Phoebe pbbhlh74 gene, protein encoded by the gene and application

Abstract

The application belongs to the field of plant molecular biology, and particularly relates to a Phoebe bourgoni PbbHLH74 gene, a protein encoded by the PbbHLH74 gene and an application of the PbbHLH74 gene.The full-length cDNA sequence of the PbbHLH74 gene is shown as SEQ ID No.1, and the amino acid sequence of a protein encoded by the PbbHLH74 gene is shown as SEQ ID No.2.A pYES2-PbbHLH74 vector is constructed to transform INVSc1 yeast cells, a pk2GW7-PbbHLH74 overexpression vector is used to infect Arabidopsis, and phenotypes, root lengths and fresh weights of transgenic plants under drought stress are determined, and the results show that the PbbHLH74 improves the tolerance of the yeast to the drought stress, the overexpression of the PbbHLH74 enhances the resistance of the Arabidopsis to the drought stress in a seed germination and seedling growth stage, improves seed germination energy and seedling root growth, and promotes plant biomass accumulation.The PbbHLH74 can be used as a candidate gene in molecular assisted breeding and creation of new drought-tolerant germplasm of the Phoebe bourgoni.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of plant biotechnology, and relates to a Phoebe bournei PbbHLH74 gene, a protein encoded by the gene and application thereof. BACKGROUND

[0002] Phoebe bournei, belonging to Lauraceae Phoebe, is a evergreen large tree, and is a second-class protected precious tree species in China, mainly distributed in southern China, such as Guangdong, Fujian, Jiangxi, southern Zhejiang, northern and northeastern Guangxi, Hubei, Hunan and eastern Guizhou. Phoebe bournei has wide uses and high economic value. Its wood has dense structure, beautiful texture, aromatic and corrosion resistance, and is not easy to deform or crack when dry, and is a good building material, high-grade furniture, carving process, and shipbuilding material. In addition, Phoebe bournei has lush branches and leaves, beautiful crown shape, and is a famous ornamental and landscaping tree species with evergreen tree body. However, the long-term growth and development process of Phoebe bournei is affected by various abiotic and biotic stresses. Among them, drought stress greatly limits the growth and development and stand productivity of Phoebe bournei.

[0003] bHLH is the second largest superfamily in plants next to MYB, and widely exists in eukaryotes, and is named for its highly conserved bHLH domain. The domain is composed of 50-60 amino acids, and is divided into basic region and helix-loop-helix region (HLH) according to its function. The basic region is located at the N-terminal of the bHLH domain, contains about 15 amino acids, and can specifically recognize and bind the cis-acting element E-box (5'-CANNTG-3'). The helix-loop-helix region is located at the C-terminal of the bHLH domain, is composed of 40-50 amino acids, and promotes protein interaction to form homodimer or heterodimer. Many studies have shown that bHLH plays an important role in regulating plant growth and development, secondary metabolism and resisting stress.

[0004] bHLH widely participates in the growth and development and secondary metabolism process at different stages of plants. AtPIF4 and AtPIF3 interact with phytochrome to control the expression of genes involved in light response regulation. Arabidopsis bHLH transcription factor SPATULA (SPT) not only plays a role in different organs (pistil, leaf and cotyledon development, etc.) of the aboveground part of the plant, but also regulates root growth by controlling the size of the root meristem. The Tb1 gene cloned from corn controls the growth and development of corn leaf axillary buds, lateral branches and male flowers. The first bHLH transcription factor identified in plants is the R gene in corn, which can regulate the expression of at least two genes in the flavonoid and anthocyanin pathways. Xinjiang poplar PalbHLH1 and PalMYB90 play a role as transcriptional activators of secondary metabolite synthesis genes in the flavonoid pathway.

[0005] bHLH plays an important role in plant response to abiotic stress such as drought, cold, high salt and iron deficiency. Arabidopsis bHLH122 positively regulates drought, NaCl and osmotic signaling in Arabidopsis by inhibiting the expression of CYP707A3 gene to promote ABA synthesis. Gossypium hirsutum GhbHLH1 gene is up-regulated by ABA and PEG induction, and may be involved in plant drought-related stress response through ABA or ABA-mediated pathway. Overexpression of PebHLH35 improves plant tolerance to drought stress by controlling stomatal density, stomatal aperture, photosynthesis and growth of transgenic Arabidopsis leaves. Rice OsbHLH148 improves plant drought tolerance by interacting with OsJAZ protein which plays a role in jasmonic acid signaling. IbbHLH79 of sweet potato binds to the CBF3 gene promoter to regulate cold stress, and overexpression of IbbHLH79 enhances the cold tolerance of transgenic plants. Overexpression of OrbHLH2 of wild rice improves the tolerance of transgenic Arabidopsis plants to salt and osmotic stress. Overexpression of PbbHLH67 of Pyrus betulaefolia enhances the salt tolerance of transgenic Arabidopsis by maintaining ion balance in plants under salt stress. Under iron deficiency conditions, overexpression of NtbHLH1 leads to changes in root length, rhizosphere pH and ferri-chelate reductase activity of transgenic tobacco, and increases the transcription level of genes related to iron deficiency response.

[0006] Based on the identification of bHLH gene family of Phyllostachys edulis and the analysis of expression patterns under PEG, ABA and MeJA treatment, drought tolerance related genes PbbHLH37 and PbbHLH74 were screened, and biological function identification under drought conditions was carried out by heterologous transformation of yeast and Arabidopsis, to provide gene resources for drought tolerance molecular breeding of Phyllostachys edulis, and PbbHLH37 has applied for a patent CN202310691797.3. SUMMARY

[0007] To solve the above problems, the present application provides Phyllostachys edulis PbbHLH74 gene, the protein encoded by the gene and the application of the gene.

[0008] Firstly, the present application provides Phyllostachys edulis PbbHLH74 protein, which is:

[0009] 1) a protein consisting of the amino acid shown in SEQ ID No. 2; or

[0010] 2) a protein derived from 1) by substituting, deleting or adding one or more amino acids in the amino acid sequence shown in SEQ ID No. 2 and having equivalent activity.

[0011] The present application also provides a gene encoding the Phyllostachys edulis PbbHLH74 protein.

[0012] Preferably, the sequence of the gene is shown in SEQ ID No. 1.

[0013] The present application also provides an expression vector containing the gene, a host cell and an engineered bacterium.

[0014] The present application also provides the use of the gene in improving the drought resistance of a plant.

[0015] In one specific embodiment of the present application, the gene is introduced into the genome of a plant and overexpressed in the transgenic plant to improve the drought resistance of the plant.

[0016] The present application also provides a method for improving the drought resistance of a plant, which comprises transforming a vector containing the gene into the genome of a plant using Agrobacterium tumefaciens-mediated transformation to obtain a transgenic plant. In one embodiment of the present application, the transgenic plant overexpresses the gene. The transgenic Arabidopsis thaliana has improved seed germination potential and root growth of seedlings and promotes the accumulation of biomass compared with a control.

[0017] The present application clones the PbbHLH74 gene of Phoebe bournei and transforms INVSc1 yeast cells. Compared with a control, the PbbHLH74-transformed yeast has significantly more clones under drought conditions and significantly improved tolerance to PEG-simulated drought in an SD / -Ura selective medium. The PbbHLH74 gene is overexpressed in transgenic Arabidopsis thaliana obtained by Agrobacterium tumefaciens-mediated transformation of inflorescences. Molecular detection results show that PbbHLH74 is significantly overexpressed in the transgenic Arabidopsis thaliana. The seed germination potential and seedling phenotype, root length and fresh weight of the PbbHLH74-overexpressing transgenic Arabidopsis thaliana under drought conditions are determined, and the results show that the germination potential of the transgenic PbbHLH74 Arabidopsis thaliana is significantly higher than that of the wild type, and the root growth and biomass accumulation of seedlings are significantly improved. In summary, we first overexpress the PbbHLH74 gene of Phoebe bournei in Arabidopsis thaliana and obtain a plant with higher drought resistance, which shows that it is feasible to artificially improve the drought resistance of a plant and has great application prospects and economic benefits. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 Total RNA of the roots of Phoebe bournei under PEG-simulated drought treatment according to the present application.

[0019] Figure 2 PCR amplification of the PbbHLH74 gene of Phoebe bournei.

[0020] Figure 3 Analysis of the drought stress tolerance of PbbHLH74-transformed yeast cells.

[0021] Figure 4 PCR detection of PbbHLH74-overexpressing plants of Phoebe bournei.

[0022] Figure 5 Analysis of the expression level of PbbHLH74 in positive lines (fluorescent quantitative PCR).

[0023] Figure 6 Analysis of seed germination potential of PbbHLH74 overexpression Arabidopsis.

[0024] Figure 7 Analysis of seed germination potential of PbbHLH74 overexpression Arabidopsis.

[0025] Figure 8 Analysis of seed germination potential of PbbHLH74 overexpression Arabidopsis. DETAILED DESCRIPTION

[0026] The experimental methods used in the following examples are conventional methods unless otherwise specified.

[0027] The materials, reagents, etc. used in the following examples can be obtained from commercial channels unless otherwise specified.

[0028] In order to make the purpose, technical scheme and advantages of the present application more clear, the present application will be further described in detail below in combination with the drawings and specific examples. It should be understood that the specific examples described herein are only used to explain the present application and are not used to limit the present application.

[0029] 1. Materials

[0030] 1.1 Experimental materials

[0031] The Phoebe zhennan material was selected from the 1.5-year-old container seedlings of the "Wuyuan No. 8" family.

[0032] Arabidopsis thaliana was used in Columbia-0 wild type and was cultured in the growth room of the Intelligent Experiment Building of Zhejiang Agriculture and Forestry University, with a growth condition of 25℃ light for 16h / d.

[0033] 1.2 Experimental reagents and instruments

[0034] Experimental reagents: DL2000 DNA Marker, 10x Loading Buffer, DNA gel recovery kit were purchased from Baorui Biotechnology Co., Ltd. (Beijing); M5 Plant RNeasy Complex Mini Kit RNA extraction kit, M5 Hiper Next III Gelred nucleic acid dye were purchased from Beijing Joinery Biotechnology Co., Ltd.; 2x TransStart FastPfu PCR SuperMix, M5 Hiper ultra-speed mix, pEASY-Blunt Zero Cloning Kit were purchased from Beijing Zoben Biotechnology Co., Ltd.; DH5α, GV3101, INVSc1 and other chemical competent cells were purchased from Shanghai Weidi Biotechnology Co., Ltd.; Reverse transcription kit, quantitative PCR detection kit, seamless cloning kit were purchased from Nanjing Novozyme Biotech Co., Ltd.; 2x Easy Taq PCR SuperMix was purchased from Zhejiang Yiside Biotechnology Co., Ltd.; Restriction endonuclease was purchased from NEB (Beijing) Co., Ltd.

[0035] Experimental instruments: NanoDrop2000 ultramicro spectrophotometer, microcentrifuge, basic electrophoresis instrument, Bio-Rad Gel Doc XR gel imager, Bio-Rad S1000 PCR thermal cycler, CFX96 real-time fluorescent quantitative PCR system, micro pipette gun (Eppendorf), super-clean workbench, water bath, ice maker, constant temperature incubation shaker and incubator, palm centrifuge, high-speed centrifuge, electronic balance, etc.

[0036] 1.3 Primer synthesis and sequencing

[0037] Primer synthesis and sequencing were completed by Zhejiang Youkang Biotechnology Co., Ltd.

[0038] 2 Method

[0039] 2.1 Extraction of total RNA from Phoebe zhennan

[0040] M5 Plant RNeasy Complex Mini Kit was used to extract total RNA from Phoebe zhennan, and the steps were as follows:

[0041] (1) Take 1 mL of lysis solution CLB into a centrifuge tube (if CLB has precipitated or precipitated, it needs to be dissolved at 65℃ water bath first), add 5% β-mercaptoethanol (1 mL CLB adds 50 μL β-mercaptoethanol) in the lysis solution CLB. Mix well by inverting and preheat in 65℃ water bath;

[0042] (2) Take about 0.1 g of sample and put it into a liquid nitrogen pre-cooled mortar, grind it into fine powder with liquid nitrogen;

[0043] (3) Add 100-200 mg of fine powder to a pre-heated lysis solution CLB (with β-mercaptoethanol already added) centrifuge tube. Immediately vortex vigorously for 30-60 s or pipette mix the lysate until a satisfactory homogenate is obtained;

[0044] (4) Place in a 65°C water bath for a short time (5-10 min) with occasional inversion 1-2 times to help lysis;

[0045] (5) Centrifuge the lysate at 13,000 rpm for 10 min to pellet the unlysed debris;

[0046] (6) Transfer the supernatant of the lysate (more supernatant can be taken if the capacity of the genomic DNA cleanup column is not exceeded, which can increase the yield) to a new centrifuge tube. Add half the volume of the supernatant of absolute ethanol (0.5 volume) at this time a pellet can form, but this does not affect the extraction process, immediately pipette mix well, do not centrifuge;

[0047] (7) Add the mixture (less than 720 μL at a time, more can be added in two portions) to a genomic cleanup column, centrifuge at 13,000 rpm for 2 min, discard the waste;

[0048] (9) Place the genomic cleanup column in a clean 2 mL centrifuge tube, add 500 μL of lysis solution RLT Plus to the genomic cleanup column, centrifuge at 13,000 rpm for 30 s, collect the filtrate, estimate the volume of the filtrate more accurately with a micropipette (usually about 450-500 μL, the volume lost during filtration should be subtracted), add 0.5 volume of absolute ethanol, immediately pipette mix well, do not centrifuge;

[0049] (10) Immediately add the mixture (less than 720 μL at a time, more can be added in two portions) to a sorbent column RA (the sorbent column is placed in a collection tube), centrifuge at 13,000 rpm for 2 min, discard the waste;

[0050] (11) Add 700 μL of deproteinization solution RW1, stand at room temperature for 1 min, centrifuge at 13,000 rpm for 30 s, discard the waste;

[0051] (12) Add 500 μL of rinse solution RW, centrifuge at 13,000 rpm for 30 s, discard the waste. Add 500 μL of rinse solution RW, repeat once;

[0052] (13) Place the sorbent column RA back in the empty collection tube, centrifuge at 13,000 rpm for 2 min, try to remove as much of the rinse solution as possible;

[0053] (14) Take out the adsorption column RA and put it into a RNase free centrifuge tube, add 30-50 μL RNase-Free H2O in the middle of the adsorption membrane according to the expected RNA yield, and let it stand at room temperature for 1 min, then centrifuge at 12,000 rpm for 1 min;

[0054] (15) If the expected RNA yield is >30 μg, repeat step 9 by adding 30-50 μL RNase-Free H2O, combine the two washing solutions, or use the first eluate to repeat step 1.

[0055] 2.2 Synthesis of reverse transcription cDNA first strand

[0056] The synthesis of reverse transcription cDNA first strand was carried out according to the instructions of PrimeScript™ RT Reagent Kit (Perfect Real Time) (TaKaRa) for total RNA.

[0057] (1) The following mixture was prepared for the removal of genomic reaction:

[0058]

[0059] The reaction program was: 42°C, 2 min; 4°C, hold.

[0060] (2) The following mixture was prepared for the reverse transcription reaction:

[0061]

[0062] The reaction program was: 37°C, 15 min; 85°C, 5 sec; 4°C, hold.

[0063] 2.3 Cloning of target genes

[0064] 2.3.1 Gene cloning

[0065] Specific primers for PbbHLH74 were designed (Table 1), and the amplification system and program are shown in Table 2.

[0066] Table 1 Cloning primers for PbbHLH74

[0067]

[0068] Table 2 Cloning system

[0069]

[0070] Reaction program: 98℃, 30 sec, 52℃, 5 sec, 72℃, 1 min, cycle 35 times; 72℃, 1 min; 16℃, hold.

[0071] 2.3.2 Purpose fragment recovery

[0072] Prepare 1% agarose gel, use agarose gel electrophoresis to detect the above PCR product, and cut the gel to recover the target fragment according to the instructions of MiniBEST Agarose Gel DNA Extraction Kit (Takara, Dalian).

[0073] (1) Prepare a sterilized 2 mL centrifuge tube, and weigh the empty tube.

[0074] (2) Cut the gel block containing the target fragment with a clean scalpel under ultraviolet light, cut the gel block into small pieces and put it into a 2 mL centrifuge tube, and weigh the gel block volume (1 mg = 1 μL as standard).

[0075] (3) Add 3 times the gel volume of Buffer GM to the gel block, dissolve the gel block at room temperature, and mix intermittently.

[0076] (3) When the gel is completely dissolved, add isopropanol with a final concentration of 20%.

[0077] (4) Add the solution in the previous step to the Spin Column adsorption column, place it on the Collection Tube, and centrifuge at 12000 rpm for 1 min, and discard the waste liquid.

[0078] (5) Add 700 μL of rinse Buffer WB to the adsorption column, and centrifuge at 12000 rpm for 1 min at room temperature, and discard the waste liquid.

[0079] (6) Repeat step (5).

[0080] (6) Empty 12000 rpm for 1 min.

[0081] (7) Place the adsorption column in a sterilized 1.5 mL centrifuge tube, add 30 μL of sterilized water (preheated to 65℃) to the adsorption membrane, and stand at room temperature for 1 min, centrifuge at 12000 rpm for 1 min, and store the collected liquid at -20℃.

[0082] 2.3.3 Purpose fragment ligation and transformation

[0083] (1) Ligation: Use pEASY Blunt Zero vector (Transgene, -Blunt ZeroCloning Kit) with the target gene, mix the following solutions, mix gently, centrifuge briefly. PCR: 25℃, 30min.

[0084]

[0085] (2) Transformation: Take Trans T1 (Transgene, Beijing) E. coli competent cells, after thawing in ice bath, add 2 μL of the above ligation product, ice bath for 30 min, 42℃ water bath heat shock for 30 s, then quickly and smoothly transfer the centrifuge tube to the ice bath for 2 min, add 500 μL of LB medium without antibiotics, 37℃ shaking bed 200 rpm for 1 h. 4000 rpm 2 min, remove part of the supernatant, leave 100 μL of bacterial solution, reblow and mix the bacteria, and plate on solid LB medium (containing 50 mg·mL -1 Kana), 37℃ inverted culture for 12 h.

[0086] (3) Bacteria detection: pick white single colonies on the plate, add 500 μL of liquid LB medium (containing 50 mg·mL -1 Kana), 37℃ shaking bed 200 rpm for 3-5 h. Take 1 μL of bacterial solution as template for PCR detection, the primers are gene cloning primers, the system and procedure are as follows.

[0087]

[0088]

[0089] Reaction procedure: 94℃, 5min; 94℃, 30sec, 52℃, 30sec, 72℃, 1min, cycle 35 times; 72℃, 5min; 16℃, hold.

[0090] (4) Agarose gel electrophoresis detection of bacterial solution PCR product, select positive clones to Zhejiang Yikang Biotechnology Co., Ltd. for sequencing.

[0091] (5) After SnapGene alignment of the sequencing results is correct, use Transgene EasyPure Plasmid MiniPrep Kit to extract plasmid, and use the obtained positive plasmid to construct the expression vector of PbbHLH74 gene.

[0092] 2.3.4 Construction of expression vector by homologous recombination

[0093] According to II One Step Cloning Kit to construct the expression vector by homologous recombination.

[0094] (1) First, add the pK2W7-eYGFPuv-3xFLAG and pYES2 linker sequence to the gene-specific primer, and use the positive plasmid obtained in the previous step as the template to prepare the following PCR system (20 μL):

[0095]

[0096] Reaction procedure: 98℃, 30 sec, 54℃, 5 sec, 72℃, 1 min, cycle 35 times; 72℃, 5 min; 16℃, hold.

[0097] (2) After ensuring that the product is a single band, refer to the MiniBEST Agarose Gel DNA Extraction Kit (Takara, Dalian) instructions to recover the target fragment.

[0098] (3) Linearize the overexpression vectors pK2W7-eYGFPuv-3xFLAG and pYES2 by double digestion, and use the pK2W7-eYGFPuv-3xFLAG and pYES2 plasmids as templates to prepare the following PCR system:

[0099]

[0100]

[0101] Reaction procedure: 37℃, 2h; 16℃, hold.

[0102] (4) Refer to the MiniBEST Agarose Gel DNA Extraction Kit (Takara, Dalian) instructions to recover the target fragment.

[0103] (5) The overexpression vector selected for genetic transformation is pK2W7-eYGFPuv-3xFLAG, and the vector selected for yeast stress verification is pYSE2, and the PbbHLH74 gene expression vector is constructed. Prepare the following mixed solution, 25℃, overnight.

[0104]

[0105] Optimal cloning vector usage = [0.02 x cloning vector base pair number] ng (0.03 pmol) Optimal insert fragment usage = [0.04 x insert base pair number] ng (0.06 pmol)

[0106] Reaction procedure: 37℃, 30min; reduce to 4℃ or immediately cool on ice.

[0107] (6) Take 2 μL for transformation of Trans T1 (Transgene, Beijing) E. coli competent cells, then pick single colonies for bacterial liquid PCR detection, the primer is gene cloning primer, and the PCR system is as follows:

[0108]

[0109] The reaction procedure is: 94℃, 5min; 94℃, 30sec, 52℃, 30sec, 72℃, 1min, cycle 35 times; 72℃, 5min; 16℃, hold.

[0110] (7) Agarose gel electrophoresis detects PCR products, and after transformation into Trans T1 (Transgene, Beijing) E. coli competent cells, positive clones are selected for sequencing in Zhejiang Yikang Biotechnology Co., Ltd. Extract the plasmid of the strain with correct sequencing results for later use.

[0111] 2.4 Lithium acetate method for transforming yeast

[0112] 1. Pretreatment of Carrier DNA: insert Carrier DNA into 95℃ metal bath for 5min or insert into 95℃ water bath for 3min, and then quickly insert into ice after heating.

[0113] 2. Take 100 μL of INVSc1 competent cells melted on ice, add 2-5 μg of pre-cooled target plasmid, 10 μL of pretreated Carrier DNA, 500 μL of PEG / LiAc, and mix well by pipetting several times, and then place in 30℃ water bath for 30min (mix well by inverting 6-8 times at 15min).

[0114] 3. Place the tube in a 42℃ water bath for 15min (mix well by inverting 6-8 times at 7.5min).

[0115] 4. Centrifuge at 5000rpm for 40s to discard the supernatant, resuspend with 400 μL of ddH2O, centrifuge for 30s to discard the supernatant.

[0116] 5. Resuspend with 50 μL of ddH2O, plate, and incubate at 29℃ for 48-96h.

[0117] 2.5 Analysis of drought stress tolerance of heterologous transformed yeast

[0118] After positive single colony was verified by PCR, it was inoculated into 50 mL of SC / -Ura liquid medium containing 2% w / v galactose to induce gene expression, and incubated at 30°C for 48 h to OD600=2.0. 100 μL of the induced yeast solution was centrifuged for 10 s to discard the supernatant, and the bacteria were resuspended in 1 mL of a solution containing 30% PEG6000 for further incubation for 36 h to test the drought tolerance of yeast cells expressing PbbHLH74. The same number of yeast cells were collected by centrifugation, resuspended in 1 mL of sterile water, and incubated at 30°C for 36 h as a control. The treated yeast cells were serially diluted with sterile water at 10-fold gradients (1x, 10x, 100x), and 2 μL was spotted on SD / -Ura medium and incubated at 30°C for 2 d to record the growth state of yeast colonies.

[0119] 2.6 Liquid nitrogen method for transforming GV1301 Agrobacterium

[0120] (1) Take the Agrobacterium competent cells stored at -80°C, and thaw on ice;

[0121] (2) Take 0.5 μg of the expression vector plasmid, add to the Agrobacterium competent cells, mix gently, and then sequentially place on ice, in liquid nitrogen, in a 37°C water bath, and in an ice-water mixture for 5 min each;

[0122] (3) Add 700 μL of YEP liquid medium without antibiotics to the centrifuge tube, and shake the bacterial solution in a shaker (28°C, 200 rpm) for 4-6 h;

[0123] (4) Centrifuge at 6000 rpm for 2 min, discard part of the supernatant, and blow the remaining 70-100 μL or so of liquid evenly;

[0124] (5) Uniformly spread the bacterial solution on YEP solid medium containing antibiotics (50 mg·mL -1 Spec and 50 mg·mL -1 Rif), and incubate in an inverted biochemical incubator at 28°C for 48-72 h;

[0125] (6) Pick single colonies on the plate for bacterial liquid PCR detection, and the primers are gene cloning primers. The PCR system is as follows:

[0126]

[0127] The reaction program is as follows: 94°C, 5 min; 94°C, 30 sec, 46°C, 30 sec, 72°C, 20 sec, for 30 cycles; 72°C, 5 min; 16°C, hold.

[0128] (7) Add the same volume of 50% glycerol to the single colony bacterial solution with a positive detection result, and store in a -80°C refrigerator.

[0129] 2.7 Agrobacterium-mediated transformation of Arabidopsis thaliana by floral dip

[0130] Agrobacterium liquid with the target gene vector was taken out from the -80°C refrigerator, plated and activated, and single colonies were picked and blown into EP tubes containing 1 mL YEP liquid medium (containing 50 mg·mL -1 of Rif and 50 mg·mL -1 of Spec), and cultured at 28°C for 16 h. 500 μL of the bacterial solution was transferred to a 500 mL conical flask containing 200 mL of the same medium, and the culture was continued at 28°C until the OD was between 0.6 and 0.8. 10 g of sucrose and 80 μL of silwet-L77 were added to the 200 mL bacterial solution. The infection solution was transferred to an open large and low container, and was drawn out with a needle tube to a large amount of foam. The Arabidopsis thaliana inflorescences with the fruit pods removed in advance were immersed in the bacterial solution for 1 min, then taken out and the excess bacterial solution on the surface was absorbed with a paper towel. It was wrapped with plastic wrap to keep it moist, and placed flat in the growth chamber for dark culture for 2 d. Then it was cultured under normal light for one week, and then re-infected. The mature seeds were collected in batches, dried at 37°C for 5 d, and stored in a 4°C refrigerator for standby use.

[0131] 2.8 Identification of positive transgenic Arabidopsis thaliana

[0132] M5 Hiper ultra-speed mix reagent was used for identification of positive Arabidopsis thaliana plants, and the method was as follows:

[0133] (1) Take out the "amplification best partner" and place it at room temperature. Observe whether there is precipitation before use. Make sure that the lysis solution has no precipitation, otherwise it should be dissolved and clarified at 37°C before use.

[0134] (2) Cut a 2 mm square transgenic Arabidopsis thaliana leaf and put it into 20 μL lysis solution. Melt the yellow gun head with a flame to form a "grinding pestle" to break the leaf tissue.

[0135] (3) PCR instrument 98°C for 5 min

[0136] (4) Centrifuge at 12000 rpm for 2 min, take 2 uL as PCR template, and the system is as follows:

[0137]

[0138] The reaction program is: 95°C for 3 min; 94°C for 25 s, 60°C for 25 s, 72°C for 1 min, 34 cycles; 72°C for 5 min.

[0139] 2.9 Fluorescent quantitative PCR identification

[0140] Total RNA was extracted from the leaves of transgenic Arabidopsis using M5 Plant RNeasy Complex Mini Kit, and reverse transcription cDNA was performed using PrimeScript™ RT Reagent Kit (Perfect Real Time) (TaKaRa, Dalian, China).

[0141] Fluorescent quantitative PCR was performed using ChamQ SYBR qPCR Master Mix (Vazyme, Nanjing, China), and Bio-Rad CFX-96 was used as the instrument. A 20-μL system mixture was prepared: 2x ChamQ SYBR qPCR Master Mix 10 μL, Primer-F 0.4 μL, Primer-R 0.4 μL, 50x ROX Reference Dye 1 0.4 μL, cDNA 1 μL, and ddH2O 7.8 μL. The reaction program was as follows: 95 °C for 30 s; 95 °C for 10 s, 60 °C for 30 s, for 40 cycles; 65 °C for 5 s, 95 °C for 5 min (dissociation curve according to the instrument type). The primers used for fluorescent quantitative PCR are shown in Table 3, and AtActin was selected as the internal reference gene.

[0142] Table 3 Primers for fluorescent quantitative PCR

[0143]

[0144] 2.10 Germination of Arabidopsis seeds under drought stress

[0145] An appropriate amount of transgenic and wild-type Arabidopsis seeds were placed in 1.5 mL EP tubes, washed once with sterilized water, washed with 75% ethanol for 90 s, washed with sterilized water for 3 times, washed with 2% NaClO for 10 min, and finally washed with sterilized water for 3 times. The seeds were then sown on MS plates and MS+200 mM mannitol plates, respectively, and then placed in a growth chamber at 4 °C in the dark for 2 d, and then placed in a growth chamber at 23 °C with 16 h light and 8 h dark. The number of seeds germinated each day was counted, and the germination rate was calculated.

[0146] 2.11 Phenotype observation, fresh weight, and root length determination of Arabidopsis seedlings under drought stress

[0147] After sterilization, wild-type and transgenic Arabidopsis seeds were sown on MS solid medium. After 5 d of growth in the Arabidopsis growth chamber, seedlings with uniform growth were moved to MS+200 mM mannitol solid medium in a sterile environment, and MS plates were used as controls. The growth state was recorded by taking photos, and the fresh weight and main root length were measured.

[0148] 3. Experimental results

[0149] 3.1 Total RNA extraction of Phoebe bourgonesis

[0150] Total RNA of Phoebe bourgonesis was extracted by M5 Plant RNeasy Complex Mini Kit Figure 1 The OD260 / 280 of the extracted RNA was between 1.8-2.1, which indicated that the total RNA was of good purity. The result of agarose gel electrophoresis showed that the 18s and 28s bands of the RNA sample were very clear, which inferred that the RNA was not degraded and met the requirements of the next experiment.

[0151] 3.2 Cloning of PbbHLH74 and construction of expression vector

[0152] The specific primer of PbbHLH74 was used to amplify the cDNA of Phoebe bourgonesis under drought stress, and the result showed that the length of the target band was 1532bp Figure 2 The specific primer PCR amplified with the linker was connected to the expression vector pK2W7-eYGFPuv-3xFLAG and pYES2 by homologous recombination method, and then transformed into E. coli. After sequencing by Zhejiang Yikang Biotechnology Co., Ltd., the single colony with correct sequence was selected to transform GV3101 Agrobacterium and INVSc1 yeast competent cells, and then the Agrobacterium liquid culture was used to infect Arabidopsis.

[0153] 3.3 Analysis of drought stress tolerance of heterologous transformed yeast

[0154] There was no significant difference in the growth state of the yeast colonies transformed with PbbHLH74 and empty plasmid pYES2. After drought treatment, the yeast colonies transformed with PbbHLH74 grew better and there were more colonies than the control Figure 3 , which indicated that PbbHLH74 significantly enhanced the drought stress tolerance of INVSc1 yeast in SD / -Ura selective medium.

[0155] 3.4 Identification of positive transgenic Arabidopsis thaliana plants of PbbHLH74

[0156] The transgenic positive lines of PbbHLH74 were obtained by PCR identification after transforming Phoebe bourgonesis into Arabidopsis thaliana Figure 4 The expression level of PbbHLH74 in positive plants was detected by fluorescent quantitative PCR Figure 5 , and the results showed that the expression levels of PbbHLH74 in L1, L2 and L3 lines were 9.5, 11.13 and 10.05 times of the control, respectively.

[0157] 3.5 Analysis of seed germination potential of PbbHLH74 transgenic Arabidopsis thaliana under drought stress

[0158] In normal medium, there was no significant difference in germination potential between wild type and PbbHLH74 transgenic Arabidopsis thaliana lines, but under drought stress, the germination potential of PbbHLH74 transgenic Arabidopsis thaliana was significantly higher than that of wild type, indicating that overexpression of PbbHLH74 enhanced the drought resistance of Arabidopsis thaliana seeds. Figure 6

[0159] 3.6 Phenotype analysis of PbbHLH74 transgenic Arabidopsis thaliana seedlings under drought stress

[0160] In normal medium, there was no significant difference in growth between PbbHLH74 transgenic Arabidopsis thaliana and wild type. Figure 7 Under drought stress, the growth of PbbHLH74 transgenic Arabidopsis thaliana was significantly enhanced, the leaves were larger, the length of main root and the density of lateral roots were increased, and the fresh weight was significantly increased. Figure 8 The results showed that PbbHLH74 transgenic Arabidopsis thaliana seedlings enhanced drought resistance.

[0161] In summary, overexpression of PbbHLH74 gene from Phoebe chekiangensis can significantly improve the drought resistance of plants.

[0162] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the technical principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the present application.​

Claims

1. The application of the gene encoding the PbbHLH74 protein in *Phoebe zhennan* in plant drought resistance, among which, The amino acid sequence of the protein is shown in SEQ ID No.

2. It transfers the gene into the plant genome and overexpresses it in transgenic plants to improve the drought resistance of the plants. The plants are either Phoebe bournei or Arabidopsis thaliana.

2. The use as described in claim 1, characterized in that, The nucleotide sequence of the gene is shown in SEQ ID No.

1.

3. A method for improving plant drought resistance, characterized in that, Using Agrobacterium tumefaciens-mediated transformation, a vector containing a gene encoding the PbbHLH74 protein of Phoebe bournei was transferred into the plant genome, and transgenic plants were obtained by screening. The amino acid sequence of the protein is shown in SEQ ID No.

2. The transgenic plants overexpress the gene, and the plants are Phoebe bournei or Arabidopsis thaliana.

4. The method as described in claim 3, characterized in that, The nucleotide sequence of the gene is shown in SEQ ID No. 1.

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