CN107881180B - Application of genes CKX2 and CKX3 in improving cold resistance of plants - Google Patents

Abstract

The invention discloses application of genes CKX2 and CKX3 in improving cold resistance of plants, wherein the genes CKX2 and CKX3 are derived from Columbia ecotype arabidopsis thaliana, and the nucleotide sequences of cDNAs of the genes are respectively shown as SEQ ID NO 1 and SEQ ID NO 2. The invention provides gene resources for cultivating new varieties of low-temperature-resistant plants, has better potential application value, and lays theoretical foundation for researching plant response stress signal mechanism and adverse environment-resistant molecular mechanism.

Description

Application of genes CKX2 and CKX3 in improving cold resistance of plants

Technical Field

The invention relates to the field of genetic engineering and molecular biology, in particular to application of genes CKX2 and CKX3 in improving cold resistance of plants.

Background

Under natural and agricultural production conditions, plants are constantly subjected to various stresses of biotic and abiotic origin. The scientific and technical means which are mastered by people can effectively overcome some adverse environmental factors such as soil nutrient deficiency, oxygen deficiency, pathogenic bacteria infection, weed competition and the like. In recent years, adverse effects of extreme temperatures on plants have become increasingly prominent with global climate abnormalities. In 2008, China in south experiences a low-temperature freezing disaster with rare history, and continuous freezing weather has great influence on agricultural production. The disaster area in China reaches 1.5 kilohm²The direct economic loss is 1590 million yuan, which is far beyond the damage caused by drought, flood, hail and other disasters in the same year.

Due to unpredictability of disasters and higher protection cost, no effective scientific and technical means for preventing damage of extreme temperatures to crops exist in actual production at present, so that excellent agronomic crop varieties with the capability of resisting disaster environment stress are obtained, and the method has important practical application value in agricultural production. Therefore, people are devoted to research the molecular mechanism of low temperature tolerance of plants, and the method aims to provide an important theoretical basis for crop stress-resistant engineering and cultivation of cold-resistant and drought-resistant varieties of crops. Under the stress condition, the plant hormone can regulate and control the growth and development of plants, influence various metabolic pathways and the response process of stress response genes, and enable the plants to adapt to the constantly changing external environment from the morphological, biochemical and cellular levels. Cytokinin is isopentene adenine substance produced in plant root, and cooperates with auxin to regulate the growth and differentiation of plant tissue and organ. Cytokinin has been used for a long time to promote plant cell proliferation, delay plant senescence and induce callus differentiation. In recent years, it has been found that cytokinins also have important physiological functions in the response process of plants to stress.

An irreversible cytokinin metabolic pathway exists in Arabidopsis thaliana. This pathway is involved in cytokinin oxidase/dehydrogenase (CKX), which is capable of degrading active cytokinins. 7 genes AtCKX1-7 encoding cytokinin oxidase were found in Arabidopsis thaliana. Transgenic plant seeds overexpressing the CKX gene are significantly increased. In rice, a major QTL gene OsCKX2 plays an important role in rice yield. The CKX gene is specifically over-expressed in roots of transgenic tobacco and arabidopsis thaliana, so that the growth and development of the root system and the drought resistance can be remarkably promoted. In 2007, researchers find that the drought resistance of sunflower can be greatly improved by applying cytokinin from an external source; meanwhile, the over-expression of AtCKX can promote the growth of root systems of Arabidopsis thaliana and also can improve the tolerance of plants to drought stress. The 2010 research shows that the single-process and multi-mutant of cytokinin receptor AHK2,3 in Arabidopsis show strong low temperature tolerance, which indicates that the signal pathway of cytokinin has negative regulation and control effect in the process of plant response to low temperature stress. Although the CKX gene has become an important target point for crop molecular genetic improvement, the role of the CKX gene in the process of responding to low-temperature stress of plants is not reported and applied at present.

Disclosure of Invention

The invention aims to provide application of genes CKX2 and CKX3 in improving cold resistance of plants.

In order to realize the purpose of the invention, the genes CKX2 and CKX3 provided by the invention are applied to improving the cold resistance of plants, and the nucleotide sequence of the gene CKX2cDNA is as follows:

i) 1, SEQ ID NO; or

ii) nucleotide sequences with the same functions, wherein one or more nucleotides are substituted, deleted and/or added in the nucleotide sequence shown in SEQ ID NO. 1; or

iii) a nucleotide sequence which hybridizes with the sequence shown in SEQ ID NO. 1 under stringent conditions, in a 0.1 XSSPE containing 0.1% SDS or a 0.1 XSSC solution containing 0.1% SDS at 65 ℃ and washing the membrane with the solution; or

iv) a nucleotide sequence having more than 90% homology with the nucleotide sequence of i), ii) or iii) and having the same function.

The nucleotide sequence of the gene CKX3cDNA is as follows:

i') the nucleotide sequence shown in SEQ ID NO. 2; or

ii') the nucleotide sequence shown in SEQ ID NO. 2 is substituted, deleted and/or added with one or more nucleotides and has the same function; or

iii') a nucleotide sequence which hybridizes with the sequence shown in SEQ ID NO. 2 under stringent conditions, in a 0.1 XSSPE containing 0.1% SDS or a 0.1 XSSC solution containing 0.1% SDS at 65 ℃ and washing the membrane with the solution; or

iv ') a nucleotide sequence having more than 90% homology with the nucleotide sequence of i'), ii ') or iii') and having the same function.

The CKX2 and CKX3 genes are derived from Columbia ecotype Arabidopsis thaliana, encode key enzymes for synthesis and metabolism of plant endogenous cytokinins, and are numbered AT2G19500 and AT5G56970 respectively in the Arabidopsis thaliana genome database. The amino acid sequences encoded by the CKX2 and CKX3 genes are shown in Seq ID Nos. 3 and 4, respectively. It is understood that one skilled in the art can substitute, delete and/or add one or several amino acids based on the disclosed amino acid sequences without affecting their activity to obtain mutant sequences of the proteins.

It is understood that, considering the degeneracy of codons and the preference of codons for different species, one skilled in the art can use codons suitable for the expression of a particular species as needed.

The invention also provides a cloning vector or various expression vectors containing the plant low temperature resistant CKX2 and/or CKX3 gene sequences or the fragments thereof, a host cell containing the vector, a transformed plant cell containing the gene sequences or the specific fragments thereof and a transgenic plant.

The plants of the present invention include monocotyledons and dicotyledons, such as Arabidopsis thaliana and the like. The plant low temperature resistant CKX2 and/or CKX3 gene is over-expressed in the plant, including any vector which can guide the over-expression of exogenous gene in the plant.

The invention also provides a method for improving the cold resistance of plants, which improves the tolerance of the plants to low temperature by over-expressing the genes CKX2 and/or CKX3 in the plants;

the invention also provides a method for constructing the low-temperature-resistant transgenic arabidopsis thaliana, which comprises the following steps: extracting total RNA of arabidopsis thaliana, carrying out reverse transcription to obtain cDNA, amplifying CKX2 or CKX3 genes by using the cDNA as a template and F and R as primers, respectively constructing amplification products on plant expression vectors, respectively transforming agrobacterium with the obtained recombinant expression vectors, respectively infecting arabidopsis thaliana inflorescences with the transformed agrobacterium, screening positive transgenic plants, and respectively obtaining low-temperature-resistant transgenic arabidopsis thaliana;

wherein, the primer sequences for amplifying the CKX2 gene are shown as Seq ID No.5 and 6;

the primer sequences for amplifying the CKX3 gene are shown in Seq ID No.7 and 8.

The plant expression vector used in the present invention is an expression vector having a 35S promoter, such as the vector pBINHygTx.

In the method, preferably, the Agrobacterium is GV 3101.

In the above method, the arabidopsis thaliana is preferably a wild type arabidopsis thaliana plant, i.e., an arabidopsis thaliana plant having a homozygous genotype.

After overexpression of the CKX2 and CKX3 genes, Arabidopsis thaliana shows a low temperature resistant phenotype. In order to facilitate the identification and screening of transgenic Arabidopsis plants, the vectors used may be processed, for example, by adding plant selectable markers or antibiotic markers having resistance.

According to the invention, through gene research of a key enzyme (cytokinin oxidase) in the metabolic pathway of cytokinin in arabidopsis thaliana, the transgenic plant over-expressing the related gene is found to be capable of generating new low-temperature stress resistance related characters, and the genes are capable of participating in regulation and control of the tolerance of the plant to low-temperature stress by changing the content and the steady state of endogenous cytokinin in the plant body. The CKX2 and CKX3 genes provided by the invention provide gene resources for cultivating new varieties of low-temperature-resistant plants, have good potential application values, and lay a theoretical basis for researching a mechanism of plant response stress signals and a molecular mechanism of adverse environment resistance.

Drawings

FIG. 1 is a restriction map of the linkage of CKX2 and CKX3 into an over-expression vector in example 1 of the present invention; wherein, 1 is a DNA Marker; 2 is a fragment of CKX 2; 3 is CKX3 fragment.

FIG. 2 is a diagram showing the semi-quantitative RT-PCR detection of the expression of a target gene in a transgenic plant in example 2 of the present invention; wherein, A is CKX2 gene expression level detection; and B is CKX3 gene expression level detection.

FIG. 3 shows the analysis of the low temperature resistant phenotype (A) and survival statistics (B) of transgenic plants of CKX2 or CKX3 under non-cold acclimation and cold acclimation conditions in example 3 of the present invention.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise indicated, the examples follow conventional experimental conditions, such as molecular cloning experiments, or conditions as recommended by the manufacturer's instructions.

The pBINHygTx cloning vector in the following examples is a commonly used cloning vector, commercially available; the arabidopsis variety is Columbia ecotype; cloning vectors commonly used for the Agrobacterium GV3101 strain.

The main reagents in the following examples are: molecular cloning related experimental products were purchased from Invitrogen corporation; taq DNA polymerase, T4 ligase, Pyrobest Taq enzyme, KOD, Bio-Inc. available from NEB, Toyobo, etc.; dNTPs were purchased from Genestar; the plasmid miniprep kit and the agarose gel recovery kit are purchased from Axygen company; MS medium, agar powder, agarose, ampicillin (Amp), kanamycin (Kan), gentamicin sulfate (Gen), rifampicin (Rif) and other antibiotics, sucrose (Glucose), Bovine Serum Albumin (BSA), nitrocellulose membrane, LB medium and others were purchased from Sigma, Bio-Rad and other companies; the various other chemicals used in the examples were all imported or home-made analytical grade reagents.

The primers used in the examples were synthesized from Hexa Huada and subjected to related sequencing.

Example 1 construction of CKX2 or CKX3 Gene overexpression vectors

To understand the effect of cytokinin content on the plant's ability to resist freezing, we targeted the oxidases CKX2 and CKX3 that could participate in the cytokinin metabolic pathway that regulates plant drought and high salt stress responses. The CKX2 and CKX3 genes were cloned from the Arabidopsis thaliana (Arabidopsis thaliana) genome. According to coding region sequence analysis, primers 1-4 are designed, coding regions of genes CKX2 and CKX3 are amplified, and the amplified coding regions are respectively connected to an overexpression vector pBINHygTx.

Primer 1: CKX2-F: 5'-gtcgacGTTGTAATTCTCTTCTTCAAAAA-3' (SEQ ID NO: 5)

Primer 2: CKX2-R: 5'-ggtaccTCAAAAGATGTCTTGCCCTGG-3' (SEQ ID NO: 6)

Primer 3: CKX3-F: 5'-gtcgacAAGAATCAAGCTATTCATAA-3' (SEQ ID NO: 7) primer 4: CKX3-R: 5'-ggtaccCTAACTCGAGTTTATTTTTT-3' (SEQ ID NO: 8)

The specific method for constructing 35S CKX2/3 is that 5 'and 3' of CKX2 or CKX3 genes are respectively added with SalI or KpnI restriction sites for amplification, PCR products (3,104 bp and 3,397bp respectively) are recovered and are connected to a cloning vector pUC 19. Obtaining positive clone bacterial plaque after passing resistance screening, extracting plasmids in small quantity by the kit and sending to sequence, and indicating that CKX2 or CKX3 genes are respectively connected in pUC19 vectors by correct sequencing. And then carrying out enzyme digestion and recovery on CKX2 and CKX3 fragments, connecting the fragments to pBINHygTx driven by an overexpression vector 35S promoter to obtain positive clone bacterial plaques after resistance screening, and extracting plasmids to carry out enzyme digestion and obtaining a gel map shown in figure 1.

Example 2 construction and detection of CKX2 or CKX3 Gene overexpressing plants

The pBINHygTx vector containing CKX2 or CKX3 gene described in example 1 was transformed into Agrobacterium GV3101 strain, and then transformed into wild Arabidopsis thaliana plant to obtain transgenic seedling of Arabidopsis thaliana. The specific method comprises the following steps: inoculating Agrobacterium containing the target vector into 100mL LB three-antibody liquid culture medium (kanamycin 50. mu.g/mL, rifampicin 50. mu.g/mL, gentamicin 50. mu.g/mL), culturing overnight at 28 deg.C with shaking until OD₆₀₀The value is 1.0-2.0, centrifuging at 5000rpm at room temperature for 10min, and collecting thallus; the cells were suspended in 200mL of the transformation solution (1/2MS, 5% sucrose, 40. mu.L Silwet L-77); soaking the arabidopsis inflorescence in the transformation liquid of the agrobacterium for 1min, covering a freshness protection package for moisturizing, placing in a dark place to ensure that the temperature is lower, taking out the plant from the freshness protection package the next day, and placing the plant back on an illumination culture rack for normal growth until the plant is harvested.

The screening resistance gene carried by the pBINHygTx vector is hygromycin, and the hygromycin resistance is used for screening arabidopsis transgenic seedlings to obtain T₁Carrying out single plant harvest on positive seedlings with hygromycin resistance, and then carrying out T-shaped seedling culture₂The seeds were tested for hygromycin resistance and a line was selected that was resistant to 3/4 but not resistant to the remaining 1/4, indicating that the overexpression vector linked to the gene of interest was inserted in a single copy in this line. And (3) removing the plants with hygromycin resistance from the strains, harvesting the plants individually, and screening the hygromycin resistance by using the obtained seeds of the T3 generation, wherein if the plants of the T4 generation are not separated, the transgenic strains are homozygotes which can be used for seed reproduction and low-temperature stress treatment experiments.

Overexpression strains were isolated in this example. And detecting the expression of the target gene in the transgenic plant by adopting semi-quantitative PCR.

1) The RNA of the transgenic plant is extracted by a Trizol method.

2) First strand cDNA Synthesis by reverse transcription (25. mu.l System)

Taking 1-2 mu g of total RNA, and adding DEPC water to 12 mu l; 2 μ l (10pM) oligo (dT) is added₁₅Mixing uniformly; ③ 70 ℃, 5 minutes, then on ice for 2-3min, then add: 5 Xbuffer 5U l, 2.5mM dNTP 5U l, 200U/. mu.l reverse transcriptase H1U l; reverse transcription reaction (first strand cDNA synthesis) was performed on a PCR instrument using the following procedure: 60 minutes at 42 ℃; fifthly, taking out after the reaction is finished, adding 10mg/ml 1 mul DNase, then standing for 20 minutes at 37 ℃, and then standing for 10 minutes at 65 ℃.

3) Semi-quantitative RT-PCR amplification. The reaction system is a PCR amplification system according to the following requirements: 10 XTaq buffer 1.5. mu.l; dNTP mixtures each 200. mu. mol/L; 10pmol of each of the upstream and downstream primers; 1 mu g of template cDNA; taq DNA polymerase 0.8. mu.l; complement ddH₂O to 15. mu.l. Amplification was performed under the following conditions: pre-denaturation at 95 ℃ for 3 min; denaturation at 94 ℃ for 30sec, annealing at 54 ℃ for 30sec, and extension at 72 ℃ for 1min for 20-35 cycles; extending for 10min at 72 ℃, and storing at 20 ℃. Tubulin as an internal reference gene. The expression of the target genes CKX2 and CKX3 is shown in FIG. 2. From FIG. 2 it can be seen that the corresponding genes of interest are up-regulated to different extents in both the over-expressed lines of CKX2 and CKX 3.

Example 3 analysis of phenotype associated with Cold temperature tolerance of plants overexpressing the CKX2 or CKX3 genes

The cytokinin plays a crucial role in regulating the growth and development of plants and responding to environmental factors. Previous literature reports that cold acclimation (cold acclimation) process can effectively improve the freezing resistance of plants. The internal mechanism is that the adaptation process of the subzero low temperature can activate the rapid rise of the plant cold response gene expression, and a large amount of osmotic substances and protective proteins are accumulated in cells, so that the plants can better adapt to the subsequent subzero low temperature. The cold acclimation conditions in this example were set to 4 days of growth at 4 ℃ under normal light.

Firstly, seedlings of transgenic plants over-expressing CKX2 and CKX3 grow for 2 weeks, then culture plates are subjected to low-temperature treatment, the treatment time is gradient cooling from-1 ℃ to-6 ℃, and the seedlings are treated for 1 hour at the temperature of-6 ℃. The treated plates were transferred to dark conditions at 4 ℃ and left overnight. Then, it was cultured under normal light conditions for 3-4 days. The injured tissue at low temperature withered by yellowing during the culture process, and the tissue capable of resisting low temperature injury gradually turns green and recovers growth (fig. 3A). The treated plates were subjected to survival statistics, depending on the criterion that the growth point was able to turn green and new leaves were grown. FIG. 3B shows that transgenic plants overexpressing CKX2 and CKX3 before cold response had a 90% survival rate after 1 hour treatment at-5 ℃ compared to only 35% wild type. The survival rate of wild type seedlings after cold response is increased to 55 percent, but still is obviously lower than the survival rate of transgenic plants of 95 percent. It can be concluded that overexpression of a key enzyme of the cytokinin metabolic pathway can enhance the anti-freezing ability of plants.

An arabidopsis transgenic plant with reduced endogenous cytokinin is obtained by constructing an overexpression vector. Then, the up-regulation of the expression level of the target gene in the transgenic plants is proved by semi-quantitative PCR. The experimental results show that the transgenic plant over-expressing the key enzyme of cytokinin metabolic pathway shows enhanced anti-freezing capability. The method of obtaining it and its function are illustrated by the following examples.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Sequence listing

<110> university of agriculture in China

Application of <120> genes CKX2 and CKX3 in improving cold resistance of plants

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Gly Gly Leu Gly Gln Phe Gly Ile Ile Thr Arg Ala Arg Ile Lys Leu

225 230 235 240

Glu Val Ala Pro Lys Arg Ala Lys Trp Leu Arg Phe Leu Tyr Ile Asp

245 250 255

Phe Ser Glu Phe Thr Arg Asp Gln Glu Arg Val Ile Ser Lys Thr Asp

260 265 270

Gly Val Asp Phe Leu Glu Gly Ser Ile Met Val Asp His Gly Pro Pro

275 280 285

Asp Asn Trp Arg Ser Thr Tyr Tyr Pro Pro Ser Asp His Leu Arg Ile

290 295 300

Ala Ser Met Val Lys Arg His Arg Val Ile Tyr Cys Leu Glu Val Val

305 310 315 320

Lys Tyr Tyr Asp Glu Thr Ser Gln Tyr Thr Val Asn Glu Glu Met Glu

325 330 335

Glu Leu Ser Asp Ser Leu Asn His Val Arg Gly Phe Met Tyr Glu Lys

340 345 350

Asp Val Thr Tyr Met Asp Phe Leu Asn Arg Val Arg Thr Gly Glu Leu

355 360 365

Asn Leu Lys Ser Lys Gly Gln Trp Asp Val Pro His Pro Trp Leu Asn

370 375 380

Leu Phe Val Pro Lys Thr Gln Ile Ser Lys Phe Asp Asp Gly Val Phe

385 390 395 400

Lys Gly Ile Ile Leu Arg Asn Asn Ile Thr Ser Gly Pro Val Leu Val

405 410 415

Tyr Pro Met Asn Arg Asn Lys Trp Asn Asp Arg Met Ser Ala Ala Ile

420 425 430

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

435 440 445

Phe Asp Asn Trp Glu Ala Phe Asp Gln Glu Asn Met Glu Ile Leu Lys

450 455 460

Phe Cys Glu Asp Ala Asn Met Gly Val Ile Gln Tyr Leu Pro Tyr His

465 470 475 480

Ser Ser Gln Glu Gly Trp Val Arg His Phe Gly Pro Arg Trp Asn Ile

485 490 495

Phe Val Glu Arg Lys Tyr Lys Tyr Asp Pro Lys Met Ile Leu Ser Pro

500 505 510

Gly Gln Asn Ile Phe Gln Lys Ile Asn Ser Ser

515 520

<210> 5

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

gtcgacgttg taattctctt cttcaaaaa 29

<210> 6

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

ggtacctcaa aagatgtctt gccctgg 27

<210> 7

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

gtcgacaaga atcaagctat tcataa 26

<210> 8

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

ggtaccctaa ctcgagttta tttttt 26

Claims (3)

1. The application of the gene CKX2 or CKX3 in improving the cold resistance of plants is characterized in that the nucleotide sequence of the gene CKX2cDNA is shown as SEQ ID NO. 1;

the nucleotide sequence of the gene CKX3cDNA is shown in SEQ ID NO. 2.

2. The use of claim 1, wherein the plant comprises a monocot and a dicot.

3. Use according to claim 2, wherein the plant comprises Arabidopsis thaliana.

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