Ananas comosus WRKY transcription factor AcWRKY6, coding gene and application

Abstract

The present application relates to a kind of gold pteridophyte 'Jin Kang No.1' WRKY transcription factor ArWRKY6, coding gene and its application, above-mentioned transcription factor ArWRKY6 includes the polypeptide with the amino acid sequence as shown in SEQ ID NO.2.The present application also provides a kind of nucleotide sequence SEQ ID NO.1 for encoding above-mentioned protein.The present application provides a theoretical basis for promoting gold pteridophyte to bloom in advance, promote leaf and seed development using genetic engineering technology, has important application value.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of genetic engineering application technology, and relates to an important WRKY structure C2-H2 protein in the growth and development process of Anoectochilus roxburghii, in particular to a WRKY transcription factor ArWRKY6 of Anoectochilus roxburghii 'Jinkang No.1' and a coding gene and application thereof. BACKGROUND

[0002] Anoectochilus roxburghii Anoectochilus roxburghii (Wall.) Lindl. is a perennial herb of Orchidaceae and Anoectochilus, also known as gold silk grass and gold thread orchid, is a rare and precious Chinese medicinal material in China; it is flat in nature and sweet in taste, has the effects of clearing heat and cooling blood, removing dampness and detoxifying, and is used for treating acute and chronic hepatitis, diabetes and other diseases, and is known as the "King of Medicine". Modern studies have shown that Anoectochilus roxburghii contains polysaccharides, flavonoids, organic acids, steroidal compounds, alkaloids, and various trace elements, and has pharmacological activities such as enhancing immunity, anti-liver injury, reducing blood sugar, and antioxidant activity. Chinese patent medicines with Anoectochilus roxburghii as the main raw material, such as compound Anoectochilus roxburghii capsules and Anoectochilus roxburghii spray, have been used in the treatment of related diseases in clinical practice. With the wide application of Anoectochilus roxburghii in medicine, health care, beauty and drinking products and many other fields, the demand for Anoectochilus roxburghii in the domestic and foreign markets is increasing. However, due to late flowering and post-fertilization embryo abortion, the natural reproduction rate of Anoectochilus roxburghii seed is low, resulting in reproductive disorders of Anoectochilus roxburghii, and the wild resources are sharply decreasing due to excessive artificial harvesting.

[0003] WRKY is a zinc finger type transcriptional regulatory factor specific to plants, which widely participates in the regulation of plant defense response and growth and development process, and is closely related to pollen development, seed development, leaf growth and senescence. However, there are few reports about the participation of WRKY protein in the early flowering, leaf and seed development of Anoectochilus roxburghii. SUMMARY

[0004] In order to fill the gap in the research on the participation of WRKY protein in the early flowering, leaf and seed development of Anoectochilus roxburghii 'Jinkang No.1', the present application discloses a WRKY transcription factor ArWRKY6 of Anoectochilus roxburghii 'Jinkang No.1', a coding gene and application thereof. ArWRKY6 The present application discloses the gene sequence of Anoectochilus roxburghii 'Jinkang No.1' and the amino acid sequence coded by the gene, protein subcellular localization, and growth phenotype of transgenic plants, which provides a theoretical basis for future regulation of gene expression and improvement of Anoectochilus roxburghii breeding efficiency by using genetic engineering technology. ArWRKY6 ArWRKY6 The present application discloses the gene sequence of Anoectochilus roxburghii 'Jinkang No.1' and the amino acid sequence coded by the gene, protein subcellular localization, and growth phenotype of transgenic plants, which provides a theoretical basis for future regulation of gene expression and improvement of Anoectochilus roxburghii breeding efficiency by using genetic engineering technology.

[0005] ​In one aspect, the present application provides a Chlona 'Jinkang No.1' WRKY transcription factor ArWRKY6 which promotes flowering, leaf and seed development, wherein the transcription factor ArWRKY6 comprises a polypeptide (protein) having an amino acid sequence as shown in SEQ ID NO. 2; or a protein having the amino acid sequence shown in SEQ ID NO. 2 with one or more amino acids being substituted, deleted or added and having the characteristics of Chlona 'Jinkang No.1' WRKY transcription factor ArWRKY6.

[0006] In another aspect, the present application provides a coding gene encoding the Chlona 'Jinkang No.1' WRKY transcription factor ArWRKY6, wherein the nucleotide sequence of the coding gene is specifically: (a) a base sequence as shown in SEQ ID NO. 1, 1-1545; or (b) a sequence having at least 70% homology with the nucleic acid shown in SEQ ID NO. 1, 1-1545.

[0007] In the present application, "isolated DNA" or "purified DNA" means that the DNA or fragment has been separated from the sequences flanking it in the natural state, and also means that the DNA or fragment has been separated from the components accompanying the nucleic acid in the natural state, and has been separated from the proteins accompanying it in the cell.

[0008] In the present application, the coding gene of Chlona 'Jinkang No.1' WRKY transcription factor ArWRKY6 refers to a nucleotide sequence encoding a polypeptide having the activity of Chlona 'Jinkang No.1' protein, such as the nucleotide sequence shown in SEQ ID NO. 1, 1-1545, and its degenerate sequence. The degenerate sequence refers to a sequence in which one or more codons in the nucleotide sequence shown in SEQ ID NO. 1, 1-1545 are replaced by degenerate codons encoding the same amino acid. Due to the degeneracy of the codon, the degenerate sequence with a homology as low as about 70% to the nucleotide sequence shown in SEQ ID NO. 1, 1-1545 can also encode the sequence shown in SEQ ID NO. 2. The coding gene can also refer to a nucleotide sequence having a homology of at least 70% to the nucleotide sequence shown in SEQ ID NO. 1.

[0009] The coding gene can also refer to a variant of the sequence shown in SEQ ID NO. 1 which can encode the same function as the natural Chlona 'Jinkang No.1' WRKY transcription factor ArWRKY6; these variants include (but are not limited to) deletions, insertions and / or substitutions of generally 1-120 nucleotides, and additions of up to 90 nucleotides at the 5' and / or 3' end.

[0010] In the present application, the Chlona 'Jinkang No.1' WRKY transcription factor ArWRKY6 can be analyzed by real-time fluorescent quantitative PCR. ArWRKY6The expression pattern of the gene product, i.e. the analysis of Anoectochilus roxburghii 'Jinkang No. 1' ArWRKY6 The presence and quantity of mRNA transcript of the gene in cells.

[0011] In addition, the Anoectochilus roxburghii 'Jinkang No. 1' ArWRKY6 Based on nucleic acid homology or expression protein homology, the Anoectochilus roxburghii 'Jinkang No. 1' ArWRKY6 Gene-related homologous genes or homologous proteins.

[0012] The Anoectochilus roxburghii 'Jinkang No. 1' ArWRKY6 The nucleotide full-length sequence or fragment thereof related to the gene can be obtained by PCR amplification, recombination or artificial synthesis. For PCR amplification, primers can be designed according to the nucleotide sequence disclosed in the present application, and a commercially available cDNA library or a cDNA library prepared according to the conventional method known to those skilled in the art is used as a template for amplification to obtain the relevant sequence. When the sequence is long, two or more PCR amplifications are often required, and then the fragments amplified in each amplification are spliced together in the correct order.

[0013] When the relevant sequence is obtained, the recombination method can be used to obtain the relevant sequence in large quantities. This is usually to clone it into a vector, then into cells, and then to separate the relevant sequence from the proliferated host cells by conventional methods.

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

[0015] In addition to being produced by recombination, fragments of the protein of the present application can also be produced by directly synthesizing peptides using solid-phase technology. Each fragment of the protein of the present application can be chemically synthesized, and then chemically linked to produce a full-length molecule.

[0016] The present application also provides a recombinant expression vector comprising the coding gene of the Anoectochilus roxburghii 'Jinkang No. 1' WRKY transcription factor ArWRKY6 described above. The recombinant expression vector described above is pCAMBIA1304- ArWRKY6 .

[0017] The present application also provides the use of the coding gene of the Anoectochilus roxburghii 'Jinkang No. 1' WRKY transcription factor ArWRKY6 described above in promoting early flowering of Anoectochilus roxburghii, promoting leaf and seed development.

[0018] The above-mentioned use includes constructing a recombinant expression vector containing the coding gene of the above-mentioned transcription factor ArWRKY6, and transforming plant hosts to cultivate and screen transgenic plants.

[0019] Beneficial effects: the market demand of Anoectochilus roxburghii, a rare and precious Chinese medicinal material, is large. The coding sequence of the important transcription factor ArWRKY6 in the growth and development process of Anoectochilus roxburghii 'Jinkang No.1' is cloned for the first time, and the expression mode of the gene is analyzed by using the method of fluorescent real-time quantitative PCR ArWRKY6 transient expression analysis of the transcription factor ArWRKY6 in tobacco leaf epidermal cells, the subcellular localization of the transcription factor ArWRKY6 is analyzed, and the theoretical basis is provided for promoting early flowering of Anoectochilus roxburghii, promoting leaf and seed development and new variety breeding in the future, and the application value is great. ArWRKY6 BRIEF DESCRIPTION OF DRAWINGS

[0020] ArWRKY6 The Ar Figure 1 gene of Anoectochilus roxburghii 'Jinkang No.1' in the present application and the WRKY protein sequence of Arabidopsis thaliana are compared in homology (DNAMAN) and the evolutionary tree analysis diagram of the homologous genes; wherein, A is the system evolution tree diagram; B is the homologous sequence alignment;

[0021] WRKY6 The localization diagram of the transcription factor ArWRKY6 of Anoectochilus roxburghii 'Jinkang No.1' in tobacco leaf epidermal cells;

[0022] Figure 2 The expression change diagram of the Ar Figure 3 gene of Anoectochilus roxburghii 'Jinkang No.1' in plant roots, stems, leaves and juvenile flower buds, mature flower buds and flowers;

[0023] ArWRKY6 The plant growth and development phenotype diagram of the wild type and the overexpression Figure 4 gene; wherein, A is the comparison diagram after planting for 35 days; B is the comparison diagram of rosette leaf number; C is the statistical diagram of rosette leaf number;

[0024] ArWRKY6 The influence diagram of the wild type and the overexpression Figure 5 gene on pistil, stamen and seed traits; wherein, A is the microscopic diagram of pistil, stamen and seed; B is the statistical diagram of hundred-grain weight; C is the statistical diagram of seed length / width; D is the statistical diagram of pistil / stamen length. DETAILED DESCRIPTION

[0025] The present application will be further described in conjunction with specific embodiments, but these embodiments are only used to illustrate the present application and not used to limit the scope of the present application.

[0026] The experimental methods not specified in the following examples are usually used according to conventional conditions, for example, the conditions described in the molecular cloning experiment guide (the fourth edition of the original book), or the conditions suggested in the reagent instruction book.

[0027] ArWRKY6 ​, Anoectochilus roxburghii 'Jinkang No. 1' Example 1 Gene cloning

[0028] 1. Acquisition of plant materials

[0029] Total RNA was extracted from the stems, leaves, buds and other tissues of Anoectochilus roxburghii 'Jinkang No. 1' during normal growth and flowering period.

[0030] 2. RNA Extraction

[0031] Total RNA was extracted using the Polysaccharide and Polyphenol Plant RNA Extraction Kit (Beijing Huayueyang Biotechnology Co., Ltd.). The integrity of the RNA was identified by gel electrophoresis, and the purity and concentration of the RNA were determined by 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, the genus Anoectochilus 'Jinkang No. 1' was obtained. ArWRKY6 The full-length gene was obtained by reverse transcription (HiScript III 1st Strand cDNA Synthesis Kit) using the extracted RNA to obtain cDNA. Using the first-strand cDNA as a template, PCR amplification was performed using primers ArWRKY6-F (5'-ATGGCGGACAAGGAGGCGAAAACCG-3') and ArWRKY6-R (5'-CTAAAGGGCTTGTGCAGACTCTGGC-3'). A 1545 bp fragment was amplified and ligated into the pMD18-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 ArWRKY6 The ORF reading frame of the gene was amplified and the full-length coding sequence (SEQ ID NO. 1) of 1545 bp was obtained. 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 Arabidopsis AtWRKY 32 (AT4G30935.1) protein, and it was preliminarily considered to be a ArWRKY6 Gene.

[0035] WRKY , Anoectochilus roxburghii 'Jinkang No. 1' Example 2 Gene sequence information and homology analysis

[0036] An anaphalis 'Jinkang No.1' ArWRKY6 The full-length open reading frame sequence of the gene is 1545 bp, and the detailed sequence is shown in SEQ ID NO. 1. According to the open reading frame sequence, the amino acid sequence of the transcription factor ArWRKY6 protein of Anaphalis 'Jinkang No. 1' is deduced, which contains 514 amino acids, the molecular weight is 55.4 kDa, and the isoelectric point (pi) is 6.29. The detailed sequence is shown in SEQ ID NO. 2.

[0037] An anaphalis 'Jinkang No. 1' ArWRKY6 The open reading frame sequence of the gene and the amino acid sequence of the encoded protein are subjected to nucleotide and protein homology search by using BLAST program in NCBI, and the results show that it has very high similarity with WRKY of Arabidopsis thaliana and Dendrobium officinale, as shown in SEQ ID NO. 3. ArWRKY6 The phylogenetic tree analysis shows that the transcription factor ArWRKY6 of Anaphalis 'Jinkang No. 1' has high homology with WRKY32 protein of Arabidopsis thaliana, as shown in SEQ ID NO. 4. Figure 1

[0038] Figure 1 Subcellular localization analysis of Anaphalis transcription factor ArWRKY6 in tobacco leaves

[0039] Specific primers ArWRKY6-F (5'-TCG GT ACC CGG GGA TCC ATG GCG GAC AAG GAG GCG-3') and ArWRKY6-R (5'-TGC TCA CAT GTC GAC AAG GGC TTG TGC AGA CTC TGG-3') are respectively designed from the start codon and the stop codon, and the restriction enzyme cutting sites of Example 3 I and BamH I are respectively introduced at both sides of the full-length sequence of the gene. The plasmid with the target fragment with enzyme cutting sites is subjected to double enzyme cutting of Spe I and BamH I, the pHB vector after enzyme cutting is recovered and ligated with the fragment, and the homologous recombination is carried out by using recombinant enzyme Exnase II at 37℃ for 30 min to construct the recombinant expression vector pHB- Spe YFP. ArWRKY6 The correct recombinant expression vector is transformed into Agrobacterium EHA105.

[0040] The identified EHA105 strain is inoculated into 5 mL LB (containing Kan 50 mg / L), and cultured at 28℃ and 180 rpm until the OD 600 is about 0.6; 1 mL of the bacterial solution is added into 25 mL of LB liquid medium, and cultured at 28℃ until the OD 600 ​about 0.6; 10 mL of the bacterial solution was centrifuged at 4500 rpm for 15 min; the bacterial body was suspended in MS liquid medium to OD 600 about 0.5, and AS and MES were added, and the solution was placed at room temperature for more than 3 h; tobacco leaves were injected, and the solution was cultured in the dark for 48 h, and was observed under a laser confocal microscope at an excitation wavelength of 514 nm, as shown in ArWRKY6- .

[0041] Figure 2 Chloranthus Example 4 Gene Expression Changes in Different Tissues of Chloranthus

[0042] 1. Material Obtaining: 0.1 g of root, stem, leaf, young flower bud, mature flower bud and flower of Chloranthus was taken. The samples were wrapped with aluminum platinum paper and placed in liquid nitrogen, and then transferred to a -80℃ ultra-low temperature freezer for storage.

[0043] 2. RNA extraction, RNA integrity, purity and concentration determination, and cDNA obtaining refer to Example 1.

[0044] 3. Specific primers were designed for real-time fluorescent quantitative PCR analysis of the expression of the gene in each tissue. According to the obtained Chloranthus ArWRKY6 gene sequence, specific primers for real-time fluorescent quantitative PCR gene quantitative analysis were designed, primer q ArWRKY6 -F (5'-GAACCATCGGGAGGAGGTGA-3'), primer q ArWRKY6 -R (5'-GGGCGGAATCAACAACCTCA-3'), and the primer for the reference gene actin was actin ArWRKY6 F (5'-GCTAGTGGCCGTACAACTGG-3'), and actin-R (5'-GCCAGCAAGGTCCAATCGAA-3'). -

[0045] 4. Standard curve of the target gene and the reference gene: the standard cDNA solution was gradiently diluted with ddH2O, and then the diluted cDNA was used as a template for real-time fluorescent quantitative PCR amplification with specific primers of the target gene and the reference gene, and a melting curve and a standard curve were drawn; the melting curve was analyzed to determine whether the melting curve of the target gene and the reference gene obtained a single peak, so as to determine whether the primers could obtain a single PCR amplification product; and the appropriate dilution multiple of the template cDNA was determined through the standard curve.

[0046] ​5. Real-time fluorescence quantitative analysis of target genes in the sample to be tested: using the synthesized cDNA first strand as a template, the specific primers of the target gene and the reference gene are used for amplification and fluorescence quantitative analysis. Bio-Rad CFX real-time fluorescence quantitative instrument is used for real-time fluorescence quantitative PCR reaction. The reaction system is 20 μL, and the reaction program is as follows: 95°C pre-denaturation for 30 s, 95°C denaturation for 5 s, 60°C annealing for 30 s, and 40 cycles.

[0047] 6. The relative quantitative analysis is performed by using 2 -△△Ct The results show that the expression level of Anubis in the tissues of Anubias barteri at different development stages is the highest at the flower bud stage in the juvenile stage, as shown in the table. ArWRKY6 ArWRKY6

[0048] Figure 3 , pCAMBIA1304- Example 5 Recombinant expression vector construction and ArWRKY6 Gene transformation of Arabidopsis thaliana

[0049] 1. Construction of pCAMBIA1304- ArWRKY6 Recombinant expression vector

[0050] The introduced enzyme cutting site is referred to in Example 3. The plasmid with the enzyme cutting site target fragment is double enzyme cut with pCAMBIA1304 binary transformation vector, and the cut vector is recovered and connected by homologous recombination to obtain the recombinant expression vector pCAMBIA1304- ArWRKY6 I and BamH I, and then transformed into Agrobacterium EHA105. Spe

[0051] 2. Transformation of Arabidopsis thaliana

[0052] Flower infection method for transforming Arabidopsis thaliana: the specific operation steps of the flower infection method for infecting Arabidopsis thaliana are as follows.

[0053] ① Wild-type Arabidopsis thaliana grows to the middle of the flowering period, and the best time for infection is when the flowers and fruit pods have been cut off the day before infection, and the remaining flower buds are watered and fertilized for infection.

[0054] ② 100 μL of positive Agrobacterium bacterial solution is taken in 25 mL of LB liquid medium containing Kana (50 mg / L) and Rif (25 mg / L) for expansion culture, and constant temperature shaking bed (28°C, 220 rpm) is used for vibration culture until the OD 600 value of the bacterial solution is 0.8-1.0.

[0055] ③ The OD 600 ​​​When the OD value is 0.8-1.0, centrifugal collection of the bacterial bodies is started, 6000 rpm, centrifugal collection for 10 min, and the supernatant is discarded.

[0056] IV. Resuspension is prepared with the MS+1 mM MES+2 mM MgCl2+200 μM AS formula, and the resuspension is prepared on site. The bacterial bodies are resuspended with the resuspension, and the OD is adjusted. 600 The OD value is adjusted to 0.8-1.0. 600 After the adjustment of the OD value is completed, Tween-20 is added to make the final concentration 0.02%, and the infection solution is obtained after shaking.

[0057] V. The whole inflorescence of each Arabidopsis is soaked in the infection solution for 10 min.

[0058] VI. After the infection is completed, the Arabidopsis is placed in a dark room for dark culture for 1 d to maintain humidity, and after the dark culture is completed, the Arabidopsis is placed in a greenhouse at 22°C, 65% humidity, 16 h light, and 8 h darkness for normal culture.

[0059] VII. After one week, the operation is repeated to perform the infection again to improve the transformation success rate.

[0060] VIII. After the second infection is completed, the Arabidopsis is normally cultured, and the conventional cultivation method is used for management until the seeds are harvested.

[0061] 3. Screening of transgenic positive lines

[0062] Genomic DNA and RNA of Arabidopsis are extracted to identify the positive plants.

[0063] ArWRKY6 Overexpression Example 6 Phenotype analysis of Arabidopsis

[0064] The selected objects for the phenotype observation and determination are the positive homozygous T3 generation plants and seeds (OE), and the Arabidopsis into which the pCAMBIA1304 empty vector is introduced is used as a negative control (CK). The indexes for the phenotype observation and determination are the plant growth cycle, plant height, seed length and width, and seed hundred-seed weight.

[0065] The plant growth cycle observation method is that three Arabidopsis plants are randomly selected from the transgenic plants on the 35th day after the sowing of the Arabidopsis, the growth cycle difference with the control plants is observed, and the photographs are recorded. The plant height determination method is that three Arabidopsis plants are randomly selected from the control plants and the transgenic plants on the 35th day after the sowing of the Arabidopsis, and the photographs are recorded. As shown in ArWRKY6 A. The rosette leaf number observation method is that the above-mentioned photographed Arabidopsis plants are selected as the plants for the observation of the rosette leaves, the whole Arabidopsis plants are pulled out by the roots, the roots are cut off, the rosette leaves are cut off one by one, and the photographs are taken and counted. As shown in Figure 4 B, Figure 4 C.

[0066] Pistil and stamen length measurement method: randomly select control plant flowers and transgenic plant flowers, dissect them on a slide, place them under an optical microscope, use a natural light source to take pictures, and record the average length of the pistil and stamen using a scale, repeat 10 times, as shown in Figure 4 A, Figure 5 D.

[0067] Seed length and width measurement method: select several control plant seeds and transgenic plant seeds, randomly spread them on a slide, place them under an optical microscope, use a natural light source to take pictures, and randomly select ten seeds from the field of view to calculate their average length and width using a scale, as shown in Figure 5 C. Seed hundred-grain weight measurement method: take out one hundred seeds from control plant seeds and transgenic plant seeds, weigh them on an analytical balance, as shown in Figure 5 Figure 5 B.

[0068] The specific embodiments of the present application are described above. It should be understood that the present application is not limited to the specific embodiments described above, and various modifications or changes can be made by those skilled in the art within the scope of the claims, which do not affect the essential content of the present application.

Claims

1. A method for increasing the number of leaves and 100-grain weight of seeds of Anoectochilus roxburghii 'Jinkang 1' by encoding a gene encoding the WRKY transcription factor ArWRKY6, wherein the encoding gene has the nucleotide sequence shown in SEQ ID NO.

1.

2. The use of the gene encoding the WRKY transcription factor ArWRKY6 of Anoectochilus roxburghii 'Jinkang No. 1' according to claim 1 in increasing the number of leaves 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 ArWRKY6, transforming the vector into a plant host, and cultivating and screening to obtain transgenic plants.

Images