CN107827963A - Application of the arabidopsis IDD14 genes in plant drouhgt stress patience is lifted - Google Patents

Abstract

The invention discloses the application of the Arabidopsis thaliana IDD14 gene in improving the drought stress tolerance of plants. The IDD14 gene encodes a protein with the amino acid sequence shown in SEQ ID No: 1 in the sequence list. The present invention is the first time to overexpress the Arabidopsis IDD14 gene in Arabidopsis thaliana to improve the drought tolerance of Arabidopsis plants, which provides a new idea for cultivating highly drought-resistant Arabidopsis varieties, and also improves drought resistance for other crops using homologous gene technology Theoretical support is provided. The applied IDD14 gene can provide support for research on drought resistance of corn, rice, wheat and other food crops and other crops.

Description

Application of Arabidopsis IDD14 Gene in Improving Plant Drought Stress Tolerance

technical field

The present invention relates to the technical field of plant genetic engineering, and more specifically, relates to a kind of Arabidopsis gene that improves plant drought stress tolerance and the application of the gene.

Background technique

Drought has become a worldwide problem. The world's arid and semi-arid areas have accounted for more than 1/3 of the land area. The impact of drought on plants ranks first among many natural adversity factors. Food losses caused by drought account for more than half of all natural disasters. Under natural conditions, drought stress not only seriously affects the growth and yield of crops, but also limits the distribution of plants. Therefore, to analyze the molecular mechanism of plant tolerance to drought stress and to cultivate new varieties of high-yield and stress-resistant crops has become a highly urgent and major issue. With the development of molecular biology technology, genetic engineering has become a powerful weapon for the innovation and improvement of germplasm resources.

The genes currently used in genetic engineering for drought resistance mainly include the following categories.

First, genes involved in the synthesis of osmoprotective substances (such as proline, mannitol, betaine, trehalose, etc.). These genes can enable plants to synthesize more osmotic adjustment substances under water stress, so as to improve the osmotic adjustment ability of plants, thereby enhancing the drought resistance of plants. For example, overexpressing the key enzyme gene (P5CS, deltal-pyrroline-5-carboxylatesynthase) in the proline biosynthesis pathway in rice improves the drought resistance of transgenic plants (Zhu et al., Plant Sci, 1998, 199: 41-48) .

Second, genes related to scavenging reactive oxygen species (ROS, Reactive oxygen species). The expression of such genes enhances the ability of plants to scavenge active oxygen free radicals, so that plants overexpress some enzymes (such as SOD, POD, CAT, etc.) under water stress to effectively eliminate harmful active oxygen free radicals, thereby improving cell Ability to withstand dehydration. For example, NTL4, a drought-responsive NAC transcription factor in Arabidopsis, can promote ROS production during drought-induced leaf senescence by directly binding to the promoter region of ROS synthase genes. In contrast, the ntl4 mutant lacking the NTL4 gene exhibited decreased ROS levels, delayed leaf senescence, and enhanced drought resistance (Lee et al., Plant Journal, 2012, 70:831-844).

Third, genes encoding drought-inducible proteins. These proteins can be mainly divided into two categories, (i) regulatory proteins that play an indirect protective role, mainly including G proteins, calmodulin, protein kinases, phospholipases, transcription factor proteins, etc.; (ii) functional proteins, including ion channels protein, LEA protein (Late Embryogenesis Abundant protein), heat shock protein, aquaporin, osmoregulatory protein, metabolic enzyme, etc. In the process of drought tolerance, these proteins can directly play an important protective role in the cell and improve the tolerance of plants to drought. For example, overexpression of the LEA gene resulted in enhanced drought tolerance in transgenic plants, although the exact mechanism remains unclear. LEA proteins can also act as molecular chaperones to protect molecules against cellular damage (Umezawa et al., Current Opinion in Biotechnology, 2006, 17: 113-122).

Fourth, regulate genes. Such genes include genes related to the ABA pathway, including genes related to ABA biological metabolism (such as NCED and ABAox) and genes related to the ABA signaling pathway (such as genes encoding bZIPs, Mybs, and zinc-finger transcription factors). For example, the isolated ERF/AP2 family of transcription factors CBF/DREB1 and DREB2 can bind to the cis-acting element DRE/CRT, and transgenic plants overexpressing CBF/DREB1 have increased tolerance to freezing, drought and salt stress (Liu et al. , Plant Cell, 1998, 10: 1391-1406). The activated form of the trans-acting factor DREB2 can activate the expression of stress-induced genes to improve the drought resistance of Arabidopsis (Sakuma et al., PNAS, 2006, 103: 18822-18827). Other endogenous ABA-regulated genes such as RD22 (Abe et al., Plant Cell, 2003, 15:63-78), RD29B (Fuiita et al., Plant Cell, 2005, 17:3470-3488), ABF3 or AREB2/ABF4 (Kang et al., 2002, PlantCell, 14: 343-357) and other overexpression can also improve the osmotic stress resistance of transgenic plants. The HRD gene (HARDY) of the model plant Arabidopsis thaliana, which encodes a protein that is an AP2/ERF like transcription factor, exhibits enhanced drought resistance and salt tolerance in the Arabidopsis gain-of-function mutant hrd-D. Transformation into the crop rice also exhibited a phenotype of increased water use efficiency consistent with drought resistance (Karaba et al., PNAS, 2007, 104: 15270-15275).

Due to the lack of understanding of the molecular mechanism of plant drought resistance, there is still a lot of blindness in molecular breeding for drought resistance. Moreover, the drought resistance of plants is usually the result of the co-expression of many drought resistance genes. Using a single gene strategy to improve the drought resistance of plants has no obvious effect in actual production and application. If changing the expression of a gene can regulate the drought tolerance response of plants as a whole, it will be great. An ideal choice.

As early as 1998, Colasanti et al. discovered a gene ID1 (INDETERMINATE1) that controls flowering in maize, and its encoded protein is a transcription factor with four zinc finger structures. Subsequent studies found that multiple transcription factors contained functional domains similar to the zinc finger structure in ID1, which were named INDETERMINATE Domain, and these transcription factors were also named IDD. IDDs are a class of transcription factors unique to plants. Maize, rice and Arabidopsis contain 21, 15 and 16 IDD genes, respectively. The protein sequence of rice OsID1/Ehd2/RID1 has a high degree of homology with maize ID1, and it is also a determinant of rice flowering. OsIDD14/LPA1(Loose PlantArchitecture1) is involved in the regulation of rice stem gravity and plant architecture. Unlike maize and rice, where few IDD functions have been resolved, nearly half of Arabidopsis IDD genes have been studied. IDD8/NUC (NUTCRACKER) regulates flowering by regulating sucrose metabolism, IDD3/MAG (MAGPIE), IDD8/NUC and IDD10/JKD (JACKDAW) regulate root development, and IDD1/ENY (ENHYDROUS) participates in regulating seed maturation and germination .

The excavation of excellent drought-resistant genes is very important for cultivating drought-resistant crops that can be used in field production.

Contents of the invention

The object of the present invention is to provide an Arabidopsis gene, an overexpression vector and the encoded protein of the Arabidopsis gene for improving the drought stress tolerance of plants.

To achieve the above object, the present invention adopts the following technical solutions:

The gene for improving drought stress tolerance of plants provided by the present invention is called IDD14, which is derived from Arabidopsis thaliana and encodes the following protein: the protein with the amino acid sequence shown in SEQ ID No: 1 in the sequence listing.

The nucleic acid sequence of the Arabidopsis thaliana IDD14 gene for improving drought stress tolerance of plants of the present invention may be the CDS sequence of the gene or a DNA sequence having more than 90% identity with the sequence and encoding the same functional protein. SEQ ID No: 2 in the sequence listing is the sequence of the IDD14 gene.

The present invention also provides an expression vector comprising the above nucleic acid sequence and an expression control sequence operably linked to the nucleic acid sequence. Further, the expression control sequence includes a constitutive high expression control sequence, which may be the cauliflower mosaic virus 35S promoter.

The invention also provides a method for improving drought stress tolerance of plants.

Expressing the IDD14 gene can improve the drought stress tolerance of plants such as Arabidopsis thaliana, the method is to introduce the IDD14 gene into plant cells, tissues or organs, and cultivate the imported plant cells, tissues or organs into plants, so that the IDD14 gene Expressed in plants to obtain plants with improved tolerance to drought stress.

Further, the IDD14 gene can be the CDS sequence of the gene or a DNA sequence having more than 90% identity with the sequence and encoding the same functional protein. The DNA sequence having more than 90% identity with the sequence and encoding the same functional protein is obtained by isolating, modifying and/or designing the CDS sequence of the gene using known methods. Those skilled in the art should understand that methods for changing or shortening gene sequences, and methods for testing the validity of these changed genes are well known to those skilled in the art.

The IDD14 gene of the present invention can be introduced into plant cells, tissues or organs through plant expression vectors. The plant expression vector is pVIPMyc (Cui et al., PLOS Genetics, 2013, 9: e1003759). pVIPMyc is a commonly used plant genetic transformation vector in the world, and is transformed on the basis of pVIP96. When using the IDD14 gene of the present invention or its homologous sequence component plant expression vector, any constitutive or inducible promoter can be added before its transcription initiation nucleotide. Constitutive promoters can be cauliflower mosaic virus (CAMV) 35S promoter, rice Actin promoter or corn Ubiquitin promoter, etc.; inducible promoters can be induced by low temperature, drought, ABA, ethylene, salinity or chemical etc. Promoter. The above-mentioned promoters can be used alone or in combination with other plant promoters.

The plant expression vector carrying the above-mentioned IDD14 gene or its homologous sequence can be used by any one or a combination of several methods in conventional biological methods such as Agrobacterium-mediated, Ti plasmid, plant virus vector, microinjection, gene gun, etc. Transforming plant cells, tissues or organs, and culturing the introduced plant cells, tissues or organs into plants; the plant tissues or organs involved in the present invention include plant seeds, flower buds, fruit pods, leaves, flower stems, etc. The plant can be corn, rice, wheat or Arabidopsis.

The present invention overexpresses the nucleic acid sequence of the IDD14 gene in Arabidopsis thaliana, and the results show that in two independent transgenic Arabidopsis plants, the IDD14 protein levels are significantly higher than the wild type.

The present invention obtains a mutant idd14-1D with enhanced drought resistance through the screening of Arabidopsis thaliana activation mutant library, and the drought resistance phenotype of the mutant is caused by the overexpression of a gene IDD14 encoding IDD transcription factor. Therefore, overexpressing the IDD14 gene in Arabidopsis is of great significance for improving the drought stress tolerance of Arabidopsis, which provides a new idea for breeding new varieties with high drought resistance.

The beneficial effects of the present invention are:

(1) The present invention provides an application of the gene IDD14 for improving the drought stress tolerance of Arabidopsis thaliana. The present invention finds that the drought stress tolerance of the mutant plants is significantly improved after overexpressing the IDD14 gene in the Arabidopsis ecotype Columbia. After dehydration and rehydration treatment under the same conditions, it was found that 100% of the mutant plants could return to normal growth, while only about 2% of the corresponding wild-type plants returned to normal growth.

(2) The present invention is the first time to overexpress the Arabidopsis IDD14 gene in Arabidopsis. It provides a new idea for breeding highly drought-resistant Arabidopsis varieties, and also provides theoretical support for other crops to use homologous gene technology to improve drought resistance.

(3) The IDD14 gene used in the present invention can provide support for research on drought resistance of corn, rice, wheat and other food crops and other crops.

Description of drawings

Fig. 1A and Fig. 1 B are the comparative figure of the drought stress tolerance of idd14-1D plant of the present invention and wild-type plant;

Fig. 2 A and Fig. 2 B are the comparative figure of the water loss rate of the blade of idd14-1D plant and wild-type plant;

Fig. 3 is a comparison chart of the stomatal density of idd14-1D plants and wild-type plants;

Fig. 4A and Fig. 4B are the drought stress tolerance comparative figure of transgenic plant and wild-type plant of overexpression IDD14 gene;

Fig. 5 is a result figure of the expression level of IDD14 protein detected in T3 generation transgenic positive plants with Western blot.

Detailed ways

In order to make the object, technical solution and advantages of the present invention clearer, the present invention will be described in further detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described here are only used to explain the present invention, not to limit the present invention.

The following examples define the invention and describe the method used by the invention in the isolation of cloned IDD14 gene and verification of its function. From the following description and these Examples, those skilled in the art can ascertain the essential characteristics of the present invention, and without departing from the spirit and scope of the present invention, various changes and modifications can be made to the present invention so that it can be used in different ways. uses and conditions.

Embodiment 1: screening overexpression IDD14 gene plant

The mutant idd14-1D used in the present invention is a mutant with abnormal leaf development screened from an Arabidopsis T-DNA mutant library containing an activation tag (Cui et al., PLOS Genetics, 2013, 9: e1003759), with obvious Leaf roll down phenotype. The previous genetic analysis showed that idd14-1D is a dominant mutant with a single T-DNA insertion, and the phenotype of mutant idd14-1D is caused by the overexpression of IDD14.

Example 2: IDD transcription factor subfamily positively regulates drought stress tolerance response in Arabidopsis

Tolerance responses to drought stress of idd14-1D mutant plants overexpressing IDD14 gene and control wild-type plants were analyzed using drought treatment experiments, determination of water loss rate of detached leaves, and comparative analysis of leaf stomatal density.

(1) The idd14-1D mutant plants overexpressing the IDD14 gene have strong drought stress tolerance

A certain amount of seeds of idd14-1D mutant plants and wild-type Arabidopsis plants were weighed, and after disinfection, the seeds were sown on MS medium for 2-3 days at a low temperature of 4°C and cultured under continuous light for 7 days, and the seedlings were transplanted. Mix vermiculite: nutrient soil at a ratio of 1:1, weigh the same weight of nutrient soil (about 70-85 grams) in a small pot with a flat bottom, water the bottom to make the soil completely wet, and plant 9 seedlings in each small pot , cover film, 12 small pots per plate. The culture conditions in the growth chamber were: 21°C (light)/18°C (dark), photoperiod: 12 h (light)/12 h (dark), light intensity 80-120 μmol·m ^-2 ·s ^-1 , relative humidity 50 %about. Remove the film after 3 days, continue to grow for 7 days, select small pots with consistent growth for drought treatment, randomly place small pots of different strains, and often change the position of small pots to reduce the position effect. The same experiment was repeated more than 3 times. The results showed that after 20 days without watering, the wild-type Arabidopsis plants wilted earlier than the idd14-1D mutant plants overexpressing the IDD14 gene. After 3 days of recovery, the statistical results showed that 100% of the idd14-1D mutant plants overexpressing the IDD14 gene could return to normal growth, while only about 2% of the corresponding wild-type plants returned to normal growth (as shown in Figure 1A-1B, wherein Figure 1A For WT (wild-type Arabidopsis plants) and idd14-1D (idd14-1D mutant plants overexpressing IDD14 gene) control, drought treatment and rehydration phenotype. Figure 1B is the survival rate of WT and idd14-1D after rehydration , ** represents a very significant difference between each genetic material and WT (p<0.01)).

(2) The water loss rate of leaves of idd14-1D mutant plants overexpressing IDD14 gene is reduced

The specific method of dehydration experiment on detached leaves is as follows: the seeds of idd14-1D mutant plants and wild-type Arabidopsis plants were sterilized and kept at low temperature for 3 days, sowed on 1/2MS medium, cultured under continuous light for 7 days, and then transplanted into soil , grow for about four weeks in a 21°C (light)/18°C (dark), 12h (light)/12h (dark) growth chamber. Before flowering, cut off the above-ground part and put it into a petri dish for weighing immediately, and put it under the condition of 22±1°C to carry out the leaf water loss test, and weigh it at regular intervals, and repeat 4 times for each line. Leaf water loss rates were calculated as percent loss of initial fresh weight. As shown in Figure 2A-2B (Fig. 2A represents the dehydration phenotype of WT and idd14-1D detached leaves, Bar=1cm; Fig. 2B represents the dehydration rate of WT and idd14-1D detached leaves. * represents each genetic material and WT There was a significant difference (p<0.05)), the results showed that the water loss rate of the detached leaves of the idd14-1D mutant plants overexpressing the IDD14 gene was significantly lower than that of the wild-type plants (p<0.05).

(3) The stomatal density of the idd14-1D mutant plants overexpressing the IDD14 gene was reduced

The fully expanded rosette leaves (grown under 12h (light)/12h (dark)) of the same parts of the idd14-1D mutant and wild-type Arabidopsis plants at 5 to 6 weeks were respectively cut and placed in the buffer. For each treatment, 3 leaves were taken for parallel experiments, the epidermis strips were torn off, the mesophyll cells were removed by brushing, and observed under a 10×20 magnification Leica optical microscope. 3 to 5 visual fields were randomly selected from each epidermis, and the number of stomata was recorded. Each treatment was repeated 4 to 6 times, and the average number of stomata and errors in parallel experiments were counted. As shown in Figure 3 (wherein idd14-1D represents the idd14-1D mutant plant overexpressing the IDD14 gene. ** represents that there is a very significant difference between each genetic material and WT (wild-type Arabidopsis plant) (p<0.01 )), the results showed that the stomatal density of leaves of idd14-1D mutant plants overexpressing IDD14 gene was lower than that of wild-type plants, and this difference was extremely significant (p<0.01).

Guard cell stomatal observation buffer:

500 mM KCl (10×): 3.7275 g was dissolved in 100 mL H ₂ O.

5.0 mM CaCl ₂ (50×): 0.0555 g was dissolved in 100 mL H ₂ O.

100mM Mes/KOH (10×): Dissolve 1.952g in 100mL double-distilled water, adjust the pH to 6.1 with 1.0M KOH.

1.0M KOH: Dissolve 5.61g in 100mL double distilled water.

Embodiment 3: Isolation and cloning are used to construct the DNA fragment of IDD14 gene plant expression vector

Total RNA was extracted from Arabidopsis leaves using TRIzol reagent (purchased from Invitrogen). The specific steps are as follows: pre-cool the centrifuge, take 50-100 mg of sample and add liquid nitrogen to grind thoroughly, add 1 mL of TRIzol solution, and let stand at room temperature for 5 minutes; add 200 μL of chloroform, shake vigorously for 15 seconds, and let stand at room temperature for 3 minutes; 4°C, 12,000 g Centrifuge for 15 minutes; draw the supernatant into a new 1.5mL centrifuge tube (RNase-free, ribonuclease-free) (purchased from AXYGEN), add 1 times the volume of isopropanol, mix well, and let stand at room temperature for 10 minutes; Centrifuge at 12,000 g at 4°C for 10 min; discard the supernatant, add 1 mL of 70% ethanol solution, and pop the pellet; centrifuge at 7,500 g at 4°C for 5 min; discard the supernatant, centrifuge for a short time to absorb the remaining ethanol; store at room temperature After 10 min, 50 μL of RNase-free water was added and fully dissolved, and the concentration of RNA was measured by an ultraviolet spectrophotometer.

Use reverse transcriptase (purchased from Invitrogen) to reverse transcribe it into cDNA, the specific steps are as follows: sequentially add 2 μg total RNA, 1 μl 10× digestion buffer, 1 μL DNase I (deoxyribonuclease I) (RNase-free ) and DEPC water (MiliQ pure water treated with diethyl pyrocarbonate and sterilized by high temperature and high pressure) to 10 μl to prepare a DNA digestion reaction solution, put it at 37°C for half an hour, add 1 μL 25mM EDTA, and inactivate it at 65°C 10min; add 1μL 50mM Oligo(dT)18 and 1μL 10mM dNTP (deoxy-ribonucleoside triphosphate, deoxyribonucleoside triphosphate) to the digested product, mix well, place at 65°C for 5min for denaturation, and place in ice bath immediately after the reaction Cool in medium for at least 1 min. Then add 4 μl 5-fold concentration first-strand buffer, 1 μl 0.1M DTT, 0.4 μl RNase (ribonuclease) inhibitor and 0.6 μL reverse transcriptase (200 U/μL), mix well and incubate at 50°C for 1 h. Then in a 70°C water bath for 15 minutes to inactivate the enzyme and terminate the reaction, the first-strand cDNA was synthesized, and the target gene was amplified using the first-strand cDNA as a template.

Use the upstream primer IDD14F (5'-gggcccccATGCATAGAAGACGACATAAAG-3' with restriction site, sequence-specific primer plus ApaI site and two protective bases, SEQ ID NO: 3) and downstream primer IDD14R (5'-gagctccTGAAGATGCTCTATCACTCG- 3', a sequence-specific primer plus an XhoI site and a guard base, SEQ ID NO: 4). Use high-fidelity Phusion DNA polymerase (purchased from Thermo Company) to amplify the target fragment, and the PCR reaction conditions are 98°C pre-denaturation for 30 seconds; 98°C for 10 seconds, 51°C for 30 seconds, 72°C for 30 seconds, 30 cycles; 72 °C for 10 minutes. Agarose gel electrophoresis was used to detect the target band, and the DNA gel recovery kit (purchased from Vigorous Company) was used to recover the corresponding target band; the sequencing carrier was connected (purchased from TransGen Corporation). Positive clones were screened and sequenced to obtain the desired DNA fragment, which was named pEASY-IDD14cDNA.

Example 4: Construction and genetic transformation of IDD14 gene 35S overexpression vector

In order to better analyze the function of the IDD14 gene, the applicant increased the expression level of the IDD14 gene in Arabidopsis through 35S overexpression technology. The function of the gene was studied according to the phenotype and physiological characteristics of the transgenic plants. The construction method of 35S overexpression IDD14 gene plant expression vector is as follows: use cauliflower mosaic virus (CAMV) 35S promoter; First the positive clone pEASY-IDD14cDNA that obtains in embodiment 3 is cut with ApaI and XhoI double enzymes, reclaims insert fragment; Similarly, the plant expression vector of pVIMyc was digested with the same method, and the vector fragment was recovered. The recovered insert fragment and the vector fragment were used for ligation reaction to transform Escherichia coli DH5α. Positive clones were screened by enzyme digestion to obtain a plant expression vector named pVIMyc-IDD14. pVIPMyc is a commonly used plant genetic transformation vector in the world, which is transformed on the basis of pVIP96. Transform pVIMyc-IDD14 into EHA105 host bacteria (EHA105 host bacteria is a kind of Agrobacterium).

It was introduced into Arabidopsis thaliana type Columbia through Agrobacterium-mediated genetic transformation, and a total of 30 independent transgenic Arabidopsis plants were obtained from the transformation. Specific steps: cut off the bloomed flowers and siliques on the bolting and flowering plants, and keep the unopened flower buds; inoculate the Agrobacterium of the plants to be transformed into LB (Luria-Bertani) liquid medium containing corresponding antibiotics, at 28°C Cultivate overnight at 220rpm until the _OD600 is about 1.8; collect the bacteria by centrifugation at 6,000rpm at room temperature for 10 minutes; pour off the supernatant, and add an equal volume of dip solution (5.0% sucrose solution + 0.025% (v/v) Silwet-L77) to resuspend the bacteria Put the resuspended bacterium solution into a petri dish, soak the flower stems of Arabidopsis thaliana in the dipping solution, and fully soak them; water the transformed plants and cover them with plastic bags to keep them moist, and remove the plastic bags for normal cultivation after about 24 hours; After 3 weeks of transformation, the materials will slowly mature, and the seeds of the transgenic materials will be harvested, and the positive transgenic seedlings of the T1 generation will be obtained by screening on 1/2 MS medium containing the corresponding carrier resistance. The ratio can determine whether it is a single-copy insertion, and obtain homozygous transgenic lines in the T3 generation.

As shown in Figures 4A-4B, it is a comparison chart of the drought stress tolerance of the above-mentioned IDD14 gene transgenic plants and wild-type plants. Wherein 35S-IDD14 represents the transgenic plant overexpressing the IDD14 gene. Figure 4A shows the drought treatment and rehydration phenotypes of WT and 35S-IDD14, 12-1 and 3-2 represent different positive transgenic lines, Bar=1cm. Figure 4B shows the survival rate of WT and 35S-IDD14 positive transgenic lines after rehydration, ** means that there is a very significant difference between each genetic material and WT (p<0.01). Transgenic plants and wild-type plants with consistent growth were selected for drought treatment, small pots of different strains were randomly placed, and the position of small pots was often changed to reduce the position effect. The same experiment was repeated more than 3 times, and the results showed that after stopping watering for 20 days, the wild-type plants wilted earlier than the transgenic plants, and after all the plants wilted, the statistical results showed that after adding water for 3 days, Figure 4B shows The recovery rate of 35S-IDD14 plants reaches 100%, that is, 100% of the transgenic plants overexpressing the IDD14 gene can recover to normal growth, while only about 2% of the corresponding wild-type plants can recover to normal growth.

The sequence of the gene IDD14 connected into the PVIPMyc vector:

IDD14ApaI F 5′-gggcccccATGCATAGAAGACGACATAAAG-3′

IDD14XhoI R 5′-gagctccTGAAGATGCTCTATCACTCG-3′

Tm 52.1℃/50.6℃PCR primer length: 999bp

Example 5: Detection of IDD14 protein levels in transgenic plants and wild-type Arabidopsis

With Arabidopsis Columbia wild type and 2 independent T3 transgenic Arabidopsis plants obtained in Example 4 as materials, the soluble protein of leaves was extracted, and Western blot was used to detect the IDD14 protein level in Arabidopsis leaves. The specific method is as follows: collect 2g leaves from the above materials, fully grind them into powder in liquid nitrogen, add an appropriate amount of protein extraction buffer (0.5M Tris-MES, pH=8.0, 0.5mM EDTA and protease inhibitor mixture), mix, Place on ice for 30 minutes. Then centrifuge at 12,000 g at 4°C for 20 minutes, filter the supernatant with a filter cloth, and freeze it at minus 80°C for future use. Protein electrophoresis was performed using 10% SDS-PAGE. After electrophoresis, transfer to PVDF membrane (Millipore, Billerica, MA) by blotting. Then, the expression of IDD14 protein was reflected by detecting the level of MYC tag protein fused with IDD14 protein. As shown in Figure 5 (wherein 35S-IDD14 represents the plant transgenic for IDD14; 12-1 and 3-2 represent different positive transgenic lines), in two independent transgenic Arabidopsis plants (12-1 and 3-2 ), the level of IDD14 protein was significantly higher than that of Arabidopsis Columbia wild-type plants.

The mutant idd14-1D used in the present invention is a mutant with abnormal leaf development obtained by screening from an Arabidopsis T-DNA mutant library containing an activation tag, and has an obvious leaf roll-down phenotype. The previous genetic analysis showed that idd14-1D is a dominant mutant with a single T-DNA insertion, and its phenotype is caused by the overexpression of IDD14. In the T3 generation plants of the mutant, it was found that the drought stress tolerance of the Arabidopsis plants was significantly higher than that of the wild-type plants: after dehydration and rehydration under the same conditions, it was found that 100% of the overexpressed IDD14 gene plants could return to normal growth , while only about 2% of the corresponding wild-type plants returned to normal growth. Therefore, overexpressing the IDD14 gene in Arabidopsis is of great significance for improving the drought stress tolerance of Arabidopsis, which provides support for the research on the drought resistance of corn, rice, wheat and other food crops and other crops.

All reagents, operation steps and methods used in the present invention are commonly used reagents, operation steps and methods in the art.

The above description is only a preferred embodiment of the present invention, and is not used to limit the implementation scope of the present invention; if it does not depart from the spirit and scope of the present invention, any modification or equivalent replacement of the present invention shall be covered by the claims of the present invention within the scope of protection.

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ataacttgcc acaattgcaa gcaacatttt caagctccgg ctgctttggt tcctcctcct 900

cctcagacgc attgtaccga tgagagcacg tctctggccg tgagctacat gtcttcggcg 960

actaccgaag gagaaaaggc gagtgataga gcatcttcat ag 1002

<210> 3

<211> 30

<212>DNA

<213> Artificial sequence

<400> 3

gggcccccat gcatagaaga cgacataaag 30

<210> 4

<211> 27

<212>DNA

<213> Artificial sequence

<400> 4

gagctcctga agatgctcta tcactcg 27

Claims (9)

1. application of the arabidopsis IDD14 genes in plant drouhgt stress patience is lifted, in the IDD14 gene coded sequences table SEQ ID No：The protein of amino acid sequence shown in 1.

2. application as claimed in claim 1, it is characterised in that the sequence of the IDD14 genes is the CDS sequences of the gene.

3. application as claimed in claim 2, it is characterised in that SEQ in the nucleotide sequence of the IDD14 genes such as sequence table ID No：Shown in 2.

4. the application as described in claim any one of 1-3, it is characterised in that by the IDD14 gene transfered plant cells, group Knit or organ, by the plant cell after importing, tissue or organ culture into plant, the IDD14 genes is expressed in plant, Obtain the plant of drought stress patience lifting.

5. application as claimed in claim 4, it is characterised in that the IDD14 genes import plant by plant expression vector Cell, tissue or organ.

6. application as claimed in claim 5, it is characterised in that the plant is corn, rice, wheat or arabidopsis.

7. application as claimed in claim 5, it is characterised in that the plant expression vector is pVIPMyc.

8. application as claimed in claim 5, it is characterised in that the plant expression vector passes through a kind of composing type or induction type Promoter drives the expression of the IDD14 genes.

9. application as claimed in claim 8, it is characterised in that cauliflower mosaic virus is used in the plant expression vector 35S promoter drives the expression of the IDD14 genes.