CN105255924A - Betula platyphylla cycloartenol synthase gene BPX3 and application for regulation and control of betula platyphylla triterpene content - Google Patents

Abstract

The invention belongs to the betula platyphylla transgenic technology field, and concretely relates to a betula platyphylla cycloartenol synthase gene BPX3 and an application for regulation and control of a betula platyphylla triterpene content. The betula platyphylla cycloartenol synthase gene BPX3 is characterized in that by utilization of designed primers BPX3R and BPX3F (shown in the graph 2), the cDNA full-length sequence of the cycloartenol synthase gene is obtained from betula platyphylla plants through clone, the sequence is shown in the graph 1 and is named after BPX3 temporarily, and the graph 1 and the graph 2 are shown in the specification. Genetic transformation of betula platyphylla clone is carried out by RNAi carriers of the constructed BPX3 gene p121-N4 and through agrobacterium tumefaciens mediation leaf disc transformation and transgenic strains are obtained through screening. In transgenic betula platyphylla saplings Y-12 and B-6, BPX3 is inhibited, the relative expression levels of BPY and BPW genes are raised substantially. The contents of betulinic acid and oleanolic acid in the transgenic betula platyphylla plant callus are higher than that of wild types substantially, which shows that BPX3 gene RNAs are inhibited and synthesis andaccumulation of object triterpene products betulinic acid and oleanolic acid are promoted. The deveopment of the betula platyphylla cycloartenol synthase gene BPX3 lays a foundation for betula platyphylla triterpene substance metabolism approach genetic modification and biotechnology utilization. The betula platyphylla cycloartenol synthase gene BPX3 can be used in research of raising the betula platyphylla triterpene effective component content by the transgenic technology and industrialization, and has good application prospects.

Description

The application of white birch ring A Qi alcohol synthase gene BPX3 and regulation and control white birch triterpene content

Art

The invention belongs to birch transgenic technical field, be specifically related to the application that white birch ring A Qi alcohol synthase gene BPX3 and BPX3RNAi regulates and controls the accumulation of white birch triterpene effective constituent.

Background technology

Containing important secondary metabolite-white birch triterpene (TBP) in white birch (Betulaplatyphyllasuk.) tree, wherein trochol, Betulinic acid and Oleanolic Acid composition are anticancer, the anti AIDS virus (HIV) of great exploitation potential for its and Cardiovarscular and the pioneer's medicine protecting liver.

Along with natural drug exploitation is day by day risen, natural plant resource is tending towards deficient, how to utilize natural plant resource also day by day urgent effectively and reasonably; Simultaneously due to the secondary metabolite of plant, content is low in its natural state, and the output how improving Secondary metabolites is the key link that can the exploitation being related to natural drug be applied to actual production.But secondary metabolism approach is usually very complicated, and usually has complicated interaction between different approaches.In order to change the content of certain secondary metabolite in plant according to the wish of people, or allow the secondary metabolite that phytosynthesis is new, except needing first to illustrate their pathways metabolism, also must understand accumulation and transport mechanism etc. after the expression regulation suffered by enzyme in pathways metabolism, the abundance of precursor, Product formation position, synthesis.In order to make these crude substance better by the mankind are utilized, genetic modification is carried out to its pathways metabolism and will become an important research direction, wherein improve the gene expression amount of target pathway by engineered method or suppress the genetic expression of other approach, promoting that plant or cell expressing secondary metabolite are a kind of new means.Several secondary metabolites existing obtain expression in transgenic plant and culturing cell at present.

In higher plant, the triterpene skeleton different more than 100 kinds is had to be in the news.These triterpene skeletons are synthesized through squalene cyclase (oxidosqualenecyclases, OSCs) by common precursor 2,3-oxidosqualene (2,3-oxidosqualene).Wherein this enzyme forms sterol (ring A Qi alcohol) and different triterpenoids respectively in substrate cyclization process.Ring A Qi alcohol synthase (cycloartenolsynthase, CAS) gene is at mangrove (Rhizophorastylosa), Arabidopis thaliana ^[69](Arabidopsisthaliana), be cloned out in the plant such as Rhizoma Paridis (Parispolyphyllavar.yunnanensis), Radix Et Caulis Acanthopanacis Senticosi (Eleutherococcussenticosus), Rhizome of Peltate Yam (Dioscoreazingiberensis).And in the research of the secondary metabolites such as molecular regulation triterpene compound, have and CAS gene in pseudo-ginseng, ginseng is carried out silence, result makes total saponin content significantly increase respectively.And about CAS gene in China white birch and after suppressing, report is had no to the research of white birch triterpene synthesis regulation.

The various activeconstituents of white birch triterpene (trochol, Oleanolic Acid, dammarenediol etc.) and sterol (ring A Qi alcohol) are all from precursor substance 2 common in MVA pathways metabolism, 3-oxidosqualene, under the regulation and control of different triterpene synthase gene, biosynthesizing is carried out along respective metabolism direction, this just causes competing between several product same precursor, and mutually suppress synthesis, cause total triterpene contents higher, but target effective composition Betulinic acid and content of oleanolic acid low, restriction apply further.

This research is according to white birch triterpene pathways metabolism, and with China's betula mandshurica Nakai for material, clone white birch ring A Qi alcohol synthase gene BPX3, carries out bioinformatic analysis, build ring A Qi alcohol synthase gene BPX3RNAi expression vector simultaneously, and genetic transformation white birch clone.After detecting BPX gene inhibition, the synthesis of white birch triterpene component, accumulation and triterpene pathway key gene express change, to the regulating effect of betulinic acid and Oleanolic Acid synthesis and the regulating and controlling effect of BPX3 gene in white birch triterpene metabolism network after blocking to clear and definite sterol synthesis branch.Carrying out of this research is laid a good foundation for the genetic modification of white birch triterpene substance pathways metabolism and the utilization of biotechnology.

Summary of the invention:

The present invention aims to provide and a kind ofly can regulate and control the synthesis of white birch triterpene effective constituent and the relevant gene accumulated, by the albumen of this genes encoding and application thereof, for Future Development gene engineering product lays the foundation.

In order to achieve the above object, technical scheme provided by the invention is:

1. the invention provides the BPX3 gene of a kind of white birch, gene order and amino acid are as shown in Figure 1.

2. present invention also offers a kind of primer pair for the white birch BPX3 gene that increases, described primer pair as BPX3R and BPX3F, as shown in Figure 2.

3. the invention provides a kind of recombinant RNA interference carrier containing BPX3 gene, be made up of the goal gene of empty carrier and this empty carrier of insertion.Specifically comprise: plant expression vector pBI121, be connected respectively to empty carrier pBI121 intron pdk both sides by BPX3 gene justice, antisense fragments, obtain recombinant plant RNAi expression vector pBI121-BPX3, i.e. p121-N4.

4. present invention also offers BPX3 gene in the synthesis of regulation and control white birch triterpene effective constituent and the application in accumulation.

Accompanying drawing illustrates:

Fig. 1 is that RT-PCR of the present invention amplification white birch BPX3 gene nucleotide and aminoacid sequence illustrate;

Fig. 2 is white birch BPX3PCR amplimer sequence;

Fig. 3 BPX3 comprises the conserved sequence (35th ~ 675aa position) of a special ISOPREN_C2_like superfamily;

Fig. 4 is that BPX3 gene coding amino acid sequence of the present invention and other plant CAS genetic homology comparison illustrate;

Fig. 5 is BPX3 gene coding amino acid sequence evolution of the present invention tree analysis chart;

Fig. 6 is BPX3 gene RNAi plant PCR qualification figure of the present invention;

Fig. 7 (A) is BPX3RNAi transgenosis white birch callus content of oleanolic acid;

Fig. 7 (B) is BPX3RNAi transgenosis white birch callus TLC Determination of Betulinic Acid.

Embodiment

Embodiment 1. white birch sterol (ring A Qi alcohol) BPX3 gene clone

1. the cDNA sequence of white birch BPX3 gene obtains

Utilize Tris-CTAB method to extract white birch suspension cell total rna, after utilizing RQ1RNase-FreeDNase DNA digestion, reverse transcription synthesis cDNA, cDNA synthetic operation is with reference to purchased from TaKaRa company premixExTaq ^tM(PerfectRealTime) test kit specification sheets.By analyzing plant ring A Qi alcohol synthase sequence in known NCBI, utilize PrimerPremier5 to design primer to obtain the sequence of China betula mandshurica Nakai ring A Qi alcohol synthase gene, primer is by Hua Da gene chemical synthesis.RT-PCR primer sequence is

BPX3R：5`-ATAGGGTACCATGTGGAAGCTGAAGATCG-3`

BPX3F：5`-CATTCTCGAGTTAGGGAGCCTGCAATACC-3`

RT-PCR system and program: 94 DEG C, 5min, 35 circulations (94 DEG C of 1min, 61 DEG C of 1min, 72 DEG C of 3min), 72 DEG C of 10min.PCR primer is detected by 1.0% agarose gel electrophoresis.

2.BPX3 gene clone, order-checking

(1) whole for PCR product is carried out agarose gel electrophoresis, under gel is placed in ultraviolet lamp, cut with the gel of scalpel by object segment portion, and unnecessary gel is cut away as far as possible, collect in the centrifuge tube of sterilizing.Reclaim test kit specification sheets according to Promega glue to reclaim, 1% agarose gel electrophoresis detects.

(2) basis t1 cloning vector specification sheets will reclaim product with t1 cloning vector connects.Reaction system is as follows: PCR reclaims product 0.5 ~ 4 μ L, t1CloningVector, 1 μ L.Mix gently, room temperature (20 DEG C ~ 37 DEG C) reaction 5min.After reaction terminates, centrifuge tube is placed on ice.

(3) above-mentioned connection product is added in 50 μ LTrans1-T1 competent cells, flicks mixing, ice bath 30min, after 42 DEG C of heat shock 45s, be placed in 2min on ice immediately.Add 800 μ L antibiotic-free LB liquid nutrient mediums, 37 DEG C, 1h is cultivated in 200r concussion.Get 8 μ L500mMIPTG and 40 μ L20mg/mLX-gal mix, being applied to containing middle concentration is equably on the LB flat board of 100mg/L ammonia benzyl mycin.Treat IPTG, after X-gal is absorbed, get 200 μ L bacterium liquid and be spread evenly across on this plate culture medium, 37 DEG C of overnight incubation.

(4) picking white colony is inoculated in the LB liquid nutrient medium containing ammonia benzyl mycin respectively, and 37 DEG C jolt cultivation 8h, carries out PCR reaction qualification.

(5) carry out recovery order-checking, checking cloning and sequencing exactness to above-mentioned PCR result, checking order is completed by Hua Da gene.

3.BPX3 gene biological bioinformatics analysis

The cDNA full length sequence of ring A Qi alcohol synthase (CAS) gene of white birch is obtained, temporary called after BPX3 (Fig. 1) by RT-PCR method.

Bioinformatics software is utilized to carry out characterization of molecules to this sequence, physico-chemical property, homology and genealogical tree evolutionary analysis, result shows that BPX3 gene cDNA total length is 2225bp, open reading frame is 2208bp, to encode 687 amino acid (Fig. 1), comprise the conserved sequence (35th ~ 675aa position) (Fig. 3) of a special ISOPREN_C2_like superfamily, the product of coding is hydrophilic protein, have by α spiral (alphahelix), extended chain (extendedstrand) and random coil (randomcoil) secondary structure and four transbilayer helixs, and with Japanese white birch (Betulaplatyphyllavar.japonica), the ring A Qi alcohol synthase homology higher (Fig. 4) of the species such as margosa tree (Azadirachtaindica).Aminoacid sequence has carried out ClustalW method sequence analysis, and the CAS sequence that result shows protein coded by its BPX3 and each species has higher similarity, and most of sequence has conservative property (Fig. 5).GO analytical results display BPX3 aminoacid sequence has ring A Qi alcohol synthase activity, catalytic activity, isomerase activity and intramolecular transfer enzymic activity on molecular function, take part in the biosynthetic process of pentacyclic triterpenoid and phytosterin compound.It is AQP-CHIP that cellular constituent (cellularcomponent) analyzes this protein of display, is distributed in vacuole.

The acquisition of embodiment 2. white birch sterol (ring A Qi alcohol) BPX3RNAi transfer-gen plant

1. the design of white birch BPX3 Gene interfere fragment and BPX3RNAi vector construction

Utilize white birch BPX3 full length gene, the 250bp interference fragment chosen is positioned at 1803rd ~ 2052 positions of BPX3 total length, finds do not have homology with white birch genes involved after BLAST comparison.And the sense fragment Sequences upstream of interference fragment synthesized by Shanghai Sheng Gong biotech firm and the downstream of antisense fragments, be added with SacI and BamHI restriction enzyme site respectively, middle hair clip spatial sequence is the pdk intron of 300bp, justice and antisense fragments insert pdk intron both sides in plant binary expression vector pBI121, by the interference carrier plasmid called after pBI121-BPX3 of gained BPX3 gene.Utilize heat shock method by pBI121-BPX3 transformation of E. coli competent cell, after single bacterium colony grows, picking list bacterium colony, joining 5mL contains in the LB liquid medium of ammonia benzyl mycin or kantlex, 37 DEG C of constant-temperature shaking culture are about 8h, extract plasmid, and carry out PCR qualification to recombinant plasmid.

2. triparental mating transformation Agrobacterium LBA4404

(1) inoculate acceptor Agrobacterium LBA4404 on the YEB solid medium containing 100mg/mL Rifampin, 28 DEG C of cultivations, after single bacterium colony grows, select a single colony inoculation in YEB liquid nutrient medium, 28 DEG C of shaken overnight.

(2) inoculation assists bacterium Helper on solid LB media, and 37 DEG C of cultivations, after waiting bacterium colony to grow, select a single colony inoculation in LB liquid nutrient medium, 37 DEG C of shaking culture are spent the night.

(3) by the single colony inoculation containing recombinant plasmid containing 50 μ g/mL kantlex LB liquid nutrient medium in, 37 DEG C of shaking culture are spent the night.

(4) when three kinds of bacteria growings are to OD ₂₆₀when being about 0.5, equal-volume mixes, and coats not containing on any antibiotic YEB solid medium, 28 DEG C of incubated overnight.

(5) bacterium colony grown is transferred on the solid YEB substratum containing 100mg/mL Rifampin and 50mg/mL kantlex with inoculating needle, cultivates 3 ~ 4 days, grow single bacterium colony for 28 DEG C.

(6) the single bacterium colony grown is transferred to again on the YEB solid medium containing 100mg/mL Rifampin and 50mg/mL kantlex, cultivates 3 ~ 4 days, until grow bacterium colony for 28 DEG C.Picking list bacterium colony is also identified.Identify with recombinant plasmid in correct Agrobacterium, by further amplification cultivation, for the conversion of plant.

3. the qualification of interference carrier transformation Agrobacterium

In order to identify conversion and the screening positive clone of plasmid, according to hairpin structure spatial sequence pdk intron sequences design primer I n-F/-R, its product is the intron sequences of 268bp (not containing the Sequence of restriction enzyme site).Utilize the purulence bacillus liquid that this primer pair contains Rifampin and kalamycin resistance to carry out PCR qualification, obtain positive colony, consistent with expected results through order-checking, obtained positive colony engineering Agrobacterium is designated as L-N4.

4. white birch BPX3RNAi genetic transformation

When carrying out the experiment of BPX3-RNAi fragment genetic transformation, induce into aseptic seedling (WPM substratum+0.5mg/LIBA, sucrose 20g/L by the excellent Betula platyphylla seed of Northeast Forestry University's breeding garden, agar 5.3g/L, pH6.5), and select stem to strengthen, the material that blade is large is tested.Adopt leaf disk method carry out white birch tissue genetic transformation, outside shade comprises the blade of white birch tissue cultured seedling, stem section and petiole, infects through engineering strain L-N4, Agrobacterium after Activation Assay, picking list bacterium colony, joins 5mL and contains in the YEB substratum of kantlex and Rifampin, to OD ₆₀₀about=0.6 ~ 0.8,28 DEG C, 220r shaking culture 12h.Get 1mL bacterium liquid next day and be added to the YEB substratum of 50mL containing kantlex (50mg/mL, 50 μ L) and Rifampin (50mg/mL, 50 μ L), 28 DEG C of constant temperature, 220r shaking culture 4 ~ 5h, to OD ₆₀₀=0.1 ~ 0.5 carries out Dual culture (IS substratum+0.8mg/L6-BA+0.6mg/LNAA, sucrose 20g/L, agar 6g/L, pH6.0) 3 ~ 5 days, microbiotic (containing 400mg/L cephamycin+40mg/L kantlex) removes Agrobacterium, to transfer again division culture medium, for NT substratum+0.5mg/L6-BA, sucrose 20g/L, agar 6g/L, pH6.0 ~ 6.5) and differentiation from the induction of raw seedling, when the indefinite bud broken up grows to more than 1cm, cut indefinite bud, and the division culture medium inserted containing Selective Pressure continues differentiation, root culture, obtain genetically modified aseptic tissue cultured seedling.Culture condition: temperature is 24 ~ 26 DEG C, intensity of illumination is 2,000lx, light application time 16h/d, and humidity is 40 ~ 50%.

5. the PCR qualification of transgenosis white birch

Extract transgenosis white birch DNA as template, with pBI121-BPX3 plasmid DNA for positive control, the white birch plant DNA of unconverted is negative control, and primer is that NptII-F/R, pdkIntron sequence carries out pcr amplification detection.Pcr amplification condition: 94 DEG C of denaturation 3min; 94 DEG C of sex change 45s, 55 DEG C of renaturation 45s, extend 45s, 35 circulations by 72 DEG C; 72 DEG C extend 7min.The PCR primer agarose gel electrophoresis of 1% detects.PCR qualification display obtains transfer-gen plant 30 strain (part NPTII gene PCR qualification result is as Fig. 6), carries out the checking of Southern trace further.

The Southern trace qualification of transgenosis white birch: with plasmid pBI121 plasmid for template, NptII-F/-R is that to carry out NptII and the 268bppdk intron sequences fragment label that PCR reaction product is about 519bp be probe to primer, transgenosis white birch DNA is template, and NptII-F/-R is that the pcr amplification reaction product of primer carries out Southern Blot experiment and identifies further and verify transfer-gen plant for hybridization template.Concrete operations are, utilize NptII-F and NptII-R primer, with pBI121 vector plasmid for template carries out pcr amplification reaction synthesis NptII fragment, reclaim purifying as the template of probe mark, carry out mark and the qualification of Southern trace of probe according to the digoxigenin labeled of Roche company and detection kit specification sheets.Transfer-gen plant and positive control all show consistent hybridising band, prove that the transgenic fragment comprising NptII, pdkIntron is incorporated in Plant Genome further.

The regulating and controlling effect of example 3:BPX3 transgenosis white birch in the synthesis of white birch triterpene effective constituent and accumulation

Extraction and the reverse transcription cDNA of wild-type and transgenosis white birch tissue cultured seedling RNA operate with example 1.According to standard fluorescence quantification PCR primer principle of design design triterpene synthesis key gene primer.Utilize real-time fluorescence quantitative PCR ( premixExTaq ^tM, TaKaRa) and method detects 4 strain positive transgenic plant ring A Qi alcohol synthase BPX3 gene relative expression quantities respectively, the transfer-gen plant that screening silence efficiency is higher.Result shows, and the BPX2 gene relative expression quantity in transfer-gen plant B-6 is the relative expression quantity of BPX2 gene in 48%, Y-12 plant of wild type control group is 3% of control group expression amount, significantly lower than wild-type white birch.Result illustrates that BPX3RNAi significantly can reduce the expression amount of BPX3 gene.

Respectively the detection of relative expression quantity is carried out to other relative enzyme genes in the triterpenoid route of synthesis of Y-12 and B-6.In transfer-gen plant, in Y-12 transfer-gen plant, the relative expression quantity of BPY is 5.55 times of control group, and BPW content also significantly improves, and is 2.89 times of wild-type; And in B-6 transfer-gen plant, except BPX3, other genes raise all to some extent, BPY relative expression is 70.92 times of wild-type, and the relative expression of BPW brings up to 84.10 times of contrast; Illustrate that the interference of white birch BPX3 gene is conducive to Betulinic acid and Oleanolic Acid synthetic gene up-regulated expression.

High performance liquid chromatography is utilized to detect the content of Oleanolic Acid and betulinic acid in the callus of transfer-gen plant induction respectively, concrete grammar is: precision takes 0.5g and organizes dry sample, add 25mL hydrochloric acid-ethanol solution (2:8, v/v), 90 DEG C of backflow 3h, shake up filtration after cooling.Precision measures filtrate 15mL and distilled water 15mL respectively, mixing is placed in 80 DEG C of water-baths and boils off ethanol, then 20mL extracted with diethyl ether is got respectively 3 times, merge supernatant ether extraction liquid in 40 DEG C of low temperature evaporates to dryness, 1mL dissolve with methanol residue, utilize 0.45 μm of organic filter membrane to filter, this is sample detection liquid, then utilizes high performance liquid chromatography to detect.

High performance liquid chromatography (HPLC) testing conditions: with Waters company 600-717-2487 chromatographic system, chromatographic column HiQsilC18V4.6mm × 250mm; Moving phase is acetonitrile: water=9:1 (v/v); Column temperature 25 DEG C; Sensitivity 16AUFS; Flow velocity 1.0mL/min; (Oleanolic Acid regression equation is determined wavelength 210nm, sample introduction 20 μ L: y=10000000x+43301, R ²=0.9993; Betulinic acid regression equation is: y=10000000x+63763, R2=0.9994).

Result shows, and the content of oleanolic acid of transfer-gen plant Y-12 callus is significantly higher than wild-type, is respectively 5.97 times and 8.92 times of wild control group; And the accumulation volume of betulinic acid significantly raises in B-6, be 2.10 times (Fig. 7 (A), Fig. 7 (B)) of control group.Show that the RNAi of BPX3 in white birch can significantly lower BPX3 expression amount, BPW and BPY gene can be impelled significantly to raise, this significantly promotes synthesis and the accumulation of triterpene downstream pathway target triterpene product betulinic acid and Oleanolic Acid.

Claims (6)

1. The application of white birch ring A Qi alcohol synthase gene BPX3 and regulation and control white birch triterpene content, is characterized in that comprising the following steps:

   1. a BPX3 gene for white birch, is characterized in that, described BPX3 gene order and proteins encoded aminoacid sequence are as shown in Figure 1.
2. 2., for a primer pair for the BPX3 gene described in claim 1 that increases, its feature as shown in Figure 2.
3. 3. a restructuring BPX3RNA interference carrier, be made up of the goal gene of empty carrier and this empty carrier of insertion, it is characterized in that, described goal gene is BPX3 gene according to claim 1.
4. 4. the transfer-gen plant containing plant RNA interference expression vector described in claim 3.
5. 5. BPX3 gene and the application of proteins encoded in the triterpene synthesis of regulation and control white birch and accumulation thereof according to claim 1.
6. 6. BPX3 gene and proteins encoded thereof are improving the application in white birch triterpene in Betulinic acid and content of oleanolic acid according to claim 1.