CN1775797A - Boea clarkeane drought-resistant and salt-tolerance related gene and its coding protein and use - Google Patents
Boea clarkeane drought-resistant and salt-tolerance related gene and its coding protein and use Download PDFInfo
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Abstract
The invention discloses the drought defying and salt proof relative gene of Boea hygrometrica and the coding albumen. The gene one of the following nucleotide sequence: 1) SEQ ID NO: 1 of the DNA sequence in sequence table; 2) SEQ ID NO:2 of the amino acid sequence in sequence table; 3)nucleotide sequence intercrossing with the DNA sequence. The BhLeal could regulate the drought defying and salt proof ability of plants.
Description
Technical field
The present invention relates to plant gene and proteins encoded thereof and application, particularly relate to one and derive from drought resisting, salt-resistant related gene and proteins encoded thereof and its application in the plant of cultivating drought resisting, salt tolerance raising of revolving capsule lettuce tongue.
Background technology
Plant except causing metabolic variation, can also swash more in vivo other expression of gene under the condition that arid salt damage is coerced, make and produce new protein accumulation in the nutritive issue of plant.These proteic accumulations can improve the adaptive faculty of plant to poor environment, as lea protein, and a kind of exactly coded product (Yu Jianing, drought resistance biotechnology progress 2002,22 (2) of mountain logical sequence .LEA albumen and plant: 10-14) that is subjected to arid salt damage induced gene.Lea protein has the wetting ability of height, coerced down by arid salt damage plant and a large amount of water moleculess can be caught in the guided cell, protect the biomacromolecule in the cell, keeps the ad hoc structure of cell, improves patience and the resistance of plant to environment-stress.In a lot of plants, finding lea protein at present, and it is being divided into 5 groups, all with the closely related (Yu Jianing of drought resisting, salt tolerant of plant according to these the constructional feature of lea protein, the drought resistance of mountain logical sequence .LEA albumen and plant, the biotechnology progress, 2002,22 (2): 10-14; Michael J Wise.LEAping to conclusions:Acomputational reanalysis of late embryogenesis abundant proteins and theirpossible roles.BMC Bioinformatics 2003,4:52; Yu Jianing, Jinsong ZHANG, mountain logical sequence, Chen Shouyi. functional analysis 2005,47 (11) in two new the 3rd group of LEA genes and the yeast in the wheat: 1372-1381).The importance of this gene pairs plant stress-resistance reaction and in the using value in plant stress-resistance genetically engineered field by common concern.Though the degeneration-resistant strain of utilizing adversity gene to cultivate crop, woods grass etc. in China has become the main thought of plant breeding, the degeneration-resistant resource gene that has China's independent intellectual property right also very lacks.Therefore understand the mechanism of action of this gene in depth, can provide fundamental basis for the Mechanism of Physiological and Biochemical of research plant drought on the one hand, on the other hand, can provide the degeneration-resistant resource gene that has independent intellectual property right for cultivating the adversity resistant plant kind.
Most plants (comprising staple crops kind and model plant) are impatient at serious arid and salt stress.Have only resurrection plant can stand the extremely condition of arid, just can the normal vital movement of very fast recovery when treating that moisture content is sufficient, therefore be drought-enduring mechanism of research and the fabulous plant resources that drought-enduring gene is provided.Resurrection plant in the known angiosperm seldom and mainly is distributed in South Africa, South America and Australia.It is first kind of resurrection plant of launching molecule and physiological level systematic study in China that the Gesneriaceae plant is revolved capsule lettuce tongue (Boeahygrometrica).The blade of this plant has very strong drought-enduring recovery ability, in room temperature, relative air humidity is implemented drought stress after 72 hours under 0 the condition, the blade relative water content reduces to about 3%, leaf-shrinkage is to below 1/3 of former leaf area, photosynthesis stops substantially, as long as feedwater again, the blade stretching, extension that just can absorb water, and revert to apparent state of blade and physiological status (comprising photosynthetic recovery) (Deng X before being untreated, Wang H, Hu Z, mRNA differential displayvisualized by silver staining tested on gene expression in resurrection plantBoea hygrometrica.Plant Moecular Biology Reporter 17:279.1999; Deng X, Hu Z, Wang H, Wen X, Kuang T.A comparison of photosynthetic apparatus of thedetached leaves of the resurrection plant Boea hygrometrica with itsnon-tolerant relative Chirita heterotrichia in response to dehydration andrehydration.Plant Science.165:851-861.2003).Utilize this plant, (not delivering data) identified and cloned to existing a plurality of gene related to drought tolerance.
Summary of the invention
The purpose of this invention is to provide one and derive from drought resisting, the salt-resistant related gene that revolves capsule lettuce tongue.
Drought resisting provided by the present invention, salt-resistant related gene, name is called BhLeal, and derive from Gesneriaceae and revolve capsule lettuce tongue and belong to and to revolve capsule lettuce tongue (Boea hygrometrica), be one of following nucleotide sequence:
1) SEQ ID № in the sequence table: 1 dna sequence dna;
2) SEQ ID № in the code sequence tabulation: 2 aminoacid sequence;
3) under the rigorous condition of height can with SEQ ID № in the sequence table: the nucleotide sequence of the 1 dna sequence dna hybridization that limits.
The rigorous condition of described height be 0.1 * SSPE (or 0.1 * SSC), in the solution of 0.1%SDS, hybridization and wash film under 65 ℃ of conditions.
SEQ ID № in the sequence table: 1 by 588 based compositions, and its encoding sequence is that coding has SEQ ID № in the sequence table: the protein of 2 amino acid residue sequence from 5 ' end 43-432 bit base.
The albumen of BhLeal coded by said gene of the present invention (BHLeal) is the protein with one of following amino acid residue sequences:
1) the SEQ ID № in the sequence table: 2;
2) with SEQ ID № in the sequence table: 2 amino acid residue sequence is through replacement, disappearance or the interpolation of one to ten amino-acid residue and have the protein of regulation and control plant drought, salt resistance ability.
SEQ ID № in the sequence table: 2 are made up of 129 amino-acid residues.
Contain expression carrier of the present invention, transgenic cell line and host bacterium and all belong to protection scope of the present invention.
Arbitrary segmental primer is to also within protection scope of the present invention among the amplification BhLeal.
Another object of the present invention provides a kind of method that improves plant drought, salt tolerance.
The method of raising plant drought provided by the present invention, salt tolerance is that described drought resisting, the salt-resistant related gene that revolves capsule lettuce tongue imported plant tissue or cell, obtains the plant of drought resisting, salt tolerance raising.
The described BhLeal gene that revolves capsule lettuce tongue can import explant by containing the described plant expression vector that revolves the BhLeal gene of capsule lettuce tongue; The carrier that sets out that is used to make up described plant expression vector can be any one double base agrobacterium vector or can be used for carrier of plant micropellet bombardment etc., as pBin19, pBI121, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300 or other plant expression vector of deriving.
When using the gene constructed plant expression vector of BhLeal, before its transcription initiation Nucleotide, can add any enhancement type, composing type, organizing specific type or inducible promoter, as cauliflower mosaic virus (CAMV) 35S promoter, general living plain gene Ubiquitin promotor (pUbi) etc., they can use separately or be used in combination with other plant promoter; In addition, when using gene constructed plant expression vector of the present invention, also can use enhanser, comprise translational enhancer or transcriptional enhancer, these enhanser zones can be ATG initiator codon or neighboring region initiator codon etc., but must be identical with the reading frame of encoding sequence, to guarantee the correct translation of whole sequence.The source of described translation control signal and initiator codon is widely, can be natural, also can be synthetic.Translation initiation region can be from transcription initiation zone or structure gene.
For the ease of transgenic plant cells or plant being identified and screening, can process used plant expression vector, can produce the enzyme of colour-change or the gene of luminophor (gus gene, GFP gene, luciferase genes etc.) as adding the coding that in plant, to express, have the antibiotic marker thing (gentamicin marker, kantlex marker etc.) of resistance or anti-chemical reagent marker gene (as anti-weedkiller gene) etc.From the security consideration of transgenic plant, can not add any selected marker, directly with adverse circumstance screening transformed plant.
Carry BhLeal gene of the present invention plant expression vector can Ti-plasmids, Ri plasmid, plant viral vector, directly DNA conversion, microinjection, electricity be led, conventional biological method transformed plant cells or tissue such as agriculture bacillus mediated by using, and the plant transformed cell or tissue is cultivated into plant.By the plant transformed host both can be farm crop such as paddy rice, wheat, soybean, tobacco, corn, rape, Chinese sorghum, cotton, can also be fruits and vegetables flower plants such as herbages such as clover, trifolium, wheatgrass and strawberry, tomato.
The gene related to drought tolerance BhLeal that revolves capsule lettuce tongue provided by the present invention, the drought-enduring recovery ability of adjustable plant, thus significantly improve the drought tolerance of plant.This gene and proteins encoded thereof have important theory and practical significance for the drought-enduring crop of cultivation, woods grass new variety, can be applicable to the cultivation and the evaluation of the required resistance plant kind of husbandry and ecological environment treatment, have higher actual application value.
The present invention will be further described below in conjunction with specific embodiment.
Description of drawings
Fig. 1 detects the result of BhLeal expression pattern for Northern hybridization
Fig. 2 is the physical map of BhLeal prokaryotic expression carrier pGEX-4T-1/BhLeal
The result of the confirmatory experiment that Fig. 3 is significantly improved for the drought-resistant ability of the prokaryotic cell prokaryocyte of overexpression BhLeal gene
The result of the confirmatory experiment that Fig. 4 is significantly improved for the salt resistance ability of the prokaryotic cell prokaryocyte of overexpression BhLeal gene
Embodiment
Method therefor is ordinary method if no special instructions among the following embodiment, and it is synthetic that the primer and probe are given birth to the worker by Shanghai.
The acquisition of embodiment 1, the drought resisting of revolving capsule lettuce tongue, salt-resistant related gene BhLeal
Extraction utilizes ZAP-cDNA through 8 hours the total RNA that revolves capsule lettuce tongue blade of drought stress
Library construction test kit (Stratagene, La Jolla, CA) construction cDNA library.4800 genes of random choose therefrom, (UK) the automatization point sample is at Hybond-N for BioRobotics Ltd, Cambridge with BioGrid robot
+Nylon membrane (Amersham Biosciences, Freiburg, Germany) on, make cDNA microarray (cDNA chip).With chip with handle normal growth without arid revolve capsule lettuce tongue blade and prepared through 8 hours the polyA-RNA that revolves capsule lettuce tongue blade of drought stress
33The probe hybridization of P mark is analyzed their dynamic changes in normal condition and drought-induced back genetic expression.Drought-induced back gene is checked order, sequencing result shows the gene clone that 1 drought-induced rise is arranged, this gene has SEQ ID № in the sequence table: 1 nucleotide sequence, SEQ ID № in the sequence table: 1 by 588 based compositions, its encoding sequence is from 5 ' end 43-432 bit base, coding has SEQ ID №: the protein of 2 amino acid residue sequence is BhLeal with this unnamed gene, with its proteins encoded called after BHLeal.
The expression pattern analysis experiment of embodiment 2, BhLeal
Extract respectively and coerced processing, through drought stress 2,8,24,72 hours, and 0.2M NaCl coerces with water logging and coerces 9 hours the total RNA that revolves capsule lettuce tongue blade and measure concentration, each sample of getting equivalent carries out 1% agarose gel electrophoresis, treat to move to gel apart from 2/3 place, point sample hole through smelling the blue painted sample of phenol, RNA is transferred on the nylon membrane, roasting film is 120 minutes under 80 ℃, again commentaries on classics there is the nylon membrane of above-mentioned RNA to put into hybrid pipe, behind 65 ℃ of prehybridization 2.5h, add purified, the fluorescein-labeled BhLeal probe of sex change (SEQ ID №: hold the 1st to 588 base position nucleotide sequence from 5 ' in 1), hybridized 24 hours for 65 ℃, pour out hybridization solution, with 2 * SSC, 0.1%SDS, 0.1 * SSC and 0.1%SDS solution are under 65 ℃, respectively wash film 30min, use Gene Images CDP-Star Detection Module (Amersham Phamacia Biotech at last, Little Chalfont Buckinghamshire, UK) position and the intensity of detection hybridization signal.The part detected result as shown in Figure 1, the mRNA that shows BhLeal be not subjected to drought stress and water logging is coerced revolves in the capsule lettuce tongue blade and detect less than expression, can induce its great expression through drought stress 8 hours and salt stress after 9 hours.
The cytology location of embodiment 3 BhLeal
Utilize PSORT computer program analysis software (Nakai and Kanehisa, Expert system for predicting protein localization sites in gram-negative bacteria.Proteins.11 (2): 95-110,1991) the coded PROTEIN B hLeal of BhLeal is carried out the cytology location, the result shows that BhLeal is positioned to revolve in the tenuigenin of capsule lettuce tongue leaf cell.
The drought resisting functional verification experiment of embodiment 4, BhLeal
Earlier being template through 8 hours the total RNA that revolves capsule lettuce tongue blade of drought stress, by synthetic its cDNA of oligo-dT reverse transcription, be template with this cDNA again, under the guiding of primer OB-44-full-f (5 '-AGAATTCATGCAGACTGCGAAGC-3 ') and OB-44-full-r (5 '-ACTCGAGCTACTGAGTCGGAGCT-3 '), the full length fragment of amplification BhLeal gene, after cutting with restriction enzyme EcoR I and Xho I enzyme, with be connected through the prokaryotic expression carrier pGEX-4T-1 of same enzyme double digestion (Pharmacia company), obtain BhLeal Prokaryotic Expression carrier, called after pGEX-4T-1/BhLeal, its physical map makes the expression of BhLeal and GST fusion gene be subjected to the regulation and control of IPTG inducible promoter tac as shown in Figure 2.The BhLeal Prokaryotic Expression carrier and the pGEX-4T-1 empty carrier that build are changed over to respectively in the e. coli bl21 (DE3), be inoculated in the 2mL LB liquid nutrient medium that contains 50 μ g/mL penbritins, 37 ℃ of shaking culture 12-16h (150 change/min) after, by dilution in 1: 100,37 ℃ of shaking culture 1-2h again are to OD
600Value is for behind the 0.3-0.6, and abduction delivering also detects the BhLeal gene overexpression is to the influence of prokaryotic cell prokaryocyte drought-resistance ability (with the PEG6000 simulating drought of different concns) in prokaryotic cell prokaryocyte, and concrete grammar is: collect 5mL OD
600Value is the bacterium liquid of 0.3-0.6, centrifugal (8000 commentaries on classics/min) 1min, collect thalline, precipitation is resuspended in respectively in the 5mL LB nutrient solution that contains 3.75%, 7.5%, 15%, 30% PEG6000 and 50 μ g/mL penbritins respectively, add final concentration simultaneously and begin inducing culture for 0.01mm/L IPTG, 37 ℃ of shaking culture (150 commentaries on classics/min), survey OD respectively after 1 hour
600Value, and collection 1.5mL thalline, it is centrifugal that (4000 change/min) collect thalline, precipitation is resuspended in 100 μ l, 2 * sds gel sample loading buffer, boiling water 10min, the centrifugal 10min of 12000g, collect supernatant, sample 5 μ l carry out the SDS-PAGE electrophoresis detection on every sample, and detected result shows that the BhLeal gene has obtained overexpression in prokaryotic cell prokaryocyte, its protein expression level is induced down at 0.01mm/L IPTG to be increased, with respect to the bacterium that changes empty carrier, the survival ability tool of bacterium in the presence of 7.5%PEG6000 that changes the BhLeal gene is improved to some extent simultaneously, and (with the bacterium that changes empty carrier pGEX-4T-1 over to is contrast as shown in Figure 3; Ordinate zou is a survival rate, represents with percentage ratio), and on protein level, the bacterium that changes the BhLeal gene compared with the control, the ability of anti-PEG also is improved.This experiment proves that in the procaryotic cell expression system BhLeal gene can improve the drought-resistance ability of cell.
The anti-salt functional confirmatory experiment of embodiment 5, BhLeal
Obtain BhLeal Prokaryotic Expression carrier with method same as described above, pGEX-4T-1/BhLeal, its physical map make the expression of BhLeal and GST fusion gene be subjected to the regulation and control of IPTG inducible promoter tac as shown in Figure 2.The BhLeal Prokaryotic Expression carrier and the pGEX-4T-1 empty carrier that build are changed over to respectively in the e. coli bl21 (DE3), be inoculated in the 2mL LB liquid nutrient medium that contains 50 μ g/mL penbritins, 37 ℃ of shaking culture 12-16h (150 change/min) after, by dilution in 1: 100,37 ℃ of shaking culture 1-2h again are to OD
600Value is for behind the 0.3-0.6, and abduction delivering also detects the BhLeal gene overexpression is to the influence of prokaryotic cell prokaryocyte drought-resistance ability in prokaryotic cell prokaryocyte, and concrete grammar is: collect 5mL OD
600Value is the bacterium liquid of 0.3-0.6, centrifugal (8000 commentaries on classics/min) 1min, collect thalline, precipitation is resuspended in respectively in the 5mL LB nutrient solution of the NaCl that contains 0.3M, 0.6M, 0.9M and 1.8M respectively and 50 μ g/mL penbritins, add final concentration simultaneously and begin inducing culture for 0.01mm/L IPTG, in (150 commentaries on classics/min), survey OD respectively after 1 hour of 37 ℃ of shaking culture
600Value, and collection 1.5mL thalline, it is centrifugal that (4000 change/min) collect thalline, precipitation is resuspended in 100 μ l, 2 * sds gel sample loading buffer, boiling water 10min, the centrifugal 10min of 12000g, collect supernatant, sample 5 μ l carry out the SDS-PAGE electrophoresis detection on every sample, and detected result shows that the BhLeal gene has obtained overexpression in prokaryotic cell prokaryocyte, its protein expression level is induced down at 0.01mm/L IPTG to be increased, with respect to the bacterium that changes empty carrier, the bacterium that changes the BhLeal gene is at 0.3M simultaneously, 0.6M be improved to some extent with the survival ability tool under the 0.9M NaCl existence, (with the bacterium that changes empty carrier pGEX-4T-1 over to is contrast as shown in Figure 4; Ordinate zou is a survival rate, represents with percentage ratio), and on protein level, the bacterium that changes the BhLeal gene compared with the control, the ability of anti-NaCl also is improved.This experiment proves that in the procaryotic cell expression system BhLeal gene can improve the salt resistance ability of cell.
Sequence table
<160>2
<210>1
<211>588
<212>DNA
<213〉revolve capsule lettuce tongue genus and revolve capsule lettuce tongue (Boea hygrometrica)
<400>1
cattacaact?ttatatttca?tattaaataa?aaccaataaa?atattcatac?ttctaatcaa 60
aatattatct?attcttcttc?ttcatccaat?tatctttttt?atttccttaa?atccaaatct 120
taatttaatt?cttataaaac?tatttcaatt?tctaattaat?acaaataatt?ttccaattaa 180
cataataatt?ctaaataatc?ttaatccaat?tttattaatc?attaatataa?ttcctataat 240
tctaccttcc?attttcattc?taatcatcat?ttacaatttt?ttcatcatta?tcttcacttt 300
tctttttctt?ctccttctac?tcaattcaat?cattatccct?catttttatc?cactcatatt 360
ccaccttatt?aatcttcttc?ccattattat?cattcttcac?cttttactaa?cccttcatct 420
cctactcatt?atcatcacct?ctctttcctt?tatttaattt?ttcatctttt?tattaattta 480
ctcttattta?ttttttctat?ttctttcttt?cctctttcca?tcttttaatt?tttctttttt 540
atatttttta?tctcttcttt?tttatttttt?aataaattca?tttattac 588
<210>2
<211>129
<212>PRT
<213〉revolve capsule lettuce tongue genus and revolve capsule lettuce tongue (Boea hygrometrica)
<400>2
Met?Gln?Thr?Ala?Lys?Gln?Lys?Met?Ser?Asp?Ala?Ala?Ala?Ser?Ala?Lys
1 5 10 15
Glu?Arg?Val?Asp?Val?Leu?Lys?Ala?Lys?Ala?Glu?Gly?Lys?Ala?Asp?Lys
20 25 30
Thr?Met?Ala?Ser?Ser?Lys?Glu?Asp?Lys?Glu?Val?Ala?Lys?Glu?Gln?Lys
35 40 45
Lys?Ala?Lys?Glu?Ala?Glu?Ala?Lys?Val?Arg?Lys?His?Glu?Glu?Lys?Ala
50 55 60
Asp?Asn?Ala?Thr?Gly?His?Leu?Gln?Ala?Lys?His?Gln?Gly?Gln?Phe?Gly
65 70 75 80
Gln?Gln?Asp?Leu?His?Gly?Ala?Gly?Ala?Ala?Pro?Ala?Thr?Gln?Gly?Asn
85 90 95
Gln?Tyr?Pro?Ala?Gly?Gly?Ala?Thr?Gln?Ile?Pro?Pro?Glu?Glu?Ala?Ala
100 105 110
Ala?Gln?Tyr?Glu?Gln?Ala?Ala?Pro?Gly?Thr?Asn?Pro?Ala?Ala?Pro?Thr
115 120 125
Gln
Claims (8)
1, revolving drought resisting, the salt-resistant related gene of capsule lettuce tongue, is one of following nucleotide sequence:
1) SEQ ID № in the sequence table: 1 dna sequence dna;
2) SEQ ID № in the code sequence tabulation: 2 aminoacid sequence;
3) under the rigorous condition of height can with SEQ ID № in the sequence table: the nucleotide sequence of the 1 dna sequence dna hybridization that limits.
2, drought resisting, the salt-resistant related gene that revolves capsule lettuce tongue according to claim 1, it is characterized in that: described gene has SEQ ID № in the sequence table: 1 dna sequence dna.
3, the described proteins encoded that revolves the drought resisting of capsule lettuce tongue, salt-resistant related gene of claim 1, it is characterized in that: described albumen is one of following amino acid residue sequences:
1) the SEQ ID № in the sequence table: 2;
2) with SEQ ID № in the sequence table: 2 amino acid residue sequence is through replacement, disappearance or the interpolation of one to ten amino-acid residue and the protein with regulation and control plant drought, salt tolerant recovery ability.
4, the proteins encoded that revolves the drought resisting of capsule lettuce tongue, salt-resistant related gene according to claim 3, it is characterized in that: described albumen has SEQ ID № in the sequence table: 2 amino acid residue sequence.
5, contain the described expression carrier of claim 1, transgenic cell line and host bacterium.
6, a kind of method that improves plant drought, salt tolerance is that described drought resisting, the salt-resistant related gene that revolves capsule lettuce tongue of claim 1 imported plant tissue or cell, obtains the plant of drought resisting, salt tolerance raising.
7, method according to claim 6 is characterized in that: described drought resisting, the salt-resistant related gene that revolves capsule lettuce tongue imports plant tissue or cell by containing the described plant expression vector that revolves the drought resisting of capsule lettuce tongue, salt-resistant related gene; The carrier that sets out that is used to make up described plant expression vector is pBin19, pBI121, pCAMBIA2301, pCAMBIA1301 or pCAMBIA1300.
8, method according to claim 6 is characterized in that: described plant host is paddy rice, wheat, soybean, tobacco, corn, rape, Chinese sorghum, cotton, clover, trifolium, wheatgrass, strawberry or tomato.
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CN102020707A (en) * | 2010-11-02 | 2011-04-20 | 中国科学院植物研究所 | Protein, coding genes and application thereof |
CN101747419B (en) * | 2008-12-08 | 2012-02-22 | 中国科学院遗传与发育生物学研究所 | Protein related to salt tolerance, coding gene thereof and application thereof |
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CN103382475A (en) * | 2012-05-03 | 2013-11-06 | 中国科学院植物研究所 | DNA fragment related to drought tolerance and alkali resistance of plant and application thereof |
CN103382475B (en) * | 2012-05-03 | 2015-01-14 | 中国科学院植物研究所 | DNA fragment related to drought tolerance and alkali resistance of plant and application thereof |
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