CN104293808A - Liriodendron hybrids LhMKK2 gene and expression protein and application thereof - Google Patents

Abstract

The invention discloses a Liriodendron hybrids LhMKK2 gene, expression protein of the Liriodendron hybrids LhMKK2 gene and application of the liriodendron hybrids LhMKK2 gene. The nucleotide sequence is showed in SEQID NO.1 and the amino acid sequence is showed in SEQID NO.2. On the basis of the completely established somatic embryogenesis system of the Liriodendron hybrids and the fact that part of the transcriptome data is already obtained, the full length of the Liriodendron hybrids LhMKK2 gene, through the adoption of homology-based cloning, is obtained and is named as LhMKK2. Through gene expression analysis, that the Liriodendron hybrids LhMKK2 gene is widely expressed in plants and is involved in the growth and development of plants, and the adversity stress response process are proved; through functional analysis of the gene, it is discovered that the overexpression LhMKK2 gene is capable of enhancing resistance of plants to salt stress response, influencing the growth and development of plants and providing theoretical basis for stress resistance molecular breeding of Liriodendron hybrids and can be further widely used in adversity resistance stress of plants.

Description

A kind of hybridized Chinese tuliptree LhMKK2 gene and expressing protein thereof and application

Technical field

The invention belongs to field of plant genetic, be specifically related to a kind of hybridized Chinese tuliptree lhMKK2gene and expressing protein thereof and application.

Background technology

Hybridized Chinese tuliptree is that the people such as famous Forest Tree Genetics and Breeding man of Nanjing Forestry University professor Ye Peizhong utilize Chinese Liriodendron chinense (L.chinese(HEMSL.) Sarg) interspecific hybrid that obtains with yellow poplar (L.tulipifera Linn.) cross experiment, there is obvious hybrid vigour.Hybridized Chinese tuliptree is because of its fast growth, and disease and insect resistance is strong, and material texture is fine and smooth, leaf peculiar, fast growth, and be the fine tree species of collection afforestation, industrial cut stock, ornamental plantation, be also used to courtyard greening, afforestation and commerical tree species, market potential is large.Current hybridized Chinese tuliptree is bred mainly through the sexual propagation mode of the conventional genderless such as cuttage, grafting propagation method and artificial hybridization, but because be subject to the impact of season and hybrid seeding efficiency, limit the breeding of superior hybrid plant, traditional nursery modes of reproduction, speed is slow, supply falls short of demand far away for good seed quantity, affects application and the popularization of hybridized Chinese tuliptree.Chen Jinhui etc. with hybridized Chinese tuliptree immature embryo for cultivated material, successful somatic embryos on solid medium, establish the Development of Somatic Embryogenesis, and successfully enter later stage factorial praluction, fundamentally improve slow nursery speed and the mode of traditional breeding way.By continuing to optimize of studying of later stage, the perfect suspension culture system of hybridized Chinese tuliptree cells,primordial, for carrying out the Basic Experiment Study such as the growth of high xylophyta, growth, regulatory pathway and resistance, provides a large amount of technical foundation and test materials basis.

Mitogen-activated protein kinase (Mitogen-Activated Protein Kinase, MAPK) cascade path is guarded at eukaryote camber, that mediated plant extracellular signal is passed in cell the signal of interest transduction path causing cellular response, in cytodifferentiation and growth course, hormone, plays a significant role in multiple biology and abiotic stress.MAPK path participates in signal transduction by three kinds of three-stage cascade kinases usually, comprises the kinases (mitogen-activated protein kinase kinase kinases, MAPKKK are called for short MEKK) of the kinases of mitogen-activated protein kinase; Kinases (the mitogen-activated protein kinase kinases of mitogen-activated protein kinase, MAPKK, be called for short MEK or MKK) and mitogen-activated protein kinase MAPK, pass through phosphorylation activation downstream signal step by step successively, outside broad variety stimulus signal is passed in nucleus, and then cause cellular response, participate in the various procedures such as growth, growth, division, differentiation, apoptosis of mediated cell, the even metabolism of growth hormone, dormin, ethene and phytokinin, collaborative involved in plant environment stress response.Therefore, growing of two-level concatenation kinases MAPKK family's involved in plant, hormone signal transduction, and stress resistance of plant stress response process.

At present, by the sequential analysis of arabidopsis gene group, 80 have been identified mAPKKKgene, 10 mAPKKgene, 20 mAPKgene, these genes are also extensively present in other Plant Genome.Such as, 21 have been identified in willow mAPKgene and 11 mAPKKgene, has 15 in paddy rice mAPKgene and 8 mAPKKgene, 75 mAPKKKgene.In addition, be separated in many species at present mAPKKfamily gene, as wheat, rape, paddy rice, parsley, tobacco etc.

Summary of the invention

Goal of the invention: for the deficiencies in the prior art, the object of this invention is to provide a kind of hybridized Chinese tuliptree mKK2gene.Another object of the present invention is to provide a kind of above-mentioned hybridized Chinese tuliptree lhMKK2the expressing protein of gene.The present invention also has an object to be to provide hybridized Chinese tuliptree lhMKK2the application of gene in Hybrid Liriodendron resistance molecular breeding.

Technical scheme: in order to realize foregoing invention object, the technical solution used in the present invention is as follows:

A kind of hybridized Chinese tuliptree lhMKK2gene, its nucleotide sequence is as shown in SEQ ID NO.1.

Described hybridized Chinese tuliptree lhMKK2the expressing protein of gene, its aminoacid sequence is as shown in SEQ ID NO.2.

Described hybridized Chinese tuliptree lhMKK2the application of gene in hybridized Chinese tuliptree resistance molecular breeding.

Described contains hybridized Chinese tuliptree lhMKK2the carrier of gene or host cell.

MAPKK family gene plays a part " forming a connecting link " in MAPK cascade path, is the point of crossing of upstream and downstream signal transduction pathway, in Arabidopis thaliana, mKK2gene participates in the hormone signal transduction such as jasmonic, Whitfield's ointment process, and MEKK1-MKK2-MPK4/MPK6; MKK2-MPK4 cascade path is the integral part to low temperature, the transduction of salt stress acknowledge signal, the also growth and development process of involved in plant.The present invention is on the basis of the hybridized Chinese tuliptree body embryo generation system of built vertical maturation, the kinases of homologous clone mitogen-activated protein kinase lhMKK2full length gene.

Useful effect: compared with prior art, the present invention is on the basis of the perfect hybridized Chinese tuliptree somatic embryo generation system set up, by the cloning and identification to hybridized Chinese tuliptree MAPKK family gene, the expression analysis of gene, the means such as the genetic transformation of gene, its function of analysis verification, demonstrates hybridized Chinese tuliptree lhMKK2gene involved in plant Somatic Embryogenesis, process LAN lhMKK2gene can improve the resistance that plant responds salt stress, affects growing of plant, therefore can be used for the resistance stress response improving plant, for the resistance molecular breeding of hybridized Chinese tuliptree provides foundation, has good prospects for commercial application.

Accompanying drawing explanation

Fig. 1 is 1% agarose gel electrophoresis figure of hybridized Chinese tuliptree blade total serum IgE;

Fig. 2 is lhMKK21% agarose gel electrophoresis figure of full length gene PCR primer;

Fig. 3 is 1% agarose gel electrophoresis figure of object fragment double digestion reaction; In figure, M:DL2000 Marker; A, b: lhMKK2with pMD ^tM19-T Vector connection carrier Xba I, Sac I double digestion reacts;

Fig. 4 is 1% agarose gel electrophoresis figure of empty expression vector pBI121 double digestion reaction; In figure, a: empty carrier PBI121 plasmid Xba I, Sac I double digestion reacts; M1:DL2000 Marker; M2: stripe size is respectively from top to bottom: 23130bp, 9416bp, 6557bp, 4361bp, 2322bp, 2027bp, 564bp;

Fig. 5 builds lhMKK2over-express vector figure;

Fig. 6 is lhMKK2gene is at the real-time quantitative result figure of different sites; In figure, 1: stamen; 2: root; 3: stem; 4: leaf; 5: bud; 6: flower; 7: petal; 8: gynoecium;

Fig. 7 is lhMKK2gene is at the real-time quantitative result figure of different times; In figure, 1: embryo callus; 2: fluid suspension culture 7d; 3: transition incubation period; 4: globular embryo; 5: torpedo embryo; 6: early stage cotyledonary embryos; 7: ripe cotyledon embryo;

Fig. 8 is that T1, T2 are for positive plant PCR detected result figure; In figure, p: lhMKK2the recombinant plasmid of+PBI121, positive control; M:DL2000 Marker; The PCR primer of 1-12 transfer-gen plant; A, b: the PCR primer of wildtype Arabidopsis thaliana amplification, negative control; C: water, blank;

Fig. 9 is T1 generation lhMKK2the growing state figure of transgenic line and wild-type Ler; In figure, A: lhMKK2transgenic line; B: wild-type Ler; C: lhMKK2the side shoot of transgenic line; The side shoot of D: wild-type Ler;

Figure 10 is T1 generation lhMKK2the growing state figure of transgenic line and wild-type Col; In figure, A, B, C: lhMKK2transgenic line; D: wild-type Col; E, F: lhMKK2the blade of transgenic line;

Figure 11 is process LAN lhMKK2transgenic arabidopsis T2 is for plant strain growth statistical graph;

Figure 12 be under salt stress T2 for the growing state figure of transfer-gen plant and wild-type Col; In figure, A: wildtype Arabidopsis thaliana contrasts; Wildtype Arabidopsis thaliana under B:200mmol/L NaCl; C: transfer-gen plant contrasts; Transfer-gen plant under D:200mmol/L NaCl;

Figure 13 is the 7th day wild-type and transfer-gen plant growing state figure under salt stress; In figure, wildtype Arabidopsis thaliana under A:200mmol/LNaCl; Transfer-gen plant under B:200mmol/LNaCl.

Embodiment

Below in conjunction with specific embodiment, the present invention is described further.

Embodiment 1

With hybridized Chinese tuliptree plant for material, extract total serum IgE, and reverse transcription becomes cDNA, designs corresponding primer and carries out PCR, after agarose gel electrophoresis detects, reclaim object band, with pMD ^tM19-T Vector(Takara D102A) carrier connection, proceed to intestinal bacteria, check order and analyze.Picking positive colony carries out plasmid extraction, add after suitable restriction enzyme site with expression vector PBI121 double digestion simultaneously, after connecting under the effect of T4 ligase enzyme, proceed in agrobacterium strains GV3101, after Arabidopis thaliana is of the right age, soak conversion method by floral organ to transform, observe T1, T2 and represent type, carry out statistical study, and carry out resistance Stress treatment, analyze its function.

(1) extraction of total serum IgE

With the spire of hybridized Chinese tuliptree for material, according to NORGEN test kit ( norgen Bioteke) operation steps carry out the extraction of RNA, the reagent used and consumptive material all make it without RNA enzyme through 0.1%DEPC water treatment.As shown in Figure 1, band is complete clear for 1% agarose gel electrophoresis result of hybridized Chinese tuliptree spire total serum IgE; With the light absorption value of determined by ultraviolet spectrophotometry total serum IgE, OD ₂₆₀/ OD ₂₈₀value is 2.05, OD ₂₆₀/ OD ₂₃₀be that 2.01, RNA integrity is better, purity is high, can be used for reverse transcription.

The detailed process that RNA extracts is as follows: 1, get appropriate hybridized Chinese tuliptree blade, be placed in mortar, add 600 μ L Lysis Solution, by sample grinding fully.2, lysate is transferred in centrifuge tube.3, put upside down mixing, place 2min, make its abundant cracking, the centrifugal 2min of 12000rpm, draw supernatant and move in new centrifuge tube.4, add isopyknic 70% ethanol, vortex mixes.5, mixed solution to be moved in centrifugal column (under connect 2mL collection tube), the centrifugal 1min of 12000rpm, abandons filtrate, puts back to collection tube.6, add 400 μ L Wash Solution, the centrifugal 1min of 12000rpm, abandons filtrate, puts back to collection tube.7, add DNA contained in appropriate DNAase sample digestion, the centrifugal 1min of 14000rpm, sucks back filtrate on pillar, 25 DEG C of-30 DEG C of standing 15min.8, add 400 μ L Wash Solution, the centrifugal 1min of 14000rpm, abandons filtrate.9, third time adds 400 μ L Wash Solution, and the centrifugal 1min of 14000rpm, abandons filtrate.10, centrifugal column is put back to collection tube, the centrifugal 2min of 14000rpm, abandons collection tube.11, centrifugal column is placed in gains in depth of comprehension 1.5mL centrifuge tube, adds 50 μ L Elution Solution eluted rnas.12, the centrifugal 1min of 200 ~ 2,000rpm centrifugal 2min, 14000rpm, volume less than 50 μ L, then uses the centrifugal 1min(of 14000rpm can wash-out repeatedly).

(2) acquisition of cDNA

With carried RNA for template, reverse transcription obtains cDNA, use the SuperScript III First-Strand Synthesis Kit of Invitrogen company.RNA usage quantity in experiment is 1 μ g, and concrete steps are as follows:

1, reaction solution (10 μ L) is configured: 1 μ L RNA(≤5 μ g), 1 μ L Primer(Oligo dT), 1 μ L 10mM dNTP mix, DEPC-Treated Water up to 10 μ L.Of short duration low-speed centrifugal, after 5min, is placed in 1 ~ 2min on ice immediately by 65 DEG C.

2, in the PCR pipe of previous step, corresponding reagent is added in the following order: 2 μ L 10 × RT Buffer, 4 μ L 25mM MgCl ₂, 2 μ L 0.1M DTT, 1 μ L RNase OUT (40U/ μ L), 1 μ L SuperScript III RT.After soft mixing, be placed in PCR instrument, response procedures is 50 DEG C of 50min, 85 DEG C of 5min.

3, after low-speed centrifugal, often add the RNase H of 1 μ L in pipe, after 37 DEG C of digestion 20min, collect to obtain cDNA ,-20 DEG C save backup.

(3) homologous clone obtains goal gene

Utilize Oligo7.0 to design primer, carry out PCR, thus obtain goal gene, carry out conversion order-checking, finally compare.The primer sequence of design is as follows:

LhMKK2-F：5’-ATTTCGGACTTACAGTGCCACAAC-3’；

LhMKK2-R：5’-ACACTGCTCTTCCTCCATGTAACC-3’。

(1) PCR amplification system:

Following component preparation PCR reaction solution is pressed, 50 μ L reaction systems: 5 μ L 10 × PCR Buffer, 5 μ L 2mMdNTPs, 3 μ L 25mMMgSO according to specification sheets ₄, 1.5 μ L Forward Primer, 1.5 μ L Reverse Primer, 1 μ L cDNA, 1 μ L KOD-Plus polymerase, 32 μ L ddH ₂o.

(2) PCR reaction conditions: 94 DEG C, 2min; 98 DEG C, 10sec, 55-60 DEG C, 30sec, 68 DEG C, 1min, 36 cycles; 4 DEG C, Forever.

Hybridized Chinese tuliptree lhMKK2full length gene PCR primer detects at 1% agarose gel electrophoresis, and result as shown in Figure 2.PCR primer is detected at 1% agarose gel electrophoresis, obtain expection object fragment, the DNA gel of AXYGEN company is used to reclaim test kit, recovery purifying is carried out to object fragment, step is as follows: under ultraviolet lamp, 1) cut the sepharose containing target DNA, exhausts gel surface liquid and shred with paper handkerchief.Calculated for gel weight (recording 1.5mL centrifuge tube weight in advance), this weight is as a gel volume (as 100mg=100 μ L volume).2) add the Buffer DE-A of 3 gel volumes, in 75 DEG C of heating (low melting-point agarose gel is in 40 DEG C of heating) after mixing, be interrupted mixing (every 2-3min), until gel piece melts (about 6-8min) completely.3) add the Buffer DE-B of 0.5 Buffer DE-A volume, mix; When the DNA fragment be separated is less than 400bp, add the Virahol of 1 gel volume.4) draw the mixed solution in 3, transfer to DNA preparation pipe (being placed in 2mL centrifuge tube), the centrifugal 1min of 12000 × g.Abandon filtrate.5) put back centrifuge tube by preparing pipe, add 0.5 mL Buffer W1, the centrifugal 30s of 12000 × g, abandons filtrate.6) put back centrifuge tube by preparing pipe, add 0.7 mL Buffer W2, the centrifugal 30s of 12000 × g, abandons filtrate.7) 2mL centrifuge tube is placed in by preparing pipe, centrifugal 1 min of 12000 × g.8) be placed in clean 1.5mL centrifuge tube by preparing pipe, prepare film centre at DNA and add 25-30 μ L water or Eluent buffer, room temperature leaves standstill 1min, the centrifugal 1min eluted dna of 12000 × g.

Object fragment tailing.KOD-PLUS high-fidelity enzyme is flat terminal enzyme, and gained PCR primer is flat end, needs first to add poly A tail and just can carry out TA clone, step is as follows: 20 μ L systems: 10 μ L DNA, 2 μ L 10 × PCR Buffer, 0.4 μ L 10mMdNTPs, 1.6 μ L 25mMMgCl ₂, 0.2 μ L rTaq polymerase, 5.8 μ L ddH ₂o.Response procedures: 72 DEG C, 30min; 4 DEG C, Forever.

By goal gene fragment and cloning vector pMD ^tM19-T Vector(Takara D102A), carry out ligation.Linked system is as follows, in the PCR pipe of sterilizing, drying, adds following solutions successively: 4 μ L PCR purified product (10ng), 5 μ L Solution I, 1 μ L pMD ^tM19-T Vector.Inhale gently with pipettor and play mixing, low-speed centrifugal, 16 DEG C of connections are spent the night.

Connection product is proceeded in e. coli jm109 bacterial strain.Concrete steps are as follows: prepare LB solid medium (adding Amp+IPTG+X-gal) (each culture medium flat plate coating 50mg/mL IPTG and each 40uL of 20mg/mL X-gal, room temperature stand for standby use) before experiment; (1) take out competent cell from-80 DEG C of Ultralow Temperature Freezers, be placed in thawed on ice, the competent cell of 100uL adds the connection product of 5uL, mixes gently, ice bath 30min; (2) 42 DEG C of heat shock 90sec, are placed in immediately and leave standstill 2min on ice; (3) add 800uLLB liquid nutrient medium (not adding Amp microbiotic), 37 DEG C, 1-2h is cultivated in 180rpm concussion; 4) the centrifugal 3min of 4000rpm, washes 800uL supernatant off, will remain the suction of bacterium liquid and beat resuspended; (5) getting appropriate re-suspension liquid is uniformly coated on LB solid medium, is inverted for 37 DEG C and cultivates 12-16h.

Detect recombinant plasmid, the positive colony obtained is carried out sequencing analysis.Interpretation of result, lhMKK2genes encoding section length is 1190bp, comprises complete open reading frame (ORF), and sequence is as shown in SEQ ID NO.1, and coded protein sequence, as shown in SEQ ID NO.2, comprises 369 amino acid.By the sequence BLAST tool analysis in NCBI obtained that checks order, find through comparison, the MAPKK family gene homology of sequence and Arabidopis thaliana, Cortex Populi Tomentosae, tomato etc. reaches more than 90%.

Embodiment 2

(1) gene function analysis

First hybridized Chinese tuliptree 35S is built: lhMKK2over-express vector, and proceeded to agrobacterium strains, transform Liang Zhong wildtype Arabidopsis thaliana Colombia Col(Columbia by inflorescence infusion method) and blue thatch Burger Ler(Landsberg), obtain process LAN lhMKK2the positive transgenic plant of gene, and for positive plant, Stress treatment is carried out to T2, research and analyse hybridized Chinese tuliptree lhMKK2function.

(2) structure of carrier

The present invention's coli strain used is that precious biotechnology (Dalian) company limited of E. coli JM109(buys); Expression vector is that pBI121(Biovector Co., LTD company buys).

Detailed process is as follows:

1. existed by PCR lhMKK2gene fragment upstream and downstream adds Xba I and Sac I double enzyme site respectively, and PCR system and reaction conditions increase with total length, and the primer is as follows:

LhMKK2+Xba I-F：5’-TCTAGAATTTCGGACTTACAGTGCCACAAC-3’；

LhMKK2+ Sac I-R：5’-GAGCTCACACTGCTCTTCCTCCATGTAACC-3’。

2. recombinant plasmid carries out double digestion reaction through checking order correctly with carrier simultaneously.Use Xba I and Sac I restriction enzyme to carry out double digestion reaction, obtain containing cohesive terminus and cover whole ORF's lhMKK2gene fragment.With the pBI121 expression vector of same endonuclease reaction process sky.

Double digestion reaction system (20 μ L): 1ug reclaims product, 2 μ L 10 × M buffer, 1 μ L Xba I, 1 μ L Sac I, ddH ₂o up to 20 μ L.

Inhale gently and play mixing, 37 DEG C of water-baths, double digestion reaction 4h, adds 10 × stop buffer and stops endonuclease reaction.

Double digestion product is carried out separation detection through 1% agarose gel electrophoresis, and double digestion result, as shown in Fig. 3, Fig. 4, is judged according to stripe size lhMKK2gene and pBI121 expression vector are correctly cut.

By goal gene fragment, the double digestion product of empty expression vector PBI121, uses AxyPrep DNA Gel Extraction Kit(AXYGEN) gel reclaims test kit and carries out recovery purifying, be dissolved in the TE damping fluid of 20 μ L.

3. detect the digestion products concentration reclaimed and purity, add each reagent (object fragments molecules number: carrier molecule number=3 ~ 5:1) by linked system, 16 DEG C of water-baths connect spends the night.20uL ligation system is as follows: 2 μ L T4 DNA ligase buffer(10 ×), 1 μ L pBI121,4 μ L object fragments, 1 μ L T4 DNA ligase, 12 μ L ddH ₂o.

4. connect the super competent cell of product conversion e. coli jm109, picking list colony inoculation is in LB liquid nutrient medium, and 37 DEG C of concussions are cultured to OD ₆₀₀value is 0.55-0.6; Use total length primer to carry out bacterium liquid PCR, with screening positive clone, use AxyPrep Plasmid Miniprep Kit(AXYGEN afterwards) extract plasmid and carry out double digestion checking.Positive colony is checked order simultaneously, detect in vector construction process whether base mutation or deficient phenomena occur.Build over-express vector as shown in Figure 5, comprise promotor, goal gene, the length of terminator and position.

(2) real-time fluorescence quantitative PCR

Select materials is respectively hybridized Chinese tuliptree different sites material, and hybridized Chinese tuliptree 1 × 5002genotype body embryonic development different times material, comprises complete somatic embryo growth material in each period, right lhMKK2gene carries out RT-PCR, and analyzing it is the differential expression situation with different development stage in different tissues portion.Detailed process is as follows:

Real-time quantitative PCR adopts the Power SYBR Green PCR Master Mix test kit of ABI company, at ABI 7500 Real time PCR Systems(Applied Biosystems) on complete, each example reaction in triplicate, data acquisition 7500 System SDS software(Applied Biosystems) extraction and analysis.

1) design of RT-PCR primer

According to the requirement of RT-PCR, devise lhMKK2the primer of gene, through the analysis of solubility curve, the 18S rRNA in final selection hybridized Chinese tuliptree is as internal reference, and primer is as follows:

18SrRNA-F：5’-ATTTCTGCCCTATCAACTTTCG-3’；

18SrRNA-R：5’-TTGTTATTTATTGTCACTACCTCCC-3’；

LhMKK2-F（qPCR）：5’-GCTTTCCTTATTCACCGCCTG-3’；

LhMKK2-R（qPCR）：5’-TGTGCTGCCTTTCTATCCTTCG-3’。

2) RT-PCR reaction system (20 μ L): 10 μ L 2 × Power SYBR Green PCR Master Mix, 1 μ L Forward Primer, 1 μ L Reverse Primer, 1 μ L cDNA, 7 μ L ddH ₂o.

3) qRT-PCR reaction conditions: 95 DEG C, 10min; 95 DEG C, 15sec, 60 DEG C, 1min, 40 Cycles.

4) test materials

Hybridized Chinese tuliptree different sites material.Be respectively the stamen of hybridized Chinese tuliptree seedling, root, stem, leaf, bud, petal, petal, gynoecium.Hybridized Chinese tuliptree different development stage material.With hybridized Chinese tuliptree 1 × 5002genotype is starting materials, and the somatic embryo that induction obtains grows the synchronization material of different times.Comprise: embryo callus, liquid suspension cell, the cell adjustment period of liquid suspension system, globular embryo, torpedo embryo, early stage cotyledonary embryos, seven periods of ripe cotyledon embryo.

5) test-results

Found that according to real-time quantitative PCR, lhMKK2gene at hybridized Chinese tuliptree different sites root, stem, leaf, bud, flower, petal, stamen, all has expression in gynoecium, inorganization specificity (as shown in Figure 6); lhMKK2the expression amount that gene grows different times at hybridized Chinese tuliptree somatic embryo has larger difference, and the expression amount in embryo callus is the highest, does not all express period at globular embryo and torpedo embryo, the expression amount in ripe cotyledon embryo all lower (as shown in Figure 7).

(3) conversion of Agrobacterium

1. the agrobacterium strains that the present invention uses buys for GV3101(Biovector Co., LTD company), adopt frozen-thawed method to build lhMKK2over-express vector proceeds to Agrobacterium.Detailed process is as follows: 1) ice bath melted Agrobacterium competent cell, and often pipe adds 1-5uL(100ng) reclaim the recombinant plasmid of purifying, inhale gently and play mixing, be placed on ice, standing ice bath 30min.2) liquid nitrogen flash freezer 1min, 37 DEG C of thermal shock 1-5min, are placed in rapidly 1-2min on ice.3) the LB liquid nutrient medium of 800ul antibiotic-free is added, 28 DEG C, 100rpm, at a slow speed shaking culture 2-4h.4) the centrifugal 3min of 4000rpm, sucks part supernatant.5) leave and take appropriate bacterium liquid, mix gently, coat on the LB solid medium containing 50mg/L Kan and 50mg/L Rif.6) cultivation 30-48h is inverted, to growing single bacterium colony for 28 DEG C.7) bacterium liquid PCR detects positive colony, and 4 DEG C save backup.

2. Arabidopis thaliana to be planted grows to blooms, and maintains its state of health.The positive colony be detected by PCR, shakes bacterium to OD ₆₀₀during for 0.6-0.8, inflorescence infusion method is adopted to be proceeded in wildtype Arabidopsis thaliana by goal gene.Detailed process is as follows: 1) by bacterium liquid 5000 rpm, 5 min are centrifugal, collects thalline, with the 1/2MS solution suspension containing 5% sucrose; 2) before immersion, add the SilwetL-77 that concentration is 0.05%, shake out foam; 3) over-ground part of Arabidopis thaliana is soaked 15 ~ 30 sec in Agrobacterium aaerosol solution, period rocks gently; 4) Arabidopis thaliana soaked is kept flat on pallet, cover moisturizing with preservative film, masking foil sealing lucifuge 24h; 5) take off preservative film, under normal condition, be cultured to seed maturity, stop after ripe watering.

(5) transfer-gen plant Phenotypic Observation

1. the seed harvested drying, adds 50mg/L card by T1 for seed and receives the 1/2MS substratum of mycin and screen, find positive plant still normal growth, and without blocking, to receive the plant of chloramphenicol resistance dead for other.

2. by the possible transgenic positive plantlet of transplant that filters out in Nutrition Soil, normally cultivate.Choose transgenic positive plant, get appropriate young leaflet tablet, extract DNA and carry out PCR detection as template, determine that it is positive plant.T2 is identical with it for positive plant detection method.As shown in Figure 8, display negative control is without band, and transfer-gen plant PCR object band is consistent with positive control, is defined as positive plant for detected result.

3. process LAN lhMKK2the T1 of gene is for the Phenotypic Observation of transfer-gen plant.Discovery turns the transfer-gen plant under wild-type Ler background, compares with wild-type Ler, and occur that plant strain growth is slow, pod is short and small, the phenotypic characteristic of abortion.Turn the transfer-gen plant under wild-type Col background, compare with wild-type Col, occur that plant strain growth is slow, the phenotypic characteristic of leaf rolling.Result as Fig. 9, shown in 10.

4. process LAN lhMKK2the T2 of gene is for the statistical study of transfer-gen plant.Add up Shoot number respectively, side shoot number, lobe numbers etc., calculating mean value, transfer-gen plant grows slow because of microbiotic impact early stage than wildtype Arabidopsis thaliana, after waiting transplanting to grow one week to Nutrition Soil, start fast growth, observe the major branch finding transgenic line, lotus throne leaf branch amount and stem leaf branch amount are all more than wild-type, and lobe numbers is also more than wildtype Arabidopsis thaliana.As table 1, shown in Figure 11.

Table 1. transfer-gen plant grows the statistics of two weeks

5. process LAN lhMKK2the T2 of gene is for the salt stress process of transfer-gen plant.Choose three strains respectively, be numbered respectively: T2-17, T2-21, T2-23, NaCl concentration gradient is: 0mmol/L, 50mmol/L, 100mmol/L, 200mmol/L; The each process of each strain each repetition three ware, each 50 strains of every ware, growth 7d, period observes plant strain growth situation, and adds up lethal plant number and lethality rate.Result shows, and under the NaCl condition of salt stress of 50mmol/L, 100mmol/L, transfer-gen plant and wildtype Arabidopsis thaliana phenotype are all without too big-difference, and plant can normal growth, does not all occur that plant is lethal.Under the NaCl salt stress of 200mmol/L, albefaction appearance is dead gradually for the 3rd day plant leaf, and substantially all lethal to the 7th day wildtype Arabidopsis thaliana, lethality rate reaches 96%, and transfer-gen plant on average still has 15 strain normal growths, and lethality rate is up to 70%.Statistics is as shown in table 2, phenotype as Figure 12, shown in 13.

The growing state of transfer-gen plant and wildtype Arabidopsis thaliana under the NaCl salt stress of table 2. 200mmol/L

As can be seen here, lhMKK2gene involved in plant growth and development process, process LAN lhMKK2gene can improve the resistance of plant to salt stress, and involved in plant environment stress responds.

SEQUENCE LISTING

<110> Nanjing Forestry University

<120> hybridized Chinese tuliptree lhMKK2gene and expressing protein thereof and application

<130> 100

<160> 10

<170> PatentIn version 3.3

<210> 1

<211> 1190

<212> DNA

<213> hybridized Chinese tuliptree lhMKK2gene order

<400> 1

atttcggact tacagtgcca caactggtga acacctaatt tccatgtccc gcgggcgatt 60

gctgctaaag gtagacgaag ccatgaaaac tggtggacgc tccaacctca acaagctcaa 120

gctccatctt cctcctccag atgaaatcta cttctccaaa ttcctgactc aaagcggtac 180

attcaaggac ggcgatctcc tcgtcaacaa ggatggcctc cgaatcgtcc ctcagaacaa 240

agaagaggag cctcctgtta taaaaccatt ggacaaccag ttaactttag ctgatgtaga 300

cactattaaa gtgattggaa agggtagtgg tggggttgtg caactagtgc gccacaagtg 360

gaccggccag ttctttgctc tgaaggttat ccaaatgaat attttggaga gtgtccgcaa 420

gcagattgcg caggaattga agattaatca gtcctcacaa tgcccatatg ttgtcgtttg 480

ttaccactct ttctacgata acggtgtcat atctatagtt ttagagtaca tggatggtgg 540

gtcgcttgcg gattttctga agaaagtgag atcaattccg gagccatatc ttgctgctat 600

ctgtaagcag gtactcaagg gtttgatgta tcttcatcat gaaaaacaca taatccatag 660

ggatttaaaa ccgtcaaatt tattgattaa tcatagagga gaagtcaaga taactgattt 720

tggtgttagt gccatattag cgagtacctc tggtcagcgg gatacttttg ttggcactta 780

caactacatg tcgccagaga gaattagtgg aggcacctat ggctacaaaa gtgacatttg 840

gagcttgggg atagttttgc tggagtgtgc cacgggcagc tttccttatt caccgcctga 900

acaagaggct ggatggacta acttttatga gcttttggaa gcaatagttg atcaaccatc 960

gccttctgca tcttctgacc atttttcgcc agagttctgc tcatttattt cagcatgtgt 1020

acaaaaagac ccgaaggata gaaaggcagc acatgtacta ttggaacatc ctttcctgag 1080

catgtatgga gacctggata gtgaacttgc gtcgtacttc accagcgctg gatctccact 1140

tgccaccttc tgaatatctt ccatctggtt acatggagga agagcagtgt 1190

<210> 2

<211> 369

<212> PRT

<213> hybridized Chinese tuliptree lhMKK2the expressing protein of gene

<400> 2

Met Ser Arg Gly Arg Leu Leu Leu Lys Val Asp Glu Ala Met Lys Thr

1 5 10 15

Gly Gly Arg Ser Asn Leu Asn Lys Leu Lys Leu His Leu Pro Pro Pro

20 25 30

Asp Glu Ile Tyr Phe Ser Lys Phe Leu Thr Gln Ser Gly Thr Phe Lys

35 40 45

Asp Gly Asp Leu Leu Val Asn Lys Asp Gly Leu Arg Ile Val Pro Gln

50 55 60

Asn Lys Glu Glu Glu Pro Pro Val Ile Lys Pro Leu Asp Asn Gln Leu

65 70 75 80

Thr Leu Ala Asp Val Asp Thr Ile Lys Val Ile Gly Lys Gly Ser Gly

85 90 95

Gly Val Val Gln Leu Val Arg His Lys Trp Thr Gly Gln Phe Phe Ala

100 105 110

Leu Lys Val Ile Gln Met Asn Ile Leu Glu Ser Val Arg Lys Gln Ile

115 120 125

Ala Gln Glu Leu Lys Ile Asn Gln Ser Ser Gln Cys Pro Tyr Val Val

130 135 140

Val Cys Tyr His Ser Phe Tyr Asp Asn Gly Val Ile Ser Ile Val Leu

145 150 155 160

Glu Tyr Met Asp Gly Gly Ser Leu Ala Asp Phe Leu Lys Lys Val Arg

165 170 175

Ser Ile Pro Glu Pro Tyr Leu Ala Ala Ile Cys Lys Gln Val Leu Lys

180 185 190

Gly Leu Met Tyr Leu His His Glu Lys His Ile Ile His Arg Asp Leu

195 200 205

Lys Pro Ser Asn Leu Leu Ile Asn His Arg Gly Glu Val Lys Ile Thr

210 215 220

Asp Phe Gly Val Ser Ala Ile Leu Ala Ser Thr Ser Gly Gln Arg Asp

225 230 235 240

Thr Phe Val Gly Thr Tyr Asn Tyr Met Ser Pro Glu Arg Ile Ser Gly

245 250 255

Gly Thr Tyr Gly Tyr Lys Ser Asp Ile Trp Ser Leu Gly Ile Val Leu

260 265 270

Leu Glu Cys Ala Thr Gly Ser Phe Pro Tyr Ser Pro Pro Glu Gln Glu

275 280 285

Ala Gly Trp Thr Asn Phe Tyr Glu Leu Leu Glu Ala Ile Val Asp Gln

290 295 300

Pro Ser Pro Ser Ala Ser Ser Asp His Phe Ser Pro Glu Phe Cys Ser

305 310 315 320

Phe Ile Ser Ala Cys Val Gln Lys Asp Pro Lys Asp Arg Lys Ala Ala

325 330 335

His Val Leu Leu Glu His Pro Phe Leu Ser Met Tyr Gly Asp Leu Asp

340 345 350

Ser Glu Leu Ala Ser Tyr Phe Thr Ser Ala Gly Ser Pro Leu Ala Thr

355 360 365

Phe

<210> 3

<211> 24

<212> DNA

<213> Artificial

<220>

<223> LhMKK2-F primer sequence

<400> 3

atttcggact tacagtgcca caac 24

<210> 4

<211> 24

<212> DNA

<213> Artificial

<220>

<223> LhMKK2-R primer sequence

<400> 4

acactgctct tcctccatgt aacc 24

<210> 5

<211> 30

<212> DNA

<213> Artificial

<220>

<223> LhMKK2+Xba I-F primer sequence

<400> 5

tctagaattt cggacttaca gtgccacaac 30

<210> 6

<211> 30

<212> DNA

<213> Artificial

<220>

<223> LhMKK2+ Sac I-R primer sequence

<400> 6

gagctcacac tgctcttcct ccatgtaacc 30

<210> 7

<211> 22

<212> DNA

<213> Artificial

<220>

<223> 18SrRNA-F primer sequence

<400> 7

atttctgccc tatcaacttt cg 22

<210> 8

<211> 25

<212> DNA

<213> Artificial

<220>

<223> 18SrRNA-R primer sequence

<400> 8

ttgttattta ttgtcactac ctccc 25

<210> 9

<211> 21

<212> DNA

<213> Artificial

<220>

<223> LhMKK2-F(qPCR) primer sequence

<400> 9

gctttcctta ttcaccgcct g 21

<210> 10

<211> 22

<212> DNA

<213> Artificial

<220>

<223> LhMKK2-R(qPCR) primer sequence

<400> 10

tgtgctgcct ttctatcctt cg 22

Claims (4)

1. a hybridized Chinese tuliptree lhMKK2gene, its nucleotide sequence is as shown in SEQ ID NO.1.

2. hybridized Chinese tuliptree according to claim 1 lhMKK2the expressing protein of gene, its aminoacid sequence is as shown in SEQ ID NO.2.

3. hybridized Chinese tuliptree according to claim 1 lhMKK2the application of gene in hybridized Chinese tuliptree resistance molecular breeding.

4. containing hybridized Chinese tuliptree according to claim 1 lhMKK2the carrier of gene or host cell.