CN106399324A - Apple auxin delivery vector gene MdPIN1 for regulating root growth, and application thereof - Google Patents
Apple auxin delivery vector gene MdPIN1 for regulating root growth, and application thereof Download PDFInfo
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Abstract
The invention relates to an apple auxin delivery vector gene MdPIN1 for regulating root growth, and an application thereof in regulation of the root growth and affection of auxin delivery. Ectopic expression of the MdPIN1 gene in Arabidopsis thaliana is carried out by using a 35S strong promoter driving principle transgenic technology to obtain transgenic Arabidopsis thaliana. Over-expression of the MdPIN1 in Arabidopsis thaliana affects plant root auxin delivery, and has no substantial influences on the synthesis of auxin. Fur phenotype observation finds that over-expression of the MdPIN1 in Arabidopsis thaliana substantially inhibits main root elongation and promotes the quantity increase of lateral roots. The above results show that the apple MdPIN1 gene plays a great role in affection of auxin delivery and regulation of plant roots.
Description
Technical field
The present invention relates to molecular biology and biological technical field are and in particular to a kind of apple regulates and controls Auxin transport and root
It is clone and its application of development gene MdPIN1.
Background technology
Auxin is a kind of important plant hormone, plays a significant role, including plant in plant growth and development process
Organ generation (Benkov á et al., 2003；Bohn-Courseau, 2010), gravimetric response and and apical dominance (Kepinski
And Leyser, 2005) and tissue differentiation (Reinhardt, 2003).Iuntercellular individual event polar translocation is that auxin is distinctive
Means of transportation, that is, auxin can only transport to lower end from the morphologic upper end of plant, and (Liu et unrelated with Action of Gravity Field
al.,1993；Lupini et al.,2014).Polar Transport of Auxin is present in the stem of plant, in root and leaf, affects auxin
Mal-distribution and plant forms (Friml, 2003).The polar translocation of auxin is carried by the Auxin transport being positioned on plasma membrane
Body be responsible for (Kepinski and Leyser, 2005；Mravec et al.,2009).
Auxin Transport Carriers, including auxin input carrier (AUX1) and auxin output carrier (PINs), they lead to
Overregulate the transport of auxin and distribution influence growth and development of plants various aspects (ek and Friml,2009；
Haga and Sakai,2012).In arabidopsis, there are eight PIN genes (PIN1-PIN8), PIN1 is identified first and grows
Element transport is related (Kramer et al., 2004).622 amino acid residues of AtPIN1 gene code, comprise 8-12 for life
The long element vital trans-membrane region of transport activity.Previous studies have shown that AtPIN1 participate in lateral root orga- nogenesis (Et al., 2014), Phototropism (Blakeslee et al., 2004), form generation and Auxin transport
(Heisler et al.,2010).The function of AtPIN1 can by auxin transport inhibitor affect (Geldner et al.,
2001), further illustrate AtPIN1 to play a significant role during Polar Transport of Auxin.Recently, paddy rice PIN1 gene,
OsPIN1 is identified to participate in adventitious root generation and the tiller (Xu et al., 2005) that auxin relies on.
Apple is fruit crop important in the world, carries out research to fruit tree growth element polar translocation and contributes to realizing plant
Morphosis and root system development are adjusted, and finally improve output of the fruit tree.In our current research, we are identified by way of homologous clone
MdPIN1 gene, result shows, in arabidopsis, ectopic expression MdPIN1 promotes plant root and develops and Auxin transport.
Content of the invention
It is an object of the invention to provide one kind is identified and a detached new regulation and control root growth element transport from apple
And the auxin output vector gene MdPIN1 of root system development.Experiment finds MdPIN1 impact root growth element transport,
MdPIN1 overexpression arabidopsis shows main root length and is suppressed, the phenotype that lateral root number increases；This gene is pointed out to plant
Play a significant role in thing Auxin transport and root system development.
First, the invention discloses a kind of detached apple development element transport agent gene M dPIN1, its nucleotide sequence
As SEQ ID NO：Shown in 1.
Further, described auxin Transport Carriers gene M dPIN1 is derived from loud, high-pitched sound apple.
Second aspect present invention discloses the albumen of aforementioned apple development element transport agent gene M dPIN1 coding, its amino
Acid sequence such as SEQ ID NO：Shown in 2.
The present invention obtains apple development element transport related gene MdPIN1, its nucleotide sequence using Homology-based cloning
As SEQ ID NO：Shown in 1, protein amino acid sequence such as SEQ ID NO:Shown in 2.
MdPIN1 gene order is as follows：
ATGATTACATTATCCGACTTCTACCACGTCATGACGGCGGTGGTGCCGCTCTACGTGGCCATGATCTTGGCCTACGG
CTCCGTGAAGTGGTGGAAGATCTTCACCCCCGACCAGTGCTCCGGCATCAACCGCTTCGTCGCCCTCTTTGCCGTCC
CCCTCCTCTCCTTCCACTTCATCTCCACCAACGACCCTTACAACATGAACACCCGCTTCATTGCCGCCGACACCCTC
CAGAAGCTGATCGTCCTCGCCGTCCTCGGCGTCTGGACCAAAGTCAGCAAAAGGGGCTGCCTGGAATGGACGATCAC
TCTCTTCTCCGTCTCCACTCTGCCCAACACTCTGGTCATGGGAATCCCATTGCTCAAGGGAATGTACGGCGATTTTT
CCGGGAGTTTGATGGTGCAGATCGTCGTCCTCCAGTGCATTATCTGGTACACTTTGATGCTTTTCATGTTCGAATAC
CGAGGAGCAAGACTCCTCATCTCGGAGCAGTTTCCCGACACTGCGGGATCCATTGTCTCCATCCACGTCGACTCCGA
TATTATGTCGCTCGACGGAAGACAGCCCCTCGAGACTGAAGCGGAGATCAAGGAGGACGGCAAACTCCACGTCACTG
TCAGAAAATCAAACGCTTCGCGCTCGGATATTTTATCGCGGCGATCTCAGGGGCTGTCGTCCACCACCCCGCGGCCG
TCGAATCTCACTAATGCGGAGATTTACTCCCTGCAGTCTTCGAGAAATCCGACGCCGAGAGGGTCAAGTTTCAACCA
CACGGACTTCTACTCCATGATGGCTGCCGGAAGGAACTCGAATTTCGGAGCTAACGATGTTTATGGGATGTCTGCGT
CCAGAGGGCCGACTCCACGGCCATCAAATTTCGAGGAAGACGGTGGCGGCGGCGCTGTCAGCTCCGCTACTGGAAAT
AAGCCACGGTTTTACCACGGCGGACAGAATAATGCAGTGGCGCATTACCCGGCCCCAAACCCAGGGATGTTTTCTCC
GACGGCGTCCAGAACCGTCACCGCTAATGCTAATAGCAATGCCATGAATGCAAAGAGAGCTAATGGGCAAGCTCAGA
AAACAGAGGACACCAATGGCGGGAAGGATCTTCATATGTTTGTTTGGAGCTCAAGTGCTTCTCCTGTTTCAGATGTG
TTTGGAAGCAATGAATACGGTGGTGCTGCCCATGATCACAAAGAAGTAAAATTGGCTGTGTCTCCAGGAAAAGTGGA
GGGGAGGAGAGAGAATCAGGAAGAGTATTTGGAGAGAGAAGATTTCAGATTTGGAAACAGAGATCAGATGAACATGA
ACAATGAGGCTGAGAAAGGAGGGGATGGGATTGGAAAAGCCAAAGTGATGCCTCCAACAAGTGTGATGACAAGGCTA
ATTCTCATCATGGTTTGGAGAAAACTCATTAGAAACCCAAACACTTACTCCAGCTTGATCGGCCTCACTTGGTCTCT
AGTCTCATTCAGGTGGCACGTTCAAATGCCAGCCATTGTAGCGAAGTCCATCGCCATACTATCTGATGCAGGACTTG
GCATGGCCATGTTCAGCCTCGGTTTGTTTATGGCTTTGCAGCCAAAGATCATAGCTTGTGGAAACTCCGTTGCAGCT
TTTGCCATGGCTGTGAGATTCCTTACAGGTCCAGCTGTCATGGCAGCTGCTTCCATTGCTGTTGGCTTAAGAGGCAC
TCTCTTACATGTTGCCATTGTACAGGCAGCCCTACCCCAAGGAATTGTTCCCTTTGTCTTTGCCAAGGAATACAATG
TACACCCTGATATTCTCAGCACGGGGGTTATATTTGGAATGTTGATTGCGTTGCCCATAACGCTTGTTTACTACATT
TTGTTGGGGCTATGA(SEQ ID NO:1)
The amino acid sequence of MdPIN1 gene code is as follows：
MITLSDFYHVMTAVVPLYVAMILAYGSVKWWKIFTPDQCSGINRFVALFAVPLLSFHFISTNDPYNMNTRFIAADTL
QKLIVLAVLGVWTKVSKRGCLEWTITLFSVSTLPNTLVMGIPLLKGMYGDFSGSLMVQIVVLQCIIWYTLMLFMFEY
RGARLLISEQFPDTAGSIVSIHVDSDIMSLDGRQPLETEAEIKEDGKLHVTVRKSNASRSDILSRRSQGLSSTTPRP
SNLTNAEIYSLQSSRNPTPRGSSFNHTDFYSMMAAGRNSNFGANDVYGMSASRGPTPRPSNFEEDGGGGAVSSATGN
KPRFYHGGQNNAVAHYPAPNPGMFSPTASRTVTANANSNAMNAKRANGQAQKTEDTNGGKDLHMFVWSSSASPVSDV
FGSNEYGGAAHDHKEVKLAVSPGKVEGRRENQEEYLEREDFRFGNRDQMNMNNEAEKGGDGIGKAKVMPPTSVMTRL
ILIMVWRKLIRNPNTYSSLIGLTWSLVSFRWHVQMPAIVAKSIAILSDAGLGMAMFSLGLFMALQPKIIACGNSVAA
FAMAVRFLTGPAVMAAASIAVGLRGTLLHVAIVQAALPQGIVPFVFAKEYNVHPDILSTGVIFGMLIALPITLVYYI
LLGL(SEQ ID NO:2)
Extract total serum IgE from loud, high-pitched sound Tissue-cultured apple seedling, reverse transcription obtains cDNA.According to plan announced in international gene pool
The conserved amino acid sequence of PIN1 in southern mustard, designs degenerate primer, carries out Conventional polymerase chain formula reaction (Polymerase
chain reaction,PCR).The PCR primer of suitable size is connected with pMD18-T carrier, conversion bacillus coli DH 5 alpha impression
State cell, screens recon, and carries out sequencing analysis confirmation, finally obtain cDNA full length sequence.
This gene open reading frame (open reading frame, ORF) is 1863bp.Thus push away, this gene code
620 amino acid, gene order number is MDP0000138035, positioned at No. 6 chromosome, includes including 6 extrons and 5
Son.Its amino acid sequence is retrieved in international gene pool, finds that the arabidopsis related gene AtPIN1 with announcement has higher
Homology, so we by this unnamed gene be MdPIN1.
Third aspect present invention discloses aforementioned apple development element transport agent gene M dPIN1 and is adjusting plant strain growth element
Purposes in transport and/or root system development.
Further, purposes in root system development for the described apple MdPIN1 gene, for improving main root length and suppressing side
Radical mesh.
From experimental result, the element transport of apple MdPIN1 effect gene root growth, but root growth element synthesis is not had
Have a significant impact.
Fourth aspect present invention discloses a kind of nucleic acid construct, comprises the aforementioned apple life being under strong promoter controls
Long element transport agent gene M dPIN1.
Further, described strong promoter is cauliflower mosaic virus 35 S promoter.
Using the transgenic technology of strong promoter (cauliflower mosaic virus 35 S promoter) driving principle, by MdPIN1 base
The overexpression carrier of cause proceeds in arabidopsis, thus obtaining transfer-gen plant.MdPIN1 impact root growth element transport,
MdPIN1 overexpression arabidopsis shows main root length and is suppressed, and the phenotype that lateral root number increases illustrates MdPIN1 gene
Plant strain growth element transport and root system development play a significant role.
In sum, the beneficial effects of the present invention is, inventor isolates a new regulation first in apple
Plant strain growth element transport and the auxin Transport Carriers gene of root system development.
Brief description
The structure of Fig. 1 carrier and the acquisition of transgenic arabidopsis
(A:Apple MdPIN1 gene PCR amplified production electrophoresis MdPIN1:MdPIN1 gene amplification product, Mark:Molecular weight
Mark DM2000；B：35S:MdPIN1 structural representation；C：RT-PCR analyzes MdPIN1 gene in transgenic arabidopsis L1, L2,
Expression in L3 strain)；
The impact to plant strain growth element transport for Fig. 2 MdPIN1
(A-B：Root system geotropism is tested, C-D：Plant phototropism is tested)；
Fig. 3 MdPIN1 has no significant effect to plant strain growth element synthesis
(A：Auxin accumulation B is observed in DR5-GUS dyeing：Quantitative PCR detection wildness arabidopsis intends south with transgenosis
Mustard and auxin synthetic gene YUC1, YUC2, YUC4, YUC6, TAA1 expression)；
Fig. 4 MdPIN1 adjusts root system development
(A：The growth wildtype Arabidopsis thaliana (Col-0) of 10 days and transgenic arabidopsis (L1, L2, L3) root system system are observed；
B-C：Compared with wildtype Arabidopsis thaliana, main root length is substantially suppressed transgenic arabidopsis, and lateral root number significantly increases, Bars
=1cm).
Specific embodiment
The present invention have studied apple development element transport related gene MdPIN1 by molecule clone technology and sends out in regulation root system
Educate, the application in impact Auxin transport.Using the transgenic technology of 35S strong promoter driving principle, MdPIN1 gene is existed
Ectopic expression in arabidopsis, obtains transgenic arabidopsis；MdPIN1 overexpression impact plant root auxin in arabidopsis
Transport, but auxin synthesis is had no significant effect.Phenotypic Observation finds further, MdPIN1 overexpression in arabidopsis
Main root elongation can be significantly inhibited, and promote lateral root number to increase.
Below in conjunction with accompanying drawing, the principle of the present invention and feature are described, example is served only for explaining the present invention, and
Non- for limiting the scope of the present invention.
The clone of embodiment 1 loud, high-pitched sound apple MdPIN1 gene
1. loud, high-pitched sound apple tissue culture blade RNA extracts
Extract the total serum IgE of loud, high-pitched sound apple tissue culture blade by CTAB method, comprise the following steps：
1) take 1.5g through the 200mM NaCl salt treatment loud, high-pitched sound Tissue-cultured apple seedling of 24 hours, put into the mortar of precooling, liquid feeding
Nitrogen grinds, and proceeds in the 50ml centrifuge tube of precooling；
2) be rapidly added 10ml be preheating to 65 DEG C Extraction buffer (CTAB 20%w/v, Tris-HCl 0.1mol/l,
EDTA 25mol/l, NaCl 2mol/l, the distilled water constant volume of mercaptoethanol 2%w/v, PVP 2%w/v, no RNase, wherein
PVP and mercaptoethanol are now added with existing), gently mix, 0.5 hour in 65 DEG C of water-baths；
3) add with upper step centrifuge tube liquid isopyknic water-saturated phenol/chloroform/isoamyl alcohol (25:24:1) mixture, ice bath
Vibration 0.5 hour, 4 DEG C, 12,000rpm be centrifuged 20 minutes；Supernatant is transferred in new 50ml centrifuge tube；
4) add the 10mol/L precooling LiCl of 1/3 supernatant volume, place 3 hours for -20 DEG C, 12,000rpm 30 points of centrifugations
Clock, abandons supernatant；
5) 500 μ l SSTE buffer solution (NaCl 1mol/l, SDS 0.5%w/v, EDTA 10mol/l, no RNase are added
Distilled water constant volume) after the precipitation that fully suspends, be averagely dispensed in 2 1.5ml centrifuge tubes；
6) it is separately added into water-saturated phenol/chloroform/isoamyl alcohol (25 isopyknic with suspension:24:1) mixture, ice bath shakes
Swing 10 minutes, 4 DEG C, 12,000rpm is centrifuged 10 minutes；Supernatant is transferred to new 1.5ml centrifuge tube；
7) it is separately added into chloroform/isoamyl alcohol (24 isopyknic with supernatant:1) mixture, ice bath vibration 10 minutes, 4 DEG C,
12,000rpm is centrifuged 10 minutes；Supernatant is transferred to new 1.5ml centrifuge tube；
8) the precooling absolute ethyl alcohol of 2.5 times of supernatant volume, -20 DEG C of placement 1-2 hours are added；
9) in 4 DEG C, 12,000rpm be centrifuged 20 minutes, 70% ethanol washes 2 times；
10) in 4 DEG C, 14,000rpm be centrifuged 10 minutes, on super-clean bench air-dry precipitation；Add 20 μ l DEPC water dissolving RNAs.
11) put -80 DEG C to be in store for, or carry out following reverse transcription experiment immediately.
2. total serum IgE reverse transcription is obtained cDNA
The reverse transcription of total serum IgE uses commercially available reverse transcription reagent box, obtains reverse transcription cDNA.
The amplification of 3.MdPIN1 full length DNA sequence
Design degenerate primer (MdPIN1-F/MdPIN1-R), enters performing PCR amplification with above-mentioned reverse transcription cDNA for template.
MdPIN1-F：5’-CCACTCAATCTCTCTGCCAAT-3’(SEQ ID NO:3)；
MdPIN1-R：5’-GACTGGTGAGAGGGAGAGTT-3’(SEQ ID NO:4).
PCR amplification system：CDNA product 25 μ l, the 5 × TdT buffer solution 10 μ l purifying, 0.1%BSA5 μ l, 10mM dCTP
2.5 μ l, TdT 15U, distilled water is settled to 50 μ l.
PCR amplification program：94 DEG C of denaturations 5 minutes；Loop parameter is 94 DEG C of denaturation 30 seconds, 56 DEG C anneal 30 seconds, 72 DEG C
Extend 90 seconds, carry out 32 circulations；72 DEG C fully extend 10 minutes.
After PCR reaction terminates, reclaim PCR primer and be connected on pMD-18T carrier, obtain pMD18-T-MdPIN1
Plasmid.Sequencing (Beijing Liuhe Huada Genomics Technology Co., Ltd) result shows, MdPIN1 gene nucleotide series such as SEQ
ID NO:Shown in 1；Its amino acid sequence such as SEQID NO:Shown in 2.Be sequenced correct monoclonal, alkalinity extraction pMD18-T-
The DNA of MdPIN1, -20 DEG C of preservations, for follow-up function confirmatory experiment (accompanying drawing 1A)
4. apple MdPIN1 bioinformatic analysis
By GDR database (http://www.rosaceae.org/) the analysis chromosome position of MdPIN1 gene and base
Because organizing structure.Result shows, MdPIN1 is positioned on No. 6 chromosome of apple genome, is included by 6 extrons and 5
Son is constituted.
MdPIN1 contains gene coding region the nucleotides of 1863bp, carries out sequence analysis, apple using DNASTAR related software
620 amino acid of fruit MdPIN1 gene code.By NCBI website (http://www.ncbi.nlm.nih.gov/) data
Storehouse and expasy website (http://www.expasy.org/) analysis software, to the function of MdPIN1 albumen forecasting sequence knot
Structure domain and conservative domain are compared analysis, it is found that：MdPIN1 prediction albumen contains 6 continuous cross-film knots in 9-615aa
Structure domain, this is most important for the film positioning of MdPIN1 albumen.
The structure of embodiment 2 MdPIN1 genophore
For studying the function of MdPIN1 gene further, MdPIN1 gene coding region will be included in interior common 1863bp piece
Section is correctly inserted on expression vector pCAMBIA 1300 (accompanying drawing 1B).
1. utilize DNAMAN software, according to the nucleotide sequence (SEQ.ID.NO of the MdPIN1 gene isolated:1), design
Primer EMdPIN1-F and EMdPIN1-R with restriction enzyme site (SEQ ID NO:18-19), the pMD18-T- being preserved with example 1
The DNA of MdPIN1 is template, enters performing PCR reaction, the MdPIN1 sequence with EcoRI and PstI restriction enzyme site for the amplification.
EMdPIN1-F：5’-CTGCAGCCACTCAATCTCTCTGCCAAT-3’(SEQ ID NO:5), drawing horizontal line part is
Pst I restriction enzyme site；
EMdPIN1-R：5’-GGATCCGACTGGTGAGAGGGAGAGTT-3’(SEQ ID NO:6), drawing horizontal line part is
BamH I restriction enzyme site.
Primer and template, with embodiment 1, are only replaced by amplification system and amplification program.
After 2.PCR reaction terminates, 1.0% agarose gel electrophoresis, PCR primer recovery, carrier connect, convert, are sequenced.Alkali
Method extracts its DNA, and with Pst I and BamH I double digestion, identification is correctly afterwards with experiment later.
3. use Pst I and I two restriction endonucleases of BamH, simultaneously double digestion previous step gained DNA and pCAMBIA 1300
Plasmid, reclaims fragment and pCAMBIA 1300 carrier segments of MdPIN1, is coupled together the two with T4 ligase.Screening is positive
Clone is sequenced, and therefrom selects correct recon pCAMBIA 1300-MdPIN1.
4. the recon pCAMBIA 1300-MdPIN1 conversion Agrobacterium LB4404 competent cell with building.Carry out
PCR identifies, picking positive bacteria drops into row sequencing.After the correct recon pCAMBIA 1300-MdPIN1 monoclonal that is sequenced is used for
The conversion of face arabidopsis.
Embodiment 3 obtains transgenic arabidopsis
The arabidopsis seed that will obtain, respectively with 70% alcohol disinfecting 3min, 4% hypochlorite disinfectant's 8-10min (period
Repeatedly rock), aqua sterilisa rinses 5 times, blots water.It is seeded on seed germination medium (being directly laid on surface), optical culture
(25-28 DEG C, the 16h long-day/8h short-day, 10d), grow to seedling.It is transplanted to matrix and cultivates and bloom.
Picking Agrobacterium monoclonal colony inoculation in 10mL YEP fluid nutrient medium (hygromycin containing 50mg/L), 28 DEG C,
200rpm, shaken cultivation to OD600For 0.6-0.8 (about 48h)；Wherein lmL bacterium solution is taken to add in 20mL YEP fluid nutrient medium,
28 DEG C, 200rpm, shaken cultivation to OD600For 0.6-0.8 (about 5h).Thalline is collected by centrifugation, with infecting liquid (sugarcane containing 0.05g/ml
Sugar, 0.03-0.05%Silweet) suspend 20 times of dilution, standby；
Arabidopsis floral is dipped into and infects 15-20s in liquid, collect Fruit pod, 50mg L-1After hygromycin resistance screening, PCR
Detection obtains positive transgenic plant, obtains T3 for homozygote through continuous 3 generations screening, collects seed, carry out phenotype analytical.
Screen the positive candidate's transgenic line of hygromycin using screening and culturing medium (hygromycin containing 100mg/L), obtain altogether
Obtain 3 35S:MdPIN1 positive strain (L1, L2, L3)；For identifying transgenic line further, when carrying out squamous subculture, often
Strain respectively take 0.1g about plantlet in vitro, extract corresponding RNA, reverse transcription simultaneously carries out semiquantitive PCR detection, to determine these strains
The expression of MdPIN1 in system.Result shows, MdPIN1 gene overexpression (accompanying drawing 1C) in L1, L2 and L3.
The root system phenotype of embodiment 4 transgenic arabidopsis
Export carrier, PIN1 gene regulation Auxin transport as auxin.In order to study MdPIN1 to Auxin transport
Impact, we implement root system geotropism experiment and phototropism experiment.
1. root system geotropism experiment
Wild type (Col-0) and transgenic arabidopsis are taken vertically to cultivate the growth seedling dark place of 6 days and tilt 135 ° of cultures, point
Not in growth 8h, observation of taking pictures when 18h, 28h, and calculate the main root bending number of degrees.It was found that compared with wildtype Arabidopsis thaliana,
35S::The MdPIN1 transgenic arabidopsis bending number of degrees are bigger (accompanying drawing 2A-B), and in hint transgenic arabidopsis, Auxin transport can
Can be affected.
2. phototropism experiment
Take wild type (Col-0) and the transgenic arabidopsis grown in darkness seedling of 5 days to cultivate under unilateral illumination, observe system
Meter plant hypocotyl angle of bend.Result shows, wildtype Arabidopsis thaliana is compared, 35S::MdPIN1 transgenic arabidopsis hypocotyl
The bending number of degrees are bigger (accompanying drawing 2C-D).These results suggest that, in transgenic arabidopsis, Auxin transport may be affected.
Embodiment 5 MdPIN1 has no significant effect to auxin synthesis
DR5, as the Mark gene of auxin, can be used to seed hybridization.Growth selection DR5-GUS in good condition turns
Gene arabidopsis (Shandong Agricultural University's Life Science College is opened constitution and saved professor's offer) and MdPIN1 transgenic arabidopsis are carried out
Hybridization.After hybridization success, collect Fruit pod, PCR detection obtains positive transgenic plant, obtain T3 for pure through continuous 3 generations screening
Zoarium, collects seed, carries out phenotype analytical.
Auxin accumulation in root system is observed in the dyeing of GUS dyeing liquor.Result shows, hybridizes in DR5-GUS/MdPIN1 and plants
In strain, the main root tip of a root is not significantly different from lateral-root primordia position GUS dyeing, illustrates that overexpression MdMIEL1 gene may not
Impact auxin accumulation (accompanying drawing 3A).
Auxin synthetic gene in quantitative PCR detection wild type and transgenic arabidopsis (AtYUC1, AtYUC2, AtYUC4,
AtYUC6, AtTAA1) (detection method is with reference to Mashiguchi K, Tanaka K, Sakai T, et al.The main for expression
auxin biosynthesis pathway in Arabidopsis[J].Proceedings of the National
Academy of Sciences,2011,108(45):18512-18517.).Result shows, arabidopsis and transgenic arabidopsis
Middle auxin synthetic gene is not significantly different from, and hint MdPIN1 gene may not affect the synthesis (accompanying drawing 3B) of auxin.
Example 6 MdPIN1 regulates and controls root system development
For further determining that the function of transfer-gen plant, arabidopsis is sowed on vertical MS culture medium, observes and intend south
Mustard root system development phenotype.
(1) Seed Treatment.The arabidopsis seed (Col-0, L1, L2, L3) that will obtain, uses 70% alcohol disinfecting respectively
3min, 4% hypochlorite disinfectant 8-10min (period repeatedly rocks), aqua sterilisa rinses 5 times, blots water.It is seeded in seed to sprout
On culture medium.4 DEG C of Stratificated treatment cultivate (21-24 DEG C, the 16h long-day/8h short-day) after 4 days to light.
(2) the consistent wildtype Arabidopsis thaliana (Col-0) of sprouting latter 4 days about, root system development and transgenic arabidopsis are selected
(L1, L2, L3), transfers to and vertically cultivates (21-24 DEG C, the 16h long-day/8h short-day) on MS culture medium.Continue culture 6 days
Afterwards, observe arabidopsis root system phenotype and take pictures.
It was found that compared with wildtype Arabidopsis thaliana, the transgenic arabidopsis main root length of overexpression MdPIN1 is obvious
It is suppressed, lateral root number significantly increases.(accompanying drawing 4A-C)
In sum, MdPIN1 gene not only affects Auxin transport, and root system development is also had a significant impact.
The foregoing is only presently preferred embodiments of the present invention, not in order to limit the present invention, all spirit in the present invention and
Within principle, any modification, equivalent substitution and improvement made etc., should be included within the scope of the present invention.
Claims (7)
1. a kind of detached apple development element transport agent gene M dPIN1, its nucleotide sequence such as SEQ ID NO：Shown in 1.
2. detached apple development element transport agent gene M dPIN1 according to claim 1 is it is characterised in that described life
Long element transport agent gene M dPIN1 is derived from loud, high-pitched sound apple.
3. the albumen of apple development element transport agent gene M dPIN1 coding described in a kind of detached claim 1, its amino acid
Sequence such as SEQ ID NO：Shown in 2.
4. apple development element transport agent gene M dPIN1 described in claim 1 is adjusting plant strain growth element transport and/or root system
Developmental purposes.
5. purposes according to claim 4 is it is characterised in that use in root system development for the described apple MdPIN1 gene
On the way, for improving main root length and suppressing lateral root number.
6. a kind of nucleic acid construct, comprises the element transport agent of apple development described in claim 1 being under strong promoter controls
Gene M dPIN1.
7. nucleic acid construct according to claim 6 is it is characterised in that described strong promoter is cauliflower mosaic virus
35S promoter.
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| CN201610754968.2A CN106399324A (en) | 2016-08-30 | 2016-08-30 | Apple auxin delivery vector gene MdPIN1 for regulating root growth, and application thereof |
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| CN107176980A (en) * | 2017-06-15 | 2017-09-19 | 山东农业大学 | Apple polypeptide MdCEP1PHypAnd its application |
| CN107245489A (en) * | 2017-06-08 | 2017-10-13 | 山东农业大学 | A kind of apple polypeptide hormone gene M dCEP7 of regulation and control root system development and its application |
| CN111793636A (en) * | 2020-07-29 | 2020-10-20 | 山东农业大学 | Apple gene MdBT2 for regulating and controlling adventitious root development and application thereof |
| CN112136689A (en) * | 2020-08-31 | 2020-12-29 | 山东大学 | Experiment system for detecting influence of rhizosphere microorganisms on plant growth and root system development and application thereof |
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| CN107245489A (en) * | 2017-06-08 | 2017-10-13 | 山东农业大学 | A kind of apple polypeptide hormone gene M dCEP7 of regulation and control root system development and its application |
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| CN111793636B (en) * | 2020-07-29 | 2021-09-28 | 山东农业大学 | Apple gene MdBT2 for regulating and controlling adventitious root development and application thereof |
| CN112136689A (en) * | 2020-08-31 | 2020-12-29 | 山东大学 | Experiment system for detecting influence of rhizosphere microorganisms on plant growth and root system development and application thereof |
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