Cold response transcription factor tcp9 conserved among different species and application thereof

Abstract

The application discloses a cold response transcription factor TCP9 conserved among different species and an application thereof, and belongs to the field of genetic engineering and plant genetic breeding.The application provides the cold response transcription factor TCP9 conserved among different species, and provides an application of the cold response transcription factor TCP9 in negative regulation of plant cold tolerance.The application provides a valuable resource for cultivating new plant varieties with low-temperature resistance, and lays a theoretical foundation for revealing a molecular mechanism of plant low-temperature response.Gene editing of TCP9 can be used for cultivating new plant varieties with cold resistance, and has potential application value.

Description

Technical Field

[0001] The invention belongs to the fields of genetic engineering and plant genetic breeding, and particularly relates to a cold-responsive transcription factor TCP9 that is conserved among different species and an application thereof. Background Art

[0002] Discovering key cold-resistant genes in plants and verifying their functions will provide important theoretical references for breeding new cold-resistant plant varieties.

[0003] In recent years, researchers have made significant progress in understanding how plants perceive and transmit low-temperature signals. Among these, the cold-responsive pathway, centered around CBFs, has been the most widely studied. Despite the crucial role CBFs play in plant responses to cold stress, preliminary statistical analysis of previous studies reveals that only approximately 10% to 20% of all cold-responsive genes are directly regulated by CBFs. This suggests that, in addition to CBFs, numerous other cold-responsive regulatory factors exist in plants. Therefore, identifying these cold-responsive regulatory factors, exploring their functions, and elucidating their molecular mechanisms will provide new evidence for understanding the key scientific question of how plants respond to cold stress.

[0004] At present, although researchers have explored the cold response mechanism of plants in many different plants, most of these studies only focus on a single species or closely related species, and lack systematic comparative studies across species, especially functional verification of related genes. This limitation has led to the functional conservation of identified potential cold-resistant genes in other species and their practical application potential not being fully demonstrated. Summary of the Invention

[0005] In view of the problems existing in the prior art, the purpose of the present invention is to provide a cold-responsive transcription factor TCP9 that is conserved among different species and a technical solution for its application.

[0006] In order to solve the above technical problems, the technical solutions adopted by the present invention are as follows:

[0007] The first aspect of the present invention provides the use of TCP9, a cold-responsive transcription factor conserved among different species, in negatively regulating plant cold tolerance.

[0008] TCP9 belongs to the TCP family of transcription factors unique to plants. Arabidopsis thaliana ) AtTCP9 and Populus trichocarpa ( Populus trichocarpa ) PtrTCP9 For example, analysis TCP9 The cold response pattern under low temperature stress and the functional application of TCP9 in plant cold resistance were revealed. AtTCP9 and PtrTCP9 can be induced to express by low temperature stress, and AtTCP9 The cold response of the human body is independent of the core cold response regulatory factors CBFs. AtTCP9 and PtrTCP9 Overexpression in Arabidopsis revealed that 35S:: AtTCP9 and 35S:: PtrTCP9 The cold tolerance of overexpressing transgenic plants was significantly reduced, indicating that TCP9 is a conserved cold-responsive transcription factor that negatively regulates plant cold tolerance.

[0009] Furthermore, the cold-responsive transcription factor TCP9 in this application includes AtTCP9 Genes and PtrTCP9 Gene.

[0010] Furthermore, the application AtTCP9 The nucleotide sequence of the gene is one of the following nucleotide sequences:

[0011] (1) The nucleotide sequence shown in SEQ ID NO. 2;

[0012] (2) A nucleotide sequence in which one or more nucleotides are added, deleted, substituted or inserted into the nucleotide sequence shown in SEQ ID NO. 2;

[0013] (3) a nucleotide sequence having an identity of 80% or more to the nucleotide sequence shown in SEQ ID NO. 2;

[0014] (4) A nucleotide sequence that differs from the nucleotide sequence shown in SEQ ID NO. 2 due to the degeneracy of the genetic code.

[0015] Furthermore, the application PtrTCP9 The nucleotide sequence of the gene is one of the following nucleotide sequences:

[0016] (1) The nucleotide sequence shown in SEQ ID NO. 4;

[0017] (2) A nucleotide sequence in which one or more nucleotides are added, deleted, substituted or inserted into the nucleotide sequence shown in SEQ ID NO. 4;

[0018] (3) a nucleotide sequence having an identity of 80% or more to the nucleotide sequence shown in SEQ ID NO. 4;

[0019] (4) A nucleotide sequence that differs from the nucleotide sequence shown in SEQ ID NO. 4 due to the degeneracy of the genetic code.

[0020] The second aspect of the present invention provides a plant cold tolerance negative regulatory factor AtTCP9 , the nucleotide sequence of the gene is one of the following nucleotide sequences:

[0021] (1) The nucleotide sequence shown in SEQ ID NO. 2;

[0022] (2) A nucleotide sequence in which one or more nucleotides are added, deleted, substituted or inserted into the nucleotide sequence shown in SEQ ID NO. 2;

[0023] (3) a nucleotide sequence having an identity of 80% or more to the nucleotide sequence shown in SEQ ID NO. 2;

[0024] (4) A nucleotide sequence that differs from the nucleotide sequence shown in SEQ ID NO. 2 due to the degeneracy of the genetic code.

[0025] Plant cold tolerance negative regulatory factor involved in the present invention AtTCP9 , derived from Columbia-type Arabidopsis thaliana, with a gene ID of AT2G45680, a full-length open reading frame comprising 1071 nucleotide sequences, encoding 356 amino acid sequences, the encoded amino acid sequence of which is shown in SEQ ID NO.1.

[0026] The third aspect of the present invention provides a plant cold tolerance negative regulatory factor PtrTCP9, The nucleotide sequence of the gene is one of the following:

[0027] (1) The nucleotide sequence shown in SEQ ID NO. 4;

[0028] (2) A nucleotide sequence in which one or more nucleotides are added, deleted, substituted or inserted into the nucleotide sequence shown in SEQ ID NO. 4;

[0029] (3) a nucleotide sequence having an identity of 80% or more to the nucleotide sequence shown in SEQ ID NO. 4;

[0030] (4) A nucleotide sequence that differs from the nucleotide sequence shown in SEQ ID NO. 4 due to the degeneracy of the genetic code.

[0031] Plant cold tolerance negative regulatory factor involved in the present invention PtrTCP9 The gene ID is Potri.002G152200, its full-length open reading frame includes 1047 nucleotide sequences, encoding 348 amino acid sequences, and its encoded amino acid sequence is shown in SEQ ID NO.3.

[0032] The fourth aspect of the present invention provides a method for reducing plant cold tolerance, which is to overexpress the cold-responsive transcription factor TCP9 in the plant, specifically to overexpress the negative regulatory factor AtTCP9 or the negative regulatory factor PtrTCP9 .

[0033] Furthermore, the plants in this method include dicotyledonous plants and monocotyledonous plants.

[0034] Furthermore, the plant in the method is Arabidopsis thaliana of the Columbia ecotype.

[0035] The beneficial effects of the present invention are as follows: the present invention discloses a cold-responsive transcription factor TCP9 that is conserved among different species; it is constructed by a plant expression vector and overexpressed in Arabidopsis thaliana. AtTCP9 and PtrTCP9 , all significantly reduced the plant's cold tolerance, indicating that TCP9 is a transcription factor that negatively regulates plant cold tolerance. This study provides valuable resources for breeding new cold-tolerant plant varieties and lays a theoretical foundation for uncovering the molecular mechanisms of plant cold response. Gene editing of TCP9 can be used to breed new cold-tolerant plant varieties and has promising application prospects in plant molecular breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] 图1 : Conserved cold response patterns of TCP9 homologous genes in different species.

[0037] 图2 : Schematic diagram of the construction of the overexpression vector of the present invention.

[0038] 图3 :35S of the present invention:: AtTCP9 and 35S:: PtrTCP9 Screening of positive overexpressing Arabidopsis plants.

[0039] 图4 :35S of the present invention:: AtTCP9 and 35S:: PtrTCP9 Identification of overexpressing plants at the DNA level.

[0040] 图5 :35S of the present invention:: AtTCP9 and 35S:: PtrTCP9 Characterization of overexpressing plants at the RNA level.

[0041] 图6 :Wild-type Arabidopsis and 35S:: AtTCP9 Cold tolerance phenotype and survival rate statistics of overexpressing Arabidopsis thaliana.

[0042] 图7 :Wild-type Arabidopsis and 35S:: PtrTCP9 Cold tolerance phenotype and survival rate statistics of overexpressing Arabidopsis thaliana. DETAILED DESCRIPTION

[0043] In order to better understand the present invention, the following is a further explanation of the invention in conjunction with specific examples, but the content of the present invention is not limited to the following examples. Where specific techniques or conditions are not specified in the examples, the techniques or conditions described in the literature in this field or the product specifications are used. Where the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased.

[0044] Example 1: Analysis of cold response patterns of TCP9 homologous genes in different species

[0045] (1) Based on the comparative genome analysis of multiple species and comparative transcriptome analysis under low temperature stress, in Arabidopsis thaliana ( Arabidopsis thaliana )、Poplar ( Populus trichocarpa ), birch ( Betula pendula ),tobacco( Nicotiana tabacum ) and rice ( Oryza sativa ) and other different species to identify conserved cold-responsive orthologous gene groups, among which TCP9 is the focus of the present invention.

[0046] (2) The expression levels of TCP9 homologous genes in different species under low temperature stress were retrieved. Based on the average expression levels of three biological replicates and the phylogenetic relationship of TCP9 homologous protein sequences in different species, a clustered heat map of the expression levels of TCP9 homologous genes in different species was drawn.

[0047] The cold response patterns of TCP9 homologous genes of different species in the present invention are as follows 图1 shown.

[0048] Example 2: Construction of overexpression vector

[0049] (1) Design of gene cloning primers

[0050] Get it on Phytozome AtTCP9 and PtrTCP9 The CDS sequence, according to AtTCP9 、 PtrTCP9 The CDS sequence information and the vector information of pCAMBIA1300-GFP were used to design the vector with modified restriction sites. AtTCP9 and PtrTCP9 The designed primer sequences for gene cloning are shown in Table 1.

[0051] Table 1 AtTCP9 and PtrTCP9 Gene cloning primers

[0052]

[0053] (2) AtTCP9 and PtrTCP9 Amplification of gene CDS sequences

[0054] The cDNA of wild type Arabidopsis Col-0 and wild type P. trichocarpa were used as templates, respectively, and the CDS sequences of the above designed primers were amplified by PCR in vitro with high-fidelity DNA polymerase. After the PCR amplification, agarose gel electrophoresis was performed for detection, and the PCR products were recovered and purified by using a gel recovery kit. AtTCP9 PtrTCP9 AtTCP9 PtrTCP9

[0055] (3) Double enzyme digestion of plant expression vector and PCR product and gel recovery

[0056] The plasmid of plant expression vector pCAMBIA1300-GFP was extracted by using a plasmid extraction kit. The pCAMBIA1300-GFP plasmid and the PCR product of the above designed primers were digested by using restriction endonuclease Sal I and Sac I, respectively, and the pCAMBIA1300-GFP plasmid and the PCR product of the above designed primers were digested by using restriction endonuclease Kpn I and Sac I, respectively. The reaction conditions were as follows: 37°C for 45 min, and 80°C for 5 min for inactivation. The enzyme digestion products were further recovered by gel recovery. AtTCP9 PtrTCP9

[0057] (4) AtTCP9 PtrTCP9

[0058] The pCAMBIA1300-GFP vector recovered by enzyme digestion was connected with the PCR products of the above designed primers recovered by enzyme digestion by using T4 DNA ligase. 5 μL of the ligation product was heat-shocked and transformed into DH5α E. coli competent cells at 42°C, and the transformed bacteria were spread on solid LB medium containing 50 mg / L kanamycin and cultured at 37°C overnight. 3-5 single colonies were screened from the resistant plate for PCR detection, and the single colonies with successful PCR amplification were sent to a company for sequencing. AtTCP9 PtrTCP9

[0059] (5) Agrobacterium transformation and positive clone screening

[0060] ​​​​​​​​​​The plasmids of the successfully sequenced pCAMBIA1300-AtTCP9 and pCAMBIA1300-PtrTCP9 recombinant vectors were transformed into GV3101 competent Agrobacterium cells. The transformed Agrobacterium was evenly spread on solid LB medium containing 50 mg / L kanamycin and 50 mg / L rifampicin and incubated inverted at 28°C for 36-48 hours. Single colonies on the resistant plates were selected for PCR testing, and the culture suspensions of positive colonies were stored at -80°C until further use.

[0061] Schematic diagram of overexpression vector construction and recombinant vector in the present invention AtTCP9 and PtrTCP9 The PCR results were as follows 图2 shown.

[0062] In this embodiment AtTCP9 The nucleotide sequence is shown in SEQ ID NO.2, PtrTCP9 The nucleotide sequence is shown in SEQ ID NO.4.

[0063] Example 3: 35S:: AtTCP9 and 35S:: PtrTCP9 Screening and verification of overexpressing Arabidopsis plants

[0064] (1) Agrobacterium-mediated genetic transformation of Arabidopsis thaliana

[0065] ① Thaw the Agrobacterium culture containing the pCAMBIA1300-AtTCP9 and pCAMBIA1300-PtrTCP9 recombinant vectors stored in a -80 freezer in an ice box. Add each culture into 5 mL of liquid LB medium containing 50 mg / L kanamycin and 50 mg / L rifampicin, and culture at 28°C with shaking at 220 rpm for about 12 to 16 hours.

[0066] ② Transfer the activated bacterial solution into 30 mL of fresh liquid LB medium at a ratio of 1:30 and continue to culture at 28°C and 220 rpm until the OD 600 Reached 0.8.

[0067] ③ Centrifuge at 3000 rpm for 20 min, discard the supernatant, collect the bacteria, and resuspend the Agrobacterium cells in a solution containing 5% Sucrose and 0.05% Silwet L-77 to form an infection solution for later use.

[0068] ④ Before the initial infection, remove the fruit pods of wild-type Arabidopsis Col-0 and soak the inflorescence in the infection solution for 10 seconds. After infection, incubate in the dark for 16 to 24 hours.

[0069] After the end of the dark treatment, the Arabidopsis is continuously placed under the normal 16 h light / 8 h dark photoperiod for continuous culture, and is infected every 5 days, and each Agrobacterium is infected three times. After the pods mature, the T0 generation seeds are collected and dried in a culture box.

[0070] (2) 35S: AtTCP9 and 35S: PtrTCP9 Screening of positive transgenic plants

[0071] 1. After the T0 transgenic seeds are dried, they are uniformly scattered on 1 / 2 MS solid medium containing 20 mg / L hygromycin after being sterilized with 75% alcohol. Then, the medium is placed in a 4°C refrigerator for 2 days, and then is placed under a 22°C 12 h / 12 h photoperiod for culture.

[0072] 2. After the screening plate is cultured at 22°C for 14 days, the T1 generation positive seedlings screened on the medium containing hygromycin are transplanted into a small flowerpot containing nutrient soil, and each single plant is numbered, and then is placed under a 22°C 12 h / 12 h photoperiod for culture.

[0073] In the present application, the 35S: AtTCP9 and 35S: PtrTCP9 overexpression positive transgenic plants are screened in the resistance plate. 图3 As shown in the table.

[0074] (3) 35S: AtTCP9 and 35S: PtrTCP9 Identification of overexpression plants at the DNA level

[0075] 1. When the T1 generation positive seedlings are cultured in the nutrient soil for about 3 weeks, the leaf blades of wild-type Arabidopsis Col-0, 35S: AtTCP9 overexpression Arabidopsis positive plants, 35S: PtrTCP9 overexpression Arabidopsis positive plants are taken into 1.5 mL centrifuge tubes, 3 steel balls are added into the centrifuge tubes, and then the centrifuge tubes are rapidly frozen in liquid nitrogen and placed in a mixing shaker for rapid shaking for 1 min.

[0076] 2. After centrifugation at 10,000 rpm for 2 min, 400 µL of DNA extraction buffer is added into each centrifuge tube, and then the mixture is vortexed, centrifuged briefly, and then is placed in a -20°C freezer for freezing for 30 min. After freezing, the mixture is boiled at 95°C for 5 min, and then is centrifuged at 10,000 rpm for 5 min.

[0077] ③ Pipette 300 µL of the supernatant into a new 1.5 mL centrifuge tube and add an equal volume of pre-chilled isopropanol. Centrifuge at 10,000 rpm for 5 min. Discard the supernatant and remove any remaining liquid. After drying, add 55-60 µL of TE buffer. Store the dissolved DNA in a -20°C refrigerator.

[0078] ④ Using the above extracted DNA as a template, amplify AtTCP9 and PtrTCP9 The primers of gene CDS sequence are respectively used to transfer the exogenous AtTCP9 and PtrTCP9 The CDS sequence was amplified by PCR. After PCR amplification, agarose gel electrophoresis was performed to detect whether the amplified band was consistent with its theoretical sequence size.

[0079] In the present invention, the exogenously introduced AtTCP9 and PtrTCP9 The PCR detection results of the sequences in 35S::AtTCP9 and 35S::PtrTCP9 overexpressing plants are shown in Figure 2. 图4 shown.

[0080] (4) 35S:: AtTCP9 and 35S:: PtrTCP9 Identification of overexpressing plants at the RNA level

[0081] ① When the T1 positive seedlings were cultured in nutrient soil for about 3 weeks, wild-type Arabidopsis Col-0, 35S:: AtTCP9 Overexpression of Arabidopsis thaliana positive plants, 35S:: PtrTCP9 Five to six leaves from each positive overexpressing Arabidopsis plant were wrapped in tin foil and quickly frozen in liquid nitrogen. The leaves of each strain were thoroughly ground in liquid nitrogen. Total RNA from each strain was extracted using a plant total RNA extraction kit. The extracted mRNA was reverse transcribed into cDNA using a reverse transcription kit. The cDNA was diluted 10-fold and stored in a -20°C freezer until ready for use.

[0082] ② After reverse transcription, dilute Col-0, 35S:: AtTCP9 Overexpression of Arabidopsis thaliana positive plants and 35S:: PtrTCP9 The cDNA of each positive plant of overexpressing Arabidopsis was used as a template. AtTCP9 and PtrTCP9 qRT-PCR was performed using the following qRT-PCR primers and internal reference primers. The PCR reaction program was set as follows: 95°C for 30 s, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. qRT-PCR results were analyzed using the 2-ΔΔCt method.

[0083] Table 2 AtTCP9 and PtrTCP9 qRT-PCR primers and internal reference primer sequences

[0084]

[0085] In the present invention, 35S:: AtTCP9 and 35S:: PtrTCP9 Overexpression plants AtTCP9 and PtrTCP9 The expression detection results of 图5 shown.

[0086] Example 4: Cold tolerance phenotype observation and survival rate statistics

[0087] (1) Freezing stress treatment

[0088] ① Plate seedling preparation. Wild-type Arabidopsis Col-0, 35S:: AtTCP9 and 35S:: PtrTCP9 The seeds of each overexpressing Arabidopsis thaliana strain were sterilized and evenly planted on 1 / 2 MS solid culture medium. After culturing in the dark at 4°C for 3 days, they were transferred to an incubator with a culture condition of 22°C and a photoperiod of 12 h light / 12 h dark for further culturing for 10 days.

[0089] ② Soil-cultured seedling preparation. Select Arabidopsis seedlings that have relatively consistent growth after 10 days of culture and transplant them into pots filled with nutrient soil. Then transfer them to a greenhouse at 22°C with a photoperiod of 12 h light / 12 h dark for another 14 days.

[0090] ③ Freeze stress treatment. During freeze stress treatment, the culture conditions in the low-temperature incubator were set as follows: 4°C for 10 minutes, 0°C for 20 minutes, and then decreased by 1°C every hour until the desired final treatment temperature was reached, where it was maintained at this treatment temperature for 6 hours. After freeze stress treatment, Arabidopsis plants were cultured at 4°C in the dark for another 12 hours and then transferred to a greenhouse at 22°C with a photoperiod of 12 hours light / 12 hours dark for recovery.

[0091] (2) Observation of cold-resistance phenotype and survival rate statistics

[0092] After freezing stress treatment, the plate-grown and soil-grown seedlings were allowed to recover for 3 days under normal culture conditions at 22°C. The growth of each strain was then observed to compare differences in tolerance to freezing stress. The cold-tolerance phenotypes of the different strains were photographed, and the survival rate of each strain after freezing stress treatment was calculated.

[0093] In the present invention, wild-type Arabidopsis and 35S:: AtTCP9 Comparison of cold tolerance phenotype and survival rate of overexpressed Arabidopsis thaliana 图6 As shown, wild-type Arabidopsis and 35S:: PtrTCP9 Comparison of cold tolerance phenotype and survival rate of overexpressed Arabidopsis thaliana 图7 By comparison, it was found that after freezing stress treatment, 35S:: AtTCP9 and 35S:: PtrTCP9 The damage degree of overexpressing Arabidopsis thaliana was more serious than that of wild-type Arabidopsis Col-0. AtTCP9 and 35S:: PtrTCP9 The survival rates of overexpressing Arabidopsis were significantly lower than those of Col-0, indicating that overexpression of 35S:: AtTCP9 and 35S:: PtrTCP9 These results indicate that TCP9 is a conserved cold-responsive transcription factor among different plants and negatively regulates plant cold tolerance.

Claims

1. Overexpression AtTCP9 Gene or PtrTCP9 Application of genes in reducing cold tolerance in Arabidopsis thaliana, the AtTCP9 The nucleotide sequence of the gene is shown in SEQ ID NO.

2. PtrTCP9 The nucleotide sequence of the gene is shown in SEQ ID NO.

4.

2. A method for reducing the cold resistance of plants, characterized in that: Overexpression of the cold-responsive transcription factor TCP9 in plants AtTCP9 Gene or PtrTCP9 gene, the plant is Arabidopsis thaliana, the AtTCP9 The nucleotide sequence of the gene is shown in SEQ ID NO.

2. PtrTCP9 The nucleotide sequence of the gene is shown in SEQ ID NO.4.

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