CN101148673B - Soybean abloom time adjusting gene GAL1 - Google Patents

Abstract

The present invention provides one kind of soybean flowering time regulating gene GAL1 and its separating and cloning process and application. The soybean flowering time regulating gene GAL1 codes protein with the amino acid sequence as shown in SEQ ID No. 2 or the amino acid sequence of SEQ ID No. 2 through the substitution, deletion or addition of one or several amino acids and with the same function. Over expressing the gene can reduce the sensitivity of flowering on light period and expand plant cultivating range.

Description

Soybean abloom time adjusting gene GAL 1

Technical field

The invention belongs to gene engineering technology field, be specifically related to the gene GAL1 of soybean blossoming time adjusting, and the application in plant breeding.

Background technology

Soybean is one of main farm crop of China, has an important effect in national economy, is mainly reflected in following several aspect: the important source of (1) plant protein; (2) edible oil; (3) main production raw material of biofuel is compared with ordinary diesel oil, and biofuel all is better than ordinary diesel oil at aspects such as cold filter clogging temperature, flash-point, combustion efficiency, sulphur content, burning oxygen-consumption, environmental protection, the more important thing is that biofuel has regenerability; (4) have good market outlook with the byproduct glycerine in soybean and the rape production biofuel process, oleic acid, Yelkin TTS etc.; (5) in addition, soybean still is typical short day plant, be to be used to one of vegetable material of studying photoperiodic reaction the earliest, thereby it is also significant in theory research.

According to Ministry of Agriculture investigation, because production-demand gap strengthens, in the first five months, 2005 oil plant and edible oil import are apparently higher than same period last year, and the imported petroleum product of estimating 2005 also will account for 50%.According to the WTO rule, soybean does not belong to the product of carrying out the Tariff Quota management, can not limit the soybean import volume with Tariff Quota access amount means, thereby import and homemade soybean contradiction will be more outstanding.Working hard on the international competitiveness of raising soybean, improve the output of soybean, is the key that solves China's soybean imbalance between supply and demand.

The soybean resource of China is very abundant, and fine quality such as many high yields, high oil, high protein and strong stress resistance are arranged.But because soybean is strict short day plant, blooming of plant can only just can be finished under specific photoperiod condition; Will (Yang Zhi climbs and Zhou Xinan, soybean photoperiod genetic breeding progress because of the output of interregional photoperiodic difference remarkably influenced soybean in the area that differs 1.5 latitudes.China's oil crops journal.1999，21(1)：67-73。The soybean of China can be divided into 3 districts according to its ecological distribution, contains 10 each subprovince (Gai Junyi, Wang Yuesheng, the research that Chinese soybean kind ecological zone is divided.Scientia Agricultura Sinica, 2001,34 (2): 139-145), the kind between different zones is difficult to mutual introducing culture.Therefore, this characteristic of soybean just limits the big area popularization of fine quality.That is to say that lacking eurytopic kind of photoperiod is the key that soybean produces, solving this, to introduce a fine variety problem imperative.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 (Zhao Cun etc. are with the soybean varieties (being) of photoperiod revulsion screening light insensitiveness all fundamentally not change the soybean blossoming habit.The soybean science.1996，15(1)：42-47。Chen Jiemin and Yang Fangren.Date of seeding, is to the influence of soybean blossoming and output.The soybean science, 1998,17 (3): 225-230.Yang Zhi climbs and Zhou Xinan, soybean photoperiod genetic breeding progress.China's 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.The 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 from solving the flowering habit problem of soybean in essence.

At present, the Arabidopis thaliana molecule mechanism of blooming there is more detailed understanding.The result of study of aspect such as molecular biology and genetics shows, blooming of Arabidopis thaliana is subjected to the control of four approach, it is the photoperiod approach, autonomous approach, vernalization approach and Plant hormones regulators,gibberellins approach (Mouradov et al, 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 thecontrol of flowering in Arabidopsis.Nature, 2001,410:1116-1120.Hepworth SR, Valverde F, Ravenscroft D, Mouradov A, Coupland G.Antagonistic regulation of flowering-time gene SOC1by CONSTANS andFLC via separate promoter motifs.EMBO is (16) J.2002.21: 4327-37).Express SOC1 and can make the phenotype of mutant such as mutant such as mutant such as photoperiod key gene cry1, cry2, co and ft, vernalization pathway key gene flc and fri and Plant hormones regulators,gibberellins pathway key gene gal be recovered (Hepworth SR as crossing, Valverde F, Ravenscroft D, Mouradov A, Coupland G.Antagonistic regulation of flowering-time geneSOC1 by CONSTANS and FLC via separate promoter motifs.EMBO is (16): 4327-37 J.2002.21.Moon?J，Suh?SS，Lee?H，Choi?KR，Hong?CB，Paek?NC，Kim?SG，Lee?I.The?SOC1MADS-box?gene?integratesvernalization?and?gibberellin?signals?for?flowering?in?Arabidopsis.Plant?J.2003.35(5)：613-23。Searle?I，He?Y，Turck?F，Vincent?C，Fornara?F，Krober?S，Amasino?RA，Coupland?G. The?transcription?factor?FLC?confersa?flowering?response?to?vernalization?by?repressing?meristem?co

Summary of the invention

The objective of the invention is to separating clone soybean GAL1 gene;

Another object of the present invention is to provide the coded protein of GAL1 gene;

A further object of the present invention is to provide the GAL1 gene regulating the especially application of soybean blossoming in the time of plant.

GAL1 is first AGL family gene that separates and obtain Function Identification from soybean, and G wherein is the initial of Glycine max (Linn) Merril, and AL is the abbreviation of AGL.

SOC1 (AGL20) gene according to Arabidopis thaliana, paddy rice etc. finds a collection of soybean est sequence by the homology comparison, wherein several EST are positioned at 5 ' end of candidate gene, therefrom design the PCR forward primer, with the total mRNA of soybean is that template is carried out reverse transcription and got cDNA,, carry out PCR and obtain the GAL1 complete sequence as the PCR reverse primer with the joint sequence on the oligo (dT).Its nucleotide sequence is shown in sequence table SEQ ID NO.1, and the aminoacid sequence of its proteins encoded is shown in sequence table SEQ ID NO.2.

The present invention also comprises the aminoacid sequence shown in the sequence table SEQ ID NO:2 through replacing, lack or add one or several amino acids formed derived protein with same function, and these proteinic genes of encoding.

GAL1 gene and Arabidopis thaliana SOC1 gene height homology, its encoded protein matter and the proteic consistence of Arabidopis thaliana SOC1 reached for 69% (as shown in Figure 1).

The GAL1 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 GAL1 gene has the similar function with Arabidopis thaliana SOC1, splits the time spent to play important regulatory role.

The present invention also comprises and contains the host cell that has above-mentioned nucleotide sequence or its segmental cloning vector or expression vector, contain described carrier, contains described nucleotide sequence or its segmental plant transformed cell and transgenic plant.

The GAL1 gene has important use to be worth, and crosses expression GAL1 and can reduce flowering of plant to photoperiodic sensitivity.A lot of good plant varieties are arranged at present, but it blooms and have the specific photoperiod, this makes improved seeds extensively to plant, and has limited its popularization.Cross expression GAL1, then can make these plants adapt to the growth of different areas.Especially be applied on the soybean.

Description of drawings

Fig. 1 is the proteic aminoacid sequence contrast of soybean GAL1 albumen and Arabidopis thaliana SOC1, and wherein GmSOC1 represents the albumen of soybean GAL1 genes encoding, and AtSOC1 represents Arabidopis thaliana SOC1 albumen;

Fig. 2 is the contrast of blooming of soc1 mutant and its commentaries on classics GAL1 gene plant, and wherein, left side plant is the soc1 mutant, and the right is a transformed plant.

Fig. 3 is the contrast of blooming of co mutant and its commentaries on classics GAL1 gene plant, and wherein, left side plant is the co mutant, and the right is a transformed plant.

Fig. 4 is the contrast of blooming of cry1cry2 double-mutant and its commentaries on classics GAL1 gene plant, and wherein, left side plant is the soc1 mutant, and the right is a transformed plant.

Embodiment

Following embodiment is used for further specifying of the present invention, but is not used for limiting the scope of the invention.

The separating clone of embodiment 1 GAL1 gene

According to Arabidopis thaliana, the mRNA sequence of SOC1 genes (AGL20) such as paddy rice, in the Genebank database, carry out the homology comparison, find a collection of soybean est sequence, CD413368 wherein, BQ273618 and BQ273771 are positioned at 5 ' end of candidate gene, therefrom design PCR forward primer F1:TTT AAA TTT CAT TCT CCA AAA CTC, with the total mRNA of soybean is template, for carrying out reverse transcription, primer gets cDNA with the oligo that adds joint (dT) (GAC TCT GAT GCT GACAAT GAC TTT TTT TTT TTT TTT TT), with joint sequence as PCR reverse primer R1:GAC TCT GAT GCT GAC AATGAC TTT, with F1, R1 is that primer carries out PCR acquisition GAL1 complete sequence.

The PCR reaction totally is 25 μ L, comprises 50ngcDNA, 50 μ mol/L dNTP, 1 μ mol/L primer, 1U rTaq enzyme and 1 * damping fluid.The PCR response procedures is: 94 ℃ of pre-sex change 5min; 94 ℃ of 30s then, 63 ℃ of 30s, 72 ℃ of 1min carry out 35 circulations; Last 72 ℃ of 10min mend flat terminal.

The Analysis and Identification of embodiment 2 GAL1 genes

The cDNA sequence total length 1065bp of GAL1, wherein CDS is 293-922, the coding 209AA albumen, with the proteic homology of Arabidopis thaliana SOC1 be 69%.The protein structure analysis revealed, its N end contains a MADS-box, and this territory is the conserved domain of AGL family transcription regulaton factor, combines with DNA by forming homology or heterodimer; A K-box is also contained at the albumen middle part, and this territory may be a spiral ring spirane structure, may form relevant with polymer on the function.

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 GAL1; And the paper of also not publishing so far that relates to its functional study.Therefore think that GAL1 is the new gene of soybean.Embodiment 3 changes the acquisition of GAL1 gene Arabidopis thaliana

According to the sequence information of GAL1, design a pair of primer GW-F:ATG ACG GCG TAC CAATCG GAG C and GW-R:TTA CAC TAA CTGGAG AGC GGT TTG GT in the ORF end positions.With the total mRNA of soybean is template, with this primer is carried out the cDNA sequence that RT-PCR obtains the GAL1 complete coding region, and the PCR reaction totally is 25 μ L, comprises 50ng cDNA, 50 μ mol/L dNTP, 1 μ mol/L primer, 1U rTaq enzyme and 1 * damping fluid.The PCR response procedures is: 94 ℃ of pre-sex change 5min; 94 ℃ of 30s then, 58 ℃ of 30s, 72 ℃ of 1min carry out 32 circulations; Last 72 ℃ of 10min mend flat terminal.The PCR product cloning to pGEM-T carrier (Promega company), after identifying correctly, order-checking was made up expression vector 35S::GAL1 and 35S::GFP:GAL1.To cross expression vector and import agrobacterium strains GV3101 (Koncz C., Schell J.The promoter of TL-DNA gene5controlsthe tissue-specific expression of chimaeric genes carried by a novel typeof Agrobacterium binary vector.Mol.Gen.Genet.1986,204:383-396.) in, with dipping in colored method (Steven J.Clough and Andrew F.Bent.Floral dip:ASimpledMethodforAgrobacterium-mediated TransformationofArabidopsis thaliana.Plant Journal.1998.16 (6), 735-743.) arabidopsis thaliana transformation, obtained to express the Arabidopis thaliana plant of GAL1.

Embodiment 4 GAL1 gene function analysis

Utilize the method for embodiment 3, the GAL1 gene is spent the type mutant respectively evenings such as arabidopsis thaliana transformation soc1, co single mutant and cry1cry2 double-mutant, found that transformed plant (Fig. 2,3,4, the right plant) all than mutant (Fig. 2,3,4, left side plant) prematurity, its florescence is approached wild-type.The GAL1 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 GAL1 gene has the similar function with Arabidopis thaliana SOC1, splits the time spent to play important regulatory role.

Sequence table

＜110〉Institute of Crop Science, Chinese Academy of Agricultural Science

＜120〉soybean abloom time adjusting gene GAL 1

<130>

<160>2

<170>PatentIn?version3.3

<210>1

<211>1065

<212>DNA

＜213〉soybean

<220>

<221>CDS

<222>(293)..(922)

<400>1

<210>2

<211>209

<212>PRT

＜213〉soybean

<400>2

Claims (8)

1. soybean blossoming time regulatory gene, its coded proteic aminoacid sequence is shown in SEQ ID NO.2.

2. gene as claimed in claim 1, its nucleotide sequence is shown in SEQ ID NO.1.

3. contain claim 1 or 2 described expression carrier.

4. the host of containing the described expression vector of claim 3.

5. host as claimed in claim 4, it is a vegetable cell.

6. claim 1 or 2 application of described gene in plant breeding.

7. application as claimed in claim 6, wherein said plant are soybean.

8. application as claimed in claim 6, wherein said plant are Arabidopis thaliana.

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