CN102181432A - Cloning method of wild pueraria lobata glucosyltransferase gene PlUGT4 - Google Patents
Cloning method of wild pueraria lobata glucosyltransferase gene PlUGT4 Download PDFInfo
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Abstract
The invention adopts the homologous gene cloning method and combines with the RACE (rapid-amplification of cDNA (complementary deoxyribonucleic acid) ends) method for designing a reverse transcription primer, and a wild pueraria lobata glucosyltransferase gene, namely PlUGT4 is obtained by cloning from wile pueraria lobata. The nucleotide sequence of the wild pueraria lobata glucosyltransferase gene and an encoding protein sequence and a gene cloning method thereof can be widely applied in improvement of quality of the wild pueraria lobata, in particular to development, utilization and other fields taking the wild pueraria lobata as a medicinal plant.
Description
Technical field
The present invention relates to a kind of gene cloning process, particularly elegant jessamine Transglucosylase gene
PlUGT4Cloning process.
Background technology
Elegant jessamine (
Pueraria lobata(Willd.) be that the leguminous plants Pueraria lobota belongs to Ohwi), its root is as the existing quite long history of Chinese traditional Chinese medicine.Root of kudzu vine flavor is sweet, and property is hot cool, returns spleen, stomach warp.Have rise sun, invigorate blood circulation, the effect of vein relaxing, its effective medicinal ingredients is isoflavonoid puerarin, Daidzin, daidzein etc.Wherein puerarin content height, the property of medicine are good, can significantly improve the patients with coronary heart disease blood supply of cardiac muscle, treatment unstable angina pectoris, premature ventricular beat; Lowering blood glucose, blood fat, anti-oxidant, increase blood fluidity, reduce pulmonary hypertension, treatment diabetic peripheral neuropathy and improve effect (Song such as insulin resistant
Et al., 1988; Xu
Et al., 2005; Huang Zhuyan etc., 2007).To the approach of elegant jessamine biosynthesizing puerarin, especially the research of regulatory enzyme obtains paying close attention to always, has cloned chalcone synthase gene (Nakajima from elegant jessamine
Et al., 1996; Yamaguchi
Et al., 1999), clone's cinnamophenone-isoflavones isomerase is at escherichia coli expression (Terai
Et al., 1996), elegant jessamine cinnamophenone reductase gene is expressed (Joung in tobacco
Et al., 2003).Correlation research between above-mentioned elegant jessamine anabolism Expression of Related Genes and puerarin production does not have report as yet.Think at present, the biosynthesizing of puerarin be isoliquiritigenin under the Transglucosylase effect, C
8The position connects a glucosyl group by C-C and forms C-glucosyl cinnamophenone intermediate, and then self dewatering forms puerarin, and its C-glycosylation occurs in the cinnamophenone stage.Several connecting key blended of catalysis Transglucosylase is arranged in known mode plant Arabidopis thaliana, the paddy rice, but the aminoacid sequence that lacks independent catalysis C-Transglucosylase, can't access the cDNA and the aminoacid sequence of C-Transglucosylase, therefore, from elegant jessamine, clone glycosyltransferase and study its function, for puerarin biosynthetic pathway and study on regulation in the understanding adjusting plant materials provide foundation, for microbial project scale operation puerarin provides the basis, significant in theory and application facet.Reported and pointed out, the not phase simultaneously of the different developmental phases of the biology of different genera, the different tissues of same biont, same tissue even same cell strain, the expression of its glycosyltransferase also has difference (Bettina
Et al.2005), for example gene order and these the glycosyltransferase overwhelming majority of lucerne potato with a series of glycosyltransferases participated in the flavonoid metabolism, and the conserved sequence that scientist will participate in the glycosyltransferase of secondary metabolism is called PSPG frame (Hughes and Hughes, 1994).The PSPG frame is by forming near about 40 amino acid of PROTEIN C end, and conservative property is very strong, reaches 60%-80%, on evolving also as a sign (Gachon who screens the gene of glycosyltransferase
Et al., 2005).Present stage, generally utilize homologous gene clone and RACE technology clone glycosyltransferase gene, if but design of primers is improper, and reaction conditions control is bad, can't clone complete purpose at all.
Elegant jessamine Transglucosylase gene
PlUGT4Be newfound a kind of glycosyltransferase gene, its Genebank number of landing is EU889122, total length 1590bp, and its sequence is shown in SEQ ID NO. 9, and its protein sequence is shown in SEQ ID NO.10.This gene can be widely used in the improvement of elegant jessamine quality, and especially elegant jessamine is as the fields such as development and utilization of medicinal plant.But lack effective cloning process at present.
Summary of the invention
The object of the present invention is to provide the cloning process of elegant jessamine Transglucosylase gene PlUGT4.
The technical solution used in the present invention is:
The cloning process of elegant jessamine Transglucosylase gene PlUGT4 may further comprise the steps:
1) total RNA of extraction elegant jessamine, reverse transcription obtains cDNA;
2) with cDNA be template, with degenerated primer UGTD-F:5 ' TTYGTNACNCAYTGYGGNTGGAA 3 ' (SEQ ID NO.1) and UGTD-R:5 ' TCCATNAGNCTNCGNCANGCYTTYTCDAT 3 ' (SEQ ID NO.2) is primer, carry out thermograde PCR, obtain cDNA, its reaction conditions is: 94 ℃ of pre-sex change 5 min, 94 ℃ of sex change 30s, 54 ℃～63 ℃ 30s, 72 ℃ are extended 30s, 20～40 circulations, last 72 ℃ are extended 10min, obtain intermediate sequence;
3) the total RNA that obtains with step 1) is a template, and 3 ' RACE T:5 ' GACTCGAGTCGACATCGATTTTTTTTTTTTTTTTT 3 ' (SEQ ID NO.3) obtains cDNA for the primer reverse transcription;
4) cDNA that a step obtains more than is a template, and nest-type PRC obtains 3 ' end sequence of elegant jessamine Transglucosylase gene PlUGT4;
5) the total RNA that obtains with step 1) is a template, adopt TaKaRa company 5 '-Full RACE Kit obtains 5 ' end sequence of elegant jessamine Transglucosylase gene PlUGT4;
6) intermediate sequence, 3 ' end sequence, 5 ' end sequence are spliced the cDNA sequence that obtains PlUGT4 with SeqMan, obtain the cDNA sequences Design primer PlUGT4F:ATGGCTGACCAGCTCACCGACGACCAG(SEQ ID NO.4 of PlUGT4 according to splicing) and PlUGT4R:TCAGTTTCCGTGAATCCGTTCAAGTC(SEQ ID NO.5), the cDNA that obtains with step 1) is a template, and PCR obtains open reading frame (ORF) the albumen cDNA sequence of elegant jessamine Transglucosylase gene PlUGT4.
Preferably, in the step 4), the primer of PCR is PlUGT4-F1:ACCATCATGGAAAGTGTGAACGC(SEQ ID NO.6), PlUGT4-F2:AGAATGGAGACCGTGGAACGACTTTGG (SEQ ID NO.7) and 3 ' RACE R:GACTCGAGTCGACATCGA(SEQ ID NO.8).
Method of the present invention can clone elegant jessamine Transglucosylase gene PlUGT4 effectively.
Embodiment
Below in conjunction with embodiment, further specify the present invention.
In following examples, the degenerated primer of use is:
UGTD-F：5′?TTYGTNACNCAYTGYGGNTGGAA?3′（SEQ?ID?NO.1）
UGTD-R：5′?TCCATNAGNCTNCGNCANGCYTTYTCDAT?3′（SEQ?ID?NO.2）。
Embodiment 1
1) total RNA of extraction elegant jessamine, reverse transcription obtains cDNA;
2) cDNA that a step obtains more than is a template, uses degenerated primer UGTD-F and UGTD-R to carry out pcr amplification, and reaction conditions is: 94 ℃ of pre-sex change 5min, 94 ℃ of sex change 30s, 54 ℃ of 30s, 72 ℃ are extended 30s, 20 circulations, 72 ℃ are extended 10min then, and amplification obtains 150 bp swimming band;
3) getting total RNA 2 μ g is template, and the primer that designs with the walking method is a primer, and reverse transcription obtains cDNA;
4) obtaining cDNA with reverse transcription is template, by walking method design for the primer reverse transcription obtains cDNA, pcr amplification can't obtain the end of elegant jessamine Transglucosylase gene.
Embodiment 2
1) total RNA of extraction elegant jessamine, reverse transcription obtains cDNA;
2) cDNA that a step obtains more than is a template, uses degenerated primer UGTD-F and UGTD-R to carry out pcr amplification, and reaction conditions is: 94 ℃ of pre-sex change 5min, 94 ℃ of sex change 30s, 60 ℃ of 30s, 72 ℃ are extended 30s, 40 circulations, 72 ℃ are extended 10min then, can't amplify RNA swimming band.
Embodiment 3
1) total RNA of extraction elegant jessamine, reverse transcription obtains cDNA;
2) cDNA that a step obtains more than is a template, use degenerated primer UGTD-F and UGTD-R to carry out pcr amplification, reaction conditions is: 94 ℃ of pre-sex change 5min, 94 ℃ of sex change 30s, 57 ℃ of 30s, 72 ℃ are extended 30s, 30 circulations, 72 ℃ are extended 10min then, and amplification obtains 250 bp swimming band, is called intermediate sequence;
3) getting total RNA 2 μ g is template, reverse transcription primer 5 ' GACTCGAGTCGACATCGATTTTTTTTTTTTTTTTT 3 ' (SEQ ID NO.3) with the design of RACE-T method is a primer, and SuperScript III ThermoScript II (Invitrogen) reverse transcription obtains cDNA;
4) in the above step reverse transcription to obtain cDNA be template, primer PlUGT4-F1:ACCATCATGGAAAGTGTGAACGC(SEQ ID NO.6) and 3 ' RACE R:GACTCGAGTCGACATCGA(SEQ ID NO.8) carry out first round pcr amplification and obtain the cDNA product; CDNA product with first round PCR is a template, PlUGT4-F2:AGAATGGAGACCGTGGAACGACTTTGG(SEQ ID NO.7) and 3 ' RACE R:GACTCGAGTCGACATCGA(SEQ ID NO.8) take turns pcr amplification for primer carries out second, obtain the PCR product, the PCR program is: 94 ℃ of pre-sex change 5min, 94 ℃ of sex change 30s, 55 ℃ of 30s, 72 ℃ are extended 1min, 30 circulations, 72 ℃ are extended 10min then;
5) the PCR product is checked order, prove that the PCR product that obtains is 3 ' end sequence of elegant jessamine Transglucosylase gene PlUGT4;
6) be template with total RNA, adopt TaKaRa company 5 '-Full RACE Kit obtains 5 ' end sequence of elegant jessamine Transglucosylase gene PlUGT4;
7) with SepMan intermediate sequence, 3 ' end sequence and 5 ' end sequence are spliced, obtain the cDNA sequence of PlUGT4, and according to the cDNA sequences Design primer PlUGT4F:ATGGCTGACCAGCTCACCGACGACCAG(SEQ ID NO.4 that obtains PlUGT4) and PlUGT4R:TCAGTTTCCGTGAATCCGTTCAAGTC(SEQ ID NO.5), the cDNA that obtains with step 1) is a template, and PCR obtains open reading frame (ORF) the albumen cDNA sequence of elegant jessamine Transglucosylase gene PlUGT4.
To the order-checking of PCR product, prove that the sequence that obtains is the orf protein cDNA sequence of PlUGT4.
Embodiment 4
1) total RNA of extraction elegant jessamine, reverse transcription obtains cDNA;
2) cDNA that a step obtains more than is a template, uses degenerated primer UGTD-F and UGTD-R to carry out pcr amplification, and reaction conditions is: 94 ℃ of pre-sex change 5min, 94 ℃ of sex change 30s, 63 ℃ of 30s, 72 ℃ are extended 30s, 30 circulations, 72 ℃ are extended 10min then, and amplification obtains 200 bp swimming band;
3) be template with total RNA 2 μ g, the primer that designs with the walking method is a primer, and reverse transcription obtains cDNA;
4) obtaining cDNA with reverse transcription is template, and pcr amplification can not obtain the end of elegant jessamine Transglucosylase gene.
＜110〉South China Normal University
＜120〉cloning process of elegant jessamine Transglucosylase gene PlUGT4
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<400> 2
tccatnagnc?tncgncangc?yttytcdat 29
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gactcgagtc?gacatcgatt?tttttttttt?ttttt 35
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＜213〉artificial sequence
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atggctgacc?agctcaccga?cgaccag 27
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＜213〉artificial sequence
<400> 5
tcagtttccg?tgaatccgtt?caagtc 26
<210> 6
<211> 23
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＜213〉artificial sequence
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accatcatgg?aaagtgtgaa?cgc 23
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<211> 27
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<400> 7
agaatggaga?ccgtggaacg?actttgg 27
<210> 8
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<212> DNA
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<400> 8
gactcgagtc?gacatcga 18
<210> 9
<211> 1590
<212> DNA
<213> Pueraria?montana?var.?lobata
<400> 9
gaaaacacat?tcatattttc?ctttgtgttt?ttcctttcat?tttccctcac?tttctcgctt 60
cccaaacgcc?cttctctctc?tcctccaatg?gctgaccagc?tcaccgacga?ccagatcgcc 120
gagttcaagg?aggccttcag?cctcttcgac?aaggacggcg?atggttgtat?tactaccaag 180
gaacttggga?ctgtgatgcg?gtctcttggt?caaaacccaa?ctgaggcaga?actgcaggat 240
atgatcaatg?aggttgatgc?tgatggcaac?ggaaccatcg?atttccccga?gttcctcaac 300
ctgatggctc?gcaagatgaa?ggacaccgac?tcagaggagg?agctcaagga?ggctttccgc 360
gtgtttgaca?aggatcagaa?tggtttcatc?tctgcggcca?agctcggtat?tcccagattg 420
atgttcctcg?gaggaagcta?cctcgcccac?tctgcccagc?attccctcaa?aaaatatggg 480
cctcacaagg?acatgcaatc?cgacacccac?aaattcgcct?tccctgactt?gccccaccac 540
ttggagatga?cacgcttgca?gctaccggac?tggcttcgag?agccaaatgg?atacacttat 600
ttgatggaca?tgattagaga?ttcagaaaaa?aagagctacg?gttctttgtt?tgataccttc 660
tatgacctcg?aaggtactta?ccaggagcat?tacaagacag?ccaccgggac?cagaacctgg 720
agcttaggac?cagtttcctt?gtgggtgaac?caagatgctt?ccgataaggc?tgcaaggggc 780
catgcaaaag?aagaagaaga?agaagggtgg?cttaaatggc?tcaattctaa?gcctgagaaa 840
tctgttctct?acgtgacttt?tgggagcatg?agcaagttcc?cttcttccca?gcttgttgaa 900
atagctcaag?cccttgaaga?atccggccac?aatttcatgt?gggtggttaa?gaagagggat 960
gatggggatg?gttttctgga?agagttcgag?aagagagtga?aagcaagcaa?taaaggttat 1020
ctaatatggg?gttgggcacc?gcaactgctg?atactggaga?attcggccat?tggaggactg 1080
gtgactcact?gcggatggaa?caccatcatg?gaaagtgtga?acgcagggtt?gcccatggcg 1140
acctggcctc?tgtttgcgga?acagttcttt?aacgaaaagc?tggtggttga?tgtgctgaaa 1200
attggagtgg?cagtgggagc?caaagaatgg?agaccgtgga?acgactttgg?gaaagaggtt 1260
gtgaaaaagg?aggacattgg?aaaggccatt?gctttgctca?tgagcagtgg?agaggagagt 1320
gcagaaatga?ggagaagagc?tgttgctctt?ggcagtgctg?ccaagagagc?tatacagttt 1380
ggagggtctt?ctcataataa?tatgttggaa?ttggtccaag?agctcaaatc?actcagactt 1440
gaacggattc?acggaaactg?agtgactaac?catgtcttgg?cccctgtgtg?tgagacgtat 1500
gttgcgtttt?gctacataaa?ctgaatttat?gtttgaattt?gcaaatttca?tttaaggaat 1560
gtacggctta?tcaaaaaaaa?aaaaaaaaaa 1590
<210> 10
<211> 457
<212> PRT
<213> Pueraria?montana?var.?lobata
<400> 10
Met?Ala?Asp?Gln?Leu?Thr?Asp?Asp?Gln?Ile?Ala?Glu?Phe?Lys?Glu?Ala
1 5 10 15
Phe?Ser?Leu?Phe?Asp?Lys?Asp?Gly?Asp?Gly?Cys?Ile?Thr?Thr?Lys?Glu
20 25 30
Leu?Gly?Thr?Val?Met?Arg?Ser?Leu?Gly?Gln?Asn?Pro?Thr?Glu?Ala?Glu
35 40 45
Leu?Gln?Asp?Met?Ile?Asn?Glu?Val?Asp?Ala?Asp?Gly?Asn?Gly?Thr?Ile
50 55 60
Asp?Phe?Pro?Glu?Phe?Leu?Asn?Leu?Met?Ala?Arg?Lys?Met?Lys?Asp?Thr
65 70 75 80
Asp?Ser?Glu?Glu?Glu?Leu?Lys?Glu?Ala?Phe?Arg?Val?Phe?Asp?Lys?Asp
85 90 95
Gln?Asn?Gly?Phe?Ile?Ser?Ala?Ala?Lys?Leu?Gly?Ile?Pro?Arg?Leu?Met
100 105 110
Phe?Leu?Gly?Gly?Ser?Tyr?Leu?Ala?His?Ser?Ala?Gln?His?Ser?Leu?Lys
115 120 125
Lys?Tyr?Gly?Pro?His?Lys?Asp?Met?Gln?Ser?Asp?Thr?His?Lys?Phe?Ala
130 135 140
Phe?Pro?Asp?Leu?Pro?His?His?Leu?Glu?Met?Thr?Arg?Leu?Gln?Leu?Pro
145 150 155 160
Asp?Trp?Leu?Arg?Glu?Pro?Asn?Gly?Tyr?Thr?Tyr?Leu?Met?Asp?Met?Ile
165 170 175
Arg?Asp?Ser?Glu?Lys?Lys?Ser?Tyr?Gly?Ser?Leu?Phe?Asp?Thr?Phe?Tyr
180 185 190
Asp?Leu?Glu?Gly?Thr?Tyr?Gln?Glu?His?Tyr?Lys?Thr?Ala?Thr?Gly?Thr
195 200 205
Arg?Thr?Trp?Ser?Leu?Gly?Pro?Val?Ser?Leu?Trp?Val?Asn?Gln?Asp?Ala
210 215 220
Ser?Asp?Lys?Ala?Ala?Arg?Gly?His?Ala?Lys?Glu?Glu?Glu?Glu?Glu?Gly
225 230 235 240
Trp?Leu?Lys?Trp?Leu?Asn?Ser?Lys?Pro?Glu?Lys?Ser?Val?Leu?Tyr?Val
245 250 255
Thr?Phe?Gly?Ser?Met?Ser?Lys?Phe?Pro?Ser?Ser?Gln?Leu?Val?Glu?Ile
260 265 270
Ala?Gln?Ala?Leu?Glu?Glu?Ser?Gly?His?Asn?Phe?Met?Trp?Val?Val?Lys
275 280 285
Lys?Arg?Asp?Asp?Gly?Asp?Gly?Phe?Leu?Glu?Glu?Phe?Glu?Lys?Arg?Val
290 295 300
Lys?Ala?Ser?Asn?Lys?Gly?Tyr?Leu?Ile?Trp?Gly?Trp?Ala?Pro?Gln?Leu
305 310 315 320
Leu?Ile?Leu?Glu?Asn?Ser?Ala?Ile?Gly?Gly?Leu?Val?Thr?His?Cys?Gly
325 330 335
Trp?Asn?Thr?Ile?Met?Glu?Ser?Val?Asn?Ala?Gly?Leu?Pro?Met?Ala?Thr
340 345 350
Trp?Pro?Leu?Phe?Ala?Glu?Gln?Phe?Phe?Asn?Glu?Lys?Leu?Val?Val?Asp
355 360 365
Val?Leu?Lys?Ile?Gly?Val?Ala?Val?Gly?Ala?Lys?Glu?Trp?Arg?Pro?Trp
370 375 380
Asn?Asp?Phe?Gly?Lys?Glu?Val?Val?Lys?Lys?Glu?Asp?Ile?Gly?Lys?Ala
385 390 395 400
Ile?Ala?Leu?Leu?Met?Ser?Ser?Gly?Glu?Glu?Ser?Ala?Glu?Met?Arg?Arg
405 410 415
Arg?Ala?Val?Ala?Leu?Gly?Ser?Ala?Ala?Lys?Arg?Ala?Ile?Gln?Phe?Gly
420 425 430
Gly?Ser?Ser?His?Asn?Asn?Met?Leu?Glu?Leu?Val?Gln?Glu?Leu?Lys?Ser
435 440 445
Leu?Arg?Leu?Glu?Arg?Ile?His?Gly?Asn
450 455
Claims (2)
1. the cloning process of elegant jessamine Transglucosylase gene PlUGT4 may further comprise the steps:
1) total RNA of extraction elegant jessamine, reverse transcription obtains cDNA;
2) with cDNA be template, with degenerated primer UGTD-F:5 ' TTYGTNACNCAYTGYGGNTGGAA 3 ' (SEQ ID NO.1) and UGTD-R:5 ' TCCATNAGNCTNCGNCANGCYTTYTCDAT 3 ' (SEQ ID NO.2) is primer, carry out thermograde PCR, obtain cDNA, its reaction conditions is: 94 ℃ of pre-sex change 5 min, 94 ℃ of sex change 30s, 54 ℃～63 ℃ 30s, 72 ℃ are extended 30s, 20～40 circulations, last 72 ℃ are extended 10min, obtain intermediate sequence;
3) the total RNA that obtains with step 1) is a template, and 3 ' RACE T:5 ' GACTCGAGTCGACATCGATTTTTTTTTTTTTTTTT 3 ' (SEQ ID NO.3) obtains cDNA for the primer reverse transcription;
4) cDNA that a step obtains more than is a template, and nest-type PRC obtains 3 ' end sequence of elegant jessamine Transglucosylase gene PlUGT4;
5) the total RNA that obtains with step 1) is a template, adopt TaKaRa company 5 '-Full RACE Kit obtains 5 ' end sequence of elegant jessamine Transglucosylase gene PlUGT4;
6) intermediate sequence, 3 ' end sequence, 5 ' end sequence are spliced the cDNA sequence that obtains PlUGT4 with SeqMan, obtain the cDNA sequences Design primer PlUGT4F:ATGGCTGACCAGCTCACCGACGACCAG(SEQ ID NO.4 of PlUGT4 according to splicing) and PlUGT4R:TCAGTTTCCGTGAATCCGTTCAAGTC(SEQ ID NO.5), the cDNA that obtains with step 1) is a template, and PCR obtains open reading frame (ORF) the albumen cDNA sequence of elegant jessamine Transglucosylase gene PlUGT4.
2. the cloning process of elegant jessamine Transglucosylase gene PlUGT4 according to claim 1, it is characterized in that: in the step 4), the primer of PCR is PlUGT4-F1:ACCATCATGGAAAGTGTGAACGC(SEQ ID NO.6), PlUGT4-F2:AGAATGGAGACCGTGGAACGACTTTGG (SEQ ID NO.7) and 3 ' RACE R:GACTCGAGTCGACATCGA(SEQ ID NO.8).
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| 《植物生理学通讯》 20090731 周文灵 等 野葛葡糖基转移酶基因PlUGT3的克隆与生物信息学分析 651-656 1-2 第45卷, 第7期 * |
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| CN103333905A (en) * | 2013-05-30 | 2013-10-02 | 吉林农业大学 | Soybean chalcone reductase gene CHR2 and applications thereof |
| CN103275996B (en) * | 2013-05-30 | 2014-05-07 | 吉林农业大学 | Soybean chalcone reductase gene CHR4 and application |
| CN107267647A (en) * | 2017-08-23 | 2017-10-20 | 广西作物遗传改良生物技术重点开放实验室 | PCR reaction system for kudzu root SCoT molecular marker |
| CN107267647B (en) * | 2017-08-23 | 2020-11-13 | 广西作物遗传改良生物技术重点开放实验室 | PCR reaction system for kudzu root SCoT molecular marker |
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