HD-ZIP transcription factor ArHDZ19, its encoding gene and application in Anoectochilus roxburghii 'Hongxia'

Abstract

The present invention discloses an HD-ZIP transcription factor, ArHDZ19, from Anoectochilus roxburghii 'Hongxia', its encoding gene, and its applications. The transcription factor ArHDZ19 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 promoting reproductive growth and enhancing seed vigor in Anoectochilus roxburghii using genetic engineering techniques, and 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 ArHDZ19, 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. They participate in plant-specific biological processes and play a crucial role in their growth and development. HD-ZIP III subfamily transcription factors regulate plant reproductive development and are closely associated with embryonic and postembryonic morphogenesis, plant cell differentiation, lateral organogenesis, vascular cell division, and polarity establishment. However, no studies have yet reported the involvement of HD-ZIP proteins in reproductive development in Anoectochilus roxburghii. Summary of the Invention

[0004] To fill the gap between Anoectochilus 'Hongxia' ArHDZ19 The present invention provides a gene cloning, expression pattern and the blank of the transcription factor ArHDZ19 of Anoectochilus roxburghii'Hongxia'. ArHDZ19 Gene sequence and encoded amino acid sequence, protein subcellular localization, and transgenic plant growth phenotype will provide a basis for future regulation using genetic engineering technology. ArHDZ19 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 ArHDZ19 having the function of promoting reproductive development and improving seed traits. The above-mentioned transcription factor ArHDZ19 includes 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 ArHDZ19 by replacing, deleting or adding one or more amino acids in the amino acid sequence 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 ArHDZ19, and the nucleotide sequence of the above-mentioned encoding gene is specifically: (a) the base sequence shown in positions 1 to 2553 of SEQ ID NO.1; or (b) a sequence with at least 70% homology to the nucleic acid shown in positions 1 to 2553 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 ArHDZ19 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 2553 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 2553 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 2553 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 above-mentioned coding gene may also refer to a variant form of the sequence shown in SEQ ID NO.1 that can encode the same function as the natural Anoectochilus roxburghii 'Hongxia' HD-ZIP transcription factor ArHDZ19; these variant forms include (but are not limited to): deletions, insertions and / or substitutions of usually 1 to 120 nucleotides, and additions of up to 120 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 ArHDZ19 Analysis of the expression patterns of gene products in Anoectochilus roxburghii 'Hongxia' ArHDZ19 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' ArHDZ19 Gene nucleotide sequence and amino acid sequence can be screened based on nucleic acid homology or expressed protein homology to identify Anoectochilus roxburghii 'Hongxia' ArHDZ19 Gene-related homologous genes or homologous proteins.

[0012] Anoectochilus roxburghii 'Hongxia' of the present invention ArHDZ19 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 comprising the gene encoding the HD-ZIP transcription factor ArHDZ19 of the aforementioned Anoectochilus roxburghii 'Hongxia'. The aforementioned recombinant expression vector is pHB-ArHDZ19.

[0017] The present invention also provides the use of a gene encoding a HD-ZIP transcription factor ArHDZ19 of anoectochilus roxburghii 'Hongxia' in promoting reproductive growth of anoectochilus roxburghii and improving seed vigor.

[0018] The above application includes: constructing a recombinant expression vector containing the above coding gene of the transcription factor ArHDZ19, transforming the vector into a plant host, and cultivating and screening to obtain transgenic plants.

[0019] As a rare and precious Chinese medicinal material, Anoectochilus roxburghii has a large market demand. This study cloned the coding sequence of ArHDZ19, 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. ArHDZ19 The expression pattern of genes, the subcellular localization of transcription factor ArHDZ19 in tobacco leaf epidermal cells transiently expressed, will provide a basis for the future regulation of gene expression using genetic engineering technology. ArHDZ19 The spatiotemporal expression of genes provides a theoretical basis for improving the vigor of Anoectochilus roxburghii seeds, promoting reproductive growth, and breeding new varieties, and has great application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Anoectochilus roxburghii 'Hongxia' HD-ZIP Homology comparison results (DNAMAN) between the gene and Arabidopsis HD-ZIP protein sequences and phylogenetic tree analysis of homologous genes; A is the phylogenetic tree analysis of ArHDZ19 protein and homologous proteins in other species; B is the multiple sequence alignment diagram;

[0021] Figure 2 This is the localization map of the transcription factor ArHDZ19 from Anoectochilus roxburghii'Hongxia' in the epidermal cells of tobacco leaves;

[0022] Figure 3 Anoectochilus roxburghii 'Hongxia' ArHDZ19 A diagram showing changes in gene expression during plant growth and development;

[0023] Figure 4 Wild type and overexpression ArHDZ19 Plant growth and development phenotype diagram of the gene;

[0024] Figure 5 Wild type and overexpression ArHDZ19 Effects of gene expression on seed traits in plants; A is a diagram of wild-type seeds; B is a diagram of overexpression ArHDZ19 A is the seed diagram of gene; C is the diagram of 100-grain weight of seeds; D is the diagram of seed length. DETAILED DESCRIPTION

[0025] 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.

[0026] 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.

[0027] Example 1 , Anoectochilus roxburghii 'Hongxia' ArHDZ19 Gene cloning

[0028] 1. Acquisition of plant materials

[0029] Total RNA was extracted from the stems, leaves, buds, flowers and other tissues of the normally growing Anoectochilus roxburghii 'Hongxia'.

[0030] 2. RNA Extraction

[0031] Beijing Quanshijin Biological Company TransZol Total RNA was extracted using the Up Plant Total RNA Extraction Kit, and the integrity of the RNA was assessed by gel electrophoresis. The purity and concentration of the RNA were determined using a spectrophotometer (Nanodrop 2000).

[0032] 3. Full-length cloning of genes

[0033] Based on the nucleotide sequence and protein function annotation results provided by the laboratory's early full-length transcriptome analysis, we obtained the ArHDZ19 The full-length gene was obtained by reverse transcription (TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix) of the extracted RNA to obtain cDNA. Using the first-strand cDNA as a template, PCR was performed using primers ArHDZ19-F (5'-ATGGCGATGATGGTGAGCGC-3') and ArHDZ19-R (5'-TCAAACAAAAGACCAGTTGATGATCATAA-3'). A 2553 bp fragment was amplified and ligated to the pMD19-T vector. M13-47 and RV-M were used as universal primers and the fragment was sent to Hangzhou Youkang for sequencing.

[0034] The sequencing results were combined with NCBI's ORF Finding (http: / / www.ncbi.nlm.nih.gov / gorf) prediction and the discovery of Anoectochilus roxburghii ArHDZ19 The ORF reading frame of the gene was amplified to obtain the full-length coding sequence (SEQ ID NO.1) of 2553bp. The sequencing results were compared with the NCBI website BLAST database (GenBank, http: / / blast.ncbi.nlm.nih.gov / ). Its nucleotide sequence and encoded protein showed high homology with the known nucleotide and protein sequences of Dendrobium officinale DcHD-Zip Ⅲ (XP_020698923.1), and it was preliminarily considered to be a HD-ZIP Gene.

[0035] Example 2 , Anoectochilus roxburghii 'Hongxia' ArHDZ19 Gene sequence information and homology analysis

[0036] Anoectochilus roxburghii 'Hongxia' of the present invention ArHDZ19 The full-length open reading frame (ORF) of the gene is 2553 bp, and the detailed sequence is shown in SEQ ID NO. 1. The amino acid sequence of the transcription factor ArHDZ19 protein from Anoectochilus roxburghii 'Hongxia' was deduced based on the ORF sequence, which consists of 850 amino acids, a molecular weight of 93.4 kDa, and an isoelectric point (pI) of 8.19. The detailed sequence is shown in SEQ ID NO. 2.

[0037] Anoectochilus roxburghii 'Hongxia' ArHDZ19The open reading frame sequence of the gene and the amino acid sequence of its encoded protein were searched for nucleotide and protein homology in NCBI using the BLAST program. The results showed that it had a very high similarity with Dendrobium officinale DcHD-Zip Ⅲ (XP_020698923.1) at the amino acid level. Figure 1 As shown in B. Phylogenetic tree analysis showed that the transcription factor ArHDZ19 of Anoectochilus roxburghii 'Hongxia' has a high homology with HD-ZIP proteins of other known species, such as Figure 1 As shown in A.

[0038] Example 3 , Construction of Anoectochilus roxburghii pHB-ArHDZ19 Recombinant Expression Vector and Analysis of the Subcellular Localization of Transcription Factor ArHDZ19 in Tobacco Leaves

[0039] Specific primers ArHDZ19-F (5'-TCGGTACCCGGGGATCCATGGCGATGGTGGGGAAGGA-3') and ArHDZ19-R (5'-TGCTCACCATGTCGACGACAAATGACCAGTTCATGAAC-3') were designed from the start codon and the stop codon, respectively, and introduced on both sides of the full-length gene sequence. BamH I and Spe I restriction enzyme site. The target fragment plasmid with restriction enzyme site is combined with pHB binary transformation vector. BamH I and Spe I double enzyme digestion, recovery of pHB vector after enzyme digestion and ArHDZ19 The fragments were ligated with T4 ligase at 16°C for 6 h to construct the recombinant expression vector pHB-ArHDZ19.

[0040] The identified recombinant expression vector pHB-ArHDZ19 was transformed into Agrobacterium EHA105. The identified EHA105 strain was inoculated into 5 mL 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 OD 600 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 mixture was about 0.6, and AS and MES were added and placed at room temperature for more than 3 h; the mixture was injected into tobacco leaves and cultured in the dark for 48 h. The leaves were observed under a laser confocal microscope with an excitation wavelength of 514 nm. Figure 2 shown.

[0041] Example 4 , golden thread lotus ArHDZ19 Gene expression changes in different tissues of Anoectochilus roxburghii

[0042] 1. Material Acquisition: Collect 0.1 g of root, stem, leaf, and flower tissue samples from Anoectochilus roxburghii at different developmental stages (leaf bud, flower bud, and flowering). Wrap the samples in aluminum foil, place them in liquid nitrogen, and then store them in a -80°C freezer until ready for use.

[0043] 2. RNA extraction, determination of RNA integrity, purity, and concentration, and acquisition of cDNA were performed as described in Example 1.

[0044] 3. Design specific primers to perform real-time fluorescence quantitative PCR to analyze the expression of genes in various tissues. ArHDZ19 Gene sequence, designed for real-time PCR ArHDZ19 Specific primers for gene quantitative analysis, primer q ArHDZ19 -F (5′-GTATACGCCGGAGCAAGTGG-3′), primer q ArHDZ19 -R (5'-TCTCTGATGAGCTGCTGCCT-3'), the internal reference gene actin primer is actin - F (5'-GCTAGTGGCCGTACAACTGG-3'), actin-R (5'-GCCAGCAAGGTCCAATCGAA-3').

[0045] 4. Standard curves for target genes and internal reference genes: Serially dilute the standard cDNA solution with ddH2O. Then, use the diluted cDNA as a template and perform real-time PCR amplification with specific primers for the target gene and internal reference gene, respectively. Draw melting curves and standard curves. Analyze the melting curves to determine whether the melting curves of the target gene and internal reference gene produce a single peak, thereby determining whether a single PCR amplification product can be obtained using these primers. Use the standard curve to determine the appropriate dilution factor of the template cDNA.

[0046] 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.

[0047] 6. Use 2 -△△Ct The relative quantitative analysis showed that in the tissues of Anoectochilus roxburghii at different developmental stages, ArHDZ19Gene expression levels increased significantly, such as Figure 3 shown.

[0048] Example 5 、 ArHDZ19 Gene transformation into Arabidopsis thaliana

[0049] 1. Construction of plant expression vector

[0050] The restriction enzyme sites introduced refer to Example 3. The target fragment plasmid with the restriction enzyme sites was transformed with the pHB binary vector. BamH I and Spe I double digestion, the digested vector was recovered and connected by homologous recombination, and the recombinant expression vector was transformed into Agrobacterium EHA105.

[0051] 2. Transformation of Arabidopsis

[0052] Floral invasion method for transformation of Arabidopsis thaliana: The specific steps for infecting Arabidopsis thaliana using the Agrobacterium-mediated inflorescence immersion method are as follows.

[0053] ① The best time for infection of wild-type Arabidopsis is when it grows to the early to middle flowering stage. The day before infection, cut off the blooming flowers and pods, leaving the flowers in bud, and water and fertilize them adequately in preparation for infection.

[0054] ②Pipette 100 μL of positive Agrobacterium culture solution into 25 mL of LB liquid medium containing Kana (50 mg / L) and Rif (25 mg / L) for expansion culture, and shake culture in a constant temperature shaker (28°C, 220 rpm) until the culture OD reaches 600 The value is 0.8-1.0.

[0055] ③OD value of Agrobacterium culture to be expanded 600 When the value reaches 0.8-1.0, centrifuge to collect the bacteria at 6000 rpm for 10 min and discard the supernatant.

[0056] ④ Resuspend the cells in MS resuspension solution (4.43 g / L MS powder + 20 g / L sucrose, pH 5.8) and adjust the OD 600 Value to 0.8-1.0, OD 600 After the value is adjusted, add Tween-20 to make the final concentration of 0.02%, shake well to obtain the infection solution.

[0057] ⑤ Soak the inflorescence of each Arabidopsis plant in the infection solution for 10 minutes.

[0058] ⑥ After infection, the Arabidopsis thaliana bags were placed flat in a dark room for dark culture for 1 day to maintain humidity. After dark culture, they were placed in a greenhouse at 22°C, 65% humidity, 16 h light and 8 h dark for normal culture.

[0059] ⑦ One week later, repeat the operation to infect it again to improve the transformation success rate.

[0060] ⑧After the secondary infection is completed, cultivate it normally and manage it with conventional cultivation methods until harvest.

[0061] 3. Screening of Transgenic Positive Lines

[0062] Genomic DNA and RNA were extracted using the kits from Quanshijin Biotechnology Co., Ltd. (Beijing) to identify positive plants.

[0063] Example 6 , overexpression ArHDZ19 Arabidopsis phenotypic analysis

[0064] The objects selected for phenotypic observation and measurement of Arabidopsis thaliana were positive homozygous T3 generation plants and positive homozygous T3 generation seeds (OE), and Arabidopsis thaliana transformed with pHB empty vector was used as negative control (CK). The indicators of phenotypic observation and measurement were: plant growth cycle, plant height, seed length and width, and 100-grain seed weight.

[0065] Plant growth cycle observation method: On the 37th day after sowing, three Arabidopsis plants were randomly selected from the transgenic plants. The growth cycle differences between them and the control plants were observed and photographed. Plant height measurement method: On the 44th day after sowing, three Arabidopsis plants were randomly selected from the control plants and three from the transgenic plants. They were removed from the seedling pots, the culture medium was washed, and they were placed on a black cloth to measure the distance from the base of the rosette leaf to the top of the inflorescence and photographed. Figure 4 shown.

[0066] Method for measuring seed length and width: Select several control plant seeds and transgenic plant seeds, randomly spread them on a glass slide, place them under an optical microscope, use natural light to take pictures and record them, and randomly select ten seeds from the field of view to calculate the average length and width using a scale. Method for measuring seed weight of 100 seeds: Count 100 seeds from each of the control plant seeds and transgenic plant seeds, and weigh them on an analytical balance. Figure 5 A- Figure 5 As shown in D.

[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 ArHDZ19 from Anoectochilus roxburghii 'Hongxia', characterized in that The amino acid sequence of the transcription factor ArHDZ19 is shown in SEQ ID NO.

2.

2. A gene encoding the HD-ZIP transcription factor ArHDZ19 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 ArHDZ19 of Anoectochilus roxburghii 'Hongxia' according to claim 2.

4. The recombinant expression vector according to claim 3, characterized in that The recombinant expression vector is pHB-ArHDZ19.

5. Use of the gene encoding the HD-ZIP transcription factor ArHDZ19 of Anoectochilus roxburghii 'Hongxia' according to claim 2 in increasing the plant height and 100-grain weight of Anoectochilus roxburghii.

6. Use of the gene encoding the HD-ZIP transcription factor ArHDZ19 of Anoectochilus roxburghii 'Hongxia' according to claim 5 in increasing the plant height and 100-grain weight of Anoectochilus roxburghii, characterized in that: The application includes: constructing a recombinant expression vector containing the coding gene of the transcription factor ArHDZ19, transforming a plant host, and cultivating and screening to obtain transgenic plants.

Images