CN115232827B - OjTPS1 gene sequence related to water fennel beta-caryophyllene synthesis and application thereof - Google Patents
OjTPS1 gene sequence related to water fennel beta-caryophyllene synthesis and application thereof Download PDFInfo
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- NPNUFJAVOOONJE-ZIAGYGMSSA-N β-(E)-Caryophyllene Chemical compound C1CC(C)=CCCC(=C)[C@H]2CC(C)(C)[C@@H]21 NPNUFJAVOOONJE-ZIAGYGMSSA-N 0.000 title claims abstract description 83
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 62
- NPNUFJAVOOONJE-UHFFFAOYSA-N beta-cariophyllene Natural products C1CC(C)=CCCC(=C)C2CC(C)(C)C21 NPNUFJAVOOONJE-UHFFFAOYSA-N 0.000 title claims abstract description 46
- NPNUFJAVOOONJE-UONOGXRCSA-N caryophyllene Natural products C1CC(C)=CCCC(=C)[C@@H]2CC(C)(C)[C@@H]21 NPNUFJAVOOONJE-UONOGXRCSA-N 0.000 title claims abstract description 46
- 230000015572 biosynthetic process Effects 0.000 title claims abstract description 25
- 238000003786 synthesis reaction Methods 0.000 title claims abstract description 23
- 241000938542 Oenanthe aquatica Species 0.000 title abstract description 7
- 241000196324 Embryophyta Species 0.000 claims abstract description 32
- 244000211187 Lepidium sativum Species 0.000 claims abstract description 19
- 235000007849 Lepidium sativum Nutrition 0.000 claims abstract description 19
- 238000000034 method Methods 0.000 claims abstract description 6
- 239000013598 vector Substances 0.000 claims description 17
- 241000219195 Arabidopsis thaliana Species 0.000 claims description 13
- 238000010367 cloning Methods 0.000 claims description 10
- 240000008881 Oenanthe javanica Species 0.000 claims description 8
- 235000000365 Oenanthe javanica Nutrition 0.000 claims description 8
- 230000009466 transformation Effects 0.000 claims description 7
- 238000012216 screening Methods 0.000 claims description 6
- YQYJSBFKSSDGFO-UHFFFAOYSA-N Epihygromycin Natural products OC1C(O)C(C(=O)C)OC1OC(C(=C1)O)=CC=C1C=C(C)C(=O)NC1C(O)C(O)C2OCOC2C1O YQYJSBFKSSDGFO-UHFFFAOYSA-N 0.000 claims description 4
- 238000002360 preparation method Methods 0.000 claims description 4
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- 230000002018 overexpression Effects 0.000 claims 3
- 239000013604 expression vector Substances 0.000 claims 2
- 239000001169 1-methyl-4-propan-2-ylcyclohexa-1,4-diene Substances 0.000 claims 1
- 244000056139 Brassica cretica Species 0.000 claims 1
- 235000003351 Brassica cretica Nutrition 0.000 claims 1
- 235000003343 Brassica rupestris Nutrition 0.000 claims 1
- QKSKPIVNLNLAAV-UHFFFAOYSA-N bis(2-chloroethyl) sulfide Chemical compound ClCCSCCCl QKSKPIVNLNLAAV-UHFFFAOYSA-N 0.000 claims 1
- 235000010460 mustard Nutrition 0.000 claims 1
- 150000007875 phellandrene derivatives Chemical class 0.000 claims 1
- 230000009261 transgenic effect Effects 0.000 abstract description 14
- 241000219194 Arabidopsis Species 0.000 abstract description 7
- 102000004169 proteins and genes Human genes 0.000 abstract description 7
- 150000003505 terpenes Chemical class 0.000 abstract description 7
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- 230000001131 transforming effect Effects 0.000 abstract description 4
- 239000000126 substance Substances 0.000 abstract description 3
- 108090000790 Enzymes Proteins 0.000 abstract description 2
- 102000004190 Enzymes Human genes 0.000 abstract description 2
- 229930004725 sesquiterpene Natural products 0.000 abstract description 2
- 150000004354 sesquiterpene derivatives Chemical class 0.000 abstract description 2
- 150000001413 amino acids Chemical class 0.000 abstract 1
- 238000010195 expression analysis Methods 0.000 abstract 1
- 238000003757 reverse transcription PCR Methods 0.000 abstract 1
- 239000002299 complementary DNA Substances 0.000 description 7
- LFJQCDVYDGGFCH-UHFFFAOYSA-N β-phellandrene Chemical compound CC(C)C1CCC(=C)C=C1 LFJQCDVYDGGFCH-UHFFFAOYSA-N 0.000 description 7
- 238000003753 real-time PCR Methods 0.000 description 6
- 238000011529 RT qPCR Methods 0.000 description 5
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- ZJPGOXWRFNKIQL-JYJNAYRXSA-N Phe-Pro-Pro Chemical compound C([C@H](N)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(O)=O)C1=CC=CC=C1 ZJPGOXWRFNKIQL-JYJNAYRXSA-N 0.000 description 3
- 125000003275 alpha amino acid group Chemical group 0.000 description 3
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- VWFJDQUYCIWHTN-YFVJMOTDSA-N 2-trans,6-trans-farnesyl diphosphate Chemical compound CC(C)=CCC\C(C)=C\CC\C(C)=C\CO[P@](O)(=O)OP(O)(O)=O VWFJDQUYCIWHTN-YFVJMOTDSA-N 0.000 description 1
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- 241000208173 Apiaceae Species 0.000 description 1
- 101710137820 Beta-caryophyllene synthase Proteins 0.000 description 1
- VWFJDQUYCIWHTN-FBXUGWQNSA-N Farnesyl diphosphate Natural products CC(C)=CCC\C(C)=C/CC\C(C)=C/COP(O)(=O)OP(O)(O)=O VWFJDQUYCIWHTN-FBXUGWQNSA-N 0.000 description 1
- 241000212324 Oenanthe <angiosperm> Species 0.000 description 1
- 102000019337 Prenyltransferases Human genes 0.000 description 1
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- 125000004030 farnesyl group Chemical group [H]C([*])([H])C([H])=C(C([H])([H])[H])C([H])([H])C([H])([H])C([H])=C(C([H])([H])[H])C([H])([H])C([H])([H])C([H])=C(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
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- 238000010353 genetic engineering Methods 0.000 description 1
- NUHSROFQTUXZQQ-UHFFFAOYSA-N isopentenyl diphosphate Chemical compound CC(=C)CCO[P@](O)(=O)OP(O)(O)=O NUHSROFQTUXZQQ-UHFFFAOYSA-N 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- CBIDRCWHNCKSTO-UHFFFAOYSA-N prenyl diphosphate Chemical compound CC(C)=CCO[P@](O)(=O)OP(O)(O)=O CBIDRCWHNCKSTO-UHFFFAOYSA-N 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- -1 terpenoid compounds Chemical class 0.000 description 1
- 230000017260 vegetative to reproductive phase transition of meristem Effects 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 108700026220 vif Genes Proteins 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
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- C12Y402/00—Carbon-oxygen lyases (4.2)
- C12Y402/03—Carbon-oxygen lyases (4.2) acting on phosphates (4.2.3)
- C12Y402/03057—Beta-caryophyllene synthase (4.2.3.57)
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Abstract
An OjTPS1 gene sequence related to water fennel beta-caryophyllene synthesis and application thereof. Beta-caryophyllene is a sesquiterpene substance in plants and has multiple functions. TPS is a key enzyme in the plant terpene synthesis process, and is responsible for forming different terpene skeletons so as to generate various terpenes. The application clones a new OjTPS1 gene from cress. The gene is 1692bp long, can code 563 amino acids, and has a DDxxD structural domain of typical TPS family proteins. The fluorescent quantitative expression analysis shows that the expression of the OjTPS1 gene has tissue specificity and the highest expression quantity in flowers. Genetically transforming the OjTPS1 gene into arabidopsis, the transgenic plants are able to produce higher levels of β -caryophyllene. The application clones an OjTPS1 gene from cress by using RT-PCR technology, and can improve the beta-caryophyllene content of plants.
Description
Technical Field
The application belongs to the technical field of plant genetic engineering, and particularly relates to an OjTPS1 gene sequence related to water fennel beta-caryophyllene synthesis and application thereof.
Background
The cress (Oenanthe javanica (BI.) DC) is an alias cress, a perennial herb perennial root plant of the genus Oenanthe in Umbelliferae, native to China, southeast Asia, etc. (Lu et al., food Chem 2015,6495819). The cress mainly takes petioles and tender stems as edible parts, has higher nutritional value, is rich in various vitamins, carbohydrates, proteins, crude fibers and the like, and is deeply favored by consumers (Feng et al, planta 2018, 247:301-315). Oenanthe Javanica is a special vegetable crop with a unique flavor due to the production of large amounts of terpenoid substances (Seo et al, J Agric Food Chem 2005, 53:6766-6770).
Beta-caryophyllene belongs to terpenoid compounds, is an important volatile sesquiterpene substance in plants, and has a faint scent taste (Skold et al Food Chem Toxicol 2006, 44:538-545). Beta-caryophyllene is also widely used in the cosmetic and fragrance industries due to its special fragrance (Bandna et al, nat Prod Res 2009, 23:1445-1450). TPS is a key enzyme in the plant terpene synthesis process and is responsible for the formation of different terpene backbones and thus different terpenes (Christianson et al, chem Rev 2017, 117:11570-11648). Farnesyl (farnesyl diphosphate, FPP) produced by prenyltransferases catalyzing the production of IPP and DMAPP is a direct substrate for the synthesis of beta-caryophyllene, which is capable of catalyzing FPP to produce beta-caryophyllene (Tholl et al, plant J2005, 42:757-771). DcTPS1 is capable of catalyzing the production of β -caryophyllene with FPP as a substrate, which is associated with the formation of a specific flavour of carrot (Yahyaa et al, J Agric Food Chem 2015, 63:4870-4878). At present, TPS genes related to beta-caryophyllene synthesis in cress are not reported yet.
Disclosure of Invention
This section is intended to outline some aspects of embodiments of the application and to briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section as well as in the description of the application and in the title of the application, which may not be used to limit the scope of the application.
The present application has been made in view of the above and/or problems occurring in the prior art.
Therefore, the application aims to overcome the defects in the prior art and provide an OjTPS1 gene sequence related to the synthesis of cress beta-caryophyllene.
In order to solve the technical problems, the application provides the following technical scheme: ojTPS1 gene sequence related to water fennel beta-caryophyllene synthesis comprises the gene sequence shown as Seq ID No. 1.
It is another object of the present application to provide an application of OjTPS1 gene sequence related to water fennel beta-caryophyllene synthesis. Which comprises the steps of the following steps of,
selecting plant material: selecting an OjTPS1 gene-derived plant and a final transformed recipient plant;
extraction of total RNA and Synthesis of cDNA: extracting total RNA of a source plant, and reversing the total RNA into cDNA;
cloning of OjTPS1 Gene: designing cloning primers, cloning by using the inverted cDNA as a template and connecting the cDNA into a vector by using PCR;
sequence analysis: sequencing, comparing the sequences of OjTPS1 genes of cress, analyzing, comparing and drawing a evolutionary tree;
qPCR amplification: designing a primer for performing a fluorescent quantitative PCR test, and detecting the expression quantity of the OjTPS1 gene in different tissues of a source plant;
construction of vectors and transformation into recipient plant cells: designing a primer, realizing the connection of the OjTPS1 gene into a vector by using a homologous recombination technology, and transforming the recombinant vector into a receptor plant cell;
identification of the transformation and determination of the content of beta-caryophyllene: screening and identifying by using a marker gene according to the screening marker on the carrier, and determining the content of the beta-caryophyllene.
As a preferred scheme for the application of the OjTPS1 gene sequence related to the synthesis of phellandrene, β -caryophyllene, according to the present application, wherein: in the plant material, the source plant is cress and the receptor plant is arabidopsis thaliana.
As a preferred scheme for the application of the OjTPS1 gene sequence related to the synthesis of phellandrene, β -caryophyllene, according to the present application, wherein: in the cloning of the OjTPS1 gene, the cloning primer is a forward primer: 5'-ATGGCTCTTAATGTTCTAGC-3', reverse primer: 5'-TCATAATGGAACTGAATCAA-3'.
As a preferred scheme for the application of the OjTPS1 gene sequence related to the synthesis of phellandrene, β -caryophyllene, according to the present application, wherein: in qPCR amplification, the primers designed were forward primers: 5'-CGCAGGTCGGCTAATTATCATCCAA-3', reverse primer: 5'-GTTGTTCCACTGTGTCTGAGTCATCTT-3'.
As a preferred scheme for the application of the OjTPS1 gene sequence related to the synthesis of phellandrene, β -caryophyllene, according to the present application, wherein: constructing a vector, transforming the vector into a receptor plant cell, connecting an OjTPS1 gene into the vector by utilizing a homologous recombination technology, and designing a forward primer: 5'-TTTACAATTACCATGGGATCCATGGCTCTTAATGTTCTAGC-3', reverse primer: 5'-ACCGATGATACGAACGAGCTCTCATAATGGAACTGAATCAA-3' the OjTPS1 gene was ligated into the vector.
As a preferred scheme for the application of the OjTPS1 gene sequence related to the synthesis of phellandrene, β -caryophyllene, according to the present application, wherein: constructing a vector and transforming the vector into a receptor plant cell, wherein the vector is pCAMBIA-1301 vector.
As a preferred scheme for the application of the OjTPS1 gene sequence related to the synthesis of phellandrene, β -caryophyllene, according to the present application, wherein: the screening mark on the carrier is hygromycin, and the transformation is identified and the content of beta-caryophyllene is measured.
As a preferred scheme for the application of the OjTPS1 gene sequence related to the synthesis of phellandrene, β -caryophyllene, according to the present application, wherein: in qPCR amplification, the OjTPS1 gene was expressed in the highest amount in flowers.
The application has the beneficial effects that:
(1) The application provides a preparation method and application of an OjTPS1 gene, the obtained OjTPS1 gene can be expressed in arabidopsis thaliana, the beta-caryophyllene content of a transgenic plant can be obviously improved, the synthesis mechanism of beta-caryophyllene can be further known, and meanwhile, the obtained plant has higher beta-caryophyllene content.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings that are needed in the description of the embodiments will be briefly described below, it being obvious that the drawings in the following description are only some embodiments of the present application, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art. Wherein:
FIG. 1 is an evolutionary analysis of OjTPS1 protein of Oenanthe javanica and terpene synthase protein of other species according to the application;
FIG. 2 shows the gene analysis of OjTPS1 protein of Oenanthe Javanica and beta-caryophyllene synthetase of other species;
FIG. 3 is an analysis of the expression of OjTPS1 gene of OjTPS in different tissues of OjTPS;
FIG. 4 shows the transgenic verification by GUS-stained leaves prepared in the transgenic lines-1 and-2 prepared in the present application;
FIG. 5 shows the beta-caryophyllene content of the over-expressed OjTPS1 gene transgenic strain-1 and strain-2 Arabidopsis and wild type Arabidopsis prepared by the application.
Detailed Description
In order that the above-recited objects, features and advantages of the present application will become more apparent, a more particular description of the application will be rendered by reference to specific embodiments thereof which are illustrated in the appended drawings.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present application, but the present application may be practiced in other ways other than those described herein, and persons skilled in the art will readily appreciate that the present application is not limited to the specific embodiments disclosed below.
Further, reference herein to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic can be included in at least one implementation of the application. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments.
Plant material used in the present application: the cress is a cress variety of cress No.1, the arabidopsis thaliana is a Columbia wild type arabidopsis thaliana, and plant materials used are all stored in aquatic vegetable test bases of Yangzhou university.
The gene sequence of the OjTPS1 gene is shown as the sequence of the Seq ID No. 1.
Example 1
Extracting total RNA of water fennel and synthesizing cDNA: total RNA of Oenanthe javanica was extracted by means of a RNA simple Total RNA Kit (Tiangen company, beijing) kit. The total Oenanthe javanica RNA was inverted to cDNA using the HiScript III 1st Strand cDNA Synthesis Kit (Vazyme, nanjing) kit.
Example 2
Cloning of OjTPS1 Gene of OjTPS family: the amino acid sequence of carrot DcTPS1 is analyzed in a genome and a transcriptome of cress to obtain an OjTPS1 gene related to beta-caryophyllene synthesis, and a pair of cloning primers are designed according to the obtained OjTPS1 gene. Among the primers designed, the forward primer: 5'-ATGGCTCTTAATGTTCTAGC-3', reverse primer: 5'-TCATAATGGAACTGAATCAA-3'. The cDNA obtained in example 1 was used as a template, the OjTPS1 gene was cloned by PCR, and the resulting mixture was subjected to agarose gel electrophoresis analysis, and then ligated with pMD19-T vector (Dalian TaKaRa Co.) and sequenced.
Example 3
Sequence analysis: the sequence of the OjTPS1 gene of cress is obtained by sequencing, and the coded amino acid sequence is analyzed. Amino acid sequence alignment was performed by DNAMAN software and the evolutionary tree was drawn using MEGA 7.0 software.
Example 4
Real-time quantitative PCR reaction: according to the sequence of the OjTPS1 gene of cress, a pair of real-time fluorescent quantitative PCR primers are designed by using Primer 6.0 software: forward primer: 5'-CGCAGGTCGGCTAATTATCATCCAA-3', reverse primer: 5'-GTTGTTCCACTGTGTCTGAGTCATCTT-3'. The amount of OjTPS1 gene expressed in different cress tissues was detected by performing a fluorescent quantitative PCR assay on a CFX96 Real-time PCR system instrument using ChamQ Universal SYBR qPCR Master Mix (Vazyme, nanjing).
Example 5
Construction of a transgenic vector and transformation of Arabidopsis thaliana: according to the sequence of the OjTPS1 gene, a forward primer is designed: 5'-TTTACAATTACCATGGGATCCATGGCTCTTAATGTTCTAGC-3', reverse primer: 5'-ACCGATGATACGAACGAGCTCTCATAATGGAACTGAATCAA-3'. The OjTPS1 gene is connected to a pCAMBIA-1301 vector by utilizing a homologous recombination technology, the genetic transformation of arabidopsis thaliana is carried out by adopting a dipping method after the agrobacterium GV3101 is transformed by the recombinant vector, and specific genetic transformation operation parameters are shown in Zhang et al, nat Protoc 2006,1:641-646.
Example 6
Identification of transgenic Arabidopsis thaliana and determination of beta-caryophyllene content: and (3) screening and identifying the transgenic arabidopsis thaliana by using hygromycin according to the screening mark on the carrier. Wild-type and transgenic arabidopsis flowers were sampled after flowering of arabidopsis, and the β -caryophyllene content was determined using gas chromatography-mass spectrometry (GC-MS).
Example 7
The protein sequence coded by the OjTPS1 gene cloned by the application and terpene synthase proteins derived from other species are analyzed, a phylogenetic tree is constructed, the specific operation is shown in an example 3, the obtained phylogenetic tree is shown in a figure 1, and the result shows that the OjTPS1 protein and other beta-caryophyllene synthases belong to TPS-a subfamily and have the latest evolutionary relationship with carrot DcTPS 1. Further sequence alignment and evolutionary analysis of OjTPS1 with other species of beta-caryophyllene synthetases is shown in FIG. 2, which shows that OjTPS1 contains the DDxxD domain of a typical TPS family protein.
For the fluorescence quantitative PCR detection of different tissues of cress, the specific operation is shown in example 4, the obtained fluorescence quantitative PCR data is shown in fig. 3, the expression of OjTPS1 gene in cress has tissue specificity according to fig. 3, the expression in flowers is obviously higher than that in other tissues, and the expression quantity in roots is the lowest. The OjTPS1 gene is genetically transformed in arabidopsis thaliana by utilizing a transgenic technology, and a transgenic strain is identified by hygromycin and GUS staining, so that a strain-1 and a strain-2 are obtained.
The beta-caryophyllene content of the obtained over-expressed OjTPS1 gene Arabidopsis and wild type Arabidopsis is further verified, the cultivated leaves are recorded in the graph in FIG. 4 together, the wild type color on the left side is yellow, the transgenic strain-1 and the strain-2 on the right side are obviously GUS blue, and meanwhile, the strain-2 is obviously darker in dyeing compared with the strain-1. The beta-caryophyllene content measurement of the wild type and the transgenic arabidopsis thaliana is shown in fig. 5, and the result shows that the beta-caryophyllene content of the transgenic arabidopsis thaliana over-expressing the OjTPS1 gene is obviously higher than that of the wild type arabidopsis thaliana.
It should be noted that the above embodiments are only for illustrating the technical solution of the present application and not for limiting the same, and although the present application has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present application may be modified or substituted without departing from the spirit and scope of the technical solution of the present application, which is intended to be covered in the scope of the claims of the present application.
Sequence listing
<110> university of Yangzhou
<120> OjTPS1 gene sequence related to Oenanthe javanica beta-caryophyllene synthesis and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1692
<212> DNA/RNA
<213> Artificial sequence (Artificial Sequence)
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atggctctta atgttctagc aacttctgct tctgccaaaa ctgttactcc tcagcttact 60
cgcaggtcgg ctaattatca tccaagcgtt tggggagaca agttcctagc atacgattgc 120
tctaatcacg ggaaagatga ctcagacaca gtggaacaac tcgaaaaact gaaagaagaa 180
attaagacga cgttggtgca aacagccgaa gaaccaaaac agctgatcaa cttgattgat 240
ggtatccaac gattgggtat ttcgtaccat ttcgaagctg agattgagac tctgttgcag 300
cacatgaacg atttctatga tgaattttgc ggtggtaatg atgttgatga ccttcatgat 360
gttgcactat gttttcgact actaaggcaa gccggacata aggtgtcctc taataagttc 420
ttcaaataca tggatgcaaa caggaagttc aaagagtgct tgcttaggga tactcgagga 480
ctgttaagct tgtatgaagc aacacatttt atggttcatg aagaagagat attggaagag 540
gcacttcagt tcaccacctt tcacctagag aatctcaagt ctgacttgaa caatcctctt 600
gcaagccaga tctctcatgc acttaagtat ccgattcgaa agaatttaaa caggcttgga 660
gtaaggcaat acatttctgc tcaagaaaag aatgattcgt cgcataatga agttcttaaa 720
tttgcgaaat tggacttcaa tcggttgcag aaaatgtatc aacaagagat aggccatatt 780
accaggtggt ggaagaattt aaattttgct gtgaaacttc catttgcaag ggatagggtg 840
gtggagtgtt acttctggat attaggggtg tactatgagc cccaatattg catagcccga 900
agatttctta cgaaagtaat tatgcttgca tcggttgttg atgacatata tgatttgtat 960
ggtaccttcg aagaactttt actcttcact gatgcaattg aaagatggga caccgatgct 1020
tttgatctgc tacccgatta catgaaagtt tgttatcagg ctctccttga tacttatagt 1080
gaaatggagg aagtgctaga aaaggaaggt ggaacaccag tataccgtgt tcacgaagca 1140
aaaaaatcat ttaataggtt agccaaggca tatctggatg aagctaagtg gtttaaagaa 1200
ggttactatc caactacaga ggagtatatg aatgtggcac tcgtgtccgc aggctatgga 1260
acgatggcta caaattcgtt tgttggaatg ggggacgagg caacaaggga agccttccaa 1320
tgggtgtcta atgatccttt gattgttcga gcttcatcct taattgcaag actatgtgat 1380
gacatgacag gccatgagtt tgagcaggag aaaggagacg taccctcagc tgttgagtgc 1440
tacatgaaac aacatggagc tacaaaacat gaggcatatt ctgagttgca gaaaagggtt 1500
acaaatgctt ggaaggatat aaatcaggag tgtctcaatc gaactaatat tccattgtct 1560
ctccttgcaa gaatcgataa tcttacgaga gccataaata tactctatga tggtgatgat 1620
ggctacacac attcatcaac aaggacaaaa gatcttataa catcggtgct cattgattca 1680
gttccattat ga 1692
Claims (6)
1. Related to the synthesis of phellandrene beta-caryophylleneOjTPS1A gene characterized in that: the saidOjTPS1The gene sequence is shown in Seq ID No. 1.
2. Overexpression of claim 1OjTPS1Gene in improving cress or south-lookingUse of the content of mustard beta-caryophyllene.
3. A method for preparing a plant having an increased β -caryophyllene content, characterized by: cloning of claim 1OjTPS1Genes, construction of the sameOjTPS1The over-expression vector of the gene is transformed into a receptor plant cell, and the plant with the increased beta-caryophyllene content is screened and identified; the plant is Arabidopsis thaliana and Oenanthe Javanica.
4. A method according to claim 3 for the preparation of plants with increased β -caryophyllene content, characterized in that:OjTPS1in the cloning of the gene, the cloning primer is a forward primer: 5'-ATGGCTCTTAATGTTCTAGC-3', reverse primer: 5'-TCATAATGGAACTGAATCAA-3'.
5. A method according to claim 3 for the preparation of plants with increased β -caryophyllene content, characterized in that: the over-expression vector is pCAMBIA-1301 vector.
6. A method according to claim 3 for the preparation of plants with increased β -caryophyllene content, characterized in that: in the identification of transformation and the content measurement of beta-caryophyllene, the screening mark on the carrier is hygromycin.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110093352A (en) * | 2018-01-29 | 2019-08-06 | 南京农业大学 | One kind transcription factor AgMYB1 gene order relevant to the synthesis of celery anthocyanidin and its application |
| CN110093328A (en) * | 2018-01-29 | 2019-08-06 | 南京农业大学 | One kind CAD gene order relevant to Chinese celery lignin synthesis and its application |
| CN111004763A (en) * | 2019-12-26 | 2020-04-14 | 中国科学院青岛生物能源与过程研究所 | Engineering bacterium for producing β -caryophyllene and construction method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110093352A (en) * | 2018-01-29 | 2019-08-06 | 南京农业大学 | One kind transcription factor AgMYB1 gene order relevant to the synthesis of celery anthocyanidin and its application |
| CN110093328A (en) * | 2018-01-29 | 2019-08-06 | 南京农业大学 | One kind CAD gene order relevant to Chinese celery lignin synthesis and its application |
| CN111004763A (en) * | 2019-12-26 | 2020-04-14 | 中国科学院青岛生物能源与过程研究所 | Engineering bacterium for producing β -caryophyllene and construction method and application thereof |
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