CN104628841A - ZmLAX3 protein, and coding gene and application thereof - Google Patents

Abstract

The invention discloses a ZmLAX3 protein, and a coding gene and application thereof. The invention provides a protein disclosed as 1) or 2): protein disclosed as Sequence 2 in the sequence table; and 2) Sequence-2-derived protein subjected to substitution and/or deletion and/or addition of one or more nucleotide residues with the same functions as the nucleotide sequence disclosed as Sequence 2 in the sequence table. The experiment proves that the cloning of the ZmLAX3 gene detects that the transgenic crop obtained from the gene has plant types, fringe character change and other phenotypes. The transgenic positive plant can improve the plant types, fringe characters and other important agronomic characters when being used for production, is used for enhancing the yield and quality of the crops, and has important economic value and social benefits.

Description

A kind of ZmLAX3 albumen and encoding gene thereof and application

Technical field

The present invention relates to field of plant genetic, particularly relate to a kind of ZmLAX3 albumen and encoding gene thereof and application.

Background technology

Along with world economy rapid growth, corn is as the important crops integrating the multiple uses such as grain, feed, industrial processes, and global demand amount increases severely.China is Maize Production big country of the world, improve corn yield become the problem that China's Maize Industry needs solution badly, be also agriculture production and socioeconomic in the urgent need to.

Yield traits is one of Main Agronomic Characters of corn, is the quantitative character of controlled by multiple genes, and there is complicated interaction between gene, genetic expression is easily affected by environment, shows as the voriability of proterties, and hereditary basis is very complicated.Yield Traits In Corn is the result of tassel row number, row grain number, grain multiple economical character comprehensive action such as heavily, and these proterties interact, influence each other.

The research of current Yield Traits In Corn mainly concentrates on the exploitation of QTL (quantitative trait locus) molecule marker, and in this, as assistant breeding means.Many scholars have done a large amount of fruitful research work in Yield Traits In Corn and hereditary basis thereof, orient a large amount of QTL relevant to yield traits.Although located on corn yield and yield component traits several QTL site (Shi Yunsu, the important self-mating system analysis of genetic diversity of corn and Correlated Yield Characters QTL study, 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 jointly detected in matter not of the same race (Temperate zone germplsam and Tropical germplsam), and and environment between have and significantly do mutually, QTL stable between environment is little, challenge (Luna et al., Mol Breed, 2006 are added to the versatility of molecular marker assisted selection, 17,227-239).Tassel row number is an important character relevant to output, and the research about tassel row number also mainly concentrates on exploitation (Li et al., Genet.Mol.Res., 2014,13, the 1707-1716 of QTL site; Zhang et al., Theor Appl Genet, 2013,126,1545-1453; ).With the research work of tassel row number genes involved, only there are (Nat.Genet., 2013 such as Bomment, 45, in corn, 334-337) find that FEA2 gene can make inflorescence meristem increase, and improve tassel row number that there are the potentiality improving corn yield.

LAX3 gene is the gene of encoding growth element input carrier albumen, and expressive site mainly concentrates on (Swarup et al., Biochem Soc T., 2000,28,481-485) in the vascular tissue of root and hollow.LAX3 carrier transport protein belongs to multigene family AUX/LAX family, has function conservative property and growth hormone absorption and transport function.

Summary of the invention

The present invention's object is to provide ZmLAX3 albumen and encoding gene thereof.

Albumen provided by the invention, called after ZmLAX3 is following 1) or 2):

1) protein shown in sequence 2 in sequence table;

2) aminoacid sequence shown in sequence in sequence table 2 is had through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation the protein that identical function derives by sequence 2.

The replacement of one or several amino-acid residue above-mentioned and/or disappearance and/or be added to the replacement and/or disappearance and/or interpolation that are no more than 10 amino-acid residues.

Above-mentioned protein DNA molecule of encoding also is the scope of protection of the invention.

Above-mentioned DNA molecular is following 1)-4) in any one DNA molecular:

1) coding region is the DNA molecular shown in sequence in sequence table 1;

2) coding region is the DNA molecular shown in sequence in sequence table 3;

2) under strict conditions with 1) DNA sequence dna that limits hybridizes and encodes and have identical function protein DNA molecule;

3) with 1) DNA sequence dna that limits at least has 70%, at least have 75%, at least have 80%, at least have 85%, at least have 90%, at least have 95%, at least have 96%, at least have 97%, at least have 98% or at least have 99% homology and encode and have identical function protein DNA molecule.

Above-mentioned stringent condition can be in the solution of 6 × SSC, 0.5%SDS, and hybridize at 65 DEG C, then use 2 × SSC, 0.1%SDS and 1 × SSC, 0.1%SDS respectively wash film once.

Recombinant vectors containing above-mentioned DNA molecular, expression cassette, transgenic cell line or recombinant bacterium are also the scope of protection of the invention.

Transgenic cell does not comprise plant propagation material.

Above-mentioned recombinant vectors is inserted in expression vector by above-mentioned DNA molecular, obtains the recombinant vectors of expressing above-mentioned albumen.

In an embodiment of the present invention, described expression vector is pZP211::UBI.

The carrier that above-mentioned recombinant vectors obtains for BamHI and Sac I enzyme of the ZmLAX3 gene replacement pZP211::UBI expression vector shown in sequence in sequence table 3 being cut the DNA fragmentation between recognition site, express ZmLAX3 because of, by this positive plasmid called after pZP211 UBI::ZmLAX3.

The primer pair of above-mentioned DNA molecular total length or its any fragment of increasing also is the scope of protection of the invention.

Above-mentioned primer pair is specially as follows:

Upstream primer: 5'-AT gGATCCtCATCGGCTGGGCGTGGTCT-3' is as shown in sequence 4;

Downstream primer: 5'-AT gAGCTCtGTCAGGCTCAGGCAGGGTC-3' is as shown in sequence 5;

The application in improvement plant plant type and/or improvement Ear Characters of above-mentioned albumen, above-mentioned DNA molecular or above-mentioned recombinant vectors, expression cassette, transgenic cell line or recombinant bacterium is also the scope of protection of the invention;

Described improvement plant plant type is embodied in and improves plant height, raising fringe position leaf area, reduction fringe position Leaf angle, raising tassel length;

Described fringe position leaf area is specially leaf area under leaf area on fringe, fringe portion leaf area and/or fringe further;

Described fringe position Leaf angle is specially Leaf angle on fringe, fringe portion Leaf angle and/or fringe portion Leaf angle further;

Described improvement Ear Characters be embodied in improve fruit ear spike length, reduce fruit ear fringe thick, reduce fruit ear tassel row number, improve fruit ear capable grain number, increase that seed grain is wide and/or to increase seed grain thick.

Described plant is monocotyledons or dicotyledons, and described monocotyledons is corn.

Another object of the present invention is to provide a kind of method of cultivating transgenic plant.

Method provided by the invention, for the DNA molecular of the above-mentioned albumen of coding is imported object plant, obtains transgenic plant,

Described transgenic plant have following 1-10) middle at least one feature:

1) plant height of described transgenic plant is greater than the plant height of described object plant;

2) the fringe position leaf area of described transgenic plant is greater than the fringe position leaf area of described object plant;

Described fringe position leaf area is specially leaf area under leaf area on fringe, fringe portion leaf area and/or fringe;

3) the fringe position Leaf angle of described transgenic plant is less than the fringe position Leaf angle of described object plant;

Described fringe position Leaf angle is specially Leaf angle and/or fringe portion Leaf angle on fringe;

4) the tassel length of described transgenic plant is greater than the tassel length of described object plant;

5) the fruit ear spike length of described transgenic plant is greater than the fruit ear spike length of described object plant;

6) to be slightly less than the fruit ear fringe of described object plant thick for the fruit ear fringe of described transgenic plant;

7) the fruit ear tassel row number of described transgenic plant is less than the fruit ear tassel row number of described object plant;

8) fruit ear of described transgenic plant capable grain number is greater than the capable grain number of fruit ear of described object plant;

9) the seed grain of described transgenic plant is wider than the capable grain number of fruit ear of described object plant;

10) the thick seed grain being greater than described object plant of the seed grain of described transgenic plant is thick.

In aforesaid method, the DNA molecular of described above-mentioned albumen imports object plant by above-mentioned recombinant vectors.

Described object plant is monocotyledons or dicotyledons, and described monocotyledons is corn.

Experiment of the present invention proves, the present invention provides a kind of gene for improveing Plant Type in Maize and panicled characters first, called after ZmLAX3, the DNA sequence dna comprising the full length fragment of this ZmLAX3 gene is utilized to build over-express vector, then by ZmLAX3 gene overexpression vector introduction agrobacterium strains, and infect maize immature embryos with agrobacterium-mediated transformation, establish transgenic corns strain.After contriver cultivates further, transgenic corns strain and WT lines are compared, find to utilize the transgenic crop of this gene gained to have plant type and panicled characters change isophenous, this transgenic positive plant is applied to production, Main Agronomic Characters such as improvement plant type and panicled characters etc., for improving the yield and quality of crop, there is important economic worth and social benefit.

Accompanying drawing explanation

Fig. 1 is plant expression vector pZP211 UBI::ZmLAX3 carrier structure figure;

Fig. 2 is the procurement process schematic diagram of transgenic corn plant,

In figure, A is that transgenic calli screens in the gradient of screening culture medium; B is Calli Differentiation candidate transgenic seedlings; C is candidate's transfer-gen plant strong sprout; D is that candidate's transfer-gen plant greenhouse is transplanted;

Fig. 3 is the PCR specific amplification result schematic diagram of candidate's transfer-gen plant,

The detected result of gene ZmLAX3 for the purpose of A in figure, B is the detected result of riddled basins NPT II; Wherein, M represents molecular weight marker, and PC represents positive plasmid, and CK represents wild type control, and L represents candidate's transfer-gen plant;

Fig. 4 is the real-time quantitative PCR detected result of candidate's transfer-gen plant,

Wherein, CK represents wild type control, and L represents candidate's transfer-gen plant;

Fig. 5 is positive transgenic strain and WT lines filling stage plant type schematic diagram;

Fig. 6 is positive transgenic strain and WT lines filling stage plant height statistical graph;

Fig. 7 is the leaf area statistical graph of leaf on positive transgenic strain and WT lines filling stage fringe, fringe portion leaf, fringe inferior lobe;

Fig. 8 is positive transgenic strain and WT lines filling stage V18 (fall a leaf), the Leaf angle statistical graph of V17 (fall two leaves), V16 (fall three leaves);

Fig. 9 is positive transgenic strain and WT lines filling stage tassel length statistical graph;

Figure 10 is positive transgenic strain and WT lines filling stage 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 graph;

Figure 13 is positive transgenic strain and the thick statistical graph of WT lines mature fruit cluster fringe;

Figure 14 is positive transgenic strain and WT lines mature fruit cluster tassel row number statistical graph;

Figure 15 is positive transgenic strain and WT lines mellow fruit head progeny row grain number statistical graph;

Figure 16 is positive transgenic strain and WT lines ripe seed grain length statistical graph;

Figure 17 is positive transgenic strain and the wide statistical graph of WT lines ripe seed grain;

Figure 18 is positive transgenic strain and the thick statistical graph of WT lines ripe seed grain;

Figure 19 is positive transgenic strain and WT lines thousand seed weight statistical graph;

Figure 20 is positive transgenic strain and WT lines list fringe output statistics figure;

In above figure, if no special instructions, asterisk represents significant difference (T checks, * * 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 strain title.

Embodiment

The experimental technique used in following embodiment if no special instructions, is ordinary method.

Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.

The present invention is defined further in following examples, describe and embodiment according to above, those skilled in the art can determine essential characteristic of the present invention, and when not departing from spirit and scope of the invention, various change and amendment can be made, to make its applicable various uses and condition to the present invention; Wherein above-mentioned expression vector imports in vegetable cell by the present invention, and introduction method is all well known in the art, and these methods include but are not limited to: Agrobacterium-medialed transformation method, particle bombardment, electrization, Ovary injection etc.The present invention's selectable marker gene used is neomycin phosphotransferase gene (NPT II), can comprise other selectable marker gene and reporter gene further.The screening microbiotic that the present invention selects is paromycin, selects the microbiotic such as kantlex and G418 also can play identical screening effect; In addition, of the present inventionly state of the art is.

Embodiment 1, ZmLAX3 gene clone

1, the Isolation and purification of corn RNA

Utilize Trizol method to extract the total serum IgE of neat 319 (hereinafter also referred to the wild-type corn) of corn inbred line, concrete grammar step is as follows:

Get the 1.5ml centrifuge tube of DEPC process, add 1ml Trizol; Get 100mg corn material, be placed in liquid nitrogen and be repeatedly ground to powder, move in above-mentioned centrifuge tube, fully mix, room temperature leaves standstill 5 minutes; 12000rpm, 4 DEG C, centrifugal 10 minutes, gets supernatant and moves in new centrifuge tube, add 200 μ l chloroforms, acutely rock 15 seconds, and room temperature leaves standstill 3 minutes; 12000rpm, centrifugal 15 minutes, gets in the new centrifuge tube of upper solution to by 4 DEG C; Add 250 μ l Virahols and 250 μ l high level salt solutions, put upside down mixing, room temperature leaves standstill 10 minutes; 12000rpm, 4 DEG C, centrifugal 10 minutes, outwells supernatant, unnecessary supernatant in sucking-off centrifuge tube, adds 75% ethanol of 1ml ice precooling, concussion; 7500rpm, centrifugal 5 minutes, absorbs the ethanol in centrifuge tube with vacuum pump, 37 DEG C of dryings 10 minutes by 4 DEG C; Add the ddH of 50 μ l DEPC process ₂o, precipitation of upspringing gently makes it dissolve; Water-bath 10 minutes in 55-60 DEG C of water-bath, rapid ice bath 5 minutes, brief centrifugation, is placed in-80 DEG C of preservations, obtains RNA.

For guaranteeing that RNA quality reaches order-checking requirement, use the RNA sample purity after spectrophotometer and agarose gel electrophoresis detection purifying and concentration respectively, its moderate purity and concentration standard are: RNA purity be OD260/280 and OD260/230 all 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 cDNA first chain

In the centrifuge tube of 0.2ml DEPC process, order adds 1 μ l, 50 μMs of Oligo dT Primer and 15.5 μ l total serum IgE, 70 DEG C of sex change 6 minutes, rapid ice bath 10 minutes.In above-mentioned centrifuge tube, order adds 2 μ l dNTP Mixture (10mM), 5 μ l 5 × PrimerScript Buffer, 0.5 μ l RNase Inhibitor (40U), 1 μ l PrimerScript RTase (200U), adds DEPC-H ₂o to 25 μ l.In PCR instrument, working procedure is 25 DEG C, 10 minutes; 42 DEG C, 90 minutes; 95 DEG C, 5 minutes.After EP (end of program), sample is frozen stand-by in-80 DEG C.

3, the clone of ZmLAX3 gene

With the cDNA of reverse transcription for template, following primer pair is adopted to carry out pcr amplification:

Upstream primer: 5'-AT gGATCCtCATCGGCTGGGCGTGGTCT-3', as shown in sequence 4;

Downstream primer: 5'-AT gAGCTCtGTCAGGCTCAGGCAGGGTC-3', as shown in sequence 5;

Wherein upstream primer draws horizontal line part is BamH I restriction enzyme site, and it is Sac I restriction enzyme site that downstream primer draws horizontal line part; PCR amplification system is 2 μ l upstream 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 solution (2.5mmol/L), 0.25 μ l Phusion DNA polymerase, 1 μ l cDNA template, adds ddH ₂cumulative volume is supplemented to 11.75 μ l by O;

Amplification condition is: 98 DEG C of denaturations 30 seconds; 98 DEG C of sex change 10 seconds, 59 DEG C of annealing 25 seconds, 72 DEG C extend 1 point 40 seconds, circulate 30 times; 72 DEG C extend 10 minutes.

Get 4 μ l PCR primer to be connected with pMD19-T Simple carrier, operation steps is carried out according to TaKaRa Products pMD19-T Simple Vector specification sheets.Then product conversion bacillus coli DH 5 alpha competent cell is connected, overnight incubation on the LB solid medium containing penbritin (100mg/L).Picking white colony, overnight incubation in the LB liquid nutrient medium containing penbritin (100mg/L).Alkalinity extraction plasmid DNA, enzyme cuts the laggard row sequencing of qualification.

Amplified production is through sequencing analysis, its nucleotides sequence is classified as sequence 3 in sequence table, unnamed gene shown in this PCR primer is ZmLAX3 gene, its coding region is sequence 1 or sequence 3 127-1689 position, the protein designations of this genes encoding is ZmLAX3, and the aminoacid sequence of this albumen is sequence 2 in sequence table.

The functional study of embodiment 2, ZmLAX3 gene

1, the structure of plant recombinant vector pZP211 UBI::ZmLAX3

Extract the RNA of corn inbred line neat 319, reverse transcription obtains cDNA as template, carries out pcr amplification with upstream primer and downstream primer, obtains the ZmLAX3 gene PCR amplified production of 1897bp.

Restriction enzyme BamHI and Sac I enzyme double digestion ZmLAX3 gene PCR amplified production, with with BamHI and Sac I double digestion pZP211::UBI expression vector " clone of corn ZmAATP gene and the structure of genetic transformation carrier ", Journal of Shandong agri.Univ's (natural science edition), 2012,43 (3) 321-327; The public can obtain from Shandong Agricultural University) connect, operation steps is carried out according to Fermentas Products T4 DNA ligase specification sheets.Then product conversion bacillus coli DH 5 alpha competent cell is connected, overnight incubation on the LB solid medium containing spectinomycin (50mg/L).Picking white colony, overnight incubation in the LB liquid nutrient medium containing spectinomycin (50mg/L).Alkalinity extraction plasmid DNA is also carried out BamHI and Sac I enzyme and is cut qualification, and what obtain 1897bp is positive plasmid.

Positive plasmid is sent to order-checking, the carrier that this positive plasmid obtains for BamHI and Sac I enzyme of the replacement of ZmLAX3 shown in sequence in sequence table 3 pZP211::UBI expression vector being cut the DNA fragmentation between recognition site, express ZmLAX3 gene, be pZP211 UBI::ZmLAX3 by this positive plasmid called after, its carrier part structural representation as shown in Figure 1.

By pZP211 UBI::ZmLAX3 transformation Agrobacterium LBA4404 competent cell, and obtain and for the agrobacterium strains transformed, LBA4404/pZP211 UBI::ZmLAX3 can be named.

Two, agriculture bacillus mediated maize immature embryos conversion and the acquisition of resistant plant

Contaminate the day before yesterday, the Agrobacterium bacterium colony LBA4404/pZP211 UBI::ZmLAX3 (carrying the single bacterium colony of Agrobacterium of recombinant plasmid) of picking activation is inoculated in the liquid YEP medium containing 50mg/L Rifampin and 50mg/L spectinomycin, and 28 DEG C of 220rpm overnight shakings are cultivated; Loaded by above-mentioned Agrobacterium solution in 80ml centrifuge tube, centrifugal 5 minutes of 6000rpm, supernatant discarded, collecting precipitation is also resuspended with AB induction broth, 28 DEG C of shaking tables is cultivated after at least 5 hours and can carry out precipitating, contaminating.

After picking pollination, neat 319 ratarias of wild-type corn of 10-12 days put into pre-dip-dye nutrient solution, and centrifugal 5 seconds of 2700rpm, discards substratum; Add pre-dip-dye substratum 2ml Eddy diffusion, centrifugal 5 seconds of 2700rpm; 46 DEG C of water-baths 3 minutes, ice bath 1 minute, centrifugal 5 seconds of 2700rpm, discards substratum, adds 2ml and contaminate nutrient solution in advance, and centrifugal 10 minutes of 4 DEG C of 14000rpm, discard nutrient solution; Move in 80ml centrifuge tube by the bacterium liquid cultivated through AB inducing culture, 6000rpm collects thalline in centrifugal 5 minutes, with dip-dye nutrient solution by thalline Eddy diffusion; Being added by 1ml bacterium liquid is equipped with in the centrifuge tube of rataria, and softly mix for several times, room temperature leaves standstill 5 minutes, discards bacterium liquid; Moved to by rataria on Dual culture substratum, 28 DEG C of light culture, after one week, are transferred to screening culture medium (Fig. 2 A), and every two weeks subcultures once; Being transferred to division culture medium (Fig. 2 B) after screening twice, proceeding to root media (Fig. 2 C) by breaking up the seedling that obtains, after growth of seedling goes out more than 3 sturdy roots, carry out hardening; Hardening, after 7 days, moves to chamber planting (Fig. 2 D), obtains T0 for turning ZmLAX3 corn.

Substratum is infected in advance for the addition of 1.5mg/L 2 in the present embodiment, MS substratum (the Murashige and Skoog of 4-D, 1962), screening culture medium is for the addition of 2.0mg/L 2, the N6 substratum (Zhu Zhiqing etc., 1974) of 4-D, wherein 2,4-D is 2,4 dichlorophenoxyacetic acid.

Three, the Molecular Identification of ZmLAX3 corn is turned

1, the PCR of candidate's transfer-gen plant detects

Adopt CTAB method (Sambrook and Russell, Molecular Cloning: A Laboratory guide, 2001) extract T0 for turning ZmLAX3 milpa genomic dna, design primer certification mark gene NPT II and Ubi-ZmLAX3 respectively and merge fragment, primer pair sequence is as follows:

Marker gene NPT II gene primer is to as follows:

Upstream primer: 5'-GTGGAGAGGCTATTCGGCTATGACTG-3', as shown in sequence 6;

Downstream primer: 5'-AGCTCTTCAGCAATATCACGGGTAGC-3', as shown in sequence 7;

Marker gene NPT II extension increasing sequence length is 650bp, and amplification condition is: 94 DEG C of denaturations 5 minutes; 94 DEG C of sex change 30 seconds, 59 DEG C of annealing 30 seconds, 72 DEG C extend 1 minute, circulate 35 times; 72 DEG C extend 10 minutes.

It is as follows to (upstream primer is promoter region sequence in expression vector, and downstream primer is ZmLAX3 gene order) that Ubi-ZmLAX3 merges fragment primer:

Upstream primer: 5'-TTTTAGCCCTGCCTTCATACGC-3', as shown in sequence 8;

Downstream primer: 5'-GTCTTGGTCCTTCTCCATTTCC-3', as shown in sequence 9;

Goal gene ZmLAX3 extension increasing sequence length is 340bp, amplification condition: amplification condition is: 94 DEG C of denaturations 5 minutes; 94 DEG C of sex change 30 seconds, 61 DEG C of annealing 30 seconds, 72 DEG C extend 1 minute, circulate 35 times; 72 DEG C extend 10 minutes.

Result as shown in Figure 3, what obtain 650bp is the T0 generation containing marker gene NPT II turn ZmLAX3 corn strain (3B), show L147, L 155, L157, L158 be positive transgenic plant also have size to be that 340bp goal gene ZmLAX3 exists (3A) simultaneously.

Wild-type corn does not have 650bp marker gene NPT II.

2, the expression level of Real-time PCR Analysis ZmLAX3 gene in positive transgenic plant

Extracting Molecular Detection is total serum IgE positive transgenic plant T0 generation turning ZmLAX3 corn strain L97, L107, L131, L133, and reverse transcription is cDNA.

Reference gene 18sRNA primer pair is as follows:

Upstream primer: 5'-GATACCGTCCTAGTCTCAACC-3', as shown in sequence 10;

Downstream primer: 5'-GCCTTGCGACCATACTCC-3', as shown in sequence 11;

Goal gene ZmLAX3 primer pair is as follows:

Upstream primer: 5'-CTGAGCGGCATACTGTTC-3', as shown in sequence 12;

Downstream primer: 5'-CCTTCTCCCTCTCCTTCC-3', as shown in sequence 13;

Take reverse transcription product as template, reaction system reference green Realtime PCR Master Mix (QPK-201) specification sheets, amplification condition is: 95 DEG C of denaturations 1 minute; 95 DEG C of sex change 10 seconds, 58 DEG C of annealing 10 seconds, 72 DEG C extend 15 seconds, 30 circulations; 95 DEG C of sex change 15 seconds, 60 DEG C of annealing 30 seconds, 95 DEG C extend 15 seconds, curve plotting.

Take wild-type corn as contrast (CK).

Result as shown in Figure 4, can find out, in L147, L155, L157 and L158, the expression amount of ZmLAX3 gene is all higher than WT lines, illustrate that ZmLAX3 gene is not only incorporated in the genome of positive transgenic plant, and transcriptional level obtains effective expression in positive transgenic Corn.L147, L155, L157 and L158 turn ZmLAX3 corn strain in positive T0 generation.

Adopting uses the same method proceeds in wild-type corn by empty carrier pZP211::UBI, obtains T0 for turning pZP211::UBI corn.Adopt the detection that uses the same method, its result and wild-type corn are without significant difference.

T0 is sowed for plant, cultivates, obtain T1 for plant.

Four, the phenotype analytical of positive transgenic plant

In T1 generation, is turned pZP211::UBI corn, T1 sows for turning ZmLAX3 corn strain L147, L155, L157 and L158 and wild-type corn (CK) simultaneously.The strain of each strain 100, experiment repetition 3 times, results averaged.

Observe the plant height of filling stage strain, result is as Fig. 5, and compared with WT lines (CK), T0 generation turns the plant height of ZmLAX3 corn strain (TL) generally higher than wild-type.

Plant height statistic data result as Fig. 6, T1 generation turn ZmLAX3 corn strain L147, L155, L157 and L158 plant height compared with WT lines, all reach pole significant difference level, illustrate that process LAN ZmLAX3 gene can increase plant height.Wherein, L147 strain plant height increases minimum, about increases by 20 centimetres, and L158 strain increases at most, about increases by 35 centimetres.

To filling stage T1 generation turn ZmLAX3 corn strain L147, the fringe position leaf of L155, L157 and L158 carries out leaf area statistical study, result as shown in Figure 7, in T0 generation, turns leaf on ZmLAX3 corn strain fringe, fringe portion leaf and leaf area obvious increase compared with WT lines of fringe inferior lobe, all reaches pole significant difference level.Wherein, the leaf area increase of the positive strain of L155 is the most remarkable.

Turn ZmLAX3 corn strain L147, V18 (a leaf), the V17 (two leaves) of L155, L157 and L158 and the Leaf angle of V16 (three leaves) to filling stage T1 generation to add up, result is as Fig. 8, find T1 generation turn ZmLAX3 corn strain V18, V17 with V16 all corresponding than the WT lines blade of Leaf angle Leaf angle reduce, wherein, V18 blade L155, L157 with the Leaf angle in L158 strain compared with WT lines, reach pole significant difference level; The Leaf angle of V17 blade in L147 with L155 strain, compared with WT lines, reaches significant difference level; The Leaf angle of V16 blade, compared with WT lines, reaches significant difference level in L158 strain, in L147, L155 with L157 strain compared with WT lines, reaches pole significant difference level.

Turned to the statistical result showed of ZmLAX3 corn strain tassel length and Tassel-Branch number T1 generation, compared with wild-type corn, in T1 generation, turns the trend (Fig. 9) that ZmLAX3 corn strain tassel length presents increase, L147 with L158 is positive, and strain reaches significant difference level compared with WT lines, L155 and L157 strain is without considerable change.

Tassel-Branch number is variation tendency inconsistent (Figure 10) in different positive transgenic strain, and the remarkable minimizing compared with WT lines of L147 strain Tassel-Branch number, reaches pole significant difference level; L155 strain Tassel-Branch number presents minimizing trend compared with WT lines, reaches significant difference level; L157 strain Tassel-Branch number compared with WT lines without considerable change; L158 strain presents the variation tendency contrary with other strains, and Tassel-Branch number increases compared with WT lines, and reaches pole significant difference level.

In T1 generation, turns pZP211::UBI corn and wild-type corn result without significant difference.

Five, positive transgenic plant species test data analysis

In T1 generation, is turned pZP211::UBI corn, T1 sows for turning ZmLAX3 corn strain L147, L155, L157 and L158 and wild-type corn (CK) simultaneously.The strain of each strain 100, experiment repetition 3 times, results averaged.

To T1 generation turn that ZmLAX3 corn strain L147, the spike length of L155, L157 and L158 mature fruit cluster, fringe are thick, the panicled characters such as tassel row number and row grain number observes and measures, and statistics.

As is illustrated by figs. 11 and 12, in T0 generation, turns ZmLAX3 corn strain L147, L155, L157 and L158 corn ear spike length increases, and compared with WT lines, the increase of L147, L157 and L158 strain Ear-Length reaches significant difference level;

As illustrated in figures 11 and 13, in T0 generation, turns ZmLAX3 corn strain L147, L155, L157 and L158 fringe slightly all presents reduction trend, and it is consistent to reduce level, compared with WT lines, all reaches pole significant difference level;

As shown in Figure 11 and Figure 14, in T0 generation, turns ZmLAX3 corn strain L147, L155, L157 and L158 tassel row number significantly reduces, minimizing level is basically identical, and compared with WT lines, L147, L155, L157 and L158 positive transgenic strain all reaches pole significant difference level;

As shown in Figure 11 and Figure 15, in T0 generation, turns ZmLAX3 corn strain L147, the increase of L155, L157 and L158 capable grain digital display work, and compared with WT lines, L147 strain reaches significant difference level, L158 strain reaches pole significant difference level, L155 and L157 is without considerable change.

To T0 generation turn ZmLAX3 corn strain L147, L155, L157 and L158 mature fruit cluster grain characters measures, and statistics, in T0 generation, turns ZmLAX3 corn strain seed grain length (Figure 16) compared with WT lines, without noticeable change; T0 generation turns ZmLAX3 corn strain L147, L155, L157 and L158 seed grain is loose presents increase trend (Figure 17), compared with WT lines, L147 strain reaches significant difference level, L155 and L157 strain reaches pole significant difference level, and L158 strain is without considerable change; T0 generation turns ZmLAX3 corn strain L147, L155, L157 and L158 seed grain is thick presents increase trend (Figure 18), and compared with WT lines, L147 and L157 strain reaches significant difference level, L155 and L158 strain is without considerable change.

The thousand seed weight of corn positive transgenic strain L147, L155, L157 and L158 and single fringe output are measured, and statistics, in T0 generation, turns ZmLAX3 corn strain L147, L155, L157 slightly increase compared with WT lines with L158 thousand seed weight, do not reach significant difference level (Figure 19); In T0 generation, turns ZmLAX3 corn strain L147, the mono-fringe output of L155, L157 and L158 presents downtrending, does not reach significant difference level (Figure 20) 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 reduces, row grain number obviously increases, grain length without considerable change, the wide remarkable increase of grain, the thick obvious increase of grain, thousand seed weight is without considerable change, single fringe output is without noticeable change, and explanation can utilize ZmLAX3 genetically modified crops panicled characters and grain characters, has higher using value than WT lines.

In addition in the present invention ZmLAX3 gene and also may be used for producing other containing the plant expression vector of this gene can the transgenic plant of improved agronomic traits, as crops such as wheat, paddy rice, Chinese sorghums, comprise the organ of these type of transgenic plant, tissue, cell and seed thereof and offspring.

Claims (10)

1. an albumen is following 1) or 2):

1) protein shown in sequence 2 in sequence table;

2) aminoacid sequence shown in sequence in sequence table 2 is had through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation the protein that identical function derives by sequence 2.

2. protein DNA molecule described in coding claim 1.

3. DNA molecular as claimed in claim 2, is characterized in that: described DNA molecular is following 1)-4) in any one DNA molecular:

1) coding region is the DNA molecular shown in sequence in sequence table 1;

2) coding region is the DNA molecular shown in sequence in sequence table 3;

2) under strict conditions with 1) DNA sequence dna that limits hybridizes and encodes and have identical function protein DNA molecule;

3) with 1) DNA sequence dna that limits at least has 70%, at least have 75%, at least have 80%, at least have 85%, at least have 90%, at least have 95%, at least have 96%, at least have 97%, at least have 98% or at least have 99% homology and encode and have identical function protein DNA molecule.

4. the recombinant vectors containing DNA molecular described in Claims 2 or 3, expression cassette, transgenic cell line or recombinant bacterium.

5. recombinant vectors as claimed in claim 4, is characterized in that:

Described recombinant vectors, for being inserted in expression vector by DNA molecular described in Claims 2 or 3, obtains the recombinant vectors of expressing albumen described in claim 1.

6. recombinant vectors according to claim 5, is characterized in that: described expression vector is pZP211::UBI.

7. the primer pair of DNA molecular total length or its any fragment described in Claims 2 or 3 of increasing.

8. the application in improvement plant plant type and/or improvement Ear Characters of recombinant vectors, expression cassette, transgenic cell line or recombinant bacterium described in DNA molecular described in albumen, Claims 2 or 3 described in claim 1 or claim 4;

Described improvement plant plant type is embodied in and improves plant height, raising fringe position leaf area, reduction fringe position Leaf angle and/or improve tassel length;

Described fringe position leaf area is specially leaf area under leaf area on fringe, fringe portion leaf area and/or fringe further;

Described fringe position Leaf angle is specially Leaf angle on fringe, fringe portion Leaf angle and/or fringe portion Leaf angle further;

Described improvement Ear Characters be embodied in improve fruit ear spike length, reduce fruit ear fringe thick, reduce fruit ear tassel row number, improve fruit ear capable grain number, increase that seed grain is wide and/or to increase seed grain thick.

9. cultivate a method for transgenic plant, for the DNA molecular of albumen described in coding claim 1 is imported object plant, obtain transgenic plant,

Described transgenic plant have following 1-10) middle at least one feature:

1) plant height of described transgenic plant is greater than the plant height of described object plant;

2) the fringe position leaf area of described transgenic plant is greater than the fringe position leaf area of described object plant;

Described fringe position leaf area is specially leaf area under leaf area on fringe, fringe portion leaf area and/or fringe;

3) the fringe position Leaf angle of described transgenic plant is less than the fringe position Leaf angle of described object plant;

Described fringe position Leaf angle is specially Leaf angle and/or fringe portion Leaf angle on fringe;

4) the tassel length of described transgenic plant is greater than the tassel length of described object plant;

5) the fruit ear spike length of described transgenic plant is greater than the fruit ear spike length of described object plant;

6) to be slightly less than the fruit ear fringe of described object plant thick for the fruit ear fringe of described transgenic plant;

7) the fruit ear tassel row number of described transgenic plant is less than the fruit ear tassel row number of described object plant;

8) fruit ear of described transgenic plant capable grain number is greater than the capable grain number of fruit ear of described object plant;

9) to be wider than the seed grain of described object plant wide for the seed grain of described transgenic plant;

10) the thick seed grain being greater than described object plant of the seed grain of described transgenic plant is thick.

10. method according to claim 9, is characterized in that: the DNA molecular of albumen described in described coding claim 1 imports object plant by the recombinant vectors described in claim 4 or 5.