Phoebe bournei PbHDZ35 gene and application thereof in promoting plant growth and development and improving plant drought resistance

Abstract

The invention discloses a phoebe bournei PbHDZ35 gene, and the phoebe bournei PbHDZ35 gene has a nucleotide sequence as shown in SEQ ID No: 1, or a sequence which is obtained by deleting, adding and / or substituting one or more nucleotides of the sequence and has an unchanged function. The invention also discloses a protein coded by the phoebe bournei PbHDZ35 gene. The invention further discloses a method for promoting plant growth and development. The invention further discloses application of the phoebe bournei PbHDZ35 gene in promoting plant growth and development. The invention also discloses an application of the phoebe bournei PbHDZ35 gene in improving the drought resistance of plants. According to the application, the phoebe bournei PbHDZ35 gene is overexpressed in arabidopsis thaliana for the first time, a plant with stronger growth and development and weakened drought resistance is obtained, a theoretical basis is provided for artificially improving the plant yield and improving the drought resistance of the plant, and the application prospect and the economic benefit are relatively great.

Description

Technical Field

[0001] The invention belongs to the field of plant biotechnology and relates to a Phoebe bournei PbHDZ35 gene and an application thereof in promoting plant growth and development and improving plant drought resistance. Background Art

[0002] Phoebe bournei, belonging to the genus Phoebe in the family Lauraceae, is a large evergreen tree endemic to China and a rare species under national second-level key protection. The species is mainly distributed in southern China, including Guangdong, Fujian, Jiangxi, southern Zhejiang, northern and northeastern Guangxi, Hubei, Hunan and eastern Guizhou. Due to its excellent wood properties, Phoebe bournei is of great economic value. Its wood has a dense structure, beautiful texture, fragrance and corrosion resistance. It is not easy to deform or crack after drying. It is widely used in high-end building materials, exquisite furniture manufacturing, handicraft carving and shipbuilding. In addition, Phoebe bournei has dense branches and leaves, beautiful crowns, upright tree shape and perennial green characteristics, so it is also favored in landscaping and garden viewing. Although Phoebe bournei has a wide range of applications in the economic and ecological fields, its long-term growth and development process faces challenges from multiple abiotic and biotic stresses. Drought stress, as one of the main environmental pressures, significantly restricts the growth and development of Phoebe bournei and the productivity of forest stands. Drought not only affects trees' water absorption, photosynthesis efficiency and nutrient acquisition, but also causes physiological metabolic disorders in trees, reduces their stress resistance, and thus affects the sustainable development of forests and the stability of ecosystems.

[0003] The HD-ZIP (Homeodomain-Leucine Zipper) gene family is a transcription factor family unique to plants. It is named because it has both highly conserved HD (homeodomain) and ZIP (leucine zipper) domains. The HD domain consists of about 61 amino acids and is located at the N-terminus of the protein. It is responsible for binding to specific cis-acting elements and regulating the transcription of downstream genes. The ZIP domain is located at the C-terminus and consists of a repeating sequence composed of multiple leucine residues. It mediates protein dimerization to form homo- or hetero-dimers, thereby enhancing its stability and functional diversity. The dual characteristics of this structure enable the HD-ZIP gene family to play a vital role in various physiological processes of plants, especially stress responses.

[0004] A large number of studies have shown that the HD-ZIP gene family plays a core role in plant drought tolerance and the regulation of abscisic acid (ABA) signaling pathways. ABA is a key hormone for plants to cope with abiotic stresses such as drought and salt stress. HD-ZIP genes regulate ABA synthesis and its signal transduction pathways, affecting important physiological processes such as stomatal opening and closing, root structure development, and water metabolism balance, thereby improving the drought resistance of plants. Under drought stress, HD-ZIP genes can respond to environmental signals and promote the synthesis and accumulation of drought-resistant related proteins by directly or indirectly regulating the expression of downstream resistance genes. At the same time, the HD-ZIP gene family also acts as an important regulatory factor in the ABA-mediated signaling network, regulating the expression of genes related to ABA response, and participating in the regulation of stomatal movement, stress-adaptive growth, and stress recovery stages. In addition, the HD-ZIP gene family also has the function of regulating development, not limited to the response to environmental stress. Some HD-ZIP genes have been found to be closely related to the morphological construction of plant tissues such as stems, leaves, and roots. Its regulatory mechanism involves complex regulation of gene networks, and exhibits diverse functions under different growth stages or environmental conditions. This multiple regulatory feature makes the HD-ZIP gene family a hot area in plant development and stress biology research. Among them, HB7 / 12 genes are widely involved in regulating the response of plants to abiotic stresses (such as drought and salt stress) in many species such as Arabidopsis thaliana, poplar (Populus spp.), and tobacco (Nicotiana tabacum), mainly affecting plant water management and stomatal behavior by regulating the ABA negative feedback signaling pathway. However, there has been no report on the function of the PbHDZ35 gene in Phoebe bournei.

[0005] Based on the identification of the HD-ZIP gene family of Phoebe bournei and the analysis of its expression patterns under PEG, ABA and natural drought treatments, the applicant combined the PEG transcriptome WGCNA results to screen out the gene PbHDZ35 that is highly positively correlated with ABA, and identified its biological function under drought conditions through heterologous transformation of yeast and Arabidopsis thaliana, providing genetic resources for the molecular breeding of Phoebe bournei for drought tolerance. Summary of the invention

[0006] In order to solve the above problems, the present invention provides a Phoebe bournei PbHDZ35 gene and its application in promoting plant growth and development and improving plant drought resistance.

[0007] The present invention provides a P. minnan PbHDZ35 gene, which has a nucleotide sequence as shown in SEQ ID No: 1, or a sequence in which one or more nucleotides are deleted, added and / or substituted but the function remains unchanged. Preferably, the nucleotide sequence of the gene is SEQ ID No: 1.

[0008] The invention also provides a protein encoded by the Phoebe bournei PbHDZ35 gene, which is composed of the amino acids shown in SEQ ID No.2.

[0009] The present invention also provides a method for promoting plant growth and development, the method comprising preparing a modified plant containing the PbHDZ35 gene of Phoebe bournei, wherein the expression of the PbHDZ35 gene of Phoebe bournei is increased in the modified plant relative to the wild-type plant; wherein the base sequence of the PbHDZ35 gene of Phoebe bournei is shown in SEQ ID NO: 1. It should be noted that in the concept of "increased expression", "expression" refers to the process in which a gene is transcribed and translated to produce a biologically active protein; an increase in the intensity of the above process is an increase in expression, and specifically, the product obtained by transcription and the product obtained by translation can be increased.

[0010] The present invention also provides an application of the PbHDZ35 gene of Phoebe bournei in promoting plant growth and development, and prepares a modified plant containing the PbHDZ35 gene of Phoebe bournei, wherein the expression of the PbHDZ35 gene of Phoebe bournei is increased in the modified plant relative to the wild-type plant; wherein the base sequence of the PbHDZ35 gene of Phoebe bournei is shown in SEQ ID NO: 1. It should be noted that in the concept of "increased expression", "expression" refers to the process in which a gene is transcribed and translated to produce a biologically active protein; an increase in the intensity of the above process is an increase in expression, and specifically, the product obtained by transcription and the product obtained by translation can be increased.

[0011] In one embodiment, the plant is Phoebe chinensis or Arabidopsis thaliana. Preferably, the plant is Arabidopsis thaliana.

[0012] In one embodiment, the PbHDZ35 gene of Phoebe fujianensis is connected to a vector, transformed into Arabidopsis thaliana by Agrobacterium-mediated transformation, screened, cultured and transgenic strains are obtained. The PbHDZ35 gene of Phoebe fujianensis is transferred into the Arabidopsis thaliana genome and overexpressed in transgenic plants to promote the overall growth and development of Arabidopsis thaliana. Compared with the control, the transgenic Arabidopsis thaliana promotes the growth of seedling roots and leaves and increases the accumulation of plant biomass.

[0013] The present invention also provides an application of the PbHDZ35 gene of Phoebe bournei in improving drought resistance of plants, and prepares a modified plant containing the PbHDZ35 gene of Phoebe bournei, wherein the expression of the PbHDZ35 gene of Phoebe bournei is reduced in the modified plant relative to the wild-type plant; wherein the base sequence of the PbHDZ35 gene of Phoebe bournei is shown in SEQ ID NO: 1. It should be noted that in the concept of "reduced expression", "expression" refers to the process in which a gene is transcribed and translated to produce a biologically active protein; an increase in the intensity of the above process is reduced expression, and specifically, the product obtained by transcription and the product obtained by translation can be reduced.

[0014] The applicant used the Agrobacterium tumefaciens-mediated method to transform the vector containing the PbHDZ35 gene of Phoebe fujian into the plant genome, so that the transgenic plants overexpressed the PbHDZ35 gene of Phoebe fujian to obtain transgenic plants. The wild type and two homozygous overexpression strains (L7 and L8) were subjected to natural drought treatment, and it was found that after 15 days of natural growth, the leaf size of the overexpression strain was significantly larger than that of the wild type, and its growth was significantly weaker than that of the wild type WT after 15 days of drought treatment. Similarly, if the PbHDZ35 gene of Phoebe fujian is knocked out in the modified plant, the drought resistance of the modified plant will be significantly improved.

[0015] In one embodiment of the present invention, the PbHDZ35 gene of P. minnan was cloned and transformed into INVSc1 yeast cells. Compared with the control, yeast transformed with PbHDZ35 had significantly fewer clones under drought conditions, which significantly inhibited the yeast's tolerance to PEG-simulated drought in SD / -Ura selective medium.

[0016] In summary, this application overexpressed the PbHDZ35 gene in Arabidopsis for the first time, and obtained plants with stronger growth and development and reduced drought resistance, which provided a theoretical basis for artificially increasing plant yield and improving plant drought resistance, and has great application prospects and economic benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the electrophoresis diagram of total RNA from the roots of Phoebe bournei under the PEG simulated drought treatment of the present invention.

[0018] Figure 2 The electrophoresis diagram of the PCR amplified PbHDZ35 gene of Phoebe bournei of the present invention.

[0019] Figure 3 This is a diagram for analyzing the drought stress tolerance of yeast cells transformed with PbHDZ35 of the present invention.

[0020] Figure 4 This is a PCR detection diagram of positive plants of the Phoebe bournei PbHDZ35 gene overexpression plant of the present invention.

[0021] Figure 5 This is a graph analyzing the expression level of PbHDZ35 in the positive strains identified in the present invention.

[0022] Figure 6 This is a graph showing the growth potential of the aboveground part of Arabidopsis thaliana overexpressing PbHDZ35 under natural growth according to the present invention.

[0023] Figure 7 This is a diagram of the phenotypic observation and root length measurement of PbHDZ35 transgenic Arabidopsis thaliana treated with mannitol-simulated drought.

[0024] Figure 8 This is a phenotypic observation diagram of PbHDZ35 transgenic Arabidopsis thaliana under natural drought treatment of the present invention.

[0025] Fig. 9 This is a diagram showing the physiological index determination of PbHDZ35 transgenic Arabidopsis thaliana under natural drought treatment according to the present invention. DETAILED DESCRIPTION

[0026] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0027] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0028] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0029] 1 Materials

[0030] 1.1 Experimental Materials

[0031] The Phoebe chinensis material selected was the two-year-old container seedlings of the “Wuyuan No. 8” family.

[0032] Arabidopsis thaliana used the Columbia-0 wild type and was cultured in the growth room of the State Key Laboratory of Zhejiang Agriculture and Forestry University under the growth conditions of 25°C and 16 h / d of light.

[0033] 1.2 Experimental reagents and instruments

[0034] Experimental reagents: DL2000 DNA Marker, 10× Loading Buffer, and DNA gel recovery kit were purchased from Bioray Biotechnology (Beijing) Co., Ltd.; M5 Plant RNeasy Complex Mini Kit RNA extraction kit and M5 Hipure Next III Gelred nucleic acid dye were purchased from Beijing Polymer Biotechnology Co., Ltd.; 2×TransStartFastPfu PCR SuperMix, M5 Hiper ultralight speed mix, and pEASY-Blunt Zero Cloning Kit were purchased from Beijing Quanshijin 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, and seamless cloning kit were purchased from Nanjing Novogene Biotechnology Co., Ltd.; 2×Easy Taq PCR SuperMix was purchased from Zhejiang Easyside Biotechnology Co., Ltd.; restriction endonucleases were purchased from NEB (Beijing) Co., Ltd. Superoxide dismutase (SOD) (NBT method) detection kit (micro method), peroxidase (POD) detection kit (micro method), and ascorbate peroxidase (APX) detection kit (micro method) were all purchased from YEAST Puinte Bioengineering Co., Ltd.

[0035] Experimental instruments: NanoDrop2000 ultra-micro spectrophotometer, micro centrifuge, basic electrophoresis instrument, Bio-RadGel Doc XR gel imager, Bio-Rad S1000 PCR thermal cycler, CFX96 real-time fluorescence quantitative PCR system, micropipette gun (Eppendorf), clean bench, water bath, ice maker, constant temperature culture shaker and incubator, handheld centrifuge, high-speed centrifuge, electronic balance, portable photosynthesis measurement system (LI-6800), scanning electron microscope, microplate reader (tecan SPARK), etc.

[0036] 1.3 Primer synthesis and sequencing

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

[0038] 2 Methods

[0039] 2.1 Extraction of total RNA from Phoebe bournei

[0040] M5 Plant RNeasy Complex Mini Kit was used to extract total RNA from Phoebe bournei. The steps are as follows:

[0041] (1) Take 1 mL of lysis buffer CLB into a centrifuge tube (if CLB has precipitation or precipitate, it needs to be placed in a 65°C water bath to re-dissolve), add 5% β-mercaptoethanol to the lysis buffer CLB (1 mL CLB plus 50 μL β-mercaptoethanol). Invert to mix and preheat in a 65°C water bath;

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

[0043] (3) Transfer 100-200 mg of fine powder to a preheated lysis buffer CLB (with β-mercaptoethanol) centrifuge tube. Immediately vortex vigorously for 30-60 seconds or pipette and mix until a satisfactory homogenate is obtained;

[0044] (4) Return to the 65°C water bath for a short period of time (5-10 min), occasionally inverting 1-2 times to aid lysis;

[0045] (5) Centrifuge the lysate at 13,000 rpm for 10 min to precipitate the insoluble fragments;

[0046] (6) Take the supernatant of the lysate (you can take more supernatant as long as it does not exceed the capacity of the genomic DNA removal column, which can increase the yield) and transfer it to a new centrifuge tube. Add half the volume of the supernatant (0.5 volume) of anhydrous ethanol. Precipitation may occur at this time, but it will not affect the extraction process. Mix it immediately by pipetting and do not centrifuge.

[0047] (7) Add the mixture (less than 720 μL each time, which can be added twice at most) to a genome cleanup column, centrifuge at 13,000 rpm for 2 min, and discard the waste liquid;

[0048] (9) Place the genomic DNA cleanup column in a clean 2 mL centrifuge tube, add 500 μL of lysis buffer RLT Plus to the genomic DNA cleanup column, centrifuge at 13,000 rpm for 30 seconds, collect the filtrate, use a micropipette to more accurately estimate the volume of the filtrate (usually about 450-500 μL, the volume lost during filtration should be subtracted), add 0.5 times the volume of anhydrous ethanol, and mix immediately by pipetting. Do not centrifuge;

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

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

[0051] (12) Add 500 μL of Rinse Buffer RW, centrifuge at 13,000 rpm for 30 s, and discard the waste liquid. Add 500 μL of Rinse Buffer RW, and repeat;

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

[0053] (14) Take out the adsorption column RA and place it in an RNase-free centrifuge tube. Add 30-50 μL RNase-free H2O to the middle part of the adsorption membrane according to the expected RNA yield (preheating in a 65°C water bath can increase the yield). Leave it at room temperature for 1 min and centrifuge at 12,000 rpm for 1 min.

[0054] (15) If the expected RNA yield is >30 μg, add 30-50 μL RNase-Free H2O and repeat step 9. Combine the two washes or use the first eluate and add it back to the column and repeat the step.

[0055] 2.2 Synthesis of the first strand of reverse transcribed cDNA

[0056] The first-strand cDNA was synthesized by reverse transcription of the total RNA according to the instructions of PrimeScriptTM RT Reagent Kit (Perfect Real Time) (TaKaRa).

[0057] (1) Prepare the following mixed solution for genome removal reaction:

[0058]

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

[0060] (2) Prepare the following mixture for reverse transcription reaction:

[0061]

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

[0063] 2.3 Target gene cloning

[0064] 2.3.1 Gene cloning

[0065] Specific primers for PbHDZ35 were designed (Table 1), and the amplification system and procedure are shown in Table 2.

[0066] Table 1 Cloning primers of PbHDZ35

[0067]

[0068] Table 2 Cloning system

[0069]

[0070] Reaction program: 98°C, 30 sec, 52°C, 5 sec, 72°C, 1 min, 35 cycles; 72°C, 1 min; 16°C, hold.

[0071] 2.3.2 Target fragment recovery

[0072] A 1% agarose gel was prepared and the PCR product was detected by agarose gel electrophoresis. If the bands were correct, the target fragments were excised and recovered 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) Use a clean scalpel to cut out the gel block containing the target fragment under ultraviolet light, chop the gel block into pieces and put it into a 2 mL centrifuge tube, weigh and calculate the volume of the gel block (with 1 mg = 1 μL as the standard);

[0075] (3) Add 3 times the volume of the gel into the gel block and dissolve the gel block at room temperature, shaking intermittently to mix;

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

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

[0078] (5) Add 700 μL of Buffer WB to the adsorption column, incubate at room temperature at 12,000 rpm for 1 min, and discard the waste liquid;

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

[0080] (6) Idle: 12000 rpm, 1 min;

[0081] (7) Place the adsorption column in a sterilized 1.5 mL centrifuge tube, add 30 μL of sterile water (preheated to 65°C) to the adsorption membrane, let it stand at room temperature for 1 min, centrifuge at 12,000 rpm for 1 min, and store the collected solution at -20°C.

[0082] 2.3.3 Target fragment ligation and transformation

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

[0084]

[0085] (2) Transformation: Take Trans T1 (Transgene, Beijing) competent E. coli, melt it in an ice bath, add 2 μL of the above ligation product, place it in an ice bath for 30 min, heat shock it in a 42°C water bath for 30 s, then quickly and steadily transfer the centrifuge tube to an ice bath for 2 min, add 500 μL of LB medium without antibiotics, and culture it at 37°C on a shaker at 200 rpm for 1 h. 4000 rpm for 2 min, remove part of the supernatant, keep 100 μL of the bacterial solution, re-pump and mix the bacteria, and spread it on a solid LB medium (containing 50 mg·mL -1 Kana) and cultured inverted at 37°C for 12 h.

[0086] (3) Bacterial test: Pick a single white colony on the plate and add 500 μL of liquid LB medium (containing 50 mg mL -1 Kana), 37℃ shaker 200rpm culture for 3-5h. Take 1μL bacterial solution as template for PCR detection, the primers are gene cloning primers, the system and procedure are as follows:

[0087]

[0088] The reaction program was: 94°C, 5 min; 94°C, 30 sec, 52°C, 30 sec, 72°C, 1 min, 35 cycles; 72°C, 5 min; 16°C, hold.

[0089] (4) Detect the PCR products of the bacterial solution by agarose gel electrophoresis, and select the positive clones and send them to Zhejiang Youkang Biotechnology Co., Ltd. for sequencing.

[0090] (5) After the SnapGene sequencing results are correct, the plasmid is extracted using the Transgene EasyPure PlasmidMiniPrep Kit, and the expression vector of the PbHDZ35 gene is constructed using the obtained positive plasmid.

[0091] 2.3.4 Construction of expression vector using homologous recombination

[0092] according to IIOne Step Cloning Kit was used to construct the expression vector by homologous recombination.

[0093] (1) First, add the target gene-specific primers containing pK2W7-eYGFPuv-3xFLAG and pYES2 linker sequences, and use the positive plasmid obtained in the previous step as a template to prepare the following PCR system (20 μL):

[0094]

[0095] Reaction program: 98°C, 30 sec, 54°C, 5 sec, 72°C, 1 min, 35 cycles; 72°C, 5 min; 16°C, hold.

[0096] (2) After ensuring that the product is a single band, the target fragment was excised and recovered according to the instructions of MiniBEST Agarose Gel DNA Extraction Kit (Takara, Dalian).

[0097] (3) Double restriction enzyme digestion and linearization of the overexpression vectors pK2W7-eYGFPuv-3xFLAG and pYES2 were performed, and the following PCR system was configured using the pK2W7-eYGFPuv-3xFLAG and pYES2 plasmids as templates:

[0098]

[0099] Reaction procedure: 37°C, 2h; 16°C, hold.

[0100] (4) Refer to the instructions of MiniBEST Agarose Gel DNA Extraction Kit (Takara, Dalian) to excise the agarose gel and recover the target fragment.

[0101] (5) Genetic transformation: The expression vector used was pK2W7-eYGFPuv-3xFLAG, and the vector used for yeast stress verification was pYSE2. The expression vectors of the PbHDZ35 gene were constructed respectively. The following mixed solutions were prepared and incubated at 25°C overnight.

[0102]

[0103] The optimal amount of cloning vector used = [0.02 × number of base pairs of cloning vector] ng (0.03 pmol) The optimal amount of insert fragment used = [0.04 × number of base pairs of insert fragment] ng (0.06 pmol)

[0104] Reaction procedure: 37°C, 30 min; cool to 4°C or immediately cool on ice.

[0105] (6) Take 2 μL to transform Trans T1 (Transgene, Beijing) competent E. coli, and then pick a single clone for bacterial liquid PCR detection. The primers are gene cloning primers, and the PCR system is as follows:

[0106]

[0107] The reaction program was: 94°C, 5 min; 94°C, 30 sec, 52°C, 30 sec, 72°C, 1 min, 35 cycles; 72°C, 5 min; 16°C, hold.

[0108] (7) Detect the PCR product by agarose gel electrophoresis, transform it into Trans T1 (Transgene, Beijing) Escherichia coli competent cells, select the positive clones and send them to Zhejiang Youkang Biotechnology Co., Ltd. for sequencing. Extract the plasmid of the strain with the correct sequencing result and set aside.

[0109] 2.4 Lithium acetate method to transform yeast, the steps are as follows:

[0110] (1) Pretreatment of carrier DNA: insert the carrier DNA into a 95°C metal bath for 5 min or into a 95°C water bath in a float for 3 min. After heating, quickly insert it into ice.

[0111] (2) Take 100 μL of INVSc1 competent cells melted on ice, add 2-5 μg of pre-cooled target plasmid, 10 μL of pre-treated carrier DNA, and 500 μL of PEG / LiAc in sequence, pipette and hit several times to mix, and incubate in a 30°C water bath for 30 min (invert 6-8 times every 15 min to mix).

[0112] (3) Place the tube in a 42°C water bath for 15 min (invert 6-8 times at 7.5 min to mix thoroughly).

[0113] (4) Centrifuge at 5000 rpm for 40 seconds and discard the supernatant. Resuspend in 400 μL ddH2O and centrifuge for 30 seconds and discard the supernatant.

[0114] (5) Resuspend in 50 μL ddH2O, apply to plates, and culture at 29°C for 48-96 h.

[0115] 2.5 Analysis of drought stress tolerance in heterologous transformed yeast

[0116] After PCR verification, the positive single colony was picked and 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 until OD600 = 2.0. Take 100 μL of the induced yeast solution and centrifuge for 10 seconds to discard the supernatant, resuspend the bacteria in 1 mL of a solution containing 30% PEG6000 and continue to incubate for 36 h to test the drought resistance of yeast cells expressing PbHDZ35. The control group centrifuged and collected the same number of yeast cells, resuspended them in 1 mL of sterile water and incubated at 30°C for 36 h. The treated yeast cells were continuously diluted with sterile water in a 10-fold gradient (1×, 10×, 100×), and 2 μL was spotted on SD / -Ura medium, cultured at 30°C for 2 days, and the growth status of the yeast colonies was recorded.

[0117] 2.6 Liquid nitrogen method to transform GV1301 Agrobacterium

[0118] (1) Take out the competent Agrobacterium stored at -80°C and thaw it on ice;

[0119] (2) Take 0.5 μg of expression vector plasmid and add it to the competent Agrobacterium. After flicking to mix, place it on ice, in liquid nitrogen, in a 37°C water bath, and in an ice-water mixture for 5 min each.

[0120] (3) Add 700 μL of antibiotic-free YEP liquid culture medium to the centrifuge tube and place the bacterial solution in a shaker (28°C, 200 rpm) for 4-6 h;

[0121] (4) Centrifuge at 6000 rpm for 2 min, discard part of the supernatant, and pipette to mix evenly the remaining 70-100 μL of liquid;

[0122] (5) Apply the bacterial solution evenly on the surface containing antibiotics (50 mg mL -1 Spec and 50mg·mL -1 Rif) on YEP solid medium and inverted culture in a 28°C biochemical incubator for 48-72h;

[0123] (6) Pick a single clone on the plate for bacterial liquid PCR detection. The primers are gene cloning primers. The PCR system is as follows:

[0124]

[0125] The reaction program was: 94°C, 5 min; 94°C, 30 sec, 46°C, 30 sec, 72°C, 20 sec, 30 cycles; 72°C, 5 min; 16°C, hold.

[0126] (7) Add the same volume of 50% glycerol to the monoclonal bacterial suspension with positive test results and store it in a -80°C refrigerator.

[0127] 2.7 Agrobacterium-mediated inflorescence infection method for transformation of Arabidopsis

[0128] Take the Agrobacterium culture containing the target gene vector out of the -80℃ refrigerator, streak the plate for activation, pick a single clone and pipette it into a 1mL YEP (containing 50mg mL -1 Rif and 50mg·mL -1 Spec) liquid culture medium in an EP tube and culture at 28℃ shaking for 16h. Take 500μL of bacterial solution and transfer it to a 500mL conical flask with 200mL of the same culture medium, and continue to culture at 28℃ shaking until the OD is between 0.6-0.8. Add 10g sucrose and 80μL silwet-L77 to 200mL bacterial solution. Transfer the infection solution to a container with a large opening and a low height, and use a syringe to pump until a lot of foam appears. After immersing the Arabidopsis inflorescence with the pods cut off in advance in the bacterial solution for 1min, take it out and absorb the excess bacterial solution on the surface with a paper towel. Wrap it with plastic wrap to keep it moist, lie flat in the growth room and culture it in the dark for 2d. Then carry out normal light culture and carry out secondary infection one week later. Collect mature seeds in batches, dry them at 37℃ for 5d, and store them in a refrigerator at 4℃ for later use.

[0129] 2.8 Identification of positive transgenic Arabidopsis

[0130] Use M5 Hiper ultrafast mix reagent to identify Arabidopsis positive plants as follows:

[0131] (1) Take out the "Best Amplification Partner" and place it at room temperature. Observe whether there is precipitation before use. Make sure there is no precipitation in the lysate. Otherwise, dissolve it at 37°C and clarify it before use.

[0132] (2) Cut off 2 square millimeters of transgenic Arabidopsis leaves and place them in 20 μL of lysis buffer. Use fire to melt the yellow tip of the gun to form a "grinding pestle" to break up the leaf tissue.

[0133] (3) PCR instrument 98℃ for 5 minutes

[0134] (4) Centrifuge at 12000 rpm for 2 min and take 2 uL as PCR template. The system is as follows:

[0135]

[0136] The reaction program was: 95°C for 3 min; 94°C for 25 s, 60°C for 25 s, 72°C for 1 min, with 34 cycles; 72°C for 5 min.

[0137] 2.9 Fluorescence quantitative PCR identification

[0138] Total RNA was extracted from transgenic Arabidopsis leaves using the M5 Plant RNeasy Complex Mini Kit, and reverse transcribed into cDNA using the PrimeScriptTM RT Reagent Kit (Perfect Real Time) (TaKaRa, Dalian, China).

[0139] ChamQ SYBR qPCR Master Mix (Vazyme, Nanjing, China) was used for fluorescence quantitative PCR. The instrument used was Bio-Rad CFX-96. A 20 μL system mixture was prepared: 2×ChamQ SYBR qPCR Master Mix 10 μL, Primer-F 0.4 μL, Primer-R 0.4 μL, 50×ROX Reference Dye 1 0.4 μL, cDNA 1 μL, and ddH2O7.8 μL; the reaction program was: 95°C 30 s; 95°C 10 s, 60°C 30 s, 40 cycles; 65°C 5 s, 95°C 5 min (melting curve was set according to the instrument type). The primers used for fluorescence quantitative PCR are shown in Table 3, and AtActin was selected as the internal reference gene.

[0140] Table 3 Fluorescence quantitative PCR primers

[0141]

[0142] 2.10 Arabidopsis phenotypic observation and aboveground height determination

[0143] Wild-type and transgenic Arabidopsis seeds were sterilized and sown on MS solid culture medium. After culturing in the Arabidopsis growth chamber for 5 days, seedlings with consistent growth were selected and moved to the matrix. They were grown under natural conditions for 21 days, and their aboveground heights were measured.

[0144] 2.11 Phenotypic observation and root length measurement of Arabidopsis seedlings under mannitol-simulated drought stress

[0145] Wild-type and transgenic Arabidopsis seeds were sterilized and sown on MS solid medium. After culturing in the Arabidopsis growth room for 5 days, seedlings with consistent growth were selected and moved to MS + 200mM mannitol solid medium under a sterile environment. MS plates were used as controls and continued to grow for 3 days. The growth status was recorded by taking photos and the taproot length was measured.

[0146] 2.12 Phenotypic observation and physiological index measurement of Arabidopsis thaliana under natural drought stress

[0147] Wild-type and transgenic Arabidopsis thaliana grown naturally for 21 days were subjected to natural drought treatment at 0d, 5d, 10d, and 15d. A control group grown naturally during the same period of time was set up to measure the photosynthetic-related indicators of each group. At the same time, samples were taken and stored at -80 for later measurement of protective enzyme activity.

[0148] 2.12.1 Protecting enzyme activity

[0149] (1) POD activity detection

[0150] Sample treatment: Weigh 0.1g tissue, add 1ml extraction solution and homogenize in an ice bath. Then centrifuge at 8000g / 4℃ for 10min, take the supernatant and place it on ice for testing.

[0151] Working solution preparation: Before use, mix reagent 1, reagent 2, and reagent 3 in a ratio of 2.6 (mL): 1.5 (μL): 1 (μL) and preheat at 25°C for 10 min.

[0152] ELISA assay: Preheat the ELISA for more than 30 minutes, adjust the wavelength to 470nm, and set the value to zero with distilled water. Add 10μL of sample and 190μL of working solution to a 96-well plate, mix well, and record the absorbance value A1 at 470nm at 1 minute and the absorbance value A2 after 2 minutes. Calculate ▲A-A2-A1.

[0153] Calculation of tissue POD activity: POD (U / g fresh weight) = ▲A × V total ÷ (W × V' sample ÷ V total) ÷ ​​0.005 ÷ T = 4000 × ▲A ÷ W (Unit definition: A470 change of 0.005 per minute per gram of tissue in per mL of reaction system is one unit of enzyme activity.)

[0154] (2) SOD activity detection

[0155] Sample treatment: Weigh 0.1g tissue, add 1ml extraction solution and homogenize in an ice bath. Then centrifuge at 8000g / 4℃ for 10min, take the supernatant and place it on ice for testing.

[0156] Working solution preparation: Dilute reagent 2 twice with distilled water, and prepare as much as needed. (Reagent 2 and distilled water are diluted 1:1). Dissolve a bottle of reagent 4 with 5mL of distilled water (use it within one week after dissolution), and then dilute it 4 times with distilled water, and prepare as much as needed (Reagent 4 and distilled water are diluted 1:3). Before the measurement, bathe reagents 1, 3 and 4 in a water bath at 37℃ (mammals) or 25℃ (other species) for more than 5 minutes.

[0157] ELISA assay: Preheat the ELISA instrument for more than 30 minutes and adjust the wavelength to 560nm. Add the following reagents to the 96-well plate in sequence, mix well, let stand at room temperature for 30 minutes, and record the absorbance value A of each tube at 560nm.

[0158]

[0159] Calculation of tissue SOD activity: SOD activity (U / g fresh weight) = [inhibition percentage ÷ (1-inhibition percentage) × V total] ÷ (W × V sample ÷ V total) = 11.11 × inhibition percentage ÷ (1-inhibition percentage) ÷ W

[0160] (3) APX activity detection

[0161] Sample treatment: Weigh 0.1g tissue, add 1ml extraction solution and homogenize in an ice bath. Then centrifuge at 13000g / 4℃ for 20min, take the supernatant and place it on ice for testing.

[0162] Working solution preparation and microplate reader determination: Preheat the microplate reader for more than 30 minutes and adjust the wavelength to 290nm. Preheat reagent 1 at 25℃ for 30 minutes. Add 20μL supernatant, 140μL preheated reagent 1, 20μL reagent 2 and 20μL reagent 3 to the 96-well plate in sequence, mix quickly and measure the light absorption A1 and A2 at 290nm for 10s and 130s, △A=A1-A2.

[0163] Calculation of tissue POD activity: APX (nmol / min / g fresh weight) = ΔA ÷ (ε × d) × V total × 10 9 ÷(W×V sample ÷V sample total)÷T=3571×ΔA÷W (Activity unit definition: 1 nmol AsA oxidized per gram of tissue per minute is 1 enzyme activity unit.)

[0164] 2.12.2 Photosynthetic measurements

[0165] The photosynthesis-related indicators of Arabidopsis thaliana were measured using the LI6800 photosynthesis meter.

[0166] (1) Instrument calibration and setup: Close the leaf chamber to preheat and self-check the instrument. After preheating, plug in the carbon dioxide cylinder and set the environmental parameters: CO2 concentration is 400 ppm, the H2O sensor uses deionized water source, and light intensity (Photosynthetically Active Radiation, PAR): set the required light intensity, usually 200-1500 μmolphotons m -2 s -1 , the temperature of the leaf chamber is set to be consistent with the laboratory temperature (usually in the range of 20-25°C). The flow rate is usually set at 500μmol / s, which is adjusted according to the volume of the leaf chamber and the size of the leaves. Wait for the instrument indicators to stabilize before starting the next measurement.

[0167] (2) Material preparation and measurement: Select healthy, undamaged and fully expanded leaves for measurement. Usually select leaves that are mature all around, and avoid leaves that have just sprouted or aged. Gently wipe the surface of the leaf to ensure that there is no dust or impurities that interfere with the measurement. Open the leaf chamber of the LI-6800, place the leaf in the small leaf chamber (3×3cm), ensure that the leaf is flat and not twisted, and cover the leaf chamber cover. After the panel value is stable, mark and record the data.

[0168] 2.13 Pore opening

[0169] Leaves from the same part of wild-type and transgenic Arabidopsis thaliana grown naturally for 21 days were placed on a scanning electron microscope to observe the stomata on the back of the leaves and take pictures. After 10 days of natural drought treatment, the stomata were observed and photographed in the same way, and the length and width of the stomata were measured using imageJ software. Stomatal aperture = width / length.

[0170] 2.14 Water loss rate of aboveground parts

[0171] The aboveground parts of wild-type and transgenic Arabidopsis thaliana grown for 21 days were placed in a 25°C constant temperature incubator, and their fresh weights were recorded every 30 minutes for a total of 12 measurements. The water loss rate was calculated as the original fresh weight minus the fresh weight measured each time / the original fresh weight.

[0172] 3. Experimental results

[0173] 3.1 Total RNA extraction and analysis of Phoebe bournei roots

[0174] M5 Plant RNeasy Complex Mini Kit was used to extract total RNA from Phoebe bournei ( Figure 1 ), and the OD260 / 280 ratio of the extracted RNA was determined by UV spectrophotometer to be between 1.8 and 2.1, indicating that the total RNA purity was good; the agarose gel electrophoresis results showed that the 18s and 28s bands of the RNA sample were very clear, which could be inferred that the RNA was not degraded and met the requirements of the next experiment.

[0175] 3.2 Cloning and vector construction of P. bournei PbHDZ35 sequence

[0176] Using the cDNA of Phoebe bournei treated with drought as a template, PCR amplification was performed using the PbHDZ35 specific primers. The result was a target band of 616 bp in length ( Figure 2 ). The specific primers of the vector linker were PCR amplified by homologous recombination method and connected to the expression vectors pK2W7-eYGFPuv-3xFLAG and pYES2 and transformed into Escherichia coli. After sequencing by Zhejiang Youkang Biotechnology Co., Ltd., the single clone with the correct sequence was selected to extract the plasmid and then transformed into GV3101 Agrobacterium and INVSc1 yeast competent cells, and the cultured Agrobacterium bacterial solution was used to infect Arabidopsis thaliana.

[0177] 3.3 Analysis of tolerance of heterologous transformed yeast to PEG-simulated drought stress

[0178] It was observed that there was no significant difference in the growth status of yeast colonies between untreated transformed PbHDZ35 and empty plasmid pYES2. After 30% PEG simulated drought treatment, the growth status of yeast colonies transformed with PbHDZ35 was worse than that of the control, and there were obviously fewer clones ( Figure 3 ). These results indicate that PbHDZ35 significantly inhibited the drought stress tolerance of INVSc1 yeast in SD / -Ura selective medium.

[0179] 3.4 Identification of positive plants of PbHDZ35 transgenic Arabidopsis

[0180] After the PbHDZ35 gene of Phoebe bournei was transformed into Arabidopsis thaliana, the transgenic positive strains were obtained by PCR identification ( Figure 4 ), and the expression level of PbHDZ35 in positive plants was detected by fluorescence quantitative PCR ( Figure 5 ), the results showed that the expression levels of PbHDZ35 in L7 and L8 strains were 14.81 and 21.15 times that of the control, respectively.

[0181] 3.5 Determination of the aboveground growth and biomass of PbHDZ35 transgenic Arabidopsis thaliana under natural growth

[0182] To explore the effect of overexpression of PbHDZ35 on Arabidopsis, the phenotypes of the wild type and two homozygous overexpression lines (L7 and L8) were compared after 21 days of natural growth. It was found that the overexpression lines grew significantly faster than the wild type plants ( Figure 6 ).

[0183] 3.6 Phenotypic observation and root length measurement of PbHDZ35 transgenic Arabidopsis thaliana treated with mannitol-simulated drought

[0184] To further explore the role of the PbHDZ35 gene in plant drought stress, the wild type and two homozygous overexpression lines (L7 and L8) were treated with mannitol to simulate drought. It was found that the overexpression lines grew better than the WT under natural growth, but showed more sensitivity under mannitol treatment ( Figure 7 ).

[0185] 3.7 Phenotypic observation and physiological index determination of PbHDZ35 transgenic Arabidopsis thaliana under natural drought treatment

[0186] The wild type and two homozygous overexpression lines (L7 and L8) were subjected to natural drought treatment. It was found that after 15 days of natural growth, the number and size of leaves of the overexpression lines were significantly greater than those of the WT, while their growth was significantly weaker than that of the WT after 15 days of drought treatment ( Figure 8a). After measuring the antioxidant enzyme activity, it was found that after 15 days of drought, the protective enzyme activities (POD, SOD, APX) of the overexpression strain were significantly lower than those of the WT ( Fig. 9 d). Photosynthetic data showed that the net photosynthesis and stomatal conductance of the overexpression plants were significantly higher than those of the WT plants under normal growth, while they showed a faster decline rate after natural drought for 10-15 days ( Figure 8 b). To further explain its phenotype, the degree of stomatal opening and closing was observed using a scanning electron microscope. It was found that the stomatal opening of the overexpression strain was significantly higher than that of the WT under natural growth, and the stomata could not be closed after drought ( Fig. 9 a, b). The aerial parts of Arabidopsis were placed in a 25℃ constant temperature box to observe the water loss rate. It was found that the aerial parts of the overexpression strain had a significantly higher water loss rate than the WT, further verifying the excessive opening of its stomata ( Fig. 9 c).

[0187] Summarizing the phenotypic comparison between the overexpression plants and WT in the above experiments, it is believed that overexpression of the PbHDZ35 gene will promote plant growth and weaken the tolerance to drought stress, that is, knocking out the PbHDZ35 gene can improve the drought resistance of plants.

[0188] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the technical principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A Phoebe bournei PbHDZ35 gene, characterized in that: The P. minnan PbHDZ35 gene has the nucleotide sequence shown in SEQ ID No: 1, or a sequence in which one or several nucleotides are deleted, added and / or substituted but the function remains unchanged.

2. The gene according to claim 1, characterized in that The nucleotide sequence of the gene is SEQ ID No:

1.

3. The protein encoded by the gene according to claim 1 or 2, characterized in that The protein consists of the amino acids shown in SEQ ID No.

2.

4. A method for promoting plant growth and development, the method comprising preparing a modified plant containing the PbHDZ35 gene of Phoebe bournei, characterized in that: Compared with the wild-type plant, in the modified plant, the expression of the Phoebe bournei PbHDZ35 gene is increased; wherein the base sequence of the Phoebe bournei PbHDZ35 gene is shown in SEQ ID NO:

1.

5. Application of Phoebe bournei PbHDZ35 gene in promoting plant growth and development, characterized in that: A modified plant containing the Phoebe bournei PbHDZ35 gene is prepared. Compared with a wild-type plant, in the modified plant, the expression of the Phoebe bournei PbHDZ35 gene is increased; wherein the base sequence of the Phoebe bournei PbHDZ35 gene is shown in SEQ ID NO:

1.

6. The use according to claim 5, characterized in that: The plant is Phoebe bournei or Arabidopsis thaliana.

7. The use according to claim 6, characterized in that: The plant is Arabidopsis thaliana.

8. The use according to claim 7, characterized in that: The Phoebe bournei PbHDZ35 gene is connected to a vector, transformed into Arabidopsis thaliana through Agrobacterium-mediated transformation, and then screened, cultured, and transgenic strains are obtained.

9. Application of Phoebe bournei PbHDZ35 gene in improving drought resistance of plants, characterized in that: A modified plant containing the Phoebe bournei PbHDZ35 gene is prepared, wherein the expression of the Phoebe bournei PbHDZ35 gene is reduced in the modified plant relative to the wild-type plant; wherein the base sequence of the Phoebe bournei PbHDZ35 gene is shown in SEQ ID NO:

1.

10. The use according to claim 9, characterized in that: The plant is Arabidopsis thaliana.

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