit), ligated into pGEM T-easy vector, and transformed into JM109 (Method Same as above), 10 white colonies were randomly picked and cultured in LB liquid medium containing 50 ⁇ g/mL ampicillin, and cultured at 37 ° C overnight, glycerol was added to a final concentration of 20%, and stored at -80 ° C for use.
- SEQ ID NO: 9 and SEQ ID NO: 10 were used for bacterial liquid PCR amplification (reaction system and reaction conditions are the same as above), and 4 positive clones were obtained and sent to Yingjie Jieji (Shanghai) Trading Co., Ltd. for sequencing. The sequence is SEQ ID. NO: 2.
- Pnos were amplified using the primers SEQ ID NO: 11 and SEQ ID NO: 12 with the plant expression vector PBI121 (purchased from Beijing Huaxia Ocean Technology Co., Ltd.) using TaKaRa's PrimeSTAR HS DNA polymerase.
- PCR reaction conditions pre-denaturation at 94 °C for 5 min, denaturation at 94 °C for 30 s, annealing at 56 °C for 30 s, extension at 72 °C for 30 s, after 33 cycles, extension at 72 °C for 10 min.
- pCAMBIA2300_l was obtained by EcoRI and Bglll digestion with pCAMBIA2300 (promega, T4 ligase cassette).
- PrimeSTAR HS DNA polymerase 50 ⁇ 1 PCR reaction system: 10 ⁇ 15 ⁇ PS Buffer, 3 ⁇ 12.5 mM dNTP, 1.0 ⁇ 1 PBI121, 1.0 ⁇ 1 PrimeSTAR, 10 ⁇ ⁇ primers SEQ ID NO: 13 and SEQ ID NO: 14 each 2.0 ⁇ 1, And 31 ⁇ l of double distilled water.
- SEQ ID NO: 15 and SEQ ID NO: 16 The Arabidopsis thaliana rd29A promoter was amplified using Arabidopsis thaliana (Columbia type, available from www. arabidopsis.org) as a template (see Zeng J., et L. 2002, Preparation). Of total DNA from "recalcit rant plant taxa", Acta Bot. Sin., 44(6): Method 694-697 for extracting Arabidopsis DNA). PrimeSTAR HS DNA polymerase using TaKaRa.
- PCR reaction system 10 ⁇ 5XPS Buffer, 3 ⁇ 1 2.5 mM dNTP, 1.0 ⁇ 1 Arabidopsis DNA, 1.0 ⁇ 1 PrimeSTAR, 10 ⁇ primers SEQ ID NO: 15 and SEQ ID NO: 16 2.0 11, and 31 ⁇ of double distilled water.
- PCR reaction conditions predenaturation at 94 °C for 5 min, denaturation at 94 °C 30 s, annealed at 58 °C for 30 s, extended at 72 °C for 30 s, after 33 cycles, extended at 72 °C for 10 min.
- the pCAMBIA2300-3 was obtained by digesting with HindIII and Pstl (connection method is the same as above) pCAMBIA2300-2.
- SEQ ID NO: 17 and SEQ ID NO: 18 amplify GhNHX1-1 (template is GhNHXl-1 obtained in Example 2) using TaKaRa's PrimeSTAR HS DNA polymerase.
- PCR reaction conditions pre-denaturation at 94 °C for 5 min, denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 2 min, after 33 cycles, extension at 72 °C for 10 min.
- the plant expression vector rd29A_GhNHXl-l_2300 was obtained by restriction enzyme digestion with Pstl and Sad (connection method as above) PCAMBIA2300-3.
- Agrobacterium LBA4404 (available from Biovector Science Lab, Inc) Competent preparation: Agrobacterium LBA4404 was plated on LB solid medium containing 50 ⁇ g/ml rifampicin and 50 ⁇ g/ml streptomycin 1-2 days in advance Single spot inoculation, culture at 28 °C for 1 to 2 days. Single colonies were picked and inoculated into 5 ml of LB liquid medium containing 50 ⁇ g/ml rifampicin and 50 ⁇ g/ml streptomycin, and cultured overnight at 28 ° C (about 12-16 h) to 0D600 value. 0. 4, forming a seed bacterial liquid.
- Transformation of Agrobacterium Thaw competent cells on ice, add 1 ⁇ l of plasmid to 40 ⁇ l of competent cells, mix and ice bath for about 10 min o Transfer the mixture of competent and DNA to pre-cool with gun
- the electric shock cup tap to bring the suspension to the bottom, taking care not to have air bubbles.
- Place the electric shock cup (purchased from bio-rad) in the electric shock room On the slide, push the slide to place the electric shock cup on the base electrode of the shock chamber.
- the program of MicroPulser purchased from bio-rad
- the program of MicroPulser purchased from bio-rad
- the electric shock is set to "Agr" and the electric shock is once. The electric shock cup was immediately taken out and the pre-warmed LB medium at 28 ° C was added.
- the leaves of the sterile seedlings were cut into 5 mm ⁇ 5 mm leaf discs, and the leaf discs were inoculated with Agrobacterium containing the expression vector rd29A-GhNHXl-l-2300 in the logarithmic growth phase for 10 min, and the bacterial liquid was absorbed in the dark condition. Co-culture for 2 days (MS medium).
- the leaves were transferred to differentiation medium (MS+1 mg/L cytokinin (BA) + 0.1 mg/L naphthaleneacetic acid (face +50 mg/L kanamycin + 500 mg/L cephalosporin) After incubation for about 45 days under light conditions, the buds should be grown and transferred to rooting medium (MS+50 mg/L kanamycin + 500 mg/L cephalosporin) for about 30 days, until the root system is developed. The seedlings were then transferred to MS medium supplemented with 500 mg/L cephalosporin for number storage.
- differentiation medium MS+1 mg/L cytokinin (BA) + 0.1 mg/L naphthaleneacetic acid (face +50 mg/L kanamycin + 500 mg/L cephalosporin)
- rooting medium MS+50 mg/L kanamycin + 500 mg/L cephalosporin
- the obtained transgenic tobacco leaves were extracted, and DNA (the Arabidopsis thaliana DNA extraction method in Example 3) was used, using SEQ ID NO: 9: and SEQ ID NO: 10 (50 ⁇ l PCR reaction system: 5 ⁇ 1 ⁇ Buffer, 3 ⁇ 1 2 ⁇ 5 mM dNTP, 2.0 ⁇ 1 DNA, 1.0 ⁇ 1 Ex Taq 10 ⁇ M primers SEQ ID NO: 9 and SEQ ID NO: 10 each 2.0 ⁇ 1, and 35 ⁇ l of double distilled water.
- H1-T H20 tobacco seeds and non-GM control tobacco seeds were immersed in sterile double distilled water for 30 min, soaked in 75% alcohol for 30 s, and washed twice with sterile double distilled water. Then, it was immersed in 0.1% liter of mercury for 8 min, and washed twice with sterilized double distilled water to complete surface sterilization.
- Group 1 T-sterilized surface.
- H1-T. H20 tobacco seed, control tobacco Seeds were germinated under sterile conditions on MS solid medium.
- Group 2 T that will be surface sterilized.
- H1-T. H20 tobacco seeds and control tobacco seeds were germinated under sterile conditions on MS solid medium supplemented with 100 mM NaCl. 25 .
- the absorbance values of total RNA at 260 nm and 280 nm were measured using a HITACHI UV spectrophotometer U-2001 to calculate the individual RNA concentrations.
- Reverse transcription was performed using the invitrogen reverse transcription kit Superscript III Reverse Transcriptase (2 ⁇ g total R A as a template, reverse transcription primer SEQ ID NO: 10) according to the method described in the specification.
- GhNHXl-1 protein was detected by amplifying GhNHXl-1 by SEQ ID NO: 9 and SEQ ID NO: 10.
- PCR was performed using TaKaRa's PrimeSTAR HS DNA polymerase with reverse transcribed cDNA as a template.
- 50 ⁇ 1 PCR reaction system 10 ⁇ ⁇ 5 X PS Buffer, 3 ⁇ 1 2.5 mM dNTP, 2.0 ⁇ 1 cDNA, 1.0 ⁇ 1 PrimeSTAR, 10 ⁇ primers SEQ ID N0:9 and SEQ ID NO: 10 each 2.0 ⁇ 1, and 30 ⁇ 1 of double distilled water.
- PCR reaction conditions pre-denaturation at 94 °C for 5 min, denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 1 min, after 29 cycles, extension at 72 °C for 10 min.
- M DNA Ladder Marker (DL2000, purchased from Shenzhen Ruizhen Biotechnology Co., Ltd.), 1-5 for control tobacco, 6-15 for salt-tolerant transgenic tobacco T1 plants, 16_21 for salt-tolerant transgenic tobacco T1 plants .
- the size of the band shown in the figure is consistent with the size of the GhNHXl-1 gene.
- control tobacco did not transcribe the mRNA of GhNHXl-1, and the T1 generation of the salt-tolerant transgenic tobacco had stronger transcription of GhNHXl-1, and the T1 generation of the salt-tolerant transgenic tobacco had no transcription or weak transcription.
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Abstract
Provided are an ion channel protein NHX1-1 from cotton and coding genes thereof, and the use in the incubation of transgenic plants with increased salt tolerance.
Description
一个棉花离子通道类蛋白及其编码基因与应用 A cotton ion channel protein and its coding gene and application
技术领域 本发明涉及离子通道类蛋白及其编码基因与应用,特别是涉及一个来源于棉花的 离子通道类蛋白 NHX1-1及其编码基因, 以及其在培育耐盐性提高的转基因植物中的 应用。 背景技术 盐胁迫是世界农业生产最重要的非生物逆境危害之一, 盐渍土壤通常以钠盐、钙 盐或镁盐为主, 成为影响植物生长、 导致粮食和经济作物减产的主要因素。世界上盐 碱土的面积约有 4亿公顷, 占灌溉农田的 1/3。 盐碱地在中国分布广泛, 现有盐碱地面 积约 0. 4亿公顷。 随着我国人口增加, 耕地减少, 盐碱地资源的开发利用有着极其重 要的现实意义。而植物抗盐碱、耐干旱能力的提高和适宜在盐碱地上生长并具有较高 经济和生态价值的植物种或品系的选育,则是利用盐碱地经济、 有效的措施。 对绝大 多数农作物来说,大多数植物对盐碱、干旱的耐受性差,只能生长在氯化钠含量为 0. 3% 以下的土壤上, 土壤中过量的 Na+会对植物体的正常的生长代谢产生毒害作用。 因此 如何在盐渍环境下提高作物产量就成为全世界农业生产中十分重要的问题。 FIELD OF THE INVENTION The present invention relates to ion channel proteins and their encoding genes and applications, and more particularly to a cotton-derived ion channel protein NHX1-1 and its encoding gene, and its application in breeding transgenic plants with improved salt tolerance . BACKGROUND OF THE INVENTION Salt stress is one of the most important abiotic stress hazards in agricultural production in the world. Salted soil is usually dominated by sodium, calcium or magnesium salts, and is a major factor affecting plant growth and causing food and economic crop yield reduction. The world's saline-alkali soil covers an area of about 400 million hectares, accounting for one-third of the irrigated farmland. Saline-alkali land is widely distributed in China, and the existing saline-alkali land area is about 0.4 million hectares. With the increase of population in China and the reduction of cultivated land, the development and utilization of saline-alkali resources has extremely important practical significance. The improvement of plant resistance to salt and alkali, drought-tolerant ability and the selection of plant species or strains suitable for growth on saline-alkali land with high economic and ecological value is an economical and effective measure to utilize saline-alkali land. For most crops, most plants are poorly tolerant to saline and alkali, and can only grow on soils with a sodium chloride content of less than 0.3%. Excess Na + in the soil will be plant Normal growth metabolism produces toxic effects. Therefore, how to increase crop yield in a salted environment has become a very important issue in agricultural production worldwide.
植物的耐盐性是一个十分复杂的数量性状,其耐盐机制涉及从植株到器官、组织、 生理生化直至分子的各个水平。各国的科学家也为此做了大量的工作, 并取得了很多 新进展,特别在利用高等模式植物拟南芥来研究植物的耐盐分子机理方面,使该领域 的研究有了突破性的进展 (Zhu JK. 2002. Sal t and drought stress s ingal transduct ion in plants. Annu. Rev. Plant Biol. 53: 1247-1273; Zhang ZL. 2011. Arabidops i s Floral Init iator SKBl Confers High Salt Tolerance by Regulat ing Transcript ion and Pre-mRNA Spl icing through Altering Hi stone H4R3 and Smal l Nuclear Ribonucleoprotein LSM4 Methylat ion. Plant Cel l, 23 : 396 - 411 )。 高 等植物细胞可有多种途径感受外界环境中物化参数的变化,从而将胞外的信号变为胞 内信号, 通过系列的信号传导最后将胁迫信号传递至细胞核内, 激活转录因子, 而激 活转录因子再作用于功能基因, 启动逆境应答基因的表达从而提高植物的耐逆性。尽 管研究者已从不同侧面开展了大量研究,但由于其机制十分复杂,植物抗盐中的许多 重要问题仍有待探索。例如, 植物抗盐的关键因子仍未找到; 植物耐盐的分子机制并
不十分清楚。 发明内容 本发明人利用 SSH与 RACE相结合的方法克隆出了棉花的一个离子通道类蛋白The salt tolerance of plants is a very complex quantitative trait, and its salt tolerance mechanism involves various levels from plants to organs, tissues, physiology and biochemistry to molecules. Scientists from various countries have also done a lot of work for this purpose, and have made a lot of new progress, especially in the use of the higher model plant Arabidopsis to study the salt-tolerant molecular mechanism of plants, making breakthroughs in research in this field ( Zhu JK. 2002. Sal t and drought stress s ingal transduct ion in plants. Annu. Rev. Plant Biol. 53: 1247-1273; Zhang ZL. 2011. Arabidops is Floral Init iator SKBl Confers High Salt Tolerance by Regulat ing Transcript ion And Pre-mRNA Spl icing through Altering Hi stone H4R3 and Smal l Nuclear Ribonucleoprotein LSM4 Methylat ion. Plant Cel l, 23: 396-411). Higher plant cells can have multiple ways to sense changes in the physical and chemical parameters in the external environment, thereby transforming extracellular signals into intracellular signals, and finally transmitting stress signals to the nucleus through a series of signal transductions, activating transcription factors, and activating transcription. The factor acts on the functional gene and initiates the expression of the stress response gene to increase the tolerance of the plant. Although researchers have conducted a large number of studies from different sides, due to the complexity of its mechanism, many important issues in plant salt resistance remain to be explored. For example, the key factors of plant salt tolerance have not been found; the molecular mechanism of plant salt tolerance Not very clear. SUMMARY OF THE INVENTION The present inventors cloned an ion channel protein of cotton using a combination of SSH and RACE.
(本文命名为 NHX1-1 ) 的编码基因的 DNA序列。 并发现将其导入转基因植株后, 可明显改善转基因植株的抗盐性, 而且这些性状可稳定遗传。 (This article is named NHX1-1) The DNA sequence of the coding gene. It was found that the introduction of the transgenic plants significantly improved the salt tolerance of the transgenic plants, and these traits were stably inherited.
本发明第一方面提供棉花的一个离子通道类蛋白 NHX1-1的编码基因,其序列为 SEQ ID NO: 2。 The first aspect of the present invention provides a gene encoding an ion channel-like protein NHX1-1 of cotton having the sequence of SEQ ID NO: 2.
本发明第二方面提供一种重组表达载体,其含有本发明第一方面所述的基因并且 所述基因的核苷酸序列与所述表达载体的表达控制序列可操作地连接; 优选地,所述 载体为附图 2所示的 rd29A-GhNHXl-l-2300载体。 A second aspect of the invention provides a recombinant expression vector comprising the gene of the first aspect of the invention, and the nucleotide sequence of the gene is operably linked to an expression control sequence of the expression vector; preferably, The vector is the rd29A-GhNHX1-1-2300 carrier shown in Fig. 2.
本发明第三方面提供一种重组细胞,其含有本发明第一方面所述的基因或者本发 明第二方面所述的重组表达载体; 优选地, 所述重组细胞为重组农杆菌细胞。 A third aspect of the invention provides a recombinant cell comprising the gene of the first aspect of the invention or the recombinant expression vector of the second aspect of the invention; preferably, the recombinant cell is a recombinant Agrobacterium cell.
本发明第四方面提供一种改善植物耐盐性的方法,包括: 将本发明第一方面所述 基因或者本发明第二方面所述的重组表达载体导入植物或植物组织并使所述基因表 达; 优选地, 所述植物是烟草。 A fourth aspect of the present invention provides a method for improving salt tolerance of a plant, comprising: introducing the gene of the first aspect of the invention or the recombinant expression vector of the second aspect of the invention into a plant or plant tissue and expressing the gene Preferably, the plant is tobacco.
本发明第五方面提供一种制备转基因植物的方法,包括: 在有效产生植物的条件 下培养含有本发明第一方面所述基因、本发明第二方面所述的重组表达载体的植物或 植物组织; 优选地, 所述植物是烟草。 A fifth aspect of the invention provides a method for producing a transgenic plant, comprising: cultivating a plant or plant tissue comprising the gene of the first aspect of the invention, the recombinant expression vector of the second aspect of the invention under conditions effective to produce a plant; Preferably, the plant is tobacco.
本发明第六方面提供本发明第一方面所述的基因、本发明第二方面所述的重组表 达载体或者本发明第三方面所述的重组细胞用于改善植物耐盐性以及用于植物育种 的用途; 优选地, 所述植物是烟草。 A sixth aspect of the present invention provides the gene according to the first aspect of the present invention, the recombinant expression vector of the second aspect of the present invention or the recombinant cell of the third aspect of the present invention for improving salt tolerance of a plant and for use in plant breeding Use; Preferably, the plant is tobacco.
本发明第七方面提供发明第一方面所述的基因编码的氨基酸序列, 如 SEQ ID ΝΟ:1所示。 附图说明 图 1是 GhNHXl-1的植物表达载体 (rd29A-GhNHXl-l-2300)的构建流程。 A seventh aspect of the invention provides the amino acid sequence encoded by the gene of the first aspect of the invention, as shown in SEQ ID NO: 1. Brief Description of the Drawings Fig. 1 is a construction flow of a plant expression vector (rd29A-GhNHX1-1-2300) of GhNHX1-1.
图 2是 GhNHXl-1的植物表达载体 (rd29A-GhNHXl-l-2300)的质粒图。 Figure 2 is a plasmid map of the plant expression vector (rd29A-GhNHX1-1-2300) of GhNHXl-1.
图 3是 GhNHXl-1转基因烟草种子和非转基因烟草种子 (对照) 在常规 MS培养基 上的生长结果。 培养皿左侧 (GM)为转烟草基因种子的生长结果; 右侧 (CK) 为非
转基因烟草种子 (对照) 的生长结果。 Figure 3 shows the growth results of GhNHXl-1 transgenic tobacco seeds and non-transgenic tobacco seeds (control) on conventional MS medium. The left side of the culture dish (GM) is the growth result of the transgenic tobacco seed; the right side (CK) is non- Growth results of transgenic tobacco seeds (control).
图 4是 GhNHXl-1转基因烟草种子和非转基因烟草种子 (对照) 在添加有 100 mM NaCl的 MS培养基上的生长结果。 培养皿左侧 (GM) 为转烟草基因种子 (TQH2) 的 生长结果; 右侧 (CK) 为非转基因烟草种子 (对照) 的生长结果。 Figure 4 shows the growth results of GhNHXl-1 transgenic tobacco seeds and non-transgenic tobacco seeds (control) on MS medium supplemented with 100 mM NaCl. The left side of the culture dish (GM) is the growth result of the transgenic tobacco seed (T Q H2); the right side (CK) is the growth result of the non-transgenic tobacco seed (control).
图 5是转基因 T1代烟草植株和非转基因对照植株在转录水平上的蛋白表达验证结 果。 M为 Marker, 1_5为对照烟草, 6_15为耐盐转基因烟草 T1代植株, 16_21为不耐盐 转基因烟草 T1代植株。 具体实施方式 下面结合非限制性实施例对本发明进行进一步说明。 实施例 1 盐胁迫下棉花 SSH文库构建: Figure 5 shows the results of protein expression verification at the transcriptional level of transgenic T1 tobacco plants and non-transgenic control plants. M is Marker, 1_5 is the control tobacco, 6_15 is the salt-tolerant transgenic tobacco T1 plant, and 16_21 is the salt-tolerant transgenic tobacco T1 plant. BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be further described below in conjunction with non-limiting examples. Example 1 Cotton SSH library construction under salt stress:
具体方法为: The specific method is:
利用 Clontech公司的 PCR-select™ cDNA Subtraction Kit试剂盒所示的方法通 过抑制差减杂交方法构建差减文库。在实验过程中以经 NaCl处理 6h的棉花幼苗的根 的 mRNA作为样品 (tester), 以未处理的棉花幼苗的根的 mRNA作为对照 (driver)。 A subtractive library was constructed by inhibition subtractive hybridization using the method shown in Clontech's PCR-selectTM cDNA Subtraction Kit. The mRNA of the roots of the cotton seedlings treated with NaCl for 6 hours was used as a tester during the experiment, and the mRNA of the roots of the untreated cotton seedlings was used as a driver.
( 1 ) 供试材料: (1) Test materials:
冀棉 14 (国家棉花中期库, 获取单位中国棉花研究所, 统一编号: ZM-30270) 播种到灭过菌的蛭石上, 在 25°C、 光暗周期 16h/8h条件下培养, 每周浇 1/2MS培养 ¾ ( 9. 39 mM KN03, 0. 625 mM KH2P04, 10. 3 mM NH^Oa, 0. 75 mM MgS04, 1. 5 mM CaCl2, 50 μ Μ ΚΙ , 100 μ Μ Η3Β03, 100 μ M MnS04, 30 μ M ZnS04, 1 μ M N¾Mo04, 0. 1 μ M CoCl2, 100 μ Μ N¾EDTA, 100 μ Μ FeS04) 一次。 当苗株长高达 25-30 cm时用于实 验。 冀棉14 (National Cotton Medium-Term Library, obtained by the China Cotton Research Institute, Uniform No.: ZM-30270) Seeded on sterilized vermiculite, cultured at 25 ° C, light dark cycle 16h/8h, weekly pouring 1/2MS culture 3⁄4 ( 9. 39 mM KN0 3 , 0. 625 mM KH2P0 4 , 10. 3 mM NH^Oa, 0.75 mM MgS0 4 , 1. 5 mM CaCl 2 , 50 μ Μ ΚΙ , 100 μ Μ Η 3 Β 0 3 , 100 μ M MnS0 4 , 30 μ M ZnS0 4 , 1 μ M N3⁄4Mo0 4 , 0.1 μM CoCl 2 , 100 μ Μ N3⁄4EDTA, 100 μ Μ FeS0 4 ) Once. It was used for experiments when the seedlings were as long as 25-30 cm.
( 2 ) 材料处理: (2) Material handling:
将供试幼苗分为 2组, 每组 4株。 第一组为对照组, 在 25°C、 光照下培养, 放 置到 1/2MS液体培养基中。 第二组为处理组, 25°C、 光照下培养, 放置到添加有终浓 度为 200 mM NaCl的 1/2MS液体培养基中, 处理 6小时, 处理完毕后及时剪取两组幼 苗的根, 用液氮迅速冷冻后, 于 -70°C冰箱中保存。 The test seedlings were divided into 2 groups of 4 plants each. The first group was a control group, cultured at 25 ° C under light, and placed in 1/2 MS liquid medium. The second group was treated with 25 ° C, light culture, placed in 1/2 MS liquid medium supplemented with a final concentration of 200 mM NaCl, and treated for 6 hours. After the treatment, the roots of the two groups were cut out in time. After rapid freezing with liquid nitrogen, it was stored in a -70 ° C refrigerator.
( 3 ) 总 RNA提取: (3) Total RNA extraction:
分别取对照和 NaCl处理的棉花根 0. 5g, 用植物 RNA提取试剂盒 ( invitrogen) 提取棉花的总 RNA。 用 HITACHI公司的紫外分光光度计 U-2001测定总 RNA在 260 nm
和 280 nm的吸光度值, 0D260/0D280比值为 1. 8-2· 0, 表明总 RNA纯度较高, 用 1. 0% 的琼脂糖凝胶电泳检测总 A的完整性, 28S条带的亮度约为 18S条带的 2倍, 表明 RNA的完整性良好。使用 Qiagen 公司的 Oligotex mRNA 纯化试剂盒(purification of polyA+ RNA from total RNA)分离 mRNA。 The total RNA of cotton was extracted with 0.5 g of control and NaCl-treated cotton roots, respectively, using a plant RNA extraction kit (invitrogen). Determination of total RNA at 260 nm using HITACHI's UV spectrophotometer U-2001 And the absorbance value at 280 nm, the ratio of 0D260/0D280 is 1. 8-2· 0, indicating that the total RNA purity is high, and the integrity of total A and the brightness of the 28S strip are detected by 1.0% agarose gel electrophoresis. It is about twice as large as the 18S band, indicating good RNA integrity. mRNA was isolated using the Qiagen Oligotex mRNA Purification Kit (purification of polyA+ RNA from total RNA).
(4) 抑制消减杂交: (4) Inhibition subtractive hybridization:
为了增加获得表达序列标签 (Expressed sequence tag, EST) (unigene)的有效 性, 避免基因无酶切位点及所获得序列在非翻译区。 本实验室用 Rsal, Haelll分别 对双链 cDNA (按照消减试剂盒中记载的方案, 由 mRNA逆转录获得)进行消化, 做两 组抑制差减,其他步骤及方法按 Clontech公司的 PCR_selectTMcDNA Subtraction Kit 试剂盒所示的方法进行抑制消减杂交, 最后合并两组正向消减杂交 cDNA片段的第二 次 PCR产物。 In order to increase the availability of the Expressed Sequence Tag (EST) (unigene), the gene is not cleavable and the obtained sequence is in the untranslated region. The laboratory uses Rsal, Haelll to digest the double-stranded cDNA (according to the protocol described in the subtractive kit, obtained by reverse transcription of mRNA), and performs two sets of suppression subtraction. Other steps and methods are based on PCR_select TM cDNA Subtraction of Clontech The method shown in the Kit was used for suppression subtractive hybridization, and finally the second PCR product of the two groups of forward subtracted hybrid cDNA fragments was combined.
( 5) cDNA消减文库的构建与初步筛选、 克隆、 鉴定 (5) Construction and preliminary screening, cloning and identification of cDNA subtractive library
正向消减杂交 cDNA片段的第二次 PCR产物 (QIAquick PCR Purification Kit 纯化,购自 Qiagen)与 pGEM_T Easy (购自 Promega试剂盒)载体连接,依照 pGEM_T Easy 试剂盒的程序, 具体步骤如下: 用 200 l PCR管依次加入下列成分: 纯化的正向差 减杂交 cDNA片段的第二次 PCR产物 3 μ 1, Τ4连接酶缓冲液 5 μ 1, pGEM-T Easy 载体 1 μ 1, T4 DNA连接酶 1 μ ΐ , 于 4°C连接过夜。 取 10 μ L连接反应产物, 加 入到 100 μ L感受态大肠杆菌 JMI09 (购自 TAKARA)中,冰浴 30 min、 热休克 60 s、 冰 浴 2 min, 另加 250 L LB培养液 (1% Tryptone购自 0X0ID, 0. 5% Yeast Extract 购自 0X0ID, 1% NaCl购自国药) 置 37°C水浴中, 以 225 r/min振荡培养 30 min, 取 200 μ L菌液种植于含 50 μ g/mL氨苄青霉素的 LB (同上) /X-gal/IPTG (X-gal/IPTG 购自 TAKARA) 培养板上, 37°C培育 18 h。 计数培养板中直径〉 1 mm的清晰白色及蓝 色菌落数, 随机挑取 300个白色菌落 (编号: Gh-S001至 Gh-S300)。 将所有白色克隆 挑于含有 50 μ g/mL氨苄青霉素的 LB 液体培养基的 96 孔细胞培养板 (CORNING)中, 37°C培养过夜后加甘油至终浓度 20%, 于 - 80°C保存备用。以巢式 PCR 引物 Primer l 和 Primer 2R (Clontech公司的 PCR- select™ cDNA Subtraction Kit试剂盒自带) 进行菌液 PCR扩增, 得到 211个阳性克隆, 对所有阳性克隆在送英潍捷基 (上海) 贸易有限公司测序 (6) 差异克隆的 cDNA测序分析: The second PCR product of the forward subtracted hybrid cDNA fragment (QIAquick PCR Purification Kit purified from Qiagen) was ligated with the pGEM_T Easy (purchased from Promega kit) vector. According to the procedure of the pGEM_T Easy kit, the specific steps are as follows: l The PCR tube is sequentially added with the following components: Purified positive subtractive hybridization cDNA fragment second PCR product 3 μ 1, Τ4 ligase buffer 5 μ 1, pGEM-T Easy vector 1 μ 1, T4 DNA ligase 1 μ ΐ , connected overnight at 4 ° C. 10 μL of the ligation reaction product was added to 100 μL of competent E. coli JMI09 (purchased from TAKARA), ice bath for 30 min, heat shock for 60 s, ice bath for 2 min, and 250 L LB medium (1%). Tryptone was purchased from 0X0ID, 0. 5% Yeast Extract was purchased from 0X0ID, 1% NaCl was purchased from Sinopharm. It was placed in a 37°C water bath, shaken at 225 r/min for 30 min, and 200 μL of bacterial solution was planted in 50 μm. LB (ibid.) / X-gal/IPTG (X-gal/IPTG purchased from TAKARA) of g/mL ampicillin was incubated at 37 ° C for 18 h. Count the number of clear white and blue colonies with a diameter > 1 mm in the culture plate and randomly pick 300 white colonies (number: Gh-S001 to Gh-S300). All white clones were picked in 96-well cell culture plates (CORNING) containing LB liquid medium containing 50 μg/mL ampicillin, cultured overnight at 37 ° C, glycerol was added to a final concentration of 20%, and stored at -80 ° C. spare. The nested PCR primers Primer l and Primer 2R (Clontech's PCR-selectTM cDNA Subtraction Kit kit) were used to carry out PCR amplification of the bacterial cells, and 211 positive clones were obtained, and all positive clones were sent to the British 潍jieji ( Shanghai) Trading Co., Ltd. sequencing (6) cDNA sequencing analysis of differential clones:
将上述 211个差异克隆的 DNA测序结果去除载体和不明确序列及冗余的 cDNA后, 共得到 153个 EST (unigene)。 经 BlastN发现其中 87条在 GenBank 中有同源序列,
29条功能未知或者为假定蛋白, 另有 37条未获得同源匹配, 推测可能是处于 3' 、 5' 末端非翻译区的较短序列。 实施例 2 棉花离子通道类编码基因 GhNHXl-1的克隆 After removing the DNA sequencing results of the above 211 differential clones and removing the vector and the ambiguous sequence and the redundant cDNA, a total of 153 ESTs (unigene) were obtained. Among BlastN, 87 of them found homologous sequences in GenBank. The 29 functions are unknown or hypothetical proteins, and another 37 have not obtained homologous matches, presumably the shorter sequences in the 3' and 5' untranslated regions. Example 2 Cloning of cotton ion channel coding gene GhNHXl-1
克隆子 Gh-S037序列为 SEQ ID No: 3, 序列分析表明该序列的编码的氨基酸序 列属于离子通道类蛋白 NHX1,本文将克隆子 Gh-S037编码的全长基因命名为 GhNHXl-1, 该基因对应的蛋白命名为 NHX1-1。 The sequence of the cloned Gh-S037 is SEQ ID No: 3. Sequence analysis indicated that the encoded amino acid sequence of the sequence belongs to the ion channel-like protein NHX1. The full-length gene encoded by the clone Gh-S037 was named GhNHXl-1. The corresponding protein was named NHX1-1.
GhNHXl-1全长基因的克隆 Cloning of the full-length gene of GhNHXl-1
根据已经获得的 Gh-S037基因片段, 设计两条特异性引物, 作为 3' RACE的 5' 端特异性引物。 Based on the Gh-S037 gene fragment that has been obtained, two specific primers were designed as the 5'-end specific primer for 3' RACE.
GhNHXl GSP1: SEQ ID NO: 4: GhNHXl GSP1: SEQ ID NO: 4:
TGGATTGCTT AGTGCTTACA TTA TGGATTGCTT AGTGCTTACA TTA
GhNHXl GSP2: SEQ ID NO: 5: GhNHXl GSP2: SEQ ID NO: 5:
CAACGGATCG CGAGGTTGCT CTC CAACGGATCG CGAGGTTGCT CTC
实验步骤按试剂盒说明书操作 (3' RACE System for Rapid Amplification of cDNA Ends试剂盒购自 invitrogen公司)。 The experimental procedure was performed according to the kit instructions (3' RACE System for Rapid Amplification of cDNA Ends kit purchased from Invitrogen).
用 SEQ ID NO: 4与 3' 端引物 AUAP (试剂盒自带), 以 mRNA逆转录的 cDNA为模 板进行第一轮 PCR扩增。 具体步骤如下: ExTaq 购自 TAKARA, 50 lPCR反应体系:
5 μ 1 ΙΟ Χ Εχ Buffer, 3 μ 1 2. 5 mM的 dNTP, 2. 0 μ 1 mRNA反转录的 cDNA, 1. 0 μ 1 Ex Taq 10 μ Μ的引物 SEQ ID NO : 4和 AUAP各 2. 0 μ 1, 以及 35 μ 1的双蒸水。 PCR反应条件: 94°C预变性 5 min, 94 °C 变性 30 s, 58°C退火 30 s, 72 °C 延伸 2 min, 33个循环后, 72°C 延伸 10 min e The first round of PCR amplification was carried out using SEQ ID NO: 4 and the 3' primer AUAP (provided by the kit), using mRNA reverse transcribed cDNA as a template. The specific steps are as follows: ExTaq is purchased from TAKARA, 50 l PCR reaction system: 5 μ 1 ΙΟ Χ Εχ Buffer, 3 μ 1 2. 5 mM dNTP, 2. 0 μ 1 mRNA reverse transcribed cDNA, 1. 0 μ 1 Ex Taq 10 μ Μ primers SEQ ID NO: 4 and AUAP 2. 0 μ 1, and 35 μ 1 of double distilled water. PCR reaction conditions: pre-denaturation at 94 °C for 5 min, denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 2 min, after 33 cycles, extension at 72 °C for 10 min e
所得的 PCR产物用双馏水稀释 50倍后取 2. 0 μ 1作为模板, 用 SEQ ID NO : 5 与 3' 端引物 AUAP进行第二轮 PCR扩增,具体步骤如下: 50 μ 1 PCR反应体系: 5 μ 1 ΙΟ Χ Εχ Buffer, 3 μ 1 2. 5 mM的 dNTP, 2. 0 μ 1稀释的第一轮 PCR产物, 1. 0 μ 1 Ex Taq 10 μ Μ的引物 SEQ ID NO : 5和 AUAP各 2. 0 μ 1, 以及 35 μ 1的双蒸水。 PCR 反应条件: 94°C预变性 5 min, 94 °C 变性 30 s, 58°C退火 30 s, 72 °C 延伸 2min, 33个循环后, 72°C 延伸 10 min。 第二次 PCR产物回收片段约为 1200bp条带 (Gel Extraction Kit购自 OMEGA)连接于 pGEM_T Easy Vector, 转化到大肠杆菌 JM109 (具 体方法同上), 随机挑取 10个白色菌落于含有 50 μ g/mL氨苄青霉素的 LB 液体培养 基中培养, 37°C培养过夜后加甘油至终浓度 20%, -80°C保存备用。 SEQ ID NO : 5与 3' 端引物 AUAP进行菌液 PCR扩增, 得到 5个阳性克隆, 送英潍捷基 (上海) 贸易 有限公司测序测序,获得该基因的 cDNA的 3' 端。 The obtained PCR product was diluted 50-fold with double-distilled water, and then 2.0 μl was used as a template, and the second round of PCR amplification was carried out using SEQ ID NO: 5 and the 3' primer AUAP, and the specific steps were as follows: 50 μ 1 PCR reaction System: 5 μ 1 ΙΟ Χ Εχ Buffer, 3 μ 1 2. 5 mM dNTP, 2. 0 μ 1 diluted first round PCR product, 1. 0 μ 1 Ex Taq 10 μ Μ primer SEQ ID NO : 5 And AUAP each of 2.0 μl, and 35 μl of double distilled water. PCR reaction conditions: pre-denaturation at 94 °C for 5 min, denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 2 min, after 33 cycles, extension at 72 °C for 10 min. The second PCR product was recovered from a fragment of approximately 1200 bp (Gel Extraction Kit from OMEGA) linked to pGEM_T Easy Vector, transformed into E. coli JM109 (specifically as above), and randomly picked 10 white colonies containing 50 μg/ The mL ampicillin was cultured in LB liquid medium, cultured at 37 ° C overnight, and then glycerin was added to a final concentration of 20%, and stored at -80 ° C until use. SEQ ID NO: 5 and 3' primers AUAP were used for PCR amplification of the bacterial cells, and 5 positive clones were obtained, which were sent to Yingjie Jieji (Shanghai) Trading Co., Ltd. for sequencing and sequencing, and the 3' end of the cDNA of the gene was obtained.
根据已经获得的 GhNHXl-1基因片段,设计三条特异性引物,作为 5' RACE的 3' 端特异性引物。 Based on the GhNHXl-1 gene fragment that has been obtained, three specific primers were designed as the 3'-end specific primer for 5' RACE.
GhNHXl-1 GSP3: SEQ ID NO : 6: GhNHXl-1 GSP3: SEQ ID NO: 6:
CCCTCGCAAC TGAGTATGGC CC CCCTCGCAAC TGAGTATGGC CC
GhNHXl-1 GSP4: SEQ ID NO : 7: GhNHXl-1 GSP4: SEQ ID NO: 7:
TGCACCACGC ATAAGACCGG CC TGCACCACGC ATAAGACCGG CC
GhNHXl-1 GSP5: SEQ ID NO : 8: GhNHXl-1 GSP5: SEQ ID NO: 8:
ACATGGAGCT TTCTTAGTCA AG ACATGGAGCT TTCTTAGTCA AG
实验步骤按试剂盒说明书操作 (5' RACE System for Rapid Amplification of cDNA Ends试剂盒购自 invitrogen公司)。 The experimental procedure was performed according to the kit instructions (5' RACE System for Rapid Amplification of cDNA Ends kit purchased from Invitrogen).
用 SEQ ID NO: 7与 5'通用引物 AAP (试剂盒自带), 以 mRNA逆转录的 cDNA (反转录引物 SEQ ID NO: 6)为模板进行第一轮 PCR扩增,具体步骤如下: Ex Taq购 自 TAKARA, 50 μΐ PCR反应体系: 5 μΐ ΙΟχΕχ Buffer, 3 μΐ 2.5 mM的 dNTP, 2.0 μΐ mRNA反转录的 cDNA, 1.0 μΐ Ex Taq、 10 μΜ的引物 SEQ ID NO: 7和 AAP各 2.0 μ1, 以及 35 μΐ的双蒸水。 PCR反应条件: 94°C预变性 5 min, 94 °C 变性 30 s, 55°C退火 30 s, 72°C 延伸 2min, 33个循环后, 72°C 延伸 10 min。 所得的 PCR产物用双蒸水 稀释 50倍后取 2.0 μΐ作为模板,用 SEQ ID NO: 8与 3'端引物 AUAP进行第二轮 PCR
扩增, 具体步骤如下: 50 μ1 ΡΟ 反应体系: 5 μΐ lOxEx Buffer, 3 μΐ 2.5 mM的 dNTP, 2.0 μ1稀释的第一轮 PCR产物, 1.0 l Ex Taq、 10 μΜ的引物 SEQ ID NO: 8和 AUAP 各 2.0 μ1, 以及 35 μΐ的双蒸水。 PCR反应条件: 94°C预变性 5 min, 94 °C 变性 30 s, 58°C退火 30 s, 72 °C 延伸 2 min, 33个循环后, 72°C 延伸 10 min。 第二次 PCR回 收片段约为 1200bp条带 (Gel Extraction Kit购自 OMEGA) 连接于 GEM-T Easy Vector, 转化到 JM109(具体方法同上),随机挑取 10个白色菌落于含有 50 g/mL氨苄 青霉素的 LB 液体培养基中培养, 37°C培养过夜后加甘油至终浓度 20%, -80°C保存备 用。 SEQ ID NO: 8与 3'端引物 AUAP进行菌液 PCR扩增(反应体系及反应条件同上), 得到 4个阳性克隆,送英潍捷基 (上海) 贸易有限公司测序测序,获得该基因的 cDNA 的 5'端。 The first round of PCR amplification was carried out using SEQ ID NO: 7 and 5' universal primer AAP (provided with the kit), and cDNA reverse transcription cDNA (reverse transcription primer SEQ ID NO: 6) was used as a template for the first round of PCR amplification. The specific steps are as follows: Ex Taq was purchased from TAKARA, 50 μΐ PCR reaction system: 5 μΐ ΙΟχΕχ Buffer, 3 μΐ 2.5 mM dNTP, 2.0 μΐ mRNA reverse transcribed cDNA, 1.0 μΐ Ex Taq, 10 μΜ primers SEQ ID NO: 7 and AAP 2.0 μl, and 35 μΐ double distilled water. PCR reaction conditions: predenaturation at 94 °C for 5 min, denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 2 min, after 33 cycles, extension at 72 °C for 10 min. The obtained PCR product was diluted 50-fold with double distilled water, and 2.0 μL was used as a template, and the second round of PCR was carried out using SEQ ID NO: 8 and the 3' primer AUAP. Amplification, the specific steps are as follows: 50 μ1 ΡΟ Reaction system: 5 μΐ lOxEx Buffer, 3 μΐ 2.5 mM dNTP, 2.0 μl diluted first round PCR product, 1.0 l Ex Taq, 10 μΜ primer SEQ ID NO: 8 and AUAP each 2.0 μl, and 35 μΐ double distilled water. PCR reaction conditions: predenaturation at 94 °C for 5 min, denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 2 min, after 33 cycles, extension at 72 °C for 10 min. The second PCR recovery fragment was approximately 1200 bp band (Gel Extraction Kit was purchased from OMEGA). GEM-T Easy Vector was ligated to JM109 (the same method as above), and 10 white colonies were randomly picked up to contain 50 g/mL ampicillin. The penicillin was cultured in LB liquid medium, cultured at 37 ° C overnight, and then glycerin was added to a final concentration of 20%, and stored at -80 ° C until use. SEQ ID NO: 8 and the 3' primer AUAP were used for PCR amplification (reaction system and reaction conditions as above), and 4 positive clones were obtained and sent to Yingji Jieji (Shanghai) Trading Co., Ltd. for sequencing and sequencing, and the gene was obtained. The 5' end of the cDNA.
所得的 5' RACE产物克隆测序后,与 3' RACE产物测序结果拼接。获得 GhNHXl_l 全长 cDNA序列 SEQ ID NO : 19。 The obtained 5' RACE product was cloned and sequenced, and spliced with the 3' RACE product sequencing result. The full-length cDNA sequence of GhNHXl_l was obtained as SEQ ID NO: 19.
SEQ ID NO : 19: SEQ ID NO: 19:
1 CTCTTAGCAA AGACTTCAAA ATCATTAAAG TTTTGTTCGT TAACACAATG GCAATCGGGA 1 CTCTTAGCAA AGACTTCAAA ATCATTAAAG TTTTGTTCGT TAACACAATG GCAATCGGGA
61 TATTAAGCAC TCTTTTAGCA AAATCAGAGA CGATTTTAGG GTCTGGTCAC AGCTCAGTAG61 TATTAAGCAC TCTTTTAGCA AAATCAGAGA CGATTTTAGG GTCTGGTCAC AGCTCAGTAG
121 TTTCAATGAA TTTATTCGTT GCTCTTCTTT GCGGGTGTAT CGTGATCGGT CATTTGCTAG121 TTTCAATGAA TTTATTCGTT GCTCTTCTTT GCGGGTGTAT CGTGATCGGT CATTTGCTAG
181 AGGAAAGCCG ATGGATGAAT GAATCCATTA CTGCCCTTGC CATTGGGCTG TGCACTGGAA181 AGGAAAGCCG ATGGATGAAT GAATCCATTA CTGCCCTTGC CATTGGGCTG TGCACTGGAA
241 TTGTAATTTT GCTTACAACA GGAGGAAAAA GCTCTCATCT TTTAGTTTTC AGTGAAGATT241 TTGTAATTTT GCTTACAACA GGAGGAAAAA GCTCTCATCT TTTAGTTTTC AGTGAAGATT
301 TGTTCTTTAA TTATTTGCTT CCACCTATTA TTTTCAATGC AGGATTCCAG GTGAAAAAGA301 TGTTCTTTAA TTATTTGCTT CCACCTATTA TTTTCAATGC AGGATTCCAG GTGAAAAAGA
361 AGCAATTTTT CCGCAACTTT ATGACCATAA TGCTGTTTGG TGCAGTTGGC ACTTTAATAT361 AGCAATTTTT CCGCAACTTT ATGACCATAA TGCTGTTTGG TGCAGTTGGC ACTTTAATAT
421 CCTTTGCCAT CATATCTCTA GGTGCCATAC ATTTTTTCAA GAAAATGAGT ATTGGTAATC421 CCTTTGCCAT CATATCTCTA GGTGCCATAC ATTTTTTCAA GAAAATGAGT ATTGGTAATC
481 TCAAGATAGG GGATTATCTT GCCATTGGGG CAATATTTTC AGCAACAGAC TCTGTTTGCA481 TCAAGATAGG GGATTATCTT GCCATTGGGG CAATATTTTC AGCAACAGAC TCTGTTTGCA
541 CTTTGCAAGT GCTTAATCAG GACGATACGC CTTTATTGTA TAGTCTGGTT TTCGGGGAGG541 CTTTGCAAGT GCTTAATCAG GACGATACGC CTTTATTGTA TAGTCTGGTT TTCGGGGAGG
601 GAGTTGTGAA TGATGCCACA GCTGTTGTTC TTTTCAAAGC AATCCAAAGC TTTGACCTTC601 GAGTTGTGAA TGATGCCACA GCTGTTGTTC TTTTCAAAGC AATCCAAAGC TTTGACCTTC
661 CTCACATCAA CACCACCATT GCTTTGCAAT TTGTTGGGAA CTTTTTATAT TTATTCATCT661 CTCACATCAA CACCACCATT GCTTTGCAAT TTGTTGGGAA CTTTTTATAT TTATTCATCT
721 CGAGTACATT GCTTGGGGTT TTGGCTGGAT TGCTTAGTGC TTACATTATT AAAAAGCTCT721 CGAGTACATT GCTTGGGGTT TTGGCTGGAT TGCTTAGTGC TTACATTATT AAAAAGCTCT
781 ATTTCGGAAG GCACTCAACG GATCGCGAGG TTGCTCTCAT GATACTCATG GCTTACCTTT781 ATTTCGGAAG GCACTCAACG GATCGCGAGG TTGCTCTCAT GATACTCATG GCTTACCTTT
841 CATATATGCT GGCTGAATTT TTCTATTTAA GTGCGATCCT CACTGTGTTC TTTTGTGGGA841 CATATATGCT GGCTGAATTT TTCTATTTAA GTGCGATCCT CACTGTGTTC TTTTGTGGGA
901 TTGTTATGTC TCATTATACA TGGCATAATG TTACCGAAAG TTCAAGAGTG ACAACCAAGC901 TTGTTATGTC TCATTATACA TGGCATAATG TTACCGAAAG TTCAAGAGTG ACAACCAAGC
961 ATGCTTTTGC CACACTATCA TTTGTTGCTG AGATTTTCAT CTTCCTCTAT GTTGGTATGG961 ATGCTTTTGC CACACTATCA TTTGTTGCTG AGATTTTCAT CTTCCTCTAT GTTGGTATGG
1021 ATGCTTTGGA CAT T GAG AAA TGGAGAGTAG T TAG T GAT AG CCCTGGGAAA TCAGTTGGGG1021 ATGCTTTGGA CAT T GAG AAA TGGAGAGTAG T TAG T GAT AG CCCTGGGAAA TCAGTTGGGG
1081 TGAGCGCAAT TCTATTAGGC TTGATTCTTG TTGGGAGAGC AGCCTTTGTT TTCCCTTTAT
1141 CTTCCATTTC CAACTTGACT AAGAAAGCTC CATGTGACAA AATTGATTTC AAACAGCAAG1081 TGAGCGCAAT TCTATTAGGC TTGATTCTTG TTGGGAGAGC AGCCTTTGTT TTCCCTTTAT 1141 CTTCCATTTC CAACTTGACT AAGAAAGCTC CATGTGACAA AATTGATTTC AAACAGCAAG
1201 TTACCATTTG GTGGGCCGGT CTTATGCGTG GTGCAGTTTC AATGGCACTT GCTTATAATC1201 TTACCATTTG GTGGGCCGGT CTTATGCGTG GTGCAGTTTC AATGGCACTT GCTTATAATC
1261 AGTTTACCAG CTTGGGCCAT ACTCAGTTGC GAGGGAATGC AATGATGATA ACCAGCACAA1261 AGTTTACCAG CTTGGGCCAT ACTCAGTTGC GAGGGAATGC AATGATGATA ACCAGCACAA
1321 TCACAGTTGT ACTTTTCAGC ACTGTGGTTT TTGGCTTGAT GACTAAACCA TTGATTAGAA1321 TCACAGTTGT ACTTTTCAGC ACTGTGGTTT TTGGCTTGAT GACTAAACCA TTGATTAGAA
1381 TCTTGCTTCC TTCACCGAAA AATCGAATGC TCTCTTCCGA ACCGTCTACA CCTAAGTCAC1381 TCTTGCTTCC TTCACCGAAA AATCGAATGC TCTCTTCCGA ACCGTCTACA CCTAAGTCAC
1441 TCACCGTGCC GCTTCTCACC AATGGCCACG ACTTAGAGGC TTACGAAAGC GACCGAAATC1441 TCACCGTGCC GCTTCTCACC AATGGCCACG ACTTAGAGGC TTACGAAAGC GACCGAAATC
1501 TAATAACCCG GCCAACAAGC TTAAGGATGT TCTTGAGCAC TCCTTCCAAC ACCGTGCACT1501 TAATAACCCG GCCAACAAGC TTAAGGATGT TCTTGAGCAC TCCTTCCAAC ACCGTGCACT
1561 ACTATTGGAG AAAATTCGAC AACGCGTTCA TGCGACCTGT ATTCGGAGGA AGGGGTTTCG1561 ACTATTGGAG AAAATTCGAC AACGCGTTCA TGCGACCTGT ATTCGGAGGA AGGGGTTTCG
1621 TACCCTTCGT TCCGGGATCG CCCATCGAAC AAAATGATCA ACAATGGCAG TGAATAATGA1621 TACCCTTCGT TCCGGGATCG CCCATCGAAC AAAATGATCA ACAATGGCAG TGAATAATGA
1681 AG AG AT C AC A GATACCAGGT ACTAAAATAC TACACTAATT TTTATAAACT TGTAGGATAG1681 AG AG AT C AC A GATACCAGGT ACTAAAATAC TACACTAATT TTTATAAACT TGTAGGATAG
1741 TGGAATGAAA ACCGGCTTTT CAGTGTGAAG TTTGCTAATA TATACGTGAG AGTTATGTTA1741 TGGAATGAAA ACCGGCTTTT CAGTGTGAAG TTTGCTAATA TATACGTGAG AGTTATGTTA
1801 CTTGGACTCG GGTTTGGGTT TTAAATATGG GTATATGTCT GACTTGGATA TATTCATAAA1801 CTTGGACTCG GGTTTGGGTT TTAAATATGG GTATATGTCT GACTTGGATA TATTCATAAA
1861 AAAAATTAAA ACAAAATAGA AGGACACAAA AGTGAGAGTT TGGCCGGTTG ATTTGATTTT1861 AAAAATTAAA ACAAAATAGA AGGACACAAA AGTGAGAGTT TGGCCGGTTG ATTTGATTTT
1921 AACAATTTTT TTCCAGCGTT TGCTGTTTAA TGTTTGATTG TATCTTACAT TGAATGCTGC1921 AACAATTTTT TTCCAGCGTT TGCTGTTTAA TGTTTGATTG TATCTTACAT TGAATGCTGC
1981 TTTGGAGTTT TTTTTTCTTT TTTCTTTTTT TTTGTGAGGG TTTTTTTTGT AAAAGGTTTA1981 TTTGGAGTTT TTTTTTCTTT TTTCTTTTTT TTTGTGAGGG TTTTTTTTGT AAAAGGTTTA
2041 ATTTTATTGA TTGTATATCT TTTTATTAAA AAAAAAAAAA AAAAAAAAAA 根据 GhNHXl-1全长 cDNA序列设计一对引物如下: 2041 ATTTTATTGA TTGTATATCT TTTTATTAAA AAAAAAAAAA AAAAAAAAAA A pair of primers were designed based on the full-length cDNA sequence of GhNHXl-1 as follows:
GhNHXl-lF: SEQ ID NO : 9: GhNHXl-lF: SEQ ID NO: 9:
ATGGCAATCG GGATATTAAG CACT ATGGCAATCG GGATATTAAG CACT
GhNHXl-lR: SEQ ID NO : 10: GhNHXl-lR: SEQ ID NO: 10:
TCACTGCCAT TGTTGATCAT TTTG TCACTGCCAT TGTTGATCAT TTTG
通过 SEQ ID NO : 9和 SEQ ID NO : 10来克隆 GhMHl-1全长。 The full length of GhMHl-1 was cloned by SEQ ID NO: 9 and SEQ ID NO: 10.
采用 TaKaRa的 PrimeSTAR HS DNA聚合酶, 以棉花的 cDNA为模板进行 PCR反应。 50 μ 1 PCR反应体系: 10 μ 1 5 X PS Buffer, 3 μ 1 2· 5 mM的 dNTP, 2. 0 μ 1 cDNA, 1. 0 μ 1 PrimeSTAR, 10 μ Μ的引物 SEQ ID NO : 9和 SEQ ID NO : 10各 2· 0 μ 1, 以 及 30 μ ΐ的双蒸水。 PCR反应条件: 94°C预变性 5 min, 94 °C 变性 30 s, 58°C退火 30 s, 72 °C 延伸 2min, 33个循环后, 72°C 延伸 10 min。 The PCR reaction was carried out using TaKaRa's PrimeSTAR HS DNA polymerase and cotton cDNA as a template. 50 μ 1 PCR reaction system: 10 μ 1 5 X PS Buffer, 3 μ 1 2· 5 mM dNTP, 2. 0 μ 1 cDNA, 1. 0 μ 1 PrimeSTAR, 10 μ Μ primer SEQ ID NO: 9 and SEQ ID NO: 10 each of 2·0 μ 1, and 30 μΐ of double distilled water. PCR reaction conditions: pre-denaturation at 94 °C for 5 min, denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 2 min, after 33 cycles, extension at 72 °C for 10 min.
PCR扩增产物加 A尾: PCR产物加 2. 5倍的无水乙醇, _20°C放置 10分钟, 离心, 去上清, 晾干, 用 21 μ ΐ双蒸水溶解。 加入 2. 5 μ 1 lO X Ex Buffer, 0. 5 μ 1 5 mM 的 dATP , 2. 5 μ 1 lO X Ex Taq。 反应条件: 70°C反应 30分钟。 将得到约 1600 bp 的 DNA片段回收(Omega回收试剂盒), 连接至 pGEM T-easy载体上,转化 JM109 (方法
同上), 随机挑取 10个白色菌落于含有 50 μ g/mL氨苄青霉素的 LB 液体培养基中培 养, 37°C培养过夜后加甘油至终浓度 20%, -80°C保存备用。 SEQ ID NO: 9与 SEQ ID NO: 10进行菌液 PCR扩增 (反应体系及反应条件同上), 得到 4个阳性克隆,送至英 潍捷基 (上海) 贸易有限公司测序,序列为 SEQ ID NO: 2。 PCR amplification product plus A tail: PCR product plus 2.5 times absolute ethanol, _20 ° C for 10 minutes, centrifuge, remove the supernatant, air dry, dissolved in 21 μ ΐ double distilled water. Add 2. 5 μ 1 lO X Ex Buffer, 0.5 μl 5 mM dATP, 2. 5 μ 1 lO X Ex Taq. Reaction conditions: The reaction was carried out at 70 ° C for 30 minutes. A DNA fragment of about 1600 bp was recovered (Omega Recovery Kit), ligated into pGEM T-easy vector, and transformed into JM109 (Method Same as above), 10 white colonies were randomly picked and cultured in LB liquid medium containing 50 μg/mL ampicillin, and cultured at 37 ° C overnight, glycerol was added to a final concentration of 20%, and stored at -80 ° C for use. SEQ ID NO: 9 and SEQ ID NO: 10 were used for bacterial liquid PCR amplification (reaction system and reaction conditions are the same as above), and 4 positive clones were obtained and sent to Yingjie Jieji (Shanghai) Trading Co., Ltd. for sequencing. The sequence is SEQ ID. NO: 2.
NHX1- -1蛋白的氨基酸序列: SEQ] Amino acid sequence of NHX1- -1 protein: SEQ]
1 MAIGILSTLL AKSETILGSG 1 MAIGILSTLL AKSETILGSG
21 HSSWSMNLF VALLCGCIVI 21 HSSWSMNLF VALLCGCIVI
41 GHLLEESRWM NESITALAIG 41 GHLLEESRWM NESITALAIG
61 LCTGIV工: LLT TGGKSSHLLV 61 LCTGIV: LLT TGGKSSHLLV
81 FSEDLFFNYL LPP工工 FNAGF 81 FSEDLFFNYL LPP Worker FNAGF
101 QVKKKQFFRN FMTIMLFGAV 101 QVKKKQFFRN FMTIMLFGAV
121 GTLISFAIIS LGAIH TKKM 121 GTLISFAIIS LGAIH TKKM
141 SIGNLKIGDY LAIGAIFSAT 141 SIGNLKIGDY LAIGAIFSAT
161 DSVCTLQVLN QDDTPLLYSL 161 DSVCTLQVLN QDDTPLLYSL
181 VFGEGWNDA 181 VFGEGWNDA
201 SFDLPHINTT 工 ALQFVGNFL 201 SFDLPHINTT work ALQFVGNFL
221 YLFISSTLLG VLAGLLSAYI 221 YLFISSTLLG VLAGLLSAYI
241 工 KKLYFGRHS TDREVALMIL 241 KKLYFGRHS TDREVALMIL
261 MAYLSYMLAE TYLSAILTV 261 MAYLSYMLAE TYLSAILTV
281 TCGIVMSHY TWHNVTESSR 281 TCGIVMSHY TWHNVTESSR
301 VTTKHAFATL SFVAEIFIFL 301 VTTKHAFATL SFVAEIFIFL
321 YVGMDALDIE KWRWSDSPG 321 YVGMDALDIE KWRWSDSPG
341 KSVGVSAILL GLILVGRAAF 341 KSVGVSAILL GLILVGRAAF
361 VFPLSSISNL TKKAPCDKID 361 VFPLSSISNL TKKAPCDKID
381 FKQQVT工丽 A GLMRGAVSMA 381 FKQQVT Gongli A GLMRGAVSMA
401 LAYNQFTSLG HTQLRGNAMM 401 LAYNQFTSLG HTQLRGNAMM
421 ITSTITWLF STWFGLMTK 421 ITSTITWLF STWFGLMTK
441 PLIRILLPSP KNRMLSSEPS 441 PLIRILLPSP KNRMLSSEPS
461 TPKSLTVPLL TNGHDLEAYE 461 TPKSLTVPLL TNGHDLEAYE
481 SDRNLITRPT SLRMFLSTPS 481 SDRNLITRPT SLRMFLSTPS
501 NTVHYYWRKF DNAFMRPVFG 501 NTVHYYWRKF DNAFMRPVFG
521 GRGFVPFVPG SPIEQNDQQW
541 521 GRGFVPFVPG SPIEQNDQQW 541
GhNHXl-1 编码基因的核苷酸序列: SEQ ID NO: 2 Nucleotide sequence of the GhNHXl-1 encoding gene: SEQ ID NO: 2
1 ATGGCAATCG GGATATTAAG CACTCTTTTA GCAAAATCAG AGACGATTTT AGGGTCTGGT 1 ATGGCAATCG GGATATTAAG CACTCTTTTA GCAAAATCAG AGACGATTTT AGGGTCTGGT
61 CACAGCTCAG TAGTTTCAAT GAATTTATTC GTTGCTCTTC TTTGCGGGTG TATCGTGATC 121 GGTCATTTGC TAGAGGAAAG CCGATGGATG AATGAATCCA TTACTGCCCT TGCCATTGGG 181 CTGTGCACTG GAATTGTAAT TTTGCTTACA ACAGGAGGAA AAAGCTCTCA TCTTTTAGTT 241 TTCAGTGAAG ATTTGTTCTT TAATTATTTG CTTCCACCTA TTATTTTCAA TGCAGGATTC 301 CAGGTGAAAA AGAAGCAATT TTTCCGCAAC TTTATGACCA TAATGCTGTT TGGTGCAGTT 361 GGCACTTTAA TATCCTTTGC CATCATATCT CTAGGTGCCA TACATTTTTT CAAGAAAATG 421 AGTATTGGTA ATCTCAAGAT AGGGGATTAT CTTGCCATTG GGGCAATATT TTCAGCAACA 481 GACTCTGTTT GCACTTTGCA AGTGCTTAAT CAGGACGATA CGCCTTTATT GTATAGTCTG 541 GTTTTCGGGG AGGGAGTTGT GAATGATGCC ACAGCTGTTG TTCTTTTCAA AGCAATCCAA 601 AGCTTTGACC TTCCTCACAT CAACACCACC ATTGCTTTGC AATTTGTTGG GAACTTTTTA 661 TATTTATTCA TCTCGAGTAC ATTGCTTGGG GTTTTGGCTG GATTGCTTAG TGCTTACATT 721 ATTAAAAAGC TCTATTTCGG AAGGCACTCA ACGGATCGCG AGGTTGCTCT CATGATACTC 781 ATGGCTTACC TTTCATATAT GCTGGCTGAA TTTTTCTATT TAAGTGCGAT CCTCACTGTG 841 TTCTTTTGTG GGATTGTTAT GTCTCATTAT ACATGGCATA ATGTTACCGA AAGTTCAAGA 901 GTGACAACCA AGCATGCTTT TGCCACACTA TCATTTGTTG CTGAGATTTT CATCTTCCTC 961 TATGTTGGTA TGGATGCTTT GGACATTGAG AAATGGAGAG TAGTTAGTGA TAGCCCTGGG 1021 AAATCAGTTG GGGTGAGCGC AATTCTATTA GGCTTGATTC TTGTTGGGAG AGCAGCCTTT 1081 GTTTTCCCTT TATCTTCCAT TTCCAACTTG ACTAAGAAAG CTCCATGTGA CAAAATTGAT 1141 TTCAAACAGC AAGTTACCAT TTGGTGGGCC GGTCTTATGC GTGGTGCAGT TTCAATGGCA 1201 CTTGCTTATA ATCAGTTTAC CAGCTTGGGC CATACTCAGT TGCGAGGGAA TGCAATGATG 1261 ATAACCAGCA CAATCACAGT TGTACTTTTC AGCACTGTGG TTTTTGGCTT GATGACTAAA 1321 CCATTGATTA GAATCTTGCT TCCTTCACCG AAAAATCGAA TGCTCTCTTC CGAACCGTCT 1381 ACACCTAAGT CACTCACCGT GCCGCTTCTC ACCAATGGCC ACGACTTAGA GGCTTACGAA 1441 AGCGACCGAA ATCTAATAAC CCGGCCAACA AGCTTAAGGA TGTTCTTGAG CACTCCTTCC 1501 AACACCGTGC ACTACTATTG GAGAAAATTC GACAACGCGT TCATGCGACC TGTATTCGGA 1561 GGAAGGGGTT TCGTACCCTT CGTTCCGGGA TCGCCCATCG AACAAAATGA TCAACAATGG 1621 CAGTGA 61 CACAGCTCAG TAGTTTCAAT GAATTTATTC GTTGCTCTTC TTTGCGGGTG TATCGTGATC 121 GGTCATTTGC TAGAGGAAAG CCGATGGATG AATGAATCCA TTACTGCCCT TGCCATTGGG 181 CTGTGCACTG GAATTGTAAT TTTGCTTACA ACAGGAGGAA AAAGCTCTCA TCTTTTAGTT 241 TTCAGTGAAG ATTTGTTCTT TAATTATTTG CTTCCACCTA TTATTTTCAA TGCAGGATTC 301 CAGGTGAAAA AGAAGCAATT TTTCCGCAAC TTTATGACCA TAATGCTGTT TGGTGCAGTT 361 GGCACTTTAA TATCCTTTGC CATCATATCT CTAGGTGCCA TACATTTTTT CAAGAAAATG 421 AGTATTGGTA ATCTCAAGAT AGGGGATTAT CTTGCCATTG GGGCAATATT TTCAGCAACA 481 GACTCTGTTT GCACTTTGCA AGTGCTTAAT CAGGACGATA CGCCTTTATT GTATAGTCTG 541 GTTTTCGGGG AGGGAGTTGT GAATGATGCC ACAGCTGTTG TTCTTTTCAA AGCAATCCAA 601 AGCTTTGACC TTCCTCACAT CAACACCACC ATTGCTTTGC AATTTGTTGG GAACTTTTTA 661 TATTTATTCA TCTCGAGTAC ATTGCTTGGG GTTTTGGCTG GATTGCTTAG TGCTTACATT 721 ATTAAAAAGC TCTATTTCGG AAGGCACTCA ACGGATCGCG AGGTTGCTCT CATGATACTC 781 ATGGCTTACC TTTCATATAT GCTGGCTGAA TTTTTCTATT TAAGTGCGAT CCTCACTGTG 841 TTCTTTTGTG GGATTGTTAT GTCTCATTAT ACATGGCATA ATGTTACCGA AAGTTCAAGA 901 GTGACAACCA AGCATG CTTT TGCCACACTA TCATTTGTTG CTGAGATTTT CATCTTCCTC 961 TATGTTGGTA TGGATGCTTT GGACATTGAG AAATGGAGAG TAGTTAGTGA TAGCCCTGGG 1021 AAATCAGTTG GGGTGAGCGC AATTCTATTA GGCTTGATTC TTGTTGGGAG AGCAGCCTTT 1081 GTTTTCCCTT TATCTTCCAT TTCCAACTTG ACTAAGAAAG CTCCATGTGA CAAAATTGAT 1141 TTCAAACAGC AAGTTACCAT TTGGTGGGCC GGTCTTATGC GTGGTGCAGT TTCAATGGCA 1201 CTTGCTTATA ATCAGTTTAC CAGCTTGGGC CATACTCAGT TGCGAGGGAA TGCAATGATG 1261 ATAACCAGCA CAATCACAGT TGTACTTTTC AGCACTGTGG TTTTTGGCTT GATGACTAAA 1321 CCATTGATTA GAATCTTGCT TCCTTCACCG AAAAATCGAA TGCTCTCTTC CGAACCGTCT 1381 ACACCTAAGT CACTCACCGT GCCGCTTCTC ACCAATGGCC ACGACTTAGA GGCTTACGAA 1441 AGCGACCGAA ATCTAATAAC CCGGCCAACA AGCTTAAGGA TGTTCTTGAG CACTCCTTCC 1501 AACACCGTGC ACTACTATTG GAGAAAATTC GACAACGCGT TCATGCGACC TGTATTCGGA 1561 GGAAGGGGTT TCGTACCCTT CGTTCCGGGA TCGCCCATCG AACAAAATGA TCAACAATGG 1621 CAGTGA
实施例 3 GhNHXl-1基因植物表达载体构建 Example 3 Construction of GhNHXl-1 Gene Plant Expression Vector
选择植物双元表达载体 PCAMBIA2300 (购自北京鼎国昌盛生物技术有限责任公
司) 作为植物表达载体, 用 Pnos启动子替换 ΝΡΤΠ基因含双增强子的 35S启动子, 以降低 ΝΡΤΠ蛋白在植物中的表达。选择诱导型启动子 rd29A及 Tnos作为 GhNHXl_l 基因的启动子和终止子。 Select plant binary expression vector PCAMBIA2300 (purchased from Beijing Dingguo Changsheng Biotechnology Co., Ltd. As a plant expression vector, the 35S promoter of the ΝΡΤΠ gene containing the double enhancer was replaced with the Pnos promoter to reduce the expression of prion protein in plants. The inducible promoters rd29A and Tnos were selected as promoters and terminators of the GhNHXl_l gene.
用引物 SEQ ID NO: 11和 SEQ ID NO: 12以植物表达载体 PBI121 (购自北京华 夏远洋科技有限公司) 为模板扩增 Pnos, 采用 TaKaRa的 PrimeSTAR HS DNA聚合酶。 50 μ 1 PCR反应体系: 10 μ 15XPS Buffer, 3 μ 12· 5 mM的 dNTP, 1.0 μ 1 PBI121, 1.0 μ 1 PrimeSTAR, 10 μΜ的引物 SEQ ID NO: 11和 SEQ ID NO: 12各 2.0 μ 1, 以及 31 μΐ的双蒸水。 PCR反应条件: 94°C预变性 5 min, 94 °C 变性 30 s, 56°C退 火 30 s, 72 °C 延伸 30 s, 33个循环后, 72°C 延伸 10 min。 通过 EcoRI、 Bglll酶 切连接到 pCAMBIA2300 (promega, T4 连接酶盒)获得 pCAMBIA2300_l。 Pnos were amplified using the primers SEQ ID NO: 11 and SEQ ID NO: 12 with the plant expression vector PBI121 (purchased from Beijing Huaxia Ocean Technology Co., Ltd.) using TaKaRa's PrimeSTAR HS DNA polymerase. 50 μ 1 PCR reaction system: 10 μ 15×PS Buffer, 3 μ 12· 5 mM dNTP, 1.0 μ 1 PBI121, 1.0 μ 1 PrimeSTAR, 10 μΜ primers SEQ ID NO: 11 and SEQ ID NO: 12 each 2.0 μ 1 , and 31 μΐ of double distilled water. PCR reaction conditions: pre-denaturation at 94 °C for 5 min, denaturation at 94 °C for 30 s, annealing at 56 °C for 30 s, extension at 72 °C for 30 s, after 33 cycles, extension at 72 °C for 10 min. pCAMBIA2300_l was obtained by EcoRI and Bglll digestion with pCAMBIA2300 (promega, T4 ligase cassette).
SEQ ID NO: 11 : SEQ ID NO: 11:
GCACGAATTCATACAAATGGACGAACGGAT SEQ ID NO: 12: GCACGAATTCATACAAATGGACGAACGGAT SEQ ID NO: 12:
ATCCAGATCTAGATCCGGTGCAGATTATTTG SEQ ID NO: 13和 SEQ ID NO: 14以 PBI121为模板扩增 Tnos, 采用 TaKaRa的 ATCCAGATCTAGATCCGGTGCAGATTATTTG SEQ ID NO: 13 and SEQ ID NO: 14 amplify Tnos with PBI121 as a template, using TaKaRa
PrimeSTAR HS DNA聚合酶。 50 μ 1 PCR反应体系: 10 μ 15XPS Buffer, 3 μ 12.5 mM的 dNTP, 1.0 μ 1 PBI121, 1.0 μ 1 PrimeSTAR、 10 μ Μ的引物 SEQ ID NO: 13 和 SEQ ID NO: 14各 2.0 μ 1, 以及 31 μ 1的双蒸水。 PCR反应条件: 94°C预变性 5 min, 94 °C 变性 30 s, 58°C退火 30 s, 72 °C 延伸 30 s, 33个循环后, 72°C 延伸 10 mine 通过 Sacl、 EcoRI酶切连接到 pCAMBIA2300-l (promega T4 连接酶盒)获得 PCAMBIA2300-2 PrimeSTAR HS DNA polymerase. 50 μ 1 PCR reaction system: 10 μ 15×PS Buffer, 3 μ 12.5 mM dNTP, 1.0 μ 1 PBI121, 1.0 μ 1 PrimeSTAR, 10 μ Μ primers SEQ ID NO: 13 and SEQ ID NO: 14 each 2.0 μ 1, And 31 μl of double distilled water. PCR reaction conditions: pre-denaturation at 94 °C for 5 min, denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 30 s, after 33 cycles, extension at 72 °C for 10 min by Sacl, EcoRI digestion Connect to pCAMBIA2300-l (promega T4 ligase cassette) to obtain PCAMBIA2300-2
SEQ ID NO: 13: SEQ ID NO: 13:
AAG dOTAATTTCCCCGATCGTTCAAA AAG dOTAATTTCCCCGATCGTTCAAA
SEQ ID NO: 14: SEQ ID NO: 14:
TCA GAA M AGTGAATTCCCGATCTAGTA TCA GAA M AGTGAATTCCCGATCTAGTA
SEQ ID NO: 15 和 SEQ ID NO: 16 以拟南芥 (哥伦比亚型 , 购自 www. arabidopsis. org)DNA为模板扩增拟南芥 rd29A启动子(参考 Zeng J. , et L. 2002, Preparation of total DNA from "recalcit rant plant taxa" , Acta Bot. Sin. , 44(6): 694-697 中的方法提取拟南芥 DNA)。 采用 TaKaRa的 PrimeSTAR HS DNA聚合 酶。 50 μ 1 PCR反应体系: 10 μΐ 5XPS Buffer, 3 μ 1 2.5 mM的 dNTP, 1.0 μ 1 拟南芥 DNA, 1.0 μ 1 PrimeSTAR, 10 μΜ的引物 SEQ ID NO: 15和 SEQ ID NO: 16 各 2.0 μ1, 以及 31 μΐ的双蒸水。 PCR反应条件: 94°C预变性 5 min, 94 °C 变性
30 s,58°C退火 30 s,72°C 延伸 30 s, 33个循环后, 72 °C 延伸 10 min。通过 HindIII、 Pstl酶切连接到 (连接方法同上) pCAMBIA2300-2获得 pCAMBIA2300-3 SEQ ID NO: 15 and SEQ ID NO: 16 The Arabidopsis thaliana rd29A promoter was amplified using Arabidopsis thaliana (Columbia type, available from www. arabidopsis.org) as a template (see Zeng J., et L. 2002, Preparation). Of total DNA from "recalcit rant plant taxa", Acta Bot. Sin., 44(6): Method 694-697 for extracting Arabidopsis DNA). PrimeSTAR HS DNA polymerase using TaKaRa. 50 μ 1 PCR reaction system: 10 μΐ 5XPS Buffer, 3 μ 1 2.5 mM dNTP, 1.0 μ 1 Arabidopsis DNA, 1.0 μ 1 PrimeSTAR, 10 μΜ primers SEQ ID NO: 15 and SEQ ID NO: 16 2.0 11, and 31 μΐ of double distilled water. PCR reaction conditions: predenaturation at 94 °C for 5 min, denaturation at 94 °C 30 s, annealed at 58 °C for 30 s, extended at 72 °C for 30 s, after 33 cycles, extended at 72 °C for 10 min. The pCAMBIA2300-3 was obtained by digesting with HindIII and Pstl (connection method is the same as above) pCAMBIA2300-2.
SEQ ID NO : 15: SEQ ID NO: 15:
ACTAAGCTTCCTTCTTGACATCATTCAATTTTA SEQ ID NO : 16: ACTAAGCTTCCTTCTTGACATCATTCAATTTTA SEQ ID NO : 16:
TGAO^ CTCCAAAGATTTTTTTCTTTCCAATAG TGAO^ CTCCAAAGATTTTTTTCTTTCCAATAG
SEQ ID NO : 17和 SEQ ID NO : 18扩增 GhNHXl_l(模板是实施例 2所获得 GhNHXl_l ), 采用 TaKaRa的 PrimeSTAR HS DNA聚合酶。50 μ 1 PCR反应体系: 10 μ 1 5 X PS Buffer, 3 μ 1 2. 5 mM的 dNTP, 1. 0 μ 1 GhNHXl- 1- pGEM, 1. 0 μ 1 PrimeSTAR, 10 μ Μ的引 物 SEQ ID NO : 17和 SEQ ID NO : 18各 2. 0 μ 1, 以及 31 μ 1的双蒸水。 PCR反应条 件: 94°C预变性 5 min, 94 °C 变性 30 s, 58°C退火 30 s, 72 °C 延伸 2min, 33个 循环后, 72 °C 延伸 10 min。 通过 Pstl、 Sad 酶切连接到 (连接方法同上) PCAMBIA2300-3, 获得植物表达载体 rd29A_ GhNHXl-l_2300。 SEQ ID NO: 17 and SEQ ID NO: 18 amplify GhNHX1-1 (template is GhNHXl-1 obtained in Example 2) using TaKaRa's PrimeSTAR HS DNA polymerase. 50 μ 1 PCR reaction system: 10 μ 1 5 X PS Buffer, 3 μ 1 2. 5 mM dNTP, 1. 0 μ 1 GhNHXl- 1- pGEM, 1. 0 μ 1 PrimeSTAR, 10 μ Μ primer SEQ ID NO: 17 and SEQ ID NO: 18 each of 2.0 μl, and 31 μl of double distilled water. PCR reaction conditions: pre-denaturation at 94 °C for 5 min, denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 2 min, after 33 cycles, extension at 72 °C for 10 min. The plant expression vector rd29A_GhNHXl-l_2300 was obtained by restriction enzyme digestion with Pstl and Sad (connection method as above) PCAMBIA2300-3.
SEQ ID NO : 17: SEQ ID NO: 17:
TGAC 7¾ i7ATGGCAATCGGGATATTAAGCACT TGAC 73⁄4 i7ATGGCAATCGGGATATTAAGCACT
SEQ ID NO : 18: SEQ ID NO: 18:
AAGGUCTCACTGCCATTGTTGATCATTTTG 实施例 4 rd29A-GhNHXl-l-2300表达载体转化农杆菌 AAGGUCTCACTGCCATTGTTGATCATTTTG Example 4 rd29A-GhNHXl-l-2300 Expression Vector Transformation Agrobacterium
农杆菌 LBA4404 (购自 Biovector Science Lab, Inc) 感受态制备: 提前 1-2天 将农杆菌 LBA4404在含 50 μ g/ml利福平和 50 μ g/ml链霉素的 LB固体培养基上划 单斑接种, 28 °C培养 1至 2天。挑取单菌落接种于 5 ml含 50 μ g/ml利福平和 50 μ g/ml 链霉素的 LB液体培养基中, 28°C下摇动培养过夜(约 12-16 h)至 0D600值为 0. 4, 形 成种子菌液。 取 5 ml活化后的菌液 (1 : 20的比例) 接种于 100 ml同样浓度抗生素 的 LB液体培养基中, 28°C摇动培养 2-2. 5 h至 0D6。。=0. 8。冰浴菌液 10 min,每隔 3 min 摇匀一次, 令细菌均匀进入休眠状态。 于 4°C下 4000 g离心 10 min, 弃上清液; 加 入一定量预冷 10%甘油重悬浮菌体, 4°C下 4000 g离心 10 min, 收集沉淀; 用 10%甘 油重复洗 3-4次; 加入适量冰浴预冷的 10%甘油重新悬浮细菌沉淀, 以 40 μ 1/管将 其分装, 于 -70°C保存备用。 Agrobacterium LBA4404 (available from Biovector Science Lab, Inc) Competent preparation: Agrobacterium LBA4404 was plated on LB solid medium containing 50 μg/ml rifampicin and 50 μg/ml streptomycin 1-2 days in advance Single spot inoculation, culture at 28 °C for 1 to 2 days. Single colonies were picked and inoculated into 5 ml of LB liquid medium containing 50 μg/ml rifampicin and 50 μg/ml streptomycin, and cultured overnight at 28 ° C (about 12-16 h) to 0D600 value. 0. 4, forming a seed bacterial liquid. 5小时至0D 6。 Take 5 ml of activated bacterial solution (1: 20 ratio) inoculated in 100 ml of the same concentration of antibiotics in LB liquid medium, shaking at 2 ° C. 2 h. . =0. 8. The ice bath solution was shaken for 10 min every 3 min to allow the bacteria to enter the dormant state evenly. Centrifuge at 4000 g for 10 min at 4 °C, discard the supernatant; add a certain amount of pre-cooled 10% glycerol to resuspend the cells, centrifuge at 4000 g for 10 min at 4 °C, collect the precipitate; repeat washing with 10% glycerol3- 4 times; add the appropriate amount of ice bath pre-cooled 10% glycerol to resuspend the bacterial pellet, dispense it in 40 μl/tube, and store at -70 °C for later use.
转化农杆菌: 在冰上融化感受态细胞, 往 40 μ 1的感受态细胞中加入 1 μ 1的 质粒, 混匀后冰浴约 10 min o 将感受态和 DNA的混合物用枪转移到预冷的电击杯中, 轻敲使悬浮液到达底部, 注意不要有气泡。 将电击杯 (购自 bio-rad)放到电击室的
滑道上, 推动滑道将电击杯放至电击室基座电极处。 使用 0. lcm 的电击杯的时候, MicroPulser (购自 bio-rad) 的程序设置为 "Agr" , 电击一次 。 立即取出电击杯, 加入 28°C预热的 LB培养基。 快速而轻柔的用枪将细胞打匀。 将悬浮液转入 1.5 ml 的离心管, 28°C, 225 rpm培养 1 h。 取 100〜200 μ 1的菌液涂布与相应的抗性筛 选培养基平板上(LB固体培养基,含 50 μ g/ml利福平、 50 μ g/ml链霉素、 50 μ g/ml 卡那霉素), 28°C培养。 实施例 5 利用农杆菌介导的转化法获得转基因烟草 Transformation of Agrobacterium: Thaw competent cells on ice, add 1 μl of plasmid to 40 μl of competent cells, mix and ice bath for about 10 min o Transfer the mixture of competent and DNA to pre-cool with gun In the electric shock cup, tap to bring the suspension to the bottom, taking care not to have air bubbles. Place the electric shock cup (purchased from bio-rad) in the electric shock room On the slide, push the slide to place the electric shock cup on the base electrode of the shock chamber. When using a 0. lcm electric shock cup, the program of MicroPulser (purchased from bio-rad) is set to "Agr" and the electric shock is once. The electric shock cup was immediately taken out and the pre-warmed LB medium at 28 ° C was added. Quickly and gently spread the cells with a gun. The suspension was transferred to a 1.5 ml centrifuge tube and incubated at 28 ° C, 225 rpm for 1 h. Take 100~200 μl of the bacterial solution and plate on the corresponding resistant screening medium (LB solid medium containing 50 μg/ml rifampicin, 50 μg/ml streptomycin, 50 μg/ Ml Kanamycin), cultured at 28 °C. Example 5 Obtaining Transgenic Tobacco by Agrobacterium-mediated Transformation
用 75%酒精浸泡烟草种子 (国家烟草中期库, 获取单位: 中国农科院烟草所, 库 编号 I5A00660) 30 s, 用灭菌双蒸水洗两次。 再用 0. 1%升汞浸泡 8 min, 用灭菌双 蒸水洗两次, 完成表面灭菌。将表面灭菌的烟草种子置于 MS (18.78 mMKN03, 1.25 mM KH2P04, 20.6 mM NH4NO3, 1.5 mM MgS04, 3.0 mM CaCl2, 50 μ M KI, 100 μΜ H3B03, 100 μ M MnS04 , 30 μ M ZnS04, 1 μ M Na2Mo04, 0. 1 μΜ CoCl2, 100 μ M Na2EDTA, 100 μΜ FeS04, 7.4 g/L琼脂, 蔗糖 30 g/L) 上于无菌条件下发芽, 制备无菌苗。 取无菌苗叶片剪成 5 mmX5 mm 大小的叶盘, 用处于对数生长期的含表达载体 rd29A-GhNHXl-l-2300的农杆菌浸染叶盘 10 min, 吸干菌液, 在黑暗条件下共培养 2 天 (MS 培养基)。 将叶片转到分化培养基 (MS+1 mg/L 细胞分裂素 (BA) +0. 1 mg/L 萘乙酸 (麵 +50 mg/L卡那霉素 +500 mg/L头孢霉素) 上, 光照条件下培养 45天左 右, 待芽长大后切下转移到生根培养基 (MS+50 mg/L卡那霉素 +500 mg/L头孢霉素) 中培养 30天左右, 待根系发达后将小苗转入仅加有 500 mg/L头孢霉素的 MS培养基 上进行编号保存。 To soak tobacco seeds with 75% alcohol (National Tobacco Medium Term Bank, obtained by: Institute of Tobacco, Chinese Academy of Agricultural Sciences, Library No. I5A00660) 30 s, washed twice with sterile double distilled water. Then, it was immersed in 0.1% liter of mercury for 8 min, and washed twice with sterilized double distilled water to complete surface sterilization. Surface-sterilized tobacco seeds were placed in MS (18.78 mM KN0 3 , 1.25 mM KH 2 P0 4 , 20.6 mM NH4NO3, 1.5 mM MgS0 4 , 3.0 mM CaCl 2 , 50 μM KI, 100 μΜ H 3 B0 3 , 100 μ M MnS0 4 , 30 μ M ZnS0 4 , 1 μ M Na 2 Mo0 4 , 0.1 μM CoCl 2 , 100 μM Na 2 EDTA, 100 μΜ FeS0 4 , 7.4 g/L agar, sucrose 30 g/L) Germinate under sterile conditions to prepare sterile seedlings. The leaves of the sterile seedlings were cut into 5 mm×5 mm leaf discs, and the leaf discs were inoculated with Agrobacterium containing the expression vector rd29A-GhNHXl-l-2300 in the logarithmic growth phase for 10 min, and the bacterial liquid was absorbed in the dark condition. Co-culture for 2 days (MS medium). The leaves were transferred to differentiation medium (MS+1 mg/L cytokinin (BA) + 0.1 mg/L naphthaleneacetic acid (face +50 mg/L kanamycin + 500 mg/L cephalosporin) After incubation for about 45 days under light conditions, the buds should be grown and transferred to rooting medium (MS+50 mg/L kanamycin + 500 mg/L cephalosporin) for about 30 days, until the root system is developed. The seedlings were then transferred to MS medium supplemented with 500 mg/L cephalosporin for number storage.
取获得的转基因烟草叶片, 提取 DNA (同实施例 3中拟南芥 DNA提取方法), 用 SEQ ID NO: 9: 和 SEQ ID NO: 10 (50 μ 1 PCR反应体系: 5 μ 1 ΙΟΧΕχ Buffer, 3 μ 1 2· 5 mM的 dNTP, 2.0 μ 1 DNA, 1.0 μ 1 Ex Taq 10 μ M的引物 SEQ ID NO: 9 和 SEQ ID NO: 10各 2.0 μ 1, 以及 35 μ 1的双蒸水。 PCR反应条件: 94°C预变性 5 min, 94 °C 变性 30 s, 58°C退火 30 s, 72V 延伸 2 min, 33个循环后, 72°C 延伸 10 min), PCR鉴定, 保存阳性植株进行编号 T。H1_T。H20。 实施例 6 过表达 GhNHXl-1转基因烟草 T1的耐盐模拟实验及功能鉴定 The obtained transgenic tobacco leaves were extracted, and DNA (the Arabidopsis thaliana DNA extraction method in Example 3) was used, using SEQ ID NO: 9: and SEQ ID NO: 10 (50 μl PCR reaction system: 5 μ 1 ΙΟΧΕχ Buffer, 3 μ 1 2 · 5 mM dNTP, 2.0 μ 1 DNA, 1.0 μ 1 Ex Taq 10 μ M primers SEQ ID NO: 9 and SEQ ID NO: 10 each 2.0 μ 1, and 35 μl of double distilled water. PCR reaction conditions: pre-denaturation at 94 °C for 5 min, denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72V for 2 min, after 33 cycles, extension at 72 °C for 10 min), PCR identification, preservation of positive plants Carry the number T. H1_T. H20. Example 6 Overexpression of GhNHXl-1 transgenic tobacco T1 salt tolerance simulation experiment and functional identification
T。H1-T。H20烟草种子、非转基因对照烟草种子用灭菌双蒸水浸泡 30min,用 75%酒精 浸泡 30 s, 用灭菌双蒸水洗两次。 再用 0. 1%升汞浸泡 8 min, 用灭菌双蒸水洗两次, 完成表面灭菌。 做两组实验: 第一组: 将表面灭菌的 T。H1-T。H20烟草种子、 对照烟草
种子置于 MS固体培养基上于无菌条件下发芽。 第二组: 将表面灭菌的 T。H1-T。H20烟草 种子、对照烟草种子置于添加有 lOOmMNaCl的 MS固体培养基上于无菌条件下发芽。 25 。C、 10小时光培养 /14小时暗培养循环, 10天后观察结果。 第一组试验结果表明, 在 MS固体培养基中 TO代各株系的种子发芽率为 90%, 生长状态良好, 均与对照无明显差 异(图 3); 第二组试验结果发现, 在含 lOOmMNaCl的 MS培养基中, T。H1_T。H20烟草种子 和对照种子均能萌发, 但对照植株生长明显受到抑制。 T1代转基因植株(T0代转基因 植物的种子长成的植株)的耐盐性鉴定表明, Ί Η2、 Ί Η5、 Ί Η7、 Ί Η13、 Ί Η15、、 Ί Η16、 Ί\Η19、 Ί\Η20转基因植株生长整体高于对照植株, 显现出明显的耐盐性(参见图 4, 以 Ί Η2为例, Ί Η5、 Ί Η7、 Ί Η13、 Ί Η15、、 Ί Η16、 Ί Η19、 Ί Η20与 Ί Η2的结果类似, 在此 未示出)。 实施例 7 在转录水平上验证 NHX1-1蛋白表达 T. H1-T. H20 tobacco seeds and non-GM control tobacco seeds were immersed in sterile double distilled water for 30 min, soaked in 75% alcohol for 30 s, and washed twice with sterile double distilled water. Then, it was immersed in 0.1% liter of mercury for 8 min, and washed twice with sterilized double distilled water to complete surface sterilization. Do two sets of experiments: Group 1: T-sterilized surface. H1-T. H20 tobacco seed, control tobacco Seeds were germinated under sterile conditions on MS solid medium. Group 2: T that will be surface sterilized. H1-T. H20 tobacco seeds and control tobacco seeds were germinated under sterile conditions on MS solid medium supplemented with 100 mM NaCl. 25 . C, 10 hours light culture / 14 hours dark culture cycle, the results were observed after 10 days. The results of the first group of experiments showed that the seed germination rate of each line of TO in the MS solid medium was 90%, and the growth state was good, and there was no significant difference between the two groups (Fig. 3). The second group of test results found that lOOmM NaCl in MS medium, T. H1_T. Both H20 tobacco seeds and control seeds were able to germinate, but the growth of control plants was significantly inhibited. Identification of salt tolerance of T1 transgenic plants (plants grown from seeds of T0 transgenic plants) showed that Ί2, Ί Η5, Ί Η7, Ί Η13, Ί Η15, Ί Η16, Ί\Η19, Ί\Η20 transgene The overall growth of the plants was higher than that of the control plants, showing obvious salt tolerance (see Figure 4, taking Ί Η 2 as an example, Ί Η 5, Ί Η 7, Ί Η 13, Ί Η 15, Ί 、 16, Ί Η 19, Ί Η 20 and Ί Η 2 The result is similar, not shown here). Example 7 Validation of NHX1-1 protein expression at the transcriptional level
分别取对照烟草、不耐盐转基因烟草 T1代植株、 耐盐转基因烟草 T1代植株(生 长状况良好) lOOmMNaCl处理 14天叶子 0.05g,用植物 RNA提取试剂盒( invitrogen) 提取的总 RNA。用 HITACHI公司的紫外分光光度计 U-2001测定总 RNA在 260 nm和 280 nm的吸光度值,计算各个 RNA浓度。使用 invitrogen反转录试剂盒 Superscript III Reverse Transcriptase并依照说明书中方法进行反转录 (2 μ g总 R A作为模板,反 转录引物 SEQ ID NO: 10)。 通过 SEQ ID N0:9和 SEQ ID NO: 10扩增 GhNHXl_l, 检测 GhNHXl-1蛋白相对表达情况。 采用 TaKaRa的 PrimeSTAR HS DNA聚合酶, 以反转录 的 cDNA为模板进行 PCR反应。 50 μ 1 PCR反应体系: 10 μ ΐ 5 X PS Buffer, 3 μ 1 2.5 mM的 dNTP, 2.0 μ 1 cDNA, 1.0 μ 1 PrimeSTAR, 10 μΜ的引物 SEQ ID N0:9 和 SEQ ID NO: 10各 2.0 μ 1, 以及 30 μ 1的双蒸水。 PCR反应条件: 94°C预变性 5 min, 94 °C 变性 30 s, 58°C退火 30 s, 72 °C 延伸 lmin, 29个循环后, 72°C 延伸 10 mine 产物电泳结果如图 5所示: M为 DNA Ladder Marker (DL2000,购自深圳瑞真 生物技术有限公司), 1-5为对照烟草, 6-15为耐盐转基因烟草 T1代植株, 16_21为 不耐盐转基因烟草 T1代植株。 图中所示条带大小与 GhNHXl-1基因的大小一致。结果 表明对照烟草没有转录 GhNHXl-1的 mRNA, 耐盐转基因烟草 T1代植株对 GhNHXl-1的 转录较强, 不耐盐转基因烟草 T1代植株没有转录或者转录很弱。
Control tobacco, salt-tolerant transgenic tobacco T1 plants, salt-tolerant transgenic tobacco T1 plants (good growth condition) lOOmM NaCl treatment 14 days leaf 0.05g, total RNA extracted by plant RNA extraction kit (invitrogen). The absorbance values of total RNA at 260 nm and 280 nm were measured using a HITACHI UV spectrophotometer U-2001 to calculate the individual RNA concentrations. Reverse transcription was performed using the invitrogen reverse transcription kit Superscript III Reverse Transcriptase (2 μg total R A as a template, reverse transcription primer SEQ ID NO: 10) according to the method described in the specification. The relative expression of GhNHXl-1 protein was detected by amplifying GhNHXl-1 by SEQ ID NO: 9 and SEQ ID NO: 10. PCR was performed using TaKaRa's PrimeSTAR HS DNA polymerase with reverse transcribed cDNA as a template. 50 μ 1 PCR reaction system: 10 μ ΐ 5 X PS Buffer, 3 μ 1 2.5 mM dNTP, 2.0 μ 1 cDNA, 1.0 μ 1 PrimeSTAR, 10 μΜ primers SEQ ID N0:9 and SEQ ID NO: 10 each 2.0 μ 1, and 30 μ 1 of double distilled water. PCR reaction conditions: pre-denaturation at 94 °C for 5 min, denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 1 min, after 29 cycles, extension at 72 °C for 10 min. M: DNA Ladder Marker (DL2000, purchased from Shenzhen Ruizhen Biotechnology Co., Ltd.), 1-5 for control tobacco, 6-15 for salt-tolerant transgenic tobacco T1 plants, 16_21 for salt-tolerant transgenic tobacco T1 plants . The size of the band shown in the figure is consistent with the size of the GhNHXl-1 gene. The results showed that the control tobacco did not transcribe the mRNA of GhNHXl-1, and the T1 generation of the salt-tolerant transgenic tobacco had stronger transcription of GhNHXl-1, and the T1 generation of the salt-tolerant transgenic tobacco had no transcription or weak transcription.
Claims
1. 棉花的一个离子通道类蛋白编码基因, 其序列为 SEQ ID NO: 2。 An cotton ion channel protein-encoding gene having the sequence of SEQ ID NO: 2.
2. 一种重组表达载体, 其含有权利要求 1所述的基因并且所述基因的核苷酸序 列与所述表达载体的表达控制序列可操作地连接。 2. A recombinant expression vector comprising the gene of claim 1 and the nucleotide sequence of the gene operably linked to an expression control sequence of the expression vector.
3. 权利要求 2所述的载体, 其为附图 2所示的 rd29A-GhNHXl-l-2300载体。 3. The vector of claim 2 which is the rd29A-GhNHX1-1-2300 carrier shown in Fig. 2.
4. 一种重组细胞, 其含有权利要求 1所述的基因或者权利要求 2或 3所述的重 组表达载体; 优选地, 所述重组细胞为重组农杆菌细胞。 A recombinant cell comprising the gene of claim 1 or the recombinant expression vector of claim 2 or 3; preferably, the recombinant cell is a recombinant Agrobacterium cell.
5. 一种改善植物耐盐性的方法,包括: 将权利要求 1所述的基因或者权利要求 2 或 3所述的重组表达载体导入植物或植物组织并使所述基因表达; 优选地,所述植物 是烟草。 A method for improving salt tolerance of a plant, comprising: introducing the gene of claim 1 or the recombinant expression vector of claim 2 or 3 into a plant or plant tissue and expressing the gene; preferably, The plant is tobacco.
6. 一种制备转基因植物的方法, 包括: 在有效产生植物的条件下培养含有权利 要求 1所述的基因或者权利要求 2或 3所述的重组表达载体的植物或植物组织。 A method for producing a transgenic plant, comprising: cultivating a plant or plant tissue comprising the gene of claim 1 or the recombinant expression vector of claim 2 or 3 under conditions effective to produce a plant.
7. 权利要求 6所述的方法, 其中所述植物是烟草。 7. The method of claim 6 wherein the plant is tobacco.
8. 权利要求 1所述的基因、权利要求 2或 3所述的重组表达载体或者权利要求 4 所述的重组细胞用于改善植物耐盐性以及用于植物育种的用途。 The gene of claim 1, the recombinant expression vector of claim 2 or 3, or the recombinant cell of claim 4 for use in improving plant salt tolerance and for use in plant breeding.
9. 权利要求 8所述的用途, 其中所述植物是烟草。 9. The use of claim 8 wherein the plant is tobacco.
10. 权利要求 1所述的基因编码的蛋白, 其序列如 SEQ ID NO: 1所示。
The gene-encoded protein according to claim 1, which has the sequence shown in SEQ ID NO: 1.
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WO2000052137A2 (en) * | 1999-03-02 | 2000-09-08 | Emerald Bioagriculture Corporation | Plant ligand-gated ion channels |
WO2001061006A2 (en) * | 2000-02-15 | 2001-08-23 | Wyeth | Two pore potassium channels, nucleotide sequences encoding them, and methods of using same |
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WO2001061006A2 (en) * | 2000-02-15 | 2001-08-23 | Wyeth | Two pore potassium channels, nucleotide sequences encoding them, and methods of using same |
Non-Patent Citations (2)
Title |
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HE, YIMIN ET AL.: "Genetic Engineering Progresses in Plant Resistance to Salt Stress", CHINA BIOTECHNOLOGY, vol. 29, no. 3, 31 December 2009 (2009-12-31), pages 100 - 104 * |
SONG, WEIYI ET AL.: "Advances in the Study of the Relationship between Calcium Channel Proteins and Plant Salt and Cold Resistance", ACTA BOTANICA BOREALI-OCCIDENTALIA SINICA, vol. 30, no. 11, 31 December 2010 (2010-12-31), pages 2351 - 2357 * |
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