CN102399273B - Soybean GmCOL5 gene and its coded protein and use - Google Patents
Soybean GmCOL5 gene and its coded protein and use Download PDFInfo
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- CN102399273B CN102399273B CN 201110033085 CN201110033085A CN102399273B CN 102399273 B CN102399273 B CN 102399273B CN 201110033085 CN201110033085 CN 201110033085 CN 201110033085 A CN201110033085 A CN 201110033085A CN 102399273 B CN102399273 B CN 102399273B
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Abstract
The invention discloses a soybean GmCOL5 gene coded protein. The soybean GmCOL5 gene coded protein is a protein 1 composed of an amino acid sequence shown by the formula of SEQ ID NO.2, or a protein 2 which is derived from the protein 1 through replacement, deletion or addition of one or more amino acids of the amino acid sequence shown by the formula of SEQ ID NO.2 and has activity same as the protein 1. The invention also discloses a soybean GmCOL5 gene for coding the soybean GmCOL5 gene coded protein. Through overexpression of the soybean GmCOL5 gene, plant anthesis is obviously promoted; anthesis is brought forward; and a breeding period is shortened. In crossbreeding, the soybean GmCOL5 gene coded protein and the soybean GmCOL5 gene can solve flowering asynchronism problems, breeding period control problems of various crops, vegetables, fruits and flowers, photoperiodic sensitivity problems and introduction problems.
Description
Technical field
The present invention relates to the genetically engineered field, particularly relate to a soybean blossoming gene, its proteins encoded and the application in plant photoperiod and flowering time adjusting thereof.
Background technology
Soybean is one of important farm crop, is the important source of secondary meta-bolitess such as plant protein, edible oil, biofuel and isoflavones and Yelkin TTS.Because soybean is short day plant, blooming is subjected to photoperiodic strict control, thereby the excellent kind between different zones can not be introduced a fine variety mutually, also be subjected to the photoperiodic restriction of environment breeding time (Zhang et al., 2008).If can reduce soybean to photoperiodic sensitivity, break through soybean blossoming to photoperiodic restriction, just can solve the problem of introducing a fine variety of soybean, thereby realize the mutual exchange of each interregional fine quality, enrich various places excellent germplasm resource, regulate soybean growth period, improve soybean yields and quality.The method that solved the soybean photoperiod sensitivity in the past and mainly be by hybridization obtains new variety, and still, this breeding method has strong and cycle of dependency to the parent and shortcoming such as grows, and does not up to the present obtain soybean photoperiod wide adaptability kind as yet.
Study on plants such as Arabidopis thaliana are shown the photoperiod of plant is (Mouradov et al., 2002 that are subjected to extremely complicated adjusting network control; Turck et al., 2008).But one of them key protein plays crucial effects to the adjusting of flowering time, and it is exactly CONSTANS (CO) gene.Putterill etc. (1995) have reported the CO gene in the Arabidopis thaliana the earliest.Discover CO between biological clock and downstream floral genes by a large amount of, change optical signal into the signal of blooming (Suarez et al., 2001).Rise and development along with genomics and information biology, CO in the different plant species is constantly by cognition, as paddy rice (Oryza sativa) (Yano et al., 2000), wheat (Tritivum aestivum) (Nemoto et al., 2003) and the CO gene of chlamydomonas (Chlamydomonas reinhardtii) (Serrano et al., 2009).In the plant of having studied, CO often is present in the organism with the form of a plurality of copies, and the function of each CO copy has notable difference.There is 17 members (Robson et al. in the CO family of Arabidopis thaliana, 2001), 16 members (Griffiths et al. is arranged in the paddy rice, 2003), also clone in the swede type rape (Brassica napus) and obtain 4 CO homologous genes (Robert et al., 1998), in temperate zone gymnosperm European spruce (Picea abies), identify 2 CO homologous genes (Holefors et al., 2009).But function and the application facet thereof of soybean CO gene are not appeared in the newspapers.Changing plant by the genetic expression of adjusting soybean blossoming does not report photoperiodic sensitivity and flowering time yet.
Summary of the invention
The purpose of this invention is to provide a kind of soybean GmCOL5 albumen, 1), the protein be made of the aminoacid sequence shown in the SEQID NO.2, or 2 it is :), in the aminoacid sequence shown in the SEQ ID NO.2, be substituted, lack or add one or several amino acid and have with isoreactivity by 1) protein of deriving.
Another object of the present invention provides the gene of the above-mentioned soybean GmCOL5 albumen of coding, and its nucleotide sequence is shown in SEQ ID NO.2.
Should be appreciated that those skilled in the art can not influence under its active prerequisite according to aminoacid sequence disclosed by the invention, replace, lack and/or increase one or several amino acid, obtain the mutant nucleotide sequence of described albumen.For example at nonactive section, (205) (N) replaced with (V), or (159) (K) lacked, or increase (S) in (114 back).
Therefore, soybean GmCOL5 albumen of the present invention comprises that also aminoacid sequence shown in the SEQ ID No.2 is substituted, replaces and/or increases one or several amino acid, have soybean GmCOL5 albumen with isoreactivity by the protein derived protein that obtains of soybean GmCOL5.Gene of the present invention comprises the nucleic acids encoding said proteins sequence.In addition, should be understood that the degeneracy of considering codon and the preferences of different plant species codon, those skilled in the art can use as required and be fit to the codon that specific species are expressed.
Another object of the present invention provides the plant expression vector that carries the GmCOL5 gene, and described plant expression vector is p35S-GW.
GmCOL5(full name of the present invention is Glycine max CONSTANS Like5) be ' to cultivate a gene of being cloned into farming 18 ' (the Glycine max L. ' Kennong18 ') from soybean, homology between the aminoacid sequence of it and Arabidopis thaliana CO albumen is 53.7%, in conservative functional domain (two B-box and a CCT structural domain) sequence height homology.So the function class of supposition and Arabidopis thaliana CO gene seemingly, and GmCOL5 also has the function that promotion is bloomed.Soybean GmCOL5 gene mainly all has expression in soybean cotyledon, stem, leaf, flower, pod, wherein expression amount is the highest in the leaf in flowering period.
The present invention is gene constructed to expression vector p35S-GW with GmCOL5, and expands numerous in bacillus coli DH 5 alpha.By the agrobacterium mediation converted method, the GmCOL5 gene that p35S-GW is carried changes Arabidopis thaliana over to, obtains the Arabidopis thaliana transformed plant.The result shows that GmCOL5 has the plant of reduction to photoperiodic sensitivity and promotes the effect that Arabidopis thaliana is bloomed.
The present invention also provides cloning vector or all kinds of expression vector that contains GmCOL5 nucleotide sequence or its fragment, the host cell that contains described carrier, the transformed plant cells that contains described nucleotide sequence and transgenic plant.
The present invention also provides soybean blossoming gene and the application of proteins encoded in plant photoperiod and flowering time adjusting thereof.
The invention provides soybean GmCOL5 gene and encoded protein thereof, and shorten plant breeding time by in plant, crossing expression GmCOL5 gene, promote flowering of plant.Therefore, GmCOL5 gene and encoded protein thereof can be regulated the florescence, can be used for solving the problem of the flowering asynchronism in the cross-breeding, problem and the photoperiod sensitivity problem of control breeding times of various crops, vegetables, fruit, flowers and introduce a fine variety problem.
Description of drawings
Fig. 1 is the comparison of the aminoacid sequence of the aminoacid sequence of soybean blossoming gene GmCOL5 proteins encoded of the present invention and CO gene coded protein.
Fig. 2 is the structural representation of clone's intermediate carrier pGWCm.
Fig. 3 A is the expression level of soybean blossoming gene GmCOL5 different tissues organ in soybean.
Fig. 3 B is the expression level of soybean blossoming gene GmCOL5 different development stage in soybean.
Fig. 4 is the location of soybean blossoming gene GmCOL5 in nucleus.
Fig. 5 is the structural representation of plant expression vector p35S-GW.
Fig. 6 is soybean blossoming gene GmCOL5 arabidopsis thaliana transformation, causes the Arabidopis thaliana prematurity.
Wherein, in Fig. 3, the developmental stage of the letter representation plant of short-term front, the letter representation organ of short-term back, U represents Dan Ye; T represents compound leaf (numeral of T back is the order of compound leaf); F represents flower; Pd represents pod; Pt represents petiole (pod of numeral different times thereafter); R represents root; HH represents epicotyl; EH represents hypocotyl; SAM represents vegetative point; St represents stem; L represents the adnation leaf; C represents cotyledon, and the Sd representative is the seedling in 1 week after planting.
Embodiment
Following examples are used for explanation the present invention, but are not used for limiting the scope of the invention.
The clone of embodiment 1 soybean blossoming gene GmCOL5
Utilize forward primer 5'-TAAACAGAAAGGCAACC-3' and reverse primer 5'-CTAGAAAGTGGGAAAAATGCTAC-3' ' to cultivate the blade amplification CO homologous gene of farming 18 ' (Glycine max L. ' Kennong18 ') by the method for RT-PCR from soybean respectively.
The PCR response procedures is: 95 ℃ of pre-sex change of 5min, and 95 ℃ of 30S, 50 ℃ of 35S, 72 ℃ of 2min, 25 circulations, 72 ℃ of 10min extend.
The PCR product that amplification is obtained directly is cloned on as shown in Figure 2 the pGWCm carrier according to the TA cloning process.Earlier the pGWCm carrier being reclaimed test kit with gel after with the hydrolysis of Ahd I restriction endonuclease reclaims enzyme and cuts product with acquisition T carrier.The PCR product spends the night in 16 ℃ with the T carrier and is connected then, will connect product transformed into escherichia coli DH5 α, and amplification therein, screening positive clone and order-checking.
The GmCOL5 gene that obtains, its gene order is shown in SEQ ID NO1; By the aminoacid sequence of its encoded protein matter shown in SEQ ID NO2.
The amino acid sequence analysis of embodiment 2 soybean blossoming gene GmCOL5 proteins encoded
GmCOL5 protein sequence and the homology between the Arabidopis thaliana of soybean blossoming gene are 53.7%, and in conservative functional domain (two B-box and a CCT structural domain) sequence height homology, as shown in Figure 1.Therefore infer the function class of GmCOL5 and Arabidopis thaliana CO seemingly, have the activity that promotes flowering of plant.
The expression level of embodiment 3 soybean blossoming gene GmCOL5 different tissues organ and different development stage in soybean
Utilize real-time quantitative fluorescence PCR (quantitative real time RT-PCR) to measure the expression of soybean blossoming gene GmCOL5 in soybean.Real-time fluorescence quantitative PCR adopts ABI StepOne to carry out, and detects fluorescent signal with SYBR Green I.Upstream primer:
5 '-GTGGTGATAAGGGTTTTTTGTTTG-3 '; Downstream primer: 5 ' AGTGTTGCTGTAATTCTGCTGGT 3 '.
Reaction system is:
SYBR Primix Ex Taq(2×)(TaKaRa) 7.5μl
Upstream primer (10 μ M) 0.3 μ l
Downstream primer (10 μ M) 0.3 μ l
ROX Reference Dye(50x) 0.3μl
cDNA 1.0μl
Sterilization distilled water 5.6 μ l
Reaction parameter is two-step approach: 95 ℃ of 10S, warm start; 95 ℃ of 5S, 60 ℃ of 1min, 40 circulations.Carry out stdn and mapping with the expression of gene of gene chip data analysis software Genesis.
Soybean blossoming gene GmCOL5 mainly all has expression in soybean cotyledon, stem, leaf, flower, pod, wherein expression amount is the highest in the leaf in flowering period, as shown in Figure 3.
Embodiment 4 location of soybean blossoming gene GmCOL5 proteins encoded in transgenic plant
Adopt the method for particle gun bombardment, the fusion gene with soybean blossoming gene GmCOL5 and YFP is converted in the soybean leaves.Concrete grammar is as follows:
1) preparation of particulate bullet: with bronze suspension (diameter is 1.0 μ m), 4 μ l 0.1mol/L spermidine (suction filtration sterilization) and the 6 μ l 2.5mol/LCaCl of 10 μ g recombinant plasmid dnas and 6 μ l 50mg/ml
2, vortex vibration mixing leaves standstill 15min on ice, and the centrifugal 10s of 12,000rpm collects the bronze precipitation, and is resuspended with 20 μ l dehydrated alcohols.
2) bombardment receptor material: the soybean young leaflet tablet is placed on the MS substratum 22 ℃ of pre-4h that cultivate.Select the pressure membrane of 1100psi for use, in bombardment film central authorities, adopt PDS 1000/He type particle gun (Bio-Rad) to bombard 20 μ l bronzes-DNA mixing object point, target distance 6cm, vacuum tightness is 25In.Hg.Behind 22 ℃ of dark cultivation 24h of epidermic cell after the bombardment, observe down at laser confocal microscope (Leica TCS SP2).
YFP fluorescent signal in the transformant is represented the location of soybean blossoming gene GmCOL5 proteins encoded, as shown in Figure 4.Fig. 4 shows that soybean blossoming gene GmCOL5 encoded protein mainly is positioned nucleus in soybean leaves.
The plant expression vector of embodiment 5 soybean blossoming gene GmCOL5
The cloning vector of the soybean blossoming gene GmCOL5 that will obtain from embodiment 1 mixes the back, and (two kinds of each 50ng of plasmid, LR enzyme 1 μ l mend ddH by the LR reaction with plant expression vector p35S-GW (available from Invitrogen) equal proportion as shown in Figure 5
2O is to final volume 5 μ l, and 25 ℃ of reactions are more than 6 hours behind the mixing), GmCOL5 is structured on the p35S-GW, called after p35S-GW-COL5 is used for crossing expression soybean blossoming gene GmCOL5 plant, studies its function.
The acquisition of the transgenic arabidopsis of embodiment 6GmCOL5
The preparation of 1 agrobacterium liquid
The agrobacterium tumefaciens GV3101::p35S-GW-COL5 that will contain the goal gene binary expression vector is inoculated in 5ml and contains in the liquid LB substratum of corresponding resistant, 28 ℃ of 200rmp cultivate, before transforming 1d be inoculated in 200ml contain corresponding antibiotic liquid LB substratum enlarged culturing to OD600 be 0.8～1.2,5, the centrifugal 15min of 000rmp collects thalline, thalline is resuspended in and infects damping fluid (it is a large amount of to contain 1/2MS, the 1/2MS trace, 5c/0 sucrose and 0.02c/0 SilweetL-77, pH 5.7), making OD600 is about 0.8.
2, Arabidopis thaliana transforms
Arabidopis thaliana is cultivated under the long day condition, treats to cut behind the stem bolting, treats can transform when most side shoot is buddingged.Inflorescence put into fill in the container that infects damping fluid, soak 10～30s, transform finish after, cover lucifuge, preserve moisture with the black plastic bag, throw off plastics bag behind 16～24h.Routine Management is to seed maturity.
3, Basta resistance screening transfer-gen plant
Seed results back is evenly broadcast in soil then in 37 ℃ of oven dry (about 1 week), and preservative film prevents from polluting and preserving moisture on the cover, treats to spray when cotyledon opens fully the Basta of dilution in 1: 1000 behind sowing 7～10d, the screening positive plant.Utilize respectively forward primer 5 '-GTTATGGGTCAACGGTTTC-3 ' and reverse primer 5 '-resistant plant of CTAGAAAGTGGGAAAAATGCTAC-3 ' evaluations acquisition, guarantee the transfer-gen plant for GmCOL5.Identify that by PCR obtaining GmCOL5 crosses express transgenic T1 for plant totally 3 strains.
Embodiment 7 soybean blossoming gene GmCOL5 promote Arabidopis thaliana to bloom
Arabidopis thaliana transformed plant and condition (photoperiod of 16 hour illumination/8 hour dark, the light intensity 80 μ molms of its mutant co-2 length day plant strain growth chamber from embodiment 6 acquisitions
-2S
-1) under cultivate simultaneously.To the T of 30 strain GmCOL5 wherein
1In generation, crosses the express transgenic plant and carries out florescence observation.The result shows, soybean blossoming gene GmCOL5 arabidopsis thaliana transformation promotes Arabidopis thaliana to bloom, and is insensitive to the photoperiod, and as shown in Figure 6, wherein the right side representative contrasts the co-2 mutant, and the left side represents transfer-gen plant.To bloom be 20～35 days to the transformation plant of GmCOL5 from being seeded into, average out to 30.5 days, and flowering time of its contrast co-2 mutant is about 55～68 days, average out to 60.8 days.The result shows that soybean GmCOL5 albumen has the activity that obvious promotion is bloomed.
Though above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements all belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
The sequence explanation:
SEQ ID NO1 is depicted as the nucleotide sequence of soybean GmCOL5; SEQ ID NO2 is depicted as the aminoacid sequence of soybean GmCOL5; It is right that SEQ IDNO3 and SEQNO4 are depicted as the primer of clone soybean GmCOL5.It is right that SEQ IDNo 5 and SEQ ID No 6 are depicted as soybean GmCOL5Real-time PCR primer; SEQ ID No 7 and SEQ ID No 8 are depicted as PCR and identify the transformant the primer.
Claims (7)
1. soybean GmCOL5 albumen is characterized in that, its aminoacid sequence is shown in SEQ ID NO.2.
2. the soybean GmCOL5 gene of coding claim 1 described albumen.
3. gene according to claim 2 is characterized in that, nucleotide sequence is shown in SEQ ID NO.1.
4. the carrier that contains claim 2 or 3 described genes.
5. carrier according to claim 4 is characterized in that, described carrier is p35S-GW-COL5.
6. the agrobatcerium cell that contains the described carrier of claim 5.
7. claim 2 or the 3 described genes application in regulating Arabidopis thaliana photoperiod and flowering time.
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| CN101148673A (en) * | 2006-09-19 | 2008-03-26 | 中国农业科学院作物科学研究所 | Soybean abloom time adjusting gene GAL1 |
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| CN101148673A (en) * | 2006-09-19 | 2008-03-26 | 中国农业科学院作物科学研究所 | Soybean abloom time adjusting gene GAL1 |
Non-Patent Citations (4)
| Title |
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| 大豆GmCOL4基因的克隆与分析;张清哲等;《作物学报》;20100412;第36卷(第04期);第539-548页 * |
| 大豆GmCOL8基因的克隆及表达分析;马锦花等;《植物生理学通讯》;20100131;第46卷(第01期);第17-23页 * |
| 张清哲等.大豆GmCOL4基因的克隆与分析.《作物学报》.2010,第36卷(第04期),第539-548页. |
| 马锦花等.大豆GmCOL8基因的克隆及表达分析.《植物生理学通讯》.2010,第46卷(第01期),第17-23页. |
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