Ancestor gene DR2 of DREB gene family and application of ancestor gene DR2

Abstract

The invention discloses an ancestor gene DR2 of a DREB gene family and application thereof, and belongs to the technical field of gene engineering. On one hand, the invention provides an ancestor gene DR2 of a DREB gene family, and on the other hand, the invention provides application of the gene. Through artificial construction, the DR2 gene is over-expressed in arabidopsis thaliana, so that seed germination can be promoted, and plant biomass accumulation and root growth are improved; new varieties with different seed germination, different biomass and different root lengths are obtained; the stable hereditary characteristic is realized; the biological function of the DREB gene in the evolution process can be clarified, and the method has a good application prospect in plant molecular improvement breeding.

Description

Technical Field

[0001] The invention belongs to the technical field of gene engineering, and specifically relates to an ancestral gene DR2 of a DREB gene family and an application thereof. Background Art

[0002] AP2 / ERF (APETALA2 / ethylene response element binding factor) transcription factors are an important family of transcription factors in plants, playing a key regulatory role in many biological and physiological processes, such as plant morphogenesis, abiotic stress, hormone signal transduction and metabolite regulation. Among them, dehydration response element binding protein (DREB) is an important subfamily of AP2 / ERF transcription factors. In the past two decades, scientists have conducted extensive research on the biological functions of DREB transcription factors in regulating plant responses to various stresses, especially in biotic and abiotic stresses. Therefore, DREB transcription factors were once considered to be a class of resistance proteins in plants. However, recent studies have shown that DREB transcription factors also have indispensable functions in the growth and development of plants.

[0003] In order to enhance environmental adaptability, plants will start their life cycle in the appropriate season. In this process, some DREBs proteins act as positive or negative regulators to regulate seed dormancy and germination by responding to internal and external environmental signals, thereby affecting the initial stage of the plant life cycle. When the seeds break through the seed coat, they will enter a juvenile vegetative growth stage. During this stage, DREBs transcription factors have been shown to be involved in the developmental regulation of various tissues and organs such as hypocotyls, roots, stems and leaves. When plants grow to a certain stage and accumulate sufficient nutrients, they enter the reproductive growth stage. During this period, DREBs transcription factors are also widely involved in related processes, including flowering induction, gametophyte development and seed development. DREBs transcription factors adapt to the complex and changing external environment by regulating plant-specific growth and development patterns. Current evidence shows that DREBs transcription regulators are crucial in regulating plant stress resistance and unique growth and development processes. Considering that the evolution and expansion of the DREBs family are related to the environmental adaptability of terrestrial plants, DREBs proteins are likely to have played an important role in plant evolution.

[0004] The DREB subfamily differentiated from the ethylene response factor (ERF) subfamily of the common ancestor of Zygnemophyceae and Embryophyta during the migration of plants to land, and then expanded to form three different subfamilies. Further studies have shown that under the stimulation of global cooling, the AP2 / ERF / III subfamily expanded through whole genome doubling (WGD) and tandem duplication (TD). And over time, the genes continued to expand and differentiate to form the DREB ancestral gene. Artificially constructing the DREB ancestral sequence through the ancestral sequence construction method is of great significance for exploring the functional differentiation during its evolution. Summary of the invention

[0005] In view of the problems existing in the prior art, the purpose of the present invention is to design and provide a technical solution for the ancestral gene DR2 of the DREB gene family and its application.

[0006] The present invention is specifically implemented by the following technical solutions:

[0007] The first aspect of the present invention provides an ancestral gene DR2 of the DREB gene family, the nucleotide sequence of which is:

[0008] 1) the nucleotide sequence shown in SEQ ID No.1;

[0009] 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.1;

[0010] 3) a nucleotide sequence having 80% or more identity with the nucleotide sequence shown in SEQ ID NO.1; or

[0011] 4) A nucleotide sequence that is different from the nucleotide sequence shown in SEQ ID NO. 1 due to the degeneracy of the genetic code.

[0012] The second aspect of the present invention provides a protein encoded by the ancestral gene DR2 of the above-mentioned DREB gene family, the amino acid sequence of the protein is:

[0013] 1) the amino acid sequence shown in SEQ ID No. 2; or

[0014] 2) An amino acid sequence with equivalent function formed by replacing, deleting and / or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID No. 2.

[0015] The third aspect of the present invention provides the use of the ancestral gene DR2 of the above-mentioned DREB gene family in regulating plant seed germination, biomass and root growth.

[0016] Furthermore, the regulation in this application specifically includes early seed germination, increased biomass and promoted root growth.

[0017] Furthermore, the plant in the application is Columbia ecotype Arabidopsis thaliana.

[0018] Furthermore, the application includes the following methods:

[0019] 1) The plant contains DR2, the ancestral gene of the DREB gene family; or

[0020] 2) Overexpression of DR2, the ancestral gene of the DREB gene family, in plants.

[0021] Furthermore, the method 2) in the application uses Agrobacterium-mediated method to transfer the ancestral gene DR2 of the DREB gene family into plants to obtain transgenic plants that overexpress the ancestral gene DR2 of the DREB gene family.

[0022] The beneficial effects of the present invention are that the present invention discloses the ancestral amino acid sequence and nucleotide sequence of the DREB gene family; through artificial construction, the DR2 gene is overexpressed in Arabidopsis, which can promote seed germination, increase the accumulation of plant biomass and the growth of the root system; thereby obtaining new varieties with different seed germination, different biomass and different root lengths; having stable genetic characteristics; and being helpful to clarify the biological function of the DREB gene in the evolutionary process, and having good application prospects in plant molecular improvement breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It is a schematic diagram of restoring the DREB ancestral gene by the ML (maximum likelihood estimation) method of the present invention;

[0024] Figure 2 It is a schematic diagram of PCR electrophoresis of the gene amplification sequence of the present invention;

[0025] Figure 3 Schematic diagram of the construction of the overexpression vector of the present invention;

[0026] Figure 4 is a schematic diagram of the expression level of DR2 in the transgenic Arabidopsis plants of the present invention;

[0027] Figure 5 This is a statistical diagram of germination of Arabidopsis seeds overexpressing DR2 according to the present invention;

[0028] Figure 6 This is a schematic diagram of the development of Arabidopsis thaliana overexpressing DR2 for 16 days;

[0029] Figure 7 This is a statistical diagram of the fresh weight of Arabidopsis thaliana overexpressing DR2 according to the present invention;

[0030] Figure 8 This is a schematic diagram of the root length phenotype of Arabidopsis thaliana overexpressing DR2 according to the present invention;

[0031] Fig. 9 This is a schematic diagram of the root length statistics of Arabidopsis thaliana overexpressing DR2 in the present invention. DETAILED DESCRIPTION

[0032] In order to better understand the present invention, the present invention is further explained below in conjunction with the embodiments, but the present invention is not limited to the following embodiments.

[0033] Example 1

[0034] Ancestral sequence reconstruction (ASR) is to restore ancient protein sequences based on existing protein sequences. Seven major representative species covering the entire land plant from lower to higher order were selected, and 116 DREB family members were obtained from the whole genome protein sequence using the hidden Markov model. Then, multiple sequence alignments were performed on the collected amino acid sequences and a phylogenetic tree was constructed. The results showed that the obtained phylogenetic tree had five main branches ( Figure 1 ). Using the constructed phylogenetic tree and the input homologous sequence data, the ancestral sequence was deduced in FastML. Through the above steps, the ancestral gene DR2 of the DREB gene family was obtained. The nucleotide sequence of the ancestral gene DR2 of the DREB gene family is shown in SEQ ID No.1, and the amino acid sequence of its encoded protein is shown in SEQ ID No.2. In order to compare the similarity of these inferred amino acid sequences with the existing DREB, the BLAST function of NCBI was used. After sorting according to the confidence interval, it was compared with the DREB proteins of currently known species and found that the similarity of DR2 was 60%-77% (Table 1). Its amino acid sequence is completely different from the currently known DREB proteins.

[0035] Table 1

[0036] Known sources of protein NCBI accession number of known proteins Maximum similarity to known proteins Dillenia turbinata KAK6923434.1 77％ Oriental tree XP_010056415.2 60％ Deschampsia antarctica AIZ97485.1 64％

[0037] Example 2

[0038] 1. Gene cloning

[0039] PCR amplification was performed using two pairs of primers (PR1) and a 2×Phanta Flash Master Mix (Novozymes, Jiangsu, China) kit. The forward primer of PR1 is shown in SEQ ID NO.3, and the reverse primer of PR1 is shown in SEQ ID NO.4. The nucleotide sequence of DR2 obtained in Example 1 was used to synthesize the DR2 fragment by the method of artificially synthesizing DNA fragments and used as a PCR template full-length genomic DNA fragment. The PCR program was: pre-denaturation at 95°C for 5 minutes, followed by 35 cycles (denaturation at 95°C for 30 seconds, annealing at 58°C for 30 seconds, and extension at 72°C for 1 minute), and finally extension at 72°C for 5 minutes. All primers used in this example were designed using Snapgene 4.1.8 software and synthesized by Qingke Biotechnology Co., Ltd. (Beijing, China). The primer information is shown in Table 1. Afterwards, the PCR product was observed by DNA electrophoresis and we found that there was a clear DNA band of about 1216 bp relative to the DNA marker and it was considered to be our target gene (see Figure 2 ) was cut off and the product was recovered by product purification kit (Novozymes, Jiangsu, China).

[0040] 2 Plasmid construction

[0041] First, the pCAMBIA1300 overexpression vector was double-digested with restriction endonucleases BamHI and EcoRI, and then the correct band was observed by DNA electrophoresis and cut out, and then the product was recovered using a product purification kit. Finally, according to the instructions of the homologous recombination kit (Novozyme, Jiangsu, China), the vector fragment and the PCR fragment were connected (see Figure 3 ).

[0042] 3. Transformation of E. coli

[0043] After taking out the DH5α competent cells from the -80℃ refrigerator, place them on ice to thaw. Add the ligated product to the competent cells and mix gently, then place them in an ice bath for 30 minutes. Quickly place the tube containing the mixture in a 42℃ water bath for heat shock for 90 seconds, then immediately place it on ice for 2 minutes. Add 900μL LB liquid culture medium and culture in a shaker at 37℃, 200rpm for 100 minutes. Centrifuge the cultured bacterial solution (3000rpm / min, 3 minutes), discard the supernatant, resuspend the bacteria with 100μL liquid LB, and then apply it on LB solid culture medium containing 50mg / L Kan. Incubate inverted in a 37℃ constant temperature incubator for 13 to 15 hours.

[0044] 4 PCR identification of positive single clones

[0045] Randomly select monoclonal colonies on the plate for detection, and place them in a PCR tube containing 8 μL ddH2O. Add 10 μL 2×PCRMix premix and 1 μL primer (PR1), mix thoroughly and perform PCR reaction. After the PCR program is completed, perform electrophoresis detection.

[0046] 5 Plant transformation and plant positive identification

[0047] The sequenced target gene plasmid was transformed into Agrobacterium. The transformation steps are as follows: Take out the GV3101 Agrobacterium competent cells stored at -70℃ and melt them in an ice water bath. Under sterile conditions, add 2-5μL of the target gene plasmid to the competent cells and mix gently. After the centrifuge tube is placed in liquid nitrogen for rapid freezing for 5 minutes, it is placed in a 37℃ water bath for 5 minutes and then placed on ice for 2 minutes. Add 500μL of LB liquid culture medium and then culture it on a shaker at 28℃ and 200rpm for 2-3 hours. Centrifuge quickly for 3 minutes, draw 100μL of bacterial solution, spread it on LB solid culture medium containing kanamycin (50mg / L) and rifampicin (25mg / L), and invert it in a 28℃ constant temperature incubator for 2-3 days. Pick from a single colony, transfer it to 5mL of resistant LB culture medium, and shake and culture it at 220rpm and 28℃ until the bacterial solution becomes turbid. Transfer the turbid bacterial solution to 30 mL of liquid LB medium containing the corresponding antibiotics, culture at 220 rpm and 28°C for 6 hours, and then centrifuge at 3000 rpm for 20 minutes to collect the bacteria. Add an equal amount of Arabidopsis infection solution to the bacterial solution, mix gently, suspend the bacteria in the inflorescence of Arabidopsis, culture in the dark for 16-24 hours, and then continue to culture in the greenhouse, and collect the seeds after the fruit pods mature.

[0048] Example 3

[0049] 1. Gene expression analysis

[0050] Wild-type Arabidopsis seedlings grown on 1 / 2MS 0.8% agar plates and positive OE seedlings screened on 1 / 2MS and 25 mg / L hygromycin 0.8% agar plates were planted in soil and grown for 3 weeks. Arabidopsis leaves were taken and total RNA was extracted for quantitative real-time PCR using the CTAB method. cDNA was synthesized using HiScript III First Strand cDNA Synthesis Kit (+gDNA Wiper) (Novozymes, Nanjing, China). Real-time fluorescence PCR was performed using PR2 as a gene quantitative primer; PR3 and PR4 as internal reference primers, the forward primer of PR2 is shown in SEQ ID NO.5, the reverse primer of PR2 is shown in SEQ ID NO.6, the forward primer of PR3 is shown in SEQ ID NO.7, the reverse primer of PR3 is shown in SEQ ID NO.8, the forward primer of PR4 is shown in SEQ ID NO.9, and the reverse primer of PR4 is shown in SEQ ID NO.10. Through expression amount calculation, we found that the expression amount of transgenic materials was higher than that of wild-type materials; and the gene expression amount of overexpression strain 1 and expression strain 5 was significantly different from that of wild-type (see Figure 4 ).

[0051] 2 Phenotypic determination

[0052] The transgenic materials and wild-type materials were disinfected with sodium hypochlorite and placed on 1 / 2MS medium using sterilized toothpicks, 50 seeds each. On the first and second days, there was no significant difference in the germination of wild-type seeds and transgenic materials, and neither of them germinated. It was not until the third day that statistics were found (see Figure 5 ), compared with wild-type plants, the germination rate of transgenic materials was significantly higher than that of wild-type plants. Among them, the germination rate of overexpression strain 5 was 3 times that of wild-type; at the same time, the germination rate of overexpression strain 1 was 2 times that of wild-type.

[0053] Transgenic materials and wild-type Arabidopsis seeds were sterilized with sodium hypochlorite and placed on 1 / 2MS medium using sterilized toothpicks, with 6 seeds in each group. After growing in 1 / 2MS medium for 16 days, the results were recorded by photographing and weighing (see Figure 6 ), the weight of the overexpression lines was significantly higher than that of the wild type (see Figure 7 ); in particular, the weight of the overexpression strain 5 was twice that of the wild type.

[0054] Transgenic materials and wild-type Arabidopsis seeds were sterilized with sodium hypochlorite and placed on 1 / 2MS medium using sterilized toothpicks, with 8 seeds in each group. After growing in 1 / 2MS medium for 8 days, photos were taken and root length was measured (see Figure 8), the root length of the overexpression lines was significantly longer than that of the wild type; especially the overexpression line 5 (see Fig. 9 ), and its root length was twice that of the wild type.

[0055] In summary, we found that overexpression of DR2 in Arabidopsis can advance seed germination, promote root growth, and increase plant biomass.

[0056] Table 2 List of primers used in the present invention

[0057]

Claims

1. An ancestral gene of the DREB gene family, DR2, It is characterized in that The nucleotide sequence of the gene is: 1) the nucleotide sequence shown in SEQ ID No.1; 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.1; 3) a nucleotide sequence having 80% or more identity with the nucleotide sequence shown in SEQ ID NO.1; or 4) A nucleotide sequence that is different from the nucleotide sequence shown in SEQ ID NO. 1 due to the degeneracy of the genetic code.

2. A protein encoded by the ancestral gene DR2 of the DREB gene family according to claim 1, It is characterized in that The amino acid sequence of this protein is: 1) the amino acid sequence shown in SEQ ID No. 2; or 2) An amino acid sequence with equivalent function formed by replacing, deleting and / or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID No.

2.

3. Use of the ancestral gene DR2 of the DREB gene family as claimed in claim 1 in regulating plant seed germination, biomass and root growth.

4. The use according to claim 3, It is characterized in that The regulation specifically includes early seed germination, increased biomass and promoted root growth.

5. The use according to claim 3, It is characterized in that The plant is Arabidopsis thaliana of the Columbia ecotype.

6. The use according to claim 3, It is characterized in that These include: 1) The plant contains DR2, the ancestral gene of the DREB gene family; or 2) Overexpression of DR2, the ancestral gene of the DREB gene family, in plants.

7. The use according to claim 4, It is characterized in that The method 2) uses Agrobacterium-mediated method to transfer the ancestral gene DR2 of the DREB gene family into plants to obtain transgenic plants with overexpression of the ancestral gene DR2 of the DREB gene family.

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