CN106047887B - Larch LkANT gene, albumen and application - Google Patents
Larch LkANT gene, albumen and application Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
Abstract
The invention discloses a kind of larch LkANT gene, albumen and applications, and the cDNA sequence of gene is as shown in SEQ ID NO:1.The protein of larch LkANT coded by said gene, it is characterised in that: the amino acid sequence of protein is as shown in SEQ ID NO:2 and it is cultivating the application in multi-branched genetically modified plants.The present invention clones the key gene of regulation larch branch and seed development, is separated and functional analysis by modern molecular biology technique.The acquisition of such key gene not only has important theoretical value, but also provides important candidate gene to improve grain yield using genetic engineering means, with important application prospects in fields such as crop genetic improvements.
Description
Technical field
The invention belongs to genetic engineering field, especially a kind of larch LkANT gene, albumen and application.
Background technique
The branch development of higher plant is to determine an important factor for phytomorph is built up, and is had with phytomass and yield
Close relationship.The plant type of aboveground vegetation part is raw derived from the shoot apical meristem in embryonic development period and the side of postembryonal development
Separate living tissue.Lateral meristem can break up generation axillary bud, and axillary bud further growth develops to form side shoot.Side shoot is the same as stem phase
Together, it may have form the ability of blade, flower, axil separate living tissue and secondary branch, but species difference branch situation also not phase
Together.Plant branching is developed mainly by the multiple regulation of inherent cause, plant hormone and environmental signal.In recent years, by a variety of
The further investigation of plant obtains a series of important gene of regulation branch developments.LAS is earliest influence axillary meristem shape
At one of gene, the mutation of LAS gene causes number of branch purpose to reduce in arabidopsis.Tiller is the gramineae plants such as rice
An important branch phenomenon and an important economical character, are directly related to the spike number of plant in growth and development process
And further influence yield.For Li Jiayang etc. by map clone technology, separation is obtained plays important work in rice tiller regulating
Gene M OC1, functional analysis find that the gene can effectively facilitate tiller and the growth of axillary bud.And TB1 gene is then branch
The negative regulatory factor of control, the overexpression of the gene can reduce the formation of side shoot.With the continuous deepening of research, Ren Menfa
The related gene of more and more regulation branch developments is showed, the people that are found to be of these genes are determined using reverse genetics means
To transformation plant forms, cultivating, there is ideotype to have established important foundation to obtain high yield new variety of plant.
Seed is important grain source for the survival of mankind, and the size and number of seed is to influence crop yield
Principal element.Consequently found that the important factor in order of control seed amount and size, and relevant functional gene is obtained, to crop
SOYBEAN IN HIGH-YIELD BREEDING has important theory significance and application value.Correlative study finds the increase of the branch amount and seed production of plant
There is a direct relation, the yield of awnless brome and Perennial Ryegrass Seed is positively correlated with unit area branch amount.GASA gene
Family is the special gene family regulated and controled by gibberellin.GASA4 gene overexpression can significantly increase transgenosis
The seed size of plant.However, ironically the seed of gasa4 mutant can become smaller, but mutant plants single-strain seed
Total output it is but higher than wild type, this is mainly due to gasa4 mutant plants generate compared with multiple-limb the reason of.Thus may be used
See, the branch of plant and the yield of plant seed have close relationship.ANT transcription factor belongs to a member of AP2 subfamily, main
It participates in adjusting the cell division mutually coordinated with organ growth.The afunction mutant of ANT gene is due to cell in arabidopsis
Number reduces and floral organ is made to become smaller, however, the overexpression of the gene increases the size of rachis and floral organ.
Herbaceous plant is concentrated mainly on for the clone of the development of regulation plant branching and seed development related gene at present, and
The clone of the genoid and also very rare to its application in xylophyta, particularly gymnosperm.Larch is needle
The main commerical tree species of the Typical Representative of tree species and China have highly important economic value and the ecological value.Meanwhile
Larch has indeterminate growth ability as xylophyta again, wherein containing genetic resources abundant.
Summary of the invention
The purpose of the present invention is to provide a kind of larch LkANT gene and application, the present invention obtains the gene for the first time, and
Behavior study has been carried out to the gene, has obtained good effect.
The present invention realizes purpose, and specific step is as follows:
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 amino acid sequence of protein is as shown in SEQ ID NO:2.
Larch LkANT gene is cultivating the application in multi-branched genetically modified plants.
Larch LkANT gene is harvest object cultivating with seed, is obtained in high yield genetically modified plants new varieties
Using.
The advantages and positive effects of the present invention are as follows:
The present invention carries out the key gene of regulation larch branch and seed development by modern molecular biology technique
Clone, separation and functional analysis, the acquisition of the key gene provided not only have important theoretical value, but also to utilize gene
Engineering means improve grain yield and provide important candidate gene, before having important application in fields such as crop genetic improvements
Scape.
The present invention observes by specific experiment and counts different growth and development period transgenic plants and control group Plant Leaf
The biological properties such as piece, branch and silique.As the result is shown: (1) with wildtype Arabidopsis thaliana compared with, genetically modified plants plant leaf
It significantly increases；(2) compared with wildtype Arabidopsis thaliana, genetically modified plants plant branch amount be increased significantly；(3) quasi- southern with wild type
Mustard compares, and genetically modified plants plant silique obviously becomes larger, and single-strain seed weight is about 2.5 times of wild type.
The present invention is by modern molecular biology technique, to the key gene of regulation larch branch and seed development
LkANT cloned, separate and functional analysis, the acquisition of LkANT gene not only have important theoretical value, but also to utilize
Genetic engineering means improve grain yield and provide important candidate gene, have important application in fields such as crop genetic improvements
Prospect.
Detailed description of the invention
Fig. 1 transgenic plant T1 is for resistance screening (Fig. 1 a) and T3 for resistance screening (Fig. 1 b).
Fig. 2 WT lines (left side) growth and development period phenotypic characteristic different from transgenic plant (right side).Fig. 2 a: wild type
Plant (left side) and transgenic plant (right side) 2 week old growing state；Fig. 2 b: WT lines (left side) and transgenic plant (right side) 3 weeks
Age growing state；Fig. 2 c: WT lines (left side) and transgenic plant (right side) 5 week old growing state；Fig. 2 d: WT lines
(left side) and transgenic plant (right side) 8 week old growing state.
Fig. 3 WT lines and transgenic plant stem leaf branch situation analysis chart.
Fig. 4 WT lines and transgenic plant Pod length analysis chart.
Fig. 5 WT lines and transgenic 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 it is not limit that following embodiment, which is descriptive,
Qualitatively, this does not limit the scope of protection of the present invention.
In the present invention, sequencing analysis is carried out to larch transcript profile using Illumina/Solexa high throughput sequencing technologies.
The unigene of a 1465bp is obtained by going deep into data mining and sequence assembly, and height is presented in the unigene in axillary bud
Expression pattern.It is therefore designed according to unigene sequence information obtained special for the functional character for analysing in depth the unigene
Different primer expands the unigene sequence using RT-PCR technology in larch mRNA.Using ORF Finder to this
The open reading frame (ORF) of unigene carries out analysis and finds that the unigene includes the ORF of a 1395bp, encodes 464 ammonia
The protein of base acid, is named as LkANT.
BLAST compares analysis and finds the species such as protein sequence and black pine, tobacco, tomato and the grape of gene order coding
The homology of ANT-Like transcription factor is only 60%-80%, this show gymnosperm it is larchen in LkANT gene long-term
Evolution in it is comparatively conservative, remain itself unique sequence structure feature.The structure of gene determines its function, therefore deep
Enter to probe into the functional character of the gene for excavating, cloning larch Fineness gene by genetic engineering means for crop genetic
Improvement has preferable application potential.
A kind of clone of larch LkANT gene and cultivate multi-branched and the method for high yield genetically modified plants is as follows:
1, larch Total RNAs extraction and the first chain cDNA synthesis
Using larch current year raw young leaflet tablet as test material, total serum IgE is extracted using CTAB method, and reverse using M-MLV
It records kit reverse transcription and obtains the first chain cDNA.
2, the separation of LKANT gene and clone
(1) 5.0 software design of primer premier 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 digestion 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 segments.
3, the recycling of target gene and cloning and sequencing
(1) connection reaction
It is gently mixed, (20 DEG C -37 DEG C) reaction 5-10min of room temperature；
After reaction, centrifuge tube is placed on ice.
(2) it converts
Add connection product in 50 μ L Trans1-T1 competent cells, flicks mixing, ice bath 30min；
42 DEG C of heat shock 30s, are immediately placed on 2min on ice；
250 μ L are added to balance to the LB of room temperature, 37 DEG C of incubation 1h of 200rpm；
8 μ L 100mmol IPTG are taken, 40 μ L 25mg/mL X-gal, 25 μ L 100mg/mL Amp mixing equably apply
In on ready plate, 37 DEG C of placement 30min；
After IPTG and X-gal are absorbed, 150 μ L bacterium solution coated plates, overnight incubation are taken.
(3) identification of positive recombinant
Random picking white colony is in the LB liquid medium containing 1mg/LAmp；
37 DEG C of 200rpm incubation about 8h；
It takes 1 μ L bacterium solution to be used as the template of PCR reaction, identifies 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 detects clip size (Fig. 6).
(4) fungi preservation and sequencing
Isometric 40% glycerol LB culture medium is added in positive colony recon bacterium solution, mixes, -20 DEG C of preservations.Positive colony
Recon bacterium solution is sent to the raw work sequencing portion in Shanghai and is sequenced, and sequencing result is sequence 1.
4, plasmid enzyme restriction
(1) cloning vector plasmids containing target gene and pRI201-AN-GUS vector plasmid use KpnI and NotI bis- simultaneously
Digestion；
(2) digestion system is as follows
(3) it mixes gently；
(4)37℃3h；
(5) agarose gel electrophoresis detects digestion result；
(6) target fragment is recycled.
5, the connection of endonuclease bamhi
(1) the pRI201-AN-GUS carrier with restriction enzyme site target gene fragment and with corresponding restriction enzyme site recycled connects
It connects；
(2) linked system
(3) it mixes gently；
(4) 16 DEG C of water-baths keep the temperature 12h.
6, recombinant plasmid transformed Agrobacterium
(1) it takes 1 μ g recombinant plasmid to be added in 100 μ L Agrobacterium competence, mixes 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) 600 μ L of YEB is added；
(6) 28 DEG C of 200rpm recovery 4h；
(7) 150 μ L recovery bacterium solutions is taken to be applied to YEB plate (anti-containing three)；
2-3d is cultivated in (8) 28 DEG C of inversions.
(9) identified positive strain is saved backup in -20 DEG C.
7, the plantation of arabidopsis
(1) arabidopsis seed is placed in 1.5mL centrifuge tube, and 70% ethyl alcohol of 1mL, oscillation washing 30s is added；
(2) 6,000rpm 1min collect seed, abandon supernatant；
(3) 1mL sterile water is added, oscillation washing 1min collects seed, is repeated 2 times；
(4) 2% sodium hypochlorite and 0.05%tween-20 oscillation disinfection 15min is added；
(5) 6,000rpm 1min collect seed, abandon supernatant；
(6) 1mL sterile water is added, oscillation 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 the completion of vernalization, 22 DEG C of illumination boxs, 16h illumination, 8h dark culturing are placed in；
(10) 4 leaf phase arabidopsis are gone in Nutrition Soil, 1, every basin, and phjytotron culture (22 DEG C, 16h illumination, 8h
Dark, relative humidity 50-70%).
8, Agrobacterium-mediated Transformation arabidopsis
(1) there is the Agrobacterium inoculation of purpose recombinant plasmid in 10mLYEB culture medium -20 DEG C of containing for preservation；
(2) 28 DEG C of 200rpm activation are overnight；
(3) 1:100 dilutes thallus in 250mLYEB culture medium；
(4) 28 DEG C of 200rpm are cultivated to OD600=1.2~1.6；
(5) room temperature 5,000rpm is centrifuged 15min and collects thallus；
(6) thallus is resuspended in conversion buffer (5% sucrose) in equal volume；
(7) arabidopsis is inverted in the conversion buffer that thallus is resuspended, disseminates 10s；
(7) plant is taken out, is wrapped plant with preservative film, dark culture is put for 24 hours in side；
(8) overcover, erect plants culture are gone；
(9) culture to arabidopsis is yielded positive results under normal condition, collects seed.
9, transgenic plant Kan resistance screening
(1) (Kan containing 50mg/L) is seeded in MS solid medium after transgenic arabidopsis seed disinfection；
Vernalization 3-4d under (2) 4 DEG C of dark conditions；
(3) 22 DEG C of illumination boxs, 16h illumination, 8h dark culturing are placed in；
(4) arabidopsis of failed transgenosis is long starts albefaction death to two panels cotyledon, and the arabidopsis of successful transgenic
Plant leaf is green, well developed root system, robust growth (Fig. 1).To resistance seedling length to 4 leaf phases, go in Nutrition Soil, 1, every basin,
Phjytotron culture (22 DEG C, 16h illumination, 8h is dark, relative humidity 50-70%).
It (5) is that positive plant extracts its genomic DNA and cDNA, further progress PCR and RT- by resistance screening
PCR identification.
10, genetically modified plants biological character is observed
It observes and counts different growth and development period transgenic plants and control group plant leaf, branch and silique etc. and is biological
It learns feature (Fig. 2-Fig. 5).As the result is shown:
(1) compared with wildtype Arabidopsis thaliana, genetically modified plants plant leaf is significantly increased；
(2) compared with wildtype Arabidopsis thaliana, genetically modified plants plant branch amount be increased significantly；
(3) compared with wildtype Arabidopsis thaliana, genetically modified plants plant silique obviously becomes larger, and single-strain seed weight is about open country
2.5 times of raw type.
Larch LkANT gene provided by the invention can cultivate the application in multi-branched genetically modified plants.
Larch LkANT gene provided by the invention can be harvest object cultivating with seed, obtain high yield and turn base
Because of the application in new variety of plant.
SEQID NO:1 larch LKANTcDNA sequence
5′-ATGTATTTTGATCACAATGTAGGCGAAGATAATAACGGTCCCTGCAAAATGATCAACGTAAATCA
GATGCCATATTCAGAGTGCAGGCGCATGGGCGTTCCATCTTCATTTCATACATACGGGGACAATGGAAACAATACA
TATGAGGCGGCAATGCACGAACAAGGGCTTAGCCAGCATAATATGTTCGCAGATTGCAGTTTACAGTTCAATCCTC
CCGGGCCAGGAATTATAGAGAACCCATCGAGCTGCATGGTGGGGATATCAGCAATGAAAACATTGCTAAGGCAATA
CCCCAACGGTGGTTCTTCTGACAAGAATTCAACCAATGAGTCTCATGAGACCCTTAATAATATTGGGGATTTGCAG
TCTCAGGCTCTGACTCTAACAATGAGTCCGGGATCCCAGTCCAGTTCGGTTACCATAGTTCCCCATTCGGGCACAA
ATACAGAATGTGTTGCAGTGGAGACGAGCAGAAAGAGAGCTGCTGGATCAAAATCTGGTAACACCAGACAGCCTGT
TCCTCGGAAATCCATTGATACATTTGGTCAACGGACATCCCAGTACCGTGGTGTCACAAGGCATCGGTGGACAGGG
CGCTACGAGGCGCACCTATGGGATAACAGTTGCAAGAAAGAAGGTCAGACTAGGAAAGGGAGACAAGTGTATTTGG
GAGGTTATGATAAAGAAGAAAAGGCTGCTAAATCTTATGACTTGGCCGCACTGAAGTACTGGGGGCCTTCAACGCA
TATTAACTTCCCGTTGAACACTTATGAGAAAGAACTTGAAGAGATGAAGCACATGACCAGGCAGGAGTACGTTGCC
AATTTGAGGAGGAAGAGTAGTGGATTTTCCAGAGGGGCATCTATGTACCGAGGAGTAACAAGGCACCACCAGCATG
GCAGATGGCAGGCGCGCATTGGCAGAGTTGCAGGGAACAAGGACCTCTACCTTGGAACTTTCAGTACTCAGGAAGA
GGCCGCAGAAGCCTACGACATCGCTGCCATCAAGTTCAGGGGAACGAATGCTGTGACAAATTTTGATATCAGCAGA
TATGACGTGAAACGCATCCTGGCAAGCAATACCTTGCTAGTTGGTGAGTTGGCCAGACTAAACAGGGATCAGCTGG
CGGAGCCATTAACGGCTGAGCCCGCTCAGGCCGACGTCCCCATCGTGATTCATCAAATTACCAACGAAAAGTACAA
CGCTAAGGAAATTGAATACGAAGATGAGGATACTAAGAACAACTGGCAGATGCAGATGAGCTGCGATGAGGAGCAA
AACCAGATTAATTCTTGTTCCAATGAGCCTTCCGATCATGACAAAATGTGGGATGATCAGAAGCAAGCCAGTCCTT
TACAGAATCTCAACATCTGGCAGCTCTTCATAACCTAA-3′
SEQ ID NO:2 larch LKANT protein sequence
MYFDHNVGEDNNGPCKMINVNQMPYSECRRMGVPSSFHTYGDNGNNTYEAAMHEQGLSQHNMFADCSLQ
FNPPGPGIIENPSSCMVGISAMKTLLRQYPNGGSSDKNSTNESHETLNNIGDLQSQALTLTMSPGSQSSSVTIVPHS
GTNTECVAVETSRKRAAGSKSGNTRQPVPRKSIDTFGQRTSQYRGVTRHRWTGRYEAHLWDNSCKKEGQTRKGRQVY
LGGYDKEEKAAKSYDLAALKYWGPSTHINFPLNTYEKELEEMKHMTRQEYVANLRRKSSGFSRGASMYRGVTRHHQH
GRWQARIGRVAGNKDLYLGTFSTQEEAAEAYDIAAIKFRGTNAVTNFDISRYDVKRILASNTLLVGELARLNRDQLA
EPLTAEPAQADVPIVIHQITNEKYNAKEIEYEDEDTKNNWQMQMSCDEEQNQINSCSNEPSDHDKMWDDQKQASPLQ
NLNIWQLFIT
Claims (3)
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 amino acid sequence of protein such as SEQ ID NO:
Shown in 2.
3. larch LkANT gene described in claim 1 is harvest object cultivating with seed, obtains high yield transgenosis and plant
Application in object new varieties.
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| 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 |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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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| Title |
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| 落叶松APETALA2-Like转录因子基因的分离及功能分析;李爱等;《植物遗传资源学报》;20140630;第15卷(第6期);1305-1311 |
| 落叶松生长素受体基因LkTIR1的克隆及功能分析;李爱等;《植物生理学报》;20160120;第52卷(第01期);92-100 |
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