CN115725625A - Application of SmTAT2 gene in preparation of transgenic plants with high phenolic acid compounds yield - Google Patents

Application of SmTAT2 gene in preparation of transgenic plants with high phenolic acid compounds yield Download PDF

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CN115725625A
CN115725625A CN202210927698.6A CN202210927698A CN115725625A CN 115725625 A CN115725625 A CN 115725625A CN 202210927698 A CN202210927698 A CN 202210927698A CN 115725625 A CN115725625 A CN 115725625A
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smtat2
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salvia miltiorrhiza
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CN115725625B (en
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张利
钟明志
廖进秋
姜媛媛
杨瑞武
王龙
邓雪雪
柴松岳
林丽
张云松
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Sichuan Agricultural University
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Abstract

The invention provides application of SmTAT2 gene in preparation of transgenic plants with high phenolic acid compounds yield, and belongs to the field of genetic engineering. The invention discovers for the first time that the SmTAT2 gene can improve the content of rosmarinic acid and salvianolic acid B in hairy roots of salvia miltiorrhiza, and the SmTAT2 gene plays a positive regulation role in the synthesis and accumulation of rosmarinic acid and salvianolic acid B in salvia miltiorrhiza. The invention transfers SmTAT2 gene into salvia miltiorrhiza, so that the content of rosmarinic acid and salvianolic acid B in salvia miltiorrhiza is obviously improved, and the medicinal value of salvia miltiorrhiza plants is improved. The SmTAT2 gene, the recombinant vector and the recombinant bacterium thereof can be used for improving the rosmarinic acid content in the salvia miltiorrhiza bunge and the synthesis and accumulation of salvianolic acid B, thereby realizing the directional improvement of the salvia miltiorrhiza bunge. The construction method of the transgenic plant provides important reference for the directional improvement of the salvia miltiorrhiza.

Description

Application of SmTAT2 gene in preparation of transgenic plant with high phenolic acid compound yield
Technical Field
The invention belongs to the field of genetic engineering, and particularly relates to application of SmTAT2 gene in preparation of transgenic plants with high phenolic acid compounds.
Background
Salvia miltiorrhiza (Salvia milirhizoza Bunge) is a perennial herb of Salvia (Salvia) of Labiatae (Lamiaceae), and its dried root and rhizome are one of the traditional bulk Chinese medicinal materials in China. Saviae Miltiorrhizae radix is bitter in taste and slightly cold in nature, and has effects of promoting blood circulation, dredging collaterals, removing blood stasis, relieving pain, cooling blood, relieving swelling, clearing heart fire, and relieving restlessness, and can be used for treating menoxenia, amenorrhea, dysmenorrhea, thoracico-abdominal pain, pyretic arthralgia, vexation, insomnia, hepatosplenomegaly, and angina pectoris.
According to the structural characteristics and physicochemical properties of the compound, the chemical components of the salvia miltiorrhiza can be divided into two types: one class is liposoluble Tanshinone compounds, such as Tanshinone I and Tanshinone II A; one is water soluble phenolic acid compound, such as rosmarinic acid, salvianolic acid B, etc. The content of salvianolic acid B in the water-soluble medicinal components of the salvia miltiorrhiza is the highest, and the salvianolic acid B is used as an index component for controlling the quality of the salvia miltiorrhiza in the pharmacopoeia of the people's republic of China in 2005 edition and 2010 edition. Rosmarinic acid has anti-inflammatory, analgesic, and analgesic effects, is widely distributed in plants, mainly exists in Labiatae, boraginaceae, cucurbitaceae, etc., has been developed into medicine, and was marketed in Germany in 1990.
In recent years, with the rapid increase of the demand of salvia medicinal materials and the reduction of wild resources, the quality of the artificially cultivated salvia is uneven, so that the content of phenolic compounds in the salvia needs to be increased from the molecular level to cultivate high-quality salvia.
Disclosure of Invention
An object of the present invention is to provide the use of the SmTAT2 gene in the preparation of transgenic plants with high phenolic acid compound yield.
The SmTAT2 gene refers to a gene that expresses tyrosine aminotransferase 2 (i.e., smTAT2 protein). It can be from Salvia miltiorrhiza, and also from other plants.
Another object of the present invention is to provide a method for producing a transgenic plant highly productive of phenolic acids.
The invention provides a recombinant vector which comprises a SmTAT2 gene.
Further, the SmTAT2 gene is derived from salvia miltiorrhiza bunge; and/or, the recombinant vector is a recombinant pCAMBIA1300 vector.
Further, the nucleotide sequence of the SmTAT2 gene is shown as SEQ ID NO. 1.
The invention also provides a recombinant bacterium, which comprises the recombinant vector; preferably, the recombinant bacterium is recombinant escherichia coli.
The invention also provides a method for preparing the transgenic plant with high phenolic acid compounds yield, which is to transfer SmTAT2 gene into the plant to obtain the plant expressing SmTAT2 protein.
Further, the amino acid sequence of the SmTAT2 protein is shown as SEQ ID NO. 2.
Further, the method for transferring a plant is one of an agrobacterium method, a particle gun method, an electroporation method, a PEG mediated method, a liposome method, and a calcium phosphate-DNA coprecipitation method.
Further, the plant is salvia miltiorrhiza.
The invention also provides SmTAT2 gene, the recombinant vector and the application of the recombinant bacterium in preparing transgenic plants with high phenolic acid compounds yield; preferably, the plant is salvia miltiorrhiza.
Further, the phenolic acid compound is rosmarinic acid and/or salvianolic acid B.
The invention discovers that the SmTAT2 gene can improve the content of rosmarinic acid and salvianolic acid B in hairy roots of the salvia miltiorrhiza for the first time, and discovers that the SmTAT2 gene plays a positive regulation role in the synthesis and accumulation of the rosmarinic acid content and the salvianolic acid B in the salvia miltiorrhiza for the first time.
The SmTAT2 gene is transferred into the salvia miltiorrhiza bunge, so that the content of rosmarinic acid and salvianolic acid B in the salvia miltiorrhiza bunge is obviously improved, and the medicinal value of salvia miltiorrhiza bunge plants is improved.
The SmTAT2 gene, the recombinant vector and the recombinant bacterium thereof can be used for improving the rosmarinic acid content in the salvia miltiorrhiza bunge and the synthesis and accumulation of salvianolic acid B, and realizing the directional improvement of the salvia miltiorrhiza bunge. The construction method of the transgenic plant provides important reference for the directional improvement of the salvia miltiorrhiza.
Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.
The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.
Description of the drawings:
FIG. 1 shows the expression of SmTAT2 in roots, stems, leaves and flowers of Salvia miltiorrhiza.
FIG. 2 is an electrophoresis diagram of SmTAT2 gene clone positive identification.
FIG. 3 is a positive identification map of transgenic hairy roots. In Panel A, NC denotes negative control, PC denotes positive control, OE45 denotes overexpression 45, OE47 denotes overexpression 47, OE48 denotes overexpression 48; in Panel B, M represents Maker, NC represents a negative control, PC represents a positive control, OE45 represents overexpression 45, OE47 represents overexpression 47, OE48 represents overexpression 48; in panel C, M represents Maker, NC represents negative control, PC represents positive control, CC1 represents CRISPR/Cas9, CC4 represents CRISPR/Cas9, CC5 represents CRISPR/Cas5.
FIG. 4 is a diagram showing the target site gene editing of a target gene.
FIG. 5 is a graph showing the content of rosmarinic acid and salvianolic acid B in transgenic hairy roots. In the figure, EV represents empty vector, OE45 represents over-expression 45, oe47 represents over-expression 47, oe48 represents over-expression 48, cc1 represents CRISPR/Cas9, cc4 represents CRISPR/Cas9, cc5 represents CRISPR/Cas5.
FIG. 6 is a graph showing the relative expression level of SmTAT2 in transgenic hairy roots.
Detailed description of the preferred embodiments
The raw materials and equipment used in the invention are known products, and are obtained by purchasing products sold in the market.
Example 1 tissue localization of SmTAT2 Gene
Fresh and tender salvia miltiorrhiza roots, stems, leaves and flowers are collected, and total RNA extraction of each tissue is carried out by using a plant total RNA extraction kit (purchased from Lanbo Biotech, inc. at Zhejiang) and referring to instructions provided by the kit. The extracted total RNA was reverse transcribed into cDNA using TransScriptone-StepRT-PCRSuperMix (purchased from Kyoto Kogyo Biotech Co., ltd.) with reference to the kit instructions. The reference genes used in qRT-PCR are Actin and GAPDH, and the reference genes and target gene primers are respectively as follows:
F-Actin：5’-AGGAACCACCGATCCAGACA-3’(SEQ ID No.3)；
R-Actin：5’-GGTGCCCTGAGGTCCTGTT-3’(SEQ ID No.4)；
F-GAPDH：5’-CCACCGTCCACTCCATCACT-3’(SEQ ID No.5)；
R-GAPDH：5’-TGGGAACTCGGAACGACATAC-3’(SEQ ID No.6)；
F-qSmTAT2：5’-ACCGTTAGGGGCGTGCTC-3’(SEQ ID No.7)；
R-qSmTAT2：5’-TGCGGAAGGAAGGGAAGG-3’(SEQ ID No.8)。
quantitative utilization of fluorescence
Figure BDA0003780360800000031
Green qPCR Supermix (purchased from Beijing Quanshi gold Biotechnology Co., ltd.) kit, a 10. Mu.L reaction system was set, wherein qMix 5. Mu.L, ddH 2 O3. Mu.L, upstream primer 0.5. Mu.L, downstream primer 0.5. Mu.L, and template 1. Mu.L. The qRT-PCR reaction procedure was 30s at 94 ℃, 5s at 94 ℃, 30s at 60 ℃ and 42 thermal cycles. Finally utilize 2 -ΔΔCT And calculating the relative expression amount. The results show that SmTAT2 is expressed in roots, stems, leaves and flowers of salvia miltiorrhiza, is 1 typical structural expression gene, and the expression level in the flowers, the leaves and the stems is obviously higher than that in the roots (figure 1).
Through identification, the nucleotide sequence of SmTAT2 gene in root, stem, leaf and flower of salvia miltiorrhiza is shown as SEQ ID NO.1, and the amino acid sequence of the expressed protein is shown as SEQ ID NO. 2.
SEQ ID No.1：
ATGGAGAATGGAGGTTCGGCGGCGGCGCGGTGGCGTTTCGAGGGGAACGAGCGGCTCATCCTGGCCGGAGGAGTCACCGTTAGGGGCGTGCTCAACACTGTTATTGGAAATCTCGACGGAAACGATACCAGACCGGTGATTCCGCTCGGCCACGGAGATCCATCCGCCTTCCCTTCCTTCCGCACCTCTCCTTTTGCGGAGGACGCCGTCTGCTCCGCGGTCCGCTCCACCAAGTTCAACGGTTACTCCTCCACCGTCGGTATTCCCGCGGCTCGCAGTGCTGTAGCAGAGTATCTTTCTAAAGACCTTCCTTACAAGTTATCACCTGATGATGTTTTCCTGACCATTGGATGCACCCAAGCGTTAGAAGCCGTTGTGACTGTCCTTGCTCGTCCCGGTGCTAACCTTTTGCTTCCAAGGCCAGGCTTCCCTTACTACGAAGCCAGGGCTGGCTTTTGTAGCCTTGAAGTCCGTCACTTTGATCTTCTCCCGGAACAAGATTGGGAAGTGGACTTGGCTTCGGTTGAAGCTCTGGCTGATGCAAATACAGTTGCTATGGTTATTATCAATCCGGGCAATCCCTGTGGAAATGTTTTCAAGTATGAACACTTGAAAAAGGTCGCGGAGACAGCTAGAAAGCTTGGAATCCTAGTGATCTCGGATGAAGTGTATGACCATCTTACCTTTGGTAGCAACCCCTTTGTCCCAATGGGAGTCTTTGCATCAATTGCCCCGATCTTGACTCTTGGATCAATATCTAAGAGATGGATCGTCCCGGGTTGGAGACTTGGCTGGCTTGTGACAAACGATCCCGATGGTATCCTTACTAAGCAAGGGATTGTTGACAGCATCAAAGGTTTCCTGAATATTTCCTCTGACCCAGCAACCTTTATGCAGGGGGCAGTTCCACAGATTCTTGAGAATACCCCGAGCGACTTCTTTCAGAAAATTGTTAGTACACTTAGAGAAACAGCAGACATATGCTATGAACGCAGCAAAGAAATTCCCTGCATAACTTGCCCGAGCAGACCTGAAGGATCCATGTTTGCTATGGTGAAGCTCAATCTGTCTCTCCTTGAAGGCATTGAGGATGACATGGACTTCTGCTGCAAGCTCGCCAAAGAGGAATCCGTGATACTCCTCCCAGGTTTTGCTGTAGGGCTCAAGAATTGGTTACGCGTTACATTTGCCATCGAACCATCATCTCTCGATGATGGCTTCCTCAGAATAAAAGCTTTTCATCAAAGGCACGCCAAGAAACAATGA.
SEQ ID No.2：
MENGGSAAARWRFEGNERLILAGGVTVRGVLNTVIGNLDGNDTRPVIPLGHGDPSAFPSFRTSPFAEDAVCSAVRSTKFNGYSSTVGIPAARSAVAEYLSKDLPYKLSPDDVFLTIGCTQALEAVVTVLARPGANLLLPRPGFPYYEARAGFCSLEVRHFDLLPEQDWEVDLASVEALADANTVAMVIINPGNPCGNVFKYEHLKKVAETARKLGILVISDEVYDHLTFGSNPFVPMGVFASIAPILTLGSISKRWIVPGWRLGWLVTNDPDGILTKQGIVDSIKGFLNISSDPATFMQGAVPQILENTPSDFFQKIVSTLRETADICYERSKEIPCITCPSRPEGSMFAMVKLNLSLLEGIEDDMDFCCKLAKEESVILLPGFAVGLKNWLRVTFAIEPSSLDDGFLRIKAFHQRHAKKQ.
Example 2 SmTAT2 Gene cloning
Using cDNA of Salvia miltiorrhiza leaf in example 1 as a template, PCR amplification was performed using 2 XTAQA PCR Mastermix reagent (purchased from Tiangen Biochemical technology, beijing, ltd.) in a system of Mix 10. Mu.L, ddH 2 O8. Mu.L, upstream primer 0.5. Mu.L, downstream primer 0.5. Mu.L, and template 1. Mu.L. The PCR reaction procedure was 94 ℃ for 3min,94 ℃ for 30s,55 ℃ for 30s,72 ℃ for 1min,34 thermal cycles, 72 ℃ for 5min, and 12 ℃ for 12 ℃. A target gene band was obtained by 2% agarose gel electrophoresis, and then PCR products were recovered and purified using a gel recovery kit (purchased from Tiangen Biochemical technology, beijing) Ltd.) with reference to the kit instructions. Double-enzyme digestion of target gene PCR product and plant expression vector pCAMBIA1300 by using restriction enzymes Kpn I and Sal I + UBQ10-GFP, digestion system Gbuffer 10. Mu.L, PCR recovery product/plant expression vector 8. Mu.L, kpn I1. Mu.L, sal I1. Mu.L. The cleavage procedure was 37 ℃ for 2h. The enzyme-cleaved product was recovered by using a clean recovery kit (purchased from Tiangen Biochemical technology, beijing) Co., ltd.) and referring to the instructions of the kit. Finally, T4 ligase (purchased from Takara Bio Inc.) is used for the purpose of clean recoveryThe gene product and the plant expression vector are linked, the linking system is 1 mu L of T4 ligase, 4 mu L of T4 buffer, 3 mu L of the target gene clean recovery product and 2 mu L of the vector clean recovery product. The reaction program is 16 ℃ for 12h.
The ligation product was transformed into E.coli competent DH 5. Alpha. By ice bath 30min, heat shock at 42 ℃ for 30s and ice bath for 2min. Adding 500mL LB liquid medium 180rpm, culturing at 37 deg.C for 1h, and spreading the bacterial liquid to belt Kan + And (3) culturing on a resistant LB solid culture medium overnight, selecting single bacteria, identifying positive clones by using a PCR method, and detecting a target gene band as shown in figure 2 to prove that the target gene clone is successful. Obtaining positive recombinant plasmid pCAMBIA1300 + -UBQ10-SmTAT2-GFP。
EXAMPLE 3 construction of transgenic hairy roots of Salvia miltiorrhiza
Extracting recombinant plasmid pCAMBIA1300 from target gene positive clone bacterial liquid by using plasmid extraction kit (purchased from Tiangen Biochemical technology (Beijing) Co., ltd.) with reference to kit specification + -UBQ10-SmTAT2-GFP. The recombinant plasmid was transformed into Agrobacterium rhizogenes Accc10060 in the same manner as in example 2. An E-CRISP Design on-line tool (http:// www.e-crisp.org/E-CRISP /) is used for designing the sgRNA of the target gene according to the 5' -NGG principle, and 2 sgRNAs with high editing efficiency, good specificity and low off-target rate are selected. Wherein sgRNA1 is CGAGCGAATCACGGTC (SEQ ID No. 9), and sgRNA2 is TTATACGAGCCAGGC (SEQ ID No. 10); . Selected sgRNAs were synthesized into complementary oligonucleotide pairs and annealed to generate dimers, then sgRNA1 was subcloned into the vector by cleaving the CRISPR/Cas9 vector with BbsI and sgRNA2 was subcloned into the CRISPR/Cas9 vector using the same method, then the vector containing AtU6 and sgRNA2 was amplified with primers carrying KpnI and XbaI cleavage sites, and the PCR product was subcloned into the sgRNA1 target editing vector by double cleavage. Finally, the pCAMBIA1300 is subjected to double enzyme digestion by utilizing Dra III and AhdI + Recombination of CRISPR/Cas9 vector with 2 sgRNAs to pCAMBIA1300 + To finally form pCAMBIA1300 + -AtU6-sgRNA1-sgRNA2-Cas9. The recombinant plasmid was transformed into Agrobacterium rhizogenes Accc10060 by the same transformation method as in example 2.
Will carry the target gene to the exteriorRif-carrying Rif of Agrobacterium rhizogenes Accc10060 of expression vector and CRISPR/Cas9 gene editing vector + And Kan + YEB medium was activated to an OD 600 of 0.6, and then the suspension was resuspended in an equal volume of MS medium. Scratching a 1-month-old salvia miltiorrhiza aseptic seedling in an ultraclean workbench, dip-dyeing the aseptic seedling with a heavy suspension bacterial liquid for 10min, putting the aseptic seedling on aseptic filter paper for draining, inoculating the aseptic seedling on a 1/2MS solid culture medium, placing the aseptic seedling in a incubator for dark culture at 25 ℃ for 2d, then cleaning an explant with sterile water, draining the aseptic filter paper, inoculating the aseptic seedling on a rooting culture medium for inducing rooting, growing about 10d of hairy roots, placing the grown overexpression primary generation hairy roots under a fluorescence microscope, observing GFP fluorescence by using blue excitation light to determine the positivity of the overexpression hairy roots, wherein as shown in figure 3A, wild hairy roots serve as Negative Controls (NC), overexpression empty vector transgenic hairy roots which do not carry target genes serve as Positive Controls (PC), and the overexpression hairy roots of 3 plants both emit strong green fluorescence and the overexpression hairy roots of 3 plants serve as the positivity. Then, a specific gene RolB of an Ri plasmid of the agrobacterium rhizogenes and a partial fragment containing a UBQ10 promoter and a target gene SmTAT2 in an overexpression vector are designed and named as UBQ10-SmTAT2. Their primers are shown below:
F-RolB：5’-GCTCTTGCAGTGCTAGATTT-3’(SEQ ID No.11)；
R-RolB：5’-GAAGGTGCAAGCTACCTCTC-3’(SEQ ID No.12)；
F-UBQ10-SmTAT2：5’-CGACGAGTCAGTAATAAACG-3’(SEQ ID No.13)；
R-UBQ10-SmTAT2：5’-CATTGGGACAAAGGGGTT-3’(SEQ ID No.14)。
then, the overexpression hairy roots are amplified by PCR, as shown in FIG. 3B, the wild type hairy roots are taken as NC, the target gene recombinant plasmid is PC, NC only can amplify RolB gene bands, and the result shows that only agrobacterium is transferred into the hairy roots, but the overexpression recombinant plasmid is not transferred, and the PC and the overexpression 3 hairy roots amplify UBQ10-SmTAT2 fragments besides RolB genes, so that the overexpression 3 hairy roots are positive, which is the repeated verification of GFP fluorescence results. For positive identification of CRISPR/Cas9 gene editing hairy roots, a PCR method is also used, and primers of a unique gene Cas9 in a gene editing vector are designed as follows:
F-Cas9：5’-CAGGACAGAGTAAGCCACC-3’(SEQ ID No.15)；
R-Cas9：5’-CATAAGCCCGAGAACATC-3’(SEQ ID No.16)。
as shown in FIG. 3C, the wild type hairy root without NC is transferred into the CRISPR/Cas9 empty vector, and the hairy root is PC, it can be seen that NC can only amplify RolB gene, and PC and CRISPR/Cas9 gene editing hairy root can both amplify RolB and Cas9 gene, indicating that 3 strains of gene editing hairy roots are positive. Then 3 positive hairy root genomes are taken as template PCR amplification double-target-point regions, PCR products TA are cloned to an intermediate vector and sent to a company Limited in biological engineering (Shanghai) to be sequenced by using a sanger method, sequencing results are shown in figure 4, and the double target points of the gene editing hairy roots are edited in different degrees.
The induced hairy roots are subcultured for 1 time for 15d, after subculture is carried out on a rooting culture medium for 2 times, the growing length of the hairy roots is about 5cm, the hairy roots are transferred into a liquid rooting culture medium for suspension culture, subculture is carried out for 1 time for the same 15d, and finally the hairy roots grow to 2 months of age, total RNA of the transgenic hairy roots is extracted, the total RNA is reversely transcribed into cDNA, unloaded hairy roots are set as a control, and the relative expression quantity of SmTAT2 in the transgenic hairy roots is detected by utilizing qRT-PCR (quantitative reverse transcription-polymerase chain reaction), as shown in figure 6, the expression quantity of over-expressed hairy root SmTAT2 of 3 strains is obviously higher than that of the control strains, and the expression quantity of 3 strains of hairy root SmTAT2 edited by a CRISPR/Cas9 gene is obviously lower than that of the control strains, which indicates that the transgenic induction is successful. Then drying the hairy roots to constant weight at 50 ℃, grinding the dried hairy roots into dry powder, and measuring the rosmarinic acid content and the salvianolic acid B content in the transgenic hairy roots by using HPLC (high performance liquid chromatography), wherein the rosmarinic acid content and the salvianolic acid B content in the transgenic hairy roots are respectively shown in figures 5A and 5B, so that the rosmarinic acid content and the salvianolic acid B content in the over-expressed hairy roots are obviously higher than those in a comparison strain, and the rosmarinic acid content and the salvianolic acid B content in the gene-edited hairy roots are obviously lower than those in the comparison strain, which shows that the target gene SmTAT2 plays a forward regulation role in the synthesis and accumulation of rosmarinic acid content and salvianolic acid B in salvia miltiorrhiza, and the SmTAT2 gene can be used for improving the synthesis and accumulation of rosmarinic acid content and salvianolic acid B in salvia miltiorrhiza, so as to realize the directional improvement of salvia miltiorrhiza.
In conclusion, the invention provides the application of SmTAT2 gene in preparing transgenic plants with high phenolic acid compounds yield. The invention discovers for the first time that the SmTAT2 gene can improve the content of rosmarinic acid and salvianolic acid B in hairy roots of salvia miltiorrhiza, and the SmTAT2 gene plays a positive regulation role in the synthesis and accumulation of rosmarinic acid and salvianolic acid B in salvia miltiorrhiza. The invention transfers SmTAT2 gene into salvia miltiorrhiza, so that the content of rosmarinic acid and salvianolic acid B in salvia miltiorrhiza is obviously improved, and the medicinal value of salvia miltiorrhiza plants is improved. The SmTAT2 gene, the recombinant vector and the recombinant bacterium thereof can be used for improving the rosmarinic acid content in the salvia miltiorrhiza bunge and the synthesis and accumulation of salvianolic acid B, thereby realizing the directional improvement of the salvia miltiorrhiza bunge. The construction method of the transgenic plant provides important reference for the directional improvement of the salvia miltiorrhiza.

Claims (10)

1. A recombinant vector, characterized in that: it includes the SmTAT2 gene.
2. The recombinant vector according to claim 1, characterized in that: the SmTAT2 gene is derived from salvia miltiorrhiza; and/or the recombinant vector is a recombinant pCAMBIA1300 vector.
3. The recombinant vector according to claim 2, characterized in that: the nucleotide sequence of the SmTAT2 gene is shown as SEQ ID NO. 1.
4. A recombinant bacterium is characterized in that: comprising the recombinant vector of any one of claims 1-3; preferably, the recombinant bacterium is recombinant escherichia coli.
5. A method for preparing a transgenic plant with high phenolic acid compound yield is characterized in that: the SmTAT2 gene is transferred into a plant to obtain the plant expressing SmTAT2 protein; wherein the SmTAT2 gene is a SmTAT2 gene as described in any one of claims 1 to 3.
6. The method of claim 5, wherein: the amino acid sequence of the SmTAT2 protein is shown as SEQ ID NO. 2.
7. The method of claim 5, wherein: the method for transferring the plant is one of an agrobacterium method, a particle gun method, an electrotransformation method, a PEG mediated method, a liposome method and a calcium phosphate-DNA coprecipitation method.
8. The method according to any one of claims 5-7, wherein: the plant is Saviae Miltiorrhizae radix.
9. Use of the SmTAT2 gene as defined in any one of claims 1 to 3, the recombinant vector as defined in any one of claims 1 to 3, the recombinant bacterium as defined in claim 4 for the preparation of a transgenic plant highly productive of phenolic compounds; preferably, the plant is salvia miltiorrhiza.
10. Use according to claim 9, characterized in that: the phenolic acid compound is rosmarinic acid and/or salvianolic acid B.
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102061301A (en) * 2010-08-04 2011-05-18 陕西师范大学 Hydroxyl cinnamon acyl transfer enzyme gene sequence of salvia miltiorrhiza bunge as well as coded amino acid sequence and application thereof
CN102876713A (en) * 2012-09-20 2013-01-16 陕西师范大学 Method for improving content of rosmarinic acid and salvianolic acid B in salvia miltiorrhiza bunge while transforming genes
CN108823143A (en) * 2018-05-24 2018-11-16 中北大学 A kind of cultural method of purple perilla high yield Rosmarinic acid suspension cell
CN110590923A (en) * 2019-10-18 2019-12-20 中国药科大学 Application of Salvia miltiorrhiza NAC1 transcription factor and coding gene thereof in regulating and controlling content of salvianolic acid compounds in Salvia miltiorrhiza

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102061301A (en) * 2010-08-04 2011-05-18 陕西师范大学 Hydroxyl cinnamon acyl transfer enzyme gene sequence of salvia miltiorrhiza bunge as well as coded amino acid sequence and application thereof
CN102876713A (en) * 2012-09-20 2013-01-16 陕西师范大学 Method for improving content of rosmarinic acid and salvianolic acid B in salvia miltiorrhiza bunge while transforming genes
CN108823143A (en) * 2018-05-24 2018-11-16 中北大学 A kind of cultural method of purple perilla high yield Rosmarinic acid suspension cell
CN110590923A (en) * 2019-10-18 2019-12-20 中国药科大学 Application of Salvia miltiorrhiza NAC1 transcription factor and coding gene thereof in regulating and controlling content of salvianolic acid compounds in Salvia miltiorrhiza

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
CHEN S. L.等: "Salvia miltiorrhiza tyrosine aminotransferase 2 (TAT2) mRNA, complete cds", GENBANK, pages 220575 *
SHUANCANG ZHANG等: "Cloning and characherization of a putative R2R3 MYB transcriptional repressor of the rosmarinic acid biosynthetic pathway from Salvia miltiorrhiza", PLOS ONE, vol. 8, no. 9 *
YUAN ZHANG等: "Pathway engineering for phenolic acid accumulations in Salvia miltiorrhiza by combinational genetic manipulation", METABOLIC ENGINEERING, vol. 21 *
侯学敏: "丹参迷迭香酸合成途径关键酶基因的鉴定与分析", 中国优秀硕士学位论文全文数据库 *
李莎莎: "丹参转录因子SmMYB111对酚酸类物质合成调控的研究", 中国优秀硕士学位论文全文数据库 *
李静: "茉莉酸甲酯对丹参酶活及酚酸化合物的含量和抗氧化活性影响", 中国优秀硕士学位论文全文数据库, pages 1 *

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