CN104628841B - A kind of ZmLAX3 albumen and its encoding gene and application - Google Patents
A kind of ZmLAX3 albumen and its encoding gene and application Download PDFInfo
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Abstract
The invention discloses a kind of ZmLAX3 albumen and its encoding gene and applications.The present invention provides a kind of albumen, be it is following 1) or 2)：1) protein shown in sequence 2 in sequence table；2) nucleotide sequence shown in sequence in sequence table 2 is passed through into the substitution of one or several nucleotide residues and/or lacks and ors add and there is identical function protein derived from sequence 2.The experiment proves that the present invention has found using the transgenic crop obtained by the gene there is plant type and panicled characters to change isophenous by cloning ZmLAX3 genes.The transgenic positive plant is applied to production, improves the Main Agronomic Characters such as plant type and panicled characters, and the yield and quality for improving crop has important economic value and social benefit.
Description
Technical field
The present invention relates to field of plant genetic more particularly to a kind of ZmLAX3 albumen and its encoding gene with answer
With.
Background technology
As world economy grows at top speed, corn is as the weight for integrating the multiple uses such as grain, feed, industrial processes
Crop, global demand amount is wanted to increase severely.China is maize production big country of the world, improve corn yield become China's Maize Industry there is an urgent need for
Solve the problems, such as and agricultural production and social economy there is an urgent need to.
Yield traits are one of Main Agronomic Characters of corn, are the quantitative character of controlled by multiple genes, exist between gene multiple
Miscellaneous interaction, gene expression is easily affected by environment, shows as the continuous variation of character, and hereditary basis is sufficiently complex.Corn
Yield traits be tassel row number, row grain number, grain again etc. multiple economical character comprehensive functions as a result, the interaction of these characters, phase
Mutually influence.
The research of Yield Traits In Corn at present focuses primarily upon QTL (quantitative trait locus) molecular labeling
Exploitation, and in this, as assistant breeding means.Many scholars have done largely in terms of Yield Traits In Corn and its hereditary basis
Fruitful research work, orient largely with the relevant QTL of yield traits.Although in corn yield and yield forming
Several QTL sites (Shi Yunsu, the important self-mating system analysis of genetic diversity of corn and Correlated Yield Characters QTL have been positioned on shape
Research, 2008；Toledo et al.,Genet.Mol.Res.,2011,10,2133-2139；Lu et al.,J
Integr.Plant Biol., 2006,48,1233-1243；.Veldboom et al.,Theor Appl Genet,1994,
89,451-458), but these QTL are seldom detected in different germplasm (Temperate zone germplsam and Tropical germplsam) jointly, Er Qieyu
There is notable interaction between environment, the QTL stablized between environment is seldom, and challenge is increased to the versatility of molecular marker assisted selection
(Luna et al.,Mol Breed,2006,17,227-239).Tassel row number is one and the relevant important character of yield, about
The research of tassel row number also focuses primarily upon exploitation (Li the et al., Genet.Mol.Res., 2014,13,1707- of QTL site
1716；Zhang et al.,Theor Appl Genet,2013,126,1545-1453；).With tassel row number related gene
In research work, only Bomment etc. (Nat.Genet., 2013,45,334-337) has found that FEA2 genes can in corn
So that inflorescence meristem is increased, and improve tassel row number, there are the potentiality for improving corn yield.
LAX3 genes are the genes of encoding growth element input carrier albumen, and expressive site is concentrated mainly on root and hollow dimension
In tubing (Swarup et al., Biochem Soc T., 2000,28,481-485).LAX3 carrier transport proteins belong to more
Gene family AUX/LAX families have function conservative and auxin absorption and transport function.
Invention content
A purpose of the invention is to provide ZmLAX3 albumen and its encoding gene.
Albumen provided by the invention, is named as ZmLAX3, be it is following 1) or 2)：
1) protein shown in sequence 2 in sequence table；
2) by amino acid sequence shown in sequence in sequence table 2 by one or several amino acid residues substitution and/or
It lacks and ors add and there is identical function protein derived from sequence 2.
It the substitution of said one or several amino acid residues and/or lacks and ors add residual to be no more than 10 amino acid
It the substitution of base and/or lacks and ors add.
The DNA molecular for encoding above-mentioned protein is also the scope of protection of the invention.
Above-mentioned DNA molecular is following 1) -4) in any DNA molecular：
1) code area is DNA molecular shown in sequence 1 in sequence table；
2) code area is DNA molecular shown in sequence 3 in sequence table；
2) hybridize under strict conditions with the DNA sequence dna 1) limited and encode the DNA molecular with identical function protein；
3) with the DNA sequence dna that 1) limits at least with 70%, at least with 75%, at least with 80%, at least have
85%, at least have with 90%, at least with 95%, at least with 96%, at least with 97%, at least with 98% or at least
There is the DNA molecular of 99% homology and coding with identical function protein.
Above-mentioned stringent condition can be to hybridize at 65 DEG C in 6 × SSC, the solution of 0.5%SDS, then with 2 × SSC,
It is primary that 0.1%SDS and 1 × SSC, 0.1%SDS respectively wash film.
Recombinant vector, expression cassette, transgenic cell line or recombinant bacterium containing above-mentioned DNA molecular are also that the present invention protects
Range.
Transgenic cell does not include plant propagation material.
Above-mentioned recombinant vector is that above-mentioned DNA molecular is inserted into expression vector, obtains the recombinant vector for expressing above-mentioned albumen.
In an embodiment of the present invention, the expression vector is pZP211::UBI.
Above-mentioned recombinant vector is that ZmLAX3 genes shown in sequence in sequence table 3 are replaced pZP211::UBI expression vectors
I digestion recognition site of BamHI and Sac between the obtained carrier of DNA fragmentation, ZmLAX3 is because naming the positive plasmid for expression
For pZP211 UBI::ZmLAX3.
The primer pair for expanding above-mentioned DNA molecular overall length or its arbitrary segment is also the scope of protection of the invention.
Above-mentioned primer pair is specially as follows：
Sense primer:5'-ATGGATCCTCATCGGCTGGGCGTGGTCT-3' is as shown in sequence 4；
Downstream primer:5'-ATGAGCTCTGTCAGGCTCAGGCAGGGTC-3' is as shown in sequence 5；
Above-mentioned albumen, above-mentioned DNA molecular or above-mentioned recombinant vector, expression cassette, transgenic cell line or recombinant bacterium are being improved
Application in plant plant type and/or improvement Ear Characters is also the scope of protection of the invention；
The improvement plant plant type, which is embodied in, to be improved plant height, improves fringe position leaf area, reduce fringe position Leaf angle, improve
Tassel length；
Fringe position leaf area is specially further leaf area under leaf area, fringe portion leaf area and/or fringe on fringe；
Fringe position Leaf angle is specially further Leaf angle, fringe portion Leaf angle and/or fringe portion Leaf angle on fringe；
The improvement Ear Characters are embodied in and improve fruit ear spike length, reduce that fruit ear fringe is thick, reduces fruit ear tassel row number, carries
High fruit ear row grain number, increase seed grain are wide and/or increase seed grain thickness.
The plant is monocotyledon or dicotyledon, and the monocotyledon is corn.
Another object of the present invention is to provide a kind of method for cultivating genetically modified plants.
Method provided by the invention, the DNA molecular for that will encode above-mentioned albumen import purpose plant, obtain transgenosis and plant
Object,
The genetically modified plants have following 1-10) at least one of feature：
1) plant height of the genetically modified plants is more than the plant height of the purpose plant；
2) the fringe position leaf area of the genetically modified plants is more than the fringe position leaf area of the purpose plant；
Fringe position leaf area is specially leaf area on fringe, leaf area under fringe portion leaf area and/or fringe；
3) the fringe position Leaf angle of the genetically modified plants is less than the fringe position Leaf angle of the purpose plant；
Fringe position Leaf angle is specially Leaf angle and/or fringe portion Leaf angle on fringe；
4) the tassel length of the genetically modified plants is more than the tassel length of the purpose plant；
5) the fruit ear spike length of the genetically modified plants is more than the fruit ear spike length of the purpose plant；
6) the fruit ear fringe of the genetically modified plants is slightly thick less than the fruit ear fringe of the purpose plant；
7) the fruit ear tassel row number of the genetically modified plants is less than the fruit ear tassel row number of the purpose plant；
8) the fruit ear row grain number of the genetically modified plants is more than the fruit ear row grain number of the purpose plant；
9) the seed grain of the genetically modified plants is wider than the fruit ear row grain number of the purpose plant；
10) the seed grain thickness of the genetically modified plants is more than the seed grain thickness of the purpose plant.
In the above method, the DNA molecular of the above-mentioned albumen imports purpose plant by above-mentioned recombinant vector.
The purpose plant is monocotyledon or dicotyledon, and the monocotyledon is corn.
The experiment proves that the present invention provides a kind of base for improveing Plant Type in Maize and panicled characters for the first time
Cause is named as ZmLAX3, builds over-express vector using the DNA sequence dna of the full length fragment comprising the ZmLAX3 genes, then will
ZmLAX3 gene overexpression vector introduction agrobacterium strains, are used in combination agrobacterium-mediated transformation to infect maize immature embryos, establish transgenosis
Corn strain.Transgenic corns strain is compared by inventor after further cultivating with WT lines, finds to utilize the base
Because there is the transgenic crop of gained plant type and panicled characters to change isophenous, which is applied to production,
The Main Agronomic Characters such as plant type and panicled characters are improved, the yield and quality for improving crop has important economic value
And social benefit.
Description of the drawings
Fig. 1 is plant expression vector pZP211 UBI::ZmLAX3 carrier structure figures；
Fig. 2 is the acquisition process schematic of transgenic corn plant,
A is that transgenic calli is screened in the gradient of screening and culturing medium in figure；B is that callus breaks up candidate transgenosis
Seedling；C is candidate transfer-gen plant strong sprout；D is the transplanting of candidate transfer-gen plant greenhouse；
Fig. 3 is the PCR specific amplification result schematic diagrams of candidate transfer-gen plant,
A is the testing result of purpose gene ZmLAX3 in figure, and B is the testing result of riddled basins NPT II；Wherein,
M indicates that molecular weight marker, PC indicate that positive plasmid, CK indicate that wild type control, L indicate candidate transfer-gen plant；
Fig. 4 is the real-time quantitative PCR testing result of candidate transfer-gen plant,
Wherein, CK indicates that wild type control, L indicate candidate transfer-gen plant；
Fig. 5 is positive transgenic strain and WT lines pustulation period plant type schematic diagram；
Fig. 6 is positive transgenic strain and WT lines pustulation period plant height statistical chart；
Fig. 7 be leaf on positive transgenic strain and WT lines pustulation period fringe, fringe portion leaf, fringe inferior lobe leaf area count
Figure；
Fig. 8 is that positive transgenic strain and WT lines pustulation period V18 (fall a leaf), V17 (fall two leaves), V16 (fall three
Leaf) Leaf angle statistical chart；
Fig. 9 is positive transgenic strain and WT lines pustulation period tassel length statistical chart；
Figure 10 is positive transgenic strain and WT lines pustulation period Tassel-Branch number statistical figure；
Figure 11 is positive transgenic strain and WT lines mature fruit cluster figure；
Figure 12 is positive transgenic strain and WT lines mature fruit cluster spike length statistical chart；
Figure 13 is positive transgenic strain and the thick statistical chart of WT lines mature fruit cluster fringe；
Figure 14 is positive transgenic strain and WT lines mature fruit cluster tassel row number statistical chart；
Figure 15 is positive transgenic strain and WT lines mellow fruit head progeny row grain number statistical chart；
Figure 16 is positive transgenic strain and WT lines ripe seed grain length statistical chart；
Figure 17 is positive transgenic strain and the wide statistical chart of WT lines ripe seed grain；
Figure 18 is positive transgenic strain and WT lines ripe seed grain thickness statistical chart；
Figure 19 is positive transgenic strain and WT lines mass of 1000 kernel statistical chart；
Figure 20 is positive transgenic strain and WT lines list fringe output statistics figure；
In the above figure, unless otherwise instructed, asterisk indicates that (T is examined significant difference, * * P (T<=t)<0.01, * P (T<=
t)<0.05).Wherein CK is wild type control, and TL is positive transgenic strain, and L147, L155, L157 and L158 are each positive
Transgenic line title.
Specific implementation mode
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
The further definition present invention in following embodiment, as described above and embodiment, those skilled in the art can be with
It determines the essential characteristic of the present invention, and without departing from the spirit and scope of the invention, the present invention can be made respectively
Kind changes and modification, so that it is applicable in various uses and condition；Wherein the present invention imports above-mentioned expression vector in plant cell,
Introduction method is all well known in the art, these methods include but are not limited to：Agrobacterium-medialed transformation method, particle gun
Method, electrization, Ovary injection etc..Selectable marker gene used in the present invention is neomycin phosphotransferase gene (NPT II),
It can further comprise other selectable marker genes and reporter gene.The screening antibiotic that the present invention selects is paromomycin, is selected
The antibiotic such as kanamycins and G418 can also play identical screening effect；In addition to this, of the present invention is this field
The prior art.
Embodiment 1, ZmLAX3 gene clonings
1, the extraction and purifying of corn RNA
Utilize the total serum IgE of Trizol methods extraction corn inbred line neat 319 (hereinafter also referred to wild-type corn), specific method
Steps are as follows：
The processed 1.5ml centrifuge tubes of DEPC are taken, 1ml Trizol are added；100mg corn materials are taken, are placed in liquid nitrogen anti-
It is ground to powder again, moves in above-mentioned centrifuge tube, mixes well, is stored at room temperature 5 minutes；12000rpm, is centrifuged 10 minutes by 4 DEG C,
It takes supernatant to move in new centrifuge tube, 200 μ l chloroforms is added, acutely rocks 15 seconds, is stored at room temperature 3 minutes；12000rpm, 4 DEG C,
Centrifugation 15 minutes, takes in upper solution to a new centrifuge tube；250 μ l isopropanols and 250 μ l high level salt solutions are added, overturn mixed
It is even, it is stored at room temperature 10 minutes；12000rpm 4 DEG C, centrifuges 10 minutes, outwells supernatant, extra supernatant in centrifuge tube is sucked out, is added
75% ethyl alcohol of 1ml ice precooling, concussion；7500rpm, is centrifuged 5 minutes by 4 DEG C, is absorbed the ethyl alcohol in centrifuge tube with vacuum pump,
37 DEG C of dryings 10 minutes；Add the 50 processed ddH of μ l DEPC2O gently bounces precipitation and makes it dissolve；In 55-60 DEG C of water-bath
Water-bath 10 minutes, rapid ice bath 5 minutes, brief centrifugation are placed in -80 DEG C of preservations, obtain RNA.
It is required to ensure that RNA mass reaches sequencing, uses spectrophotometer and agarose gel electrophoresis detection purifying respectively
RNA sample purity afterwards and concentration, moderate purity and concentration standard are：RNA purity is that OD260/280 and OD260/230 exists
Within the scope of 1.8-2.0, RNA concentration is within the scope of 1.0-2.0 μ g/ μ l.
2, the synthesis of the first chains of cDNA
It is total that 1 μ l 50 μM of Oligo dT Primer and 15.5 μ l are sequentially added in the centrifuge tube of 0.2ml DEPC processing
RNA, 70 DEG C are denaturalized 6 minutes, rapid ice bath 10 minutes.2 μ l dNTP Mixture are sequentially added in above-mentioned centrifuge tube
(10mM), 5 μ 5 × PrimerScript of l Buffer, 0.5 μ l RNase Inhibitor (40U), 1 μ l PrimerScript
RTase (200U), adds DEPC-H2O to 25 μ l.It is 25 DEG C, 10 minutes that program is run in PCR instrument；42 DEG C, 90 minutes；95 DEG C,
5 minutes.After EP (end of program), sample freezes for use in -80 DEG C.
3, the clone of ZmLAX3 genes
Using the cDNA of reverse transcription as template, PCR amplification is carried out using following primer pair：
Sense primer:5'-ATGGATCCTCATCGGCTGGGCGTGGTCT-3', as shown in sequence 4；
Downstream primer:5'-ATGAGCTCTGTCAGGCTCAGGCAGGGTC-3', as shown in sequence 5；
It is I restriction enzyme sites of BamH that wherein sense primer, which draws horizontal line part, and it is I digestions of Sac that downstream primer, which draws horizontal line part,
Site；PCR amplification system is 2 μ l sense primers (5 μm of ol/ μ L), 2 μ l downstream primers (5 μm of ol/ μ L), 5 μ l5 × Phusion
HF buffer (GC), 5 μ l dNTP mixed liquors (2.5mmol/L), 0.25 μ l Phusion DNA polymerase, 1 μ l
CDNA templates, add ddH2Total volume is supplemented to 11.75 μ l by O；
Amplification condition is：98 DEG C of pre-degenerations 30 seconds；98 DEG C are denaturalized 10 seconds, and 59 DEG C are annealed 25 seconds, and 72 DEG C extend 1 point 40 seconds,
Cycle 30 times；72 DEG C extend 10 minutes.
4 μ l PCR products are taken to be connect with pMD19-T Simple carriers, operating procedure is according to TaKaRa Products
PMD19-T Simple Vector specifications carry out.Then connection product convert bacillus coli DH 5 alpha competent cell, containing
Overnight incubation on the LB solid mediums of ampicillin (100mg/L).Picking white colony is containing ampicillin
Overnight incubation in the LB liquid medium of (100mg/L).Alkalinity extraction Plasmid DNA carries out sequencing after digestion identification.
For amplified production through sequencing analysis, nucleotides sequence is classified as sequence 3 in sequence table, and gene shown in the PCR product is ordered
Entitled ZmLAX3 genes, code area are sequence 1 or sequence 3 127-1689, and the albumen of the gene code is named as
The amino acid sequence of ZmLAX3, the albumen are sequence 2 in sequence table.
The functional study of embodiment 2, ZmLAX3 genes
1, plant recombinant vector pZP211 UBI::The structure of ZmLAX3
The RNA of corn inbred line neat 319 is extracted, reverse transcription obtains cDNA as template, with sense primer and downstream primer
PCR amplification is carried out, the ZmLAX3 gene PCR amplified productions of 1897bp are obtained.
I enzyme double digestion ZmLAX3 gene PCR amplified productions of restriction enzyme BamHI and Sac, and with BamHI and Sac
I double digestion pZP211::UBI expression vectors《The clone of corn ZmAATP genes and the structure of genetic transformation carrier》, Shandong agricultural
College journal (natural science edition), 2012,43 (3) 321-327；The public can obtain from Shandong Agricultural University) connection, operating procedure
It is carried out according to Fermentas Products T4 DNA ligase specifications.Then connection product conversion bacillus coli DH 5 alpha impression
State cell, the overnight incubation on the LB solid mediums containing spectinomycin (50mg/L).Picking white colony, containing grand
Overnight incubation in the LB liquid medium of mycin (50mg/L).Alkalinity extraction Plasmid DNA simultaneously carries out BamHI and I digestions of Sac mirror
Fixed, it is positive plasmid to obtain 1897bp.
Positive plasmid is sent into sequencing, which is that ZmLAX3 shown in sequence in sequence table 3 is replaced pZP211::
The carrier that DNA fragmentation between the I digestion recognition site of BamHI and Sac of UBI expression vectors obtains expresses ZmLAX3 genes, will
The positive plasmid is named as pZP211 UBI::ZmLAX3, carrier part structural schematic diagram are as shown in Figure 1.
By pZP211 UBI::ZmLAX3 converts Agrobacterium LBA4404 competent cell, and obtains and used for conversion
Agrobacterium strains, name LBA4404/pZP211 UBI::ZmLAX3.
Two, the conversion of agriculture bacillus mediated maize immature embryos and the acquisition of resistant plant
It disseminates the previous day, the Agrobacterium bacterium colony LBA4404/pZP211 UBI of picking activation::ZmLAX3 (carries recombination matter
The Agrobacterium single bacterium colony of grain) it is inoculated in the liquid YEP medium containing 50mg/L rifampins and 50mg/L spectinomycins, 28 DEG C
220rpm overnight shaking cultures；Above-mentioned Agrobacterium solution is fitted into 80ml centrifuge tubes, 6000rpm is centrifuged 5 minutes, is discarded
Clearly, it collects precipitation and is resuspended with AB induction broths, can be precipitated, be disseminated after being cultivated at least 5 hours on 28 DEG C of shaking tables.
10-12 days neat 319 ratarias of wild-type corn are put into pre- dip dyeing culture solution after picking pollination, 2700rpm centrifugations 5
Second, discard culture medium；Pre- dip dyeing culture medium 2ml is added to suspend again, 2700rpm is centrifuged 5 seconds；46 DEG C of water-baths 3 minutes, ice bath 1 divides
Clock, 2700rpm are centrifuged 5 seconds, discard culture medium, and 2ml is added and disseminates culture solution in advance, 4 DEG C of 14000rpm are centrifuged 10 minutes, discarded
Culture solution；Bacterium solution Jing Guo AB inducing culture cultures is moved into 80ml centrifuge tubes, 6000rpm is centrifuged 5 minutes and is collected thalline,
Thalline is suspended again with dip dyeing culture solution；By 1ml bacterium solutions be added equipped with rataria centrifuge tube in, soft mixing for several times, room temperature
5 minutes are stood, bacterium solution is discarded；Rataria is moved to and is co-cultured on culture medium, 28 DEG C of light cultures after a week, are transferred to screening and culturing medium
(Fig. 2A), subculture is primary every two weeks；Screening is transferred to differential medium (Fig. 2 B) afterwards twice, and the seedling that differentiation obtains is transferred to life
Root culture medium (Fig. 2 C) carries out hardening after growth of seedling goes out 3 or more sturdy roots；After hardening 7 days, chamber planting is moved to
(Fig. 2 D), obtain T0 generation turn ZmLAX3 corns.
The pre- culture medium that infects in the present embodiment is MS culture mediums (the Murashige and for being added to 1.5mg/L 2,4-D
Skoog, 1962), screening and culturing medium is to be added to 2.0mg/L 2, the N6 culture mediums of 4-D (Zhu Zhi clear etc., 1974), therein 2,
4-D is 2,4 dichlorophenoxyacetic acid.
Three, turn the Molecular Identification of ZmLAX3 corns
1, the PCR detections of candidate transfer-gen plant
Turned using CTAB methods (Sambrook and Russell, Molecular Cloning:A Laboratory guide, 2001) extraction T0 generations
ZmLAX3 plant genomic DNAs separately design primer detection marker gene NPT II and Ubi-ZmLAX3 fusion segments, draw
Object is as follows to sequence：
II gene primers of marker gene NPT are to as follows：
Sense primer：5'-GTGGAGAGGCTATTCGGCTATGACTG-3', as shown in sequence 6；
Downstream primer：5'-AGCTCTTCAGCAATATCACGGGTAGC-3', as shown in sequence 7；
II extension increasing sequence length of marker gene NPT is 650bp, and amplification condition is：94 DEG C of pre-degenerations 5 minutes；94 DEG C of denaturation
30 seconds, 59 DEG C were annealed 30 seconds, and 72 DEG C extend 1 minute, recycle 35 times；72 DEG C extend 10 minutes.
Ubi-ZmLAX3 merges fragment primer to (sense primer is promoter region sequence in expression vector, and downstream primer is
ZmLAX3 gene orders) it is as follows：
Sense primer：5'-TTTTAGCCCTGCCTTCATACGC-3', as shown in sequence 8；
Downstream primer：5'-GTCTTGGTCCTTCTCCATTTCC-3', as shown in sequence 9；
Target gene ZmLAX3 extension increasing sequence length is 340bp, amplification condition：Amplification condition is：94 DEG C of pre-degenerations 5 are divided
Clock；94 DEG C are denaturalized 30 seconds, and 61 DEG C are annealed 30 seconds, and 72 DEG C extend 1 minute, recycle 35 times；72 DEG C extend 10 minutes.
The results are shown in Figure 3, and it is to turn ZmLAX3 corn strains in the T0 generations containing marker gene NPT II to obtain 650bp
(3B) shows that L147, L 155, L157, L158 are positive transgenic plant, while it is 340bp target gene also to have size
There is (3A) in ZmLAX3.
Wild-type corn does not have 650bp marker gene NPT II.
2, expression of the Real-time PCR Analysis ZmLAX3 genes in positive transgenic plant
Extraction Molecular Detection is to turn ZmLAX3 corn strains L97, L107, L131, L133 in positive transgenic plant T0 generations
Total serum IgE, and reverse transcription is cDNA.
Reference gene 18sRNA primer pairs are as follows：
Sense primer：5'-GATACCGTCCTAGTCTCAACC-3', as shown in sequence 10；
Downstream primer：5'-GCCTTGCGACCATACTCC-3', as shown in sequence 11；
Target gene ZmLAX3 primer pairs are as follows：
Sense primer：5'-CTGAGCGGCATACTGTTC-3', as shown in sequence 12；
Downstream primer：5'-CCTTCTCCCTCTCCTTCC-3', as shown in sequence 13；
Using reverse transcription product as template, reaction system referenceGreen Realtime PCR Master Mix
(QPK-201) specification, amplification condition are：95 DEG C of pre-degenerations 1 minute；95 DEG C are denaturalized 10 seconds, and 58 DEG C are annealed 10 seconds, 72 DEG C of extensions
15 seconds, 30 cycles；95 DEG C are denaturalized 15 seconds, and 60 DEG C are annealed 30 seconds, and 95 DEG C extend 15 seconds, draw curve.
It is control (CK) with wild-type corn.
The results are shown in Figure 4, it can be seen that the expression quantity of ZmLAX3 genes is above in L147, L155, L157 and L158
WT lines illustrate that ZmLAX3 genes are not only integrated into the genome of positive transgenic plant, but also in positive transgenic
Transcriptional level obtains effective expression in Corn.L147, L155, L157 and L158 are to turn ZmLAX3 corn strains in positive T0 generations.
Using same method by empty carrier pZP211::UBI is transferred in wild-type corn, obtain T0 generation turn pZP211::
UBI corns.It is detected using same method, result is with wild-type corn without significant difference.
T0 is sowed for plant, culture obtains T1 for plant.
Four, the phenotypic analysis of positive transgenic plant
In T1 generations, are turned into pZP211::In UBI corns, T1 generations, turn ZmLAX3 corn strain L147, L155, L157 and L158 and open country
Raw type corn (CK) is sowed simultaneously.Each 100 plants of strain, experiment is repeated 3 times, and results are averaged.
The plant height of pustulation period strain is observed, as a result such as Fig. 5, compared with WT lines (CK), T0 generations turn
The plant height of ZmLAX3 corns strain (TL) is generally higher than wild type.
In plant height statistical data result such as Fig. 6, T1 generation, turns the plant height of ZmLAX3 corn strains L147, L155, L157 and L158
Compared with WT lines, reach pole significant difference level, illustrates that plant height can be increased by being overexpressed ZmLAX3 genes.Its
In, L147 strain plant heights increase at least, about increase by 20 centimetres, L158 strains increase at most, about increase by 35 centimetres.
The fringe position leaf for turning ZmLAX3 corn strains L147, L155, L157 and L158 to generation pustulation period T1 carries out leaf area system
Meter analysis, the results are shown in Figure 7, T0 generations turn on ZmLAX3 corn strain fringes the leaf area of leaf, fringe portion leaf and fringe inferior lobe with it is wild
It is horizontal to reach pole significant difference compared to obviously increasing for type plant.Wherein, the leaf area increase of L155 positives strain is the most aobvious
It writes.
V18 (leaf), the V17 for turning ZmLAX3 corn strains L147, L155, L157 and L158 to generation pustulation period T1 (fall
Two leaves) and the Leaf angle of V16 (fall three leaves) counted, as a result such as Fig. 8, it is found that T1 generations turn ZmLAX3 corn strains V18, V17
Reduce with the Leaf angle of the Leaf angle of V16 blade more corresponding than WT lines, wherein V18 blades in L155, L157 and
It is horizontal to reach pole significant difference compared with WT lines for Leaf angle in L158 strains；V17 blades are in L147 and L155 strains
In Leaf angle compared with WT lines, reach significant difference level；The Leaf angle of V16 blades compared with WT lines,
Reach significant difference level in L158 strains, in L147, L155 and L157 strain compared with WT lines, reaches extremely aobvious
Write level of difference.
The statistical result showed for turning ZmLAX3 corn strain tassel length and Tassel-Branch number to T1 generations, with wild type jade
Rice is compared, and T1 generations turn ZmLAX3 corn strain tassel length and increased trend (Fig. 9), L147 and L158 positives strain and open country is presented
Raw type plant is compared to significant difference level is reached, L155 and L157 strains are without significant change.
Tassel-Branch number variation tendency in different positive transgenic strains is inconsistent (Figure 10), L147 strain tassels point
Branch number substantially reduces compared with WT lines, and it is horizontal to reach pole significant difference；L155 strain Tassel-Branch numbers with it is wild
Type plant reaches significant difference level compared to reduction trend is presented；L157 strain Tassel-Branch numbers are compared with WT lines
Without significant change；The variation tendency opposite with other strains, the Tassel-Branch number compared with WT lines is presented in L158 strains
Increase, and it is horizontal to reach pole significant difference.
In T1 generations, turn pZP211::UBI corns and wild-type corn result are without significant difference.
Five, positive transgenic plant species test data analysis
In T1 generations, are turned into pZP211::In UBI corns, T1 generations, turn ZmLAX3 corn strain L147, L155, L157 and L158 and open country
Raw type corn (CK) is sowed simultaneously.Each 100 plants of strain, experiment is repeated 3 times, and results are averaged.
Turn that the spike lengths of ZmLAX3 corn strain L147, L155, L157 and L158 mature fruit clusters, fringe be thick, tassel row number to T1 generations
It is observed and is measured with panicled characters such as row grain numbers, and statistical result.
As is illustrated by figs. 11 and 12, in T0 generations, turn ZmLAX3 corn strain L147, L155, L157 and L158 corn ear spike lengths
Increase, compared with WT lines, L147, L157 and L158 strain Ear-Length increase up to significant difference level；
As illustrated in figures 11 and 13, for ZmLAX3 corn strain L147, L155, L157 and L158 fringe is turned, slightly presentation subtracts T0
Small trend, and reduce level unanimously, compared with WT lines, it is horizontal to reach pole significant difference；
As shown in Figure 11 and Figure 14, in T0 generations, turn ZmLAX3 corn strain L147, L155, L157 and L158 tassel row numbers and significantly subtract
Few, reduction is horizontal almost the same, and compared with WT lines, L147, L155, L157 and L158 positive transgenic strain reach
Pole significant difference is horizontal；
As shown in Figure 11 and Figure 15, in T0 generations, turn ZmLAX3 corn strain L147, L155, L157 and L158 row grain numbers and significantly increase
Add, compared with WT lines, L147 strains reach significant difference level, and L158 strains reach pole significant difference level, L155
With L157 without significant change.
Turn ZmLAX3 corn strain L147, L155, L157 and L158 mature fruit cluster grain characters to T0 generations to be measured, and
In statistical result, T0 generations, turn ZmLAX3 corn strain seed grain lengths (Figure 16) compared with WT lines, no significant changes；In T0 generations, turn
The loose presentation increase trend (Figure 17) of ZmLAX3 corn strain L147, L155, L157 and L158 seed grains, with WT lines phase
Than L147 strains reach significant difference level, L155 and L157 strains reach pole significant difference level, and L158 strains become without apparent
Change；In T0 generations, turn ZmLAX3 corn strain L147, L155, L157 and L158 seed grain thickness and increase trend (Figure 18) are presented, and wild
Type plant is compared, L147 and L157 strains reach significant difference level, L155 and L158 strains are without significant change.
Mass of 1000 kernel and single fringe yield to corn positive transgenic strain L147, L155, L157 and L158 are measured, and
In statistical result, T0 generations, turn ZmLAX3 corn strain L147, L155, L157 and L158 mass of 1000 kernel and slightly increase compared with WT lines
Add, it is horizontal (Figure 19) that significant difference is not achieved；In T0 generations, turn the mono- fringe yield of ZmLAX3 corn strains L147, L155, L157 and L158
Downward trend is presented, it is horizontal (Figure 20) that significant difference is not achieved compared with WT lines.
On the whole, positive transgenic strain mature fruit cluster spike length rises appreciably, and fringe slightly significantly reduces, and tassel row number significantly subtracts
Few, row grain number obviously increases, and grain length is without significant change, and grain is wide to be dramatically increased, and grain thickness obviously increases, mass of 1000 kernel without significant change,
Single fringe yield can utilize ZmLAX3 genetically modified crops panicled characters and grain characters, be planted than wild type without significant changes, explanation
Strain has higher application value.
In addition to this ZmLAX3 genes and the plant expression vector containing the gene can be used for producing it in the present invention
It can include the device of such genetically modified plants with the genetically modified plants of improved agronomic traits, such as wheat, rice, sorghum crop
Official, tissue, cell and its seed and offspring.
Claims (4)
1. following any application in improvement plant plant type and/or in improveing Ear Characters；
1）A kind of albumen is protein shown in sequence 2 in sequence table；
2）Coding 1）The DNA molecular of the albumen, the DNA molecular are following a）Or b）DNA molecular：
a）Code area is DNA molecular shown in sequence 1 in sequence table；
b）Code area is DNA molecular shown in sequence 3 in sequence table；
3）Contain 2）Recombinant vector, expression cassette or the recombinant bacterium of the DNA molecular；
The recombinant vector is by 2）The DNA molecular is inserted into expression vector, obtains expression 1）The recombinant vector of the albumen；
The improvement plant plant type, which is embodied in, to be improved plant height, improves fringe position leaf area, reduces fringe position Leaf angle and/or raising
Tassel length；
Fringe position leaf area is leaf area under leaf area, fringe portion leaf area and/or fringe on fringe；
Fringe position Leaf angle is Leaf angle and/or fringe portion Leaf angle on fringe；
The improvement Ear Characters are improving fruit ear spike length, are reducing thick fruit ear fringe, reduction fruit ear tassel row number, raising fruit ear row grain
Number, increase seed grain are wide and/or increase seed grain thickness.
2. application according to claim 1, it is characterised in that：The expression vector is pZP211::UBI.
3. a kind of method for cultivating genetically modified plants, for that will encode 1 in claim 1）The DNA molecular of the albumen imports purpose
Plant obtains genetically modified plants,
The genetically modified plants have following 1-10）At least one of feature：
1）The plant height of the genetically modified plants is more than the plant height of the purpose plant；
2）The fringe position leaf area of the genetically modified plants is more than the fringe position leaf area of the purpose plant；
Fringe position leaf area is specially leaf area on fringe, leaf area under fringe portion leaf area and/or fringe；
3）The fringe position Leaf angle of the genetically modified plants is less than the fringe position Leaf angle of the purpose plant；
Fringe position Leaf angle is specially Leaf angle and/or fringe portion Leaf angle on fringe；
4）The tassel length of the genetically modified plants is more than the tassel length of the purpose plant；
5）The fruit ear spike length of the genetically modified plants is more than the fruit ear spike length of the purpose plant；
6）The fruit ear fringe that the fruit ear fringe of the genetically modified plants is slightly less than the purpose plant is thick；
7）The fruit ear tassel row number of the genetically modified plants is less than the fruit ear tassel row number of the purpose plant；
8）The fruit ear row grain number of the genetically modified plants is more than the fruit ear row grain number of the purpose plant；
9）The seed grain that the seed grain of the genetically modified plants is wider than the purpose plant is wide；
10）The seed grain that the seed grain thickness of the genetically modified plants is more than the purpose plant is thick.
4. according to the method described in claim 3, it is characterized in that：1 in the coding claim 1）The DNA of the albumen points
Son passes through in claim 13）The recombinant vector imports purpose plant.
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| CN109022450B (en) * | 2018-08-15 | 2021-09-24 | 河南农业大学 | ZmCL 2-1 gene for regulating and controlling included angle of corn leaves and application thereof |
| CN112457386A (en) * | 2021-01-19 | 2021-03-09 | 中国科学院遗传与发育生物学研究所 | Protein EAD1 related to control of ear length and row grain number of corn as well as coding gene and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000078965A2 (en) * | 1999-06-22 | 2000-12-28 | E.I. Du Pont De Nemours And Company | Plant auxin proteins |
| CN102492718A (en) * | 2011-11-23 | 2012-06-13 | 山东大学 | Application of genes of auxin influx carrier AUX1/LAX family in breeding of corns and broomcorns |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000078965A2 (en) * | 1999-06-22 | 2000-12-28 | E.I. Du Pont De Nemours And Company | Plant auxin proteins |
| CN102492718A (en) * | 2011-11-23 | 2012-06-13 | 山东大学 | Application of genes of auxin influx carrier AUX1/LAX family in breeding of corns and broomcorns |
Non-Patent Citations (1)
| Title |
|---|
| 玉米ZmAATP基因的克隆及遗传转化载体的构建;于林卉等;《山东农业大学学报(自然科学版)》;20121231;第43卷(第3期);第323页2.2.5节 * |
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