CN107827963B

CN107827963B - Application of arabidopsis IDD14 gene in improving drought stress tolerance of plants

Info

Publication number: CN107827963B
Application number: CN201711133291.1A
Authority: CN
Inventors: 崔大勇; 刘婧; 束德峰; 张晓�; 刘艳; 王雪茹; 张璐瑶; 刘佳
Original assignee: Qilu Normal University
Current assignee: Qilu Normal University
Priority date: 2017-11-15
Filing date: 2017-11-15
Publication date: 2020-05-15
Anticipated expiration: 2037-11-15
Also published as: CN107827963A

Abstract

The invention discloses an application of an Arabidopsis IDD14 gene in improving drought stress tolerance of plants, wherein the IDD14 gene codes SEQ ID No: 1. According to the invention, the arabidopsis IDD14 gene is overexpressed in arabidopsis thaliana to improve the drought tolerance of arabidopsis thaliana plants for the first time, so that a new thought is provided for cultivating high-drought-resistant arabidopsis thaliana varieties, and a theoretical support is provided for improving the drought resistance of other crops by utilizing a homologous gene technology. The IDD14 gene can provide support for the research of drought resistance of grain crops such as corn, rice, wheat and the like and other crops.

Description

Application of arabidopsis IDD14 gene in improving drought stress tolerance of plants

Technical Field

The invention relates to the technical field of plant genetic engineering, in particular to an arabidopsis gene for improving plant drought stress tolerance and application of the gene.

Background

Drought is a worldwide problem, arid and semiarid regions of the world already occupy more than 1/3 of the land area, and the influence of drought on plants is the first of various natural adversity factors. Grain loss caused by drought accounts for more than half of grain loss caused by all natural disasters. Under natural conditions, drought stress not only severely affects crop growth and yield, but also limits the distribution of plants. Therefore, the analysis of the molecular mechanism of drought stress tolerance of plants and the cultivation of new varieties of high-yield and stress-resistant crops become a serious problem with high urgency. 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 drought-resistant genetic engineering mainly include the following classes.

First, genes involved in the synthesis of osmoprotective substances (e.g., proline, mannitol, betaine, trehalose, etc.). The genes can enable plants to synthesize more osmoregulation substances under the water stress so as to improve the osmoregulation capability of the plants and further enhance the drought resistance of the plants. For example, overexpression of key enzyme genes in the proline biosynthetic pathway in rice (P5CS, deltal-pyroline-5-carboxylateesynthase) increases drought resistance in transgenic plants (Zhu et al, Plant Sci, 1998, 199: 41-48).

Second, genes associated with Reactive Oxygen Species (ROS) scavenging. The expression of the gene enhances the scavenging ability of the plant to active oxygen free radicals, and the plant can over-express some enzymes (such as SOD, POD, CAT and the like) under the water stress to effectively eliminate harmful active oxygen free radicals, thereby improving the dehydration resistance of cells. Such as the arabidopsis thaliana drought-responsive NAC transcription factor NTL4, can promote reactive oxygen species production during drought-induced leaf senescence by binding directly to the promoter region of the ROS synthase gene. In contrast, ROS levels were shown to be reduced in NTL4 mutants lacking the NTL4 gene, to retard leaf senescence, and to enhance drought resistance (Lee et al, Plant Journal, 2012, 70: 831-844).

Third, a gene encoding a drought-induced protein. These proteins can be divided into two categories, (i) regulatory proteins with indirect protective effects, mainly including G-proteins, calmodulin, protein kinases, phospholipases, transcription factor proteins, etc.; (ii) functional proteins include ion channel proteins, LEA proteins (Late Embryogenesis Abundat proteins), heat shock proteins, aquaporins, osmoregulatory proteins, metabolic enzymes, and the like. In the drought tolerance process, the proteins can directly play an important protection role in cells, and the drought tolerance of plants is improved. For example, overexpression of the LEA gene results in increased tolerance of transgenic plants to drought, although the exact mechanism is not known. LEA proteins may also act as chaperones to protect molecules against cell damage (Umezawa et al, Current Opinion in Biotechnology, 2006, 17: 113-.

Fourth, a regulatory gene. Such genes include genes associated with the ABA pathway, including ABA biological metabolism-related genes (e.g., NCED and ABAox) and ABA signaling pathway-related genes (e.g., genes encoding bZIP-type, Myb-type, zinc-finger-type transcription factors). For example, the isolated ERF/AP2 transcription factor families CBF/DREB1 and DREB2 can bind to the cis acting element DRE/CRT, and the tolerance of the over-expressed CBF/DREB1 transgenic plants to freezing, drought and salt stress is increased (Liu et al, Plant Cell, 1998, 10: 1391-. The activation form of the trans-acting factor DREB2 can activate the expression of a stress-induced gene to improve the drought resistance of Arabidopsis (Sakuma et al, PNAS, 2006, 103: 18822-18827). Overexpression of other endogenous ABA-regulated genes such as RD22(Abe et al, Plant Cell, 2003, 15: 63-78), RD29B (Fuiiita et al, Plant Cell, 2005, 17: 3470-. The encoded protein of the HRD gene (HARDY) of the model plant Arabidopsis is AP2/ERF like transcription factor, the gene shows enhanced drought resistance and salt tolerance in the Arabidopsis thaliana function-gain mutant HRD-D, and the gene also shows a phenotype with improved water utilization efficiency consistent with the drought resistance when being transformed into crop rice (Karaba et al, PNAS, 2007, 104: 15270-.

Due to the lack of understanding of the molecular mechanism of drought resistance of plants, the breeding of drought-resistant molecules is also highly blind. And the drought resistance of the plant is usually the result of the joint expression of a plurality of drought resistance genes, the effect of improving the drought resistance of the plant by adopting a single-gene strategy is not obvious in the practical production application, and if the drought resistance reaction capability of the plant can be integrally regulated and controlled by changing the expression of one gene, the drought resistance effect is an ideal choice.

As early as 1998, Colasanti et al found a gene ID1(INDETERMINATE1) that controls flowering in maize, the protein encoded by which is a transcription factor containing four zinc finger structures. Subsequent studies found that several transcription factors, named INDETERMINATE Domain, contain domains resembling the zinc finger of ID1, and these transcription factors are also named IDD. IDDs are transcription factors specific to plants, and maize, rice and arabidopsis contain 21, 15 and 16 IDD genes, respectively. The protein sequence of the rice OsID1/Ehd2/RID1 has high homology with the maize ID1 and is also a determinant for controlling rice flower formation. OsIDD14/LPA1(Loose plant architecture1) is involved in the regulation of the stem gravity and plant type of rice. Unlike few IDD genes in maize and rice, the IDD gene of Arabidopsis thaliana has been studied. IDD8/NUC (NUTCRACKER) regulates flowering by regulating sucrose metabolism, IDD3/MAG (MAGPIE), IDD8/NUC and IDD10/JKD (JACKDAW) regulate root development, while IDD1/ENY (ENHYDROUS) is involved in regulating seed maturation and germination.

The excavation of the excellent drought-resistant gene is important for cultivating the drought-resistant crops which can be produced and applied in the field.

Disclosure of Invention

The invention aims to provide an arabidopsis gene for improving plant drought stress tolerance, an overexpression vector and an arabidopsis gene coding protein.

In order to achieve the purpose, the invention adopts the following technical scheme:

the gene for improving the drought stress tolerance of the plant, which is provided by the invention, is named IDD14, is derived from arabidopsis thaliana and encodes the following proteins: SEQ ID No: 1.

The nucleic acid sequence of the arabidopsis IDD14 gene for improving the drought stress tolerance of the plant can be a CDS sequence of the gene or a DNA sequence which has more than 90 percent of consistency with the sequence and encodes the same functional protein. SEQ ID No: shown in FIG. 2 is the sequence of IDD14 gene.

The invention also provides an expression vector comprising the nucleic acid sequence and an expression control sequence operatively linked to the nucleic acid sequence. Further, the expression control sequence comprises a constitutive high-expression control sequence, and can be a cauliflower mosaic virus 35S promoter.

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

The IDD14 gene is expressed to improve the drought stress tolerance of plants such as arabidopsis thaliana, and the like, and the method comprises the steps of introducing the IDD14 gene into plant cells, tissues or organs, culturing the introduced plant cells, tissues or organs into plants, and expressing the IDD14 gene in the plants to obtain the plants with improved drought stress tolerance.

Further, the IDD14 gene 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. The DNA sequence having 90% or more identity to the sequence and encoding the same functional protein is obtained by isolating, modifying and/or designing the CDS sequence of the gene by a known method. It will be appreciated by those skilled in the art that methods for altering or shortening gene sequences, as well as methods for testing the effectiveness of such altered 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 a plant expression vector. The plant expression vector is pVIPMyc (Cui et al, PLOS Genetics, 2013, 9: e1003759), which is a plant genetic transformation vector commonly used in the world and is formed by transformation on the basis of pVIP 96. When the IDD14 gene or its homologous sequence component of the present invention is used as a plant expression vector, any constitutive or inducible promoter may be added before its transcription initiation nucleotide. The constitutive promoter can be a cauliflower mosaic virus (CAMV)35S promoter, a rice Actin promoter or a maize Ubiquitin promoter and the like; the inducible promoter can be a promoter induced by low temperature, drought, ABA, ethylene, saline or chemical and the like. The above promoters may be used alone or in combination with other plant promoters.

The plant expression vector carrying the IDD14 gene or its homologous sequence can be used for transforming plant cells, tissues or organs by any one or combination of several of the conventional biological methods such as agrobacterium mediation, Ti plasmid, plant virus vector, microinjection, gene gun and the like, and culturing the introduced plant cells, tissues or organs into plants; the plant tissue or organ referred to in the present invention includes seeds, flower buds, fruit pods, leaves, flower stems and the like of the plant. The plant may be maize, rice, triticale or arabidopsis.

The invention leads the nucleic acid sequence of the IDD14 gene to be over-expressed in Arabidopsis thaliana, and the result shows that the level of the IDD14 protein is obviously higher than that of the wild type in 2 independent transgenic Arabidopsis thaliana plants.

The invention obtains a mutant IDD14-1D with enhanced drought resistance by screening an arabidopsis thaliana activation mutant library, wherein the drought resistance phenotype of the mutant is caused by overexpression of a gene IDD14 for coding an IDD transcription factor. Therefore, the over-expression of the IDD14 gene in the arabidopsis thaliana has important significance for improving the drought stress tolerance of the arabidopsis thaliana, and a new thought is provided for cultivating a new variety with high drought resistance.

The invention has the beneficial effects that:

(1) the invention provides application of a gene IDD14 for improving drought stress tolerance of arabidopsis thaliana. After the IDD14 gene is over-expressed in arabidopsis ecological Columbia, the drought stress tolerance of a mutant plant is obviously improved. After dehydration and rehydration under the same conditions, 100% of mutant plants were found to be able to recover normal growth, while only about 2% of corresponding wild type plants recovered normal growth.

(2) The invention overexpresses the Arabidopsis IDD14 gene in Arabidopsis for the first time. Provides a new idea for cultivating high drought-resistant arabidopsis thaliana varieties and also provides theoretical support for improving drought resistance of other crops by utilizing homologous gene technology.

(3) The IDD14 gene applied in the invention can provide support for the research of drought resistance of grain crops such as corn, rice, wheat and the like and other crops.

Drawings

FIGS. 1A and 1B are graphs comparing drought stress tolerance of idd14-1D plants of the invention with wild type plants;

FIGS. 2A and 2B are graphs comparing the rate of water loss from leaves of idd14-1D plants and wild type plants;

FIG. 3 is a graph comparing stomatal density of idd14-1D plants and wild type plants;

FIGS. 4A and 4B are graphs comparing the drought stress tolerance of transgenic plants overexpressing the IDD14 gene and wild-type plants;

FIG. 5 is a graph showing the results of Western blot detection of the expression level of IDDl4 protein in T3 transgenic positive plants.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

The following examples define the invention and describe the methods used in the invention for isolating and cloning the IDD14 gene and for verifying function. From the following description and these examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions.

Example 1: screening over-expression IDD14 gene plant

The mutant idd14-1D used in the invention is a mutant with abnormal leaf development, which is obtained by screening an arabidopsis T-DNA mutant library (Cui et al, PLOS Genetics, 2013, 9: e1003759) containing an activation tag, and has an obvious leaf roll-down phenotype. It was shown by previous genetic analysis that IDD14-1D is a dominant mutant with a single T-DNA insertion, and that the phenotype of mutant IDD14-1D is caused by overexpression of IDD 14.

Example 2: IDD transcription factor subfamily forward-regulated arabidopsis drought stress tolerance reaction

The IDD14-1D mutant plants of over-expression IDD14 gene and the tolerance reaction of the control wild plants to drought stress are analyzed by applying a drought treatment experiment, measuring the water loss rate of the leaves in vitro and comparing the leaf stomatal density.

Firstly, IDD14-1D mutant plants of over-expression IDD14 gene have stronger drought stress tolerance

Weighing a certain amount of idd14-1D mutant plants and seeds of wild type Arabidopsis plants, sterilizing, then, carrying out low temperature 2-3 days at 4 ℃, sowing the seeds on an MS culture medium, continuously culturing for 7 days by illumination, and transplanting seedlings. Mixing vermiculite and nutrient soil according to a ratio of 1: 1, weighing the same weight of nutrient soil (about 70-85 g) in small pots with flat bottoms, watering the bottoms to completely wet the soil, planting 9 seedlings in each small pot, covering with a film, and covering 12 small pots in each disc. The culture conditions of the growth chamber are as follows: 21 ℃ (light)/18 ℃ (dark), photoperiod: 12h (light)/12 h (dark), and the illumination intensity is 80-120 mu mol.m^-2·s^-1And the relative humidity is about 50%. And 3, uncovering the film after 3 days, continuing to grow for 7 days, selecting small pots with consistent growth for drought treatment, randomly placing the small pots of different strains, frequently changing the positions of the small pots, and reducing the position effect. The same experiment was repeated 3 times more, and the results showed that after 20 days of watering stopped, wild type arabidopsis plants underwent wilting earlier than IDD14-1D mutant plants overexpressing IDD14 gene, and statistical results showed that one hundred percent of IDD14-1D mutant plants overexpressing IDD14 gene could recover normal growth, while the corresponding wild type plants recovered normal growth by only about 2% after water addition for 3 days (as shown in fig. 1A-1B, where fig. 1A is WT (wild type arabidopsis plants) and IDD14-1D (IDD 14-1D mutant plants overexpressing IDD14 gene) controls, drought treatment and rehydration phenotypes fig. 1B is the survival rate after rehydration of WT and IDD14-1D, representing that each heritable material has a very significant difference from WT (p < 0.01)).

(II) the water loss rate of the IDD14-1D mutant plant leaves overexpressing IDD14 gene is reduced

The in vitro leaf water loss experiment method comprises the following steps: idd14-1D mutant plants and wild type Arabidopsis thaliana plants seeds were sterilized, cold for 3 days, sown in 1/2MS medium, cultivated in continuous light for 7 days, transferred to soil, and grown for about four weeks in a 21 deg.C (light)/18 deg.C (dark), 12h (light)/12 h (dark) growth chamber. Before flowering, the overground part is cut off and immediately placed into a culture dish for weighing, the leaf dehydration experiment is carried out at the temperature of 22 +/-1 ℃, the weighing is carried out at certain intervals, each strain is repeated for 4 times, and the leaf dehydration rate is calculated according to the percentage of losing the initial fresh weight. As shown in fig. 2A-2B (fig. 2A shows the water loss phenotype of WT and IDD14-1D excised leaves, Bar 1 cm; fig. 2B shows the water loss rate of WT and IDD14-1D excised leaves represents a significant difference (p < 0.05) between the respective genetic materials and WT), the results indicate that the water loss rate of the excised leaves of IDD14-1D mutant plants overexpressing the IDD14 gene is significantly lower than that of the wild-type plants (p < 0.05).

(III) reduction of stomatal density in IDD14-1D mutant plants overexpressing IDD14 Gene

The same parts of idd14-1D mutant and wild type Arabidopsis thaliana plants, which are completely expanded, are cut off for 5-6 weeks (grown at 12h (light)/12 h (dark)), and placed in buffer. For each treatment, 3 leaves were taken for parallel experiments, and the epidermal strips were torn off, the mesophyll cells were brushed off and observed under a 10X 20 times Leica optical microscope. From 3 to 5 fields per epidermis were randomly taken and the number of stomata was recorded. Each treatment is repeated for 4-6 times, and the average value of the number of air holes and the error in a parallel experiment are counted. As shown in fig. 3 (where IDD14-1D represents IDD14-1D mutant plants overexpressing IDD14 gene, representing a very significant difference (p < 0.01) in the respective genetic materials compared to WT (wild-type arabidopsis plants)), 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 that the difference was very significant (p < 0.01).

Guard cell pore observation buffer:

500mM KCl (10 ×): 3.7275g dissolved in 100mL H₂And (4) in O.

5.0mM CaCl₂(50X): 0.0555g dissolved in 100mL H₂And (4) in O.

100mM Mes/KOH (10X): 1.952g was dissolved in 100mL double distilled water and the pH was adjusted to 6.1 with 1.0M KOH.

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

Example 3: DNA fragment for constructing IDD14 gene plant expression vector by isolated cloning

Total RNA was extracted from leaves of Arabidopsis thaliana using TRIzol reagent (purchased from Invitrogen). The method comprises the following specific steps: a precooling centrifuge, adding 50-100 mg of sample into liquid nitrogen, fully grinding, adding 1mL of TRIzol solution, and standing for 5min at room temperature; adding 200 μ L chloroform, shaking vigorously for 15s, and standing at room temperature for 3 min; centrifuging at 12,000g for 15min at 4 ℃; sucking the supernatant into a new 1.5mL centrifuge tube (RNase-free, ribonuclease-free) (purchased from AXYGEN), adding 1 volume of isopropanol, mixing, and standing at room temperature for 10 min; centrifuging at 12,000g for 10min at 4 deg.C; the supernatant was decanted off and 1mL of 70% ethanol solution was added to bounce the precipitate; centrifuging at 7,500 g for 5min at 4 deg.C; the supernatant was decanted off, centrifuged for a short time and the remaining ethanol was aspirated off; after leaving the mixture at room temperature for 10 minutes with an opening, 50. mu.L of RNase-free water was added and dissolved sufficiently, and the concentration of RNA was measured by an ultraviolet spectrophotometer.

It was reverse-transcribed into cDNA using reverse transcriptase (purchased from Invitrogen corporation) according to the following specific steps: adding 2. mu.g of total RNA, 1. mu.l of 10 Xdigestion buffer, 1. mu.l of DNase I (deoxyribonuclease I) (RNase-free) and DEPC water (MilliQ pure water treated with diethyl pyrocarbonate and sterilized at high temperature and high pressure) to 10. mu.l in this order to prepare a DNA digestion reaction solution, digesting at 37 ℃ for half an hour, adding 1. mu.l of 25mM EDTA, and inactivating at 65 ℃ for 10 min; to the digestion product, 1. mu.L of 50mM Oligo (dT)18 and 1. mu.L of 10mM dNTP (deoxyribose-nucleoside triphosphate) were added, mixed well, denatured at 65 ℃ for 5min, and rapidly cooled in an ice bath for at least 1min after the reaction. Then, 4. mu.l of 5-fold concentration primary strand buffer, 1. mu.l of 0.1M DTT, 0.4. mu.l of RNase (ribonuclease) inhibitor and 0.6. mu.l of reverse transcriptase (200U/. mu.l) were added in this order, mixed well and incubated at 50 ℃ for 1 hour. Then, the reaction was terminated by inactivating the enzyme in a 70 ℃ water bath for 15 minutes, thereby synthesizing the first strand cDNA and amplifying the desired gene using the first strand cDNA as a template.

The forward primer IDD14F (5'-gggcccccATGCATAGAAGACGACATAAAG-3', sequence specific primer plus ApaI site and two protecting bases, SEQ ID NO: 3) and the reverse primer IDD14R (5'-gagctccTGAAGATGCTCTATCACTCG-3', sequence specific primer plus XhoI site and one protecting base, SEQ ID NO: 4) with restriction sites were used. Amplifying a target fragment by using high-fidelity Phusion DNA polymerase (purchased from Thermo company), wherein the PCR reaction condition is that the pre-denaturation is carried out for 30 seconds at 98 ℃; 10 seconds at 98 ℃, 30 seconds at 51 ℃, 30 seconds at 72 ℃ and 30 cycles; and the temperature is 72 ℃ for 10 minutes. Detecting a target band by agarose gel electrophoresis, and recovering the corresponding target band by using a DNA gel recovery kit (purchased from Vigorous company); ligation sequencing vector

(purchased from TransGen). Positive clones were selected and sequenced to obtain the desired DNA fragment, which was designated pEASY-IDD14 cDNA.

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

In order to better analyze the function of IDD14 gene, the applicant increased the expression level of IDD14 gene in Arabidopsis thaliana by 35S overexpression technique, and studied the function of the gene according to the phenotype and physiological characteristics of transgenic plants.35S overexpression IDD14 gene plant expression vector was constructed by using cauliflower mosaic virus (CAMV)35S promoter, first double digesting the positive clone pEASY-IDD14cDNA obtained in example 3 with ApaI and XhoI, recovering insert fragment, similarly digesting pVIPMyc plant expression vector by the same method, recovering vector fragment, using the recovered insert fragment and vector fragment for ligation reaction, transforming E.coli DH5 α, screening positive clones by enzymatic cleavage, obtaining plant expression vector named pVIPMyc-IDD 14. pVIPMyc is a commonly used plant genetic transformation vector in International, which is transformed on the basis of pVIP 96. pPMyc-IDD 14 is transformed into EHA105 (a host of Agrobacterium tumefaciens, a host).

The transgenic arabidopsis thaliana is introduced into an arabidopsis thaliana Columbia type through agrobacterium-mediated arabidopsis thaliana genetic transformation, and 30 independent transgenic arabidopsis thaliana plants are obtained through transformation. The method comprises the following specific steps: cutting off bloomed flowers and siliques on the bolting and flowering plants, and reserving unopened flower buds; agrobacterium of the plant to be transformed was inoculated into LB (Luria-Bertani) liquid medium containing the corresponding antibiotic and cultured overnight at 220rpm at 28 ℃ to OD₆₀₀About 1.8; centrifuging at room temperature at 6,000rpm for 10min to collect thallus; the supernatant was decanted off and an equal volume of the staining solution (5.0% sucrose solution + 0.025% (v/v) Silwet-L77) was added to resuspend the cells; putting the resuspended bacterial liquid into a culture dish, and soaking the flower stem of arabidopsis thaliana in the staining solution for sufficient soaking; watering the transformed plants, covering the plants with a plastic bag for moisturizing, and removing the plastic bag for normal culture after about 24 hours; and (3) slowly maturing the transformed material after 3 weeks, collecting transgenic material seeds, screening on an 1/2MS culture medium containing corresponding vector resistance to obtain T1 generation transgenic positive seedlings, further selfing T1 generation materials, determining whether the single copy insertion is achieved through a T2 generation resistance segregation ratio, and obtaining a homozygous transgenic line at T3 generation.

As shown in FIGS. 4A-4B, the drought stress tolerance of the IDD14 gene transgenic plant and the wild-type plant is compared. Wherein 35S-IDD14 represents a transgenic plant overexpressing the IDD14 gene. FIG. 4A shows WT and 35S-IDD14 drought treatment and rehydration phenotypes, 12-1 and 3-2 representing different positive transgenic lines, Bar 1 cm. FIG. 4B shows the survival rate after rehydration of different positive transgenic lines WT and 35S-IDD14, representing a very significant difference (p < 0.01) between the respective genetic material and WT. Selecting transgenic plants and wild plants with consistent growth for drought treatment, randomly placing small pots of different strains, frequently changing the positions of the small pots, and reducing the position effect. The same experiment is repeated for more than 3 times, and the result shows that after the watering is stopped for 20 days, the wild plants have a wilting phenomenon earlier than the transgenic plants, and after all the plants have the wilting phenomenon, the statistical result shows that after the watering is recovered for 3 days, fig. 4B shows that the recovery rate of the 35S-IDD14 plants reaches 100%, namely, the transgenic plants with hundred percent over-expression of the IDD14 gene can recover normal growth, and the corresponding wild plants recover normal growth only by about 2%.

The gene IDD14 was ligated into the sequence of the PVIPMyc vector:

IDD14ApaI F 5′-gggcccccATGCATAGAAGACGACATAAAG-3′

IDD14XhoI R 5′-gagctccTGAAGATGCTCTATCACTCG-3′

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

Example 5: detecting IDD14 protein level of transgenic plant and wild arabidopsis thaliana

Soluble proteins of leaves are extracted from arabidopsis thaliana columbia wild type and 2 independent T3 transgenic arabidopsis thaliana plants obtained in example 4, and the level of IDD14 protein in arabidopsis thaliana leaves is detected by Western blot. The specific method comprises the following steps: 2g of leaf pieces were collected from the above material, ground well in liquid nitrogen to powder, added with an appropriate amount of protein extraction buffer (0.5M Tris-MES, pH 8.0, 0.5mM EDTA and protease inhibitor mixture), mixed well, and left on ice for 30 minutes. Then, 12000g of the mixture is centrifuged at 4 ℃ for 20 minutes, and the supernatant is filtered by a filter cloth and then frozen at minus 80 ℃ for standby. Protein electrophoresis was performed by 10% SDS-PAGE. After the electrophoresis was completed, the gel was transferred to a PVDF membrane (Millipore, Billerica, MA) by blotting. Expression of IDD14 protein was then reflected by measuring the level of MYC tag protein fused to IDD14 protein. As shown in FIG. 5 (where 35S-IDD14 represents plants transgenic for IDD 14; 12-1 and 3-2 represent different positive transgenic lines), the level of IDD14 protein was significantly higher in 2 independent transgenic Arabidopsis plants (12-1 and 3-2) than in Arabidopsis Columbia wild-type plants.

The mutant idd14-1D used in the invention is a mutant with abnormal leaf development, which is obtained by screening an arabidopsis T-DNA mutant library containing an activation tag, and has an obvious leaf rolling phenotype. It was shown by preliminary genetic analysis that IDD14-1D is a dominant mutant with a single T-DNA insertion, and its phenotype is caused by overexpression of IDD 14. In the mutant T3 generation plants, the drought stress tolerance of the Arabidopsis plants is found to be significantly higher than that of the wild type plants: after dehydration and rehydration under the same conditions, 100% of over-expressed IDD14 gene plants can recover normal growth, while only about 2% of corresponding wild plants recover normal growth. Therefore, the over-expression of the IDD14 gene in Arabidopsis has important significance for improving the drought stress tolerance of Arabidopsis, and provides support for the research on the drought resistance of grain crops such as corn, rice, wheat and the like and other crops.

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

The above description is only for the preferred embodiment of the present invention, and is not intended to limit the scope of the present invention; it is intended that the following claims be interpreted as including all such alterations, modifications, and equivalents as fall within the true spirit and scope of the invention.

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gaaagtttca tagagcacca agacacttgc accgtacgcc gatcccaacc ctccaaccac 360

cgtttacatg agcagcaaca acataccaca aacgctacac aaaccgcttc aaccgcggaa 420

aacaacgaga acggggacct ctccattggt cctatattgc ctggacatcc tttacaaaga 480

agacaatccc caccgtcgga acaacaacca tccactttgc tctatccctt cgttactaat 540

ggtagtatcg agcttcagct acttccatcg aggaattgtg ctgatgagac cagccttagt 600

ctgtctatag ggacaatgga tcaaaagaca atgtcggaag ttgagaagaa gagctacgag 660

aagggagaaa cgagcctaga aagagaggag gcgagaagag aaacaaagag gcagatcgaa 720

atcgcggaat tggagtttgc tgaagccaag agaataaggc aacatgcgag agctgagctt 780

cacaaagctc atctttttag agaagaagca agtaggagga ttagtgcaac gatgatgcaa 840

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

1. An application of an Arabidopsis IDD14 gene in improving the drought stress tolerance of Arabidopsis, wherein the IDD14 gene encodes a protein of an amino acid sequence shown by SEQ ID No. 1 in a sequence table.

2. The use of claim 1, wherein the sequence of the IDD14 gene is the CDS sequence of the gene.

3. The use of claim 2, wherein the nucleotide sequence of IDD14 gene is shown as SEQ ID No. 2 in the sequence Listing.

4. The use according to any one of claims 1 to 3, wherein the IDD14 gene is introduced into a plant cell, tissue or organ of Arabidopsis thaliana, the introduced plant cell, tissue or organ of Arabidopsis thaliana is cultured into a plant, and the IDD14 gene is expressed in Arabidopsis thaliana to obtain Arabidopsis thaliana with improved drought stress tolerance.

5. The use according to claim 4, wherein the IDD14 gene is introduced into a plant cell, tissue or organ of Arabidopsis thaliana by means of a plant expression vector.

6. The use of claim 5, wherein the plant expression vector is pVIPMyc.

7. The use according to claim 5, wherein said plant expression vector drives the expression of said IDD14 gene through a constitutive or inducible promoter.

8. The use as claimed in claim 7 wherein the expression of the IDD14 gene is driven in the plant expression vector using the cauliflower mosaic virus 35S promoter.