CN101921753B - Soybean blossoming regulator gene GmCIB5, encoding protein and application thereof - Google Patents
Soybean blossoming regulator gene GmCIB5, encoding protein and application thereof Download PDFInfo
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Abstract
The invention discloses a soybean blossoming regulator gene GmCIB5 and an encoding protein thereof. The soybean blossoming regulator gene has a nucleotide sequence represented by SEQ ID No.1 and an amino acid sequence represented by SEQ ID No.2. The overexpression soybean blossoming regulator gene GmCIB5 can obviously promote plants (Arabidopsis) to blossom and shorten the blossoming time and growth season. The soybean blossoming regulator gene GmCIB5 can be used for solving the blossoming of flowers at different times in cross breeding, the growth season control problem, the photoperiodic sensitivity problem and the species introduction problem of all crops, vegetables, fruits and flowers.
Description
Technical field
The present invention relates to the genetically engineered field, particularly relate to soybean blossoming regulatory gene GmCIB5, 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 a 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.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.Though change the habit of soybean to a certain extent by the method for traditional cultivation and genetic breeding, also obtain the wider kind of some photoperiod adaptability, but all fundamentally do not change soybean blossoming habit (Zhao Cun etc., soybean varieties (being) with photoperiod revulsion screening light insensitiveness. soybean science .1996,15 (1): 42-47; Chen Jiemin and Yang Fangren, date of seeding is to the influence of soybean blossoming and output, soybean science, 1998,17 (3): 225-230; Yang Zhi climbs and Zhou Xinan, soybean photoperiod genetic breeding progress, Chinese oil crops journal, 1999,21 (1): 61-73; Luan Xiaoyan, Man Weiqun, Du Weiguang, Chen Yi, Liu Xinlei, the research of soybean light insensitiveness germplasm innovation and photoperiod breeding approach, soybean science, 2004,23 (3): 196-199).Its major cause is at present very few to the understanding of soybean blossoming molecule mechanism, thereby causes being difficult to fundamentally solve the flowering habit problem of soybean.
To Arabidopis thaliana studies show that of molecule mechanism of blooming, blooming of plant is subjected to the control of four approach, it is the photoperiod approach, autonomous approach, vernalization approach and Plant hormones regulators,gibberellins approach (Mouradov etc., 2002), the signal of these approach all is aggregated into two main integron FT and SOC1 at last, thereby promote to bloom (Suarez-Lopez P, Wheatley K, Robson F, Onouchi H, Valverde F, Coupland G, CONSTANS mediates between the circadian clock and the control of fowering in Arabidopsis.Nature, 2001,410:1116-20.; Hepworth SR, Valverde F, Ravenscroft D, Mouradov A, Coupland G.Antagonistic regulation of flowering-time gene SOC1 by CONSTANS and FLC via separate promoter motifs.EMBO is (16) J.2002.21: 4327-37).Cryptochrome (cryptochrome) is the blue light receptor that all exists in three species from the bacterium to the plant and animal, regulation and control growth and development of plant and vegeto-animal physiological clock (Cashmore AR., Cell 114:537-543 (2003)..Lin?C.，Shalitin?D.，Annu?Rev?Plant?Biol?54：469-496(2003))。At least contain two kinds of cryptochrome: CRY1 and CRY2 (Guo H, Yang H, Mockler TC, Lin C., Science279:1360-1363 (1998)) in the plant.CRY1 mainly regulates and control the de-etiolation effect of blue light control in Arabidopis thaliana, and CRY2 mainly regulates the photoperiod activity (KoornneefM, Heynh., Z Pflanzenphysiol Bd 100:147-160 (1980)) of blooming.Except that Arabidopis thaliana, also the cryptochrome in algae, liver moss, pteridophyte, tomato, rape, pea and the paddy rice has been carried out correlative study.These studies show that, in angiosperm, and identical (ImmelnD, Schlesinger R, J Biol Chem 282:21720-21728. (2007) in the mode that the cryptochrome regulation and control are grown and the Arabidopis thaliana; Imaizumi T, Kanegae T, WadaM., Plant Cell 12:81-96. (2000).; Imaizumi T, Kadota A, Plant Cell 14:373-386. (2002); Ninu L, etc., Plant J18:551-556. (1999); Giliberto L, etc., Plant Physiol 137:199-208. (2005).; Chatterjee M, Sharma P, Plant Physiol 141:61-74. (2006).; Platten JD, etc., Plant Physiol 139:1472-1482. (2005); Matsumoto N, Hirano T, Iwasaki T, Plant Physiol 133:1494-1503. (2003).; Zhang YC, etc., Plant J 46:971-983. (2006)).Similar to other Photoreceptors, cryptochrome and its target protein interact the expression of regulatory gene and physiological response (H.-Q.Yang etc., Cell 103,815 (2000); X.Yu etc., Proc Natl Acad Sci US A 104,7289 (2007); M.F.Ceriani etc., Science 285,553 (1999); M.Ni, J.M.Tepperman, P.H.Quail, Cell 95,657 (1998)).In plant materials, the interaction that CRYs and CIB1 (cryptochrome-interacting basic-helix-loop-helix) blue light relies on is an early stage Photoreceptors signaling mechanism.CIB1 albumen interacts with blue light special mode and CRY2 in yeast and Arabidopis thaliana, and the albumen relevant with other CIB1 starts the flower formation that CRY2 relies on jointly.The FTmRNA that crosses the Arabidopis thaliana transfer-gen plant of expressing CIB1 significantly improves, and shows as early blossoming (H Liu etc., Science 269,968 (2008)).The expression of finding two the cryptochrome GmCRY1a of soybean and GmCRY2a is similar to the cryptochrome of Arabidopis thaliana with function, and the both influences the elongation that blue light suppresses cell, still has only the degraded of GmCRY1a to depend on blue light and 26S proteoplast.Opposite with the Arabidopis thaliana cryptochrome, GmCRY1a but not GmCRY2a shows the activity that very strong promotion is bloomed, and the protein expression level of GmCRY1a fluctuates with the rhythm and pace of moving things of physiological clock.GmCRY1a is the main regulon (Q.Z.Zhang., etc., Proc Natl Acad Sci 2046 (2008)) of flower cycle regulating in soybean.Therefore infer that CIBs gene in the soybean that belongs to dicotyledons together may interact with GmCRY1a and regulate blooming of soybean.
Summary of the invention
The purpose of this invention is to provide a kind of soybean blossoming regulatory gene GmCIB5, its proteins encoded and the application in plant photoperiod and flowering time adjusting thereof.
In order to realize the object of the invention, a kind of soybean blossoming regulatory gene GmCIB5 encoded protein of the present invention, its aminoacid sequence shown in SEQ ID No.2 or this sequence through replacing, lack or adding one or several amino acids formed aminoacid sequence with same function.
The present invention also provides coding above-mentioned proteic soybean blossoming regulatory gene GmCIB5.
Aforesaid soybean blossoming regulatory gene GmCIB5, its nucleotide sequence is shown in SEQ IDNo.1.
The present invention also provides the carrier that contains soybean blossoming regulatory gene GmCIB5.
The present invention also provides the host cell that contains above-mentioned carrier.
The present invention also provides the transformed plant cells that contains soybean blossoming regulatory gene GmCIB5.
The present invention further provides the described gene of soybean blossoming regulatory gene GmCIB5 in the application of regulating in plant, especially soybean and Arabidopis thaliana photoperiod and the flowering time.
In addition, the invention provides the primer of a kind of soybean blossoming regulatory gene GmCIB5 that is used to increase, it comprises forward primer F1:5 ' ATGGAAAACCAATTCTTTCTCAATG-3 ' and reverse primer R1:5 '-TCATAGGGGTTCTTGATTTGAGC-3 '.
GmCIB5 is the bHLH family gene that separates and obtain Function Identification from soybean, and Gm wherein is the initial of Glycine max (Linn) Merril, and CIB is the abbreviation of cryptochrome-interacting basic-helix-loop-helix.
CDS (code sequence) sequence according to Arabidopis thaliana CIB1 gene, in http://www.phytozome.net website, carry out the homology comparison, find a collection of soybean homologous sequence, wherein the position of CDS sequence on the soybean gene group of GmCIB5 is Glyma08g46040.1, design pcr amplification primer, forward primer F1:5 '-ATGGAAAACCAATTCTTTCTCAATG-3 ' and reverse primer R1:5 '-TCATAGGGGTTCTTGATTTGAGC-3 '.With the total cDNA of soybean is template, carries out PCR and obtains the GmCIB5 complete sequence, and its nucleotide sequence is shown in SEQ ID NO.1, and the aminoacid sequence of its proteins encoded is shown in SEQ ID NO.2.
GmCIB5 gene and Arabidopis thaliana CIB1 gene height homology, its encoded protein matter and the proteic consistence of Arabidopis thaliana CIB1 reach 36.4%.(as shown in Figure 1).
The GmCIB5 gene is crossed expression in the Arabidopis thaliana wild-type, transformed plant reduces photoperiodic sensitivity, all early blossoming under long day and short day.This shows that the GmCIB5 gene has the similar function with Arabidopis thaliana CIB1, splits the time spent to play important regulatory role.
The GmCIB5 gene has important use to be worth, and crosses expression GmCIB5 and can reduce flowering of plant to photoperiodic sensitivity, can obviously promote flowering of plant, makes flowering time shorten reduction in the life period.Can be used for solving control problem breeding time, the photoperiod sensitivity problem of the flowering asynchronism problem in the cross-breeding, various crop, vegetables, fruit, flowers and introduce a fine variety problem.
Description of drawings
Fig. 1 is soybean CIB5 albumen of the present invention and the proteic aminoacid sequence contrast of Arabidopis thaliana CIB1, and wherein GmCIB5 represents soybean CIB5 albumen, and AtCIB1 represents Arabidopis thaliana CIB1 albumen;
Fig. 2 is the contrast of blooming of Arabidopis thaliana Col-0 wild-type and its commentaries on classics GmCIB5 gene T1 plant, and wherein, Col-0 wild-type plant is represented in the left side, and transformed plant is represented on the right side;
Fig. 3 is the contrast of blooming of Arabidopis thaliana Col-0 wild-type and its commentaries on classics GmCIB5 gene T2 plant under long day (LD) condition, wherein, Col-0 wild-type plant is represented in the left side, and transformed plant is represented on the right side;
Fig. 4 is the contrast of blooming of Arabidopis thaliana Col-0 wild-type and its commentaries on classics GmCIB5 gene T2 plant under short day (SD) condition, wherein, Col-0 wild-type plant is represented on the right side, and transformed plant is represented in the left side.
Embodiment
Following examples are used to illustrate the present invention, but are not used for limiting the scope of the invention.
The separating clone of embodiment 1 GmCIB5 gene
CDS (code sequence) sequence according to Arabidopis thaliana CIB1 gene, in http://www.phytozome.net website, carry out the homology comparison, find a collection of soybean homologous sequence, wherein the position of CDS sequence on the soybean gene group of GmCIB5 is Glyma08g46040.1, design pcr amplification primer, forward primer F1:5 '-ATGGAAAACCAATTCTTTCTCAATG-3 ' and reverse primer R1:5 '-TCATAGGGGTTCTTGATTTGAGC-3 '.With the total cDNA of soybean is template, carries out PCR and obtains the GmCIB5 complete sequence, and its nucleotide sequence is shown in SEQ ID NO.1.
The PCR reaction totally is 25 μ L, comprises soybean cDNA (50ng) 1 μ L; DNTP (2.5mM) 2.5 μ L; Primers F 1 (10 μ M) 1 μ L; Primer R1 (10 μ M) 1 μ L; LATaq enzyme (5U/ μ L) 0.3 μ L; 10 * damping fluid, 2.5 μ L; DdH
2O 16.7 μ L, totally 25 μ L.The PCR response procedures is: 94 ℃ of pre-sex change 5min, 95 ℃ of 30s, 53.5 ℃ of 30s, 72 ℃ of 2min, 30 circulations; Last 72 ℃ of 10min.
The Analysis and Identification of embodiment 2 GmCIB5 genes
The CDS sequence total length 1761bp of GmCIB5, the coding 586AA albumen, with the proteic homology of Arabidopis thaliana CIB1 be 36.4%.The protein structure analysis revealed, its C end contains a basic helix-loop-helix structure, and this structural domain is the conserved domain of bHLH family transcription regulaton factor, by combining with E-box among the DNA, comes transcribing of regulatory gene; Albumen n end also contains a NLS, this structural domain with go into nuclear phase and close.
According to the sequence Query Result on NCBI (www.ncbi.nlm.nih.gov), up to the present, still do not have in the soybean and the similar sequence information of CIB1; And the paper of also not publishing so far that relates to its functional study.Therefore think that GmCIB5 is the new gene of soybean.
Embodiment 3 changes the acquisition of GmCIB5 gene Arabidopis thaliana
According to the sequence information of GmCIB5, at its CDS two ends design pcr amplification primer, forward primer F 1:5 '-ATGGAAAACCAATTCTTTCTCAATG-3 ' and reverse primer R1:5 '-TCATAGGGGTTCTTGATTTGAGC-3 '.With the total cDNA of soybean is template, carries out PCR and obtains the GmCIB5 complete sequence.
The PCR reaction totally is 25 μ L, comprises soybean cDNA (50ng) 1 μ L; DNTP (2.5mM) 2.5 μ L; Primers F 1 (10 μ M) 1 μ L; Primer R1 (10 μ M) 1 μ L; LATaq enzyme (5U/ μ L) 0.3 μ L; 10 * damping fluid, 2.5 μ L; DdH
2O 16.7 μ L, totally 25 μ L.The PCR response procedures is: 95 ℃ of pre-sex change 5min, 94 ℃ of 30s, 53.5 ℃ of 30s, 72 ℃ of 2min, 30 circulations; Last 72 ℃ of 10min.Repeat above-mentioned PCR program three times, three PCR products are merged laggard row agarose gel electrophoresis, cut glue then and reclaim the PCR product that obtains purifying.
With the PCR product cloning to the pGWC carrier (Chen Qijun, the Wang Xuechen etc. that contain the Gateway joint, use the ABC of clone of T carrier acquisition with the Gateway technical compatibility, 2004.31 (10), 951-954) on, identify through order-checking to obtain identical sequence with purpose GmCIB5.By the LR reaction GmCIB5 was built up on the expression vector pLeela (available from Invitrogen), obtained carrier 35S::GmCIB5.To cross expression vector 35S::GmCIB5 imports among the agrobacterium strains GV3101:90RK (available from Invitrogen), by agriculture bacillus mediated colored method (the Steven J.Clough and Andrew F.Bent.Floral dip:A Simpled Method for Agrobacterium-mediated Transformation of Arabidopsis thaliana.Plant Journal.1998.16 (6) that dips in, 735-743.) transform the Arabidopis thaliana of full-bloom stage, the seed of transformed plant results is sowed, treat behind the sowing 7-10d that the weedicide Basta (available from Chem Service Lot:395-85A) that sprays 1000 times of dilutions when cotyledon opens fully screens, the mistake that obtains anti-Basta is expressed Arabidopis thaliana transfer-gen plant 20 strains of GmCIB5.
Embodiment 4 GmCIB5 gene function analysis
According to the 20 strain GmCIB5 Arabidopis thaliana transfer-gen plants that the method for embodiment 3 obtains, its T1 plant shows as early blossoming (Fig. 2).Select 5 strain transfer-gen plants wherein to be planted in respectively under long day and the short day, the T2 plant was early blossoming (Fig. 3 and Fig. 4) under long day and short day.Show that transformed plant reduces photoperiodic sensitivity, illustrated that the GmCIB5 gene has the similar function with Arabidopis thaliana CIB1, split the time spent to play important regulatory role.
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.
Claims (7)
1. the primer of the soybean blossoming regulatory gene GmCIB5 that is used to increase, it comprises forward primer F1:5 '-ATGGAAAACCAATTCTTTCTCAATG-3 ' and reverse primer R1:5 '-TCATAGGGGTTCTTGATTTGAGC-3 '.
2. soybean blossoming regulatory gene GmCIB5 encoded protein is characterized in that, its aminoacid sequence is shown in SEQ ID No.2.
3. coding claim 2 described proteic gene.
4. gene as claimed in claim 3 is characterized in that its nucleotide sequence is shown in SEQID No.1.
5. the carrier that contains claim 3 or 4 described genes.
6. the host cell that contains the described carrier of claim 5.
7. claim 3 or the 4 described genes application in regulating Arabidopis thaliana photoperiod and flowering time.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6949694B2 (en) * | 2001-07-12 | 2005-09-27 | Pioneer Hi-Bred International, Inc. | Methods and means for modification of plant characteristics |
| CN101148672A (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 GAL2 and application thereof |
| US7396979B2 (en) * | 2004-06-30 | 2008-07-08 | Ceres, Inc. | Nucleotide sequences and polypeptides encoded thereby useful for modifying plant characteristics and phenotypes |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6949694B2 (en) * | 2001-07-12 | 2005-09-27 | Pioneer Hi-Bred International, Inc. | Methods and means for modification of plant characteristics |
| US7396979B2 (en) * | 2004-06-30 | 2008-07-08 | Ceres, Inc. | Nucleotide sequences and polypeptides encoded thereby useful for modifying plant characteristics and phenotypes |
| CN101148672A (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 GAL2 and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| Hongtao Liu等.Photoexcited CRY2 Interacts with CIB1 to Regulate Transcription and Floral Initiation in Arabidopsis.《SCIENCE》.2008,第322卷第1535-1539页. * |
| 朱春利等.植物隐花色素结构与功能研究进展.《基因组学与应用生物学》.2009,第28卷(第l期),第174-178页. * |
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