Dendrobium officinale kimura et migo flavanonol-4-reductase gene regulated and controlled by illumination intensity

Abstract

The invention discloses a dendrobium officinale kimura et migo flavanonol-4-reductase gene regulated and controlled by illumination intensity, and relates to the technical field of gene engineering. The gene has a nucleotide sequence as shown in SEQ ID NO. 1, and an amino acid sequence as shown in SEQ ID NO. 2. The sequence is a novel dendrobium candidum flavanonol-4-reductase gene, is a flavanonol-4-reductase gene which is found in dendrobium candidum and is regulated and controlled by illumination intensity, and is a basis for researching illumination intensity regulation and control of dendrobium candidum anthocyanin synthesis. The method has important significance in improving the medicinal quality and ornamental quality of the dendrobium officinale, and the expression of a key enzyme gene (DoDFR gene) of the dendrobium officinale can be improved, so that the content of anthocyanin in the dendrobium officinale is improved.

Description

Technical Field

[0001] The invention relates to the field of genetic engineering, and in particular to a Dendrobium officinale dihydroflavonol-4-reductase gene regulated by light intensity. Background Art

[0002] Dendrobium officinale Kimura et Migo is a perennial epiphytic herb of the genus Dendrobium in the family Orchidaceae. It is a national second-level protected plant and a precious Chinese medicinal material in my country. It was included in the national catalog of medicine and food homology in 2019. Dendrobium officinale contains a variety of chemical components such as polysaccharides, alkaloids, flavonoids, phenols, amino acids, trace elements, etc., and has rich pharmacological effects, such as improving immunity, anti-tumor, anti-oxidation, anti-fatigue, anti-aging, anti-radiation, lowering blood sugar, blood pressure, blood lipids, protecting the liver and stomach, etc. Among them, polysaccharides and flavonoid compounds in Dendrobium officinale are considered to be important active substances, and the synthesis of these substances directly affects the medicinal value of Dendrobium officinale. Analyzing the synthesis pathway of secondary metabolites is the premise and basis for conducting relevant synthetic biology research.

[0003] Flavonoids are a class of natural organic compounds widely found in plants, with multiple biological activities and potential medicinal value. Flavonoids have diverse structures, including flavonoids, flavonols, anthocyanins and other subclasses. Anthocyanins are water-soluble pigments that are widely found in the vacuoles of various tissue cells in plants, such as flowers, stems, leaves, and fruits, making them appear in different colors such as red, purple, and blue. The biosynthetic pathway of anthocyanins is an important branch of the metabolic pathway of flavonoid compounds, and it is also one of the most widely studied metabolic pathways of plant secondary metabolites. Dihydroflavonol-4-reductase (DFR) is one of the key enzymes in the anthocyanin biosynthetic pathway. It has substrate specificity and can selectively catalyze dihydrokaempferol, dihydroquercetin and dihydromyricetin to produce colorless pelargonidin, colorless cyanidin and colorless delphinidin, respectively. Studies have found that the differences in plant color phenotypes are related to the different catalytic efficiencies of DFR for different substrates. The cloning and study of the dihydroflavonol-4-reductase (DFR) gene in Dendrobium officinale is the basis for studying the anthocyanin synthesis pathway in Dendrobium officinale, and is of great significance in improving the medicinal and ornamental qualities of Dendrobium officinale. Summary of the invention

[0004] The purpose of the present invention is to provide a Dendrobium officinale dihydroflavonol-4-reductase gene regulated by light intensity, a protein encoded by the gene and a recombinant plasmid containing the gene, so as to provide a basis for the metabolic synthesis of anthocyanins in Dendrobium officinale.

[0005] In order to solve the above technical problems, the technical solution adopted by the present invention is: A Dendrobium officinale dihydroflavonol-4-reductase gene regulated by light intensity, the nucleotide sequence of the dihydroflavonol-4-reductase gene is shown in SEQ ID NO: 1.

[0006] Furthermore, the CDS sequence of the dihydroflavonol-4-reductase gene is obtained by splicing transcriptome sequencing, wherein the expression of the dihydroflavonol-4-reductase gene can be increased by light.

[0007] Furthermore, the amino acid sequence of the dihydroflavonol-4-reductase gene is shown in SEQ ID NO:2.

[0008] Furthermore, the dihydroflavonol-4-reductase gene contains a recombinant plasmid.

[0009] Furthermore, the recombinant plasmid is pMD18-T.

[0010] By adopting the above technical scheme, the beneficial effects of the present invention are as follows: The dihydroflavonol-4-reductase gene is cloned from Dendrobium officinale for the first time. The present invention can be used to genetically engineer the Dendrobium officinale plant, increase the anthocyanin content in the plant through transgenic, or use the gene to identify and screen plant varieties, etc., to obtain plant varieties with high anthocyanin content. Cloning the DFR gene in Dendrobium officinale is the basis for studying the anthocyanin synthesis metabolic pathway of Dendrobium officinale, which can provide an important basis for the selection and breeding of Dendrobium officinale varieties, and also provide a basis for the study of transgenic strains with high anthocyanin content. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 This is a diagram showing the electrophoresis detection results of the CDS sequence of the DFR gene of Dendrobium officinale in the present invention.

[0012] Figure 2 The diagram is a protein tertiary structure diagram predicted by the DFR gene of Dendrobium officinale in the present invention.

[0013] Figure 3 The figure is a schematic diagram of the relationship tree analysis of the DFR gene protein of Dendrobium officinale in the present invention.

[0014] Figure 4 It is a schematic diagram of the expression of the DFR gene of Dendrobium officinale in different varieties of Dendrobium officinale and under different light treatments in the present invention. DETAILED DESCRIPTION

[0015] In order to make the purpose, technical solution and advantages of the embodiment of the present invention clearer, the technical solution of the embodiment of the present invention will be described clearly and completely below. Figure 1-Figure 4A specific embodiment of the present invention will be described.

[0016] A dihydroflavonol-4-reductase gene from Dendrobium officinale that is regulated by light intensity. The dihydroflavonol-4-reductase gene from Dendrobium officinale is abbreviated as DoDFR. Through this specific implementation method, a dihydroflavonol-4-reductase sequence (as shown in SEQ ID NO.1) derived from Dendrobium officinale and its corresponding amino acid sequence (as shown in SEQ ID NO.2) are finally obtained.

[0017] This specific implementation method includes the following steps: 1) Extraction of total RNA from Dendrobium officinale, using the pine tree method (such as the evolutionary tree method) to extract total RNA from Dendrobium officinale leaves; specifically includes the following steps: (1) 100 mg of Dendrobium officinale leaves are quickly and fully ground with liquid nitrogen, placed in a centrifuge tube (such as a 2 mL RNA-free centrifuge tube), and 20 μL of β-mercaptoethanol is added, followed by 1 mL of EB (i.e., ethidium bromide solution), and then the centrifuge tube is inverted to mix and then placed in a 60-70°C (optimal temperature is 65°C) water bath for water bath treatment, the water bath treatment time is controlled within 14-16 minutes (optimal time is 15 minutes), and the tube is inverted every 2 minutes. (2) After the water bath treatment, the centrifuge tube is centrifuged for 10 minutes using a low-temperature centrifuge, the maximum centrifugal force of which is 12000g, and then the supernatant of the centrifuge tube is taken out and placed in a new centrifuge tube. (3) Add 200 μL of chloroform (i.e., chloroform) to a new centrifuge tube and mix it upside down for about 3 minutes. Then use a low-temperature centrifuge to centrifuge the new centrifuge tube for 10 minutes, and then take out the supernatant from the centrifuge tube and put it into a new centrifuge tube. (4) Repeat step (3) several times (such as three to five times) to obtain the final supernatant. (5) Add 1 / 4 of the supernatant volume (relative to the supernatant in the centrifuge tube) of lithium chloride solution (such as 10M pre-cooled LiCl) to the centrifuge tube, mix it upside down, and place it at -20℃ for several hours for precipitation. (6) Use a low-temperature centrifuge to centrifuge the centrifuge tube for 10 minutes. The maximum centrifugal force of the low-temperature centrifuge is 12000g, and its temperature is 4℃. Discard the supernatant from the centrifuge tube, add 1ml of 75% ethanol solution to the precipitate, flick up the precipitate, and then invert it for 1 minute. (7) Centrifuge the centrifuge tube for 10 minutes using the low-temperature centrifuge, discard the supernatant in the centrifuge tube and dry the precipitate for several minutes. (8) After the precipitate is slightly dry, add 50 μl of depc water to dissolve it and let it stand to dissolve, and then obtain the total RNA of Dendrobium officinale. (9) Use a spectrophotometer (such as NanoDropOne micro-spectrophotometer) to measure the A260 value to calculate the concentration of the total RNA of Dendrobium officinale, and read the OD260 / OD280 value to estimate the purity and integrity of the total RNA; use 1.2% agarose gel electrophoresis to accurately determine the RNA quality, and control the voltage at 125V-145V (the optimal voltage is 135V).

[0018] 2) Transcriptome sequencing of Dendrobium officinale. The transcriptome sequencing method is as follows: first, according to the above-mentioned steps for extracting total RNA from Dendrobium officinale, extract total RNA from Dendrobium officinale leaves, and detect the extraction quality of total RNA from Dendrobium officinale to meet the requirements for library construction (RNA concentration>250ng / μL, total amount>20μg, OD260 / OD280 between 1.8 and 2.2, good integrity, RIN>6.5). Secondly, the magnetic bead enrichment method is used, that is, magnetic beads are used to enrich poly(A) mRNA to break it into fragments, and this is used as a template to synthesize the first cDNA chain and the second cDNA chain in sequence, and after purification, elution, end repair, and addition of poly(A), the sequencing adapter is connected; thirdly, a fragment of 200bp to 700bp is selected for PCR amplification to establish a Dendrobium officinale cDNA sequencing library, which is sequenced using IIIumina HiSeq 2000 (i.e., a high-throughput sequencer). This part of the experiment can be entrusted to Beijing Biomarker Biotechnology Co., Ltd. De novo assembly was performed using the short reads assembly software Trinity (v2.4.0). After obtaining the Contig assembly fragments without N, tgicl (v2.1) was used to remove redundancy, remove low-quality and uncertain sequences in the sequence, and retain sequences greater than 200 bp for subsequent analysis. Transdecoder (v2.0.1) (commonly used bioinformatics software) was used to predict the gene structure of the above splicing results and obtain the CDS sequence of the DFR gene.

[0019] 3) Extraction of the first strand of cDNA from Dendrobium officinale. The total RNA of Dendrobium officinale obtained by the extraction of total RNA from Dendrobium officinale is used as a template, and a reverse transcription kit (such as a Yeasen reverse transcription kit) is used to synthesize the first strand of cDNA. Specifically, the following steps are included: (1) Prepare a mixed solution in an RNase free centrifuge tube according to Table 1, gently pipette to mix, and then incubate at 42°C for 2 minutes. Table 1 is as follows: Components Usage <![CDATA[RNase-free H 2 The]]> To 15 μL 5×gDNA digester Mix 3 μL Total RNA 10 pg-500 ng (2) According to Table 2, add 4×HifairTM Ⅲ SuperMixplus to the reaction tube (i.e., centrifuge tube) in the first step, mix it by gently blowing with a pipette, and then reverse transcribe it for 25 minutes (i.e., the temperature and time period are controlled at 25℃ for 5 minutes, 55℃ for 15 minutes, and 85℃ for 5 minutes) to obtain a cDNA solution. Table 2 is as follows: Components Usage Reaction solution of step 1 15 μL 4×HifairTM Ⅲ SuperMix plus 5 μL 4) Cloning of the DoDFR gene, the cloning method comprises the following steps: (1) primer design, based on the CDS sequence of the DFR gene obtained by transcriptome sequencing in the above step 2) of Dendrobium officinale, primers are designed using PCR primer design software (such as Primer Premier 5.0 software), and the specificity of the designed primers is analyzed in Oligo6.0. Upstream primer DFRF: 5'-ATGGAGAAAGAGAGGAAGG-3'; downstream primer DFRR: 5'-TTACGTACGTACCCAACCG-3'.

[0020] (2) PCR reaction, the reaction procedure is: 95℃ pre-denaturation for 5 minutes; then denaturation at 95℃ for 30 seconds, annealing at 55℃ for 30 seconds, and extension at 72℃ for 1 minute for 35 cycles (i.e. 35 PCR cycles). The PCR reaction adopts a 50μL system, as shown in Table 3: Reagents volume Green Master Mix 25 μL Upstream primer 2 μL Downstream primer 2 μL Diluted cDNA 2 μL <![CDATA[ddH 2 The]]> 19 μL

[0021] Note: After the first strand of cDNA of Dendrobium officinale obtained in step 3) was diluted 10 times, 5 μL of PCR product was taken for detection by 1% agarose gel electrophoresis, and the remaining product was used for subsequent recovery. The results are shown in the attached manual. Figure 1 shown.

[0022] (3) Recovery of target fragments: Detect the remaining PCR product by 1% agarose gel electrophoresis. Cut the target band of expected length with a clean blade under ultraviolet light and use a gel recovery kit (such as GeneJET Plasmid Miniprep Kit) to purify and recover the PCR product.

[0023] (4) Ligation, transformation, identification and preservation of target fragments. The target fragments were linked using pMD18-T cloning vector (TaKaRa), named pMD18-T-DFR ligation system (10 μL): pMD18-T Vector 0.5 μL, gel recovery product 4.5 μL, Solution I 5 μL. Place at 4°C for overnight ligation. The ligation product was transformed into E. coli DH5α competent cells (TaKaRa). The specific operation steps are as follows: A. Take out the DH5α competent cells from -80C and quickly insert them into ice. After 5 minutes, the bacterial block melts, add the ligation product, and gently mix it by hand at the bottom of the centrifuge tube (avoid using a gun to suck it), and let it stand in ice for 25 minutes. B. Heat shock in a 42°C water bath for 45 seconds, quickly put it back on ice and let it stand for 2 minutes. C. Add 700 μL of LB liquid culture medium without antibiotics to the centrifuge tube, mix it well, and incubate it in a shaker at 37°C and 200 rpm for 60 minutes. D. After incubation, centrifuge at 5000 rpm for 1 minute to collect the bacteria, take 100 μL of the supernatant, gently blow and resuspend the bacteria, and spread it on the LB medium containing ampicillin (Amp) antibiotics. F. Place the plate upside down in a 37°C incubator for overnight culture. Pick the monoclonal colony and detect it by bacterial liquid PCR, and select the positive clone to send to Zhejiang Shangya Biotechnology Co., Ltd. for sequencing identification. The obtained sequence is compared and analyzed in NCBI's BLastn to verify that the cloned fragment is correct. The amplified product is the DoDFR gene CDS sequence, and the nucleotide sequence of the dihydroflavonol-4-reductase gene is shown in SEQ ID NO: 1; the amino acid sequence of the dihydroflavonol-4-reductase gene is shown in SEQ ID NO: 2. E. After the positive bacterial liquid is expanded, it is mixed with 50% glycerol at a ratio of 1:1 and frozen in liquid nitrogen in a -80°C refrigerator.

[0024] 5) Protein bioinformatics analysis of DoDFR gene. The analysis method includes the following steps: (1) Use the protein basic property analysis tool (ProtParam) (https: / / predictprotein.org / ) to analyze the physical and chemical properties of DoDFR gene. The full length of D. officinale DFR gene is 501 bp, encoding 166 amino acids. The online tool ProtParam provided by ExPASy ProteomicsServer was used to predict the physical and chemical properties of the protein encoded by DFR gene. The relative molecular mass of the protein was estimated to be 18.3 kDa, and the isoelectric point (pI): 8.55. (2) Use online protein structure and function prediction tools (such as Predictprotein) to predict the secondary structure of DFR gene. The results of online prediction of secondary structure showed that alpha helix accounted for 27.7% of the total amino acids, extended strand accounted for 24.7% of all amino acids, and random coil accounted for 47.6% of the total amino acids. (3) The tertiary structure of the protein was predicted by using fully automatic online software (such as SWISS-MODEL) using homology modeling to predict the tertiary structure of proteins. The three-dimensional model was drawn using PyMOL Viewer. Figure 2 (4) Finally, find the DFR amino acid sequences of plants with high homology, use the progressive multiple sequence alignment method (such as ClustalW method) to align the amino acid sequences, and then use MEGA7.0 to construct a neighbor-joining phylogenetic tree as shown in the instruction manual. Figure 3 shown.

[0025] 6) Transcriptome sequencing data and qRT-PCR verification of DoDFR gene. The verification method includes the following steps: (1) Material treatment, different light intensity treatment: The plant materials are two varieties of Dendrobium officinale preserved by the Institute of Medicinal Plants of Sanming Academy of Agricultural Sciences, namely 'Jiuxianhu-5' with red leaves and red stems and '1-8#' with green leaves and green stems. Plant tissues were cultured in MS medium, with 6-BA (2.0 mg / L), sucrose (20 g / L) and agar (6 g / L) added to the medium, and the pH value was 5.8. The photoperiod was 16 hours of light and 8 hours of darkness, and the culture temperature was 24°C. Uniform growth of 1-8# and Jiuxianhu-5 Dendrobium officinale tissue culture seedlings were selected for different light intensity treatments. The treatment conditions were as follows: the tissue culture seedlings of Dendrobium officinale were placed in a light incubator and treated at 1000 Lux, 2000 Lux, and 4000 Lux (25°C, 12 h / 12 ​​h) for 30 days, and then samples were immediately taken out and quickly frozen in liquid nitrogen and stored at -80°C.

[0026] (2) RNA extraction: Take the leaves of Dendrobium officinale tissue culture seedlings treated with different light intensities, freeze them in liquid nitrogen and grind them separately, and use the pine tree method (such as the evolutionary tree method) to extract the total RNA of Dendrobium officinale leaves. The RNA extraction method is carried out in the same way as the extraction of total RNA of Dendrobium officinale in step 1) above, and will not be described in detail again.

[0027] (3) Synthesis of the first strand of cDNA: The extracted RNA was used as a template and the first strand of cDNA was synthesized using the Yeasen reverse transcription kit Hifair™ Ⅲ1st Strand cDNA Synthesis SuperMix for qPCR (gDNA Digester Plus). The synthesis of the first strand of cDNA was carried out in the same manner as in the above step 3) of the synthesis of the first strand of cDNA of Dendrobium officinale, and will not be described in detail again.

[0028] (4) Specificity detection of qRT-PCR primers. The cDNA synthesized from the sample treated with 1-8#1000Lux was diluted 10 times and used as a reaction template for qRT-PCR. The qRT-PCR reaction was performed using an annealing temperature gradient of 50 to 65°C to determine the optimal annealing temperature. The 20 μL reaction system shown in Table 4 was used.

[0029] Reagents volume SYBRII 10 μL Upstream primer 0.8 μL Downstream primer 0.8 μL Diluted cDNA 1μL <![CDATA[ddH 2 The]]> 7.4 μL

[0030] The qRT-PCR primer sequences are shown in Table 5: Primer name Primer sequence (5'-3') DoDFR-F GTGAAGCCTTTGCTGGAT DoDFR-R AAGACCTCAAGATGCCCA Den_actin-F TATGTGGGAGATGAAGCCC Den_actin-R TCTGTTTGCCTTGGGATTG

[0031] Note: The reaction tube uses a 0.2ml PCR plate siliconized by Bio-Rad. qRT-PCR is performed in a gradient thermal cycler (such as iQTM5 thermal cycler). The reaction program is: pre-denaturation at 95℃ for 10s; then denaturation at 95℃ for 10s, annealing at 50-65℃ for 20s, extension at 72℃ for 20s, and collecting fluorescence at this temperature. The plate read program is performed for 46 cycles. Then, starting from 50℃, the temperature is increased to 95℃ with a temperature gradient of 0.5℃ per step, and the temperature is maintained for 5 seconds at each step.

[0032] (5) qRT-PCR reaction. The reverse transcribed cDNA samples of different samples in the extraction of the first chain of cDNA of Dendrobium officinale in step 3) were diluted 10 times and used as templates for qRT-PCR amplification. The reaction tube used a siliconized 0.2mL PCR plate and a 20μL reaction system (Table 4 above). A single reverse transcribed cDNA sample was repeated three times. The qRT-PCR reaction program was: pre-denaturation at 95℃ for 10s, then denaturation at 95℃ for 10s, annealing at the optimal annealing temperature for 20s, extension at 72℃ for 20s, and collecting fluorescence at this temperature. The plate read program was performed for 46 cycles. Finally, starting from 50℃, the temperature was increased to 95℃ at a rate of 0.5℃ per step, and each temperature was maintained for 5s to draw a melting curve.

[0033] (6) qRT-PCR data analysis: In this experiment, the double standard curve method was used to perform relative quantification of the expression level of the key enzyme gene (DoDFR gene) in the anthocyanin metabolic synthesis pathway. The qRT-PCR fluorescence quantitative data analysis method (2-ΔΔCT method) was used to calculate the relative expression of the gene. The expression of the DFR gene in different varieties of Dendrobium officinale and under different light treatments is shown in the attached manual. Figure 4 As shown. Under different light intensity treatments, the DFR genes in both types of Dendrobium officinale showed an up-regulated trend under light intensity treatment, and were highly expressed in 'Jiuxianhu-5', which was the same as the expression trend in the transcriptome data. In summary, light intensity within the appropriate range of 1000 Lux -4000 Lux can greatly increase the expression of the DFR gene in Dendrobium officinale, thereby increasing the content of anthocyanins in Dendrobium officinale.

[0034] The above is only a specific implementation of the present invention, but the design concept of the present invention is not limited thereto. Any non-substantial changes to the present invention using this concept shall be deemed as an infringement of the protection scope of the present invention.

[0035] SEQ ID NO: 1 atggagaaag agaggaaggg tccagtagtg gtgactggag ccagtggcta cgtgggttca tggctggtga tgaagcttct tcagaagggt tatgaggtta gggcaacagt gagagatcca acaaatctca agaaagtgaa gcctttgctg gatctcccgc gctccaatga actgctcagc atttggaaag cagatctaaa tggcaccgaa ggaagcttcg acgaggtgat acgtggcagc attggagtgt tccacgtcgc tactcccatg aattttcaat ccaaagaccc tgagaatgaa gtgatacaac cggcaatcaa cggtttgctg ggcatcttga ggtcttgcaa aaatgccggc agcgtaaagc gagtgatatt cacgtcttct gcaggaacag tgaacgtgga ggaacaccaa gcagcagcgt atgacgagac ctgctggagat gaccttgact ttgtgaaccg agtcaagatg accggttggg tacgtacgta a

[0036] SEQ ID NO: 2 MET Glu Lys Glu Arg Lys Gly Pro Val Val Val Thr Gly Ala Ser Gly TyrVal Gly Ser

[0037] Trp Leu Val MET Lys Leu Leu Gln Lys Gly Tyr Glu Val Arg Ala Thr ValArg Asp Pro Thr Asn Leu Lys Lys Val Lys Pro Leu Leu Asp Leu Pro Arg Ser Asn GluLeu Leu Ser Ile Trp Lys Ala Asp Leu Asn Gly Thr Glu Gly Ser Phe Asp Glu Val IleArg Gly Ser Ile Gly Val Phe His Val Ala Thr Pro MET Asn Phe Gln Ser Lys Asp ProGlu Asn Glu Val Ile Gln Pro Ala Ile Asn Gly Leu Leu Gly Ile Leu Arg Ser Cys LysAsn Ala Gly Ser Val Lys Arg Val Ile Phe Thr Ser Ser Ala Gly Thr Val Asn Val GluGlu His Gln Ala Ala Ala Tyr Asp Glu Thr Cys Trp Ser Asp Leu Asp Phe Val Asn ArgVal Lys MET Thr Gly Trp Val Arg Thr。

Claims

1. A light intensity-regulated Dendrobium officinale dihydroflavonol-4-reductase gene, characterized in that: The nucleotide sequence of the dihydroflavonol-4-reductase gene is shown in SEQ ID NO:

1.

2. The light intensity-regulated Dendrobium officinale dihydroflavonol-4-reductase gene according to claim 1, characterized in that: The CDS sequence of the dihydroflavonol-4-reductase gene is obtained by splicing transcriptome sequencing, wherein the expression of the dihydroflavonol-4-reductase gene can be increased by light irradiation.

3. The light intensity-regulated Dendrobium officinale dihydroflavonol-4-reductase gene according to claim 1, characterized in that: The amino acid sequence of the dihydroflavonol-4-reductase gene is shown in SEQ ID NO:

2.

4. The light intensity-regulated Dendrobium officinale dihydroflavonol-4-reductase gene according to claim 1, characterized in that: The dihydroflavonol-4-reductase gene contains a recombinant plasmid.

5. The light intensity-regulated Dendrobium officinale dihydroflavonol-4-reductase gene according to claim 1, characterized in that: The recombinant plasmid is pMD18-T.

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