CN106636124A - Seed-weight-reducing white birch gene AP2 and encoding protein thereof - Google Patents
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Abstract
The invention relates to a seed-weight-reducing white birch gene AP2 and an encoding protein thereof, and belongs to the technical field of gene in molecular biology. The seed-weight-reducing white birch gene AP2 is characterized in that the synthesis of free fatty acid and soluble protein is reduced, so that the weight of Arabidopsis seeds is reduced; the sequence of the white birch AP2 is shown as SEQ ID NO:1 in a sequence table. The amino acid sequence of the encoding protein of the gene is shown as SEQ ID NO:2 in the sequence table. The invention provides the gene AP2 relevant to the white birch flower development; a plant expression vector of the white birch gene AP2 is built; the built plant expression vector is subjected to Arabidopsis plant invasion by agrobacterium, so that the transgenic Arabidopsis plant is obtained; in addition, the free fatty acid content and the soluble protein content in the seeds of the transgenic pure plants are reduced; the result shows that the gene has the function of reducing the free fatty acid content and the soluble protein content in the Arabidopsis seeds, and can be used for regulating the biosynthesis path of the free fatty acid and the soluble protein. The gene provided by the invention has the advantages that the gene resources and theoretical foundation is provided for regulating and controlling the seed development by using the gene engineering technology; great application values are realized.
Description
Technical field：The present invention relates to one reduces free fatty and the white birch AP2 of soluble protein synthesis in seed
Gene and its encoding proteins, belong to the gene technology field in molecular biology.
Background technology：
Find that most of genes belong to MADs-box families in flower development related gene by the research to arabidopsis
Gene, only AP2 genes there is unique AP2 domains, the domain is a DNA binding structural domain, by 60 to 70
3 antiparallel beta sheets and an amphipathic alpha-helix of the conservative amino acid residue composition in left and right are constituted.AP2/
EREBP transcription factor families can be divided three classes according to the difference of its contained AP2 domains quantity：Containing 2 AP2 domains
AP2 subfamilies, the EREBP subfamilies containing an AP2 domain and containing an AP2 domain and B3 domain
RAV1 and RAV2.Jofuku in 1994 etc. is found that AP2 (APETALA2) transcription factor in arabidopsis, while first identified goes out
The AP2/EREBP domains of AP2 genes.Nineteen ninety-five Ohme-Talagi etc. successfully isolates 4 ethylene reaction knots from tobacco
Hop protein EREBPs.Relevant report shows that AP2 subfamilies have crucial regulation in the development of reproductive organs and nutrition organs
Effect.During the development of floral organs of arabidopsis, A class functional gene AP2 can suppress C classes functional gene AG (AGAMOUS)
Expression in foreign steamer floral organ sepal and petal, the development to sepal and petal plays an important role, and AP2 genes not only exist
There is expression in floral organ, also have expression in the non-flower tissue such as stem, leaf and seed.LIP1 and LIP2 in toad's-mouth is belonged to
AP2 transcription factor families, all have certainly as they of A genoids in the growth course of toad's-mouth sepal, petal and ovule
Qualitatively act on.But from unlike AP2 genes, LIP genes do not suppress the expression of C genoids.AP2 genes sending out in seed
Also play an important role during educating, it may decide that size, weight and the oily substance of seed and the accumulation of protein.Apple
The MAP2A genes of fruit have expression in the floral organs such as bud, sepal, petal, Pistil And Stamen, ovary and non-floral organ blade,
Illustrate that MAP2A can regulate and control the development of floral organ and nutrition organs.The nutrition organs of the Vv-AP2 gene pairs grapes of grape and life
The development for growing organ has different effects.The existing ability for constantly differentiating new histoorgan of separate living tissue of plant,
The meristematic capacity of itself is maintained again.AP2 genes can maintain the meristematic capacity of plant meristem.Paddy rice SNB
(SUPERNUMERARY BRACT) is AP2 subtribe transcription factors, it can regulate and control fringe separate living tissue to floral meristem change with
And development of floral organs.Corn IDS1 (INDETERMINATE SPIKELET1) is the homologous gene of arabidopsis AP2 genes, can be with
Regulate and control the development of inflorescence by regulating and controlling small ear meristem identity.At present, it is still unclear for the function of the AP2 genes of white birch
Chu, also reports without reference to the Patents of white birch AP2 gene orders and its function.
The content of the invention：
The present invention provides a kind of method that white birch flower development related gene AP2 is cloned from white birch：
(1) it is anti-with ThermoScriptTM RT-PCR System first using the CTAB methods extraction white birch female flower RNA of improvement
Transcript reagent box, synthesizes the chains of cDNA first；
(2) PCR primer of white birch AP2 code areas total length is cloned in design containing restriction enzyme site (XbaI and SmaI)；Primer sequence
It is classified as：Forward primer：AP2-F：5′-GCGCTTCTAGAATGTGGGATCTCAATGACTCG-3′；Reverse primer：AP2-R：5′-
GTATCCCGGGATAGAGGGTCTCATGAGAGAAT-3′
(3) enter performing PCR amplification to the cDNA for synthesizing using primer AP2-F and AP2-R, obtain an AP2 transcription factor base
The full-length cDNA of cause, is named as white birch AP2；
Pcr amplification product Jing is sequenced, its nucleotide sequence such as sequence table SEQ ID NO:Shown in 1；The ammonia of its encoding proteins
Base acid sequence such as sequence table SEQ ID NO:Shown in 2；
The present invention constructs the plant expression vector of white birch AP2 genes, the plant expression vector (pBI121-AP2) of structure
Jing During Agrobacterium Arabidopsis plants, obtain transgenic Arabidopsis plants and transgenosis pure lines plant bear seeds middle free-fat
Acid content and soluble protein content have declined, and showing that the gene has reduces arabidopsis seed free fatty and solvable
The function of property protein content and the biosynthesis pathway of two kinds of materials can be regulated and controled.
The technical scheme for building white birch AP2 gene plant expression vectors is as follows：
PBI121 carriers are selected as basic plant expression vector, two digestion positions of XbaI and SmaI are selected on pBI121
Point, white birch AP2 primer of the design with restriction enzyme site and protection base, and clone white birch AP2 full length genes.Double digestion plasmid
With white birch AP2 genes, then it is attached with T4 ligases, detects the carrier pBI121-AP2 for building.
The white birch AP2 genes that the present invention is provided are adjusting arabidopsis seed free fatty acid content and soluble protein content
In application.Above-mentioned plant expression vector pBI121-AP2 is converted into Agrobacterium EHA105, Jing agriculture bacillus mediated flower-dipping method leaching
Dye, obtains the transgenic Arabidopsis plants of white birch AP2 overexpression, and Jing resistance screenings and PCR detections finally give 4 transgenosis sun
Property homozygous lines：Line5, line6, line9, line15, white birch AP2 can reduce transgenic arabidopsis seed free fatty
With the content of soluble protein, compared with Wild-type non-transgenic plant, transgenic homozygous arabidopsis seed free fatty contains
Amount reduces 28.48%-32.68%；Soluble protein content reduces 8.42%-40.64%.The plant expression vector of structure
(pBI121-AP2) content of free fatty and soluble protein in transgenic arabidopsis seed can be reduced, is sharp from now on
With transgenic technology adjust Betula platyphylla seed in free fatty and soluble protein content research provide theoretical foundation with
Genetic resources.
The present invention has the advantages that compared with prior art：
1st, the invention provides reducing the white birch AP2 albumen of seed free fatty and soluble protein content, can use
In improvement and plant free fatty and soluble protein content, and can be used to search to regulate and control in other plant free fat
Fat acid and the related gene of soluble protein synthesis.
2nd, the present invention provide containing white birch AP2 gene recombined vectors, can in effective control seed free fatty and
The content of soluble protein.
3rd, the white birch AP2 genes that the present invention is provided have the content of free fatty and soluble protein in regulation and control seed
Function, it is in can be used for the related development research of content of plant free fatty and soluble protein and free for plant
The functional study of aliphatic acid and soluble protein synthesis related gene provides reference resources.
Description of the drawings
Fig. 1 shows figure for the PCR amplifications of white birch AP2 full length gene cDNA.
Fig. 2 is that plant expression vector pBI121-AP2 builds flow chart.
Fig. 3 is plant expression vector pBI121-AP2PCR detection figures.
Fig. 4 is plant expression vector pBI121-AP2 plasmid extractions detection figure.
Fig. 5 is transgenosis overexpression white birch AP2 Arabidopsis plant PCR proof diagrams.
Fig. 6 is wildtype Arabidopsis thaliana seed free fatty in weight, the seed of overexpression white birch AP2 arabidopsis seeds
And it is solvable
Property protein content statistical chart.
Specific embodiment：
With reference to specific embodiment, the invention will be further described.
The experimental technique of unreceipted actual conditions in the following example, generally according to normal condition, or according to manufacturer
Proposed condition.
Embodiment 1：The clone of white birch AP2 genes
(1) acquisition of vegetable material
Experiment material white birch female flower, picks up from Northeast Forestry University's forest of white birch.Refrigeration immediately is to -80 DEG C of Refrigerator stores after collection
It is standby.(2) extracting of white birch RNA
White birch total serum IgE is carried out using the CTAB methods step of improvement, is surveyed using agarose gel electrophoresis, ultraviolet specrophotometer
The purity and concentration of total serum IgE.Use ThermoScriptTMRT-PCR System reverse transcription reagent box synthesizes the chains of cDNA first, so
Enter performing PCR amplification, PCR reaction systems with AP2-F and AP2-R afterwards：The μ l of cDNA templates 1,10 × Buffer 5 μ l, dNTP
(2.5mM) 4 μ l, each 2 μ l (10 μM) of forward and reverse primer, the μ l of Taq Plus DNA polymerase 1, sterile purified water supplies 50
μl.PCR amplification conditions are：94 DEG C of denaturations 5min；94 DEG C of denaturation 30s, 60 DEG C of annealing 30s, 72 DEG C of extension 1min30s, 35
Circulation；72 DEG C of extension 10min.The agarose gel electrophoresis of PCR primer Jing 1.0% is separated, and is reclaimed and connect with pMD19T after purpose band
Connect, linked system is as follows：The μ l of genes of interest 4, T easy vector 1 μ l, the μ l of Solution I 5.Connection product converts large intestine
Bacillus, extracts plasmid and is sequenced.
White birch AP2 nucleotide sequence analysises
The a length of 1554bp of white birch AP2 gene orders, sequence is shown in sequence table SEQ ID NO:1, by BLASTN sequence analyses
As a result show, white birch AP2cDNA and arabidopsis (Arabidopsis thaliana), comospore poplar (Populus
Trichocarpa) similitude is up to more than 70%.
The protein sequence analysis of white birch AP2 gene codes
White birch AP2 genes encode 517 amino acid altogether, and sequence is shown in sequence table SEQ ID NO:2, carried out by BLASTP
Sequence alignment, as a result shows, with white birch AP2 amino acid sequences and comospore poplar (Populus trichocarpa), castor-oil plant
The affiliation of (Ricinus communis) is closest.
Embodiment 2：The plant expression vector construction of white birch AP2 genes
(1) double digestion of white birch AP2 and detected
Plant expression vector double digestion step is as follows：
Cloning vector and plant expressing vector pBI121 use XbaI and SmaI double digestions, and digestion system is as follows：DNA 2μ
The μ l of g, 10 × FD buffer 2, XbaI 1 μ l, the μ l of SmaI 1, sterile purified water supplies 20 μ l.37 DEG C of 2～3h of incubation after mixing,
65 DEG C of incubation 5min.The agarose gel electrophoresis of double digestion product Jing 0.8%, by OMEGA QIAquick Gel Extraction Kits step purpose piece is reclaimed
Section.
(2) connection of double digestion product
16 DEG C overnight connect, and linked system is as follows：The μ l of genes of interest 10, the μ l of pBI121 carrier segments 4, the μ l of T4 ligases 1,
The μ l of T4buffer 2, sterile purified water supplies 20 μ l
(4) Escherichia coli and detection are converted
Connection product after double digestion is passed directly in Escherichia coli so as to great expression, be enriched with the dense of expression vector
Degree.Then converted product is inoculated on the LB solid mediums containing kanamycins (50 μ g/mL), flat board is inverted in 37 DEG C of perseverances
12-18h is cultivated in warm incubator.The bacterium solution of Amplification Culture carries out the identification of bacterium solution PCR.
Embodiment 3：White birch AP2 genes overexpression functional analysis in arabidopsis
(1) culture of arabidopsis aseptic seedling：
A) take appropriate seed in centrifuge tube, add appropriate sterile distilled water, abundant whirlpool concussion, in short-term wink is from will float
Floating seed and sterilized water is suctioned out together.
B) ethanol of 1ml 70%, whirlpool concussion 10s is added seed is fully suspended in ethanol solution, suction out ethanol
(the ethanol disinfection time is unsuitable long, it is to avoid ethanol penetrates into, and reduces seed activity), adds aseptic ddH2O, concussion cleaning 2 times.
C) sodium hypochlorite of 1ml 10%, whirlpool fully shaking 5min, 12000rpm centrifugation 1min, superclean bench are added
In, with the aseptic ddH of 1ml2O, concussion cleaning 3-5 time, until solution becomes colorless in centrifuge tube, reduces remaining sodium hypochlorite to planting
The impact of son.
D) washed aseptic seed is uniformly laid on into MS media surfaces, after media surface moisture is slightly dry, seals envelope
Membrana oralis, and carry out mark.
E) culture medium of good seal is put into into vernalization treatment 2-3 days in 4 DEG C of refrigerators, it is therefore an objective to break seed dormancy.
F) take out the flat board after vernalization and be put into illumination cultivation on culturing rack (16h light/8h dark)
G) treat growth of seedling 10d or so, when growing two panels true leaf by it is transplanted in a large number to high-temperature sterilization and was poured
Cultivate in the soil of saturation nutrient solution.
(2) agriculture bacillus mediated flower-dipping method arabidopsis thaliana transformation
A) Agrobacterium culture：A single bacterium colony of picking EHA105 bacterial strains, is inoculated into 20m1 and adds corresponding antibiosis on flat board
In Bacteria Culture liquid culture medium (pH7.0) of element, on constant-temperature table, in 27 DEG C, 180r/min cultivates most 0.6-1.0.
By bacterium solution proceed to new preparation without hormone respective liquid culture medium in, be diluted to OD600For 0.2-0.3 when can be used to convert；
B) optional step：After arabidopsis grows main inflorescence, inflorescence is all cut from its bottom, inflorescence can be again after 4-6d
New to extract out, this step ensure that the inflorescence growth situation of each plant is essentially identical and late blooming.
C) first plant is watered to saturation before infecting, the flower and silique for having opened then is deducted, by inflorescence and lotus throne
Leaf together invades Agrobacterium and infects 30s in solution, and to plant surface one layer of liquid film is covered.
D) Agrobacterium of plant excess surface is infected into solution with blotting paper after infecting to blot, with preservative film plant is wrapped up.
E) all arabidopsis for infecting are covered with brown paper, is put in light culture 24h under the conditions of lucifuge.
F) remove under conditions of plant is placed on the 16h long-day by the brown paper for covering and normally cultivate, normally water, plant to be planted
Plant is bonded on bamboo let and prevents plant from lodging by pluggable bamboo let after growing tall, and can be collected when siliqua turns yellow will be burst out
Seed.
(3) screening of resistant plant and PCR are detected
By the seed collected, on the MS culture mediums containing final concentration of 50 μ g/ml Kan, method is with plan for sterile culture
The culture of southern mustard aseptic seedling, by the resistant plant for filtering out performing PCR detection is entered, and turns the PCR detections institute of white birch AP2 gene arabidopsis
Use primer is respectively BplAP2 specific primer (AP2-F2：5 '-ACTGCCTCTGCTGGTGC-3 ' and AP2-R2：5′-
GAATGCTGTCCCGTTGC-3 '), the primers of the NPT II (- F of NPT II：5 '-GGGCACAACAGACAATCG-3 ' and-the R of NPT II：5′-
ATCACGGGTAGCCAACG-3 '), PCR reaction systems are 20 μ l, and each volume of constituent is respectively：Template (DNA moulds
Plate), 2 μ l；Primer-F (10 μM), 1 μ l；Primer-R (10 μM), 1 μ l；2 × Taq PCR MasterMix, 10 μ l；Plus
ddH2O is mended to 20 μ l.PCR amplification conditions are：94 DEG C of denaturations 5min；94 DEG C of denaturation 30s, 55 DEG C of annealing 30s, 72 DEG C of extensions,
45s, 30 circulations；72 DEG C of extension 10min.The method detected using resistance screening and PCR filters out resistance homozygous lines, carries out
Subsequent experimental.
The transgenic Arabidopsis plants for obtaining are extracted into DNA, is made as template with sterilized water and the DNA for not carrying out genetic transformation
Negative control, positive control is made with the pBI121-AP2 plasmids that confirmation is sequenced as template.Respectively take 5 μ l PCR primers after amplification to exist
Electrophoresis in 1% gel, enters performing PCR detection.Homozygosis Arabidopsis plant and wild-type Arabidopsis plants to test positive
24 plants, the measurement of seed biomass is carried out, as a result as shown in fig. 6, intending in wild type for concrete analysis white birch AP2 transgenic seeds
The conspicuousness of southern canola seed biomass difference, enters to white birch AP2 transfer-gen plants and wild-type Arabidopsis plants seed biomass
Row quantitative analysis, statistics is as shown in table 1.Compared with Wild-type non-transgenic plant, transgenic homozygous arabidopsis seed weight
Amount declines 10%-15%；Free fatty acid content reduces 28.48%-32.68% in seed；Soluble protein content in seed
Reduce 8.42%-40.64%.
The above results show, white birch AP2 gene overexpressions can reduce in arabidopsis seed free fatty acid content and solvable
Property protein content, render transgenic arabidopsis seed weight reduces, and illustrate that white birch AP2 genes can be by free fat in regulation and control seed
Fat acid and the synthesis of soluble protein can provide certain section adjusting the weight of seed for follow-up study raising crop seed yield
Learn foundation.
Table 1：White birch AP2 transgenic arabidopsis compare with wildtype Arabidopsis thaliana seed biomass
Note：Weight and two kinds of content of material are Mean ± SD in table, rate value for turn white birch AP2 gene plants with it is wild
Type plant content difference.
Claims (1)
1. a kind of white birch AP2 genes and its encoding proteins for reducing seed weight, it is characterised in that reduce arabidopsis seed and dissociate
The synthesis of aliphatic acid and soluble protein, so as to reduce the weight of seed, sequence such as sequence table SEQ ID of white birch AP2 genes
NO:Shown in 1；The amino acid sequence such as list SEQ ID NO of the albumen of its coding:Shown in 2.
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| CN111118033A (en) * | 2020-02-20 | 2020-05-08 | 南京林业大学 | Hybrid liriodendron LhAP2L gene and application thereof |
| CN111118033B (en) * | 2020-02-20 | 2020-12-15 | 南京林业大学 | Hybrid liriodendron LhAP2L gene and application thereof |
| CN113061172A (en) * | 2021-05-20 | 2021-07-02 | 湖州松泉农业科技有限公司 | Plant salt tolerance related LIP1 protein and related biological material and application thereof |
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