CN111139252B

CN111139252B - Si4CL gene and its coding product and application

Info

Publication number: CN111139252B
Application number: CN201911411085.1A
Authority: CN
Inventors: 祖明艳; 付春祥; 吴振映; 韩杰; 孙震; 赵思怡; 徐悦
Original assignee: Yantai Jinrui Female Product Co ltd; Qingdao Institute of Bioenergy and Bioprocess Technology of CAS
Current assignee: Yantai Jinrui Female Product Co ltd; Qingdao Institute of Bioenergy and Bioprocess Technology of CAS
Priority date: 2019-12-31
Filing date: 2019-12-31
Publication date: 2022-04-29
Anticipated expiration: 2039-12-31
Also published as: CN111139252A

Abstract

The invention relates to a Si4CL gene and its coding product and application, belonging to the technical field of medicinal plant genetic engineering, wherein the Si4CL gene has a nucleotide sequence shown in SEQ ID NO. 1; or the nucleotide sequence shown in SEQ ID NO.1 adds, substitutes, inserts or deletes one or more nucleotide homologous sequences or alleles thereof and derived nucleotide sequences thereof, and also provides the protein coded by the gene, wherein the protein has the amino acid sequence shown in SEQ ID NO. 2; or a homologous sequence of SEQ ID NO.2 with addition, substitution, insertion or deletion of one or more amino acids. The Si4CL of the invention can carry out enzymolysis reaction on three substrates of FA, CA and pCA, and products are generated.

Description

Si4CL gene and its coding product and application

Technical Field

The invention belongs to the technical field of medicinal plant genetic engineering, and relates to cloning of saussurea involucrate 4-coumaroyl coenzyme A ligase (Si4CL) gene by using a full-length cDNA library, a coding product and application thereof, in particular to an organic acid, flavonoid and phenylpropyl enzyme gene for biosynthesis of pharmacologically active components, and a coding product and application thereof.

Background

Dried aerial parts of saussurea involucrata (Saus-surea involucrata (Kar. et Kir.) Sch. -Bip.) belonging to Compositae family. 2015 edition of Chinese pharmacopoeia records that saussurea involucrate has effects of invigorating kidney, promoting blood circulation, strengthening tendons and bones, nourishing nerves, regulating abnormal body fluid, and the like, and is mainly used for treating wind-cold-dampness arthralgia, lower abdomen pain, irregular menstruation, and the like in clinic. The main chemical components of saussurea involucrate are alkaloid, flavonoid and phenylpropanoid compounds, which are closely related to the medicinal value of the saussurea involucrate. Saussurea involucrate has special requirements on the growth environment, which determines that it mainly grows in Tianshan mountain of Xinjiang. The saussurea involucrate has diverse chemical components, the reported components comprise polysaccharide, flavone, alkaloid, terpenoid, lignin, sterol, volatile oil and the like, the saussurea involucrate is rich in amino acid, the main chemical component is flavonoid compound, and 14 types of the flavonoid compounds are identified at present. The formation of active components of medicinal plants is the product of a unique gene group in the secondary metabolic pathway of plants.

Coumaryl-CoA ligase (4-coumarate-CoA ligase, 4CL) is a key enzyme in the divergent pathway of the synthesis of plant natural phenylpropyl derivative products. The main function of 4CL is to generate corresponding coa esters using cinnamic acid and its hydroxy or methoxy derivatives such as 4-coumaric acid (pCA), Caffeic Acid (CA), Ferulic Acid (FA), 5-hydroxyferulic acid (5-OHFA), Sinapic Acid (SA) and the like as substrates. The intermediate products enter a phenylpropanoid derivative branch synthesis way, wherein generated coumaroyl CoA, feruloyl CoA and sinapoyl CoA are converted into cinnamyl alcohol derivatives, can be further converted into coumarin, chlorogenic acid and the like, can also form CoA ester, and are further converted into compounds such as trifolitin, chlorogenic acid and the like. Thus, 4CL is also a key enzyme in the biosynthesis of chlorogenic acid and trifolioside. Cloning of saussurea involucrate 4-coumaroyl coenzyme A ligase (Si4CL) gene provides important basis for improving the content of active ingredients of saussurea involucrate by using genetic engineering.

Disclosure of Invention

The invention aims to solve the technical problem of providing a saussurea involucrate 4-coumaroyl coenzyme A ligase (Si4CL) gene and a coding product thereof, and application of the product in medicines.

The saussurea involucrate 4-coumaroyl coenzyme A ligase (Si4CL) gene provided by the invention is one of the following nucleotide sequences:

(1) has a nucleotide sequence shown as SEQ ID NO. 1;

(2) the nucleotide sequence shown in SEQ ID NO.1 is added, substituted, inserted or deleted with one or more nucleotide homologous sequences or alleles thereof and nucleotide sequences derived from the alleles.

The invention also provides a protein coded by the Si4CL gene, which is saussurea involucrate 4-coumaroyl coenzyme A ligase and is one of the following amino acid sequences:

(1) has an amino acid sequence shown as SEQ ID NO. 2;

(2) a homologous sequence of SEQ ID No.2 with addition, substitution, insertion or deletion of one or more amino acids.

The invention also provides a recombinant vector pET32a-Si4CL containing the gene of claim 1.

The invention also provides the application of the gene in regulating and controlling the synthesis and genetic improvement of active substances of the saussurea involucrate, including organic acid, flavone, polysaccharide, alkaloid and phenylpropanoid.

The invention also provides application of the recombinant vector pET32a-Si4CL in regulating and controlling synthesis and genetic improvement of active substances of saussurea involucrate including organic acid, flavone, polysaccharide, alkaloid and phenylpropanoid.

Compared with the prior art, the invention has the following beneficial effects:

the Si4CL gene obtained in the invention is a key gene for regulating and controlling the synthesis of organic acid, polysaccharide, phenylpropanoid and flavonoid substances of the saussurea involucrate, which has important significance for obtaining high-flavone saussurea involucrate plants by a molecular breeding means; the molecular regulation of the Si4CL gene can increase the content of the medicinal components of the saussurea involucrate, and has important reference significance for improving the content of the effective active components of the medicinal plants. The saussurea involucrate 4-coumaroyl coenzyme A ligase coded by the Si4CL gene reacts with three substrates of FA, CA and pCA, and products are generated.

Drawings

FIG. 1: detecting the result of the PCR of the Si4CL gene;

FIG. 2: functional domain prediction analysis of Si4CL gene;

FIG. 3: e, prokaryotic expression protein electrophoresis pattern of pET32a-Si4 CL: m, marker, 1, supernatant, 2, precipitation, 3, purified protein;

FIG. 4: and (4) detecting the enzyme activity of Si4 CL.

A, single marking of p-coumaric acid (p-CA); the reaction result of the Si4CL prokaryotic protein and p-CA shows that the substrate is p-CA and the product is p-coumaroyl-CoA; c, Caffeic Acid (CA) single label; the reaction result of the Si4CL prokaryotic protein and CA shows that the substrate is CA and the product is caffeoyl-CoA (caffeoyl-CoA); e, Ferulic Acid (FA) single label; the reaction result of the Si4CL prokaryotic protein and FA shows that the substrate is FA and the product is feruloyl-CoA (feruloyl-CoA).

Detailed Description

Materials, reagents, vectors, E.coli and the like used in the following examples are commercially available from companies such as whole gold organism (catalog No. BM101-01) for PCR amplification reaction Marker, TaKaRa for PCR amplification reaction enzyme (catalog No. DR044A) for PCR amplification reaction, and whole gold organism for protein electrophoresis experiment (catalog No. DM111-02), unless otherwise specified.

Example 1: construction of full-length cDNA library of saussurea involucrate

Taking tender leaf parts of saussurea involucrate seedling, extracting tender stem total RNA by TransZol method (Total Jinbiol, catalog number: ET101-01), detecting total RNA content and purity by using nucleic acid analyzer, taking total RNA for reverse transcription reaction, and using kit

One-Step gDNA Removal anD cDNA Synthesis SuperMix (all-purpose gold Biotech, Cat. No.: AE 311-02). The method comprises the following specific steps:

1.1 homogenate

Taking a tissue sample of saussurea involucrate preserved at-80 ℃, transferring the tissue sample into a mortar precooled by liquid nitrogen, and continuously grinding plant tissues by using a pestle until the sample is ground into powder. Load into a 2mL fully pre-cooled centrifuge tube. The liquid nitrogen in the tube is removed to prevent cover collapse, 50-100mg (about 1/5 parts of the tube) of the ground sample is taken, 1mL of TransZol is added, and the mixture is shaken and mixed evenly.

1.2 layers separation

The homogenate was kept on ice for 5min (complete separation of the nucleoprotein material). 200mL of chloroform was added, the vial cap was closed, shaken vigorously for 3s, and left on ice for 3 min. Centrifuging at 4 deg.C for about 15min to separate layers, and collecting colorless supernatant, middle white protein layer and lower red organic phase.

1.3 precipitation of RNA

At room temperature, the supernatant was taken at about 550-650mL (best possible aspiration with guaranteed quality) and transferred to a new 2mL centrifuge tube (this tube was fully pre-cooled).

Add 500. mu.L of isopropanol and mix by gentle inversion, and let stand at-20 ℃ for 10 min.

Centrifugation was carried out at high speed for 10min at 4 deg.C (1% agarose gel was used in the meantime), and the supernatant was discarded (isopropanol was removed as much as possible).

1.4RNA Wash

At least 1mL of 75% ethanol was added, vortexed, mixed, and centrifuged at 7500g at 4 ℃ for 5 min.

1.5RNA redissolution

Discard the supernatant, suck dry the ethanol, open the lid and air dry at room temperature for 10 min.

Adding 30-40 μ L of RNase-free ddH₂And dissolving RNA for 2-3min by using O.

1.6 Total RNA content and purity were determined using agarose gel electrophoresis and nucleic acid Analyzer

1.7 reverse transcription of RNA

Sequentially adding the components into a microcentrifuge tube according to the following system

1.7.1 the RNA template, adsorbed Oligo (dT) and RNase-free Water were mixed well, incubated at 65 ℃ for 5min, ice-bathed for 2min and then the other reaction components were added.

1.7.2 mix gently and incubate at 42 ℃ for 15 min.

1.7.3 heating at 85 ℃ for 5s inactivated Trans Script RT with gDNA Remover.

Example 2: si4CL gene clone and prokaryotic expression vector construction

2.1 taking saussurea involucrate cDNA as a template, and utilizing a forward primer and a reverse primer:

F：5′-ATGGGTTCATCAGCTTTGGAT-3′，

r: 5'-CTATTTGGTAAACTCCATGACCCA-3', performing PCR amplification reaction, adding 5 μ L of amplification product into 1 μ L of loading buffer running electrophoresis, and observing gel electrophoresis pattern after 15min by using a gel imager, wherein the amplified fragment is about 1700bp (FIG. 1). Enlarging the reaction system, and recovering the gene fragment by using a gel recovery kit. In the PCR amplification reaction, the reaction system is 20 μ L: 2 XBuffer 10 uL, forward primer 1 uL, reverse primer 1 uL, DMSO 1 uL, cDNA 1 uL, dNTP 0.8 uL, water 5 uL, enzyme 0.2 uL.

The reaction conditions of the PCR amplification procedure were: pre-denaturation at 98 deg.C for 5min, denaturation at 98 deg.C for 10s, annealing at 56 deg.C for 5s, extension at 72 deg.C for 1.5min, 35 cycles, extension at 72 deg.C for 10min, and cooling at 16 deg.C.

2.2 sequencing and verifying the amplified fragment to obtain a nucleic acid sequence shown as SEQ ID NO.1, wherein the coded amino acid sequence is shown as SEQ ID NO. 2. Conserved domain analysis of the encoded amino acid sequence revealed that it was a typical conserved sequence of 4CL ligase (fig. 2). Subsequently, the pET32a vector was digested with EcoRI at 37 ℃ for 12 hours, loading buffer was added for agarose gel electrophoresis, the gel image was observed, and the empty vector fragment was recovered using the recovery kit.

The enzyme digestion reaction system is as follows: EcoRI 1. mu.L, 2 XBuffer 2. mu.L, vector 10. mu.L, water 7. mu.L. The saussurea involucrate 4CL fragment was ligated into pET32a vector by infusion method. The ligation products were transformed into E.coli DH 5. alpha. by heat shock method after 1h reaction at 37 ℃ under the action of ExnaseII, and plated. The reaction system is as follows: pET32a vector 2. mu.L, enzyme 1. mu.L, fragment 1. mu.L, 5 × CE II buffer 1. mu.L.

2.3 selecting the single clone to culture in LB culture solution, and carrying out PCR detection on the bacterial solution. 20 μ L reaction: 10 μ L buffer, 1 μ L each of forward and reverse primers, 1 μ L template and 7 μ L ddH₂And O. Reaction conditions are as follows: pre-denaturation at 95 ℃ for 5min, denaturation at 95 ℃ for 30s, annealing at 56 ℃ for 30s, extension at 72 ℃ for 1.5min for 29 cycles, extension at 72 ℃ for 10min, and cooling at 16 ℃. And (3) carrying out agarose gel electrophoresis detection on the PCR product, sequencing the PCR positive monoclonal bacteria liquid, and correctly sequencing, wherein the expression vector is named as pET32a-Si4 CL.

Example 3: induced expression of engineering bacteria

3.1 according to 1: transferring original bacteria liquid with correct sequencing into 50mL LB liquid culture medium in 100 proportion, and shaking to OD₆₀₀The value was 0.6-0.8, and then 15. mu.L of IPTG (1M) was added and the mixture was incubated overnight at 18 ℃ on a shaker.

3.2 the cells were collected by centrifugation, suspended in 30mL of precooled 10mM imidazole buffer, and repeated twice on ice, and in the second resuspension, 120. mu.L of saturated PMSF solution was added.

3.3 ultrasonic crushing, opening for 5s, closing for 4s, and crushing for 15min in total.

3.4 the crushed thallus is centrifuged at high speed for 30min in a centrifuge at 4 ℃.

3.5 pipette the supernatant through the column. (crude supernatant)

3.6 purification of protein: in a refrigerator at 4 ℃. 1mL of packing was added to the column, and the column packed with Ni-NTA was equilibrated twice with 15mL of lysine buffer, and 500. mu.L of crude protein was used for the running protein gel electrophoresis control before passing through the column.

3.7 the supernatant of the protein sample was applied to a well-balanced column and after completion, washed two or three times with Washing buffer.

3.8 elute His-tagged protein with Elution buffer, join eluted protein with 1.5mL tube, one tube per 250. mu.L, join 8-10 tubes, measure concentration.

3.9 boiling sample: 50 mu L of the supernatant, the precipitate and the purified protein sample are boiled for 10min at the temperature of 95 ℃.

3.10 adding 10 μ L of loading buffer protein gel to the cooked protein sample, adjusting the voltage to 80V at the beginning stage, and increasing the voltage to 120V after Marker separation.

3.11 after running electrophoresis, staining the protein gel, and decolorizing after 1 h.

The results showed that a distinct specific protein band appeared at a molecular weight of 70kDa, consistent with a theoretical value of 69.1kDa, as shown in FIG. 3.

Example 4: detection of Si4CL gene bioactivity

The enzyme activity of Si4CL prepared in example 3 was detected by HPLC (high performance liquid chromatography), using FA, pCA, CA, and SA as substrates, adding saussurea involucrate 4-coumaroyl-CoA ligase (Si4CL) prepared in example 3, and the enzyme activity reaction system was buffer 80. mu.L, water 110. mu.L, CoA (1mM) 40. mu.L, ATP (10mM) 60. mu.L, protein 80. mu.L, and substrate (2mM) 30. mu.L. One blank was added for each substrate. Mixing, placing in 30 deg.C water bath, reacting for 30min, adding methanol solution to terminate reaction, filtering, adding into sample bottle, and performing liquid phase detection. As shown in FIG. 4, Si4CL reacted with three substrates, FA, CA and pCA, and a product was produced.

Sequence listing

<110> Nicotiana tinctoria and stamen women products Co., Ltd

Qingdao Institute of Bioenergy and Process, Chinese Academy of Sciences

<120> Si4CL gene and its coding product and application

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1620

<212> DNA

<213> saussurea involucrate (Saus-surea involverata Kar.et. Sch. -Bip.)

<400> 1

atggaggcaa agtcggagac cattttccgg tcaaggctgc cggacattta tatacccaaa 60

catctctctt tgcattccta ttgctttgaa aacatttcaa atttcggtga ccgaccgtgt 120

ttgattaacg gcgccaccgg tgacgtacac acctatgctg acgtggagct cacctcacga 180

aaagtcggtt ctgcccttca caaacacggc atcaacaaag gtgacgtcat catgatttta 240

ctcccgaatt cgccggaatt tgtctactcg ttcctcggcg cctccttcat cggcgccgtc 300

tccaccatgg ccaatccgtt cttcaccgcc gctgagatca tcaagcaagc caaagcgtcc 360

gccgcgaaaa ttattattac tcaatcggta aatgtagcga aggtgaagga atttgcatcg 420

gagaactcta ttaagctcgt ctgcatagat tcagctccgg aaaagtgctt gcacttttcc 480

gatttgattt ccggcgatga gaatgagctt ccgaaggttg agatctcgtc ggacgacgtc 540

gtagcactgc cgtattcctc cgggactacc ggactaccaa aaggagtcat gttgactcac 600

ttagggctcg taacgagcgt agcgcagcaa gtcgacggag aaaaccctaa tttatggatt 660

cacagcgagg atgtgttgat gtgcgtgttg ccattgtttc acatctactc gttgaattcg 720

atcttgttgt gcggattacg cgcaggtgca gcaatcctga ttatgcagaa attcgatata 780

gttccatttc tggagttgat tcagcagtac aaagtgacga ttggaccatt cgtgccgcct 840

atcgttctca ccattgcgaa aaacgaagaa attgtggata agtacgatct gacgtcaatt 900

cgaacggtga tgtccggcgc tgcgccgtta ggtaaagagc ttgaagacac cgttaggatg 960

aaattcccaa atgcgaaact cggacagggt tacgggatga cggaggcagg gccagtgctg 1020

gcgatgtgtt tggcttttgc taaagaacca tccgaaatca aatcaggagc ttgtggcacc 1080

gtcgtccgta atgcagagat gaagatcgtg gatccagaca ccggtgcttc tcttcctcgg 1140

aatcaacgcg gcgagatctg cattcgcggt gatcagatca tgaaaggtta ccttaatgat 1200

ccggaagcaa ccaagagaac gatcgtagac ggttggttgc acactggaga catcgggtac 1260

attgatgacg atgacgagct tttcatagtt gatcgattga aggaattaat caagtacaaa 1320

ggatttcaag ttgcgccagc tgagctcgaa gcattgctac tcacacatcc cgacatctca 1380

gatgccgctg ttgtagcaat gatagatgaa ggtgcgggtg aggttcccgt tgcttttgtg 1440

gtaaaatcga acggttccac cgtcacggaa gctgaactca agcaatttat ctccaaacag 1500

gtggtatttt acaagagatt aaaccgggtg ttcttcgttg acacgatccc taaatcccca 1560

gctgggaaga ttcttcgtaa ggatctaaga gcgaagctag cagctggtat tccaaaataa 1620

<210> 2

<211> 539

<212> PRT

<213> saussurea involucrate 4-coumaroyl-CoA ligase (Saus-surea invocata Kar.et. Kir. Sch. -Bip.)

<400> 2

Met Glu Ala Lys Ser Glu Thr Ile Phe Arg Ser Arg Leu Pro Asp Ile

1 5 10 15

Tyr Ile Pro Lys His Leu Ser Leu His Ser Tyr Cys Phe Glu Asn Ile

20 25 30

Ser Asn Phe Gly Asp Arg Pro Cys Leu Ile Asn Gly Ala Thr Gly Asp

35 40 45

Val His Thr Tyr Ala Asp Val Glu Leu Thr Ser Arg Lys Val Gly Ser

50 55 60

Ala Leu His Lys His Gly Ile Asn Lys Gly Asp Val Ile Met Ile Leu

65 70 75 80

Leu Pro Asn Ser Pro Glu Phe Val Tyr Ser Phe Leu Gly Ala Ser Phe

85 90 95

Ile Gly Ala Val Ser Thr Met Ala Asn Pro Phe Phe Thr Ala Ala Glu

100 105 110

Ile Ile Lys Gln Ala Lys Ala Ser Ala Ala Lys Ile Ile Ile Thr Gln

115 120 125

Ser Val Asn Val Ala Lys Val Lys Glu Phe Ala Ser Glu Asn Ser Ile

130 135 140

Lys Leu Val Cys Ile Asp Ser Ala Pro Glu Lys Cys Leu His Phe Ser

145 150 155 160

Asp Leu Ile Ser Gly Asp Glu Asn Glu Leu Pro Lys Val Glu Ile Ser

165 170 175

Ser Asp Asp Val Val Ala Leu Pro Tyr Ser Ser Gly Thr Thr Gly Leu

180 185 190

Pro Lys Gly Val Met Leu Thr His Leu Gly Leu Val Thr Ser Val Ala

195 200 205

Gln Gln Val Asp Gly Glu Asn Pro Asn Leu Trp Ile His Ser Glu Asp

210 215 220

Val Leu Met Cys Val Leu Pro Leu Phe His Ile Tyr Ser Leu Asn Ser

225 230 235 240

Ile Leu Leu Cys Gly Leu Arg Ala Gly Ala Ala Ile Leu Ile Met Gln

245 250 255

Lys Phe Asp Ile Val Pro Phe Leu Glu Leu Ile Gln Gln Tyr Lys Val

260 265 270

Thr Ile Gly Pro Phe Val Pro Pro Ile Val Leu Thr Ile Ala Lys Asn

275 280 285

Glu Glu Ile Val Asp Lys Tyr Asp Leu Thr Ser Ile Arg Thr Val Met

290 295 300

Ser Gly Ala Ala Pro Leu Gly Lys Glu Leu Glu Asp Thr Val Arg Met

305 310 315 320

Lys Phe Pro Asn Ala Lys Leu Gly Gln Gly Tyr Gly Met Thr Glu Ala

325 330 335

Gly Pro Val Leu Ala Met Cys Leu Ala Phe Ala Lys Glu Pro Ser Glu

340 345 350

Ile Lys Ser Gly Ala Cys Gly Thr Val Val Arg Asn Ala Glu Met Lys

355 360 365

Ile Val Asp Pro Asp Thr Gly Ala Ser Leu Pro Arg Asn Gln Arg Gly

370 375 380

Glu Ile Cys Ile Arg Gly Asp Gln Ile Met Lys Gly Tyr Leu Asn Asp

385 390 395 400

Pro Glu Ala Thr Lys Arg Thr Ile Val Asp Gly Trp Leu His Thr Gly

405 410 415

Asp Ile Gly Tyr Ile Asp Asp Asp Asp Glu Leu Phe Ile Val Asp Arg

420 425 430

Leu Lys Glu Leu Ile Lys Tyr Lys Gly Phe Gln Val Ala Pro Ala Glu

435 440 445

Leu Glu Ala Leu Leu Leu Thr His Pro Asp Ile Ser Asp Ala Ala Val

450 455 460

Val Ala Met Ile Asp Glu Gly Ala Gly Glu Val Pro Val Ala Phe Val

465 470 475 480

Val Lys Ser Asn Gly Ser Thr Val Thr Glu Ala Glu Leu Lys Gln Phe

485 490 495

Ile Ser Lys Gln Val Val Phe Tyr Lys Arg Leu Asn Arg Val Phe Phe

500 505 510

Val Asp Thr Ile Pro Lys Ser Pro Ala Gly Lys Ile Leu Arg Lys Asp

515 520 525

Leu Arg Ala Lys Leu Ala Ala Gly Ile Pro Lys

530 535

Claims

1. A Si4CL gene is characterized in that the nucleotide sequence of the gene is shown in SEQ ID NO. 1.

2. The protein encoded by the Si4CL gene of claim 1, wherein the protein is saussurea involucrate 4-coumaroyl-CoA ligase, and the amino acid sequence of the protein is shown in SEQ ID No. 2.

3. A recombinant vector pET32a-Si4CL containing the gene of claim 1.

4. The application of the gene of claim 1 in regulating and controlling the synthesis and genetic improvement of flavone and phenylpropanoid of saussurea involucrate.

5. The use of the recombinant vector pET32a-Si4CL of claim 3 in regulating and controlling the synthesis and genetic improvement of flavone and phenylpropanoid of saussurea involucrate.