CN103014019A - Flowering gene GmCOL1b of soybean and coding protein thereof - Google Patents
Flowering gene GmCOL1b of soybean and coding protein thereof Download PDFInfo
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- CN103014019A CN103014019A CN2012105123691A CN201210512369A CN103014019A CN 103014019 A CN103014019 A CN 103014019A CN 2012105123691 A CN2012105123691 A CN 2012105123691A CN 201210512369 A CN201210512369 A CN 201210512369A CN 103014019 A CN103014019 A CN 103014019A
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Abstract
The invention relates to a flowering gene GmCOL1b of soybean and a coding protein of the gene, solving the technical problem that the flowering time of soybean is not controlled. The generic sequence of the flowering gene GmCOL1b of soybean is shown as a sequence table Seq ID No:1. An amino acid sequence of the coding protein of the flowering gene GmCOL1b of soybean is shown as a sequence table Seq ID No:2. Expression of the flowering gene GmCOL1b of soybean in the soybean can remarkably inhibit flowering of soybean, so that the flowering time is delayed, and the growth period is prolonged. The invention creates a condition for culturing crop varieties in wide application by molecular means, and has an important effect in genetic improvement of plants. The flowering gene GmCOL1b of soybean and the coding protein thereof provided by the invention are applied to the field of generic engineering of soybean.
Description
Technical field
The present invention relates to soybean blossoming gene GmCOL1b 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 soybean culture is all arranged, 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 the Wide-adaptive breed breeding, utilizes the molecular breeding means to be expected to orientation adjustment soybean photoperiod and breeding time, accelerates the seed selection of Wide-adaptive kind.The result of study of model plant Arabidopis thaliana and paddy rice shows that CONSTANS (CO) gene plays the role of a nucleus in Photoperiod.In recent years, the investigator is cloned into similar gene such as GmCOL4, GmCOL9, GmCOL10 and the GmCOL11 etc. of CO from soybean, but the regulating effect that its expression pattern is subjected to light a little less than, be not soybean homologous gene (Zhang Qingzhe etc., 2010 of CO; Huang etc., 2011; Jiang etc., 2011; Liu etc., 2011).In soybean, verify the function of soybean CO gene by the genetic transformation means, change also report not of soybean photoperiod and soybean blossoming time.
Summary of the invention
The present invention will solve uncontrolled technical problem of soybean blossoming time, thereby soybean blossoming gene GmCO1b and proteins encoded thereof are provided.
The gene order of soybean blossoming gene GmCOL1b of the present invention is shown in sequence table Seq ID No:1.
The aminoacid sequence of the proteins encoded of soybean blossoming gene GmCOL1b of the present invention is shown in sequence table Seq ID No:2.
Beneficial effect of the present invention:
The present invention successfully clones soybean blossoming gene GmCOL1b first on molecular level.
The present invention utilizes PCR method to clone soybean blossoming gene GmCOL1b from soybean.The Glyma08g28370 that announces among the GmCOL1b gene coding region full length sequence that the present invention obtains and the phytozome is corresponding.
The present invention expresses the GmCOL1b gene by the genetic transformation means in soybean, verified that the GmCOL1b gene has the physiological function of strongly inhibited soybean blossoming.
Description of drawings
Fig. 1 is that the Realtime quantitative fluorescent PCR shows the expression level graphic representation of GmCOL1b gene under different illumination conditions, wherein TUB is reference gene GmTubulin, ▲ represent the GmCOL1b gene at the expression level graphic representation of short day, ◆ expression GmCOL1b gene is at the expression level graphic representation of long day;
Fig. 2 is pTF101.1-GmCOL1b plasmid over-express vector structural representation;
Fig. 3 is not for changing the soybean blossoming state graph of GmCOL1b gene over to, and wherein, arrow refers to open soybean flower;
Fig. 4 is the enlarged view at arrow indication place among Fig. 3;
Fig. 5 is the soybean blossoming state graph that changes over to behind the GmCOL1b gene;
Fig. 6 is the soybean blossoming time diagram after not changing the GmCOL1b gene over to and changing the GmCOL1b gene over to, and wherein, DN50 is not for changing the soybean of GmCOL1b gene over to, and GmCOL1b-OX changes the soybean behind the GmCOL1b gene over to.
Embodiment
Embodiment one: the gene order of the soybean blossoming gene GmCOL1b of present embodiment is shown in sequence table Seq IDNo:1.
Present embodiment is successfully to clone first soybean blossoming gene GmCOL1b on molecular level.
Present embodiment utilizes PCR method to clone soybean blossoming gene GmCOL1b from soybean.The G1yma08g28370 that announces among the GmCOL1b gene coding region full length sequence that the present invention obtains and the phytozome is corresponding.
Present embodiment is expressed the GmCOL1b gene by the genetic transformation means in soybean, verified that the GmCOL1b gene has the physiological function of strongly inhibited soybean blossoming.
Embodiment two: the aminoacid sequence of the proteins encoded of the soybean blossoming gene GmCOL1b of present embodiment is shown in sequence table Seq ID No:2.
By following verification experimental verification effect of the present invention:
(1) acquisition of soybean blossoming gene GmCOL1b
One, take eastern agricultural 50 the blade of soybean varieties as material, extracts the total RNA of blade with the operational manual of buying from the TRIzol of Invitrogen company test kit; Two, the total RNA that adopts DNase I treatment step one to extract; Three, the total RNA that gets after 1 μ g step 2 is processed is used for the synthetic of cDNA, and the synthetic operation of cDNA is according to the BD SMART that buys from BD BiosciencesClontech company
TMThe service manual of RACE cDNAAmplification Kit test kit carries out, and obtains cDNA; Four, take the cDNA that obtains as template by F1 and R1 primer amplification GmCOL1b gene, the 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, 72 ℃ are extended 10min again, and the PCR product is checked order at ABI3130 sequenator (ABI company);
Wherein, the sequence of primers F 1 is GCCATCAAAA CACCACTCTG AC; The sequence of primer R1 is GCCCGATTAG TACAACTGCA AG.
Sequencing result shows that soybean blossoming gene GmCOL1b has the nucleotide sequence of Seq ID No:1 in the sequence table, and the Seq ID No:1 in the sequence table is by 1220 based compositions, and coding has the protein of the aminoacid sequence of Seq ID No:2 in the sequence table.
(2) functional verification of soybean blossoming gene GmCOL1b
1, the Real-time quantitative fluorescent PCR carries out functional verification
One, take eastern agricultural 50 the blade of soybean varieties as material, extracts the total RNA of blade with the operational manual of buying from the TRIzol of Invitrogen company test kit; Two, the total RNA that adopts DNase I treatment step one to extract; Three, the total RNA that gets after 1 μ g step 2 is processed is used for the synthetic of cDNA, and the synthetic operation of cDNA is according to the BD SMART that buys from BD Biosciences Clontech company
TMThe service manual of RACE cDNA Amplification Kit test kit carries out, and obtains cDNA; Four, get cDNA after 5 times of the 2 μ l step 3 dilutions as the RT-PCR template, take F1Q and R1Q as primer, operation steps is undertaken by buy the operation steps of introducing in the SYBR of BioRad company Green Supermix kit test kit, and the total reaction volume of each sample is 20 μ L; Five, adopt purchase to detect from the iCycle of BioRad company iQ PCR in real time detection system;
Wherein, the sequence of primers F 1Q is CATCAAAACA CCACTCTGAC; The sequence of primer R1Q is GCGTCAGCCT TGCAGAGGAA.
The expression level graphic representation of GmCOL1b gene under different illumination conditions as shown in Figure 1, as shown in Figure 1, GmCOL1b gene of the present invention (LD) expression peak value under the long day condition is very high, and presenting the biorhythm formula expresses, express after 4 hours in illumination and to present peak value, but under the short day condition (SD), expression amount is but very low.
2, transgenosis is carried out functional verification
One, take eastern agricultural 50 the blade of soybean varieties as material, extracts the total RNA of blade with the operational manual of buying from the TRIzol of Invitrogen company test kit; Two, the total RNA that adopts DNase I treatment step one to extract; Three, the total RNA that gets after 1 μ g step 2 is processed is used for the synthetic of cDNA, and the synthetic operation of cDNA is according to the BD SMART that buys from BD BiosciencesClontech company
TMThe service manual of RACE cDNAAmplification Kit test kit carries out, and obtains cDNA; Four, respectively take GmCOL1b 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 to contain the soybean blossoming gene GmCOL1b of the two enzyme restriction enzyme sites of XbaI-SacI; The soybean blossoming gene GmCOL1b that contains restriction enzyme site that five, will obtain is cloned in the pTF101.1 plasmid, obtain the pTF101.1-GmCOL1b plasmid, take the farming 50 of soybean varieties east as material, utilize agrobacterium-mediated transformation to carry out genetic transformation, obtain genetically engineered soybean, genetically engineered soybean and the soybean that does not change the GmCOL1b gene over to are planted under the long day condition, observed soybeans they grow and bloom; Wherein, the name that the pTF101.1 plasmid is published in 2006 " Plant CellReports " is called in the article of " Improved cotyledonary node method using an alternative explantderived from mature seed for efficient Agrobacterium-mediated soybean transformation " open, is so kind as to give by the author of article; Soybean varieties east farming 50 is so kind as to give by professor Zhang Huizhen of Northeast Agricultural University;
Wherein, the sequence of primers F 2 is GCTCTAGAGC CATCAAAACA CCACTCTGAC; The sequence of primer R2 is GCGAGCTCGC CCGATTAGTA CAACTGCAAG.
The pTF101.1-GmCOL1b plasmid over-express vector structural representation that step 5 obtains as shown in Figure 2;
Do not change the soybean blossoming state graph of GmCOL1b gene over to as shown in Figure 3, wherein, arrow refers to open soybean flower, the enlarged view at arrow indication place as shown in Figure 4 among Fig. 3, change over to behind the GmCOL1b gene the soybean blossoming state graph as shown in Figure 5, can find out from Fig. 3, Fig. 4 and Fig. 5, when not changing the soybean blossoming of GmCOL1b gene over to, the soybean that changes over to behind the GmCOL1b gene does not also bloom, and shows that the soybean that changes over to behind the GmCOL1b gene presents flower-shaped attitude in obvious evening;
Soybean blossoming time diagram after not changing the GmCOL1b gene over to and changing the GmCOL1b gene over to as shown in Figure 6, wherein, DN50 is not for changing the soybean of GmCOL1b gene over to, GmCOL1b-OX changes the soybean behind the GmCOL1b gene over to, as can be seen from Figure 6, the flowering time of DN50 is 30 days, the flowering time of GmCOL1b-OX is 40 days, show soybean blossoming time of changing over to behind the GmCOL1b gene than late 10 days of the soybean blossoming time that does not change over to behind the GmCOL1b gene, thereby explanation GmCOL1b gene has the function that suppresses soybean blossoming.
Claims (2)
1. soybean blossoming gene GmCOL1b is characterized in that the gene order of soybean blossoming gene GmCOL1b is shown in sequence table Seq ID No:1.
2. the proteins encoded of soybean blossoming gene GmCOL1b as claimed in claim 1 is characterized in that the aminoacid sequence of proteins encoded of soybean blossoming gene GmCOL1b is shown in sequence table Seq ID No:2.
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| CN2012105123691A CN103014019A (en) | 2012-12-04 | 2012-12-04 | Flowering gene GmCOL1b of soybean and coding protein thereof |
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| CN2012105123691A CN103014019A (en) | 2012-12-04 | 2012-12-04 | Flowering gene GmCOL1b of soybean and coding protein thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104357442A (en) * | 2014-10-25 | 2015-02-18 | 中国科学院东北地理与农业生态研究所 | QTL mapping region for soybean flowering stage and obtaining method as well as application |
| CN104560975A (en) * | 2015-01-04 | 2015-04-29 | 中国科学院东北地理与农业生态研究所 | Soybean flowering date QTL chromosome mapping interval as well as obtaining method and application thereof |
| CN112390867A (en) * | 2020-11-17 | 2021-02-23 | 西南大学 | Chimonanthus praecox CpCO-L2 gene and protein coded by same and application of gene |
| CN114644704A (en) * | 2022-02-24 | 2022-06-21 | 东北农业大学 | Protein with plant stress regulating and controlling function and cultivation method for improving plant stress |
-
2012
- 2012-12-04 CN CN2012105123691A patent/CN103014019A/en active Pending
Non-Patent Citations (3)
| Title |
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| EMBL: "DQ371243.1", 《EMBL》 * |
| SUSAN LEDGER ET AL.: "Analysis of the function of two circadian-regulated", 《THE PLANT JOURNAL》 * |
| 张清哲 等: "大豆GmCOL4 基因的克隆与分析", 《作物学报》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104357442A (en) * | 2014-10-25 | 2015-02-18 | 中国科学院东北地理与农业生态研究所 | QTL mapping region for soybean flowering stage and obtaining method as well as application |
| CN104560975A (en) * | 2015-01-04 | 2015-04-29 | 中国科学院东北地理与农业生态研究所 | Soybean flowering date QTL chromosome mapping interval as well as obtaining method and application thereof |
| CN112390867A (en) * | 2020-11-17 | 2021-02-23 | 西南大学 | Chimonanthus praecox CpCO-L2 gene and protein coded by same and application of gene |
| CN112390867B (en) * | 2020-11-17 | 2022-02-01 | 西南大学 | Chimonanthus praecox CpCO-L2 gene and protein coded by same and application of gene |
| CN114644704A (en) * | 2022-02-24 | 2022-06-21 | 东北农业大学 | Protein with plant stress regulating and controlling function and cultivation method for improving plant stress |
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Application publication date: 20130403 |