Rose RcMYB14 transcription factor and its application

Abstract

The present invention discloses a rose RcMYB14 transcription factor and its application; the nucleotide sequence of the rose 'Yueyuefen' RcMYB14 transcription factor is shown in SEQ ID No.1; the sequence of the corresponding protein is shown in SEQ ID NO:2. The RcMYB14 gene can be induced to express by salt stress and high temperature stress. The gene is transformed into tobacco and Arabidopsis thaliana by Agrobacterium-mediated genetic transformation, which can significantly reduce the damage of plants caused by salt and high temperature stress, and can increase the growth of transgenic Arabidopsis thaliana, and significantly enhance the salt tolerance and heat tolerance of transgenic plants. The present invention lays a foundation for the study of the molecular mechanism of plant salt tolerance and high temperature resistance, and provides excellent genes for the future breeding of salt-tolerant and high temperature-resistant plant varieties by molecular technology; in addition, the transcription factor of the present invention provides an important possibility for breeding stress-resistant rose varieties, which has practical significance for the production of roses.

Description

Technical Field

[0001] The invention relates to the technical field of plant genetic engineering, and in particular to a rose RcMYB14 transcription factor and an application thereof. Background Art

[0002] Soil salinization, temperature stress and drought are the main environmental factors that affect the geographical distribution of plants in nature and limit crop production. In addition, with the global warming, the greenhouse effect is becoming more and more significant. Plants are frequently subjected to various stress conditions during growth, such as high temperature, drought, floods, etc., which aggravates the adverse effects of abiotic stress on plant growth and development, resulting in reduced plant yield and deterioration of quality (Liu Ling et al., 2018). Therefore, how to enhance plant tolerance to abiotic stress, alleviate abiotic stress damage, and improve crop yield and quality has always been a hot research issue.

[0003] When roses are subjected to salt stress, their growth is inhibited, the plants become dwarfed, and the survival rate is reduced, which is manifested by yellowing, drying, and even falling leaves; blackening of the root system and decreased root vitality; reduced number of flowers, smaller flowers, and severely reduced ornamental quality (Fan Hua et al., 2007). Under high temperature stress conditions, rose plants are stunted, the flowering time is shortened and the residual flower period is prolonged, the color of the flowers becomes lighter, the pedicels grow longer, the buds become shorter, and the flower shape is irregular (Shi Liyun et al., 2018). At present, physical and chemical approaches are mainly used in production to alleviate the damage suffered by plants from salt stress and high temperature (Ma Shaoyun, 2017; Zhang Lei et al., 2018; Zhang Huiling et al., 2019; Jia Yechun et al., 2022). In terms of physical approaches, for example, by using cooling equipment, increasing air humidity, CO 2Jiafu et al. alleviate the damage of high temperature stress to plants (Ma Shaoyun, 2017); the chemical pathway mainly improves the salt and heat tolerance of plants through the exogenous application of organic and inorganic substances, such as calcium chloride, sugars, spermidine, plant hormones (abscisic acid, gibberellin, salicylic acid, melatonin, etc.) (Wang Qinghua et al., 2018; Zhao Xiaomeng, 2020; Liu Zheng et al., 2020; Bao Ying et al., 2020; Lu Jianguo et al., 2022); However, the above-mentioned approaches require a large amount of manpower, material and financial resources in production, and most of the impacts on plants are temporary and limited. In addition, in terms of salt and heat tolerance breeding of roses, the focus is mainly on the evaluation of germplasm resource stress resistance and the screening of stress-resistant varieties, in order to obtain varieties through hybrid breeding (Yang Yonghua et al., 2007; Li Ronghua et al., 2017; Zhao Bingxue et al., 2021). However, the hybrid breeding cycle is long, the operation is cumbersome, and the hybrid offspring cannot show the excellent traits of the parents. Therefore, the use of transgenic technology to fundamentally improve the genotype of plants and improve plant traits has become one of the effective means of breeding varieties. Transgenic technology mainly improves plant stress resistance by overexpressing or knocking out genes related to abiotic stress response to improve plant yield and quality (Shen et al., 2019; Zhang et al., 2019). Transcription factors act as a bridge between stress signals and related genes in the signal transduction process of plant response to adverse stress. They receive upstream signals and regulate plant stress resistance by regulating the expression of downstream resistance-related genes. It has been reported that overexpression or knockout of stress-responsive MYB transcription factors in model plants, crops and ornamental plants, such as Arabidopsis, wheat, soybean, Pyrus betulaceae, and rose, can improve the salt tolerance and heat tolerance of plants. Therefore, enhancing the stress resistance of plants through transgenic technology has become a quick and effective measure (Liao et al., 2017; Zhao et al., 2017; Ran Kun et al., 2018; Jiang et al., 2018; Jiang Jing et al., 2019; Bao Ying et al., 2020; Qiu Wenyi et al., 2020; Wang Qingyu, 2021). Summary of the invention

[0004] The purpose of the present invention is to overcome the deficiencies of the prior art, and to provide a rose RcMYB14 transcription factor and its application; the application is the cloning of the nucleotide coding sequence of the RcMYB14 gene (i.e., the RcMYB14 transcription factor), the construction of an expression vector, the complete translation region of the RcMYB14 gene (RcMYB14 transcription factor) is combined with the cauliflower mosaic virus promoter and then transferred into the plant body (host tobacco and Arabidopsis), and the RcMYB14 transgenic plant is obtained, and the transgenic plant is molecularly identified and phenotypic observed to evaluate the application prospect of the gene in regulating plant adverse stress. The salt tolerance and heat tolerance of the transgenic plant are significantly improved under salt stress (200mM NaCl) and high temperature (42°C) conditions. The present invention enhances the tolerance of the plant to salt stress and high temperature stress by means of transgenic technology, and alleviates the damage of salt and high temperature to the plant.

[0005] To achieve the above-mentioned purpose, the present invention designs a rose RcMYB14 transcription factor, whose nucleotide sequence is shown in SEQ ID No.1.

[0006] The present invention also provides a protein RcMYB14 encoded by the above transcription factor, and its amino acid sequence is shown in SEQ ID No.2.

[0007] The present invention also provides a primer pair for obtaining the above-mentioned RcMYB14 transcription factor, wherein the primer pair is:

[0008] RcMYB14 forward primer: 5′-CGGGGTACCATGGTGAGAGCTCCTTGC-3′, RcMYB14 reverse primer: 5′-GCTCTAGATCAAAACTCTGGTAGTTCTGGTGTGTC-3′.

[0009] The present invention also provides a method for obtaining the RrMYB14 transcription factor using the above primer pair, comprising the following steps:

[0010] Using the cDNA of the leaves of rose 'Yueyuefen' as a template and the primer pair of the RcMYB14 transcription factor, PCR amplification was performed and the RcMYB14 transcription factor was purified; wherein the PCR system:

[0011] 10×Buffer 2μl 10mM dNTP 0.4μl Forward Primer 1μl Reverse primer 1μl Taq DNA polymerase 0.2μl template 1μl <![CDATA[ddH 2 The]]> 14.4μl total 20μl

[0012] PCR conditions:

[0013]

[0014] The present invention also provides a recombinant expression vector, which contains the plant expression vector of the RcMYB14 transcription factor, wherein the plant expression vector is pCAMBIA2300s.

[0015] The present invention also provides a method for constructing the above-mentioned recombinant expression vector, comprising the following steps:

[0016] Introduce the target fragment into the cloning vector 18T, and then insert the cloning vector containing the target gene 18T and pCAMBIA2300s were double-digested with KpnⅠ and XbaⅠ, and the obtained target fragment was ligated to the digested pCAMBIA2300s vector to obtain the recombinant expression vector pCAMBIA2300s-RcMYB14.

[0017] The present invention also provides a host cell containing the above recombinant expression vector, wherein the host cell is Agrobacterium GV3101.

[0018] Use of any of the following for enhancing salt tolerance and heat tolerance of plants, comprising

[0019] (1) the above-mentioned RcMYB14 transcription factor;

[0020] (2) the above-mentioned recombinant expression vector;

[0021] (3) The above-mentioned host cells.

[0022] The plant is tobacco or Arabidopsis thaliana.

[0023] Use of any one of the following in breeding salt-tolerant and heat-tolerant plant varieties, comprising:

[0024] (1) the above-mentioned RcMYB14 transcription factor;

[0025] (2) the above-mentioned recombinant expression vector;

[0026] (3) The host cell Agrobacterium GV3101 described above.

[0027] The plant is tobacco, Arabidopsis thaliana or rose.

[0028] Beneficial effects of the present invention:

[0029] The transcription factor of the present invention is named RcMYB14. By constructing an overexpression vector pCAMBIA2300s-RcMYB14 and introducing it into tobacco and Arabidopsis, transgenic plants are obtained. The transgenic plants show stronger salt tolerance and heat tolerance than the control plants. The RcMYB14 transcription factor provides a molecular biological basis for us to regulate the salt resistance and high temperature resistance of plants by means of plant genetic engineering. The application of the transcription factor will help to enhance the stress resistance of plants, increase crop yields, improve plant quality, and cultivate varieties with excellent traits. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Schematic diagram of the structure of the original plant overexpression vector pCAMBIA2300s (vector size is 11630 bp);

[0031] Figure 2 This is the expression of RcMYB14 gene in rose 'Yueyuefen' after salt stress and high temperature stress treatment;

[0032] In the figure: Figure 2 A is the expression of RcMYB14 gene in ‘Yueyuefen’ after salt stress treatment;

[0033] Figure 2 B is the expression of RcMYB14 gene in Yueyuefen after salt stress treatment.

[0034] Figure 3 Analysis of salt tolerance of wild-type tobacco and RcMYB14 transgenic tobacco of the present invention;

[0035] In the figure: Figure 3 A is the positive detection gel image of RcMYB14 transgenic tobacco resistant seedlings;

[0036] Figure 3 B is the gel image of the target gene expression detection in wild-type and RcMYB14 transgenic tobacco;

[0037] Figure 3 C is a phenotypic comparison of 4-week-old wild-type and RcMYB14 transgenic tobacco before and after salt stress treatment;

[0038] Figure 3 D is the MDA content detection diagram of 4-week-old wild-type and RcMYB14 transgenic tobacco before and after salt stress treatment;

[0039] Figure 3 E is a comparison of the phenotypes of 6-week-old wild-type and RcMYB14 transgenic tobacco after water and salt stress treatment;

[0040] Figure 3 F is the chlorophyll content detection diagram of 6-week-old wild-type and RcMYB14 transgenic tobacco after salt stress treatment;

[0041] Figure 3 G is the MDA content detection diagram of 6-week-old wild-type and RcMYB14 transgenic tobacco after salt stress treatment;

[0042] Figure 4 This is a diagram showing the high temperature resistance of wild-type tobacco and the RcMYB14 transgenic tobacco of the present invention;

[0043] In the figure: Figure 4 A is a comparison of the phenotypes of wild-type and RcMYB14 transgenic tobacco before, after and after high temperature treatment;

[0044] Figure 4 B is a comparison of the phenotypes of the middle leaves of wild-type and RcMYB14 transgenic tobacco before, after and after high temperature treatment;

[0045] Figure 4 C is the chlorophyll content detection diagram of wild-type and RcMYB14 transgenic tobacco before, after and after high temperature treatment;

[0046] Figure 4 D is the relative conductivity detection diagram of leaves of wild type and RcMYB14 transgenic tobacco before, after and after high temperature treatment;

[0047] Figure 5 This is a diagram for analyzing salt tolerance of wild-type tobacco and RcMYB14 transgenic Arabidopsis thaliana of the present invention;

[0048] In the figure: Figure 5 A is a comparison of the phenotypes of wild type and RcMYB14 transgenic Arabidopsis thaliana under salt stress;

[0049] Figure 5 B is the gel image of the target gene expression detection in wild type and RcMYB14 transgenic Arabidopsis;

[0050] Figure 5 C is the statistical result of the main root length of wild-type and RcMYB14 transgenic Arabidopsis plants after salt stress;

[0051] Figure 5 D is the statistical result of fresh weight of wild type and RcMYB14 transgenic Arabidopsis plants after salt stress;

[0052] Figure 6 Construction map for the expression vector pCAMBIA2300s-RcMYB14 (insert size 771 bp). DETAILED DESCRIPTION

[0053] The present invention is further described in detail below in conjunction with specific embodiments so that those skilled in the art can understand.

[0054] Example 1: Isolation of RcMYB14 transcription factor

[0055] The RcMYB14 transcription factor was isolated from the rose 'Yueyuefen', and its sequence is shown in SEQ ID No.1:

[0056] ;

[0057] The amino acid sequence of the protein RcMYB14 encoded by the RcMYB14 transcription factor is shown in SEQ ID No.2:

[0058] MVRAPCCEKMGLKKGPWTTEEDQILISYIQNYGHGNWRALPKHAGLLRCGKSCRLRWINYLRPDIKRGNFTREEEDAIINLHEMLGNRWSTIAARLPGRTDNEIKNVWHTHLKKRLLKHNNTTSPTCS TSNLTSQYAEPEDVNFESPTSHASMSPQPSSSDDFSSVTEASAATGQNMIKDEKIELDATFLEIDDSFWSEALSADNSGDNPLEFLEGTNDQAQLQLLLPSSIDDGMDFWYDLFIKAGYGDTPELPEF.

[0059] The cDNA of the leaves of rose 'Yueyuefen' was used as a template to amplify the required fragment. The specific steps are as follows:

[0060] The primer pair of RcMYB14 transcription factor was designed according to the RcMYB14 sequence shown in SEQ ID No.1:

[0061] RcMYB14 forward primer: 5′-CGGGGTACCATGGTGAGAGCTCCTTGC-3′, as shown in SEQ ID No. 3;

[0062] RcMYB14 reverse primer:

[0063] 5'-GCTCTAGATCAAAACTCTGGTAGTTCTGGTGTGTC-3', as shown in SEQ ID No.4.

[0064] PCR was performed using cDNA extracted from the leaves of rose 'Yueyuefen' as a template and the primer pair of the RcMYB14 transcription factor. The PCR system was:

[0065] 10×Buffer 2μl 10mM dNTP 0.4μl Forward Primer 1μl Reverse primer 1μl Taq DNA polymerase 0.2μl template 1μl <![CDATA[ddH 2 The]]> 14.4μl total 20μl

[0066] PCR conditions:

[0067]

[0068] The amplified product was purified, namely the RcMYB14 transcription factor;

[0069] The amplified RcMYB14 transcription factor was linked into 18-T vector (purchased from Takara Biotech Dalian Co., Ltd.), screen the positive clones and sequence them to obtain the required full-length gene; the clone was named 18-RcMYB14 plasmid.

[0070] Example 2: Detection of RcMYB14 gene expression in rose 'Yueyuefen' after salt stress and high temperature stress treatment

[0071] 1) Salt stress treatment of rose 'Yueyuefen' tissue culture seedlings

[0072] In order to detect whether the RcMYB14 gene responds to salt stress, the tissue culture seedlings of rose 'Yueyuefen' were inoculated into rose subculture medium (MS + 0.5 mg / L BA + 0.1 mg / L NAA + 100 μM ABA + 30 g / L Sucrose + 7.5 g / L Agar, PH 5.7) containing 200 mM NaCl, cultured at room temperature, and then sampled at 0, 1h, 6h, 24h and 48h, respectively. The samples treated at the same time point were mixed and collected, quick-frozen in liquid nitrogen, and stored in a -80℃ ultra-low temperature refrigerator for later use.

[0073] 2) High temperature stress treatment of rose 'Yueyuefen' tissue culture seedlings

[0074] In order to detect whether the RcMYB14 gene responds to high temperature stress, the rose 'Yueyuefen' tissue culture seedlings were placed in 45℃ high temperature stress treatment, and then samples were taken at 0, 4h, 9h, 12h and 24h, respectively. The samples treated at the same time point were mixed, quick-frozen in liquid nitrogen, and stored in a -80℃ ultra-low temperature refrigerator for later use.

[0075] 3) Detection of RcMYB14 gene expression in rose 'Yueyuefen' after different stress treatments

[0076] The total RNA extraction method of rose was carried out by CTAB method, and RNA of samples at different time points after 1) salt stress and 2) high temperature stress treatment was extracted. Reverse transcription was performed using TAKARA reverse transcription kit. For specific steps, please refer to the operation manual (RR047A, TaKaRa). The reverse transcription product was stored at -20℃ for future use. The expression of RcMYB14 gene was detected by qRT-PCR method ( Figure 2 qRT-PCR reaction was performed using the TAKARA fluorescent quantitative PCR kit (RR430A, TaKaRa) and the instrument model used was ABIQuantStudio 3 (Applied Biosystems, CA, USA). Each sample was repeated three times, using 2 -△△CT The relative expression levels were calculated by SPSS software, and the raw data were statistically analyzed.

[0077] The specific primer sequences for the qRT-PCR reaction are:

[0078] qRcMYB14-F: 5'-CAAGATTGCCAGGAAGAACTGATAA-3';

[0079] qRcMYB14-R: 5'-CATGACTTGTTGGACTCTCAAAATTC-3'.

[0080] The RcGAPDH gene was selected as the internal reference gene, and the primer sequences were:

[0081] qGAPDH-F: 5'-TGAAGGGTGGTGCCAAGAA-3';

[0082] qGAPDH-R: 5'-AAGGGGAGCAAGACAGTTGG-3'.

[0083] Real-time fluorescence quantitative detection found that after salt stress treatment, the expression level of RcMYB14 gene first increased and then decreased, and the gene expression reached a peak at 6h of treatment ( Figure 2 A); After high temperature stress treatment, the expression level of RcMYB14 gene showed a trend of first increasing, then decreasing, and then increasing again ( Figure 2 B). In summary, the expression of RcMYB14 gene is induced by salt stress and high temperature, and may be involved in the response process of plant to salt stress and high temperature stress.

[0084] Example 3: Construction of RcMYB14 transcription factor overexpression vector, transformation

[0085] In order to better clarify the function of the gene, the cloned gene sequence was overexpressed in tobacco and Arabidopsis, and its function was verified by observing the phenotype of transgenic plants and measuring physiological indicators. The specific steps are:

[0086] First, the positive clones obtained in Example 1 The 18-RcMYB14 plasmid was double-digested with KpnⅠ and XbaⅠ to recover the target fragment; at the same time, the genetic transformation vector pCAMBIA2300s( Figure 1 ). After the enzyme digestion, the enzyme fragment containing RcMYB14 transcription factor and the enzyme-digested pCAMBIA2300s ( Figure 1 ) vector for ligation reaction and transformed into Escherichia coli DH5α (Escherichia coli strain purchased from Takara Biotechnology Dalian Co., Ltd.). Positive clones were screened by enzyme digestion to obtain the transformation vector, which was named pCAMBIA2300s-RcMYB14( Figure 6 ).

[0087] Through the Agrobacterium-mediated tobacco genetic transformation method, pCAMBIA2300s-RcMYB14 was introduced into wild-type tobacco and Arabidopsis thaliana, respectively. After infection, co-cultivation and screening, resistant transformed seedlings were obtained, and then transgenic plants were obtained through conventional steps such as rooting, seedling training and transplanting.

[0088] The main steps and reagents used in the above genetic transformation are as follows:

[0089] 1) Abbreviations of reagents and solutions

[0090] The abbreviations of the plant hormones used in the culture medium are as follows: 6-BA (6-BenzylaminoPurine); NAA (Naphthalene acetic acid); Kan (Kanamycin); Cef (Cefotaxime).

[0091] 2) Culture medium formulation for tobacco genetic transformation

[0092] Table 1 lists the components and amounts of the culture medium for tobacco genetic transformation.

[0093] Table 1 Design of relevant culture medium for transgenic tobacco

[0094]

[0095] 3) Culture medium formulation for Arabidopsis genetic transformation

[0096] Table 2 lists the components and amounts of the culture medium for genetic transformation of Arabidopsis thaliana.

[0097] Table 2 Design of relevant culture medium for transgenic Arabidopsis

[0098]

[0099] 4) Agrobacterium-mediated genetic transformation steps

[0100] (1) Cultivation of Agrobacterium

[0101] First, Agrobacterium was pre-cultured on a solid LB medium (10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, 100 mg / L Kan; 1.5 g / L agar) with corresponding resistance selection for 48 hours at a culture temperature of 28°C; a single colony of pre-cultured Agrobacterium was picked and tested positive using the primer pair of the RcMYB14 transcription factor above, and then the positive clone was inoculated into a liquid LB medium (10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, 100 mg / L Kan) with corresponding resistance selection, and cultured overnight at 28°C 200 rpm shaking incubator until the bacterial solution concentration OD 600 The value is approximately 0.6.

[0102] (2) Agrobacterium transformation of plant tissues and acquisition of resistant seedlings

[0103] 2.1 Tobacco leaf disc transformation method

[0104] a. Cut the fully expanded young leaves from the upper part of the wild-type tobacco sterile seedlings, cut the leaves into small pieces of 1cm×1cm, and put them into a sterile beaker;

[0105] b. Pour the prepared bacterial solution into the beaker and shake the beaker gently. Soak the leaves in the bacterial solution for 8 minutes;

[0106] c. Remove the leaves from step b, transfer them to sterilized filter paper and dry them; then place them on the co-cultivation medium described in Table 2 and culture them in the dark for three days at a culture temperature of 28°C;

[0107] d. Three days later, the leaves were transferred to the budding selection medium as described in Table 1, and cultured alternately under light and dark conditions (light intensity 1000-1500 lx, light duration: 16 h / d, dark duration: 8 h / d) to screen and differentiate Kan-resistant buds at a culture temperature of 28°C;

[0108] e. After the resistant buds are formed, they are cut off and transferred to the seedling selection medium as described in Table 1. They are cultured alternately in light and dark (light intensity 1000-1500 lx, light time: 16 h / d, dark time: 8 h / d) to screen Kan-resistant seedlings. The culture temperature is 28°C.

[0109] f. The resistant seedlings obtained by screening were transferred to the rooting selection medium described in Table 1 to make them take root, and cultured alternately in light and dark (light intensity 1000-1500lx, light time: 16h / d, dark time: 8h / d) at a culture temperature of 28°C.

[0110] 2.2 Arabidopsis thaliana—Inflorescence Dip Method

[0111] a. Cultivate the above culture to a bacterial concentration of OD 600 The Agrobacterium bacterial solution with a value of about 0.6 was centrifuged at 5000 rpm for 8 min to collect the Agrobacterium strain, which was then resuspended with the resuspension solution described in Table 2 above for use in the next transformation step.

[0112] b. Soak all the inflorescences above the stem of Arabidopsis in the bacterial solution for 1 minute. After infection, cover the Arabidopsis with a black plastic bag or film and incubate in the dark for 24 hours. One week after infection, perform a second infection and transformation on this batch of plants. Collect the infected Arabidopsis siliques after they naturally crack. 0 seed.

[0113] c. The received T 0After surface disinfection, the seeds of the first generation were sown in the selection medium of Table 2 for screening. After the resistant seedlings took root, they were moved to culture soil and placed in a light culture box for growth (22°C, long daylight conditions (16 / 8h, day / night)).

[0114] Example 4: Detection of salt and heat tolerance of transgenic tobacco RcMYB14

[0115] 1) Salt stress treatment of transgenic tobacco

[0116] a: Wild-type tobacco and transgenic tobacco at 4 weeks old were placed in distilled water containing 200 mM NaCl for salt stress treatment. After the roots were immersed for 12 h, phenotypes were observed and photographed, and samples were collected to determine the malondialdehyde (MDA) content before and after treatment ( Figure 3 AD).

[0117] MDA content determination method: (1) Weigh 0.2 g of the shredded sample, add 2 ml of 5% TCA to grind into a homogenate, then add 1 ml of 5% TCA to wash slightly, pour all into a centrifuge tube, and then centrifuge the sample at 4500 rpm, 40°C, 20 min; (2) Take 2 ml of the supernatant after centrifugation, add 2 ml of 0.67% TBA solution and mix well, then boil in a boiling water bath for 10 min. After the end, take the test tube and cool it quickly. Use 0.67% TBA solution as a blank to determine A 450 , A 532 , A 600 The OD value under the condition of 100 μmol / g was used to calculate the content of malondialdehyde. 532 -A 600 )-0.56×A450]×V×10 -3 / W, (V: supernatant volume; W: sample fresh weight). The measurement results showed that after salt stress treatment, the MDA content in the leaves of RcMYB14 transgenic tobacco was significantly lower than that in normal tobacco leaves, indicating that the degree of damage to the transgenic plants was lower than that of the control plants. Overexpression of the RcMYB14 gene can improve plant salt tolerance ( Figure 3 D).

[0118] b: 6-week-old wild-type tobacco and transgenic tobacco were irrigated with 200 mM NaCl. After 6 days of salt stress treatment, phenotypic differences appeared between the wild-type and transgenic tobacco. Then, the phenotypic observations and photos of the treated seedlings were taken and the chlorophyll and MDA contents were determined ( Figure 3 EG).

[0119] The chlorophyll content was determined by ultraviolet spectrophotometry: (1) 0.2 g of sample was ground in liquid nitrogen and then added with 2 ml of 95% anhydrous ethanol. The sample was kept in dark for 4-5 h, inverted and mixed every 2 h, and centrifuged at 12,000 rpm for 5 min. The supernatant was carefully pipetted into a new centrifuge tube for determination. (2) Read the A and B values ​​respectively. 665 and A 649 The OD value under the condition was used to calculate the corresponding Chla, Chlb and total chlorophyll contents by the formula. Chla = 13.95A 665 -6.88A 649 ; Chlb=24.96A 649 -7.32A 665 ; Total chlorophyll content = Chla + Chlb. See Arnon 1949. The measurement results showed that after salt stress treatment, the chlorophyll content in the leaves of RcMYB14 transgenic tobacco was significantly higher than that in the leaves of normal tobacco ( Figure 3 F).

[0120] 2) High temperature stress treatment of transgenic tobacco

[0121] The temperature treatment was carried out in a light incubator with a high temperature of 42°C and a photoperiod of 16 h light / 8 h dark. The treatment lasted for 6 days until the wild-type tobacco and the transgenic tobacco showed phenotypic differences ( Figure 4 A), and then place the treated seedlings in a 22°C, 16h light / 8h dark environment for 5 days to recover ( Figure 4 A and Figure 4 B). Phenotypic observations and photographic records of the seedlings were taken before treatment, 6 days after treatment, and 5 days after recovery, and the chlorophyll content and relative conductivity were measured ( Figure 4 ).

[0122] The chlorophyll content of transgenic tobacco was determined by the same experimental method as in 1)b of Example 4 above. The measurement results were consistent with the results after the treatment of 4-week-old tobacco seedlings. The chlorophyll content in the leaves of transgenic tobacco after salt stress treatment was significantly higher than that in normal tobacco leaves ( Figure 4 C).

[0123] Determination of relative conductivity of transgenic tobacco: 1) Take 0.1g sample, cut into pieces, add 5ml distilled water, turn over and mix, shake at 80r / min, shake for 2h, and measure the value C with a conductivity meter 1 ; 2) The centrifuge tube was placed in a water bath at 100°C for 15 minutes, then taken out and cooled to room temperature, and the conductivity C was measured. 2 . Relative conductivity calculation formula: C (%) = (C 1 -C 0 ) / (C2 -C 0 );C 0 is the conductivity of double distilled water. The measurement results show that the relative conductivity of RcMYB14 transgenic tobacco after salt stress treatment is significantly lower than that of the control tobacco, indicating that the degree of damage to the transgenic plants is lower than that of the control plants. Overexpression of the RcMYB14 gene can improve the heat tolerance of transgenic plants ( Figure 4 D).

[0124] Example 5: Salt tolerance test of transgenic Arabidopsis thaliana RcMYB14

[0125] Salt stress treatment of transgenic Arabidopsis: The seeds of transgenic Arabidopsis RcMYB14 and wild-type Arabidopsis Col were surface-sterilized and sown in Arabidopsis germination medium (MS+30 g / L Sucrose+7.5 g / L Agar, PH 5.7). After 3 days of dark treatment at 4°C, they were placed at room temperature with a photoperiod of 16 h light / 8 h dark. After 3 days, the germinated Arabidopsis seedlings were transferred to MS+30 g / L Sucrose+7.5 g / L Agar (PH 5.7) medium containing 0 mM and 200 mM NaCl for salt stress treatment. The treatment lasted for 15 days until phenotypic differences between wild-type and transgenic Arabidopsis appeared ( Figure 5 A), and then the phenotypes of transgenic and wild-type seedlings were observed, photographed, and root length and fresh weight were measured ( Figure 5 The growth results of Arabidopsis showed that under normal conditions, there was no significant difference in the growth of wild-type and transgenic Arabidopsis, but under salt stress, the root length and fresh weight of transgenic Arabidopsis were significantly higher than those of wild-type. The above results showed that overexpression of RcMYB14 can improve the adaptability of Arabidopsis under salt stress.

[0126] Although the above embodiments have been described in detail, they are only a part of the embodiments of the present invention, not all of them. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all belong to the protection scope of the present invention.

Claims

1. Use of any one of the following for enhancing salt tolerance and heat tolerance of plants, characterized in that: It includes: (1) RcMYB14 transcription factor, the nucleotide sequence of which is shown in SEQ ID No. 1; (2) a recombinant expression vector, wherein the recombinant expression vector contains the plant expression vector of the RcMYB14 transcription factor, wherein the plant expression vector is pCAMBIA2300s; (3) A host cell, wherein the host cell is Agrobacterium GV3101 containing the above-mentioned recombinant expression vector.

2. The use according to claim 1, characterized in that: The plant is tobacco or Arabidopsis thaliana.

3. Use of any one of the following for breeding salt-tolerant and heat-tolerant plant varieties, characterized in that: It includes: (1) RcMYB14 transcription factor, the nucleotide sequence of which is shown in SEQ ID No. 1; (2) a recombinant expression vector, wherein the recombinant expression vector contains the plant expression vector of the RcMYB14 transcription factor, wherein the plant expression vector is pCAMBIA2300s; (3) a host cell, wherein the host cell is Agrobacterium GV3101 containing the above-mentioned recombinant expression vector; The plant is tobacco or Arabidopsis thaliana.

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