CN105420221A

CN105420221A - Chimonanthus praecox de-adenylated enzyme gene CpCAF1 and coded protein and application thereof

Info

Publication number: CN105420221A
Application number: CN201610019112.0A
Authority: CN
Inventors: 眭顺照; 周仕清; 李志能; 马婧; 刘道凤; 李名扬
Original assignee: Southwest University
Current assignee: Southwest University
Priority date: 2016-01-13
Filing date: 2016-01-13
Publication date: 2016-03-23
Anticipated expiration: 2036-01-13
Also published as: CN105420221B

Abstract

The invention discloses a chimonanthus praecox de-adenylated enzyme gene CpCAF1 and a coded protein and application thereof. The chimonanthus praecox de-adenylated enzyme gene is (1) the protein consisting of the amino acid shown by SEQ ID No.2 or (2) the protein derived by the (1), having the same activity and formed through substitution, deletion or one or more amino acid addition conducted on an amino acid sequence shown by SEQ ID No.2. The chimonanthus praecox de-adenylated enzyme gene CpCAF1 has the preferential expression features and a floral development delay function of a floral organ, performs overexpression in arabidopsis thaliana, can delay arabidopsis thaliana flowering, increase the leaf area, silique number and branch number and delay ageing and shows that the chimonanthus praecox CpCAF1 gene can promote plant growth and development.

Description

The albumen of wax met AMP ase gene C pCAF1 and coding thereof and application

Technical field

The present invention relates to genetically engineered field, specifically, relate to clone and the application thereof of wax met AMP ase gene C pCAF1.

Background technology

Wax plum (Chimonanthuspraecox), Calycanthaceae (Calycanthaceae), wax-cakes bait (Chimonanthus), calls golden plum, snuff, wax wood, chimonanthi,flos.Machaka, often grows thickly.March in November at florescence to next year.The famous flower and tree that La Meishi China is traditional.Wax plum originates in China, with west place in Hubei, Chuan Dong, Shan Nan for distribution center.On the south Beijing, to the north of Hengyang, to the west of Shanghai, to the east of Chengdu, various places are generally cultivated, and cultivate more with Nanjing, hubei baokang, Yanling, Hebei, Chongqing.Wintersweet immature flower Winter-Spring emits cold in full bloom, graceful bearing is unique, and full of fragrance, charm is unusual, and gardening moulding is a lot of, signifies that happiness is lucky in addition, is not only successive dynasties imperial palace influential officials and likes, be also poet, the thing be deeply in love of artist.Wax plum horticultural use and economic use are very extensive, and be not only desirable flowering shrub, also can do cut-flower, miniature gardening, its flower can make tea, makes wine, is used as medicine, extracts essence, and rhizome can do cough-relieving, blood circulation promoting medicine.Especially in germ plasm resource, typoiogical classification, Lycopersicum esculentum, Landscape Application, gene library etc., achieve a large amount of achievement over past 20 years, people are also further extensive to the application of wax plum.

CCR4/POP2, has another name called CCR4/CAF1 (Tuckeretal., 2001), and POP2 system is the relevant title of closing research and using in early days, existing multiplex CAF1 (CCR4-associatedfactor1).CCR4 and CAF1 monomer is also de-AMP ase, possesses corresponding avtive spot and digestion activity (MartineAetal., 2012).CAF1 is one of main de-AMP ase participating in mRNA degraded, at regulate gene expression with affect biological character important role (CuiYetal, 2008; LiangWetal, 2009; MartineAetal., 2012).

Summary of the invention

In order to solve the problem, the invention provides a kind of wax met AMP ase and application thereof.

Wax met AMP ase is, 1) protein be made up of amino acid shown in SEQIDNo.2; Or 2) aminoacid sequence shown in SEQIDNo.2 be substituted, lack or add one or several amino acid and have same isoreactivity by 1) derivative protein.

The present invention also provides the gene of above-mentioned albumen of encoding, and nucleotide sequence is as shown in SEQIDNo.1.Wax met AMP ase gene provided by the invention is that the method checked order by Random clones is obtained from wintersweet immature flower cDNA library, its open reading frame sequence 807bp.The similarity of the de-AMP ase gene coded protein of wax met AMP ase gene coded protein and Arabidopis thaliana (Arabidopsisthaliana), plum (Prunusmume), grape (Vitisviifera), cocoa (Theobromacacao), oil palm (Elaeisguineensis), lotus (Nelumbonucifera), castor-oil plant (Ricinuscommunis) is up to 98%, and the homology of other species is also between 95% ~ 97%, structure comparison is guarded.

Should be appreciated that those skilled in the art according to aminoacid sequence disclosed by the invention, under the prerequisite not affecting its activity, can replace, lack and/or increase one or several amino acid, obtain the mutant nucleotide sequence of described albumen.

Therefore, wax met AMP ase of the present invention also comprises aminoacid sequence shown in SEQIDNo.2 and is substituted, lacks or adds one or several amino acid, has wax met AMP ase and derives by wax met AMP ase the protein obtained with isoreactivity.Wax met AMP ase gene of the present invention comprises the nucleotide sequence of encoding said proteins.In addition, should be understood that the preferences of degeneracy and the different plant species codon considering codon, the codon that those skilled in the art can use applicable specific species to express as required.

The present invention also provides the carrier containing above-mentioned wax met AMP ase gene or its fragment, and the host cell containing this carrier; Described carrier is the cloning vector of described wax met AMP ase gene or all kinds of expression vector.

Wax plum CpCAF1 gene of the present invention has floral organ predominant expression characteristic sum flower development delay feature, overexpression in Arabidopis thaliana, Arabidopis thaliana can be postponed bloom, increase leaf area, angle fruit number and branch amount, delay senility, shows that wax plum CpCAF1 gene of the present invention can be grown by Promoting plant growth.

Accompanying drawing explanation

Figure 1 shows that the GUS dyeing of transgenic arabidopsis.A: wildtype Arabidopsis thaliana; B: transgenic arabidopsis.

Figure 2 shows that the PCR of CpCAF1 gene in transgenic arabidopsis detects.M:DNA molecular weight standard, DL2000; 1: wildtype Arabidopsis thaliana; 2-9: in transgenic arabidopsis, the PCR of CpCAF1 gene detects; 10: negative control (ddH ₂o)

Fig. 3 turns the relative expression analysis of CpCAF1 gene in CpCAF1 gene Arabidopsis leaf.1: wildtype Arabidopsis thaliana; 2,4,6,7,10: turn the different individual plant system of CpCAF1 gene Arabidopis thaliana.

Embodiment

Following examples for illustration of the present invention, but are not used for limiting the scope of the invention.

The separation of embodiment 1 wax plum CpCAF1 gene

Random choose wintersweet immature flower cDNA library is cloned, extract plasmid DNA, with 5 ' pTriplEx-Sequenceprimer for sequencing primer, carry out forward sequencing and obtain est sequence, then the SeqMan of DNAStar software package is used to carry out EST cluster, the est sequence obtained is compared by the BlastN (Nucleotide-nucleotideBLAST) of NCBI (http://www.ncbi.nlm.nih.gov/) blast program and BlastX (Translatedqueryvs.proteindatabase), obtain target clone, and preliminary its functional attributes of confirmation.

Carry out twice forward and reverse sequencing with SP5 and T7 for sequencing primer, obtain the cDNA sequence of wax plum gtp binding protein gene C AF1.According to the wax plum CAF1 gene cDNA sequence obtained, design 1 with software Primerprimer5.0 and carry out pcr amplification to the special primer across maximum ORF frame, primer sequence is as follows:

CpCAF1-F：5‘-GAATTCATGGGGAGTTTTCCCAAAGGTG-3’SEQIDNo.3

CpCAF1-R：5‘-GGATCCTTACCACCGAGACCATTCCAAGACAC-3’SEQIDNo.4

Respectively with wax plum Library plasmid DNA and wax plum cDNA for template, amplification wax plum CpCAF1 gene, PCR reaction system and reaction conditions as follows:

Be connected to carrier T, transformation of E. coli competent cell, or recon after PCR primer being reclaimed, picking recon checks order, and wax plum CAF1 gene cDNA sequence is as shown in SEQIDNo.1.

Blastp contrast is carried out at ncbi database, result display wax plum CpCAF1 gene coded protein (SEQIDNo.2) is higher with the similarity of other species CAF1 gene coded protein, with Arabidopis thaliana (Arabidopsisthaliana), plum (Prunusmume), grape (Vitisviifera), cocoa (Theobromacacao), oil palm (Elaeisguineensis), lotus (Nelumbonucifera), castor-oil plant (Ricinuscommunis), similarity is up to 98%, and the homology of other species is also between 95% ~ 97%, structure comparison is guarded.CpCAF1 albumen belongs to the DEDDh subtribe of DEDD nuclease family, and its active zone is made up of 3 sweet acid of Radix Asparagi (D), 1 L-glutamic acid (E) and contiguous Histidine (h).Compare with Arabidopis thaliana, there are about 50 amino acid whose Mutations.

The structure of embodiment 2 wax plum CpCAF1 gene plant overexpression vector

In conjunction with the characteristic distributions of the self limited restriction enzyme site of CpCAF1 gene ORF frame sequence and the multiple clone site feature of plant overexpression vector pCAMBIA2301G used; design one pair of genes special primer; and add restriction enzyme site BamHI and EcoRI in primer upstream and downstream respectively and protect base accordingly, carry the CpCAF1 gene coding region of suitable restriction enzyme site for increasing and be cloned into the multiple clone site of plant expression vector.Primer and sequence as follows:

CAF1-FGAATTCATGGGGAGTTTTCCCAAAGGTGSEQIDNo.5

CAF1-RGGATCCTTACCACCGAGACCATTCCAAGACACSEQIDNo.6

PCR amplification system and response procedures as follows:

Get 5 μ LPCR products after 1% agarose gel electrophoresis, reclaim object fragment, be transformed in competent escherichia coli cell TOP10 after being connected to cloning vector pMD19-T, dull and stereotyped upper 37 DEG C of the LB being coated on Amp is inverted incubated overnight, picking mono-clonal carries out PCR qualification, is accredited as positive plasmid and delivers to Hua Da gene sequencing.By positive plasmid called after pT-CpCAF1 correct for order-checking.With BamHI and EcoRI respectively enzyme cut pT-CpCAF1 plasmid and plant expression vector pCAMBIA2301G plasmid, reaction system is as follows:

37 DEG C of thermostat container enzymes cut 2-3h, 80 DEG C of deactivation 5min.Agarose gel electrophoresis reclaims pCAMBIA2301G carrier large fragment and CpCAF1 small segment in digestion products.The CpCAF1 gene fragment of recovery is connected according to Fermentas company's T 4 ligase enzyme specification sheets with expression vector pCAMBIA2301G fragment.22 DEG C of water-baths are spent the night and are connected more than 6h.Linked system is as follows:

To connect product conversion intestinal bacteria TOP10, dull and stereotyped upper 37 DEG C of the LB be coated on containing 50mg/LKan is inverted incubated overnight, and picking mono-clonal carries out PCR detection, is that the positive rear plasmid that extracts of bacterium liquid activation carries out double digestion checking by electrophoresis detection.PCR qualification and all correct positive plasmid of double digestion checking are checked order, determines without after base mutation, called after pCAMBIA2301G-CpCAF1, be CpCAF1 gene plant overexpression recombinant vectors.

By recombinant plasmid pCAMBIA2301G-CpCAF1 freeze-thaw method transformation Agrobacterium EHA105 competent cell, picking list bacterium colony, be placed in 650ul and contain the aseptic 1.5mL centrifuge tube (50mg/LKan and 34mg/LChl) of YEB+Kan+Chl, 28 DEG C of shaking culture 36-48h, with bacterium liquid for template carries out PCR detection.The sub-plasmid of onestep extraction Agrobacterium-mediated Transformation of going forward side by side, reverse in competent escherichia coli cell TOP10, dull and stereotyped upper 37 DEG C of the LB be coated on containing Kan is inverted incubated overnight, and picking mono-clonal carries out PCR detection, the bacterium liquid of test positive is extracted plasmid again and carries out double digestion qualification.Verify that correct Agrobacterium bacterium liquid is the engineering bacteria containing plant expression vector pCAMBIA2301G-CpCAF1 recombinant plasmid.

Embodiment 3 wax plum CpCAF1 arabidopsis thaliana transformation

Inflorescence infestation method arabidopsis thaliana transformation

(1) cultivation of Arabidopis thaliana

By appropriate Arabidopis thaliana seed collection in 1.5mL centrifuge tube, add clorox (NaClO) the fully vibration sterilization 10min that concentration is 17%, then broadcast or be spread out on MS culture medium flat plate with liquid-transfering gun uniform column afterwards 5-6 time with aseptic water washing, cultivate under culture plate being placed in the environment of the photoperiod of the dark 8h of illumination 16h/, 2000Lux lighting condition and 22 DEG C, 70% humidity after 4 DEG C of refrigerator vernalization 2-3d, after about 2 weeks, transplant in culture medium that (earthworm is native: vermiculite=1:1).Initial terminal inflorescence is cut to promote the growth of side shoot in culturing process.Cultivate about 3 weeks in matrix, during to scape about length 5cm, Agrobacterium is carried out to Arabidopis thaliana inflorescence and infects.

(2) inflorescence infestation method arabidopsis thaliana transformation

YEB solid medium containing microbiotic (Chl:34mg/L, Kan:50mg/L) is drawn the Agrobacterium EHA105 of single line activation containing plant expression vector pCAMBIA2301G-CpCAF1 recombinant plasmid, 28 DEG C of light culture 36-48h;

With sterile toothpick picking list bacterium colony, be seeded to 650ul and contain in the YEB liquid nutrient medium of microbiotic (Chl:34mg/L, Kan:50mg/L), 28 DEG C, 200rpm shaking culture 36-48h;

Carry out PCR qualification to the Agrobacterium bacterium liquid special primer cultivated, get the above-mentioned bacterium liquid of 500ul after checking is correct and be inoculated in the YEB liquid nutrient medium of 25mL nonreactive, 28 DEG C, 200rpm shakes bacterium to OD ₆₀₀for subsequent use between 1-1.2;

Collect above-mentioned bacterium liquid with the sterile centrifugation tube of 10mL, 28 DEG C, the centrifugal 15min of 5000rpm, abandons supernatant liquor, collecting precipitation, precipitates with now joining that to infect liquid (sterilized water+5% sucrose+0.5 ‰ L-77) resuspended, is diluted to OD ₆₀₀≈ 0.8

Primary infection cuts off flower open on Arabidopis thaliana scape, only retains the bud just showed money or valuables one carries unintentionally, and it is moistening to put forward maintenance matrix of watering the day before yesterday.Infecting front watering can, that carton inwall is squirted to make light culture is for subsequent use, scape head is immersed and infects liquid (0.5cm), 3-5 takes out after second and puts into carton, black cloth (shading) is coverd with outside outside carton, take out after cultivating 24h under being placed in 22 DEG C of temperature condition, remain in the environment of dark and spend the night, then forward in normal growing environment.

Can again infect to improve transformation efficiency according to the growth conditions of Arabidopis thaliana after Arabidopis thaliana infects 1 week.

The Screening and Identification of transgenic arabidopsis

(1) the Kan resistance screening of transgenic arabidopsis

Transform about after 1 month, collect fruit pod that is ripe, cracking, be T ₀for seed.Be placed on environment more than the 1 week sowing of cryodrying after being dried in 37 DEG C of constant temperature ovens by seed again, carry out antibiotic-screening.

T ₀be seeded in the enterprising row filter of MS culture medium flat plate containing 50mg/LKan for after the sterilization of transgenic seed preceding method, transforming successful Arabidopis thaliana can containing normal growth on the antibiotic substratum of Kan.

Obtain some different strains, to the T of each strain ₁, T ₂kan resistance screening is proceeded for seed, until be green resistance seedling entirely after the planting seed of all strains results, the T generally obtained ₃transgenic homozygous strain is for seed.

(2) transgenic arabidopsis GUS histochemical stain detects

By the T4 of resistance screening gained for the sowing of transgenic arabidopsis strain, the plant of getting about 2 weeks sizes carries out GUS dyeing qualification, with wildtype Arabidopsis thaliana in contrast.Result shows, 8 transgenic arabidopsis strains that screening obtains all can be dyed to blueness, and wildtype Arabidopsis thaliana then can not be dyed to blueness (Fig. 1).

(3) PCR of transgenic arabidopsis detects

Extract the leaves genomic DNA of 8 transgenic arabidopsis strains and wild-type Arabidopsis plants, carry out PCR qualification with the special primer of CpCAF1 gene, the primer is to being CpCAF1-F and CpCAF1-R.Result shows, the transgenic arabidopsis strain of gained all can amplify the object band of the same size with positive control, and wildtype Arabidopsis thaliana does not amplify object band, show that goal gene CpCAF1 is successfully inserted into (Fig. 2) in arabidopsis gene group.

The real-time fluorescence quantitative PCR of embodiment 4 transgenic arabidopsis detects

For verifying the expression of CpCAF1 gene in transgenic arabidopsis further, extract the total serum IgE of transgenic arabidopsis and wildtype Arabidopsis thaliana young leaflet tablet, with cDNA first chain after reverse transcription for template, Arabidopis thaliana Actin gene is as reference gene, quantitative fluorescent PCR is carried out to CpCAF1 gene in transgenic arabidopsis and analyzes expression, the primer is as shown in table 1 below, and result as shown in Figure 3.

Table 1 transfer-gen plant RT-PCR the primer

In 6 the transgenic arabidopsis strains detected, the differential expression comparatively large (Fig. 3) of CpCAF1 gene.When the later stage is analyzed transgenic arabidopsis, according to real time fluorescent quantitative result, choose the strain that the highest and slightly low strain of expression amount of expression amount represents transgenosis variable expression and carry out Phenotypic Observation and relevant treatment.

Embodiment 5 transgenic arabidopsis Phenotypic Observation

Expression analysis is carried out for transgenic arabidopsis strain to being accredited as positive T4, choose 2 transgenic lines (1 strongly expressed+1 slightly weak expression), and with wildtype Arabidopsis thaliana (WT) for contrast, each transgenic line and WT respectively select 5-10 strain to be Phenotypic Observation record plant, every day timing observation and comparison from seedling stage to taking out Roripa productive phase again to the phenotype in period of decaying, line correlation data statistics of going forward side by side.

For transgenic arabidopsis strain and wildtype Arabidopsis thaliana, Phenotypic Observation and index of correlation mensuration are carried out to T4, found that, the bolting time of transgenic arabidopsis strain, lotus throne leaf side shoot, with the stem leaf Lateral shoot formation time, first inflorescence, first flower and first fruit pod time of occurrence are all slightly later to WT lines (table 2), when growth 30 days, transgenic arabidopsis strain CpCAF1-3, the height of CpCAF1-5 is respectively 1.08 times of WT lines, 1.19 doubly, transgenic arabidopsis strain CpCAF1-3, the lotus throne leaf leaf area of CpCAF1-5 is respectively 1.02 times of WT lines, 1.46 doubly.60 days time, the lotus throne leaf number of branch of transgenic arabidopsis strain CpCAF1-3, CpCAF1-5 is respectively 1.33 times, 1.75 times of WT lines; Stem leaf number is respectively 1.32 times, 1.79 times of WT lines; Pod number is respectively 1.28 times, 1.83 times of WT lines.The yellow leaf time of occurrence of the first of transgenic arabidopsis strain CpCAF1-3, CpCAF1-5 is obviously later than WT lines in addition, 55 days time, yellowing leaf degree does not have wild-type serious, 65 days time, transgenic line still major part can keep green, and WT lines occurs that large-area yellow is dried up.Illustrate, the process LAN of CpCAF1 gene can regulate and control growing of transgenic arabidopsis, makes postponement of blooming, enhancing development, delay senility.

In table 2T4 generation, turns CpCAF1 gene Arabidopis thaliana strain index of correlation and observes

Index/the strain of observing	WT	CpCAF1-3	CpCAF1-5
				There is the first lotus throne leaf side shoot (d)	38.00±1.58 ^a	39.00±1.61 ^a	34.33±1.15 ^a
There is the first stem leaf side shoot (d)	32.67±0.58 ^b	35.67±0.68 ^bc	33.00±1.00 ^b
				There is (d) in first inflorescence	28.00±0.50 ^c	29.00±0.10 ^c	29.00±0.30 ^c
First the flowers are in blossom puts (d)	33.33±2.31 ^bc	36.00±1.00 ^c	33.00±1.73 ^bc
				There is (d) in first pod	36.00±2.00 ^b	37.00±1.20 ^c	35.67±1.15 ^b
The yellow leaf (d) of first	36.67±2.08 ^ab	40.00±1.00 ^c	38.67±1.53 ^bc

Often organizing number is mean+SD; A, b, c, d represent significant difference in P<0.05 level

This patent of invention is subject to state natural sciences fund (regulation and control that de-AMP ase gene C pCAF1 responds wintersweet immature flower growth and abiotic stress, item number: 31370698) subsidize.

The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the prerequisite not departing from the technology of the present invention principle; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.

Claims

1. a wax met AMP ase, it is:

1) protein be made up of the amino acid shown in SEQIDNo.2; Or

2) in the aminoacid sequence shown in SEQIDNo.2, be substituted, lack or add one or several amino acid and have same isoreactivity by 1) derivative protein.

2. the wax met AMP ase gene of albumen described in coding claim 1.

3. gene according to claim 2, is characterized in that, nucleotide sequence is as shown in SEQIDNo.1.

4. the carrier containing gene described in Claims 2 or 3.

5. the host cell containing carrier described in claim 4.

6. the transformed plant cells containing gene described in Claims 2 or 3.

7. prepare the method for wax met AMP ase described in claim 1, it is characterized in that, by gene constructed for wax met AMP ase in prokaryotic expression carrier, standby by fermentation in intestinal bacteria.

8. the gene described in Claims 2 or 3 is in the developmental application of regulation and control growth of transgenic plants.

9. application according to claim 8, is characterized in that, described gene overexpression in transgenic plant, and render transgenic plant is postponed and blooming, and Promoting plant growth is grown.