CN107502614A - A kind of screening and functional verification of the carotenoid cleavage dioxygenases encoding gene for participating in the synthesis of cape jasmine crocin - Google Patents
A kind of screening and functional verification of the carotenoid cleavage dioxygenases encoding gene for participating in the synthesis of cape jasmine crocin Download PDFInfo
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Abstract
The present invention relates to carotenoid cleavage dioxygenases CCD4a, its encoding gene and its function for participating in the synthesis of cape jasmine crocin.Belong to gene engineering technology field.The invention discloses the open reading frame sequence of cape jasmine carotenoid cleavage dioxygenases CCD4a genes and the amino acid sequence of its coding.The present invention obtains CCD4a genes based on transcription group screening, by building pET28a CCD4a prokaryotic expression carriers, verifies CCD4a function in vitro using zeaxanthin as substrate.As a result prove that CCD4a has the function of carotenoid cleavage dioxygenases, the parsing and synthetic biology research for crocin biological relations lay the foundation.
Description
Technical field
The invention belongs to field of plant genetic, and in particular to the screening of crocin biosynthesis gene and work(
It is able to verify that method.
Background technology
Crocin is carotene compounds, is mainly accumulated in west safflower column cap and cape jasmine fruit.Crocin exists
Suppress cancer, improve memory, anti-melancholy, treatment angiocardiopathy etc., there is the effect of certain.In view of crocin
Medicinal and economic value, its platymiscium west safflower have often been added many rings of light, " the red gold " being described as in medicinal material, retail
Valency is up to the ￡/kg of price 2,000 to 7,000.
China traditional Chinese medicine cape jasmine (Gardenia jasminoides Ellis) is also enriched in crocin, it was reported that western red
Flower glycosides I and crocin the II content in pulp is up to 27.54mg g-1.Meanwhile the chemistry and pharmacological research of cape jasmine are more deep
Enter, cape jasmine aboundresources, fruit is easier to draw materials compared with west safflower, so it is considered that cape jasmine is the research of crocin synthesis regulation
Potential desirable plants.
In recent years, the biosynthesis pathway research of crocin receives significant attention, and participates in crocin route of synthesis and closes
Although key enzyme is substantially clear, these key genes are all multigene families, and the gene specifically to function is unknown.To western red
The biosynthesis research of flower glycosides is related to downstream CCD, ALDH and UGT gene, and wherein CCD researchs are the most extensive, and multiple genes are
It is verified function, such as CsCCD1a, CsCCD1b, CsCCD2, CaCCD2, CsCCD4a, CsCCD4b, CsCCD4c.However, only
CsCCD2 and CaCCD2 is proved to participate in the synthesis of crocin.Whether CCD genes participate in crocin synthesis not yet in cape jasmine
Know, therefore be extremely necessary to excavate cape jasmine CCD genes and verify its function, laid the foundation for the synthesis of crocin.
The content of the invention
It is an object of the invention to provide it is a kind of participate in crocin synthesis carotenoid cleavage dioxygenases gene and
Its protein encoded.
Another object of the present invention is to provide a kind of screening technique of cape jasmine crocin synthesis key gene.
Third object of the present invention is to provide the functional verification side to the above-mentioned key gene CCD4a filtered out
Method.
CCD4a genes provided by the invention, its nucleotides sequence is classified as shown in SEQ ID No.1, or its mutant nucleotide sequence.
The protein of CCD4a gene codes provided by the invention, its amino acid sequence as shown in SEQ ID No.2, or its
Mutant nucleotide sequence.
The object of the invention can be achieved through the following technical solutions, technical scheme one：Cape jasmine crocin based on transcript profile
Key gene screening is synthesized, step is as follows：
1) UPLC detects the content of crocin in cape jasmine blade, green fruit and haw.
2) Illumina microarray datasets are based on, and according to the Standard Operating Procedure of microarray dataset, structure cape jasmine blade, green fruit
It is sequenced with haw CDNA libraries and upper machine, sequencing result is assembled and annotated.Analyze differential gene expression, primary dcreening operation west safflower
Glycosides route of synthesis key enzyme albumen sequence.
3) using qRT-PCR detection candidate gene expressions, structure phylogenetic analysis cape jasmine and other species candidate's egg
Evolutionary relationship between white, further screen crocin route of synthesis key gene.
Technical scheme two：Key gene CCD4a functional verification.Using coli expression system, with zeaxanthin
For substrate, the external function of identifying carotenoid cleavage dioxygenases CCD4a.
System is improved the invention discloses a whole set of of crocin biosynthesis gene CCD4a screenings and functional verification,
Checking CCD4a has the function of carotenoid cleavage dioxygenases, can be catalyzed zeaxanthin, to illustrating crocin synthesis
Approach provides molecular basis, while has great application value, can pass through technique for gene engineering means biosynthesis west safflower
Glycosides.
Brief description of the drawings
Fig. 1：Crocin I and crocin II UPLC are detected
Fig. 2：Candidate's crocin approach gene expression is composed
Fig. 3：Candidate CCDs, ALDHs and UGTs qRT-PCR analyses (actin is internal reference)
Fig. 4：Candidate CCDs, ALDHs and UGTs qRT-PCR analyses (GAPDH is internal reference)
Fig. 5：Candidate's CCDs, ALDHs and UGTs evolutionary relationship
Fig. 6：CCD4a external functional verification
Embodiment
Below in conjunction with the example in detail present invention.Implementation is for a better understanding of the present invention, but to be not limited to the present invention.
Experimental method in following implementation is conventional method, and involved experiment reagent is routine biochemistry reagent.
Crocin content detection in the green fruit of the cape jasmine of embodiment 1, haw and blade
1.1 experimental method
Blade, green fruit and haw sample vacuum freeze drying, mill are pulverized and sieved, every part of sample accurate weighing
0.25g adds 50% hplc grade methanol 25mL ultrasonic extractions 45 minutes in triangular flask.After extraction, a small amount of 50% is carefully added into
Methanol filling-in weight loss.0.22 μm of filter membrane, which is crossed, after extract solution filtering obtains test sample.Standard items are prepared：Accurate weighing standard
Product crocin I and crocin II make mixing mark product, respective concentration be respectively 0.035mg/ml crocins I and
0.015mg/ml crocins II.Chromatographic condition：Using Waters BEH-C18 chromatographic columns (100mm × 2.1mm, 1.7 μm), post
35 DEG C of temperature, it is that mobile phase carries out gradient elution with acetonitrile (A) and water (B), elution program is：0-6min, 5-12%A；6-
25min, 12-48%A.Flow velocity 0.3mL/min, sample size 2 μ L, Detection wavelength 440nm.
1.2 results and analysis
Crocin composition is not detected in blade, crocin I and crocin II are only accumulated in fruit, explanation
The accumulation of the two has tissue specificity.From content, with the maturation of fruit, crocin I and crocin II contents
Gradually increase, the two increase multiple are slightly different, and contents of the crocin I in haw is about three times of green fruit, and west safflower
Contents of the glycosides II in haw is about 7 times (Fig. 1) of green fruit.Difference distribution and key gene of the crocin in different parts
The uniformity of expression is that we screen the main screening principle of crocin route of synthesis gene.
Transcript profile sequencing, assembling and the annotation of the green fruit of the cape jasmine of embodiment 2, haw and blade
2.1 experimental method
2.1.1 cape jasmine RNA extractions, CDNA library constructions and upper machine
Cape jasmine plant is planted in the Fen Suo botanical gardens of China Medical Sciences Academy Medical Plants Institute Chongqing, is adopted October
Collect leaf and fruit, cleaned at once after collection and with being stored in after liquid nitrogen flash freezer in -80 DEG C of refrigerators.Using QIAGEN RNeasy
Plus Mini Kit (74134) are extracted, and concrete operations are with reference to its specification.The total serum IgE of extraction is through detected through gel electrophoresis
RNA integralities, RNA concentration is detected through NanoDrop particle fluorescences spectrophotometer, total amount should be greater than 3.5 micrograms.Pass through
Agilent2100Bioanalyzer is detected, and RIN (RNA integrity numbe) value answers ＞ 1.7.The intact RNA of 1 μ g mass
For library construction, specific steps with reference to specification (UltraTM RNA Library Prep Kit for)。
2.1.2 the filtering splicing of initial data and annotation
Raw reads sequences are filtered, obtain high quality clean reads.Carried out using Trinity composite softwares
The from the beginning assembling of sequence.Sequence after assembling obtains unigenes through cd-hit-est software de-redundancy.Pass through blastx programs
By Unigene sequence alignments to albumen database nr, KO (KEGG Ortholog database), GO (Gene Ontology)
With Pfam (Protein family), the albumen that there is highest serial similitude with target Unigene sequences is obtained, so as to obtain
The protein function annotation information of the Unigene.
2.2 results and analysis
2.2.1 the assembling of sequencing data and annotation
The transcript profile of cape jasmine blade, green fruit and haw is sequenced using the microarray datasets of Illumina HiSeq 2000,
Situation such as table 1 is sequenced in each sample.Sequence is spliced with TRINITY softwares, obtains 156,658contigs altogether, average length
For 906bp, N50 1,632bp.With the further de-redundancy of CD-HIT softwares obtain 141,665unigenes (total 122,469,
676bp), average length 864bp, N50 1,574bp (table 2).Protein sequence is compared to Uniprot, Pfam, KEGG and
GO databases, the gene dosage annotated and ratio such as table 3.
Situation brief summary is sequenced in 1 different samples of table
The sequencing data amount of table 2 and assembling situation
The disparate databases of table 3 annotate ratio
The identification of the crocin route of synthesis key gene of embodiment 3 and Differential expression analysis
3.1 experimental method
Other species crocin pathway key enzyme amino acid sequences downloaded using NCBI are as QUERY, in transcript profile number
According to BLASTP comparisons are carried out in storehouse, threshold value 1E-10, key enzyme albumen sequence is obtained, then each egg of results verification is annotated with Pfam
White domain.BLASTP comparisons are carried out in ncbi database again to obtained result, verify correct sequence.It is soft using RSEM
Part is estimated all gene expression doses of transcript profile, passes through DESeq Bioconductor package (1.14.0) software
The gene of differential expression, parameter meet log2fold changes ＞=1 (FDR value ＜ 0.001) between analysis Different Organs
Gene belong to difference expression gene.
3.2 results and analysis
Found according to gene expression trend, 5 MEP pathway genes (DXS1, DXR, IDI1, GGPPS2 and GGPPS4), 4
The CCD4a of carotenoid pathway gene (PSY1, PDS2, LCYB2 and CYP97A51) and downstream crocin approach, 3
ALDHs and 14 UGTs expression trend is consistent in the distribution of blade and fruit with crocin, predicts that it participates in crocin
Synthesis (Fig. 2).
The evolutionary relationship and expression analysis of the candidate key enzyme gene of embodiment 4
4.1 experimental method
Using QIAGEN RNeasy Plus Mini Kit (74134) kit extraction blade, green fruit and haw RNA.Profit
With Takara PrimeScriptTMII 1st Strand cDNA Synthesis Kit (6210A) kit reverse transcription obtains
One chain cDNA.Utilize SYBR premix Ex Taq kit (TaKaRa, China) kits and 7500 fluorescent quantitation systems
(Life Technologies, USA) carries out quantitative analysis to candidate gene, and each 3 technologies of reaction repeat, Actin and
GAPDH genes are as reference gene.Cape jasmine and other species CCD, ALDH and UGT sequence are compared using MEGA5.0, entered
Change tree structure and select NJ methods (neighbor-joining statistical method), Poisson model, number of repetition is
1000。
4.2 results and analysis
4.2.1 the expression of crocin pathway key enzyme gene
Because we more pay close attention to expression and the function of crocin pathway gene, so we are directed to CCD4a, 3 ALDHs
QRT-PCR experimental verifications are carried out with 14 UGTs encoding gene.From the results of view, compared with green fruit and blade, CCD4a, 2
ALDH genes and 4 UGT genes high expression (Fig. 3 and Fig. 4) in haw.Therefore, we predict that these genes may participate in
The synthesis of crocin.
4.2.2 candidate CCD4a, ALDHs and UGTs achievements result
Contribute with candidate albumen and other known functional protein and analyze CCD4a, ALDH and UGT evolutionary relationship (Fig. 5).Wait
Sortilin CCD4a gathers at one group with citrus CitCCD4 albumen, and this albumen is proved that the position of zeaxanthin 7,8 or 7 ', 8 ' can be cracked
Point.According to CCD4a gene expression and analysis of contributing, it is believed that cape jasmine CCD4a probably cracks the He of zeaxanthin 7,8
7 ', 8 ' sites generate crocetin dialdehyde.
ALDHs achievement results show that ALDH12 gathers with participating in the synthesis AaALDH1 and BoBADH of qinghaosu and bixin
Together.The BALDH of synthesis of the ALDH14 with participating in benzoic acid gathers at one group.ALDH12 and ALDH14 may participate in cape jasmine fruit
Crocin approach, catalysis crocetin dialdehyde generation crocetin.
UGTs albumen is divided into two big branch, includes 5 groups.4 UGTs gather for one with terpene synthesis associated protein
Group.So, it is believed that UGT60, UGT67, UGT86 and UGT89 may have the function of glycosylation crocetin.
The functional verification of the CCD4a genes of embodiment 5
5.1 experimental method
5.1.1CCD4a Escherichia coli and protein expression are converted
PET28a-CCD4a carriers and pACCAR25 Δ crtX carriers are transformed into competent cell BL21, gently rotated
To mix content, 30min is placed in ice, puts it to pre-heating heat shock 90s into 42 DEG C of thermostat water bath.Quickly will
It is transferred in ice bath, cell is cooled down 2min30s.Add 700 μ L LB nutrient solutions, then transfer on 37 DEG C of shaking tables, incubate 1h
Make bacteria resuscitation.The transformed bacteria solution of appropriate amount is taken to be coated on LB agar plates (34mg/mL chloromycetin+50mg/mL
Kanamycin).Plate is inverted, 37 DEG C of cultures, 12h in constant incubator.Picking individual colonies and the (34mg/ containing corresponding antibiotic
ML chloromycetin+50mg/mL Kanamycin) LB fluid nutrient mediums in, cultivate 4h.After expanding and cultivating, add
1.0mM IPTG, in 25 DEG C of shaking tables, lucifuge induction 24h, rotating speed is set to 180R.
5.1.2 chemical composition detects
After the completion of induction, by sample at 4 DEG C, 3000rpm, 15min is centrifuged, removes supernatant, retains precipitation.Use distilled water
Eluted, avoid impurity effect subsequent experimental in nutrient solution.Addition chloroform-methanol (2: 1), ultrasonic extraction 15min, at 4 DEG C,
3000rpm, 15min is centrifuged, collect supernatant.After supernatant volatilizes, dissolved with methanol, cross 0.22 μm of filter membrane, utilized
HPLC detects its chemical composition.INSTRUMENT MODEL, Agilent Technologies 1260Infinity.The μ L of sample size 10, temperature
Degree：25℃.Chromatographic condition：UV 450nm, (A)：Methanol/MTBE/ water (81/15/4), (B)：Methanol/MTBE/ water (6/90/4),
0-100%B (0-90min), flow velocity：1.0mL/min, YMC Carotenoid (5 μm) 250 × 4.6mm.
5.1.3 result and analysis
In crocin route of synthesis, precursor of the zeaxanthin as crocin, first add in carotenoid cracking pair
Double bond cracks to form crocetin dialdehyde in the presence of oxygenase CCD (carotenoid cleavage dioxygenase)
(crocetin dialdehyde), work of the crocetin dialdehyde in aldehyde dehydrogenase ALDH (aldehyde dehydrogenase)
Under, crocetin (crocetin) is generated, finally, crocetin generates crocin in the presence of glycosyl transferase.Knot
Fruit shows, after pET28a-CCD4a carriers and pACCAR25 Δ crtX carrier cotransformation Escherichia coli, relative to blank control
(the Escherichia coli body of pACCAR25 Δs crtX conversions) bacterium solution lighter, chemical detection are shown relative to blank control, corn
The content of yellow matter significantly reduces, it was demonstrated that zeaxanthin is cracked (Fig. 6) by CCD4a.
Described above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, without departing from the technical principles of the invention, some improvements and modifications can also be made, these improvements and modifications
Also it should be regarded as protection scope of the present invention.
Claims (6)
- A kind of 1. carotenoid cleavage dioxygenases CCD4a genes in cape jasmine, it is characterised in that its be following gene (a) or Gene (b)：Gene (a)：Gene with nucleotide sequence shown in SEQ ID No.1.Gene (b)：With nucleotide sequence shown in SEQ ID No.1 through replacement, lack or add one or more bases and The obtained gene with carotenoid cleavage dioxygenases function.
- 2. the protein of carotenoid cleavage dioxygenases CCD4a gene codes in a kind of cape jasmine, it is characterised in that it is as follows Protein (a) or gene (b)：Protein (a)：Protein with amino acid sequence shown in SEQ ID No.2.Protein (b)：With amino acid sequence shown in SEQ ID No.2 by replacing, lacking or add one or more amino There is the protein of carotenoid cleavage dioxygenases function obtained from acid.
- 3. one kind utilizes high throughput sequencing technologies, the method for screening crocin synthesis key gene in cape jasmine, specific steps It is as follows：1) UPLC detects the content of crocin in cape jasmine blade, green fruit and haw.2) Illumina microarray datasets are based on, and according to the Standard Operating Procedure of microarray dataset, structure cape jasmine blade, green fruit and red Fruit cDNA library and the sequencing of upper machine, are assembled and are annotated to sequencing result.Differential gene expression is analyzed, primary dcreening operation crocin closes Into pathway key zymoprotein sequence.3) using qRT-PCR detection candidate gene expressions, structure phylogenetic analysis cape jasmine and other species candidate albumens it Between evolutionary relationship, further screen crocin route of synthesis key gene.
- 4. cape jasmine CCD4a function verification method, is comprised the following steps that：By pET28a-CCD4a carriers and pACCAR25 Δs crtX carriers cotransformation into e. coli bl21 (DE3), it is mould to block that Element screens with chloramphenicol, after expanding and cultivating, adds 1.0mM IPTG, in 25 DEG C of shaking tables, lucifuge induction 24h.Induction is completed Afterwards, by sample at 4 DEG C, 3000rpm, 15min is centrifuged, removes supernatant, retains precipitation.Add chloroform-methanol (2: 1), ultrasound 15min is extracted, at 4 DEG C, 3000rpm, 15min is centrifuged, collects supernatant.After supernatant volatilizes, dissolved with methanol, mistake 0.22 μm of filter membrane, its chemical composition is detected using HPLC.INSTRUMENT MODEL, Agilent Technologies1260 Infinity. The μ L of sample size 10, temperature：25℃.Chromatographic condition：UV 450nm, (A)：Methanol/MTBE/ water (81/15/4), (B)：Methanol/ MTBE/ water (6/90/4), 0-100%B (0-90min), flow velocity：1.0mL/min, YMC Carotenoid (5 μm) 250 × 4.6mm。
- 5. the gene or its mutator of claim 1 encoding carotenoid cracking dioxygenase are in plant genetic engineering Using.It is characterized in that carotenoid cleavage dioxygenases CCD4a genes pass through gene in bacterium, fungi and higher plant Engineering means participate in the synthesis of terpenoid.
- 6. in the application described in claim 6, foreign gene imports host's needs one kind, and for carrying, carotenoid cracking is double to be added The plasmid of oxygenase CCD4a genes, it is characterised in that：Plasmid may be selected from the prokaryotic expression carriers such as pET series, pGEX series, The plant expression vector such as the Yeast expression carriers such as pPIC9, pHIL-D2, pPIC3.5 and PBI series, pCAMBIA series, and contain The nucleotide sequence or mutant nucleotide sequence of claim 1.
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