CN116041461A - Dendrobium KNOX transcription factor gene DoSTM and application thereof - Google Patents

Abstract

The invention discloses a dendrobe KNOX transcription factor gene DoSTM and application thereof. The transcription factor DoSTM or the coding gene DoSTM thereof is applied to regulating and controlling the proliferation of plant seeds in dendrobe protocorms, and the amino acid sequence of the transcription factor DoSTM is shown as SEQ ID NO. 2. The coding gene KNOX transcription factor gene DoSTM which belongs to the dendrobium nobile KNOX transcription factor DoSTM is cloned from the dendrobium nobile protocorm and is taken as a positive control factor to participate in the regulation and control of proliferation of the dendrobium nobile protocorm, and the transgenic and functional identification prove that the proliferation capacity of the transgenic dendrobium nobile is obviously improved. Therefore, the KNOX transcription factor DoSTM or the coding gene thereof has important theoretical significance and application value in the fields of orchid plant micropropagation, genetic transformation, cell engineering and the like.

Description

Dendrobium KNOX transcription factor gene DoSTM and application thereof

Technical field:

the invention belongs to the field of plants, and particularly relates to a KNOX transcription factor or a coding gene thereof and application thereof in regulating and controlling development of orchid protocorms.

The background technology is as follows:

orchid is the second major family of plants and has important commercial, medicinal and ornamental values. Plants of the genera butterfly orchid, paphiopedilum and cattleya have significant ornamental value (Cardoso et al 2020), whereas dendrobium etc. are used as herbal medicines, with important medicinal value for the treatment of some chronic diseases such as diabetes and tumours etc. (Zhang et al 2017). Dendrobium nobile (Dendrobium nobile Lindl.) recorded in the pharmacopoeia of the people's republic of China, the dendrobium nobile (D.huoshanense) Dendrobium chrysotoxum Lindl), dendrobium frigidum (D.fimbriatum Hook) and Dendrobium officinale (D.officinium Kimum et Migo) are included. At present, the medicine and food homologous test points of the dendrobium candidum are developed in the most provincial markets in China, and the rapid development of the dendrobium candidum industry is further promoted. At present, products developed by taking dendrobium as raw materials include medicines, health-care products and cosmetics, and the yield value exceeds one hundred billion RMB.

The seed of orchid contains no endosperm and has very simple structure. Upon germination, the embryo develops into a spherical nodule, called a protocorm, and forms a shoot apical meristem at the tip. The protocorm-like shape is similar to that of protocorm, and is obtained by inducing protocorm or other explants under the condition of isolated culture, and has stronger proliferation capability, can also differentiate into seedlings, and can maintain the excellent characteristics of the parent body. The protocorm-like body has wide application prospect in the research fields of plant development biology, plant efficient regeneration, genetic transformation, secondary metabolic substance production, cell engineering and the like. However, the molecular mechanism by which orchids establish this unique, unparalleled developmental process remains unclear. The cytological features and cell wall markers of PLB are similar to zygotic embryo development and are considered to be somatic embryos of the orchid family (Lee et al, 2013). However, studies have shown that some marker genes such as SERK2, BBM2, CLV2, WOX formed with somatic embryos do not exhibit high expression at the PLBs stage of paphiopedilum (Guo et al, 2021). The molecular mechanisms of the development of the protocorm, PLB and zygotic embryos of butterfly orchid were found to be different, PLB regeneration not following embryogenesis procedures (Fang et al, 2016). This suggests that the PLB developmental machinery of orchids is distinct from plant embryogenesis.

The invention comprises the following steps:

the invention aims to provide a transcription factor DoSTM or a coding gene thereof and application thereof in regulating proliferation of orchid protocorms.

The coding gene KNOX transcription factor gene DoSTM of the dendrobium candidum KNOX transcription factor DoSTM is cloned from dendrobium candidum protocorms, and takes the KNOX transcription factor DoSTM as a positive control factor to participate in development of the dendrobium candidum protocorms, and the transgenic and functional identification prove that the KNOX transcription factor DoSTM is over-expressed, so that the formation of the transgenic dendrobium candidum protocorms is promoted.

A first object of the present invention is to provide a transcription factor DoSTM, the amino acid sequence of which is shown in SEQ ID NO. 2.

A second object of the present invention is to provide a coding gene DoSTM which codes for a transcription factor DoSTM.

The nucleotide sequence of the coding gene DoSTM is shown as SEQ ID NO. 1.

The third purpose of the invention is to provide an application of a transcription factor DoSTM or a coding gene DoSTM thereof in regulating proliferation of dendrobe protocorms, wherein the amino acid sequence of the transcription factor DoSTM is shown as SEQ ID NO. 2.

Preferably, the application of the coding gene of the transcription factor DoSTM in the dendrobium to the improvement of the proliferation of the transgenic protocorm is provided.

The nucleotide sequence of the coding gene of the transcription factor DoSTM is shown as SEQ ID NO. 1.

The dendrobium is dendrobium candidum.

The coding gene KNOX transcription factor gene DoSTM which belongs to the KNOX transcription factor DoSTM of the dendrobium candidum is cloned from the dendrobium candidum protocorm and is taken as a positive control factor to participate in proliferation of the dendrobium candidum protocorm, and the transgenic and functional identification prove that the proliferation capacity of the transgenic protocorm is improved. Therefore, the KNOX transcription factor DoSTM or the coding gene thereof has important theoretical guiding significance and application value in the fields of the microsomal rapid propagation, genetic transformation, cell engineering and the like of orchid plants.

Description of the drawings:

FIG. 1 shows the full length of DoSTM, wherein 1 is DL2000 DNA Marker,2 is negative control without cDNA template, and 3 is the fragment of DoSTM.

FIG. 2 is an analysis of the expression pattern of DoSTM at different developmental stages, 1 being protocorm-like; 2 is a bud induced from a protocorm-like body.

FIG. 3 is a DoSTM subcellular localization analysis.

FIG. 4 shows the expression level of the DoSTM gene in the over-expressed dendrobium strain by fluorescence quantitative PCR analysis; wherein WT is wild-type dendrobe, and DoSTM-OX is expressed as a screened DoSTM over-expressed transgenic dendrobe.

FIG. 5 shows the proliferation of wild-type and DoSTM overexpressing dendrobe protocorms in a medium without plant growth regulator; wherein WT is wild dendrobium, and DoSTM-OX is expressed as screened DoSTM over-expressed transgenic dendrobium.

FIG. 6 is a vector map of pSAT6-EYFP-N1 vector.

The specific embodiment is as follows:

the following examples are further illustrative of the invention and are not intended to be limiting thereof.

Example 1

The following examples, in which no particular experimental procedure is noted, may be carried out in accordance with conventional methods. Conditions as described in J.Sam Brooks et al, guidelines for molecular cloning experiments, F.Osbert et al, guidelines for precision-compiled molecular biology experiments, or according to the instructions of the manufacturer of the product used.

The dendrobe used in the examples is stored in a tissue culture room of a plant garden in south China academy of sciences. Extracting RNA by taking protocorm-like materials. Reverse transcriptase M-MLV was purchased from Promega corporation under the designation: m1701; DNase I (RNase Free) was purchased from baori doctor materials technology (beijing) limited (TaKaRa china) under the product number D2215; the usual PCR reaction buffer was 2X TSINGKE Master Mix (blue) and was purchased from the department of Optimago under the trade designation TSE004-5ML; the super-expression vector is constructed by adopting high-fidelity enzyme for amplification, wherein the high-fidelity enzyme is KOD-401, and the product number is as follows, and is purchased from Toyobo (Shanghai) biotechnology Co., ltd: KOD-401; pMD18-T vector and Nco I enzyme were purchased from Takara doctor materials technology (Beijing) Inc. (TaKaRa China) with the product numbers D101A and 1160BH, respectively; MS and LB medium are common medium in the art, and the formula is referred to J.Sam Brooks et al, guidance for molecular cloning experiments. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

Extracting dendrobe protocorm RNA by an SDS method, and carrying out genome DNA removal treatment on the extracted RNA by DNase I, wherein the specific operation is carried out according to the instruction of use. Performing agarose gel electrophoresis on the obtained purified RNA, and detecting the integrity of the RNA; then detecting the concentration and purity of RNA in an ultra-trace nucleic acid protein determinator NanoDrop2000, and placing the detected qualified RNA in a refrigerator at-80 ℃ for standby. cDNA was obtained by reverse transcription using Promega reverse transcriptase M-MLV. The CDS nucleotide sequence of the DoSTM is obtained by searching in a CDS splicing file of the whole genome of the dendrobium candidum, a forward primer (5'-ATGGAAAACTGGAGCGGGGG-3') and a reverse primer (5'-TCAAAGTAGAGGCGTGCAGTC-3') are designed according to sequence information, cDNA is used as a template, and conventional PCR amplification is carried out, wherein the PCR reaction conditions are as follows: 94 ℃ for 4min;94℃30sec,55℃30sec,72℃1min,40 cycles; and at 72℃for 5min. After agarose gel electrophoresis, the fragment of interest was recovered (FIG. 1), ligated to the ligation pMD18-T vector, ligation reaction: PCR product 4.5. Mu.L, pMD18-T vector 0.5. Mu.L, solution I5. Mu.L, total 10. Mu.L volume, 16℃overnight. All ligation products were transferred to 100. Mu.L of E.coli DH 5. Alpha. Competent cells (TaKaRa, D9057A), heat-shocked on ice for 30min, at 42℃for 45sec, left on ice for 5min, and then added with 800mL of LB liquid medium, resuscitated for 45min, plated on LB plates containing 100mg/L Amp, and incubated at 37℃until colonies developed (approximately 16 h). Single colony is picked up and amplified and cultured in LB liquid medium containing 100mg/L Amp, PCR amplification is carried out, the specific operation is consistent with CDS amplification method, and detected positive recombinants deliver large genes for sequencing. The amplified target fragment has a nucleotide sequence shown as SEQ ID NO.1, an open reading frame is formed by 1 st to 909 th bases and is 909bp long, the open reading frame is named as a dendrobium candidum KNOX transcription factor gene DoSTM, the code of the open reading frame is 302 amino acids, the amino acid sequence of the open reading frame is shown as SEQ ID NO.2, and the open reading frame is named as the dendrobium candidum KNOX transcription factor gene DoSTM.

Example 2: analysis of the expression patterns of DoSTM at different developmental stages

Dendrobium candidum seeds germinate on a solid culture medium (pH value is 5.4) culture medium of 1/2MS+0.5g/L NAA+20g/L sucrose+6 g/L agar powder, and then are induced to form protocorm-like stems. The protocorm-like bodies obtained were transferred to a seedling growth medium (1/2MS+0.1 g/L NAA+1 mg/L6-BA+20 g/L sucrose+6 g/L agar powder) as a solid medium (pH 5.4) at 25.+ -. 1 ℃ C., 40. Mu. Mol m ^-2 s ^-1 12-h light/12-h dark, 60% relative humidity in a culture chamber. Collecting protocorm-like and buds induced from the protocorm-like. Excess liquid was aspirated with filter paper and the medium was removed, wrapped with aluminum foil paper, snap frozen with liquid nitrogen for 5min, RNA extraction with SDS modified version and genomic DNA removal with DNase I. Performing agarose gel electrophoresis on the obtained purified RNA, and detecting the integrity of the RNA; then detecting the concentration and purity of RNA in an ultra-trace nucleic acid protein determinator NanoDrop2000, and placing the detected qualified RNA in a refrigerator at-80 ℃ for standby. cDNA was obtained by reverse transcription using Promega reverse transcriptase M-MLV. The dendrobium candidum actin gene (NCBI accession number: JX 294908) is used as an internal reference, and the internal reference qPCR upstream primer is as follows: 5'-GCGGACGTTGATGATATTCAGCCTC-3', the downstream primer is: 5'-GAATGTGCTGAGGGAGGCAAGGATAG-3'. The qPCR primers of DoSTM are: 5'-CGGAGGAGATGCAGTTTGTT-3' and 5'-GCAGTCTATGGCGAATGGATTA-3'. Fluorescent quantitative PCR was performed using the reagent iTaq Universl SYBR Green supermix available from BIO-RAD, USA, and PCR plates and membranes were prepared using the product of BIO-RAD, USA. The reaction system and the reaction procedure are carried out according to the operation steps of the specification. 3-4 technical replicates were set for each sample, and each reaction tube reaction system was as follows:

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the reaction was performed on an ABI 7500Real-time PCR apparatus with a reaction program of 95℃for 2min of pre-denaturation; denaturation at 95℃for 15sec, annealing at 60℃and extension for 1min,40 cycles. The resulting data were processed using ABI 7500Real-time PCR system software, using 2 ^-ΔΔCT The method (Livak and Schmittgen 2001) calculates the relative expression level. DoSTM is strongly expressed in dendrobe protocorms (fig. 2).

Example 3: subcellular localization analysis of DoSTM

1. Construction of pSAT6-EYFP-N1-DoSTM vector

The cDNA synthesized by reverse transcription of dendrobe protocorm is used as a template, two adaptor primers are designed according to the sequence on a pSAT6-EYFP-N1 vector (which can be purchased from http:// www.biovector.net/product/428881.Html, the vector map is shown in figure 6), and an upstream primer adaptor: 5'-AGCTCAAGCTTCGAATTC-3', downstream primer adaptors: 5'-CCGTCGACTGCAGAATTC-3'. Underlined indicates EcoRI cleavage sites. Primers (excluding stop codons) are designed near the start codon and the stop codon of the DoSTM, and the primers are designed according to the full length of the KNOX transcription factor gene DoSTM, so that the triplet codons of the gene are ensured not to shift. The cDNA of the protocorm of dendrobium candidum is used as a template, the full length of the KNOX transcription factor gene DoSTM is obtained through high-fidelity amplification, and the amplification primers are STM-YFPF (5'-AGCTCAAGCTTCGAATTCATGGAAAACTGGAGCGGGGG-3') and STM-YFPR (5'-CCGTCGACTGCAGAATTCAAGTAGAGGCGTGCAGTCTA-3'). The amplified product is subjected to agarose gel electrophoresis, and the target DNA fragment is recovered. The pSAT6-EYFP-N1 plasmid vector was digested singly with EcoRI to give a linear plasmid vector containing cohesive ends, and the procedure was carried out according to the instruction. The linear plasmid vector was recovered and stored at-20℃until use. By the company of the Optimus of Optimim the soso homologous recombination kit is constructed, immediately placing on ice after reacting for 15min at 50 ℃, the vector with the target fragment is transformed into DH5 alpha and smeared on an LB solid culture plate containing 100mg/L Amp, and the culture is inverted and carried out for 16 hours at 37 ℃. After colonies were grown, the colonies were picked and cultured on a liquid LB medium containing 100mg/L Kan at 37℃and 220rpm for 16 hours, and then subjected to bacterial liquid PCR to verify positive recombinants. The positive clone is sent to Huada gene sequencing, bacterial liquid with correct sequencing is inoculated on a liquid LB culture medium containing 100mg/L Amp, and cultured for 16 hours at 37 ℃ and 220rpm, and plasmids are extracted and stored in a refrigerator at-20 ℃ for standby. Both the preparation of arabidopsis leaf protoplasts and the PEG-method transformation of the protoplasts were referred to the methods of epigenetic study group preservation. Fluorescence was observed on a confocal fluorescence microscope and photographed. The results showed that the YFP fluorescence overlapped with that of the nuclear localization plasmid NSL-mCherry, indicating that DoSTM is a nuclear localization protein (FIG. 3).

Example 4: acquisition and identification of DoSTM over-expression transgenic dendrobium nobile

1. Construction of KNOX transcription factor gene DoSTM overexpression vector

Two adapter primers, the upstream primer adapter, were designed based on the sequence on the pOx vector (provided by the national center for agricultural resource protection and utilization, national center laboratory Liu Yaoguang, team of academy of sciences, see, e.g., shuoshi Li Chao, cloning and functional analysis of rice SDG711 and SDG 723): 5'-TACCGGCGCGCCAAGCTT-3', downstream primer adaptors: 5'-ACGCGTACTAGTAAGCTT-3'. Primers (not including a stop codon) are designed near the start codon and the stop codon of the DoSTM, the primers are designed according to the full length of the DoSTM, cDNA of the protocorm of the dendrobium candidum is used as a template, the full length of the DoSTM is obtained through high-fidelity amplification, and the amplification primers are STMOxF (5'-GGATCCACGCGTAAGCTTATGGAAAACTGGAGCGGGGG-3') and STMOxR (5'-CTGCAGGCATGCAAGCTTAAGTAGAGGCGTGCAGTCTA-3'). The amplified product is subjected to agarose gel electrophoresis, and the target DNA fragment is recovered. The pOx plasmid vector was subjected to single Hand II cleavage to give a linear plasmid vector containing cohesive ends, which was carried out according to the instructions. The linear plasmid vector was recovered and stored at-20℃until use. By the company of the Optimus of Optimim the soso homologous recombination kit is constructed, immediately placing on ice after reacting for 15min at 50 ℃, the vector with the target fragment was transformed into DH 5. Alpha. And spread on LB solid medium plate containing 50mg/L Kan, and cultured upside down at 37℃for 16 hours. After colonies were grown, single colonies were picked up and cultured on a liquid LB medium containing 50mg/L Kan at 37℃and 220rpm for 14 hours, followed by bacterial liquid PCR to verify positive recombinants. The positive clone is sent to Huada gene sequencing, the bacterial liquid with correct sequencing is inoculated on a liquid LB culture medium containing 50mg/L Kan, cultured for 14h at 37 ℃ and 220rpm, and plasmids are extracted and stored in a refrigerator at-20 ℃ for standby. pOx the vector is a Ubi promoter (Ubi promoter) containing tobacco ubiquitin, the KNOX transcription factor gene DoSTM (shown as 1 st to 906 th bases of SEQ ID NO. 1) is connected behind the promoter to construct a plant expression vector of the KNOX transcription factor gene DoSTM, and the recombinant expression vector is named pOx-DoSTM.

2. Transformation of recombinant plasmid pOx-DoSTM into Agrobacterium EHA105

The recombinant plasmid was transferred into agrobacterium EHA105 using a freeze-thawing method. 1 μl (1 μg/μl concentration) of recombinant expression vector pOx-DoSTM was mixed with competent cells of Agrobacterium EHA105, placed on ice for 30min, quick frozen with liquid nitrogen for 1min, and rapidly transferred to 37℃for 5min. Mu.l of LB medium without antibiotics was added and incubated at 28℃for 3h at 100 rpm. The culture was spread on a solid plate containing 50mg/L Kan LB, and the plate was cultured at 28℃in an inverted state until colonies were grown (about 48 hours). Monoclonal was picked for colony PCR identification, and colony PCR identification primers were STMOxF (5'-GGATCCACGCGTAAGCTTATGGAAAACTGGAGCGGGGG-3') and STMOxR (5'-CTGCAGGCATGCAAGCTTAAGTAGAGGCGTGCAGTCTA-3'). The colony capable of amplifying the target band is positive clone, and is cultured on a liquid culture medium containing 50mg/L Kan LB until OD ₆₀₀ About 1, adding a proper amount of sterile glycerol (the final concentration is 25-30%), quick-freezing with liquid nitrogen for 2min, and preserving at-80 ℃ for standby. The agrobacterium containing the recombinant plasmid pOx-DoSTM is obtained.

3. Agrobacterium-mediated genetic transformation of dendrobe protocorms

Taking out the preserved strain from refrigerator at-80deg.C, streaking on solid plate containing 50mg/L Kan LB to activate Agrobacterium containing recombinant plasmid pOx-DoSTM, picking single colony in 100ml LB liquid medium (containing 50mg/L Kan), shake culturing at 28deg.C and 180rpm overnight (about 16 h), and reaching OD ₆₀₀ =0.8-1.0. And (5) centrifuging at 5000rpm at room temperature for 10min to collect bacterial liquid. Agrobacterium was resuspended in 100ml of infiltration medium (1/2MS+0.5 g/L MES+5% sucrose, pH 5.7). The dendrobe protocorms were cut into 5mm diameter materials and pre-cultured for 2 days. Soaking the pre-cultured dendrobium protocorm in agrobacterium heavy suspension for 3 timesAfter 0min, 2 days of dark co-culture, the culture was performed under light conditions. Resistant transformants were then selected on medium containing 30mg/mL hygromycin B.

4. Identification of transgenic plants

The hygromycin resistant transgenic protocorms were obtained by 6 (2 months each) screening. Extracting total RNA of wild-type protocorm and hygromycin-resistant transgenic protocorm, and reversely transcribing into cDNA, using ddH ₂ O ₂ The same concentration is adjusted, dendrobium is used as an internal reference, and the primers are ActinF (5'-TCCCAAGGCAAACAGAGAAA-3') and ActinR (5'-GGCCACTAGCATATAGGGAAAG-3'). The fluorescence quantitative PCR primers of the DoSTM are DoSTMF (5'-CGGAGGAGATGCAGTTTGTT-3') and DoSTMR (5'-GCAGTCTATGGCGAATGGATTA-3'), and as shown in FIG. 4, the expression level of the DoSTM in the transgenic lines DoSTM-OX1-5 and DoSTM-OX8 can be obviously higher than that of the wild type, which indicates that the DoSTM has realized over-expression in the transgenic protocorm.

Example 5: effect of overexpressed DoSTM on proliferation of transgenic protocorms

The transgenic strain and the wild type protocorm are cut into protocorm explants with the diameter of 2mm, and the protocorm explants are weighed in a sterile environment to obtain the weight when the protocorm explants are inoculated into a solid culture medium (pH value is 5.4) of 1/2MS+20g/L sucrose+6g/L agar powder, and are cultured under the conditions of 25+/-2 ℃ and 12-h illumination/12-h darkness. After 40 days of culture, the cultured protocorm-like bodies were photographed and weighed. Protocorm-like proliferation rate = (fresh weight of protocorm-like before culture)/(fresh weight of protocorm-like before culture). The DoSTM overexpressing lines were all significantly higher in proliferation than the wild-type, wild-type protocorms, which readily differentiated into buds, and proliferated less in culture medium without plant growth regulators (fig. 5).

SEQ ID NO.1

ATGGAAAACTGGAGCGGGGGAAATAACCCTCTAATGATCATGCCTCTCCTCTCTTCCACC

GCCGCTGGGGCCAACAACCCTCTCCTGCTCCTCCCGCAACCACCACCTCCGCCGCCGGC

CGACGCCTACGACACCACCCTCATCAAAGCCAAAATCATTTCTCACCCTCAATACCCCCG

TCTCCTCTCCGCCTACGTTAACTTACACAAAGTGGGCGCTCCACCGGAGGTAGTTGCCA

GACTTGAAGAAGCCTGCGCTTCTTCCTTAATATCCGGCGGCGGCGGGGGCGGCGGCGGA

GGAGAAGACCCAGCGTTAGATCAGTTCATGGAAGCTTATTGTGAGATGTTGACTAAGTAT

GAGCAAGAGCTTTCAAAGCCTTTTAAAGAAGCTATGTTGTTTTTATCTCGCATTGATTCG

CAGTTTAAGTCGCTCTCGCTTTCTTTGCCTCCTGCTCCTTCTCAAGTGTGTGCTGACCTA

GAGAAAAACGGTTCGTCTGAGGACGACATCGACTTGTGTGATAACTATGTCGACCCTGA

AGCCGGCGACCGTGAACTCACAGGCCAGCTCCTTCGCAAATACAGTGGTTACTTGGGA

AGTCTGAAGCAGGAGTTCCTCAAAAAGAGGAAGAAAGGAAAGCTCCCCAAGGAAGCT

AGGCAACAACTGCTTGACTGGTGGACTCGCCACTACAAATGGCCATATCCTTCAGAATC

TCAGAAGCTAGCACTTGCTGAGTCCACAGGCCTTGATCAAAAACAGATAAACAACTGGT

TCATCAACCAGCGAAAGCGTCACTGGAAGCCATCGGAGGAGATGCAGTTTGTTGTAATG

GATGCAGCTCATCCACATTATTTCATGGATGGCGGCCTCGCCGGTAATCCATTCGCCATAGACTGCACGCCTCTACTTTGA。

SEQ ID NO.2

MENWSGGNNPLMIMPLLSSTAAGANNPLLLLPQPPPPPPADAYDTTLIKAKIISHPQYPRLLS

AYVNLHKVGAPPEVVARLEEACASSLISGGGGGGGGGEDPALDQFMEAYCEMLTKYEQEL

SKPFKEAMLFLSRIDSQFKSLSLSLPPAPSQVCADLEKNGSSEDDIDLCDNYVDPEAGDRELT

GQLLRKYSGYLGSLKQEFLKKRKKGKLPKEARQQLLDWWTRHYKWPYPSESQKLALAESTGLDQKQINNWFINQRKRHWKPSEEMQFVVMDAAHPHYFMDGGLAGNPFAIDCTPLL。

Claims (7)

1. The transcription factor DoSTM is characterized in that the amino acid sequence is shown as SEQ ID NO. 2.

2. A coding gene DoSTM encoding the transcription factor DoSTM of claim 1.

3. The coding gene DoSTM according to claim 2, wherein the coding gene DoSTM has a nucleotide sequence as shown in SEQ ID No. 1.

4. The use of the transcription factor DoSTM or the coding gene DoSTM thereof as claimed in claim 1 for regulating proliferation of dendrobe protocorms.

5. The method of claim 4, wherein the gene encoding the transcription factor DoSTM is overexpressed in Dendrobium nobile for increasing proliferation of the transgenic protocorm.

6. The use according to claim 5, wherein the nucleotide sequence of the gene encoding the transcription factor DoSTM is shown in SEQ ID No. 1.

7. The use according to claim 4, 5 or 6, wherein said dendrobe is dendrobium candidum.

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