CN110791506B - Nitcipk 11 gene of tangut bur, and expression protein and application thereof - Google Patents

Abstract

The invention discloses a Nitcipk 11 gene and an expression protein and application thereof, belonging to the technical field of plant genetic engineering. The nucleotide sequence of the CIPK11 gene of Tangut cibotium leucotrichum disclosed by the invention is shown as SEQ ID NO.1, and the amino acid sequence of the expressed protein is shown as SEQ ID NO. 2. According to the salt-tolerant, drought-resistant and other stress-resistant characteristics of Nitraria tangutorum bobr, the full length of CIPK gene related to Nitraria tangutorum bobr stress resistance is cloned in a homologous manner by utilizing the leaf tissue of Nitraria tangutorum bobr on the basis of the existing partial transcriptome data, and the gene is named as NtCIPK11 according to the homologous gene in Arabidopsis. Salt tolerance and drought resistance analysis of homozygous NtCIPK11 gene Arabidopsis plants prove that the NtCIPK11 gene transformed plants of the Tanggute Nitraria have salt tolerance and drought resistance, and resources are increased for a plant stress resistance gene bank.

Description

Nitcipk 11 gene of tangut bur, and expression protein and application thereof

Technical Field

The invention belongs to the technical field of plant genetic engineering, and particularly relates to an NtCIPK11 gene of Tanggute nitraria tangutorum bobr, and an expression protein and application thereof.

Background

Tangut white thorn (Nitraria tangutorum) belongs to the genus Nitraria (Zygophylaceae) and is a deciduous shrub. The Nitraria tangutorum bobr has the characteristics of light preference, cold resistance, drought resistance, saline-alkali resistance, barren resistance, low land standing index and the like, and can be used for improving saline-alkali soil, improving soil fertility, preventing wind and fixing sand, maintaining oasis and protecting ecological balance. In addition, researches show that the Nitraria tangutorum bobr is rich in amino acids, vitamins, flavones, saponins, mineral elements and other components, and has high economic value and medicinal value. The environment of soil salinization and drought seriously threatens the water transportation of plants due to environmental deterioration, thereby resulting in the decrease of plant productivity and the increase of desertification. Nitraria tangutorum bobr has gradually attracted attention as a drought-tolerant, salt-tolerant plant. At present, the research on the Nitraria tangutorum bobr is mainly focused on the aspects of drought resistance, salt tolerance, physiological and biochemical properties, fruit nutrient component analysis, breeding and the like, provides a large amount of data support for the molecular mechanism research of the Nitraria tangutorum bobr, and lays a foundation for the application of resistance genes and other functional genes.

CIPKs (CBL-interacting protein kinases) are a class of proteins that interact with calcineurin-like B subunit proteins (CBLs) and are capable of transmitting Ca²⁺A protein of a signal. Such proteins contain a conserved SNF kinase domain at the N-terminus and a NAF domain at the C-terminus. There are 26 CIPK-like homeobox genes in the model plant Arabidopsis, 30 CIPK-like homeobox genes in rice, and 27 CIPK-like homeobox genes in poplar. In addition, since no analogous gene has been found in species other than plants so far, CIPK gene is Ca specific to plants²⁺Ser/Thr phosphoprotein kinase gene in the signal path. The overexpression of OsCIPK12 can improve the cold and drought resistance of rice by accumulating the content of proline and soluble sugarAnd tolerance to salt stress. The CaCPK 6 in the chickpeas mediates auxin transport and regulates the salt tolerance of tobacco seedlings. Overexpression of BrCIPK1 enhances abiotic stress tolerance by increasing the biosynthesis of proline in rice. In addition, active oxygen scavengers, such as peroxidases, are regulated by CIPKs to improve anti-stress capabilities. In addition, CIPKs have also been found to mediate responses of arabidopsis thaliana to salt stress conditions by regulating ion and water homeostasis. A plurality of researches show that the CIPK gene special for plants plays an important role in the stress-resistant processes of salt resistance, low potassium resistance, cold resistance, drought resistance and the like of the plants.

Disclosure of Invention

In view of the above problems in the prior art, the present invention provides a NitCIPK 11 gene. The invention also provides application of the NitCIPK 11 gene to improving the salt tolerance or drought resistance of plants.

In order to solve the technical problems, the technical scheme adopted by the invention is as follows:

the NitCIPK 11 gene of Tanggute Nitraria is disclosed, and the nucleotide sequence is shown in SEQ ID NO. 1.

The amino acid sequence of the expression protein of the Nitcipk 11 gene of Tangut white spine is shown in SEQ ID NO. 2.

A vector, recombinant bacterium or host cell comprising the NitCIPK 11 gene of Nitraria tangutorum bobr according to claim 1.

Preferably, the vector of the NitCIPK 11 gene of the Nitraria tangutorum bobr is a plant recombinant expression vector.

Preferably, the plant recombinant expression vector is pBI121+ NtCIPK 11.

Preferably, the promoter of the gene pBI121+ NtCIPK11 and NtCIPK11 is 35S.

The application of the NitCIPK 11 gene of Tangut Nitraria tangutorum bobr in improving the salt tolerance or drought resistance of plants.

Preferably, the application of the Nitcipk 11 gene in the Tangut Nitraria tangutorum bobr in improving the salt tolerance or drought resistance of plants comprises the following steps:

1) constructing a recombinant vector of NitCIPK 11 gene of Tangut Nitraria tangutorum bobr;

2) transforming the constructed recombinant vector of the NitCIPK 11 gene of the Nitraria tangutorum bobr into plant cells;

3) and cultivating and screening to obtain plants with salt tolerance or drought resistance.

Has the advantages that: compared with the prior art, the invention has the advantages that:

the invention clones the full length of CIPK gene related to Tangut white spine resistance to stress in a homologous way on the basis of the existing partial transcriptome data, is named as NtCIPK11 according to the homologous gene in Arabidopsis, and has the nucleotide sequence shown as SEQ ID NO.1 and the amino acid sequence of the expression protein shown as SEQ ID NO. 2. In a normal culture environment, the growth and development of Arabidopsis T3 generation homozygous plants with the NitCIPK 11 gene overexpressed in Tangut white spine have no obvious difference with wild type. However, salt tolerance and drought resistance analysis of a homozygous NtCIPK11 gene Arabidopsis plant shows that under salt stress and drought stress, an over-expressed NtCIPK11 plant has higher germination rate, longer roots, more leaves and more roots than a WT plant in the same period, and proves that the NtCIPK11 gene transformed plant has good salt tolerance and drought resistance, and the NtCIPK11 gene discloses that resources are added to a plant stress resistance gene bank, so that the method has important significance and value for research on improving the salt tolerance and the cold resistance of the plant.

Drawings

FIG. 1 is a diagram of an expression vector of the NtCIPK11 gene;

FIG. 2 is a graph of the results of qPCR analysis of the Arabidopsis thaliana NtCIPK11 gene;

FIG. 3 is a graph of seed germination for WT and three transgenic lines overexpressing NtCIPK11 (OX-1, OX-2, and OX-3);

FIG. 4 is a graph of germination rates of WT and transgenic plant seeds five days after illumination;

FIG. 5 is a graph of root length on salt medium at different concentrations 20 days after germination of WT and transgenic plants;

FIG. 6 is a graph of leaf number on various concentrations of salt medium after WT and transgenic plants germinated for 20 days;

FIG. 7 is a graph of root mass on various concentrations of salt medium 20 days after germination of WT and transgenic plants;

FIG. 8 is a picture of seed germination for NtCIPK11 and WT under different mannitol treatments;

FIG. 9 is a graph of the percentage of seedlings with two cotyledons after germination of WT and transgenic plants;

FIG. 10 is a phenotype plot of WT and over-expressing NtCIPK11 plants cultured on media of different mannitol concentrations for 20 days;

FIG. 11 is a graph of primary root length of WT and transgenic plants treated with different concentrations of mannitol;

FIG. 12 is a graph of changes in free proline content in WT and transgenic plants under drought stress.

Detailed Description

The invention is further described with reference to specific examples.

Example 1:

the Nitraria tangutorum seeds were vernalized in a relative water content of 7% at4 ℃ for 4 weeks. After vernalization, seeds are placed in a pot with the ratio of soil to sand being 1: 1 and germinate in an environment with the humidity of 55-60%, the temperature of 26-28 ℃ and the temperature of 16-h-light/8-h-dark. Two month old seedlings were irrigated with 500mM NaCl and 200mM mannitol. Treating for 2 hr, freezing the leaf of Nitraria tangutorum bobr in liquid nitrogen, storing at-80 deg.C, extracting RNA, inverting to cDNA, designing corresponding primer for PCR, agarose gel electrophoresis, recovering target band, connecting with pMD19-T vector, transferring to colibacillus, sequencing and analyzing. After the target sequence is determined, RACE primers are designed according to the sequence, 5 'and 3' sequences are obtained through PCR, and after sequencing analysis, the full-length sequence of NtCIPK11 is obtained through splicing. Designing a primer according to a full-length sequence, obtaining a full-length fragment by PCR, connecting the full-length fragment with a pMD19-T vector, transferring into escherichia coli, sequencing and analyzing again, after determining the full-length fragment, selecting a positive clone to perform plasmid extraction, adding an enzyme cutting site, performing double enzyme cutting with a vector pBI121 at the same time, connecting under the action of T4 ligase, transferring into agrobacterium tumefaciens EHA105 and GV3101, after the arabidopsis thaliana is suitable for age, performing transformation by a flower organ soaking transformation method to obtain seeds of T1 generation and T2 generation, screening homozygous T3 generation, performing salt treatment, observing phenotype and analyzing salt tolerance.

(1) Extraction of Total RNA

The RNA extraction was carried out by following the procedures of the NORGEN kit (Norgen Biotek) on the leaves of Nitraria tangutorum collected beforehand. The 1% agarose gel electrophoresis result of the total RNA of the Nitraria tangutorum leaves is clear; determination of the Absorbance, OD, of Total RNA₂₆₀/OD₂₈₀The value is 2.13, OD₂₆₀/OD₂₃₀At 2.05, the RNA quality was found to be better.

The specific process of RNA extraction is as follows:

1) add 800. mu.L of lysine Solution grind.

2) After homogenization the lysate was transferred to a new tube.

3) Mix well for 2min to lyse completely, centrifuge at 12000rpm for 2min, and transfer the supernatant to a new tube.

4) Add equal volume of 70% ethanol, vortex and mix well.

5) The mixture was transferred to a column (2 mL collection tube below), centrifuged for 1min, the filtrate was decanted, and the collection tube was replaced.

6) Adding 400uL of Wash Solution, centrifuging for 1min, discarding the filtrate, and returning to the collecting pipe.

7) Adding DNA I working solution, centrifuging at 12000rpm for 1min, sucking the filtrate back to the column, and standing at 25-30 deg.C for 15 min.

8) Adding 400 μ L of Wash Solution, centrifuging at 12000rpm for 1min, and discarding the filtrate.

9) Adding 400 μ L of Wash Solution for the third time, centrifuging at 12000rpm for 1min, and discarding the filtrate.

10) The column was returned to the collection tube, centrifuged at 12000rpm for 2min, and the tube was discarded.

11) The column was placed in a 1.7mL tube and 50. mu.L of the Elution Solution was added.

12) Centrifuging at 200-2000 rpm for 2min, at 12000rpm for 1min, with the volume less than 50 μ L, and centrifuging at 14000rpm for 1 min.

(2) Obtaining of cDNA

cDNA was obtained by reverse transcription using the RNA as a template, and Invitrogen corporation was used

III First-Strand Synthesis Kit. The amount of RNA used in the experiment was 1. mu.g, and the specific procedure was as follows:

1) the reaction solution is prepared in the following sequence, and after short-time low-speed centrifugation, the mixture is immediately placed on ice for 1-2 mm at 65 ℃ for 5 min.

Reaction solution: mu.L of RNA (. ltoreq.5. mu.g), 1. mu.L of Primer (Oligo dT), 1. mu.L of 10mM dNTPmix, DEPC-Treated Water make up to 10. mu.L.

2) The corresponding reagents are added into the tube in the previous step according to the following sequence and are placed on a PCR instrument, and the reaction program is 50min at 50 ℃ and 5min at 85 ℃. The order of addition of reagents: 2 μ L of 10 × RT Buffer, 4 μ L of 25mM MgCl₂，2μL 0.1M DTT，1μL RNaseOUT(40U/μl)，1μL SuperScript III RT。

3) The reaction solution from the previous step was centrifuged, and 1. mu.L of RNase H was added to each tube at 37 ℃ for 20 min.

(3) Homologous cloning of a Gene of interest

According to partial transcriptome data of tangut nitraria, carrying out conservative specific sequence analysis by NCBI Blast, designing a primer by utilizing Oligo7, cloning a NtCIPK11 gene fragment, and then carrying out ligation transformation sequencing and sequence analysis to determine the gene as a target gene. Cloning primers, PCR system and PCR procedure are shown below.

The NtCIPK11 fragment cloning primers are as follows:

the PCR reaction system (20. mu.L) was: 2 μ L10 XPCR Buffer, 1.2 μ L Mg²⁺(25mM/L)、0.4μL 10×dNTP、0.1μL Tag(5.0U/μL)、1μL Forward primer(10μM/L)、1μL Reverse primer(10μM/L)、1μL Template cDNA(100ng/μL)、13.3μL ddH₂O。

The PCR reaction program is: denaturation at 95 ℃ for 5min, annealing at 55 ℃ for 30s, extension at 72 ℃ for 1min, 35 cycles.

(4) Cloning of target Gene 5 'and 3' sequences

Designing RACE primers by utilizing Oligo7, cloning the 3 'end and the 5' end of a CIPK9 gene, cutting gel, recovering and obtaining a target fragment, then connecting the target fragment with a vector pMD19-T, transforming escherichia coli, selecting a single clone, sequencing and analyzing sequences, determining two terminal sequences of an NtCIPK9 gene, and splicing to obtain the full-length sequence of the NtCIPK9 gene. RACE primers, PCR reaction system and procedure are shown below.

The RACE primers are as follows:

the PCR reaction system (20. mu.L) for RACE A item was: 2 μ L10 XPCR Buffer, 1.2 μ L Mg²⁺(25mM/L)、0.4μL 10×dNTP、0.1μL Tag(5.0U/μL)、1μL CIPK11：3’/5’race primerA(10μM/L)、1μL10×Universal Primer A Mix、1μL Template cDNA(100ng/μL)、13.3μL ddH₂O。

PCR reaction program for RACE a item: denaturation at 95 ℃ for 5min, annealing at 67 ℃/69 ℃ (3 'end/5' end) for 30s, extension at 72 ℃ (3 'end/5' end) for 40s/35s, and 35 cycles.

PCR reaction systems (20. mu.L) for RACE B and C item were: 2 μ L10 XPCR Buffer, 1.2 μ L Mg²⁺(25mM/L)、2μL dNTP、0.4μL Kode、0.6μL Universal Primer A Mix、0.6μL CIPK11：3’/5’race primer B/C、1μL Diluent of race A product、12.2μL ddH₂O。

PCR reaction program for RACE B and C item: denaturation at 98 ℃ for 10s, annealing at 67 ℃/66 ℃/68 ℃/68 ℃ (3 'race B/3' race C/5 'race B/5' race C) for 30s, extension at 72 ℃ (3 'end/5' end) for 40s/35s, and 35 cycles.

(5) Gene full-Length acquisition

According to the full-length sequence of the CIPK11 gene, full-length primers are designed by utilizing Oligo7, the full-length gene of NtCIPK11 is obtained by cloning, a target band is recovered by cutting gel, the target band is connected with pMD19-T and then transferred into escherichia coli, and the complete ORF of the CIPK11 gene is determined through sequencing analysis. The CIPK11 gene of Nitraria tangutorum bobr has the overall length of 1677bp and is named as NtCIPK11, the specific sequence is shown as SEQ ID NO.1, the expressed protein sequence is shown as SEQ ID NO.2, and the expressed protein sequence comprises an Open Reading Frame (ORF) of 438 amino acids. Primers, PCR reaction system and procedure for full-length gene cloning are shown below.

The full-length gene cloning primer is as follows:

the PCR reaction system (20. mu.L) was: 2 μ L10 XPCR Buffer, 1.2 μ L Mg²⁺(25mM/L)、0.4μL 10×dNTP、0.1μL Tag(5.0U/μL)、1μL Forward primer(10μM/L)、1μL Reverse primer(10μM/L)、1μL Template cDNA(100ng/μL)、13.3μL ddH₂O。

PCR reaction procedure: denaturation at 95 deg.C for 5min, annealing at 63 deg.C for 30s, extension at 72 deg.C for 1min30s, and 35 cycles.

Example 2: nitcipk 11 gene function verification of Tanggute white thorn

Construction of 35S: the NtCIPK11 expression vector is transferred into wild Columbia arabidopsis thaliana, whether the Tangut white spine NtCIPK11 gene can enhance the salt tolerance and the drought tolerance of arabidopsis thaliana is observed, the phenotype difference of the transgenic arabidopsis thaliana and the wild arabidopsis thaliana is compared, and the function of the Tangut white spine CIPK11 gene is presumed.

(1) Construction of vectors

Coli JM109 (available from baozhijie, inc); the expression vector was pBI121 (Biovector co., purchased from LTD).

The specific process is as follows:

1. adding BamH I and SmaI enzyme cutting sites to the upstream and downstream of CIPK11 gene fragment by PCR, amplifying the PCR system and reaction conditions with the same full length, wherein the primers are respectively:

2. after the sequencing is correct, the vector can be constructed under the action of T4 ligase. The enzyme digestion was performed using BamH I and Sma I endonucleases. The empty pBI121 expression vector was treated with the same cleavage reaction.

The double digestion reaction system (20. mu.L) was: 2 μ L of 10 XK buffer, 0.5 μ L of BamH I, 0.5 μ L of SmaI, 1 μ g of recovered product, ddH₂O make up to 20. mu.L.

Water bath at 37 ℃ and enzyme digestion for 4 h. The digestion reaction was stopped by adding 10 Xstop solution, and the product was separated by 1% agarose gel electrophoresis. The digested product was recovered and purified using AxyPrep DNA Gel Extraction Kit (AXYGEN), and dissolved in 20. mu.L of TE buffer.

3. And detecting the concentration of the recovered enzyme digestion product, adding reagents (the number of target fragment molecules: the number of carrier molecules is 3: 1-5: 1) according to a connection system, and connecting at 16 ℃ overnight. The connection reaction system is as follows: 2.5. mu. L T4 DNA ligase buffer (10X), 5. mu.L of the digested expression vector, 15.5. mu.L of the digested PCR product, 2. mu. L T4 DNA ligase, ddH₂O make up to 25. mu.L.

4. Transforming Escherichia coli JM109 hypersensitive cells by the ligation product, selecting a single colony, inoculating the single colony into an LB liquid culture medium, and performing shake culture at 37 ℃ overnight; bacterial suspension PCR was performed using full-length primers to screen positive clones, after which the plasmids were extracted with AxyPrep Plasmid miniprep (AXYGEN) for enzyme validation. And simultaneously, sequencing to detect whether mutation or deletion occurs in the construction process of the vector. The expression vector was constructed as shown in FIG. 1:

(2) transformation of Agrobacterium

1. The Agrobacterium strain used in the present invention is GV3101(Biovector Co., LTD). The constructed NtCIPK11 expression vector is transferred into agrobacterium by adopting a liquid nitrogen freeze-thaw method. The specific process is as follows:

1) melting GV3101 competent cells in an ice bath, adding at least 100ng of recovered and purified expression vector plasmid, gently mixing uniformly, and carrying out ice bath for 20-30 min;

2) quickly freezing for 1min by using liquid nitrogen, thermally shocking for 3min at 37 ℃, and quickly placing on ice for 1-2 min;

3) adding 800 μ L LB culture medium without antibiotics, resuscitating at 28 deg.C and 200rpm for 3.5 h;

4) centrifuging at 4000rpm for 3min, and sucking off the culture medium;

5) mixing the rest bacteria solution, and smearing on the mixture containing 50mg.L of bacteria solution^-1On a solid LB medium of kanamycin;

6) carrying out inverted culture at 28 ℃ for 30-48 h;

7) detecting positive clone by PCR, and storing at4 ℃ for later use.

2. Healthy arabidopsis thaliana to be planted is grown to flower. And (4) carrying out soaking transformation on the arabidopsis flower organ when the positive clone detected by the PCR is shaken to OD 0.75. The specific process is as follows:

1) centrifuging the bacterial liquid at 5000rpm for 5min, collecting thalli, and suspending with 5% sucrose solution;

2) before soaking, Silwet L-77 is added, the concentration is 0.05% (500 mu L/L), and foam is shaken out;

3) soaking the overground part of arabidopsis thaliana in the agrobacterium suspension solution for 15-30 sec, slightly shaking the overground part of arabidopsis thaliana during the soaking period,

4) laying the soaked arabidopsis thaliana in a tray, covering the tray with a preservative film for moisture preservation, and sealing the tray with tinfoil paper for 24 hours in a dark place;

5) uncovering the tin foil paper, culturing under normal conditions, and stopping watering when the seeds are mature;

3. the dried seeds were harvested and T1 seed generations were screened.

4. And then moving the seeds into soil for continuous culture, collecting T1 generation arabidopsis seeds, and continuously screening the seeds to obtain T2 generation plants. Then, after collecting T2 generation Arabidopsis thaliana plants, the seeds are continuously screened to obtain homozygous screened T3 generation homozygous lines.

(3) Real-time fluorescent quantitative PCR

To determine the response of NtCIPK11 to salt and drought stress, transcription analysis was performed using real-time fluorescent quantitative pcr (qpcr) technique, total RNA extracted using nitraria tangutorum roots, stems and leaves, NtCIPK11 overexpressing plants and WT treated with 200mM NaCl and 200mM mannitol for 2 hours.

Total RNA reverse transcription was as described in gene cloning. qPCR Using SYBR-Green PCR Master batches

480 real-time PCR detection system(Roche, Basel, Switzerland) according to the manufacturer's instructions. The expression level of the target gene was normalized by transcription of the housekeeping gene actin (NsActin2, No.: AB617805) in Arabidopsis (Wang et al, 2012) and ubiquitin 10(AtUBQ10, No.: At4g05320.2) in Arabidopsis (Arabidopsis, Norris et al, 1993).

1) Design of RT-PCR primers

Primers for the NtCIPK11 gene were designed according to the RT-PCR requirements

2) RT-PCR reaction system

The reaction system is 20 μ L: 10 μ L of 2 XPower SYBR Green PCR Master Mix, 1 μ L Forward Primer, 1 μ L Reverse Primer, 1 μ L cDNA, 7 μ L ddH₂O.

3) Conditions for RT-PCR reaction

95℃10min；95℃ 15sec，60℃ 1min，40Cycles。

4) Test materials

Total RNA was extracted using Nitraria tangutorum roots, stems and leaves, and the NtCIPK11 over-expressed plants and WT were treated with 200mM NaCl and 200mM mannitol for 2 hours.

5) Test results

qPCR assays showed (fig. 2) that NtCIPK11 only expressed at higher levels in transgenic plants, confirming ectopic expression of the gene.

(4) Salt resistance test of transgenic NtCIPK11 Arabidopsis thaliana

50 seeds of each of 4 homozygous transgenic arabidopsis lines and wild type arabidopsis are respectively put on 1/2MS culture media containing 0mM, 100mM and 150mM NaCl for germination, the seeds are placed under the light for germination after vernalization for 2-3 days, and the germination rate is counted after 4 days. The 4 homozygous transgenic lines and the wild type Arabidopsis line were designated OX-1, OX-2, OX-3, OX-4 and WT, respectively. Six replicates were performed simultaneously. The phenotype of 4 days of germination is shown in FIG. 3, and the statistics of germination rates for 5 days of light are shown in FIG. 4.

The statistical results show (FIG. 4) that the seed germination rates of 4 overexpression lines transformed with NtCIPK11 gene are obviously different from the seed germination rate of WT in 0mM NaCl environment after 4 days of germination. In the 100mM NaCl environment, the germination rate of the transgenic lines is 43 percent higher than that of WT respectively, and the difference is very significant (P is less than 0.01). Under 150mM salt treatment conditions, the transgenic lines were 20% higher than WT, respectively, with the differences between OX-2 and OX-3 and WT being very significant (P < 0.01). From the results of the salt germination experiment, NtCIPK11 can improve the germination rate of Arabidopsis seeds in a salt stress environment.

After the transgenic plants and WT seeds were germinated and grown for 1 week on 1/2MS culture, 30 of each line were transferred to 1/2MS medium supplemented with 0mM, 100mM and 150mM NaCl, respectively, and the growth phenotypes of the transgenic plants and wild-type plants were observed. Three replicates were performed simultaneously. By observing the growth states of the transgenic arabidopsis thaliana and the wild arabidopsis thaliana, the NtCIPK11 can promote the root growth of plants under normal conditions. Under the environment of 100mM and 150mM NaCl, the NtCIPK11 can promote the growth of leaves and roots of arabidopsis thaliana and shows a better growth state than WT.

To further analyze the saline tolerance of transgenic plants, we observed that after 20 days after seedling germination, plants overexpressing NtCIPK11 had longer roots, more leaves, and more roots than WT plants in saline media at different concentrations, as shown in fig. 5, 6, and 7, especially under 100mM NaCl treatment, suggesting that overexpression of NtCIPK11 can improve the salt tolerance of arabidopsis thaliana.

(5) Transgenic NtCIPK11 gene Arabidopsis thaliana drought resistance test

50 seeds of 4 homozygous transgenic arabidopsis lines and 50 seeds of wild type arabidopsis are respectively put on 1/2MS culture media containing 0mM, 100mM, 150mM and 200mM mannitol for germination, the seeds are placed under the light for germination after vernalization for 2-3 days, and the germination rate is counted after 4 days. The 4 homozygous transgenic lines and the wild type Arabidopsis line were designated OX-1, OX-2, OX-3, OX-4 and WT, respectively. Six replicates were performed simultaneously. The phenotype of 4 days of germination is shown in FIG. 8.

Experimental studies found that mannitol treatment had no effect on seed germination by comparison with salt treatment. Both WT and transgenic plants were able to germinate under different mannitol treatments. However, we found that WT seedlings developed at a slower rate than the transgenic plants, and the phenotype is shown in FIG. 8. Moreover, this is reflected in the seedling proportion of the two cotyledons. As shown in FIG. 9, transgenic plants and WT did not differ much in the seedling ratio of both cotyledons in the absence of mannitol treatment; however, after treatment with mannitol, the difference began to manifest, especially at 200 mM. The average proportion of the three transgenic plants at 100mM, 150mM and 200mM is 91%, 87% and 70% respectively; higher than 31%, 20% and 5% of wild type.

Thus, these results indicate that NtCIPK11 can promote the development of seedlings in the early growth stage of plants and improve the drought resistance of plants under mannitol-simulated drought stress.

To further investigate the effect of NtCIPK11 under drought stress, we observed the growth of plants treated with different mannitol concentrations for 20 days. As shown in figure 10, plants overexpressing NtCIPK11 grew better than WT plants under different mannitol treatments. FIG. 11 shows that the primary roots of the transgenic lines were longer than the WT lines, especially on 150mM and 200mM mannitol dishes. To find beneficial factors for the growth of plants overexpressing NtCIPK11 under drought stress, we measured the content of free proline in wild type and transgenic plants. Normally, the proline content of transgenic lines is not different from that of wild type, but drought stress increases proline accumulation of wild type and transgenic plants. The free proline content in transgenic plants was significantly higher than in WT plants, as shown in figure 12. These results indicate that NtCIPK11 is involved in drought and salt stress signaling pathways by affecting the expression of plant osmoregulators.

Sequence listing

<110> Nanjing university of forestry

<120> Nitcipk 11 gene of Tanggute Nitraria, and expression protein and application thereof

<130> 100

<160> 20

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1677

<212> DNA

<213> Nitraria tangutorum

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gttgtaaaac gacggccagt gaattcgagc tcggtacccg gggatcctct agagattcta 60

atacgactca ctatagggca agcagtggta tcaacgcaga gtacatgggg accttcattt 120

tcgctctcta gtttcagtct tctccattca tatctaaccg agttttccaa ttctgagtta 180

catttttcct ttttaatttc cccgcctttc tgtttcgatc tctggccatt tcacttatgc 240

cggagattga acatgtcccc gcggattacg acagcaattg caaagctgcc aacggtgcct 300

tgtttggaaa gtacgagctc ggcaagctcc tcggctgcgg agccttcgct aaggtatacc 360

atgcgcgtga cgtccgtacg aaccagagcg tggcgattaa gatcattagc aagaagaaga 420

tcaacgttaa tctgatgtcg aacatcaagc gtgagatctc gatcatgagg cggttgaacc 480

atcgccatat cgtgaagctc cacgaggttc tggcgtcgaa aacgaagatt tatttcgtcg 540

tggagttcgc caagggcggc gagttgttcg ccagggtggc gaaaggaagg ttcagcgagg 600

atctcagcag gaagtacttc cagcagttga tatccgccgt tggttattgc cattcgcgcg 660

gcgtctatca ccgtgatctg aagccggaga atctcctgat cgacgagaac gggaatttga 720

aagtttcaga tttcggactc agcgctctga cggatcagat ccgaaccgac gggttgttgc 780

acacgctgtg tgggacccct gcttacgtgg ccccagagat attgtcgaag aaaggatacg 840

acggagccaa ggtggatatc tggtcatgcg gcgtcattct gtttgtttta acggccggtt 900

acctgccgtt taacgacccg aatctcatgg ccatgtacaa gaagatatac aaaggcgaat 960

tccggtgtcc gaaatggatg tccaacgatc ttaaacggct gttaaaccgt ctccttcata 1020

tcaatcctaa tacaaggatt accgtcgatc agattctcgg agatccatgg ttcagaaggg 1080

gcggggtcaa ggaaatcaaa ttccacgacg acgaaaacgc cgccgttccc gataaaaccg 1140

gtaaggaggg gttcggtgcg aggaatttga acgcgtttga tataatctca ttttcgtccg 1200

gtttggacct gtctggtttg ttcgatacgt cgggcaactc gttcgagaat aatactggcg 1260

aacgtttcat ctcgcgagag tcgcctgata atttgttgga gacggtgacg gagttcgcca 1320

aggttgagaa attaaggttg aagacgaaga aagaatgggg ggtggagttg gaagaacaaa 1380

acggtaattt catcatcggg gtggacgttt accggttaac ggaggaacta gtggtcgtgg 1440

aggccaacag aagagcgggt gacaccgcat gttacactga ggtgtggaag tcgaatctga 1500

gaccgcaact tcttgtgcgt caacaggaag cttcggtttc tggtaatcat taaaattgta 1560

gagagagaga gagagagaga gagagatagc aattaggagt acaaatcttt aattggattg 1620

ggttttcttt catgaaatta ggatacattc catatgaaaa aaaaaaaaaa aaaaaaa 1677

<210> 2

<211> 438

<212> PRT

<213> Nitraria tangutorum

<400> 2

Met Pro Glu Ile Glu His Val Pro Ala Asp Tyr Asp Ser Asn Cys Lys

1 5 10 15

Ala Ala Asn Gly Ala Leu Phe Gly Lys Tyr Glu Leu Gly Lys Leu Leu

20 25 30

Gly Cys Gly Ala Phe Ala Lys Val Tyr His Ala Arg Asp Val Arg Thr

35 40 45

Asn Gln Ser Val Ala Ile Lys Ile Ile Ser Lys Lys Lys Ile Asn Val

50 55 60

Asn Leu Met Ser Asn Ile Lys Arg Glu Ile Ser Ile Met Arg Arg Leu

65 70 75 80

Asn His Arg His Ile Val Lys Leu His Glu Val Leu Ala Ser Lys Thr

85 90 95

Lys Ile Tyr Phe Val Val Glu Phe Ala Lys Gly Gly Glu Leu Phe Ala

100 105 110

Arg Val Ala Lys Gly Arg Phe Ser Glu Asp Leu Ser Arg Lys Tyr Phe

115 120 125

Gln Gln Leu Ile Ser Ala Val Gly Tyr Cys His Ser Arg Gly Val Tyr

130 135 140

His Arg Asp Leu Lys Pro Glu Asn Leu Leu Ile Asp Glu Asn Gly Asn

145 150 155 160

Leu Lys Val Ser Asp Phe Gly Leu Ser Ala Leu Thr Asp Gln Ile Arg

165 170 175

Thr Asp Gly Leu Leu His Thr Leu Cys Gly Thr Pro Ala Tyr Val Ala

180 185 190

Pro Glu Ile Leu Ser Lys Lys Gly Tyr Asp Gly Ala Lys Val Asp Ile

195 200 205

Trp Ser Cys Gly Val Ile Leu Phe Val Leu Thr Ala Gly Tyr Leu Pro

210 215 220

Phe Asn Asp Pro Asn Leu Met Ala Met Tyr Lys Lys Ile Tyr Lys Gly

225 230 235 240

Glu Phe Arg Cys Pro Lys Trp Met Ser Asn Asp Leu Lys Arg Leu Leu

245 250 255

Asn Arg Leu Leu His Ile Asn Pro Asn Thr Arg Ile Thr Val Asp Gln

260 265 270

Ile Leu Gly Asp Pro Trp Phe Arg Arg Gly Gly Val Lys Glu Ile Lys

275 280 285

Phe His Asp Asp Glu Asn Ala Ala Val Pro Asp Lys Thr Gly Lys Glu

290 295 300

Gly Phe Gly Ala Arg Asn Leu Asn Ala Phe Asp Ile Ile Ser Phe Ser

305 310 315 320

Ser Gly Leu Asp Leu Ser Gly Leu Phe Asp Thr Ser Gly Asn Ser Phe

325 330 335

Glu Asn Asn Thr Gly Glu Arg Phe Ile Ser Arg Glu Ser Pro Asp Asn

340 345 350

Leu Leu Glu Thr Val Thr Glu Phe Ala Lys Val Glu Lys Leu Arg Leu

355 360 365

Lys Thr Lys Lys Glu Trp Gly Val Glu Leu Glu Glu Gln Asn Gly Asn

370 375 380

Phe Ile Ile Gly Val Asp Val Tyr Arg Leu Thr Glu Glu Leu Val Val

385 390 395 400

Val Glu Ala Asn Arg Arg Ala Gly Asp Thr Ala Cys Tyr Thr Glu Val

405 410 415

Trp Lys Ser Asn Leu Arg Pro Gln Leu Leu Val Arg Gln Gln Glu Ala

420 425 430

Ser Val Ser Gly Asn His

435

<210> 3

<211> 23

<212> DNA

<213> CIPK11 forward primer sequence (Artificial)

<400> 3

cgctaaggta taccatgcgc gtg 23

<210> 4

<211> 25

<212> DNA

<213> CIPK11 reverse primer sequence (Artificial)

<400> 4

cttccacacc tcagtgtaac atgcg 25

<210> 5

<211> 27

<212> DNA

<213> CIPK11:3' race primer A primer sequence (Artificial)

<400> 5

catggttcag aaggggcggg gtcaagg 27

<210> 6

<211> 28

<212> DNA

<213> CIPK11:3' race primer B primer sequence (Artificial)

<400> 6

acgaagaaag aatggggggt ggagttgg 28

<210> 7

<211> 26

<212> DNA

<213> CIPK11:3' race primer C primer sequence (Artificial)

<400> 7

aacggaggaa ctagtggtcg tggagg 26

<210> 8

<211> 24

<212> DNA

<213> CIPK11:5' race primer A primer sequence (Artificial)

<400> 8

acgccgcatg accagatatc cacc 24

<210> 9

<211> 23

<212> DNA

<213> CIPK11:5' race primer B primer sequence (Artificial)

<400> 9

ctgaaccttc ctttcgccac cct 23

<210> 10

<211> 26

<212> DNA

<213> CIPK11:5' race primer C primer sequence (Artificial)

<400> 10

tggagcttca cgatatggcg atggtt 26

<210> 11

<211> 28

<212> DNA

<213> CIPK11 wl forward primer sequence (Artificial)

<400> 11

gctctagaat gccggagatt gaacatgt 28

<210> 12

<211> 31

<212> DNA

<213> CIPK11 wl reverse primer sequence (Artificial)

<400> 12

tcccccggga tgattaccag aaaccgaagc t 31

<210> 13

<211> 25

<212> DNA

<213> NtCIPK 11F + BamH primer sequence (Artificial)

<400> 13

cggatccgct ctagaatgcc ggaga 25

<210> 14

<211> 27

<212> DNA

<213> NtCIPK 11R + Sma I primer sequence (Artificial)

<400> 14

cccgggtccc ccgggacgtg atttctt 27

<210> 15

<211> 24

<212> DNA

<213> NtCIPK 11F primer sequence (Artificial)

<400> 15

atacaaggat taccgtcgat caga 24

<210> 16

<211> 21

<212> DNA

<213> NtCIPK 11R primer sequence (Artificial)

<400> 16

ttcaaattcc tcgcaccgaa c 21

<210> 17

<211> 22

<212> DNA

<213> NsActin F primer sequence (Artificial)

<400> 17

catccctcat cggaatggaa gc 22

<210> 18

<211> 25

<212> DNA

<213> NsActin R primer sequence (Artificial)

<400> 18

ggtagaccca ccactaagca caatg 25

<210> 19

<211> 21

<212> DNA

<213> primer sequence AtUBQ 10F (Artificial)

<400> 19

ccggaaagac catcaccctt g 21

<210> 20

<211> 21

<212> DNA

<213> primer sequence AtUBQ 10R (Artificial)

<400> 20

tgtagtcggc caaagtacgt c 21

Claims (8)

1. Tanggute white thornNtCIPK11The nucleotide sequence of the gene is shown in SEQ ID NO. 1.

2. The Nitraria tangutorum bobr of claim 1NtCIPK11The amino acid sequence of the gene expression protein is shown in SEQ ID NO. 2.

3. Comprises the Nitraria tangutorum bobr of claim 1NtCIPK11A vector or recombinant bacterium of the gene.

4. The tangut white thorn according to claim 3NtCIPK11The gene vector is characterized in that the vector is a plant recombinant expression vector.

5. The plant recombinant expression vector as claimed in claim 4, wherein the vector is pBI121+NtCIPK11。

6. The plant recombinant expression vector according to claim 5,NtCIPK11the promoter of the gene is 35S.

7. The Nitraria tangutorum bobr of claim 1NtCIPK11The gene is applied to improving the salt tolerance or drought resistance of plants.

8. The tangut white thorn of claim 7NtCIPK11The application of the gene in improving the salt tolerance or drought resistance of plants is characterized by comprising the following steps:

1) constructing Tanggute white thornNtCIPK11Recombinant vectors of genes;

2) the constructed Tanggute white thornNtCIPK11Transforming the recombinant vector of the gene into a plant cell;

3) and cultivating and screening to obtain plants with salt tolerance or drought resistance.

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