TmERF11 protein and TmERF11 gene capable of influencing drought tolerance of taxodium distichum and application of TmERF11 protein and TmERF11 gene

Abstract

The invention provides a TmERF11 protein and a TmERF11 gene influencing the drought tolerance of cypress and application of the TmERF11 protein and the TmERF11 gene, and belongs to the technical field of plant genetic engineering. The invention provides a TmERF11 protein, and the amino acid sequence of the protein is as shown in SEQ ID NO. 10. According to the invention, the TmERF11 gene related to the drought tolerance of the cypress in Mexican is found for the first time, the TmERF11 gene is transferred into the cypress to be over-expressed, and the TmERF11 gene is subjected to drought stress in the natural environment for 14 days, so that the transgenic cypress is good in growth vigor and is not influenced by stress, and the leaf of the non-transgenic cypress is yellow, and the plant is gradually dried and dead. It is shown that the TmERF11 gene is an important gene responding to drought stress and has important application value in the field of forest tree genetic engineering. The specific function of the TmERF11 in the drought stress process is analyzed, and a theoretical foundation is laid for molecular breeding of high-drought-tolerance cypress trees based on genetic improvement.

Description

Technical Field

[0001] The invention belongs to the technical field of plant genetic engineering, and particularly relates to a TmERF11 protein and a TmERF11 gene affecting the drought resistance of Baldcypress and applications thereof. Background Art

[0002] Taxodium mucronatum (Taxodium mucronatum) is a species of the genus Taxodium in the family Taxaceae. It is an ancient relict plant that is currently naturally distributed in the United States, Mexico and Guatemala. It was introduced to my country by Han An and William Borden in 1917. Taxodium mucronatum has the advantages of fast growth, high ornamental value, tolerance to low temperatures, water and humidity, tolerance to salt and alkali, and resistance to diseases and insect pests. It is often used as an excellent tree species for greening courtyards, roads, and rivers. It is also a special tree species for beach mudflats and saline-alkali land and an important shelterbelt tree species. It has both ecological value and industrial utilization value.

[0003] However, there are no reports on the drought tolerance regulation mechanism of Baldcypress and how to exert its drought tolerance. Summary of the invention

[0004] In view of the defects in the prior art, the purpose of the present invention is to provide a TmERF11 protein and a TmERF11 gene. The TmERF11 protein and the TmERF11 gene are important genes that affect the drought resistance of Baldcypress. Overexpression of the TmERF11 gene can improve the drought resistance of the plant.

[0005] The objective of the present invention is achieved through the following technical solutions:

[0006] The present invention provides a TmERF11 protein, the amino acid sequence of the protein is shown in SEQ ID NO.10.

[0007] The present invention provides a TmERF11 gene, the nucleotide sequence of the TmERF11 gene is shown as SEQ ID NO.9.

[0008] The present invention provides a recombinant vector, which contains the TmERF11 gene described in the above technical solution.

[0009] Preferably, the recombinant vector overexpresses the TmERF11 gene.

[0010] The present invention provides a recombinant bacterium or a recombinant cell, wherein the recombinant bacterium or the recombinant cell contains the TmERF11 gene described in the above technical solution.

[0011] The present invention provides the use of the TmERF11 protein, the TmERF11 gene, the recombinant vector or the recombinant bacteria or the recombinant cell in the above technical solution in regulating plant drought resistance and / or regulating plant ethylene synthesis.

[0012] Preferably, overexpression of the TmERF11 gene can enhance the drought tolerance of the plant and / or promote ethylene synthesis in the plant.

[0013] The present invention provides the use of the TmERF11 protein, the TmERF11 gene, the recombinant vector or the recombinant bacteria or the recombinant cell in the above technical solution in plant breeding.

[0014] The present invention provides a method for improving drought tolerance of plants, comprising the following steps:

[0015] Increasing the expression of the TmERF11 gene in the plant or increasing the content of the TmERF11 protein in the plant can improve the drought resistance of the plant.

[0016] Preferably, the method for increasing the expression of the TmERF11 gene in a plant or increasing the content of the TmERF11 protein in a plant comprises:

[0017] The recombinant vector described in the above technical solution or the recombinant bacteria or recombinant cells described in the above technical solution are introduced into the plant body.

[0018] Beneficial effects of the present invention:

[0019] The present invention provides a TmERF11 protein, the amino acid sequence of which is shown in SEQ ID NO. 10. Through research, it is found that the drought resistance of the Chinese fir is relatively outstanding. Baldcypress (T.distichum), Chinese fir, pond cypress (T.distichumvar.imbricatum) and their hybrid offspring T.hybrid "Zhongshanshan"302, T.hybrid "Zhongshanshan"407 and T.hybrid "Zhongshanshan"118 were taken as the research objects, and the responses of biomass, photosynthetic parameters and antioxidant enzyme system to natural drought stress and rewatering were studied, as well as the response mechanism of proline and root morphological characteristics of T.distichum and the adaptability to soil in different regions were studied. The conclusion was that the drought tolerance of plants was in the order of Chinese fir>Chinese fir 118>Chinese fir 407>Chinese fir 302>pond cypress>baldcypress. Among the genus Baldcypress, T.hybrid "Zhongshanshan"302 was relatively drought-resistant, and the drought tolerance of T.hybrid was significantly lower than that of T.hybrid. Based on the transcriptome data of drought stress of baldcypress, the present invention found that the root, stem and leaf tissues of the baldcypress ERF11 (Ethylene responsive factor 11) gene can respond to drought stress, and show continuous high expression under natural drought environment. The present invention first discovered the TmERF11 gene related to drought resistance of baldcypress. By transferring the TmERF11 gene into baldcypress for overexpression, the transgenic baldcypress grew well and was almost unaffected by the stress under drought stress in the natural environment for 14 days, while the leaves of non-transgenic baldcypress turned yellow and fell off, and the plants gradually dried up and died. It shows that the TmERF11 gene is an important gene that responds to drought stress and will have important application value in the field of forest genetic engineering. The present invention uses molecular biological methods such as genetic transformation to analyze the specific function of TmERF11 in the process of drought stress, laying a theoretical foundation for molecular breeding of highly drought-resistant baldcypress trees based on genetic improvement. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0021] Figure 1 is a schematic diagram of vector pBI122;

[0022] Figure 2The figure shows the GUS staining results of pBI122-TmERF11 Baldcypress transgenic plants and their control plants;

[0023] Figure 3 The figure shows the PCR test results of pBI122-TmERF11 Baldcypress transgenic plants and their control plants;

[0024] Figure 4 The expression change diagram of the target gene in the pBI122-TmERF11 Baldcypress transgenic plants and their control plants;

[0025] Figure 5 This is a comparison of ACC synthase activity in pBI122-TmERF11 Baldcypress transgenic plants and their control plants;

[0026] Figure 6 This is a diagram showing the phenotypic changes of pBI122-TmERF11 transgenic Baldcypress and control plants after 14 days of drought. DETAILED DESCRIPTION

[0027] The present invention provides a TmERF11 protein, the amino acid sequence of which is shown in SEQ ID NO. 10. The present invention provides a TmERF11 gene, the nucleotide sequence of which is shown in SEQ ID NO.9.

[0028] Based on the drought stress transcriptome data of Baldcypress, the present invention discovered that the root, stem and leaf tissues of the Baldcypress ERF11 (Ethylene responsive factor 11) gene can respond to drought stress, and show sustained high expression under natural drought environment. The present invention discovered for the first time the TmERF11 gene related to drought resistance of Baldcypress. By transferring the TmERF11 gene into Baldcypress for overexpression, the transgenic Baldcypress grew well and was almost unaffected by the drought stress in the natural environment for 14 days, while the leaves of the non-transgenic Baldcypress turned yellow and fell off, and the plants gradually dried up and died. This shows that the TmERF11 gene is an important gene that responds to drought stress and will have important application value in the field of forest genetic engineering.

[0029] The present invention preferably obtains the complete coding reading frame of the TmERF11 gene by 3'RACE and 5'RACE nested PCR, as shown in SEQ ID NO. 9. The present invention compares the TmERF11 gene in NCBI and finds that the TmERF11 gene has the highest homology with the gene of C. zhongshanensis ThERF11 (homology 92%).

[0030] In the present invention, the primer pair for amplifying the TmERF11 gene preferably includes TmERF11 ORF F and TmERF11ORF R; the TmERF11 ORF F is as shown in SEQ ID NO.11, specifically: 5'-ATGTGTGGAGGAAGCATCAT-3', and the TmERF11 ORF R is as shown in SEQ ID NO.12, specifically: 5'-TTAAAGATTGCAGTCCACAGACT-3'.

[0031] The present invention provides a recombinant vector, which contains the TmERF11 gene described in the above technical solution. The present invention does not specifically limit the vector used when constructing the recombinant vector, and the conventional vector in the art can be used to express the target gene. In the present invention, the recombinant vector preferably includes the complete coding reading frame of the TmERF11 gene. In the present invention, the recombinant vector is preferably capable of achieving overexpression of the TmERF11 gene in plants, and more preferably capable of achieving overexpression of the TmERF11 gene in baldcypress plants. In the present invention, the backbone vector of the recombinant vector preferably includes a pBI122 vector. In the present invention, in the recombinant vector, the TmERF11 gene is preferably located after the promoter P35S, and under the drive of the promoter P35S, TmERF11 is efficiently expressed in plants. One of the advantages of the pBI122 vector selected in the present invention is that the pBI122 vector has a GUS gene fragment, and the transgenic plants and control plants are stained with GUS dye, and combined with PCR identification, it can be determined whether the plant is a transgenic positive plant. The present invention has no particular limitation on the method for constructing the recombinant vector, and any conventional construction method in the art may be used.

[0032] The present invention provides a recombinant bacterium or a recombinant cell, wherein the recombinant bacterium or the recombinant cell contains the TmERF11 gene described in the above technical solution. The present invention preferably transforms the recombinant vector described in the above technical solution into bacteria to obtain a recombinant bacterium containing the TmERF11 gene; and transforms the recombinant vector described in the above technical solution into cells to obtain a recombinant cell containing the TmERF11 gene. In the present invention, the recombinant cell includes a cell that can develop into a complete plant; the cell may also include a competent cell. In the present invention, the bacterium preferably includes Agrobacterium. In the present invention, the Agrobacterium preferably includes Agrobacterium competent GV3101. The present invention preferably transfers the recombinant vector into Agrobacterium by liquid nitrogen freeze-thaw method to obtain a recombinant strain. The present invention does not specifically limit the method of liquid nitrogen freeze-thaw method, and the transformation can be carried out by conventional liquid nitrogen freeze-thaw methods in the art.

[0033] The present invention provides the use of the TmERF11 protein, the TmERF11 gene, the recombinant vector or the recombinant bacteria or the recombinant cell in the above technical solution in regulating plant drought resistance and / or regulating plant ethylene synthesis.

[0034] The present invention provides the use of the TmERF11 protein, the TmERF11 gene, the recombinant vector or the recombinant bacteria or the recombinant cell in regulating the drought resistance of plants. The present invention can improve the drought resistance of plants by overexpressing the TmERF11 gene. As an optional embodiment, the plant includes baldcypress. The present invention transfers the TmERF11 gene into baldcypress to make it overexpressed. After 15 days of drought stress in the natural environment, the transgenic baldcypress still grows well, while the leaves of the wild-type baldcypress turn yellow and dry, and the plant dies.

[0035] The present invention provides the use of the TmERF11 protein, the TmERF11 gene, the recombinant vector or the recombinant bacteria or the recombinant cell in regulating plant ethylene synthesis. The present invention can promote plant ethylene synthesis and increase the ethylene synthesis rate by overexpressing the TmERF11 gene.

[0036] The present invention provides the use of the TmERF11 protein, the TmERF11 gene, the recombinant vector or the recombinant bacteria or the recombinant cell in plant breeding. The present invention constructs a TmERF11 overexpression vector through cloning technology, and the TmERF11 gene is efficiently expressed in plants through the TmERF11 overexpression vector, which is beneficial to improving the drought resistance of the plant, and drought-resistant plants can be obtained, which is beneficial to the cultivation of drought-resistant plants.

[0037] The present invention provides a method for improving drought tolerance of plants, comprising the following steps:

[0038] Drought-resistant plants are obtained by increasing the expression of the TmERF11 gene in plants or increasing the content of the TmERF11 protein in plants. The method of increasing the expression of the TmERF11 gene in plants or increasing the content of the TmERF11 protein in plants of the present invention may include: introducing the recombinant vector described in the above technical scheme or the recombinant bacteria described in the above technical scheme into the plant. The present invention preferably introduces the recombinant vector into the recombinant bacteria, and then introduces the recombinant bacteria into the plant, so that the TmERF11 gene is overexpressed in the plant, thereby improving the drought resistance of the plant. The present invention does not specifically limit the method of introducing the recombinant vector into the recombinant bacteria, and conventional methods in the art can be used. The present invention does not specifically limit the method of introducing the recombinant bacteria into the plant, and conventional methods in the art can be used. As an optional embodiment, the plant includes baldcypress.

[0039] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0040] Example 1 Cloning of the full-length TmERF11 gene

[0041] 1) RNA extraction and cDNA inversion

[0042] The total RNA of baldcypress was extracted using RNeasyPlantMini Kit (QIAGEN), and the total RNA concentration and purity were determined using NanoDrop 1000. TM PCR cDNA Synthesis Kit, 2PCRKit was used for reverse transcription.

[0043] 2) 3'RACE and 5'RACE nested PCR of TmERF11 gene

[0044] Two-year-old Mexican baldcypress was subjected to drought stress, and a control group was set up at the same time. All Mexican baldcypress potted plants were moved into the greenhouse, and the drought stress treatment group was subjected to natural drought stress for 10 days, and no watering was continued during the natural drought stress process. The control group was supplemented with water to drain the bottom of the pot at 6 pm every day, and the relative soil moisture content was maintained at 75% to 80%. When the drought stress lasted for 10 days, the drought stress treatment group and the control group were sampled, quickly frozen in liquid nitrogen, and then stored in an ultra-low temperature refrigerator at -80℃, and then sent to Beijing Qinglian Bio Company for transcriptome sequencing.

[0045] The plants treated with drought stress and the control group plants were subjected to the method described in step 1) to obtain cDNA. The obtained cDNA was extracted and transcriptome sequencing was performed. Genes with significant differential expression under drought stress were screened according to the transcriptome data to obtain the Unigene sequence of TmERF11.

[0046] The Unigene sequence of TmERF11 was obtained by transcriptome data screening, and primers were designed using Oligo 8 software (see Table 1). 3'RACE PCR and 5'RACE PCR were amplified using Takara's cDNA endrapid amplification kit. The specific operation process was completed according to the kit instructions, including 3'RACE nested PCR: reverse transcription reaction, 3'RACEOuterPCR, 3'RACE InnerPCR; 5'RACE nested PCR: dephosphorylation reaction, decapping reaction, reverse transcription reaction, 5'RACE OuterPCR, 5'RACE InnerPCR. The final PCR products were recovered, connected, transformed, and sequenced.

[0047] Table 1 3'RACE, 5'RACE PCR amplification primer sequences

[0048]

[0049] 3) Obtaining the coding reading frame sequence of TmERF11 gene

[0050] The 3'RACE and 5'RACE PCR sequencing products of TmERF11 obtained by sequencing were spliced ​​using Bioedit software, and the target gene sequence was obtained for Blast comparison on the NCBI official website, confirming that the target gene had the highest gene homology with the Zhongshan fir TheRF11 gene (homology 92%), so it was named Taxodium mucronatum ethyleneresponsive factor 11 gene, abbreviated as TmERF11, which contains a complete coding reading frame of 1002bp, the nucleotide sequence of the coding reading frame is shown in SEQ ID NO.9, and the amino acid sequence encoded by it is shown in SEQ ID NO.10.

[0051] SEQ ID NO.9:

[0052]

[0053] SEQ ID NO.10:

[0054] MCGGSIISEFIAGKTNGRKTTVRDIWTDFDKFSEYHLGKVPHEAPCQKEEEERVVKKVVEKKRKAHHLYRGVRQRPSGKWAAEIRDPIKGSRVWLGTYNSVEDAAIAYDHEARKIRGKKAKLNFPTRPITTDKSTDKVNVSVQSVSDPWKSYSSQISEEESVVVPAA NYEKKTEWLSPEVSEAAVHEYMENLERVLELKPQSATQQCCSMGFHDNNGGLSCYQSQDMTSSSSSSINRNKTHESFCESESSVSSLDSILEEASERRTQLESPRSYESVEGLIESEMGLFESPYFGARQNECPEEACLGGLPFLKRANQLDQAWIESEESLWTAIF.

[0055] Example 2 Construction of TmERF11 gene plant expression vector

[0056] The coding reading frame (ORF) sequence of TmERF11 was further obtained for vector construction, and specific primers were designed using Oligo8 software to amplify the ORF sequence of the TmERF11 gene, wherein the primers were: TmERF11 ORF F as shown in SEQ ID NO.11, specifically: 5'-ATGTGTGGAGGAAGCATCAT-3', TmERF11 ORF R as shown in SEQ ID NO.12, specifically: 5'-TTAAAGATTGCAGTCCACAGACT-3'. PCR reaction system: TakaRa LA Taq (5Μ / μL) 0.5μL, 10×LAPCR Bμffer (MgCl2) 2+ Free) 5.0μL, MgCl2 (25mM) 5.0μL, dNTP Mixtμre (2.5mM each) 8.0μL, Primer F (10μM) 2.0μL, Primer R (10μM) 2.0μL, cDNA template 1.0μL, Milli-Q Water 26.5μL. The reaction program is: 94℃3min; 94℃30sec, 56℃30sec, 72℃1min30sec, 25cycles; 72℃10min; 4℃Forever.

[0057] After the PCR reaction is completed, a PCR product is obtained, and the PCR product is sequenced to verify that the amplified target gene is the ORF fragment of the TmERF11 gene.

[0058] Using Gateway directional cloning technology, the ORF fragment of the TmERF11 gene was transferred to the target expression vector pBI122 (pBI122 is a commercial vector purchased from Shanghai Zeye Biotechnology Co., Ltd.). The reaction includes two parts: BP reaction and LR reaction. The structure of the pBI122 vector used in this example is shown in Figure 1 shown.

[0059] 1) The purpose of BP reaction is to transfer the gene fragment to the entry vector. The reaction system is: vector TmERF11ORF fragment 10-20ng, Salt solution 1μL, pCRTM8 / GW / TOPOTM vector (entry vector) 1μL, add Nuclease-free Water to a total volume of 6μL, gently mix, react at 22℃ for 60min and then transfer to ice; 6μL of the reaction product in the previous step is transformed into TOP10 competent cells, applied to LB screening culture plates containing 100mg / L spectinomycin, and cultured at 37℃ for 12h before picking single clones for bacterial liquid PCR detection. The primers used are gene-specific upstream primers, TmERF11 ORFF as shown in SEQ ID NO.11, specifically: 5'-ATGTGTGGAGGAAGCATCAT-3', and T7 primers on the vector, as shown in SEQID NO.13, specifically: 5'-TAATACGACTCACTATAGGG-3'. The positive clones after detection are further sequenced for verification. The plasmids of the positive clones were extracted and purified to obtain the purified plasmid of the entry vector, namely the recombinant entry vector.

[0060] 2) The purpose of the LR reaction is to clone the target gene of the recombinant entry vector into the destination vector. The reaction system is as follows: 100 ng of the purified plasmid of the entry vector, 1.5 μL of the destination vector pBI122 (100 ng / μL), 2 μL of LR Clonase II enzyme mix, add 1×TE (pH 8.0) to a total volume of 8 μL, vortex and briefly centrifuge, incubate at 25°C for 1 hour, add 1 μL of Proteinase K solution, mix well, and incubate at 37°C for 10 minutes to terminate the reaction; 2 μL of the LR reaction product was taken to transform Top10 competent cells, spread on an LB screening culture plate containing 100 mg / L ampicillin, pick a single clone for bacterial liquid PCR detection, the primers are M13F and M13R, the sequence of M13F is shown in SEQ ID NO.14, specifically: TGTAAAACGACG GCCAGT; the sequence of M13R is shown in SEQ ID NO.15, specifically: CAG GAAACAGCTATGACC. The positive clones after detection were further sequenced and verified. After verification, the recombinant vector after LR reaction was recovered and purified, and the expression vector pBI122-TmERF11 of the target gene was obtained, which was stored at -20°C for use in the genetic transformation of Baldcypress.

[0061] Example 3 Genetic transformation of Baldcypress with TmERF11 gene

[0062] 1) The pBI122-TmERF11 expression vector was transferred into Agrobacterium using the liquid nitrogen freeze-thaw method. The steps are as follows:

[0063] (1) Take the competent Agrobacterium GV3101 stored at -80℃ and thaw it on ice; (2) Add 200ng of the purified expression vector to 50μL competent cells, gently stir the bottom of the tube to mix, and then let it stand on ice for 5min, in liquid nitrogen for 5min, in a 37℃ water bath for 5min, and in an ice bath for 5min; (3) Add 800μL of antibiotic-free LB medium, gently shake at 28℃ and 100rpm, and resuscitate for 3h; (4) Centrifuge at 6000rpm for 1min, take about 100μL of supernatant, gently blow to resuspend the bacterial block, and spread it on an LB solid medium plate containing 50mg / Lkan (kanamycin); (5) Invert and culture at 28℃ for 30-48h, detect positive clones by PCR, and store at 4℃ for later use.

[0064] 2) Baldcypress seedlings infection

[0065] The positive clones (positive Agrobacterium) were placed in transformation solution (1 / 2MS + 0.5mg / L KT + 0.5mg / L NAA + 120mM / LAS + 200mg / L DTT + 0.02% Tween20 + 40mM / L CaCl2 + 20g / L sucrose) for propagation (28°C, 300r / min) until the bacterial solution OD 600 The value was 0.7, and the positive Agrobacterium infection bacterial solution was obtained. The positive Agrobacterium infection bacterial solution was stored at 4°C for later use.

[0066] In the clean bench, the seed shells of the bald cypress seeds were removed using scissors and tweezers, and the bald cypress tissue culture seedlings were obtained by tissue culture using MS medium using conventional tissue culture methods. The bald cypress tissue culture seedlings grown to 14 days were placed in the transformation solution containing the overexpression vector prepared in advance (i.e., the positive Agrobacterium infection solution), and the vacuum device was used for 15 minutes, and then the shaker was used for transformation at 90r / min for 25 minutes, and the positive Agrobacterium solution was allowed to infect the bald cypress, and then the washing solution (40mM CaCl2+30g / L sucrose+0.5mg / L NAA+2mg / L 6-BA+200mg / LDTT) for 3 times, clean the infected tissue culture seedlings, place them on sterile absorbent paper, dry the surface moisture and culture them in co-culture medium (MS+150μM AS+200mg / LDTT+10g / L sucrose+2g / L gel) at 25°C in the dark for 60h to obtain transgenic explants.

[0067] 3) Screening of transgenic plants

[0068] GUS detection was performed immediately after the co-cultivation. The transgenic explants and control plant explants (the control plant explants were wild-type plant explants, specifically: the seed coats of Baldcypress seeds were removed, and the plants with the same growth stage as the transgenic explants were obtained by tissue culture in MS medium using conventional tissue culture methods) were all immersed in the GUS reaction solution. After being stored at 37°C in the dark for 6 hours, they were decolorized with 75% anhydrous ethanol, observed and photographed. Since the pBI122 vector has a GUS gene fragment, the transgenic Baldcypress and the control plants were stained with GUS dye. The results are as follows Figure 2 shown. Figure 2 WT in the table is the control plant, and pBI122-TmERF11 is the pBI122-TmERF11 transgenic plant, the same below. Figure 2 It can be seen that the control plants were not stained, while the hypocotyls and leaves of the pBI122-TmERF11 transgenic plants were blue.

[0069] The DNA of the leaves of the transgenic baldcypress and the control baldcypress was further extracted for PCR detection. The primers were 35S and TmERF11 ORF R. The sequence of the 35S primer was as shown in SEQ ID NO.16, specifically: 5'-TGGTCCCCACAAGCCAGCTGCTCGA-3', and the sequence of TmERF11 ORF R was as shown in SEQ ID NO.12, specifically: 5'-TTAAAGATTGCAGTCCACAGACT-3'. The electrophoresis imaging results showed that the target band could be detected in the pBI122-TmERF11 transgenic plants, while the target DNA fragment was not amplified in the control (WT) ( Figure 3 The results of GUS and PCR tests showed that the transgenic plants of Baldcypress were positive.

[0070] Example 4 Molecular Detection of Taxodium truncatum Transgenic with pBI122-TmERF11 Gene

[0071] After obtaining positive plants by the method of Example 3, the positive plants were cultured on 200 mg / L kanamycin selection MS medium in a light incubator with long-day illumination (16 h of light and 8 h of darkness) and a temperature of 25° C. After growing for 14 days, they were used for real-time fluorescence quantitative detection.

[0072] The control plants were wild-type plants, specifically: the seed coats of Baldcypress seeds were removed, and tissue culture was performed in MS medium using conventional tissue culture methods to obtain plants at the same growth stage as the transgenic explants.

[0073] RNA was extracted from positive plants and control plants of Baldcypress for real-time quantitative molecular detection. Real-time quantitative primers were designed using Oligo8 software, the length of the amplified product was 123 bp, the primer Tm value was 57°C, and the primer sequences were: q TmERF11 F as shown in SEQ ID NO.17, specifically: 5'-TTGGAGAGGGTTTTGGAATTGA-3', qTmERF11 R as shown in SEQ ID NO.18, specifically: 5'-CGAGCTCGAGCTCGTCA-3'. The internal reference gene for real-time quantitative PCR was 18s gene (GenBank gene number: Z28335), 18s-F as shown in SEQ ID NO.19, specifically: 5'-TCAACTTTCGATGGTAGGATAGTG-3', 18s-R as shown in SEQ ID NO.20, specifically: 5'-CCGTGTCAGGATTGGGTAATTT-3'. qRT-PCR was performed using the Analitik Jenaq TOWER2.2 (Germany) system. According to the instructions of the SYBR Green kit (Rox), the amplification program was: 55°C for 2 min, 95°C for 10 min; 95°C for 15 s, 60°C for 1 min, and 40 cycles. Then the temperature was set from 60°C to 95°C to generate a melting curve. Each sample was technically replicated 3 times, and the 20 μL reaction system contained: 2 μL of diluted cDNA (cDNA concentration was approximately 500 ng·μL -1 ), 10 μL FarstStart Μniversal SYBR Green Master (Rox), 6 pmol upstream primer, 6 pmol downstream primer and 6.8 μL ddH2O. The relative expression of genes was calculated according to 2 -ΔΔCt The experimental data were statistically analyzed and plotted using SPSS 19.0 software. The quantitative PCR results are shown in Table 2 and Figure 4 Among them, CK1, CK2, CK3 and CK4 are 4 control plants, and ERF11-1, ERF11-2, ERF11-3 and ERF11-4 are the test results of 4 positive plants of Baldcypress.

[0074] Table 2 Detection results of relative expression of TmERF11 gene

[0075] name Expression name Expression CK1 1.0231 ERF11-1 259.6979983 CK2 1.0332 ERF11-2 524.3955427 CK3 1.0983 ERF11-3 203.0033415 CK4 1.07983 ERF11-4 824.4412213

[0076] From Table 2 and Figure 4 It was found that the expression level of the target gene TmERF11 in the transgenic plants was 200 to 800 times that of the non-transgenic plants, indicating that TmERF11 was overexpressed in the pBI122-TmERF11 gene-transfected Baldcypress plants.

[0077] Example 5 Physiological detection of baldcypress transformed with pBI122-TmERF11 gene

[0078] After obtaining positive plants by the method of Example 3, the positive plants were cultured on 200 mg / L kanamycin selection MS medium in a light incubator with long-day illumination (16 h of light and 8 h of darkness) and a temperature of 25° C. After growing for 14 days, they were used for ACC synthase activity detection.

[0079] The control plants were wild-type plants, specifically: the seeds of Baldcypress were removed from the seed coat, and the plants were cultured in MS medium using conventional tissue culture methods to obtain plants at the same growth stage as the transgenic explants. 1-aminocyclopropane-1-carboxylic acid (ACC) is a direct precursor for ethylene biosynthesis. Studies have reported that the release of ethylene is positively correlated with the content of ACC in plants. Therefore, the detection of ACC content and the rate of ACC generation can reflect the rate of ethylene synthesis in plants.

[0080] Material extraction: (1) Prepare a 15 mL centrifuge tube, add 15 mg PVP (polyvinyl pyrrolidone), and freeze it quickly with liquid nitrogen; (2) Accurately weigh about 1.0 g of the transgenic positive plant sample and the control sample of Baldcypress, grind them in liquid nitrogen until they are crushed, transfer them to the prepared 15 mL centrifuge tube (the centrifuge tube where PVP was added in step (1)), add 3 mL of Tricine (tris(hydroxymethyl)methylglycine) extract solution, shake for 1 min, and let it stand for 10 min; (3) Centrifuge at 4°C, 10,000 rpm for 3 min, take all the supernatant and add it to a new pre-cooled 15 mL centrifuge tube; (4) Pass the GE Sephadex desalting column, elute the column with 3 mL of tricine column wash solution, collect it in a pre-cooled centrifuge tube to obtain a crude enzyme solution; (5) Take 2 mL of the crude enzyme solution, add 200 μL of reaction buffer and 200 μL of SAM solution, and incubate at 25°C for 1 h; (6) Add 200 μL The reaction was terminated with HgCl2 solution (7) and filtered through a 0.22 μm filter membrane for HPLC-MS / MS detection.

[0081] Liquid quality testing:

[0082] The experiment used the AB Qtrap4500 mass spectrometer, the mass analyzer of which was a triple quadrupole-ion hydrazine mode. The ESI-HPLC-MS / MS method was used to quantitatively analyze plant hormones. The sample substances were separated by an Agilent 1290 high performance liquid chromatograph, with electrospray ionization (ESI) as the ion source, and scanned in multi-channel detection (MRM) mode. That is, multiple SIM (selected ion monitoring) mode scans were performed simultaneously.

[0083] (1) Preparation of standard solution: Use water as solvent to prepare ACC standard solution with a gradient of 0.5 ng / mL, 2 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 50 ng / mL, and 100 ng / mL.

[0084] (2) Liquid phase conditions: Chromatographic column: Agilent poroshell 120SB-C18 reverse phase column (2.1×150, 2.7 μm); column temperature: 30°C; mobile phase: A:B = acetonitrile: water = 30%:70%; isocratic elution, 165 μL / min; injection volume: 2 μL.

[0085] (3) Mass spectrometry conditions Curtain gas: 15 psi; Spray voltage: 4500 v; Nebulizer gas pressure: 65 psi; Auxiliary gas pressure: 70 psi; Nebulizer temperature: 400° C. The selective reaction monitoring conditions for ACC protonation are shown in Table 3. The detection of ACC activity in the present invention was entrusted to Nanjing Ruiyuan Biotechnology Co., Ltd.

[0086] Table 3 Selected reaction monitoring conditions for ACC protonation ([M+H] + )

[0087]

[0088] ACS activity (ng / (g·h)) = detection concentration (ng / mL) × dilution volume (mL) × dilution factor / (mass coefficient (g) × reaction time (h)).

[0089] Comparison of ACC synthase activity in pBI122-TmERF11 Baldcypress transgenic plants and their control plants is shown in Tables 4 and Figure 5 Y1-1, Y1-2 and Y1-3 represent three control plants, and Y4-1, Y4-2 and Y4-3 represent three pBI122-TmERF11 Baldcypress transgenic plants.

[0090] Table 4 ACC synthase activity in pBI122-TmERF11 Baldcypress transgenic plants and control plants

[0091]

[0092] From Table 4 and Figure 5 It can be seen that the ACC synthase activity of the transgenic positive plants grown to 14 days was significantly higher than that of the control plants, and its activity was 4.25 times that of the control plants. Overexpression of TmERF11 in Baldcypress can promote the synthesis of ethylene in plants.

[0093] Example 6 Drought stress treatment of pBI122-TmERF11 gene-transfected baldcypress

[0094] After obtaining positive plants by the method of Example 3, the positive plants were placed on 200 mg / L kanamycin selection MS medium for culture, and cultured in a light incubator, the light conditions were long day (light 16h, dark 8h), and the temperature was 25° C. After growing to 14 days, transplanted tissue culture seedlings were obtained; the transplanted tissue culture seedlings were transferred to a cultivation matrix, the cultivation matrix composition was V (peat soil): V (perlite) = 1:1, and drought stress treatment was performed after culturing for 7 days at 25° C., light 16h, dark 8h.

[0095] The control plants were wild-type Baldcypress at the same growth stage.

[0096] Four transgenic positive Baldcypress plants and four control plants at the same growth stage were cultured in a light incubator with long-day lighting (16 hours of light and 8 hours of darkness) and a temperature of 25°C. They were kept without watering and subjected to natural drought treatment. They were observed and photographed every day. Under drought stress, the leaves of non-transgenic Baldcypress plants began to turn yellow gradually from the 7th day of drought, and the leaves dried up and fell off on the 10th day of drought. On the 15th day of drought, all the leaves dried up and died. However, the transgenic pBI122-TmERF11 Baldcypress plants were significantly less affected. Figure 6 The left side of the figure shows the pBI122-TmERF11 transgenic baldcypress, and the right side of the figure shows the control non-transgenic baldcypress.

[0097] Depend on Figure 6 It can be seen that overexpression of TmERF11 improved the drought resistance of the plant, and TmERF11 is a gene that can improve the drought resistance of Baldcypress.

[0098] In summary, the TmERF11 gene can regulate the synthesis of ethylene in plants and is also an important gene for responding to drought stress. Overexpression of the TmERF11 gene can promote the synthesis of ethylene in plants and improve the drought resistance of plants.

[0099] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A TmERF11 protein, characterized in that The amino acid sequence of the protein is shown in SEQ ID NO.

10.

2. A TmERF11 gene, characterized in that: The nucleotide sequence of the TmERF11 gene is shown in SEQ ID NO.

9.

3. A recombinant vector, characterized in that: The recombinant vector contains the TmERF11 gene according to claim 2.

4. The recombinant vector according to claim 3, characterized in that: The recombinant vector overexpresses the TmERF11 gene.

5. A recombinant bacterium or recombinant cell, characterized in that: The recombinant bacteria or recombinant cells contain the TmERF11 gene according to claim 2.

6. Use of the TmERF11 protein according to claim 1, the TmERF11 gene according to claim 2, the recombinant vector according to claim 3 or 4, or the recombinant bacteria or recombinant cells according to claim 5 in regulating plant drought resistance and / or regulating plant ethylene synthesis.

7. The use according to claim 6, characterized in that: Overexpression of the TmERF11 gene can enhance plant drought tolerance and / or promote plant ethylene synthesis.

8. Use of the TmERF11 protein according to claim 1, the TmERF11 gene according to claim 2, the recombinant vector according to claim 3 or 4, or the recombinant bacteria or recombinant cells according to claim 5 in plant breeding.

9. A method for improving drought tolerance of plants, characterized in that: The following steps are involved: Increasing the expression of the TmERF11 gene in the plant or increasing the content of the TmERF11 protein in the plant can improve the drought resistance of the plant.

10. The method according to claim 9, characterized in that: The method for increasing the expression of the TmERF11 gene in a plant or increasing the content of the TmERF11 protein in a plant comprises: The recombinant vector according to claim 3 or 4 or the recombinant bacteria or recombinant cells according to claim 5 are introduced into a plant.

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