CN110791506B - A kind of NtCIPK11 gene of Thorn tanggut and its expression protein and application - Google Patents

Abstract

The invention discloses a NtCIPK11 gene, an expression protein and an application thereof, belonging to the technical field of plant genetic engineering. For the CIPK11 gene disclosed by the present invention, its nucleotide sequence is shown in SEQ ID NO.1, and the amino acid sequence of its expressed protein is shown in SEQ ID NO.2. According to the stress resistance characteristics such as salt tolerance and drought resistance of T. tangute, the present invention utilizes the leaf tissue of T. tangute, and on the basis of the existing partial transcriptome data, homologously clones the stress-resistance-related strain of T. tangute. The full-length CIPK gene was named NtCIPK11 according to the homologous gene in Arabidopsis. Through the salt tolerance and drought resistance analysis of homozygous NtCIPK11 gene Arabidopsis plants, it was proved that the plants transformed with the NtCIPK11 gene of Tanggut were possessed of salt tolerance and drought resistance, adding resources to the plant stress resistance gene pool.

Description

A kind of NtCIPK11 gene of Thorn tanggut and its expression protein and application

technical field

The invention belongs to the technical field of plant genetic engineering, and more particularly relates to a NtCIPK11 gene of Thorn tanggut and its expression protein and application.

Background technique

Nitraria tangutorum belongs to the genus Nitraria of the family Zygophyllaceae and is a deciduous shrub. Tanggut white thorn has the characteristics of liking light, cold resistance, drought resistance, salinity resistance, barren resistance, low site index, etc. It can be used to improve saline-alkali soil, improve soil fertility, prevent wind and sand, maintain oasis, and protect ecological balance. In addition, studies have shown that Tanggut white thorn fresh fruit is rich in amino acids, vitamins, flavonoids, soaps, mineral elements and other components, and has high economic and medicinal value. Due to environmental degradation, soil salinization and arid environment seriously threaten the water transport of plants, resulting in a decline in plant productivity and an increase in desertification. As a drought-tolerant and saline-alkali-tolerant plant, Tanggut white thorn has gradually attracted attention. At present, the research on T. tanggut mainly focuses on the physiological and biochemical properties of drought resistance and salt tolerance, the analysis of fruit nutrients and breeding, etc., providing a large amount of data support for the molecular mechanism research of T. tanguticum, and for the resistance genes and other functions. The use of genes lays the foundation.

CIPKs (CBL-interacting protein kinase) is a class of proteins that can interact with calcineurin B-like protein (CBL) and can transmit Ca ²⁺ signals. These 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, so far, no similar genes have been found in species other than plants, so the CIPK gene is a Ser/Thr-like phosphoprotein kinase gene in the plant-specific Ca ²⁺ signaling pathway. Overexpression of OsCIPK12 can improve the tolerance of rice to cold, drought and salt stress by accumulating the content of proline and soluble sugar. CaCIPK6 in chickpea mediates auxin transport and regulates salt tolerance in tobacco seedlings. Overexpression of BrCIPK1 enhances abiotic stress tolerance by increasing proline biosynthesis in rice. In addition, reactive oxygen species scavengers, such as peroxidases, are regulated by CIPKs to improve stress resistance. Furthermore, CIPKs were also found to mediate Arabidopsis responses to salt stress conditions by regulating ion and water homeostasis. Numerous studies have shown that plant-specific CIPK genes play an important role in the stress resistance processes of plants such as salt tolerance, low potassium tolerance, cold resistance and drought resistance.

SUMMARY OF THE INVENTION

Aiming at the above-mentioned problems existing in the prior art, the technical problem to be solved by the present invention is to provide a kind of NtCIPK11 gene of T. tanguticum. Another technical problem to be solved by the present invention is to provide the application of the NtCIPK11 gene of T. tanguticum in improving the salt tolerance or drought resistance of plants.

In order to solve the above-mentioned technical problems, the technical scheme adopted in the present invention is as follows:

A tangut white thorn NtCIPK11 gene, the nucleotide sequence of which is shown in SEQ ID NO.1.

The amino acid sequence of the expressed protein of the NtCIPK11 gene of T. tanguti is shown in SEQ ID NO.2.

A vector, recombinant bacteria or host cell containing the NtCIPK11 gene of T. tanguti according to claim 1.

Preferably, the vector of the NtCIPK11 gene of T. tanguticum is a plant recombinant expression vector.

Preferably, the plant recombinant expression vector is pBI121+NtCIPK11.

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

The application of the NtCIPK11 gene of T. tanguti in improving the salt tolerance or drought resistance of plants.

Preferably, the application of the NtCIPK11 gene of P. tanguensis in improving the salt tolerance or drought resistance of plants comprises the following steps:

1) construct the recombinant vector of Tanggut white thorn NtCIPK11 gene;

2) transforming the constructed recombinant vector of the NtCIPK11 gene into plant cells;

3) Cultivate and screen to obtain plants with salt tolerance or drought tolerance.

Beneficial effect: Compared with the prior art, the advantages of the present invention are:

On the basis of the existing partial transcriptome data, the present invention homologously clones the full length of the CIPK gene related to stress resistance of T. tanggut, and is named NtCIPK11 according to the homologous gene in Arabidopsis thaliana. Its nucleotide sequence As shown in SEQ ID NO.1, the amino acid sequence of the expressed protein is shown in SEQ ID NO.2. In normal culture environment, the growth and development of Arabidopsis thaliana T3 homozygous plants overexpressing NtCIPK11 gene were not significantly different from those of the wild type. However, the salt tolerance and drought resistance analysis of homozygous NtCIPK11 gene Arabidopsis plants showed that under salt stress and drought stress, NtCIPK11 overexpressed plants had higher germination rate, longer roots, more leaves and roots than WT plants in the same period. The number is also more, which proves that the plants transformed with the NtCIPK11 gene of T. tanguensis have good salt tolerance and drought resistance. The disclosure of the NtCIPK11 gene increases the resources for the plant stress resistance gene pool, and is useful for the research on improving the salt tolerance and cold tolerance of plants. significant and valuable.

Description of drawings

Fig. 1 is the expression vector map of NtCIPK11 gene;

Fig. 2 is the qPCR analysis result of Arabidopsis NtCIPK11 gene;

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

Figure 4 is a graph of the germination rate of seeds of WT and transgenic plants after five days of illumination;

Figure 5 is a graph of root lengths of WT and transgenic plants 20 days after germination on salt medium with different concentrations;

Figure 6 is a graph of the number of leaves on different concentrations of salt medium 20 days after germination of WT and transgenic plants;

Figure 7 is a graph of root mass of WT and transgenic plants 20 days after germination on salt medium with different concentrations;

Figure 8 is a picture of seed germination of NtCIPK11 and WT under different mannitol treatments;

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

Figure 10 is a phenotype diagram of WT and NtCIPK11 overexpressing plants cultured on media with different mannitol concentrations for 20 days;

Figure 11 is a graph of primary root lengths of WT and transgenic plants treated with different concentrations of mannitol;

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

Detailed ways

The present invention will be further described below with reference to specific embodiments.

Example 1:

Tanggut white thorn seeds were vernalized for 4 weeks in a relative water content of 7% at 4°C. After vernalization, the seeds were placed in soil and sand 1:1 pots and germinated in an environment with a humidity of 55% to 60%, a temperature of 26°C to 28°C, and 16-h-light/8-h-dark. . Two-month-old seedlings were irrigated with 500 mM NaCl and 200 mM mannitol. After 2 hours of treatment, the leaves of Thorn tangut were taken, immediately frozen in liquid nitrogen and stored at -80°C. After RNA was extracted and reversed into cDNA, corresponding primers were designed for PCR, and after agarose gel electrophoresis, the target band was recovered. , ligated with pMD19-T vector, transformed into E. coli, sequenced and analyzed. After the target sequence was determined, RACE primers were designed according to the sequence, and the 5' and 3' sequences were obtained by PCR. After sequencing analysis, the full-length sequence of NtCIPK11 was obtained by splicing. Primers were designed according to the full-length sequence, and the full-length fragment was obtained by PCR, ligated with the pMD19-T vector, transferred into E. coli, sequenced and analyzed again, and after confirming that it was a full-length fragment, positive clones were picked for plasmid extraction, and restriction enzymes were added. After spotting, it was double-enzymatically digested with the vector pBI121. After ligation under the action of T4 ligase, it was transferred into Agrobacterium EHA105 and GV3101. After the Arabidopsis reached the appropriate age, it was transformed by the flower organ soaking transformation method to obtain T1 generation and T2 generation. After the homozygous T3 generation was screened, salt treatment, phenotype observation and salt tolerance analysis were performed.

(1) Extraction of total RNA

The previously collected leaves of Thorn tangut were used for RNA extraction according to the operating procedure of the NORGEN kit (Norgen Biotek). The 1% agarose gel electrophoresis results of the total RNA of T. tanggut leaves were clear; the absorbance of total RNA was measured, the OD ₂₆₀ /OD ₂₈₀ value was 2.13, and the OD ₂₆₀ /OD ₂₃₀ value was 2.05, indicating that the RNA quality was good.

The specific process of RNA extraction is as follows:

1) Add 800 μL Lysis Solution to grind the sample.

2) Transfer the lysate to a new tube after homogenization.

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

4) Add an equal volume of 70% ethanol and vortex to mix.

5) Transfer the mixture to a column (connected to a 2mL collection tube below), centrifuge for 1 min, pour off the filtrate, and put it back into the collection tube.

6) Add 400uL Wash Solution, centrifuge for 1 min, discard the filtrate and put it back into the collection tube.

7) Add DNA I working solution, centrifuge at 12000rpm for 1min, suck the filtrate back onto the column, and let stand at 25°C-30°C for 15min.

8) Add 400 μL Wash Solution, centrifuge at 12000 rpm for 1 min, and discard the filtrate.

9) Add 400 μL Wash Solution for the third time, centrifuge at 12000 rpm for 1 min, and discard the filtrate.

10) Put the column back into the collection tube, centrifuge at 12,000 rpm for 2 min, and discard the collection tube.

11) Put the column into a 1.7mL tube and add 50μL of Elution Solution.

12) Centrifuge at 200-2000 rpm for 2 min, 12,000 rpm for 1 min, the volume is less than 50 μL, and then centrifuge at 14,000 rpm for 1 min.

(2) Obtaining cDNA

III First-Strand Synthesis Kit。实验中的RNA使用量为1μg，具体过程如下：Using the extracted RNA as a template, reverse transcription was used to obtain cDNA, which was obtained by Invitrogen.

III First-Strand Synthesis Kit. The amount of RNA used in the experiment is 1 μg, and the specific process is as follows:

1) Prepare the reaction solution in the following order, after a brief low-speed centrifugation, 65°C for 5 min, and immediately place it on ice for 1-2 mm.

Reaction solution: 1 μL RNA (≤5 μg), 1 μL Primer (Oligo dT), 1 μL 10 mM dNTP mix, and DEPC-Treated Water to make up 10 μL.

2) Add the corresponding reagents in the following order to the tube in the previous step, place it on the PCR machine, and the reaction program is 50°C for 50 minutes and 85°C for 5 minutes. The order of adding reagents: 2 μL 10×RT Buffer, 4 μL 25mM MgCl ₂ , 2 μL 0.1M DTT, 1 μL RNaseOUT (40 U/μl), 1 μL SuperScript III RT.

3) Centrifuge the reaction solution in the previous step, add 1 μL of RNase H to each tube, and set it at 37°C for 20 min.

(3) Homologous cloning of the target gene

According to the partial transcriptome data of Tanggut white thorn, conservative specific sequence analysis was carried out by NCBI Blast, primers were designed by Oligo7, NtCIPK11 gene fragment was cloned, and then ligation transformation sequencing and sequence analysis were carried out to determine the target gene. Cloning primers, PCR systems and PCR procedures are shown below.

The NtCIPK11 fragment cloning primers are:

PCR reaction system (20μL): 2μL 10×PCR Buffer, 1.2μL Mg ²⁺ (25mM/L), 0.4μL 10×dNTP, 0.1μL Tag (5.0U/μL), 1μL Forward primer (10μM/L), 1 μL Reverse primer (10 μM/L), 1 μL Template cDNA (100 ng/μL), 13.3 μL ddH ₂ O.

The PCR reaction program was: denaturation at 95 °C for 5 min, annealing at 55 °C for 30 s, and extension at 72 °C for 1 min, for 35 cycles.

(4) Sequence cloning of the 5' and 3' ends of the target gene

Use Oligo7 to design RACE primers, clone the 3' end and 5' end of CIPK9 gene, cut the gel to recover the target fragment, and then ligate it with the vector pMD19-T, transform E. After the two end sequences of the NtCIPK9 gene, the full-length sequence of the NtCIPK9 gene was obtained by splicing. RACE primers, PCR reaction systems and procedures are shown below.

The RACE primers are:

The PCR reaction system (20μL) of RACE A item is: 2μL 10×PCR Buffer, 1.2μL Mg ²⁺ (25mM/L), 0.4μL 10×dNTP, 0.1μL Tag (5.0U/μL), 1μL CIPK11: 3'/5'race primerA (10 μM/L), 1 μL 10×Universal Primer A Mix, 1 μL Template cDNA (100 ng/μL), 13.3 μL ddH ₂ O.

The PCR reaction program of RACE A item: denaturation at 95°C for 5min, annealing at 67°C/69°C (3'end/5'end) for 30s, extension at 72°C (3'end/5'end) for 40s/35s, 35 cycles.

The PCR reaction system (20μL) of RACE B and C item is: 2μL 10×PCR Buffer, 1.2μL Mg ²⁺ (25mM/L), 2μL dNTP, 0.4μL Kode, 0.6μL Universal Primer A Mix, 0.6μL CIPK11:3 '/5' race primer B/C, 1 μL Diluent of race A product, 12.2 μL ddH ₂ O.

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

(5) Obtaining the full length of the gene

According to the full-length sequence of CIPK11 gene, use Oligo7 to design full-length primers, clone the full-length NtCIPK11 gene, cut the gel to recover the target band, connect it with pMD19-T and transfer it into E. coli. After sequencing analysis, it is confirmed that the ORF of the CIPK11 gene is complete of. The full length of CIPK11 gene of Tanggut white thorn is 1677bp, named as NtCIPK11, the specific sequence is shown in SEQ ID NO.1, and the expressed protein sequence is shown in SEQ ID NO.2, including 438 amino acid open reading frame (ORF ). Primers, PCR reaction systems and procedures for full-length gene cloning are shown below.

The full-length gene cloning primers are:

PCR reaction system (20μL): 2μL 10×PCR Buffer, 1.2μL Mg ²⁺ (25mM/L), 0.4μL 10×dNTP, 0.1μL Tag (5.0U/μL), 1μL Forward primer (10μM/L), 1 μL Reverse primer (10 μM/L), 1 μL Template cDNA (100 ng/μL), 13.3 μL ddH ₂ O.

PCR reaction program: denaturation at 95 °C for 5 min, annealing at 63 °C for 30 s, extension at 72 °C for 1 min for 30 s, 35 cycles.

Example 2: Functional verification of the NtCIPK11 gene of T. tangutei

Construct 35S:NtCIPK11 expression vector and transfer it into wild-type Colombian Arabidopsis thaliana to observe whether NtCIPK11 gene can enhance the salt tolerance and drought tolerance of Arabidopsis thaliana, and compare transgenic Arabidopsis thaliana and wild-type Arabidopsis thaliana The phenotypic differences, speculate the function of the CIPK11 gene in T. tangut.

(1) Construction of the vector

The Escherichia coli strain used in the present invention is E.coli JM109 (purchased by Bao Bioengineering (Dalian) Co., Ltd.); the expression vector is pBI 121 (purchased by Biovector Co., LTD.).

The specific process is as follows:

1. Add BamH I and SmaI restriction sites to the upstream and downstream of the CIPK11 gene fragment by PCR. The PCR system and reaction conditions are the same as the full-length amplification. The primers are:

2. After the sequencing is correct, the vector can be constructed under the action of T4 ligase. Digestion was performed using BamHI and Sma I endonucleases. The empty pBI 121 expression vector was treated with the same digestion reaction.

The double-enzyme digestion reaction system (20 μL) was: 2 μL of 10×K buffer, 0.5 μL of BamH I, 0.5 μL of SmaI, 1 μg of recovered product, and ddH ₂ O to make up 20 μL.

37°C water bath for 4h digestion. Add 10× stop solution to stop the digestion reaction, and separate by 1% agarose gel electrophoresis. The digestion product was recovered and purified with AxyPrep DNA Gel Extraction Kit (AXYGEN) and dissolved in 20 μL of TE buffer.

3. Detect the concentration of the recovered enzyme cleavage product, add each reagent according to the ligation system (number of target fragment molecules: number of carrier molecules=3:1-5:1), and connect overnight at 16°C. The ligation reaction system was: 2.5 μL T4 DNA ligase buffer (10×), 5 μL digested expression vector, 15.5 μL digested PCR product, 2 μL T4 DNA ligase, and ddH ₂ O to make up 25 μL.

4. The ligation product was transformed into E. coli JM109 supersensitive cells, and a single colony was picked and inoculated into LB liquid medium, and incubated overnight at 37°C with shaking; bacterial liquid PCR was performed using full-length primers to screen for positive clones, and then AxyPrep Plasmid Miniprep Kit (AXYGEN ) to extract the plasmid for verification by enzyme digestion. At the same time, sequencing was used to detect whether mutation or deletion occurred during the construction of the vector. The construction of the expression vector is shown in Figure 1:

(2) Transformation of Agrobacterium

1. The Agrobacterium strain used in the present invention is GV3101 (purchased from Biovector Co., LTD). The liquid nitrogen freeze-thaw method was used to transfer the constructed NtCIPK11 expression vector into Agrobacterium. The specific process is as follows:

1) Melt the GV3101 competent cells in an ice bath, add at least 100ng of the recovered and purified expression vector plasmid, mix gently, and take an ice bath for 20-30 minutes;

2) Quick-freeze in liquid nitrogen for 1 min, heat shock at 37°C for 3 min, and quickly place on ice for 1-2 min;

3) Add 800 μL of antibiotic-free LB medium, recover at 28°C and 200rpm for 3.5h;

4) Centrifuge at 4000rpm for 3min, and suck off the medium;

5) Mix the remaining bacterial liquid and apply it on the solid LB medium with 50 mg.L ^-1 kanamycin added;

6) Inverted culture at 28°C for 30-48h;

7) The positive clones detected by PCR were stored at 4°C for future use.

2. The healthy Arabidopsis to be planted is grown to flowering. When the positive clones detected by PCR were shaken to OD0.75, the Arabidopsis flower organs were immersed and transformed. The specific process is as follows:

1) Centrifuge the bacterial liquid at 5000 rpm for 5 min, collect the bacterial cells, and suspend with 5% sucrose solution;

2) Before soaking, add SilwetL-77, the concentration is 0.05% (500μL/L), and shake out the foam;

3) Soak the aerial part of Arabidopsis thaliana in the Agrobacterium suspension solution for 15-30 sec, and shake gently during the period.

4) The immersed Arabidopsis thaliana was laid flat on the tray, covered with plastic wrap to keep moisture, and sealed with tin foil to protect from light for 24h;

5) Uncover the tin foil, cultivate under normal conditions, and stop watering when the seeds are mature;

3. Harvest the dried seeds and screen the T1 generation seeds.

4. Move to the soil to continue culturing, and after collecting the T1 generation Arabidopsis seeds, continue to screen the seeds to obtain the T2 generation plants. Then, after harvesting the T2 generation Arabidopsis plants, the seeds were screened to obtain a homozygous T3 generation homozygous line.

(3) Real-time fluorescence quantitative PCR

To determine the response of NtCIPK11 to salt and drought stress, real-time quantitative PCR (qPCR) technology was used to extract total RNA from T. tanguensis roots, stems, and leaves, and NtCIPK11 overexpression was treated with 200 mM NaCl and 200 mM mannitol for 2 hours. Plants and WT were subjected to transcriptional analysis.

480实时PCR检测系统(Roche，Basel，Switzerland)上按照制造商的说明进行。通过管家基因actin基因(NsActin2，编号：AB617805)在拟南芥(Wang et al.，2012)和泛素10(AtUBQ10，编号：At4g05320.2)在拟南芥(Arabidopsis，Norris et al.，1993)中的转录，使靶基因的表达水平正常化。Total RNA was reverse transcribed as described in Gene Cloning. qPCR using SYBR-Green PCR masterbatch in

480 real-time PCR detection system (Roche, Basel, Switzerland) according to the manufacturer's instructions. Through the housekeeping gene actin gene (NsActin2, accession: AB617805) in Arabidopsis (Wang et al., 2012) and ubiquitin 10 (AtUBQ10, accession: At4g05320.2) in Arabidopsis (Arabidopsis, Norris et al., 1993) ) to normalize the expression levels of target genes.

1) Design of RT-PCR primers

According to the requirements of RT-PCR, the primers of NtCIPK11 gene were designed

2) RT-PCR reaction system

The reaction system was 20 μL: 10 μL 2×Power SYBR Green PCR Master Mix, 1 μL ForwardPrimer, 1 μL Reverse Primer, 1 μL cDNA, 7 μL ddH ₂ O.

3) RT-PCR reaction conditions

95℃ for 10min; 95℃ for 15sec, 60℃ for 1min, 40Cycles.

4) Test material

Total RNA was extracted from the roots, stems and leaves of Thorn, and NtCIPK11 overexpressing plants and WT were treated with 200 mM NaCl and 200 mM mannitol for 2 hours.

5) Test results

qPCR assay showed (Fig. 2) that NtCIPK11 had a high expression level only in transgenic plants, which confirmed the ectopic expression of this gene.

(4) Salt tolerance test of NtCIPK11 transgenic Arabidopsis

50 seeds each of 4 homozygous transgenic Arabidopsis lines and wild-type Arabidopsis thaliana were germinated on 1/2MS medium containing 0 mM, 100 mM and 150 mM NaCl, and the seeds were vernalized for 2-3 days and then exposed to light. After germination, the germination rate was counted after 4 days. Four homozygous transgenic lines and wild-type Arabidopsis lines were named OX-1, OX-2, OX-3, OX-4 and WT, respectively. Six replicates were run simultaneously. The phenotype of 4 days of germination is shown in Figure 3, and the statistical results of the germination rate of 5 days of light are shown in Figure 4.

The statistical results (Fig. 4) showed that, after 4 days of germination, in the environment of 0 mM NaCl, the seed germination rate of the four overexpressed lines transfected with the NtCIPK11 gene was significantly different from that of the WT seed. In the 100mM NaCl environment, the germination rate of the transgenic lines was 43% higher than that of the WT, showing a very significant difference (P<0.01). Under 150 mM salt treatment, transgenic lines were 20% higher than WT, respectively, with OX-2 and OX-3 significantly different from WT (P<0.01). From the results of salt germination experiments, NtCIPK11 can improve the germination rate of Arabidopsis seeds under salt stress.

After the transgenic plants and WT seeds were germinated and grown on 1/2MS culture for 1 week, 30 plants from each line were transferred to 1/2MS medium supplemented with 0 mM, 100 mM and 150 mM NaCl, respectively. growth phenotype. Three replicates were performed simultaneously. Observing the growth status of transgenic Arabidopsis and wild-type Arabidopsis, it can be seen that NtCIPK11 can promote the root growth of plants under normal conditions. In the environment of 100mM and 150mM NaCl, NtCIPK11 can promote the growth of Arabidopsis leaves and roots, showing a better growth state than WT.

To further analyze the salinity tolerance of transgenic plants, we observed that after 20 days after seedling germination in different concentrations of salt media, plants overexpressing NtCIPK11 had longer roots, more leaves, and more roots than WT plants. More, as shown in Figure 5, Figure 6 and Figure 7, especially under 100 mM NaCl treatment, which indicates that overexpression of NtCIPK11 can improve the salt tolerance of Arabidopsis.

(5) Drought resistance test of Arabidopsis thaliana transgenic NtCIPK11

50 seeds each of 4 homozygous transgenic Arabidopsis lines and wild-type Arabidopsis thaliana were germinated on 1/2MS medium containing 0mM, 100mM, 150mM and 200mM mannitol, and the seeds were vernalized for 2-3 days. Germination was performed under light, and the germination rate was counted after 4 days. Four homozygous transgenic lines and wild-type Arabidopsis lines were named OX-1, OX-2, OX-3, OX-4 and WT, respectively. Six replicates were run simultaneously. The phenotype at 4 days of germination is shown in Figure 8.

The experimental study found that mannitol treatment had no effect on seed germination compared with salt treatment. Both WT and transgenic plants were able to germinate under different mannitol treatments. However, we found that WT seedlings developed slower than transgenic plants, and the phenotypes are shown in Figure 8. Moreover, this is reflected in the proportion of seedlings with two cotyledons. As shown in Figure 9, in the absence of mannitol treatment, there was little difference between transgenic plants and WT in the proportion of seedlings in both cotyledons; but after treatment with mannitol, differences began to appear, especially in the 200mM. The average proportions of the three transgenic plants at 100 mM, 150 mM and 200 mM were 91%, 87% and 70%, respectively; they were higher than those of the wild type at 31%, 20% and 5%.

Therefore, these results suggest that NtCIPK11 can promote the development of early seedlings and improve the drought resistance of plants under mannitol-simulated drought stress.

To further investigate the role 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. Figure 11 shows that the primary roots of the transgenic lines were longer than the WT lines, especially on 150 mM and 200 mM mannitol dishes. To find favorable factors for the growth of NtCIPK11-overexpressing plants under drought stress, we determined the content of free proline in wild-type and transgenic plants. Under normal conditions, the proline content of transgenic lines was no different from wild type, but drought stress increased proline accumulation in wild type and transgenic plants. The free proline content in transgenic plants was significantly higher than that in WT plants, as shown in Figure 12. These results suggest that NtCIPK11 is involved in drought and salt stress signaling pathways by affecting the expression of plant osmotic regulators.

sequence listing

<110> Nanjing Forestry University

<120> A kind of NtCIPK11 gene of Thorn tangut, its expression protein and application

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<170> SIPOSequenceListing 1.0

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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 agatctcgg 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 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 primer sequence (Artificial)

<400> 3

cgctaaggta taccatgcgc gtg 23

<210> 4

<211> 25

<212> DNA

<213> CIPK11 reverse primer 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 primer sequence (Artificial)

<400> 11

gctctagaat gccggagatt gaacatgt 28

<210> 12

<211> 31

<212> DNA

<213> CIPK11:wl reverse primer primer sequence (Artificial)

<400> 12

tccccccggga tgattaccag aaaccgaagc t 31

<210> 13

<211> 25

<212> DNA

<213> NtCIPK11 F+BamH primer sequence (Artificial)

<400> 13

cggatccgct ctagaatgcc ggaga 25

<210> 14

<211> 27

<212> DNA

<213> NtCIPK11 R+Sma I primer sequence (Artificial)

<400> 14

cccgggtccc ccgggacgtg atttctt 27

<210> 15

<211> 24

<212> DNA

<213> NtCIPK11 F primer sequence (Artificial)

<400> 15

atacaaggat taccgtcgat caga 24

<210> 16

<211> 21

<212> DNA

<213> NtCIPK11 R 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> AtUBQ10 F primer sequence (Artificial)

<400> 19

ccggaaagac catcaccctt g 21

<210> 20

<211> 21

<212> DNA

<213> AtUBQ10 R primer sequence (Artificial)

<400> 20

tgtagtcggc caaagtacgt c 21

Claims (8)

1. A Tanggut white thorn NtCIPK11 gene, the nucleotide sequence of which is shown in SEQ ID NO.1. 2. The expression protein of the NtCIPK11 gene of T. tanguensis according to claim 1, the amino acid sequence of which is as shown in SEQ ID NO.2. 3. A carrier or recombinant bacteria containing the NtCIPK11 gene of T. tangutes according to claim 1. 4 . The vector of the NtCIPK11 gene of T. tangutei according to claim 3 , wherein the vector is a plant recombinant expression vector. 5 . 5. The plant recombinant expression vector according to claim 4, wherein the vector is pBI121+ NtCIPK11 . 6. The plant recombinant expression vector according to claim 5, wherein the promoter of the NtCIPK11 gene is 35S. 7. The application of the NtCIPK11 gene of Thorn tanguti according to claim 1 in improving the salt tolerance or drought resistance of plants. 8. the application of Tanggut white thorn NtCIPK11 gene according to claim 7 in improving plant salt tolerance or drought resistance, is characterized in that, comprises the following steps: 1) Constructing the recombinant vector of NtCIPK11 gene of Tanggut white thorn; 2) Transform the constructed recombinant vector of the NtCIPK11 gene into plant cells; 3) Cultivate and screen to obtain plants with salt tolerance or drought tolerance.

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