CN112410362A - Rhododendron micranthum TPS gene, primer for cloning Rhododendron micranthum TPS gene, cloning method and application thereof - Google Patents
Rhododendron micranthum TPS gene, primer for cloning Rhododendron micranthum TPS gene, cloning method and application thereof Download PDFInfo
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Abstract
The invention discloses a rhododendron parviflorum TPS gene, a primer for cloning the rhododendron parviflorum TPS gene, a cloning method and application thereof, wherein the rhododendron parviflorum TPS gene is a terpene synthase gene RhTPS, the nucleotide sequence of a conserved domain of the terpene synthase gene RhTPS is shown as SEQ ID No.1, the full-length nucleotide sequence is shown as SEQ ID No.2, and the full-length amino acid sequence is shown as SEQ ID No. 3. The rhododendron terpene synthase gene RhTPS is cloned from rhododendron pulchrum for the first time, is one of key genes for forming the fragrance of the rhododendron, can be cultured by biotechnological methods such as transgenosis and the like, provides an important theoretical basis for improving the quality of the rhododendron by utilizing a genetic engineering technology in the future, and has wide application prospect and great economic value. The invention lays a foundation for the subsequent researches on the exact functions of the TPS gene of the rhododendron yunnanensis petals, the involved metabolic regulation and control approaches of the terpene compounds and the like.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a rhododendron yunnanensis TPS gene, a primer for cloning the rhododendron yunnanensis TPS gene, a cloning method and application thereof.
Background
The azalea is evergreen or deciduous shrub in the Ericaceae and the Rhododendron, is also a famous ornamental flower in the world, is widely applied to garden planting and indoor beautification, but the research on the aroma and aroma forming mechanism of the azalea is rarely reported at present. China has abundant azalea resources, but only a few azalea varieties can generate flower fragrance, such as alpine azalea variety-brocade azalea. The flower fragrance is an important character of ornamental plants and is one of the main bases for evaluating the quality of flowers. Floral scents are volatile low molecular weight compounds emitted by plants, which emit aromatic substances in parts of their vegetative and floral organs. The flower fragrance emission amount is different according to the growth stage of the flower, and the flower fragrance can attract insects to complete pollination, resist biotic stress, natural stress caused by environmental change and the like. The study on flower fragrance is relatively lagged at present compared with plant traits such as flower type and flower color which are easy to observe.
According to the analysis of the components of the floral aroma compounds, the main substances in the plant aroma components are terpene compounds, and the terpene compounds in the plant aroma are various. The arabidopsis genome sequence analysis result shows that 75% of terpene synthase genes in different terpene synthase genes have similarity in gene structure and sequence correlation, and the difference in structure and function is reflected. Terpene compounds are the main components of aromatic compounds of plants with fragrance such as osmanthus fragrans, lily, carnation and the like, and Terpene synthase (TPS) is an important enzyme for biosynthesis of Terpene compounds.
Researches show that TPS genes of fragrant plants have important influence on the formation and fragrance release of fragrant compounds, but researches on related TPS genes in rhododendron parvifolium and the synthesis relation of the TPS genes and terpene compounds are not reported, and the research on the cloning and other molecules of the TPS genes of rhododendron parvifolium is necessary, so that the invention provides a primer for cloning the TPS genes of rhododendron parvifolium, a cloning method and application thereof.
Disclosure of Invention
The invention aims to solve the technical problem of providing a rhododendron yunnanensis TPS gene, a primer for cloning the rhododendron yunnanensis TPS gene, a cloning method and application thereof aiming at the defects of the prior art.
The technical scheme adopted by the invention for solving the technical problems is as follows: the rhododendron parviflora TPS gene is a terpene synthase gene RhTPS, and the nucleotide sequence of a conserved domain of the terpene synthase gene RhTPS is shown as SEQ ID No. 1.
The Rhododendron micranthum TPS gene is terpene synthase gene RhTPS, and the full-length nucleotide sequence of the terpene synthase gene RhTPS is shown as SEQ ID NO. 2.
Furthermore, the full-length nucleotide sequence of the terpene synthase gene RhTPS is a gene sequence which is obtained by substituting, deleting and/or adding one or more bases to the nucleotide sequence shown in SEQ ID NO.2 and has terpene synthase activity.
The Rhododendron micranthum TPS gene is terpene synthase gene RhTPS, and the full-length amino acid sequence of the terpene synthase gene RhTPS is shown as SEQ ID NO. 3.
Furthermore, the full-length amino acid sequence of the terpene synthase gene RhTPS is a protein which is derived from the amino acid sequence shown in SEQ ID NO.3, is subjected to substitution, deletion and/or addition of one or more amino acids, has terpene synthase activity and is derived from the amino acid sequence shown in SEQ ID NO. 3.
A primer group for cloning a rhododendron parvifolium TPS gene comprises TPS-F1, TPS-R1, TPS-F2, TPS-R2, TPS-F3 and TPS-R3, wherein TPS-F1 and TPS-R1 are used for amplifying a nucleotide sequence shown in SEQ ID No.1, TPS-F2 and TPS-R2 are used for amplifying a nucleotide sequence shown in SEQ ID No.2, TPS-F3 and TPS-R3 are nucleotide sequences designed according to the nucleotide sequence shown in SEQ ID No.1 and used for qRT-PCR amplification, and the sequences of the primer group are as follows:
TPS-F1：CTCATTGACWCWATKCAAMGGY；
TPS-R1：CTTRGAAGTKCCCAAATCATCA；
TPS-F2：CCAGGTCCATCCACCACTTGGCTAGTTCTTGTA；
TPS-R2：ACGCTTGCCTCTTGCTAGGGTCTCCGATAA；
TPS-F3：CGCCAGAACTTTCAGTGTCAAGCATTT；
TPS-R3：TCGTGCTTCTAACCTCGGCATTCT。
the method for cloning the Rhododendron brocade TPS gene comprises the following steps:
step one, extracting total RNA of rhododendron marianum petals by adopting a polysaccharide polyphenol plant total RNA extraction kit, determining the integrity and concentration of the total RNA by using agarose gel electrophoresis with the mass concentration of 1.5% and a nucleic acid protein analyzer respectively, and synthesizing a cDNA first chain according to the instruction of a TransScript Reverse Transcriptase Reverse transcription kit;
step two, downloading the full-length sequence of the TPS gene of a plant with close relationship with the rhododendron plant from GenBank, carrying out multi-sequence comparison by adopting ClustalW, designing degenerate primers TPS-F1 and TPS-R1 by utilizing Primer 5.0 in a sequence with higher homology, wherein the sequences of Primer groups TPS-F1 and TPS-R1 are as follows:
TPS-F1：CTCATTGACWCWATKCAAMGGY；
TPS-R1：CTTRGAAGTKCCCAAATCATCA；
taking the first cDNA chain as a template, and carrying out PCR amplification reaction by using degenerate primers TPS-F1 and TPS-R1, wherein the reaction procedure of the PCR amplification reaction is as follows: 3min at 94 ℃; 30Cycles at 94 ℃ for 30s, 55 ℃ for 30s, and 72 ℃ for 1 min; obtaining a PCR product at 72 ℃ for 5 min; and (3) performing sequence determination on the PCR product after recovery, pMD18-T Vector Cloning Kit connection, DH5 alpha competent cell transformation, recombinant screening and bacterial liquid PCR identification to obtain the nucleotide sequence of the rhododendron yunnanensis terpene synthase gene RhTPS conserved domain, namely SEQ ID No. 1.
Further, RACE amplification primers TPS-F2 and TPS-R2 are designed according to the nucleotide sequence of the conserved domain of rhododendron yunnanensis terpene synthase gene RhTPS, and the sequences of TPS-F2 and TPS-R2 are as follows:
TPS-F2：CCAGGTCCATCCACCACTTGGCTAGTTCTTGTA；
TPS-R2：ACGCTTGCCTCTTGCTAGGGTCTCCGATAA；
respectively amplifying the 5 'end full length and the 3' end full length of the terpene synthase gene RhTPS by the following method: synthesizing RACE-cDNA according to the instruction of SMARTer RACE 5 '/3' Kit RACE Kit to respectively obtain 5'cDNA and 3' cDNA; taking 5' cDNA as a template, and carrying out PCR amplification reaction on the 5' tail end full length by utilizing RACE universal primers UPM and TPS-R2 to obtain a specific primer of the 5' tail end full length; taking 3' cDNA as a template, and carrying out PCR amplification reaction on the full length of the 3' tail end by utilizing RACE universal primers UPM and TPS-F2 to obtain a specific primer of the full length of the 3' tail end; comparing and splicing the conserved domain sequence, the 5 'terminal full-length sequence and the 3' terminal full-length sequence by using DNAMAN software to obtain a full-length nucleotide sequence with a full-length 1826bp sequence of SEQ ID NO. 2; coding 491 amino acids to obtain a full-length amino acid sequence with a sequence of SEQ ID NO. 3;
wherein, the reaction program for carrying out PCR amplification reaction on the 5 'end full length and the 3' end full length is as follows: 94 ℃ for 30s, 72 ℃ for 3min, 5 Cycles; 94 ℃ for 30s, 70 ℃ for 30s, 72 ℃ for 3min, 5 Cycles; 30Cycles at 94 ℃ for 30s, 68 ℃ for 30s, and 72 ℃ for 3 min; and (6) ending.
The fluorescence quantitative qRT-PCR method for the TPS gene of rhododendron micranthum comprises the following steps: designing primers TPS-F3 and TPS-R3 according to the nucleotide sequence SEQ ID NO.1 of a terpene synthase gene RhTPS conserved domain, wherein the sequences of TPS-F3 and TPS-R3 are as follows:
TPS-F3：CGCCAGAACTTTCAGTGTCAAGCATTT；
TPS-R3：TCGTGCTTCTAACCTCGGCATTCT；
a rhododendron EF1 alpha is used as an internal reference, internal reference gene primers RhEF1 alpha-F and RhEF1 alpha-R are designed, and the sequences of the RhEF1 alpha-F and the RhEF1 alpha-R are as follows:
RhEF1α-F：TGTGCCATCCTCATTATTGACTCC；
RhEF1α-R：ATGGGATCTTCTCGGGATTGTATC；
extracting total RNA of petals of rhododendron parvifolium at different flowering phases by adopting a polysaccharide polyphenol plant total RNA extraction kit, synthesizing qRT-PCR template cDNA according to the specification of a NovoScript Plus All-in-one1st Strand cDNA Synthesis Supermix (gDNA Purge) kit, respectively taking the cDNA as a template, referring to the specification of a Transstart Tip Green qPCRSuperMixS kit, and performing qRT-PCR amplification reaction by utilizing TPS-F3, TPS-R3, RhEF1 alpha-F and RhEF1 alpha-R, wherein the reaction program of the qRT-PCR amplification reaction is as follows: 30 ℃ at 94 DEG Cs; 94 ℃ for 5s, 59 ℃ for 30s, 42 Cycles; 5s at 65 ℃; 5s at 95 ℃; finishing; when each sample is amplified, internal references are amplified simultaneously, the relative gene expression quantity of each sample is the average value of 3 biological repeats, and 2 are adopted－ΔΔCTThe method is used for analyzing data and analyzing the expression quantity of terpene synthase gene RhTPS in different tissues.
The application of the rhododendron yunnanensis TPS gene in regulating and controlling the fragrance of rhododendron yunnanensis flowers is as follows: and (3) transforming the terpene synthase gene RhTPS into a flowerless rhododendron cell, and then culturing the transformed rhododendron cell.
Compared with the prior art, the invention has the following advantages:
(1) according to the invention, a primer for amplifying the conserved sequence of the TPS gene of the rhododendron yunnanensis is designed and obtained according to the nucleotide sequence of the TPS gene of other known species, and then 5 'and 3' end sequences of the TPS gene are amplified by utilizing an RACE technology according to the conserved sequence obtained by amplification, so that the full-length sequence of the TPS gene of the rhododendron yunnanensis is finally obtained for the first time. Fluorescent quantitative PCR analysis shows that the expression level of TPS gene of rhododendron micranthum petals basically rises and reaches a maximum value in a decay period, which is basically consistent with the trend of the content of volatile matters with flower fragrance in a flowering process, and is consistent with the change of the content of terpene compounds in the rhododendron micranthum petals, so that positive regulation and control effects of TPS on the synthesis of the terpene compounds can be indirectly proved. The invention lays a foundation for the subsequent researches on the exact functions of the TPS gene of the rhododendron yunnanensis petals, the involved metabolic regulation and control approaches of the terpene compounds and the like.
(2) The rhododendron terpene synthase gene RhTPS is cloned from rhododendron pulchrum for the first time, is one of key genes for forming the fragrance of the rhododendron, can be cultured by biotechnological methods such as transgenosis and the like, provides an important theoretical basis for improving the quality of the rhododendron by utilizing a genetic engineering technology in the future, and has wide application prospect and great economic value.
Drawings
FIG. 1 shows conserved domain of TPS gene of rhododendron yunnanensis petals;
FIG. 2 shows the 5' terminal region of the floral rose petal TPS gene;
FIG. 3 shows the 3' terminal region of the floral Rhododendron petal TPS gene;
FIG. 4 is a diagram of the alignment of the TPS amino acid sequence of the rhododendron yunnanensis petals with the TPS amino acid sequence of other species;
FIG. 5 is a diagram showing the expression level of TPS gene in petals of Rhododendron micranthum at different flowering stages.
Detailed Description
The invention is described in further detail below with reference to the accompanying examples.
Example 1: the rhododendron parviflora TPS gene is terpene synthase gene RhTPS, the nucleotide sequence of the conserved domain of the terpene synthase gene RhTPS is shown as SEQ ID NO.1, the full-length nucleotide sequence is shown as SEQ ID NO.2, and the full-length amino acid sequence is shown as SEQ ID NO. 3. The method for cloning the Rhododendron brocade TPS gene comprises the following steps:
firstly, selecting petals of a rhododendron yunnanensis bud period, grinding the petals with liquid nitrogen, weighing 0.1g of the petals, extracting total RNA of the rhododendron yunnanensis petals by adopting a total RNA extraction kit suitable for plants with higher polysaccharide and polyphenol content, respectively determining that the quality meets the requirements by agarose gel electrophoresis with the mass concentration of 1.5% and a nucleic acid protein analyzer (Shanghai Saimei Feishell science), and freezing and storing the petals at the temperature of-80 ℃ for later use;
step two, downloading full-length sequences of TPS genes of plants close to the relativity of rhododendron plants from GenBank, performing multi-sequence alignment by using ClustalW, selecting sequences with high homology, and designing degenerate primers according to a degenerate Primer design method (wherein R & ltA/G & gt, Y & ltC/T & gt, M & ltA/C & gt, K & ltG/T & gt, S & ltC/G & gt, W & ltA/T & gt, H & ltA/C/T & gt, B & ltC/G/T & gt, V & ltA/C/G & gt, D & ltA/G/T & gt, N & ltA/C/G/T & gt), and designing degenerate primers TPS-F1 and TPS-R1 by using Primer 5.0, wherein the sequences of Primer groups TPS-F1 and TPS-R1 are as follows:
TPS-F1：CTCATTGACWCWATKCAAMGGY；
TPS-R1：CTTRGAAGTKCCCAAATCATCA；
step three, synthesizing a first chain of petal cDNA of the rhododendron micranthum in the bud stage according to the instruction of a TransScript Reverse Transcriptase Transcriptase Reverse transcription kit (Beijing all-style gold biotechnology limited); taking the first strand of cDNA as a template, and carrying out PCR amplification reaction by using degenerate primers TPS-F1 and TPS-R1, wherein the reaction procedure of the PCR amplification reaction is as follows: 3min at 94 ℃; 30Cycles at 94 ℃ for 30s, 55 ℃ for 30s, and 72 ℃ for 1 min; obtaining a PCR product at 72 ℃ for 5min, and detecting the PCR product by electrophoresis, wherein the result is shown in figure 1; after the PCR product is recovered, connected with pMD18-T Vector Cloning Kit, transformed by DH5 alpha competent cells, screened by recombinants and identified by bacteria liquid PCR, the sequence is determined by the company of Biotechnology engineering (Shanghai) GmbH to obtain the nucleotide sequence of the conserved domain of rhododendron yunnanensis terpene synthase gene RhTPS, namely SEQ ID NO. 1.
Designing RACE amplification primers TPS-F2 and TPS-R2 according to the nucleotide sequence of a rhododendron yunnanensis terpene synthase gene RhTPS conserved domain, wherein the sequences of TPS-F2 and TPS-R2 are as follows:
TPS-F2：CCAGGTCCATCCACCACTTGGCTAGTTCTTGTA；
TPS-R2：ACGCTTGCCTCTTGCTAGGGTCTCCGATAA；
respectively amplifying the 5 'end full length and the 3' end full length of the terpene synthase gene RhTPS by the following method: RACE-cDNA was synthesized according to the instructions of SMARTer RACE 5 '/3' Kit RACE Kit (Takara Shuzo Co., Ltd.) to obtain 5'cDNA and 3' cDNA, respectively; taking 5' cDNA as a template, and carrying out PCR amplification reaction on the 5' tail end full length by utilizing RACE universal primers UPM and TPS-R2 to obtain a specific primer of the 5' tail end full length; taking 3' cDNA as a template, and carrying out PCR amplification reaction on the full length of the 3' tail end by utilizing RACE universal primers UPM and TPS-F2 to obtain a specific primer of the full length of the 3' tail end; comparing and splicing the conserved domain sequence, the 5 'terminal full-length sequence and the 3' terminal full-length sequence by using DNAMAN software to obtain a full-length nucleotide sequence with a full-length 1826bp sequence of SEQ ID NO. 2; encodes 491 amino acids, and obtains a full-length amino acid sequence with a sequence of SEQ ID NO. 3. The PCR product was detected by electrophoresis, and the results of amplified 5 'ends are shown in FIG. 2 and the results of amplified 3' ends are shown in FIG. 3. The alignment chart of the TPS amino acid sequence of rhododendron yunnanensis petals and other species TPS amino acid sequences is shown in figure 4.
The above reaction procedure for performing PCR amplification reaction on the 5 'end full length and the 3' end full length was: 94 ℃ for 30s, 72 ℃ for 3min, 5 Cycles; 94 ℃ for 30s, 70 ℃ for 30s, 72 ℃ for 3min, 5 Cycles; 30Cycles at 94 ℃ for 30s, 68 ℃ for 30s, and 72 ℃ for 3 min; and (6) ending.
Example 2: fluorescent quantitative PCR detection of TPS expression quantity in petals of rhododendron micranthum at different flowering stages
Designing primers TPS-F3 and TPS-R3 according to the nucleotide sequence SEQ ID NO.1 of a terpene synthase gene RhTPS conserved domain, wherein the sequences of TPS-F3 and TPS-R3 are as follows:
TPS-F3：CGCCAGAACTTTCAGTGTCAAGCATTT；
TPS-R3：TCGTGCTTCTAACCTCGGCATTCT；
with rhododendron EF1 alpha (DUH018457.1) as an internal reference, designing sequences of internal reference gene primers RhEF1 alpha-F and RhEF1 alpha-R, and RhEF1 alpha-F and RhEF1 alpha-R as follows:
RhEF1α-F：TGTGCCATCCTCATTATTGACTCC；
RhEF1α-R：ATGGGATCTTCTCGGGATTGTATC；
extracting total RNA of petals of rhododendron parvifolium at different flowering stages (bud stage, half-bloom stage, full-bloom stage and decay stage) by adopting the method in the step one in the embodiment 1, synthesizing qRT-PCR template cDNA according to the instruction of NovoScript Plus All-in-one1st Strand cDNA Synthesis SuperMix (near bank protein technology Co., Ltd.), respectively taking the cDNA as a template, referring to the instruction of Transstart Tip Green qPCRSUPERMixS kit (Beijing All-gold biotechnology Co., Ltd.), and performing qRT-PCR amplification reaction by utilizing TPS-F3, hETPS-R3, RhEF1 alpha-F and RF 1 alpha-R, wherein the reaction program of the qRT-PCR amplification reaction is as follows: 30s at 94 ℃; 94 ℃ for 5s, 59 ℃ for 30s, 42 Cycles; 5s at 65 ℃; 5s at 95 ℃; and (6) ending. When each sample is amplified, internal references are amplified simultaneously, the relative gene expression quantity of each sample is the average value of 3 biological repeats, and 2 are adopted－ΔΔCTThe method is used for analyzing data, and analyzing the expression quantity of the TPS gene in petals of the rhododendron micranthum at different flowering phases, wherein the result is shown in an expression level chart of figure 5.
Sequence listing
<110> Zhejiang Wanli college
<120> rhododendron yunnanensis TPS gene, primer for cloning rhododendron yunnanensis TPS gene, cloning method and application thereof
<160> 3
<170> PatentIn version 3.1
<210> 1
<211> 1143
<212> DNA
<213> nucleotide sequence of conserved domain of terpene synthase gene RhTPS (SEQ ID NO. 1)
<400>1
CTCATTGACTCAATGCAACGGTTCGGAGTAGCTTACCATTTTGAGGAAGAGATTAAGGACGCTCTTAATCTTGCTCGATTTGACTACTTTACGGCCAACCTTTACACAACTTCACTGCAATTTCGACTTCGAAGAGACCATGGTTGTACCATCGGCTCAGAGGTGTTCAACAAATTCAAAAACGGAGATGGGAAATTCAAGGAGGGCCTAAGCAAAGATGCAGAAGGTCTTTTGAGTTTGTATGAAGCTTCACACTACGGAATGCAATGCGAAATTGACTTGGAAGCCGCCAGAACTTTCAGTGTCAAGCATTTGAACTCGTTAACTGGGAAAACGGAGATCGAATTAGCAGAGCAAGTTCAACAGTCAGTACTGGAATGTCCCATACGTTGGAGAATGCCGAGGTTAGAAGCACGAAACTTCATCGATCTCTACGAATCGGACAGCGCAAGAAACCCGCTGTTGCTGGAATTCGCTAAGTTGGATTACAACCTGGTGCAGTCAGTACACCAGAAAGAAGTACAAGAACTAGCCAAGTGGTGGATGGACCTGGGCTTGAAAGAAAAGCTTGGCTTTTCTAGGGACCGATCAATGGAGAATTACTTGTGGGCAATGGGAATGGTTTCCGAGTCCCATTTCTCCAATTGCAGGAAAGGCCTTACTAAATTTGTGTGCATACTATCAGCAATAGATGATATGTACGACATCTATGGATCTATCGACGAGCTTGAACGCTTTACTGATGCAGTGAACCGGTGGAGCGTCGAGGCCACAGATGATCTACCGGAGTACATGAAAATATGCTATTTGGCCATGTTCAACTTTGGAAATGAAATTGCTTACAATGTTCTGAGAGACCATGGCTTGAACGTGGTATCCTACATTAAGGAAGAGTGGTCAAATCTTTGCGGAGCATATTTGGTGGAAGCGCGGTGGTTTTACAGTGGGTTTATACCGACGTTGGAAGAGTACTTGAAAAACGCATGGAAATCAGTGGGTGGTCCTGCAGCCATAGCCCACGCTTGCCTCTTGCTAGGGTCTCCGATAACGAGAACTTCTCTTGATAGCTACAAAGCTAGCTCTGAGCTAATTTATTGGTCATCTATCATAACCAGACTAAGTGATGATTTGGGCACTTCCAAG
<210> 2
<211> 1826
<212> DNA
<213> full-Length nucleotide sequence of terpene synthase Gene RhTPS (SEQ ID NO. 2)
<400>2
ACATGGGGACAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAATGGCGGCAGAAGAAAACAAAGAAGAAGAAGATCTTCCATACGATGGAAGAAGAAGAAGAAGACGACGATCTGCTGTGCGTTCACGACCTGAGGTGTTCAACAAATTCAAAGACGGAGATGGGAACTTCAAGGAGGGCCTAAGCAAAGATGCAGAAGGTCTTTTGAGTTTGTATGAAGCTTCACACTACGGAATGCAATGCGAAATTGACTTGGAAGCCGCCAGAACTTTCAGTGTCAAGCATTTGAACTCGTTAACTGGGAAAATGGAGATCGAATTAGCGGAGCAAGTTCAACAGTCACTGGAATGTCCCATACGTTGGAGATTGCCGAGGTTAGAAGCACGAAACTTCATCGATCTCTACGAATCGGACAGCGCAAGAAACCCGCTGTTGCTGGAATTTGCTAAGTTGGATTACAACCTGGTGCAGTCAGTACACCAGAAAGAAGTACAAGAACTAGCCAAGTGGTGGATGGACCTGGGCTTGAAAGAAAAGCTTGGCTTTTCTAGGGACCGATCAATGGAGAATTACTTGTGGGCAATGGGAATGGTTTCCGAGTCCCATTTCTCCAATTGCAGGAAAGGCCTTACTAAATTTGTGTGCATACTATCAGCAATAGATGATATGTACGACATCTATGGATCTATCGACGAGCTTGAACGCTTTACTGATGCAGTGAACCGGTGGAGCGTCGAGGCCACAGATGATCTACCGGAGTACATGAAAATATGCTATTTGGCCATGTTCAACTTTGGAAATGAAATTGCTTACAATGTTCTGAGAGACCATGGCTTGAACGTGGTATCCTACATTAAGGAAGAGTGGTCAAATCTTTGCGGAGCATATTTGGTGGAAGCGCGGTGGTTTTACAGTGGGTTTATACCGACGTTGGAAGAGTACTTGAAAAACGCATGGAAATCAGTGGGTGGTCCTGCAGCCATAGCCCACGCTTGCCTCTTGCTAGGGTCTCCGATAACGAGAACTTCTCTTGATAGCTACAAAGCTAGCTCTGAGCTAATTTATTGGTCATCTATCATAACCAGACTAAGTGATGATTTGGGCACTTCCAAGGATGAGATTATGAGAGGAGACGTGGCGAAATCAATTCAATGTTATATGAACGAGCGGAGCGTGACAGAAGAACAAGCGCGAGATCACATCAAAGGTATTGTGCATGAATCATGGAGGAAACTTAATGAGACGATTGCACAAAATTCTCACCCTACACCAATGATTTCTATGTCATTAAACATGGCAAGGACTGCTCAGTGCATCTATCAACATGGAGATGGTATCGGTACGTCGCTTGGCGTGACCAAAGATCGTTTGACCTCATTAATCGTCGATCCCATTCCAATCGAGCGGCTTACTCATGGAGAAGTACGAGGAAGAACCCATAGTTATGCAGTGGAAAACGCGTACTCCATTAGCAAGTGAAGAAAAGAGAGCTATGTAAGATCTCTTTCTTTCTTTTTCTAAATGATCAAAAAATAAATTGTTTTCAAGTCACAAGATGATTGTGATACTAGTCAAGTCACAAGCATCAAAATTAAGTCGAACCAATCATCGACGTCTGATGTCAACTGATTTCATGATAATATCGTCGATGATTGTGGGGAATATGGCGCGAAAATCTAGTGGTAAAATGTTTTGTCTTTTCATGGTCCATGTTAAAAAATCTCTTGTTGAATATTATGATGAGACACTGTAAACTAATAAGTGAGTTTGTAATTTTTATGCGTCAATTTATGGTATCAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
<210> 3
<211> 491
<212> RNA
<213> full-Length amino acid sequence of terpene synthase Gene RhTPS (SEQ ID NO. 3)
<400> 3
MGTERERERERERMAAEENKEEEDLPYDGRRRRRRRSAVRSRPEVFNKFKDGDGNFKEGLSKDAEGLLSLYEASHYGMQCEIDLEAARTFSVKHLNSLTGKMEIELAEQVQQSLECPIRWRLPRLEARNFIDLYESDSARNPLLLEFAKLDYNLVQSVHQKEVQELAKWWMDLGLKEKLGFSRDRSMENYLWAMGMVSESHFSNCRKGLTKFVCILSAIDDMYDIYGSIDELERFTDAVNRWSVEATDDLPEYMKICYLAMFNFGNEIAYNVLRDHGLNVVSYIKEEWSNLCGAYLVEARWFYSGFIPTLEEYLKNAWKSVGGPAAIAHACLLLGSPITRTSLDSYKASSELIYWSSIITRLSDDLGTSKDEIMRGDVAKSIQCYMNERSVTEEQARDHIKGIVHESWRKLNETIAQNSHPTPMISMSLNMARTAQCIYQHGDGIGTSLGVTKDRLTSLIVDPIPIERLTHGEVRGRTHSYAVENAYSISK
Claims (10)
1. The rhododendron parviflora TPS gene is characterized by being terpene synthase gene RhTPS, and the nucleotide sequence of the conserved domain of the terpene synthase gene RhTPS is shown as SEQ ID No. 1.
2. The rhododendron parviflora TPS gene is characterized by being terpene synthase gene RhTPS, wherein the full-length nucleotide sequence of the terpene synthase gene RhTPS is shown as SEQ ID No. 2.
3. The rhododendron parvifolium TPS gene as claimed in claim 2, characterized in that the full-length nucleotide sequence of terpene synthase gene RhTPS is a gene sequence with terpene synthase activity, which is obtained by substituting, deleting and/or adding one or more bases to the nucleotide sequence shown in SEQ ID No. 2.
4. The Rhododendron micranthum TPS gene is characterized in that the gene is a terpene synthase gene RhTPS, and the full-length amino acid sequence of the terpene synthase gene RhTPS is shown as SEQ ID No. 3.
5. The Rhododendron palustre TPS gene according to claim 4, wherein the full-length amino acid sequence of terpene synthase gene RhTPS is a protein derived from SEQ ID No.3 with terpene synthase activity, wherein the amino acid sequence is the amino acid sequence shown in SEQ ID No.3, and one or more amino acids are substituted, deleted and/or added.
6. A primer group for cloning a rhododendron parvifolium TPS gene is characterized by comprising TPS-F1, TPS-R1, TPS-F2, TPS-R2, TPS-F3 and TPS-R3, wherein TPS-F1 and TPS-R1 are used for amplifying a nucleotide sequence shown in SEQ ID NO.1, TPS-F2 and TPS-R2 are used for amplifying a nucleotide sequence shown in SEQ ID NO.2, TPS-F3 and TPS-R3 are nucleotide sequences designed according to the nucleotide sequence shown in SEQ ID NO.1 and used for qRT-PCR amplification, and the sequences of the primer group are as follows:
TPS-F1：CTCATTGACWCWATKCAAMGGY；
TPS-R1：CTTRGAAGTKCCCAAATCATCA；
TPS-F2：CCAGGTCCATCCACCACTTGGCTAGTTCTTGTA；
TPS-R2：ACGCTTGCCTCTTGCTAGGGTCTCCGATAA；
TPS-F3：CGCCAGAACTTTCAGTGTCAAGCATTT；
TPS-R3：TCGTGCTTCTAACCTCGGCATTCT。
7. the method for cloning TPS gene of rhododendron micranthum is characterized by comprising the following steps:
step one, extracting total RNA of rhododendron marianum petals by adopting a polysaccharide polyphenol plant total RNA extraction kit, determining the integrity and concentration of the total RNA by using agarose gel electrophoresis with the mass concentration of 1.5% and a nucleic acid protein analyzer respectively, and synthesizing a cDNA first chain according to the instruction of a TransScript Reverse Transcriptase Reverse transcription kit;
step two, downloading the full-length sequence of the TPS gene of a plant with close relationship with the rhododendron plant from GenBank, carrying out multi-sequence comparison by adopting ClustalW, designing degenerate primers TPS-F1 and TPS-R1 by utilizing Primer 5.0 in a sequence with higher homology, wherein the sequences of Primer groups TPS-F1 and TPS-R1 are as follows:
TPS-F1：CTCATTGACWCWATKCAAMGGY；
TPS-R1：CTTRGAAGTKCCCAAATCATCA；
taking the first cDNA chain as a template, and carrying out PCR amplification reaction by using degenerate primers TPS-F1 and TPS-R1, wherein the reaction procedure of the PCR amplification reaction is as follows: 3min at 94 ℃; 30Cycles at 94 ℃ for 30s, 55 ℃ for 30s, and 72 ℃ for 1 min; obtaining a PCR product at 72 ℃ for 5 min; and (3) performing sequence determination on the PCR product after recovery, pMD18-T Vector Cloning Kit connection, DH5 alpha competent cell transformation, recombinant screening and bacterial liquid PCR identification to obtain the nucleotide sequence of the rhododendron yunnanensis terpene synthase gene RhTPS conserved domain, namely SEQ ID No. 1.
8. The cloning method of Rhododendron micranthum TPS gene as claimed in claim 7, wherein RACE amplification primers TPS-F2 and TPS-R2 are designed according to nucleotide sequence of Rhododendron micranthum terpene synthase gene RhTPPS conserved domain, and TPS-F2 and TPS-R2 have the following sequences:
TPS-F2：CCAGGTCCATCCACCACTTGGCTAGTTCTTGTA；
TPS-R2：ACGCTTGCCTCTTGCTAGGGTCTCCGATAA；
respectively amplifying the 5 'end full length and the 3' end full length of the terpene synthase gene RhTPS by the following method: synthesizing RACE-cDNA according to the instruction of SMARTer RACE 5 '/3' Kit RACE Kit to respectively obtain 5'cDNA and 3' cDNA; taking 5' cDNA as a template, and carrying out PCR amplification reaction on the 5' tail end full length by utilizing RACE universal primers UPM and TPS-R2 to obtain a specific primer of the 5' tail end full length; taking 3' cDNA as a template, and carrying out PCR amplification reaction on the full length of the 3' tail end by utilizing RACE universal primers UPM and TPS-F2 to obtain a specific primer of the full length of the 3' tail end; comparing and splicing the conserved domain sequence, the 5 'terminal full-length sequence and the 3' terminal full-length sequence by using DNAMAN software to obtain a full-length nucleotide sequence with a full-length 1826bp sequence of SEQ ID NO. 2; coding 491 amino acids to obtain a full-length amino acid sequence with a sequence of SEQ ID NO. 3;
wherein, the reaction program for carrying out PCR amplification reaction on the 5 'end full length and the 3' end full length is as follows: 94 ℃ for 30s, 72 ℃ for 3min, 5 Cycles; 94 ℃ for 30s, 70 ℃ for 30s, 72 ℃ for 3min, 5 Cycles; 30Cycles at 94 ℃ for 30s, 68 ℃ for 30s, and 72 ℃ for 3 min; and (6) ending.
9. The fluorescence quantitative qRT-PCR method for the TPS gene of rhododendron micranthum is characterized by comprising the following steps of: designing primers TPS-F3 and TPS-R3 according to the nucleotide sequence SEQ ID NO.1 of a terpene synthase gene RhTPS conserved domain, wherein the sequences of TPS-F3 and TPS-R3 are as follows:
TPS-F3：CGCCAGAACTTTCAGTGTCAAGCATTT；
TPS-R3：TCGTGCTTCTAACCTCGGCATTCT；
a rhododendron EF1 alpha is used as an internal reference, internal reference gene primers RhEF1 alpha-F and RhEF1 alpha-R are designed, and the sequences of the RhEF1 alpha-F and the RhEF1 alpha-R are as follows:
RhEF1α-F：TGTGCCATCCTCATTATTGACTCC；
RhEF1α-R：ATGGGATCTTCTCGGGATTGTATC；
extracting total RNA of petals of rhododendron parvifolium at different flowering phases by adopting a polysaccharide polyphenol plant total RNA extraction kit, synthesizing qRT-PCR template cDNA according to the specification of a NovoScript Plus All-in-one1st Strand cDNA Synthesis Supermix (gDNA Purge) kit, respectively taking the cDNA as a template, referring to the specification of a Transstart Tip Green qPCRSuperMixS kit, and performing qRT-PCR amplification reaction by utilizing TPS-F3, TPS-R3, RhEF1 alpha-F and RhEF1 alpha-R, wherein the reaction program of the qRT-PCR amplification reaction is as follows: 30s at 94 ℃; 94 ℃ for 5s, 59 ℃ for 30s, 42 Cycles; 5s at 65 ℃; 5s at 95 ℃; finishing; when each sample is amplified, internal references are amplified simultaneously, the relative gene expression quantity of each sample is the average value of 3 biological repeats, and 2 are adopted－ΔΔCTThe method is used for analyzing data and analyzing the expression quantity of terpene synthase gene RhTPS in different tissues.
10. The application of rhododendron yunnanensis TPS gene of claim 1 or 2 in regulating and controlling rhododendron fragrance, which comprises the following steps: and (3) transforming the terpene synthase gene RhTPS into a flowerless rhododendron cell, and then culturing the transformed rhododendron cell.
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| CN115058439A (en) * | 2022-05-31 | 2022-09-16 | 浙江万里学院 | Rhododendron micranthum SAMT gene overexpression vector and construction method and application thereof |
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