CN101302523B

CN101302523B - Application of physiological clock regulation protein LHY to stress-tolerant plant cultivation

Info

Publication number: CN101302523B
Application number: CN2008101163072A
Authority: CN
Inventors: 陈蕾; 吴茜; 李钦清
Original assignee: BEIFANGJIESHI BIOLOGICAL SCIO-TECH Co Ltd BEIJING
Current assignee: BEIFANGJIESHI BIOLOGICAL SCIO-TECH Co Ltd BEIJING
Priority date: 2008-07-08
Filing date: 2008-07-08
Publication date: 2011-05-18
Anticipated expiration: 2028-07-08
Also published as: CN101302523A

Abstract

The invention discloses application of regulating protein LHY by a biological clock in culture for a reverse resistance plant. The protein LHY is regulated by the biological clock, which is applied to the culture for the reverse resistance plant. The invention provides a method for providing culture reverse resistance plant and comprises the following steps of: introducing an encoding gene that the protein LHY is regulated by the biological clock in a plant cell; and obtaining the reverse resistance plant. An experiment shows that the encoding gene which the protein LHY is regulated by the biological clock is introduced in a plant cell, thereby regulating the expression by a constitutive promoter without regulation of light, improving adversity of the plant, in particular to stress tolerance of drought/salt stress, improving biomass of the plant, and having profound theoretical meaning and wide practical meaning.

Description

The application of physiological clock regulation protein LHY in cultivating plant with adverse resistance

Technical field

The present invention relates to the application of physiological clock regulation protein LHY in cultivating plant with adverse resistance.

Background technology

The gene expression profile of plant can change under the environment stress, to studies show that of Arabidopis thaliana, about 30% gene expression amount changes above 2 times of (transcriptome Changes for Arabidopsis in Response toSalt, Osmotic, and Cold Stressl.Joel A.Kreps, Yajun Wu, Hur-Song Chang, Tong Zhu, Xun Wang, and Jeff F.Harper Plant Physiology, December 2002, Vol.130 pp.2129-2141), comprises following proteic encoding gene: 1) participate in the ionophorous protein that plant stress is replied directly, aquaporin, the osmoregulation factor (proline(Pro) and trimethyl-glycine etc.) synthetic enzyme; 2) participate in coercing the protein factor (protein kinase, transcription factor) that relevant signal transmission and genetic expression are regulated, as DREB class transcription factor family, bZIP (basic region/leucine zipper motif transcription factors) the class transcription factor that contains alkaline zone and leucine zipper, the MYC family of containing alkaline helix-loop-helix (bHLH) and leucine zipper and MYB family etc. with tryptophane bunch (Trp cluster).

LHY belongs to the protein-bonded encoding gene of MYB class DNA, is plant physiology clock regulation gene.The blooming of plant physiology clock regulation gene regulating photoperiodic induction, the growth of plumular axis, the motion of cotyledon and switch of pore etc.LHY constitutes the reverse feedback gene ring of regulating and control Arabidopis thaliana light, dark cycle physiological rhythm with another gene TOC1, be in the relative upstream position of plant physiology regulation and control, up to now, be considered to blooming period of plant, postpone the period of blooming that LHY crosses the express transgenic Arabidopis thaliana, vegetative growth stage prolongs (Light-regulatedtranslation mediates gated induction of the Arabidopsis clock protein LHY.The EMBO Journal, 2003,22 (4): 935-944).

Summary of the invention

The purpose of this invention is to provide the application of physiological clock regulation protein LHY in cultivating plant with adverse resistance.

The invention provides a kind of method of cultivating plant with adverse resistance, is with the encoding gene importing vegetable cell of physiological clock regulation protein LHY, obtains plant with adverse resistance.

Described physiological clock regulation protein LHY is following (a) or protein (b):

(a) protein of forming by the aminoacid sequence shown in the sequence in the sequence table 1;

(b) with the aminoacid sequence of sequence 1 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and have the physiological clock regulation function by sequence 1 deutero-protein.

The plant physiology clock regulation protein LHY (GenBank Accession NumberAJ006404) that derives from Arabidopis thaliana is made up of 645 amino-acid residues, seeing the sequence 1 of sequence table, is MYB class DNA binding domains from the 27th of aminoterminal to the 83rd amino acids residue in the sequence 1.

The replacement of described one or several amino-acid residue and/or disappearance and/or interpolation are meant replacement and/or the disappearance and/or the interpolation of no more than 30 amino-acid residues that carry out beyond described structural domain.

The encoding gene of described physiological clock regulation protein LHY can be following 1) or 2) or 3) or 4) dna molecular:

1) its encoding sequence be in the sequence table sequence 2 from the dna molecular shown in the deoxyribonucleotide of 5 ' terminal 60-1997 position;

2) its nucleotide sequence is the dna molecular shown in the sequence 2 in the sequence table;

3) under stringent condition with 1) or 2) the dna sequence dna hybridization that limits and the dna molecular of encoding said proteins;

4) with 1) or 2) dna sequence dna that limits has 90% above homology, and the dna molecular of encoding said proteins.

Above-mentioned stringent condition can be at 5 * SSC, 5 * Denhardt ' S solution, 0.05mg/mL milt DNA, in the 50% deionized formamide solution, 65 ℃ of down hybridization, then in room temperature with 2 * SSC, 0.1%SDS, 42 ℃ with 0.25 * SSC, 0.1%SDS respectively washes film 15 minutes, twice.

The encoding gene LHY (GenBank AccessionNumber AJ006404) that derives from the plant physiology clock regulation protein of Arabidopis thaliana is made up of 1938 deoxynucleotides, for the sequence 1 of sequence table from 5 ' terminal 60-1997 position deoxyribonucleotide.

The encoding gene of described physiological clock regulation protein LHY can import in the vegetable cell by any method, as importing in the vegetable cell by expression vector shown in Figure 1.

Described resistance of reverse specifically can be salt tolerant and/or drought-enduring.

The present invention also protects expression vector shown in Figure 1.

The transgenic cell line or the reorganization bacterium that contain expression vector shown in Figure 1 also belong to protection scope of the present invention.

Described reorganization bacterium specifically can import Agrobacterium LBA4404 with expression vector shown in Figure 1 and obtain.

11 genes in the photosynthetic light collection reaction (light-harvesting reactions) and 10 genes of photosystem reactive center all are subjected to the regulation and control of biologic clock.Involved in plant carbon, nitrogen, sulphur are synthetic, the gene of metabolic exhaustion and transportation storage approach also is subjected to the regulation and control of biologic clock, gene as 6 involved in sugar decomposition and oxidation pentose phosphate path, 2 participations are the gene in Triphosaden path with conversion of glucose, 9 genes that participate in the adjusting of nitrogen, 5 genes that participate in the sulphur digestion and metabolism etc.Therefore we can say that plant physiology clock regulation gene also regulated and control the basal metabolism process of the sugar of vegetable cell, nitrogen, sulphur etc.The contriver thinks that anti-contrary and these two physiological regulating control processes of vegetable cell basal metabolism of plant are close association.

Experiment shows, the overexpression of coding LHY protein gene can strengthen plant to adverse circumstance, particularly the resistance of reverse of drought/salt stress shows that LHY albumen plays a significant role, and has also proved anti-contrary and these two physiological regulating control process close association of basal metabolism of plant in the anti-contrary mechanism of plant.The proteic overexpression of LHY can also improve the biological total amount of plant simultaneously.LHY albumen is applied to plant breeding, cultivates transgenic plant, can strengthen the resistance of reverse of plant and improve the biological total amount of plant, have far-reaching theory significance and reach practice significance widely.

Following embodiment is convenient to understand better the present invention, but does not limit the present invention.

Description of drawings

Fig. 1 is a LHY plant expression vector physical map

Fig. 2 is the upgrowth situation of LHY transgenic arabidopsis under normal culture condition

Fig. 3 is the drought-enduring experimental result of LHY transgenic arabidopsis

Fig. 4 is a LHY transgenic arabidopsis salt tolerant experimental result

Fig. 5 is the PCR checking photo of LHY transgenic arabidopsis

Embodiment

Experimental technique among the following embodiment if no special instructions, is ordinary method.

The test method that adopts among the embodiment is ordinary method, referring to following document: Sambrook andRussell " Molecular Cloning:A Laboratory Manual " (2001); Cloning:APractical Approach, " Volumes I and II (D.N.Glover, ed., 1985); " Wang Guanlin, Fang Hongjun, " plant genetic engineering " second edition, 2002 the 2nd edition.

The structure of embodiment 1, plant expression vector

One, the acquisition of LHY encoding gene

1, extracts RNA

Liquid nitrogen grinds Arabidopis thaliana (Arabidopsis thaliana) blade of-70 ℃ of preservations, adds 1ml TRIZOL RNA extracting solution (ancient cooking vessel state biotech company) in the blade of every 0.1g, extracts total RNA.(Prmega, America) remaining DNA is removed in digestion, with the concentration of total RNA in spectrophotometer (Eppendorf company, the Germany) test sample with the DNase enzyme with the total RNA that obtains.

2, the acquisition of LHY encoding gene

As follows according to a pair of primer of the LHY gene order on the NCBI (GenBank Accession Number AJ006404) design:

LHY-S：5’GGGATACTTTACTTGTTAGAGAGGATTTG；

LHY-A：5’-AAGATATTGATAAAAATGTGGATGT。

Get the total RNA of 5 μ g, (precious biotechnology (Dalian) company limited) carries out reverse transcription with the reverse transcription test kit, is that template is carried out pcr amplification reaction with the cDNA fragment that obtains.

25 μ l PCR reaction systems are: 0.1 μ gcDNA, 1.5mM MgCl ₂, 20mM Tris-HCl (pH8.4), 50mMKCl, 0.2mM dNTP mixture, 0.2 μ M primer LHY-S and 0.2 μ M primer LHY-A, 1U Taq polysaccharase (Shen, Shanghai can lottery industry biotech company).

In PCR-thermal cycler (Eppendorf company, Germany), carry out PCR circulation: 94 ℃ of pre-sex change 5 minutes; Again 94 ℃ 30 seconds, 52 ℃ 30 seconds, 72 ℃ 2 minutes, totally 30 circulations; Last 72 ℃ 10 minutes.

Obtained the dna fragmentation of about 2kb behind the pcr amplification, reclaimed test kit (sky, Beijing latitude Time Technology company limited) with gel and reclaim this fragment and order-checking (three rich polygala root biotech companies).Sequencing result shows that this clip size is 2044bp, and nucleotide sequence contains LHY gene order (the 60th to the 1997th Nucleotide of sequence 1) shown in the sequence in the sequence table 1.

Sma I site with the dna fragmentation that obtains inserts plasmid pUC18 (Genbank Accession L09136) obtains recombinant plasmid, with recombinant plasmid called after pUC18::ATLHY.

Two, plant expression vector construction

1, the structure of pBI121m

1) BstXI and XhoI double digestion plasmid pCAMBIA2300 (Genebank:AF234315) reclaim the long 35S promoter of about 780bp, are connected to through having on the identical complementary terminal plasmid pUC18 that the SphI-SalI enzyme is cut, obtain plasmid pUC18-35S.

2) plasmid pUC18-35S of obtaining of HindIII and XbaI double digestion step 1) reclaims the 795bp small segment, is connected with pBI121 (Genebank:AF485783) carrier framework that same enzyme is cut, with the recombinant plasmid called after pBI121ml that obtains.

3) SmaI and SacI double digestion pBI121 insert the 1887bp fragment that contains the gusA gene that obtains on the SmaI site of plasmid pUC18 after mending flat the processing, obtain pUC18-gusA.

4) plasmid pUC18-gusA of obtaining of SmaI and SacI double digestion step 3) inserts the 1895bp fragment that obtains in the carrier framework of the pBI121ml that same enzyme cuts, with the recombinant plasmid called after pBI121m that obtains.

Two, the structure of plant expression vector pBI121m::ATLHY

KpnI and SmaI double digestion plasmid pUC18:: ATLHY, reclaim the LHY gene fragment of 2.04kb, be inserted on the plasmid pBI121m of same double digestion, with the recombinant plasmid called after pBI121m::ATLHY that obtains, the structure of plasmid pBI121m::ATLHY as shown in Figure 1, gene is subjected to the control of promotor 35S, and terminator is Tnos.

Change pBI121m::ATLHY over to Agrobacterium LBA4404, obtain containing the agrobacterium strains of pBI121m::ATLHY, with this bacterial strain called after TpBI-ATLHY.

The acquisition of embodiment 2, transfer-gen plant and transfer-gen plant resistance of reverse

One, the acquisition of transfer-gen plant

Infect Arabidopis thaliana (Arabidopsis thaliana) with the TpBI-ATLHY Agrobacterium respectively, to obtain the changeing LHY gene plant, concrete operations are as follows:

Arabidopis thaliana is cultivated according to a conventional method, and culture condition is: light application time 16/8 hour, intensity of illumination is not less than 5000Lux, 22 degrees centigrade of temperature, humidity 70%.

Developing medium is vermiculite and turfy soil by 1: 1 mixing, (per 2 premium on currency add 1 gram to water once " spending intact " nutritive medium weekly, Shanghai Wintong Chemicals Co., Ltd.), behind about 25 days of the growth of seedling, long the pinching from base portion during to high 8 centimetres of left and right sides of first inflorescence removed, after week age secondary bolting, when just will having showed money or valuables one carries unintentionally, infects in bud with Agrobacterium.Agrobacterium working concentration OD6001.2-1.4, bacterium liquid 4000 leaves the heart and collected thalline in 10 minutes, suspend with isopyknic 5% sucrose solution, survey its OD600 value, the back sucrose with 5% that converts is diluted to the working concentration OD6000.8 when contaminating, and adds the Silwet L-77 solution of ten thousand/three volumes, contaminates behind the mixing, the dip-dye method is taked " dipping in colored method ", promptly draws the Agrobacterium drop with the rifle head and dips in titbit.Shading normal growth again after 24 hours.Carry out secondary infection after 5-6 days, contaminate altogether 3 times.Infecting about about one month receipts in end back plants.

Gained T1 filters out transgenic seedling for seed on the resistance substratum, method is: 70% alcohol surface sterilization 1 minute, the chlorine bleach liquor (5% clorox+0.5%SDS), sterilized 15 minutes, sterilized water washing 5 times is seeded in 1/2MS solid medium (adding kantlex 30mg/l+ carboxylic Bian 125mg/l).4 ℃ of refrigerators are placed and were placed on illumination cultivation between normal cultivation in 3 days, after ten days the resistance seedling are moved in the nutraceutical matrix of vermiculite and turfy soil, the conventional cultivation, receive behind two first quarter moons kind for T2 for seed.

The T2 that will obtain with aforesaid method screening becomes seedling for the cultivating seeds of plant, has obtained the commentaries on classics LHY gene Arabidopis thalianas of 30 strain systems altogether.

Under normal culture condition, cultivated two months, observe and take pictures, see Fig. 2.Among Fig. 2, a left side is for changeing LHY gene Arabidopis thaliana, and the right side is the wild-type Arabidopis thaliana.The result shows, changes the LHY gene Arabidopis thaliana growing way of nourishing and growing and obviously is better than the wild-type Arabidopis thaliana, and reproductive growth is delayed.

Two, drought-enduring test

The commentaries on classics LHY gene Arabidopis thaliana of 30 strain systems and the wild-type Arabidopis thaliana of not screening are carried out drought-enduring experiment.Concrete grammar is as follows:

Commentaries on classics LHY gene Arabidopis thaliana that will be through 30 strains of that screening of card and the wild-type Arabidopis thaliana that does not screen, each strain is each 7 strain, is transplanted to the both sides of same little alms bowl simultaneously symmetrically.Each strain system establishes 3 repetitions.Routine Management, watering stops to water after 3 times, three week the back rehydrations, observe after the week and take pictures.

Drought-enduring experimental example is seen Fig. 3.Among Fig. 3, a left side is for changeing LHY gene Arabidopis thaliana, and the right side is the wild-type Arabidopis thaliana, separates with the person who is not a member of any political party.

Experimental result shows that the wild-type Arabidopis thaliana is all withered, recovers well-grown and change LHY gene Arabidopis thaliana.

Three, salt tolerant test

The commentaries on classics LHY gene Arabidopis thaliana of 30 strain systems and the wild-type Arabidopis thaliana of not screening are carried out the salt tolerant experiment.Concrete grammar is as follows:

Commentaries on classics LHY gene Arabidopis thaliana that will be through 30 strains of screening and the wild-type Arabidopis thaliana that does not screen, each strain is each 6 strain, is transplanted to the both sides of same little alms bowl simultaneously symmetrically.Each transgenic line is established 3 repetitions.First week watered, second week and water the NaCl salt solution of 200mM and 300mM the 3rd week respectively.Observe around the and take pictures.

The salt tolerant experimental example is seen Fig. 4.Among Fig. 4, a left side is for changeing LHY gene Arabidopis thaliana, and the right side is the wild-type Arabidopis thaliana, separates with the person who is not a member of any political party.

The result shows that the wild-type Arabidopis thaliana is dead gradually, though and change the growth of LHY gene Arabidopis thaliana and be affected, it is green that plant still keeps.

Four, PCR identifies

All the commentaries on classics LHY gene strain of survival is 4 in the test of step 2 and step 3.

Getting these 4 strains is that T2 is for the blade of plant and the blade of wild-type Arabidopis thaliana, extract genomic dna (Steiner JJ respectively with the CTAB method, Poklemba CJ, Fjellstrom RG, Elliott LF., A rapidone-tube genomic DNA extraction process for PCR and RAPD analyses.NucleicAcids Res.1995 Jul 11; 23 (13): 2569-70.).The genomic dna that extracts with 500ng is that template is carried out the PCR reaction respectively.Primer is as follows:

Primers F: 5 '-CCCACTATCCTTCGCAAGACC (coming from the 35s promoter sequence)-3 ';

Primer R:5 '-CATGAGAAGCCCACCAAGCA-3 '.

Reaction system is 25 μ l, contains 500ng genomic dna, 1.5mM MgCl ₂, 20mM Tris-HCl (pH8.4), 50mM KCl, 0.2mM dNTP mixture, 0.2 μ M primers F and 0.2 μ M primer R, 1U Taq polysaccharase (Shen, Shanghai can lottery industry biotech company).

In PCR-thermal cycler (Eppendorf company, Germany), carry out PCR circulation: earlier 94 ℃ 5 minutes; Again 94 ℃ 30 seconds, 52 ℃ 30 seconds, 72 ℃ 1 minute, totally 30 circulations; Last 72 ℃ 10 minutes.

Amplified production is carried out electrophoresis, and the result as shown in Figure 5.Among Fig. 5,1:1Kb Marker; 2: the wild-type Arabidopis thaliana; 3-6: change LHY gene Arabidopis thaliana.

The result shows that the pcr amplification product that changes LHY gene Arabidopis thaliana has the clear band of 1.2kb, with estimate identical, and the wild-type Arabidopis thaliana does not have the 1.2kb band.

Sequence table

＜110〉Beijing North Jieshi Biological Technology Co.,Ltd

＜120〉application of physiological clock regulation egg LHY in cultivating plant with adverse resistance

<130>CGGNARY81461

<160>2

<210>1

<211>645

<212>PRT

＜213〉Arabidopsis Arabidopis thaliana (Arabidopsis thaliana)

<400>1

Met?Asp?Thr?Asn?Thr?Ser?Gly?Glu?Glu?Leu?Leu?Ala?Lys?Ala?Arg?Lys

1 5 10 15

Pro?Tyr?Thr?Ile?Thr?Lys?Gln?Arg?Glu?Arg?Trp?Thr?Glu?Asp?Glu?His

20 25 30

Glu?Arg?Phe?Leu?Glu?Ala?Leu?Arg?Leu?Tyr?Gly?Arg?Ala?Trp?Gln?Arg

35 40 45

Ile?Glu?Glu?His?Ile?Gly?Thr?Lys?Thr?Ala?Val?Gln?Ile?Arg?Ser?His

50 55 60

Ala?Gln?Lys?Phe?Phe?Thr?Lys?Leu?Glu?Lys?Glu?Ala?Glu?Val?Lys?Gly

65 70 75 80

Ile?Pro?Val?Cys?Gln?Ala?Leu?Asp?Ile?Glu?Ile?Pro?Pro?Pro?Arg?Pro

85 90 95

Lys?Arg?Lys?Pro?Asn?Thr?Pro?Tyr?Pro?Arg?Lys?Pro?Gly?Asn?Asn?Gly

100 105 110

Thr?Ser?Ser?Ser?Gln?Val?Ser?Ser?Ala?Lys?Asp?Ala?Lys?Leu?Val?Ser

115 120 125

Ser?Ala?Ser?Ser?Ser?Gln?Leu?Asn?Gln?Ala?Phe?Leu?Asp?Leu?Glu?Lys

130 135 140

Met?Pro?Phe?Ser?Glu?Lys?Thr?Ser?Thr?Gly?Lys?Glu?Asn?Gln?Asp?Glu

145 150 155 160

Asn?Cys?Ser?Gly?Val?Ser?Thr?Val?Asn?Lys?Tyr?Pro?Leu?Pro?Thr?Lys

165 170 175

Gln?Val?Ser?Gly?Asp?Ile?Glu?Thr?Ser?Lys?Thr?Ser?Thr?Val?Asp?Asn

180 185 190

Ala?Val?Gln?Asp?Val?Pro?Lys?Lys?Asn?Lys?Asp?Lys?Asp?Gly?Asn?Asp

195 200 205

Gly?Thr?Thr?Val?His?Ser?Met?Gln?Asn?Tyr?Pro?Trp?His?Phe?His?Ala

210 215 220

Asp?Ile?Val?Asn?Gly?Asn?Ile?Ala?Lys?Cys?Pro?Gln?Asn?His?Pro?Ser

225 230 235 240

Gly?Met?Val?Ser?Gln?Asp?Phe?Met?Phe?His?Pro?Met?Arg?Glu?Glu?Thr

245 250 255

His?Gly?His?Ala?Asn?Leu?Gln?Ala?Thr?Thr?Ala?Ser?Ala?Thr?Thr?Thr

260 265 270

Ala?Ser?His?Gln?Ala?Phe?Pro?Ala?Cys?His?Ser?Gln?Asp?Asp?Tyr?Arg

275 280 285

Ser?Phe?Leu?Gln?Ile?Ser?Ser?Thr?Phe?Ser?Asn?Leu?Ile?Met?Ser?Thr

290 295 300

Leu?Leu?Gln?Asn?Pro?Ala?Ala?His?Ala?Ala?Ala?Thr?Phe?Ala?Ala?Ser

305 310 315 320

Val?Trp?Pro?Tyr?Ala?Ser?Val?Gly?Asn?Ser?Gly?Asp?Ser?Ser?Thr?Pro

325 330 335

Met?Ser?Ser?Ser?Pro?Pro?Ser?Ile?Thr?Ala?Ile?Ala?Ala?Ala?Thr?Val

340 345 350

Ala?Ala?Ala?Thr?Ala?Trp?Trp?Ala?Ser?His?Gly?Leu?Leu?Pro?Val?Cys

355 360 365

Ala?Pro?Ala?Pro?Ile?Thr?Cys?Val?Pro?Phe?Ser?Thr?Val?Ala?Val?Pro

370 375 380

Thr?Pro?Ala?Met?Thr?Glu?Met?Asp?Thr?Val?Glu?Asn?Thr?Gln?Pro?Phe

385 390 395 400

Glu?Lys?Gln?Asn?Thr?Ala?Leu?Gln?Asp?Gln?Thr?Leu?Ala?Ser?Lys?Ser

405 410 415

Pro?Ala?Ser?Ser?Ser?Asp?Asp?Ser?Asp?Glu?Thr?Gly?Val?Thr?Lys?Leu

420 425 430

Asn?Ala?Asp?Ser?Lys?Thr?Asn?Asp?Asp?LysIle?Glu?Glu?Val?Val?Val

435 440 445

Thr?Ala?Ala?Val?His?Asp?Ser?Asn?Thr?Ala?Gln?Lys?Lys?Asn?Leu?Val

450 455 460

Asp?Arg?Ser?Ser?Cys?Gly?Ser?Asn?Thr?Pro?Ser?Gly?Ser?Asp?Ala?Glu

465 470 475 480

Thr?Asp?Ala?Leu?Asp?Lys?Met?Glu?Lys?Asp?Lys?Glu?Asp?Val?Lys?Glu

485 490 495

Thr?Asp?Glu?Asn?Gln?Pro?Asp?Val?Ile?Glu?Leu?Asn?Asn?Arg?Lys?Ile

500 505 510

Lys?Met?Arg?Asp?Asn?Asn?Ser?Asn?Asn?Asn?Ala?Thr?Thr?Asp?Ser?Trp

515 520 525

Lys?Glu?Val?Ser?Glu?Glu?Gly?Arg?Ile?Ala?Phe?Gln?Ala?Leu?Phe?Ala

530 535 540

Arg?Glu?Arg?Leu?Pro?Gln?Ser?Phe?Ser?Pro?Pro?Gln?Val?Ala?Glu?Asn

545 550 555 560

Val?Asn?Arg?Lys?Gln?Ser?Asp?Thr?Ser?Met?Pro?Leu?Ala?Pro?Asn?Phe

565 570 575

Lys?Ser?Gln?Asp?Ser?Cys?Ala?Ala?Asp?Gln?Glu?Gly?Val?Val?Met?Ile

580 585 590

Gly?Val?Gly?Thr?Cys?Lys?Ser?Leu?Lys?Thr?Arg?Gln?Thr?Gly?Phe?Lys

595 600 605

Pro?Tyr?Lys?Arg?Cys?Ser?Met?Glu?Val?Lys?Glu?Ser?Gln?Val?Gly?Asn

610 615 620

Ile?Asn?Asn?Gln?Ser?Asp?Glu?Lys?Val?Cys?Lys?Arg?Leu?Arg?Leu?Glu

625 630 635 640

Gly?Glu?Ala?Ser?Thr

645

<210>2

<211>2044

<212>DNA

＜213〉Arabidopsis Arabidopis thaliana (Arabidopsis thaliana)

<400>2

gggatacttt?acttgttaga?gaggatttga?agcagcgaat?agctgcaacc?ggtcctgtta 60

tggatactaa?tacatctgga?gaagaattat?tagctaaggc?aagaaagcca?tatacaataa 120

caaagcagcg?agagcgatgg?actgaggatg?agcatgagag?gtttctagaa?gccttgaggc 180

tttatggaag?agcttggcaa?cgaattgaag?aacatattgg?gacaaagact?gctgttcaga 240

tcagaagtca?tgcacaaaag?ttcttcacaa?agttggagaa?agaggctgaa?gttaaaggca 300

tccctgtttg?ccaagctttg?gacatagaaa?ttccgcctcc?tcgtcctaaa?cgaaaaccca 360

atactcctta?tcctcgaaaa?cctgggaaca?acggtacatc?ttcctctcaa?gtatcatcag 420

caaaagatgc?aaaacttgtt?tcatcggcct?cttcttcaca?gttgaatcag?gcgttcttgg 480

atttggaaaa?aatgccgttc?tctgagaaaa?catcaactgg?aaaagaaaat?caagatgaga 540

attgctcggg?tgtttctact?gtgaacaagt?atcccttacc?aacgaaacag?gtaagtggcg 600

acattgaaac?aagtaagacc?tcaactgtgg?acaacgcggt?tcaagatgtt?cccaagaaga 660

acaaagacaa?agatggtaac?gatggtacta?ctgtgcacag?catgcaaaac?tacccttggc 720

atttccacgc?agatattgtg?aacgggaata?tagcaaaatg?ccctcaaaat?catccctcag 780

gtatggtatc?tcaagacttc?atgtttcatc?ctatgagaga?agaaactcac?gggcacgcaa 840

atcttcaagc?tacaacagca?tctgctacta?ctacagcttc?tcatcaagcg?tttccagctt 900

gtcattcaca?ggatgattac?cgttcgtttc?tccagatatc?atctactttc?tccaatctta 960

ttatgtcaac?tctcctacag?aatcctgcag?ctcatgctgc?agctacattc?gctgcttcgg 1020

tctggcctta?tgcgagtgtc?gggaattctg?gtgattcatc?aaccccaatg?agctcttctc 1080

ctccaagtat?aactgccatt?gccgctgcta?cagtagctgc?tgcaactgct?tggtgggctt 1140

ctcatggact?tcttcctgta?tgcgctccag?ctccaataac?atgtgttcca?ttctcaactg 1200

ttgcagttcc?aactccagca?atgactgaaa?tggataccgt?tgaaaatact?caaccgtttg 1260

agaaacaaaa?cacagctctg?caagatcaaa?ccttggcttc?gaaatctcca?gcttcatcat 1320

ctgatgattc?agatgagact?ggagtaacca?agctaaatgc?cgactcaaaa?accaatgatg 1380

ataaaattga?ggaggttgtt?gttactgccg?ctgtgcatga?ctcaaacact?gcccagaaga 1440

aaaatcttgt?ggaccgctca?tcgtgtggct?caaatacacc?ttcagggagt?gacgcagaaa 1500

ctgatgcatt?agataaaatg?gagaaagata?aagaggatgt?gaaggagaca?gatgagaatc 1560

agccagatgt?tattgagtta?aataaccgta?agattaaaat?gagagacaac?aacagcaaca 1620

acaatgcaac?tactgattcg?tggaaggaag?tctccgaaga?gggtcgtata?gcgtttcagg 1680

ctctctttgc?aagagaaaga?ttgcctcaaa?gcttttcgcc?tcctcaagtg?gcagagaatg 1740

tgaatagaaa?acaaagtgac?acgtcaatgc?cattggctcc?taatttcaaa?agccaggatt 1800

cttgtgctgc?agaccaagaa?ggagtagtaa?tgatcggtgt?tggaacatgc?aagagtctta 1860

aaacgagaca?gacaggattt?aagccataca?agagatgttc?aatggaagtg?aaagagagcc 1920

aagttgggaa?cataaacaat?caaagtgatg?aaaaagtctg?caaaaggctt?cgattggaag 1980

gagaagcttc?tacatgacag?acttggaggt?aaaaaaaaaa?catccacatt?tttatcaata 2040

tctt 2044

Claims

1. a method of cultivating plant with adverse resistance is with the encoding gene importing vegetable cell of physiological clock regulation protein LHY, obtains plant with adverse resistance; Described anti-contrary be salt tolerant and/or drought-enduring;

The protein that described physiological clock regulation protein LHY is made up of the aminoacid sequence shown in the sequence in the sequence table 1.

2. the method for claim 1, it is characterized in that: the encoding gene of described physiological clock regulation protein LHY is following 1) or 2) dna molecular:

2) its nucleotide sequence is the dna molecular shown in the sequence 2 in the sequence table.