CN106047887A - Dahurian larch LkANT gene, protein and applications - Google Patents
Dahurian larch LkANT gene, protein and applications Download PDFInfo
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- CN106047887A CN106047887A CN201610288134.7A CN201610288134A CN106047887A CN 106047887 A CN106047887 A CN 106047887A CN 201610288134 A CN201610288134 A CN 201610288134A CN 106047887 A CN106047887 A CN 106047887A
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- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
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Abstract
A dahurian larch LkANT gene, a protein and applications are disclosed. The cDNA sequence of the gene is shown as SEQ ID NO:1. The protein encoded by the gene is characterized in that the amino acid sequence of the protein is shown as SEQ ID NO:2, and is characterized by applications of the protein in culture of multi-branch transgenic plants. By utilization of the modern molecular biological technique, the gene critical to regulation and control of branches and seed development for dahurian larch is cloned, separated and subjected to functional analysis. The acquisition of the critical gene of this type has important theoretical value, provides important candidate genes for increasing grain yields by utilizing genetic engineering means, and has an important application prospect in the field of crop genetic improvement and the like.
Description
Technical field
The invention belongs to genetic engineering field, especially a kind of larch LkANT gene, albumen and application.
Background technology
It is to determine the key factor that phytomorph is built up that the branch of higher plant is grown, and has with phytomass and yield
Close relationship.The side that the plant type of aboveground vegetation part comes from the shoot apical meristem in fetal development period and postembryonal development is raw
Separate living tissue.Lateral meristem can break up generation axillalry bud, and axillalry bud further growth is grown and formed side shoot.Side shoot is with stem phase
With, it may have formation blade, flower, axil separate living tissue and the ability of secondary branch, but species difference branch situation the most not phase
With.Plant branching grows the main multiple regulation and control by inherited genetic factors, phytohormone and ambient signal.In recent years, by multiple
The further investigation of plant, it is thus achieved that the important gene that a series of regulation and control branches are grown.LAS is to affect axillary meristem shape the earliest
One of gene become, in arabidopsis, the sudden change of LAS gene causes number of branch purpose to reduce.Tiller is the grasses such as Oryza sativa L.
An important branch phenomenon in growth and development process, is also an important economical character, is directly connected to the spike number of plant
And affect yield further.Li Jiayang etc., by collection of illustrative plates clone technology, separate to obtain and play important work in rice tiller regulating
Gene M OC1, functional analysis finds that this gene can effectively facilitate the growth of tiller and axillalry bud.TB1 gene is then branch
The negative regulatory factor controlled, the overexpression of this gene can decrease the formation of side shoot.Along with deepening continuously of research, Ren Menfa
Having showed the related gene that increasing regulation and control branch is grown, the people that are found to be of these genes utilize reverse genetics means fixed
To transformation plant forms, cultivation has ideotype and has established important foundation to obtain high yield new variety of plant.
Seed is the important grain source that the mankind depend on for existence, and the size and number of seed is to affect crop yield
Principal element.Consequently found that control seed amount and the important factor in order of size, and obtain relevant functional gene, to crop
SOYBEAN IN HIGH-YIELD BREEDING has important theory significance and using value.Correlational study finds the branch amount of plant and the increase of seed production
Direct relation, awnless brome is had to be proportionate with unit are branch amount with the yield of Perennial Ryegrass Seed.GASA gene
Family is the special gene family regulated and controled by gibberellins.GASA4 gene overexpression can significantly increase transgenic
The seed size of plant.But, ironically the seed of gasa4 mutant can diminish, but mutant plants single-strain seed
Total output but higher than wild type, this produces the reason of relatively multiple-limb mainly due to gasa4 mutant plants.Thus may be used
Seeing, the branch of plant and the yield of plant seed have close relationship.ANT transcription factor belongs to a member of AP2 subfamily, main
Participate in the cell division that regulation is coordinated mutually with organ growth.In arabidopsis, the afunction mutant of ANT gene is due to cell
Number reduces and makes floral organ diminish, but, the overexpression of this gene adds the size of rachis and floral organ.
At present regulation and control plant branching is grown and the clone of seed development related gene is concentrated mainly on herbaceous plant, and
The clone of this genoid and the most rare to its application in xylophyta, particularly gymnosperm.Larch is needle
The Typical Representative of seeds, the commerical tree species that Ye Shi China is main, there is highly important economic worth and the ecological value.Meanwhile,
Larch has again indeterminate growth ability as xylophyta, wherein contains abundant genetic resources.
Summary of the invention
It is an object of the invention to provide a kind of larch LkANT gene and application, the present invention obtains this gene first, and
This gene is carried out behavior study, it is thus achieved that good effect.
The present invention realizes specifically comprising the following steps that of purpose
Larch LkANT gene, the cDNA sequence of gene is as shown in SEQ ID NO:1.
The protein of larch LkANT coded by said gene, the aminoacid sequence of protein is as shown in SEQ ID NO:2.
The application in cultivating multi-branched transgenic plant of the larch LkANT gene.
Larch LkANT gene is being cultivated with seed for results object, it is thus achieved that in high yield transgenic plant new varieties
Application.
Advantages of the present invention and good effect are as follows:
The key gene of regulation and control larch branch and seed development, by modern molecular biology technique, is carried out by the present invention
Clone, separate and functional analysis, it is provided that the acquisition of key gene not only there is important theory value, and for utilizing gene
Engineering means improve the candidate gene that grain yield provides important, before the fields such as crop genetic improvement have important application
Scape.
The present invention passes through specific experiment, observes and add up different growth promoter transfer-gen plant in period and matched group Plant Leaf
The biological properties such as sheet, branch and angle fruit.Result shows: (1) compared with wildtype Arabidopsis thaliana, transgenic plant plant leaf
Significantly increase；(2) compared with wildtype Arabidopsis thaliana, transgenic plant plant branch amount showed increased；(3) south is intended with wild type
Mustard compares, and transgenic plant plant angle fruit substantially becomes big, and single-strain seed weight is about 2.5 times of wild type.
The present invention is by modern molecular biology technique, to regulation and control larch branch and the key gene of seed development
LkANT carries out cloning, separating and functional analysis, and the acquisition of LkANT gene not only has important theory value, and for utilizing
Genetic engineering means improves the candidate gene that grain yield provides important, has important application in fields such as crop genetic improvements
Prospect.
Accompanying drawing explanation
Fig. 1 transfer-gen plant T1 for resistance screening (Fig. 1 a) and T3 for resistance screening (Fig. 1 b).
Fig. 2 WT lines (left) and transfer-gen plant (right) different growth promoter phenotypic characteristic in period.Fig. 2 a: wild type
Plant (left) and transfer-gen plant (right) 2 week old growing state；Fig. 2 b: WT lines (left) and transfer-gen plant (right) 3 weeks
Age growing state；Fig. 2 c: WT lines (left) and transfer-gen plant (right) 5 week old growing state；Fig. 2 d: WT lines
(left) and transfer-gen plant (right) 8 week old growing state.
Fig. 3 WT lines and transfer-gen plant stem leaf branch situation analysis chart.
Fig. 4 WT lines and transfer-gen plant Pod length analysis chart.
Fig. 5 WT lines and transfer-gen plant single-strain seed gravimetric analysis figure.
Fig. 6 Positive recombinant clones PCR qualification result figure.MIII:DNA markerIII；1-5: Positive recombinant clones.
Specific implementation method
Below by specific embodiment, the invention will be further described, and following example are illustrative, is not limit
Qualitatively, it is impossible to limit protection scope of the present invention with this.
In the present invention, utilize Illumina/Solexa high throughput sequencing technologies that larch transcript profile is carried out sequencing analysis.
By going deep into data mining and sequence assembly obtains the unigene of a 1465bp, and this unigene presents height in axillalry bud
Expression pattern.For analysing in depth the functional character of this unigene, therefore, according to the unigene sequence information design spy obtained
Different primer, uses RT-PCR technology to expand this unigene sequence in larch mRNA.Utilize ORF Finder to this
The open reading frame (ORF) of unigene is analyzed finding the ORF that this unigene comprises a 1395bp, encodes 464 ammonia
The protein of base acid, by its named LkANT.
The species such as protein sequence and Pollen pini thunbergii, Nicotiana tabacum L., Fructus Lycopersici esculenti and the Fructus Vitis viniferae of discovery this gene order coding are analyzed in BLAST comparison
The homology of ANT-Like transcription factor is only 60%-80%, this show gymnosperm larchen at LkANT gene for a long time
Evolution in the most conservative, remain the sequence structure feature of self uniqueness.Its function of the structures shape of gene, the most deeply
Enter to probe into the functional character of this gene for excavating, clone larch Fineness gene by genetic engineering means for crop genetic
Improvement has preferable application potential.
The clone of a kind of larch LkANT gene and the method for cultivation multi-branched and high yield transgenic plant are as follows:
1, larch Total RNAs extraction and the first chain cDNA synthesis
Give birth to young leaflet tablet then with larch as test material, use CTAB method to extract total serum IgE, and utilize M-MLV to reverse
Record test kit reverse transcription obtains the first chain cDNA.
2, the separation of LKANT gene and clone
(1) primer premier 5.0 software design band restriction enzyme site primer is utilized
Forward primer: 5 '-GACGGTACCATGTATTTTGATCACAATGTAGG-3 ' (underscore is KpnI restriction enzyme site)
Reverse primer X:ba5I '-TCGCGGCCGC(underscore is NotI enzyme action to TTAGGTTATGAAGAGCTGCCAG-3 '
Site)
(2) PCR reaction system
(3) pcr amplification reaction parameter
94℃1min；
94 DEG C of 20s, 52 DEG C of 20s, 72 DEG C of 30s, 35cycles；
72℃5min；
(4) 1% agarose gel electrophoresis testing goal fragments.
3, the recovery of genes of interest and cloning and sequencing
(1) coupled reaction
It is gently mixed, room temperature (20 DEG C-37 DEG C) reaction 5-10min；
After reaction terminates, centrifuge tube is placed on ice.
(2) convert
Add connection product in 50 μ L Trans1-T1 competent cells, flick mixing, ice bath 30min；
42 DEG C of heat shock 30s, are immediately placed on 2min on ice；
Add 250 μ L balances and hatch 1h to the LB, 200rpm 37 DEG C of room temperature；
Take 8 μ L 100mmol IPTG, 40 μ L 25mg/mL X-gal, 25 μ L 100mg/mL Amp mixing, be coated with equably
On ready flat board, place 30min for 37 DEG C；
After IPTG and X-gal is absorbed, take 150 μ L bacterium solution coated plates, overnight incubation.
(3) qualification of positive recombinant
Random picking white colony is in the LB fluid medium containing 1mg/LAmp；
200rpm 37 DEG C hatches about 8h；
Take 1 μ L bacterium solution and be used as the template of PCR reaction, identify recon with LKANT special primer；
PCR reaction system is as follows
PCR reaction condition
94℃10min；
94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 90s；72℃10min.
Agarose gel electrophoresis detection clip size (Fig. 6).
(4) fungi preservation and order-checking
Positive colony recon bacterium solution adds equal-volume 40% glycerol LB culture medium, mixing ,-20 DEG C of preservations.Positive colony
Recon bacterium solution is sent to raw work order-checking portion, Shanghai and checks order, and sequencing result is sequence 1.
4, plasmid enzyme restriction
(1) use KpnI and NotI double containing genes of interest cloning vector plasmids and pRI201-AN-GUS vector plasmid simultaneously
Enzyme action；
(2) enzyme action system is as follows
(3) mix gently；
(4)37℃3h；
(5) agarose gel electrophoresis detection enzyme action result；
(6) purpose fragment is reclaimed.
5, the connection of endonuclease bamhi
(1) the band restriction enzyme site genes of interest fragment reclaimed is to the pRI201-AN-GUS carrier with corresponding restriction enzyme site even
Connect；
(2) linked system
(3) mix gently；
(4) 16 DEG C of water bath heat preservation 12h.
6, recombinant plasmid transformed Agrobacterium
(1) take 1 μ g recombiant plasmid to add in 100 μ L Agrobacterium competence, mix gently；
(2) ice bath 30min；
(3) liquid nitrogen flash freezer 5min；
(4) 37 DEG C of water bath heat preservation 5min are gone to rapidly；
(5) YEB 600 μ L is added；
(6) 28 DEG C of 200rpm recovery 4h；
(7) take 150 μ L recovery bacterium solution and be applied to YEB flat board (resisting containing three)；
It is inverted for (8) 28 DEG C and cultivates 2-3d.
(9) identified positive strain saves backup in-20 DEG C.
7. the plantation of arabidopsis
(1), during arabidopsis seed is placed in 1.5mL centrifuge tube, 1mL 70% ethanol, vibration washing 30s are added；
(2) 6,000rpm 1min collects seed, abandons supernatant；
(3) 1mL sterilized water is added, vibration washing 1min, collects seed, is repeated 2 times；
(4) 2% sodium hypochlorite and 0.05%tween-20 vibration sterilization 15min are added；
(5) 6,000rpm 1min collects seed, abandons supernatant；
(6) 1mL sterilized water is added, vibration washing 1min, collects seed, is repeated 5 times；
(7) seed is uniformly seeded in MS solid medium；
Vernalization 3-4d under (8) 4 DEG C of dark conditions；
(9), after vernalization completes, it is placed in 22 DEG C of illumination boxs, 16h illumination, 8h dark culturing；
(10) 4 leaf phase arabidopsiss are gone in Nutrition Soil, 1, every basin, phjytotron cultivation (22 DEG C, 16h illumination, 8h
Dark, relative humidity 50-70%).
8, Agrobacterium-mediated Transformation arabidopsis
(1) by-20 DEG C of Agrobacterium inoculation containing purpose recombiant plasmid preserved in 10mLYEB culture medium；
(2) 28 DEG C of 200rpm activate overnight；
(3) 1:100 dilution thalline is in 250mLYEB culture medium；
(4) 28 DEG C of 200rpm cultivate to OD600=1.2～1.6；
(5) room temperature 5,000rpm is centrifuged 15min and collects thalline；
(6) equal-volume converts buffer (5% sucrose) resuspended thalline；
(7) arabidopsis is inverted in the conversion buffer of resuspended thalline, contaminates 10s；
(7) taking out plant, wrapped by plant with preservative film, light culture 24h is put in side；
(8) removing overcover, erect plants is cultivated；
(9) cultivate under normal condition to arabidopsis and yield positive results, collect seed.
9, transfer-gen plant Kan resistance screening
(1) it is seeded in after transgenic arabidopsis seed disinfection in MS solid medium (containing 50mg/L Kan)；
Vernalization 3-4d under (2) 4 DEG C of dark conditions；
(3) 22 DEG C of illumination boxs, 16h illumination, 8h dark culturing it are placed in；
(4) to start albefaction dead for the arabidopsis length of unsuccessful transgenic to two panels cotyledon, and the arabidopsis of successful transgenic
Plant leaf is green, well developed root system, robust growth (Fig. 1).Treat that resistance Seedling length, to 4 leaf phases, goes in Nutrition Soil, 1, every basin,
Phjytotron cultivation (22 DEG C, 16h illumination, 8h is dark, relative humidity 50-70%).
(5) plant being positive through resistance screening extracts its genomic DNA and cDNA, carries out PCR and RT-further
PCR identifies.
10, transgenic plant biological character is observed
Observe and add up the different biology such as growth promoter transfer-gen plant in period and matched group plant leaf, branch and angle fruit
Learn feature (Fig. 2-Fig. 5).Result shows:
(1) compared with wildtype Arabidopsis thaliana, transgenic plant plant leaf significantly increases；
(2) compared with wildtype Arabidopsis thaliana, transgenic plant plant branch amount showed increased；
(3) compared with wildtype Arabidopsis thaliana, transgenic plant plant angle fruit substantially becomes big, and single-strain seed weight is about open country
2.5 times of raw type.
The larch LkANT gene that the present invention provides can be in the application in cultivating multi-branched transgenic plant.
The larch LkANT gene that the present invention provides can cultivated with seed for results object, it is thus achieved that high yield turns base
Because of the application in new variety of plant.
SEQID NO:1 larch LKANTcDNA sequence
5′-ATGTATTTTGATCACAATGTAGGCGAAGATAATAACGGTCCCTGCAAAATGATCAACGTAAATCAG
ATGCCATATTCAGAGTGCAGGCGCATGGGCGTTCCATCTTCATTTCATACATACGGGGACAATGGAAACAATACATA
TGAGGCGGCAATGCACGAACAAGGGCTTAGCCAGCATAATATGTTCGCAGATTGCAGTTTACAGTTCAATCCTCCCG
GGCCAGGAATTATAGAGAACCCATCGAGCTGCATGGTGGGGATATCAGCAATGAAAACATTGCTAAGGCAATACCCC
AACGGTGGTTCTTCTGACAAGAATTCAACCAATGAGTCTCATGAGACCCTTAATAATATTGGGGATTTGCAGTCTCA
GGCTCTGACTCTAACAATGAGTCCGGGATCCCAGTCCAGTTCGGTTACCATAGTTCCCCATTCGGGCACAAATACAG
AATGTGTTGCAGTGGAGACGAGCAGAAAGAGAGCTGCTGGATCAAAATCTGGTAACACCAGACAGCCTGTTCCTCGG
AAATCCATTGATACATTTGGTCAACGGACATCCCAGTACCGTGGTGTCACAAGGCATCGGTGGACAGGGCGCTACGA
GGCGCACCTATGGGATAACAGTTGCAAGAAAGAAGGTCAGACTAGGAAAGGGAGACAAGTGTATTTGGGAGGTTATG
ATAAAGAAGAAAAGGCTGCTAAATCTTATGACTTGGCCGCACTGAAGTACTGGGGGCCTTCAACGCATATTAACTTC
CCGTTGAACACTTATGAGAAAGAACTTGAAGAGATGAAGCACATGACCAGGCAGGAGTACGTTGCCAATTTGAGGAG
GAAGAGTAGTGGATTTTCCAGAGGGGCATCTATGTACCGAGGAGTAACAAGGCACCACCAGCATGGCAGATGGCAGG
CGCGCATTGGCAGAGTTGCAGGGAACAAGGACCTCTACCTTGGAACTTTCAGTACTCAGGAAGAGGCCGCAGAAGCC
TACGACATCGCTGCCATCAAGTTCAGGGGAACGAATGCTGTGACAAATTTTGATATCAGCAGATATGACGTGAAACG
CATCCTGGCAAGCAATACCTTGCTAGTTGGTGAGTTGGCCAGACTAAACAGGGATCAGCTGGCGGAGCCATTAACGG
CTGAGCCCGCTCAGGCCGACGTCCCCATCGTGATTCATCAAATTACCAACGAAAAGTACAACGCTAAGGAAATTGAA
TACGAAGATGAGGATACTAAGAACAACTGGCAGATGCAGATGAGCTGCGATGAGGAGCAAAACCAGATTAATTCTTG
TTCCAATGAGCCTTCCGATCATGACAAAATGTGGGATGATCAGAAGCAAGCCAGTCCTTTACAGAATCTCAACATCT
GGCAGCTCTTCATAACCTAA-3′
SEQ ID NO:2 larch LKANT protein sequence
MYFDHNVGEDNNGPCKMINVNQMPYSECRRMGVPSSFHTYGDNGNNTYEAAMHEQGLSQHNMFADCSLQ
FNPPGPGIIENPSSCMVGISAMKTLLRQYPNGGSSDKNSTNESHETLNNIGDLQSQALTLTMSPGSQSSSVTIVPHS
GTNTECVAVETSRKRAAGSKSGNTRQPVPRKSIDTFGQRTSQYRGVTRHRWTGRYEAHLWDNSCKKEGQTRKGRQVY
LGGYDKEEKAAKSYDLAALKYWGPSTHINFPLNTYEKELEEMKHMTRQEYVANLRRKSSGFSRGASMYRGVTRHHQH
GRWQARIGRVAGNKDLYLGTFSTQEEAAEAYDIAAIKFRGTNAVTNFDISRYDVKRILASNTLLVGELARLNRDQLA
EPLTAEPAQADVPIVIHQITNEKYNAKEIEYEDEDTKNNWQMQMSCDEEQNQINSCSNEPSDHDKMWDDQKQASPLQ
NLNIWQLFIT
Claims (4)
1. larch LkANT gene, it is characterised in that: the cDNA sequence of gene is as shown in SEQ ID NO:1.
2. the protein of larch LkANT coded by said gene, it is characterised in that: the aminoacid sequence of protein such as SEQ ID NO:
Shown in 2.
3. larch LkANT gene application in cultivating multi-branched transgenic plant.
4. larch LkANT gene is being cultivated with seed for results object, it is thus achieved that answering in high yield transgenic plant new varieties
With.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110951746A (en) * | 2019-10-30 | 2020-04-03 | 山东大学 | Application of MtFRUITFULLc gene in regulation and control of leaf yield and protein content of leguminous plants |
| CN111732648A (en) * | 2020-07-21 | 2020-10-02 | 蒙树生态建设集团有限公司 | Xingan larch LgCCHC-20045 and coding gene and application thereof |
| CN112322635A (en) * | 2020-11-17 | 2021-02-05 | 南开大学 | Coding sequence of larch growth and development regulating gene and its application |
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| CN1860231A (en) * | 2003-06-06 | 2006-11-08 | 阿博根有限公司 | Transcription factors |
| CN104450770A (en) * | 2013-09-25 | 2015-03-25 | 中国林业科学研究院林业研究所 | Application of larch miR166a in plant development regulation and control |
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| CN1860231A (en) * | 2003-06-06 | 2006-11-08 | 阿博根有限公司 | Transcription factors |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110951746A (en) * | 2019-10-30 | 2020-04-03 | 山东大学 | Application of MtFRUITFULLc gene in regulation and control of leaf yield and protein content of leguminous plants |
| CN111732648A (en) * | 2020-07-21 | 2020-10-02 | 蒙树生态建设集团有限公司 | Xingan larch LgCCHC-20045 and coding gene and application thereof |
| CN111732648B (en) * | 2020-07-21 | 2021-08-24 | 蒙树生态建设集团有限公司 | Xingan larch LgCCHC-20045 and coding gene and application thereof |
| CN112322635A (en) * | 2020-11-17 | 2021-02-05 | 南开大学 | Coding sequence of larch growth and development regulating gene and its application |
| CN112322635B (en) * | 2020-11-17 | 2022-10-28 | 南开大学 | Coding sequence of larch growth and development regulation gene and application thereof |
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