CN102994516B

CN102994516B - Soybean flowering gene ft2a-1 and protein coded by the gene

Info

Publication number: CN102994516B
Application number: CN201210534973.4A
Authority: CN
Inventors: 孔凡江; 刘宝辉
Original assignee: Northeast Institute of Geography and Agroecology of CAS
Current assignee: Northeast Institute of Geography and Agroecology of CAS
Priority date: 2012-12-12
Filing date: 2012-12-12
Publication date: 2014-04-02
Anticipated expiration: 2032-12-12
Also published as: CN102994516A

Abstract

A Soybean flowering gene ft2a-1 and a protein coded by the gene. The invention relates to the soybean flowering gene ft2a-1 and protein coded by the gene. The invention aims to provide the soybean flowering gene ft2a-1 and the protein coded by the gene. The soybean flowering gene ft2a-1 provided by the invention has a gene sequence shown as a Seq ID No:1 in the sequence table. An amino acid sequence of the protein coded by the soybean flowering gene ft2a-1 is shown as a Seq ID No:2 in the sequence table. According to Arabidopsis genetic transformation verification, it illustrates that the ft2a-1 gene has obviously decreased function of promoting flowering compared with an FT2a gene. The invention can be applied to soybean gene engineering.

Description

Soybean blossoming gene ft2a-1 and proteins encoded thereof

Technical field

The present invention relates to soybean blossoming gene ft2a-1 and proteins encoded thereof.

Background technology

Soybean provides important plant protein and oil content for the mankind.Worldwide, north is to Northern Europe Sweden and the North America Canada of high latitude, reach in the south in the extensive region such as Brazil and Argentina and all have soybean culture, but the latitude span of single kind or the general suitable planting of germ plasm resource is less, this Regional suitability and soybean photoperiod and breeding time gene or quantitative trait locus (Quantitative Trait Locus, QTL) closely related.The wide good soybean varieties of seed selection ecological suitability is the fundamental way that realizes high yield, high-quality, efficient Soybean Industry.But conventional breeding is made slow progress in Wide-adaptive breed breeding, utilize molecular breeding means to be expected to orientation adjustment soybean photoperiod and breeding time, accelerate the seed selection of Wide-adaptive kind.The result of study of model plant Arabidopis thaliana and paddy rice shows, the element of blooming (Florigen, FT albumen) is transported to formation (Corbesier etc., 2007 of stem apex induction flower from blade; Tamaki etc., 2007).We have cloned 10 FT genes from soybean, being replicated in soybean linkage group of this 10 pair of genes.The result of gene expression analysis shows, in induction of flowering condition, be under short day condition, there are two FT genes (GmFT2a and GmFT5a) to form the phase (about 25～30 days) at bud, great expression in ternately compound leaf, and other gene expression amounts are extremely low or do not express.And these two genes present the genetic expression type of physiological clock rule, in illumination, start 4 hours expression intensities and weaken gradually after the highest.Under the long day of non-induction condition, to only have a FT gene (GmFT5a) relative expression, thereby caused that soybean bloom is more late with this understanding, extend breeding time.Utilize double-mutant (Harosoy-e3e4) research of soybean phytochrome A gene to find, under long day condition, the flowering period of Harosoy-e3e4 is consistent the flowering period under short day condition with its wild-type plant.And, in double-mutant, at bud, forming the phase, the expression of GmFT2a and GmFT5a is induced in a large number and is presented the genetic expression with the identical physiological clock rule of short day.These results of study have tentatively disclosed the molecule mechanism of soybean bloom and breeding time from molecular level, (1) is under the short day condition of induction, having two FT genes to participate in expressing causes the florescence very short, yet under the long day of non-induction condition, thereby only have a FT gene to participate in expressing, cause flowering period elongated; (2) soybean adapts in the existence of high latitude region phytochrome A gene inactivation through its own evolution, and this evolutionary process is finally by regulating and controlling FT genetic expression main in two soybean, to realize; (3) under long day condition, the expression of the gene induced FT gene of phytochrome A of long day plant Arabidopis thaliana, and the phytochrome A gene of short day plant soybean suppresses the expression of FT gene, illustrate that short day plant contains the photoperiodic reaction regulatory mechanism different from long day plant (Kong etc. 2010).In addition,, by Genetic Transformation of Soybean technology, investigator has further verified that GmFT2a has soybean blossoming promotion functions (Sun etc. 2011).But how the sudden change of GmFT2a gene affects soybean growth period (flowering period and ripening stage) is a very important problem in science, also the correlative study on molecular level is not reported at present.

Summary of the invention

The object of this invention is to provide soybean blossoming gene ft2a-1 and proteins encoded thereof.

The gene order of soybean blossoming gene ft2a-1 of the present invention is as shown in sequence table Seq ID No:1.

The proteins encoded of soybean blossoming gene ft2a-1 of the present invention, is characterized in that this albumen encoded by soybean blossoming gene ft2a-1 as claimed in claim 1, and the aminoacid sequence of proteins encoded is as shown in sequence table Seq ID No:2.

Beneficial effect of the present invention:

The present invention is cloning soybean blossoming gene GmFT2a(Kong etc. 2010) basis on, further GmFT2a gene order is goed deep into systematic research.We have confirmed that GmFT2a gene has the function that promotes soybean blossoming.The present invention is studied GmFT2a gene order in different varieties, and has determined ft2a-1 gene, and it is in close relations to soybean growth period.

Ft2a-1 gene and soybean blossoming gene GmFT2a(Kong etc. 2010) compare, both sequences only have a Nucleotide (No. 506) difference DNA level, but cause the missense mutation from glycine to aspartic acid of 169 of aminoacid sequences, thereby changed the function of original GmFT2a gene.The genetic transformation of Arabidopis thaliana is tested and is shown, ft2a-1 gene of the present invention is compared with soybean blossoming gene GmFT2a, and the function of its Accelerate bloom obviously weakens.

Accompanying drawing explanation

Fig. 1 is pMDC100IG-ft2a-1 plasmid expression vector structural representation;

Fig. 2 is the flowering time figure that proceeds to ft2a-1 gene and proceed to GmFT2a gene Arabidopis thaliana and wild-type Arabidopis thaliana; Wherein, Col-0 is wild-type Arabidopis thaliana, and GmFT2a-OX is the Arabidopis thaliana proceeding to after GmFT2a gene, and ft2a-1-OX is the Arabidopis thaliana proceeding to after ft2a-1 gene.

Embodiment

Embodiment one: the gene order of the soybean blossoming gene ft2a-1 of present embodiment is as shown in sequence table Seq ID No:1.

Present embodiment is studied GmFT2a gene order in different varieties, and has determined ft2a-1 gene, and it is in close relations to soybean growth period.

Present embodiment by Arabidopis thaliana genetic transformation means, is expressed GmFT2a gene and ft2a-1 gene in Arabidopis thaliana, has verified that ft2a-1 gene of the present invention compares with soybean blossoming gene GmFT2a, and the function of its Accelerate bloom obviously weakens.

Embodiment two: the proteins encoded of the soybean blossoming gene ft2a-1 of present embodiment, it is characterized in that this albumen encoded by soybean blossoming gene ft2a-1 as claimed in claim 1, the aminoacid sequence of proteins encoded is as shown in sequence table Seq ID No:2.

By following verification experimental verification effect of the present invention:

(1) acquisition of gene ft2a-1 sequence

One, take the blade of soybean varieties K3 is material, with the operational manual of buying from the TRIzol of Invitrogen company test kit, extracts the total RNA of blade; Two, the total RNA that adopts DNase I treatment step one to extract; Three, get synthetic for cDNA of total RNA after 1 μ g step 2 is processed, the synthetic operation of cDNA is according to the BD SMART buying from BD Biosciences Clontech company ^tMthe service manual of RACE cDNA Amplification Kit test kit carries out, and obtains cDNA; Four, take the cDNA that obtains as template is by F1 and R1 primer amplification ft2a-1 gene, PCR reaction conditions is as follows: 94 ℃ of denaturation 5min, 94 ℃ of sex change 30s, 56 ℃ of annealing 30s, 72 ℃ are extended 1min, totally 35 circulations, then 72 ℃ of extension 10min are checked order PCR product on ABI3130 sequenator (ABI company);

Wherein, the sequence of primers F 1 is 5 ' ccatgcctagtggaagtagg; The sequence of primer R1 is gagtgtgggagattgccaat-3 '.

Sequencing result shows that soybean blossoming gene ft2a-1 has the nucleotide sequence of Seq ID No:1 in sequence table, and the Seq ID No:1 in sequence table is comprised of 622 Nucleotide, and coding has the protein of the aminoacid sequence of Seq ID No:2 in sequence table.

(2) functional verification of soybean blossoming gene ft2a-1

One, take the blade of soybean varieties K3 is material, with the operational manual of buying from the TRIzol of Invitrogen company test kit, extracts the total RNA of blade; Two, the total RNA that adopts DNase I treatment step one to extract; Three, get synthetic for cDNA of total RNA after 1 μ g step 2 is processed, the synthetic operation of cDNA is according to the BD SMART buying from BD Biosciences Clontech company ^tMthe service manual of RACE cDNA Amplification Kit test kit carries out, and obtains cDNA; Four, take respectively ft2a-1 gene F2 and R2 as primer, the cDNA that step 3 is obtained carries out 5 ' and 3 ' amplification by PCR, PCR reaction conditions: 94 ℃ of denaturation 10min, 94 ℃ of sex change 30s, 60 ℃ of annealing 30s, 72 ℃ are extended 5min, totally 35 circulations, 72 ℃ are extended 10min again, obtain the soybean blossoming gene ft2a-1 that contains the two enzyme restriction enzyme sites of XbaI-SacI; Five, the soybean blossoming gene ft2a-1 that contains restriction enzyme site obtaining is cloned in pMDC100IG plasmid, obtain pMDC100IG-ft2a-1 plasmid, the Arabidopis thaliana (Col-0) of take is material, utilize agrobacterium-mediated transformation to carry out genetic transformation, acquisition turns ft2a-1 gene Arabidopis thaliana, to turn ft2a-1 gene Arabidopis thaliana, and turn GmFT2a gene Arabidopis thaliana and wild-type Arabidopis thaliana (Col-0) illumination in 16 hours and under dark condition, plant for 8 hours, observe their bloom; Wherein, pMDC100IG plasmid and turn GmFT2a gene Arabidopis thaliana, we are called in the article of " Two Coordinately Regulated Homologs of FLOWERING LOCUS T Are Involved in the Control of Photoperiodic Flowering in Soybean " open in the name of publishing in < < Plant Physiology > > for 2010; A Cultivar of soybean varieties K3Shu Thailand, in 2006 < < Kasetsart J > > " Molecular marker analysis of days to flowering in vegetable soybean (Glycine max) ", publication, is so kind as to give by professor Abe of Hokkaido University.

Wherein, the sequence of primers F 2 is 5 ' gctctagaccatgcctagtggaagtagg; The sequence of primer R2 is gcgagctcgagtgtgggagattgccaat-3 '.

The pMDC100IG-ft2a-1 plasmid over-express vector structural representation that step 5 obtains as shown in Figure 1;

The flowering time figure that proceeds to ft2a-1 gene and proceed to GmFT2a gene and wild-type Arabidopis thaliana as shown in Figure 2, wherein, Col-0 is wild-type Arabidopis thaliana, and GmFT2a-OX is the Arabidopis thaliana proceeding to after GmFT2a gene, and ft2a-1-OX is the Arabidopis thaliana proceeding to after ft2a-1 gene.As can be seen from Figure 2, the flowering time of Col-0 is 31 days, the flowering time of GmFT2a-OX is 24.8 days, show to proceed to Arabidopis thaliana flowering time after GmFT2a gene than the flowering time of wild-type Arabidopis thaliana early than 6 days, thereby explanation GmFT2a gene has the function of Accelerate bloom; And the flowering time of the flowering time of ft2a-1-OX and wild-type Arabidopis thaliana is basic identical, thereby illustrate that ft2a-1 gene of the present invention compares with soybean blossoming gene GmFT2a, the function of its Accelerate bloom obviously weakens or afunction.

Claims

1. soybean blossoming gene ft2a-1, is characterized in that the gene order of soybean blossoming gene ft2a-1 is as shown in sequence table Seq ID No:1.

2. the proteins encoded of soybean blossoming gene ft2a-1, is characterized in that this albumen encoded by soybean blossoming gene ft2a-1 as claimed in claim 1, and the aminoacid sequence of proteins encoded is as shown in sequence table Seq ID No:2.