HD-ZIP transcription factor ArHDZ22, its encoding gene and application in Anoectochilus roxburghii 'Hongxia'

Abstract

The present invention relates to a HD-ZIP transcription factor, ArHDZ22, encoding a gene, and uses thereof in Anoectochilus roxburghii 'Hongxia'. The transcription factor ArHDZ22 comprises a polypeptide having the amino acid sequence set forth in SEQ ID NO. 2. The present invention also provides a nucleotide sequence, SEQ ID NO. 1, encoding the protein. This invention provides a theoretical basis for utilizing genetic engineering techniques to promote reproductive growth and enhance osmotic stress tolerance in Anoectochilus roxburghii, and thus has significant application value.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering application technology, and relates to an important homeodomain-leucine zipper protein in the reproductive growth process of Anoectochilus roxburghii, and specifically to the Anoectochilus roxburghii 'Hongxia' HD-ZIP transcription factor ArHDZ22, its encoding gene, and application. Background Art

[0002] Golden thread lotus[ Anoectochilus roxburghii A perennial herbaceous plant of the genus Anoectochilus in the Orchidaceae family, also known as golden thread grass and golden thread orchid, is a rare and precious Chinese medicinal material. The entire plant is used as medicine, with a sweet taste and a neutral nature. It has the effects of clearing heat and cooling blood, removing dampness and detoxifying, earning it the title of "King of Medicine." In recent years, the widespread application of Anoectochilus roxburghii in various fields has led to a continuous increase in demand in both domestic and international markets. However, the viability of Anoectochilus roxburghii flower powder and stigma is relatively short, resulting in a low natural reproduction rate from seeds. Anoectochilus roxburghii seeds also suffer from embryo abortion after fertilization, leading to reproductive problems in the plant.

[0003] Homeodomain-leucine zipper (HD-ZIP) proteins are a class of transcription factors unique to higher plants, playing key roles in specific plant biological processes. HD-ZIP III subfamily transcription factors play a crucial role in the growth and development of higher plants, particularly in regulating reproductive development. They are closely associated with processes such as embryonic and postembryonic morphogenesis, plant cell differentiation, lateral organogenesis, vascular cell division, and polarity establishment. However, there are currently no reports on the involvement of HD-ZIP proteins in reproductive development and osmotic stress tolerance in Anoectochilus roxburghii. Summary of the Invention

[0004] To fill the gap between Anoectochilus 'Hongxia' ArHDZ22 The present invention discloses the nucleotide coding sequence and amino acid sequence of the HD-ZIP transcription factor ArHDZ22 of Anoectochilus roxburghii 'Hongxia', protein subcellular localization, and transgenic plant growth phenotype, which will provide a basis for the future regulation of the HD-ZIP transcription factor ArHDZ22 by genetic engineering technology. ArHDZ22 Gene expression provides a theoretical basis for improving the breeding efficiency of golden thread vine.

[0005] On the one hand, the present invention provides a roxburghii 'Hongxia' HD-ZIP transcription factor ArHDZ22 having the function of promoting reproductive development and improving seed traits, wherein the transcription factor ArHDZ22 comprises a polypeptide (protein) having an amino acid sequence as shown in SEQ ID NO.2; or a protein having the characteristics of the roxburghii 'Hongxia' HD-ZIP transcription factor ArHDZ22 by substituting, deleting or adding one or more amino acids in the amino acid sequence as shown in SEQ ID NO.2.

[0006] On the other hand, the present invention provides a gene encoding the above-mentioned Anoectochilus roxburghii 'Hongxia' HD-ZIP transcription factor ArHDZ22, and the nucleotide sequence of the above-mentioned encoding gene is specifically: (a) the base sequence shown in positions 1 to 2523 of SEQ ID NO.1; or (b) a sequence with at least 70% homology to the nucleotide sequence shown in positions 1 to 2523 of SEQ ID NO.1.

[0007] In the present invention, "isolated DNA" and "purified DNA" mean that the DNA or fragment has been separated from the sequences located on both sides of it in its natural state, and also means that the DNA or fragment has been separated from the components accompanying the nucleic acid in its natural state and has been separated from the proteins accompanying it in the cell.

[0008] In the present invention, the gene encoding the HD-ZIP transcription factor ArHDZ22 of Anoectochilus roxburghii 'Hongxia' refers to a nucleotide sequence encoding a polypeptide having the activity of the Anoectochilus roxburghii 'Hongxia' protein, such as the nucleotide sequence from positions 1 to 2523 of SEQ ID NO.1 and its degenerate sequences. This degenerate sequence refers to a sequence resulting from the replacement of one or more codons within nucleotides 1 to 2523 of SEQ ID NO.1 with degenerate codons encoding the same amino acid. Due to codon degeneracy, a degenerate sequence with as little as approximately 70% homology to the nucleotide sequence from positions 1 to 2523 of SEQ ID NO.1 can also encode the sequence of SEQ ID NO.2. The aforementioned encoding gene may also refer to a nucleotide sequence that is at least 70% homologous to the nucleotide sequence of SEQ ID NO.1.

[0009] The aforementioned encoding gene may also refer to variants of the sequence shown in SEQ ID NO. 1 that encode the same function as the native Anoectochilus roxburghii 'Hongxia' HD-ZIP transcription factor ArHDZ22. These variants include (but are not limited to): deletions, insertions, and / or substitutions, typically of 1 to 120 nucleotides, and additions of up to 90 nucleotides at the 5' and / or 3' ends.

[0010] In the present invention, the real-time fluorescence quantitative PCR method can be used to analyze the ArHDZ22 Analysis of the expression patterns of gene products in Anoectochilus roxburghii 'Hongxia' ArHDZ22 The presence or absence and quantity of a gene's mRNA transcript in a cell.

[0011] In addition, according to the present invention, the rosmarinus 'Hongxia' ArHDZ22 Gene nucleotide sequence and amino acid sequence can be screened based on nucleic acid homology or expressed protein homology to identify Anoectochilus roxburghii 'Hongxia' ArHDZ22 Gene-related homologous genes or homologous proteins.

[0012] Anoectochilus roxburghii 'Hongxia' of the present invention ArHDZ22 The full-length nucleotide sequence of a gene or its fragments can generally be obtained using PCR amplification, recombinant methods, or synthetic methods. For PCR amplification, primers can be designed based on the nucleotide sequences disclosed herein, and commercially available cDNA libraries or cDNA libraries prepared by conventional methods known to those skilled in the art can be used as templates to amplify the relevant sequences. When the sequence is long, two or more PCR amplifications are often required, followed by splicing the fragments amplified in the correct order.

[0013] Once the relevant sequence is obtained, it can be obtained in large quantities using recombinant methods. This is usually done by cloning it into a vector, then transferring it into cells, and then isolating the relevant sequence from the proliferated host cells using conventional methods.

[0014] In addition, mutations can also be introduced into the protein sequence of the present invention by chemical synthesis.

[0015] In addition to recombinant production, fragments of the protein of the invention can also be produced by direct peptide synthesis using solid phase techniques. Each fragment of the protein of the invention can be chemically synthesized separately and then chemically linked to produce the full-length molecule.

[0016] The present invention also provides a recombinant expression vector, which contains the coding gene of the HD-ZIP transcription factor ArHDZ22 of the above-mentioned Anoectochilus roxburghii 'Hongxia'; the above-mentioned recombinant expression vector is pCAMBIA1300-ArHDZ22.

[0017] The present invention also provides a recombinant bacterium obtained by transforming the above-mentioned recombinant expression vector into a host cell; the above-mentioned host cell is Agrobacterium EHA105.

[0018] The present invention also provides a use of the gene encoding the HD-ZIP transcription factor ArHDZ22 of the above-mentioned Anoectochilus roxburghii 'Hongxia' in promoting the reproductive growth of Anoectochilus roxburghii and improving the seed vigor.

[0019] The above application includes: constructing a recombinant expression vector containing the above coding gene of the transcription factor ArHDZ22, transforming the vector into a plant host, and cultivating and screening to obtain transgenic plants.

[0020] Beneficial effects: As a rare and precious Chinese medicinal material, Anoectochilus roxburghii has a large market demand. This invention cloned the coding sequence of ArHDZ22, an important regulatory transcription factor in the growth and development of Anoectochilus roxburghii 'Hongxia', for the first time and analyzed it using real-time fluorescence quantitative PCR. ArHDZ22The expression pattern of genes, the subcellular localization of transcription factor ArHDZ22 in tobacco leaf epidermal cells transiently expressed, will provide a basis for the future regulation of gene expression using genetic engineering technology. ArHDZ22 The spatiotemporal expression of genes provides a theoretical basis for promoting the reproductive growth of Anoectochilus roxburghii, tolerance to osmotic stress, and breeding of new varieties, and has great application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Anoectochilus roxburghii 'Hongxia' HD-ZIP Homology comparison results (DNAMAN) between the gene and the Arabidopsis HD-ZIP protein sequence and the phylogenetic tree analysis of the homologous genes; A is the multiple sequence alignment of Anoectochilus roxburghii ArHDZ22 and homologous proteins; B is the phylogenetic tree analysis;

[0022] Figure 2 This is the localization map of the transcription factor ArHDZ22 from Anoectochilus roxburghii'Hongxia' in the epidermal cells of tobacco leaves;

[0023] Figure 3 Anoectochilus roxburghii 'Hongxia' ArHDZ22 Graph showing changes in gene expression under osmotic stress;

[0024] Figure 4 Wild type and overexpression ArHDZ22 Growth phenotype of plants with the gene under long-day conditions (16 h / 8 h light / dark);

[0025] Figure 5 For overexpression ArHDZ22 A diagram showing the effects of genes on flower morphology and plant tolerance to osmotic stress, where A is a diagram showing flower morphology and B is a diagram showing plant tolerance to osmotic stress. DETAILED DESCRIPTION

[0026] The present invention will be further described below with reference to specific examples. However, these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0027] For experimental methods in the following examples where specific conditions are not specified, they were generally used according to conventional conditions, such as those described in the Molecular Cloning Laboratory Manual (fourth edition), or the conditions recommended in the reagent instructions.

[0028] Example 1 , Anoectochilus roxburghii 'Hongxia' ArHDZ22 Gene cloning

[0029] 1. Acquisition of plant materials

[0030] Total RNA was extracted from the stems, leaves, buds, and flowers of the normally growing Anoectochilus roxburghii 'Hongxia'.

[0031] 2. RNA Extraction

[0032] Beijing Quanshijin Biological Company TransZol Total RNA was extracted using the Up Plant Total RNA Extraction Kit, and the integrity of the RNA was determined by gel electrophoresis. The purity and concentration of the RNA were determined by a spectrophotometer (Nanodrop 2000).

[0033] 3. Full-length cloning of genes

[0034] Based on the nucleotide sequence and protein function annotation results provided by the laboratory's early full-length transcriptome analysis, we obtained the ArHDZ22 Full-length gene.

[0035] The extracted RNA was reverse transcribed (TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix) to obtain cDNA. The first-strand cDNA was used as a template and primers ArHDZ22 were used. - F (5'-ATGGCGATGATGGTGAGCGC-3') and ArHDZ22-R (5'- TCAAACAAAAGACCAGTTGATGATCATAA-3') were amplified by PCR to obtain a 2611 bp fragment, which was recovered and ligated into the pMD19-T vector. M13-47 and RV-M were used as universal primers and the fragment was sent to Hangzhou Youkang Biological Company for sequencing.

[0036] The sequencing results were combined with NCBI's ORF Finding (http: / / www.ncbi.nlm.nih.gov / gorf) prediction to obtain the ArHDZ22 The ORF reading frame of the gene was amplified by PCR to obtain 2523 bp of the gene of Anoectochilus roxburghii 'Hongxia' ArHDZ22 The nucleotide sequence (SEQ ID NO.1) was compared with the BLAST database (GenBank, http: / / blast.ncbi.nlm.nih.gov / ) on the NCBI website. The nucleotide sequence and the encoded protein were highly homologous to the known Arabidopsis AtHDZ22 gene, and it was initially believed to be a HD-ZIP Gene.

[0037] Example 2 , Anoectochilus roxburghii 'Hongxia' ArHDZ22 Gene sequence information and homology analysis

[0038] Anoectochilus roxburghii 'Hongxia' of the present invention ArHDZ22The full-length open reading frame (ORF) of the gene is 2523 bp, and the detailed sequence is shown in SEQ ID NO. 1. The amino acid sequence of the transcription factor ArHDZ22 protein from Anoectochilus roxburghii 'Hongxia' was deduced based on the ORF sequence, which contains 840 amino acids, a molecular weight of 217.12 kDa, and an isoelectric point (pI) of 4.83. The detailed sequence is shown in SEQ ID NO. 2.

[0039] Anoectochilus roxburghii 'Hongxia' ArHDZ22 The open reading frame sequence of the gene and the amino acid sequence of the encoded protein were searched for nucleotide and protein homology in NCBI using BLAST program. The results showed that it was similar to Arabidopsis thaliana. AtHDZ22 Genes have a high degree of similarity at the amino acid level, such as Figure 1 As shown in A. Phylogenetic tree analysis showed that the transcription factor ArHDZ22 of Anoectochilus roxburghii 'Hongxia' has a high homology with HD-ZIP proteins of other known species, such as Figure 1 As shown in B.

[0040] Example 3 , Construction of recombinant expression vector pCAMBIA1300-ArHDZ22 and analysis of subcellular localization of transcription factor ArHDZ22 in tobacco leaves

[0041] Specific primers F (5'-TCGGTACCCGGGGATCCATGGCGATGATGGTGAGCGC-3') and R (5'-TGCTCACCATGTCGACAACAAAAGACCAGTTGATGATCATAA-3') were designed from the start codon and the stop codon, and restriction sites were introduced on both sides of the full-length gene sequence. The target fragment plasmid with restriction sites was transformed with the pCAMBIA1300 binary vector. BamH I and Pst I double enzyme digestion, recovery of the digested vector and ArHDZ22 The fragments were ligated using T4 ligase at 16°C for 12-14 hours to construct the pCAMBIA1300-ArHDZ22 recombinant expression vector.

[0042] The correct recombinant expression vector was screened by PCR and sequencing, and the recombinant plasmid was transformed into Agrobacterium competent EHA105 by freeze-thaw method to obtain recombinant bacteria Agrobacterium EHA105 containing the recombinant expression vector.

[0043] The identified EHA105 strain was inoculated into 5 mL of YEP (containing 50 mg / L Kan) and cultured at 28°C and 180 rpm until the OD 600 1 mL of bacterial solution was added to 25 mL of YEP liquid medium and cultured at 28°C until OD600 Take 10 mL of bacterial solution and centrifuge at 4500 rpm for 15 min; suspend the bacteria in MS liquid medium until the OD 600 The concentration of AS and MES was about 0.6, and AS and MES were added and placed at room temperature for more than 3 hours; then injected into tobacco leaves, cultured in dark conditions for 48 hours, and observed using a laser confocal microscope, such as Figure 2 shown.

[0044] Example 4 , golden thread lotus ArHDZ22 Gene expression changes in different tissues of Anoectochilus roxburghii and under osmotic stress

[0045] 1. Material Acquisition: Anoectochilus roxburghii was subjected to sorbitol osmotic stress treatment. Samples were collected at 0, 3, 6, 12, 24, and 48 hours. The samples were wrapped in aluminum foil, placed in liquid nitrogen, and then stored in a -80°C ultra-low temperature freezer until use.

[0046] 2. RNA extraction, determination of RNA integrity, purity, and concentration, and acquisition of cDNA were performed as described in Example 1.

[0047] 3. Design specific primers to perform real-time fluorescence quantitative PCR to analyze the expression of genes in various tissues. ArHDZ22 Gene sequence, designed for real-time PCR ArHDZ22 Specific primers for gene quantitative analysis, primer q ArHDZ22 -F (5′-IndexTermGAGATACACGCCGGAGCAAG-3′), primer q ArHDZ22 -R (5'-CTCGATGTTGGCGAGGATGG-3'), internal reference gene ArActin Primers are ArActin- F(5'-GCTAGTGGCCGTACAACTGG-3'), ArActin -R(5'-GCCAGCAAGGTCCAATCGAA-3').

[0048] 4. Standard curves for target genes and internal reference genes: Serially dilute the standard cDNA solution with ddH2O. Using the diluted cDNA as a template, perform real-time PCR amplification with primers specific for the target gene and internal reference gene. Develop a melting curve and a standard curve. Analyze the melting curves to confirm that the melting curves for the target gene and internal reference gene exhibit a single peak, thereby verifying primer specificity and the uniqueness of the PCR amplification product. Use the standard curve to determine the appropriate dilution factor for the cDNA template, providing an accurate basis for subsequent quantification.

[0049] 5. Real-time fluorescence quantitative analysis of the target gene in the test sample: Using the first-strand synthesized cDNA as a template, specific primers for the target gene and the internal reference gene were used for amplification and fluorescence quantitative analysis. Real-time PCR reactions were performed on a Bio-Rad CFX real-time fluorescence quantitative instrument. The reaction system was 20 µL and the reaction procedure was as follows: pre-denaturation at 94°C for 20 s, followed by 40 cycles of 94°C for 15 s, 55°C for 15 s, and 72°C for 15 s.

[0050] 6. Use 2 -△△Ct The relative quantitative analysis was performed by the method. The results showed that under different developmental stages of Anoectochilus roxburghii tissues and osmotic stress treatments, ArHDZ22 Gene expression levels increased significantly, such as Figure 3 shown.

[0051] Example 5 、 ArHDZ22 Gene transformation into Arabidopsis thaliana

[0052] (1) Pre-shake Agrobacterium: Pick a positive single colony and transfer it to 5 mL of YEP liquid medium containing 50 mg / L Kan, 50 mg / L gentamicin, and 25 mg / L R-Rex, and culture at 28°C and 180 rpm for 24 h;

[0053] (2) Propagation of Agrobacterium: The pre-shaken Agrobacterium culture solution was propagated at a dilution of 1:100 into YEP medium containing the same resistance, cultured at 28°C, 200 rpm, for 13-16 h until the OD 600 When the concentration reaches about 0.6, the mixture is collected at 18°C ​​and 3500 rpm for 15 min.

[0054] (3) Transformation of Arabidopsis thaliana by inflorescence immersion method: The specific steps for infecting Arabidopsis thaliana using the Agrobacterium-mediated inflorescence immersion method are as follows.

[0055] ① The best time to infect Arabidopsis is when it reaches the early to mid-flowering stage. The day before infecting, cut off the flowers that have already opened and formed pods, leaving only the unopened flowers. Water and fertilize the plants adequately to prepare for infection.

[0056] ② Prepare resuspension solution MS + 20 g / L sucrose (pH 5.8). Resuspend Agrobacterium cells in the resuspension solution and adjust the OD 600 Adjust the value to 0.8-1.0, then add Silwet L-77 to make the concentration reach 0.02%, shake well to obtain the infection solution.

[0057] ③ Select wild-type Arabidopsis that has been treated the day before, completely immerse the inflorescence of each Arabidopsis plant in the infection solution, and vacuum for 10 minutes.

[0058] ④ Place the bagged Arabidopsis plants flat in a dark room for one day of dark culture. After dark culture, place them in a greenhouse at 22°C, 65% humidity, 16 hours of light and 8 hours of darkness for normal culture.

[0059] ⑤ Cultivate the Arabidopsis thaliana normally and manage it using conventional cultivation methods until harvest.

[0060] (4) Screening of transgenic positive lines

[0061] Genomic DNA and RNA were extracted using kits from Quanshijin Biotechnology Co., Ltd. (Beijing) to identify positive plants.

[0062] Example 6 , overexpression ArHDZ22 Arabidopsis phenotypic analysis

[0063] Experimental methods for early flowering

[0064] The subjects for the phenotypic observation and measurement of Arabidopsis thaliana included positive homozygous T3 plants and positive homozygous T3 seeds (OE), while Arabidopsis thaliana transformed with the pCAMBIA1300 empty vector served as a negative control (CK). The measured indicators included plant growth cycle, plant height, and flower anatomical morphology. The plant growth cycle was observed by randomly selecting three transgenic plants 37 days after sowing, observing the growth cycle differences between them and the control plants, and recording photos, such as Figure 4 The method for observing the anatomical morphology of flowers is to randomly select three flowers with similar opening degrees after the control plants and transgenic plants grow to the flowering stage, carefully cut them open with a blade and tweezers, and neatly place the pistils, stamens, corolla and sepals in order on a black cloth, and take photos to record them, as shown in the figure below. Figure 5 As shown in A.

[0065] Osmotic stress treatment experiment

[0066] Wild-type and transgenic Arabidopsis seeds were placed in 1 / 2 MS medium containing 0 and 100 mM sorbitol and cultured for 10 days for phenotypic observation and photography. Figure 5 As shown in B.

[0067] The above describes the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art may make various variations or modifications within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A HD-ZIP transcription factor ArHDZ22 from Anoectochilus roxburghii 'Hongxia', characterized in that The amino acid sequence of the transcription factor ArHDZ22 is shown in SEQ ID NO.

2.

2. A gene encoding the HD-ZIP transcription factor ArHDZ22 of Anoectochilus roxburghii 'Hongxia' according to claim 1, characterized in that: The coding gene is the nucleotide sequence shown in SEQ ID NO.

1.

3. A recombinant expression vector, characterized in that: The invention comprises the coding gene of the HD-ZIP transcription factor ArHDZ22 of the roxburghii 'Hongxia' according to claim 2.

4. The recombinant expression vector according to claim 3, characterized in that The recombinant expression vector is pCAMBIA1300-ArHDZ22.

5. A recombinant bacterium, characterized in that The method is obtained by transforming a host cell with the recombinant expression vector according to claim 4.

6. The recombinant bacterium according to claim 5, characterized in that The host cell is Agrobacterium EHA105. 7 . Use of the gene encoding the HD-ZIP transcription factor ArHDZ22 of Anoectochilus roxburghii 'Hongxia' according to claim 2 in improving the osmotic stress resistance of Anoectochilus roxburghii.

8. Use of the gene encoding the HD-ZIP transcription factor ArHDZ22 of Anoectochilus roxburghii 'Hongxia' according to claim 7 in improving the osmotic stress resistance of Anoectochilus roxburghii, characterized in that: The application includes: constructing a recombinant expression vector containing the coding gene of the transcription factor ArHDZ22, transforming the vector into a plant host, and cultivating and screening to obtain transgenic plants.

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