CN101148671B - Spartina anglica sodium hydrogen pump protein gene SaNHX and application thereof - Google Patents
Spartina anglica sodium hydrogen pump protein gene SaNHX and application thereof Download PDFInfo
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- CN101148671B CN101148671B CN200610069038XA CN200610069038A CN101148671B CN 101148671 B CN101148671 B CN 101148671B CN 200610069038X A CN200610069038X A CN 200610069038XA CN 200610069038 A CN200610069038 A CN 200610069038A CN 101148671 B CN101148671 B CN 101148671B
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Abstract
The present invention is cord grass sodium hydrogen pump protein gene SaNHX and its application. The sodium hydrogen pump protein gene SaNHX has the DNA sequence as shown in sequence 1 of the sequence list or the DNA sequence with over 90 % homology with the sequence 1 limited DNA sequence and coding protein with the same function. The gene SaNHX coded cord grass sodium hydrogen pump protein is protein with the amino acid residue sequence as shown in sequence 2 of the sequence list or derivative protein of the sequence 2 through the substitution, deletion or addition of one or several amino acid residues and with the same activity as the protein with the amino acid residue sequence in sequence 2. The gene of the present invention is significant in cultivating new variety of salt tolerant crop, especially salt tolerant rice.
Description
Technical field the present invention relates to fields such as molecular biology, zymetology, physiology and genetically engineered.Particularly, the present invention relates to a kind of sodium hydrogen pump albumen of in rice grass (Spartinaanglica), expressing (rice grass Na
+/ H
+Antiporter is SaNHX) with nucleotide sequence and application thereof.
There is 1,000,000,000 hectares saline-alkali soil in the background technology whole world, accounts for 7% (Szabolcs, 1994) of land area; 7,700,000 hectares arable land is influenced by the salt branch, accounts for 5% (Munns etc., 1999) of cultivated area.Salt damage is very big to the yield effect of crop, causes declining to a great extent of output.
Can produce salt stress to plant when the salt content in the soil is too high, normal growth and the growth of harm plant mainly show two aspects: one, and the water deficit that higher solute concentration causes in the soil; Two, absorbed the too much ion that salt ion brought in the time of plant absorbing moisture and poisoned, particularly Na
+Poison.Plant is eliminated Na
+The strategy of poisoning comprises: the absorption, the Na that reduce Na+
+Efflux and Na
+Separating.The applied research development of sodium hydrogen pump albumen aspect biology in recent years is rapid, and the characteristics of its maximum are that it can be with Na
+Efflux and pump into vacuole " storage ", and relevant with the alkalify of vacuole, this albumen in recovering the cell plasma equilibrium process, playing the part of considerable role (Plant Physiol, 1985,78:163-167).
Apse, M.P.etal. finds the sodium hydrogen pump albumen of a vacuole skin of AtNHX1 genes encoding of Arabidopis thaliana, it can be with Na
+Compartmentation is in vacuole.And the overexpression of finding the AtNHX1 gene can improve Arabidopis thaliana salt tolerance (Science, 1999,285:1256-1258).
Na on the plasma membrane of while Arabidopis thaliana
+/ H
+Antiporter albumen can be with Na
+Discharge cytolemma, with salt tolerance (Shi Huazhongetal.Nature Biotechnology, 2003 of finding behind its overexpression to improve Arabidopis thaliana, 21 (1): 81-85).
Hamada A etc. has separated a Na from the halophytes saltbush
+/ H
+Counter transport protein gene AgNHX1, AgNHX1 express in yeast NHX mutant and can remedy the responsive phenotype of zymic salt.
The above-mentioned sodium hydrogen pump albumen that studies show that is having important effect aspect the salt tolerance of plant.Yet the research that belongs to the proteic gene of sodium hydrogen pump of halophytes gramineous with paddy rice together yet there are no report.Before the present invention comes forth, any Gramineae halophytes rice grass sodium hydrogen pump protein sequence and nucleotide sequence of mentioning in the present patent application thereof that disclose or reported do not arranged as yet.
Summary of the invention first purpose of the present invention provides a kind of new rice grass sodium hydrogen pump protein gene (SaNHX).
Second purpose of the present invention provides a kind of new rice grass sodium hydrogen pump albumen (SaNHX).
The 3rd purpose of the present invention provides this rice grass sodium hydrogen pump albumen and encoding sequence is utilizing transgenic technology to improve the salt tolerance of paddy rice, the paddy rice salt tolerant intermediate materials that cultivation makes new advances.
Rice grass sodium hydrogen pump protein gene name provided by the invention is called SaNHX, is one of following nucleotide sequences:
1) dna sequence dna of sequence 1 in the sequence table;
2) with sequence table in the dna sequence dna that limits of sequence 1 have 90% above homology, and the identical function protein DNA sequence of encoding.
The DNA of sequence 1 is by 2089 based compositions in the sequence table, and the reading frame of this gene is from 5 ' end the 96th to the 1697th bit base.Arbitrary segmental primer is to also within protection scope of the present invention among the amplification SaNHX, and wherein, the distance between upstream primer and the downstream primer is between 50 to 2000 bases; The length of each primer of this primer centering is 15 to 30 bases.
A kind of rice grass sodium hydrogen pump protein s aNHX provided by the invention by gene SaNHX coding, be have the protein of sequence 2 amino acid residue sequences in the sequence table or with the amino acid residue sequence of sequence 2 through replacement, disappearance or the interpolation of one or several amino-acid residue and have identical active by sequence 2 deutero-protein with the amino acid residue sequence of sequence 2.
Utilize any carrier that can guide foreign gene in plant, to express,, can obtain transgenic cell line and transfer-gen plant that salt tolerance is improved SaNHX gene transfered plant cell provided by the present invention.Gene of the present invention can add any enhancing promotor or inducible promoter in being building up to plant expression vector the time before its transcription initiation Nucleotide.For the ease of transgenic plant cells or plant being identified and screening, can process employed carrier, as the antibiotic marker thing that adds the alternative mark of plant or have resistance.By the plant transformed host can be monocotyledons both, as paddy rice; It also can be dicotyledons.
Sodium hydrogen pump protein gene is cloned in the present invention first from the rice grass, and has confirmed that by transgenic experiments this gene can improve the saline-alkaline tolerance of paddy rice.Can cultivate the transgenic paddy rice new variety of anti-salt on this basis, make its can normal growth at saline soil on the ground.Gene of the present invention also can be widely used in cultivates other salt tolerant crop kinds, and huge economic and application prospect are arranged.
Description of drawings Fig. 1 strides the film district for rice grass sodium hydrogen pump albumen and predicts the outcome.
Fig. 2 is a rice grass SaNHX gene Southern results of hybridization.
Fig. 3 is a Northern results of hybridization under the rice grass SaNHX gene salt stress.Wherein 1～6 be respectively NaCl and coerced 0,1,3,6,10 and 24 hour.Last figure is hybridization figure, and figure below is the painted rRNA of EB.
Fig. 4 is transgenic paddy rice salt tolerant test photo.Before wherein last figure is brine treatment, after figure below is brine treatment; The left side is for after handling 15 days, and the right is for after handling 1 month.Ck1 is a fresh water contrast tissue cultured seedling, and ck2 is the saline control tissue cultured seedling, and t3 is a transfer-gen plant.
Embodiment is further set forth the present invention below in conjunction with specific embodiment.Should be understood that these embodiment only are used to illustrate the present invention, limit the scope of the invention and be not used in.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, for example the J.Sambrook equimolecular is cloned: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
Embodiment 1: clone and the sequence information and the homology analysis of rice grass SaNHX gene
1. tissue and RNA's separates
The rice grass derives from Fujian Province's Luoyuan Bay.Get portion of tissue, grind, add the 50mL pipe that fills lysate with mortar, after fully vibrating, extracted total RNA (TRIzol Reagents, Invitrogen, NY, USA).
2.SaNHX the cDNA full-length clone
According to paddy rice sodium hydrogen pump Argine Monohydrochloride conserved sequence, the design primer utilizes homologous genes clone principle, adopts RACE method (Invitrogen test kit) to carry out the cDNA full-length clone, divides three phases to carry out:
(1)3′-RACE
PCR (AUAP+GSP3-3) obtains 2003SN3 ' (793bp), reclaims, clones and order-checking.Sequencing result is BLAST on NCBI, knows that the homology of its nucleotide sequence and proteins encoded and known model plant Arabidopis thaliana (Arabidopsis thaliana) sodium hydrogen pump protein gene is higher, so think that tentatively it is a sodium hydrogen pump protein gene.
(2)5′-RACE
First round PCR (AAP+GSP5-2)
Second takes turns PCR (AUAP+GSP5-3) obtains 2003SN5 ' (553bp) (process is with (1))
(3) the 96-121 and the 1672-1697 dna sequence dna with sequence in the sequence table 1 is that primer carries out pcr amplification, obtains 2003SN coding region (1608bp).(process is with (1))
The gene that result's proof of BLAST newly obtains from the rice grass really is a plant sodium hydrogen pump protein gene.Because known homology sodium hydrogen pump albumen, as Arabidopis thaliana sodium hydrogen pump protein gene have stronger salt tolerant alkali effect (Science, 1999,285:1256-1258), the rice grass proteic gene of sodium hydrogen pump (SaNHX) of therefore encoding also has identical functions.
By being used in combination above-mentioned 3 kinds of methods, obtained candidate's the proteic complete encoding sequence of rice grass sodium hydrogen pump.With 3 ', 5 ' and the encoding sequence splicing obtain the dna sequence dna shown in the sequence 1 in the sequence table.
3.SaNHX sequence information and homology analysis
The length of the rice grass sodium hydrogen pump albumen full-length cDNA that the present invention is new is 2089bp, and detailed sequence is seen sequence 1 in the sequence table, and wherein open reading frame is positioned at 96～1697 Nucleotide.Derive the proteic aminoacid sequence of rice grass sodium hydrogen pump according to full-length cDNA, totally 533 amino-acid residues, molecular weight is 58656.13 dalton, iso-electric point (pI) is 8.563.Detailed sequence is seen sequence 2 in the sequence table.
Rice grass SaNHX has been carried out striding the film differentiation analysed, the result as shown in Figure 1.Stride film and distinguish and to analyse the SaNHX that shows the rice grass and have 12 to stride the film district, visible rice grass SaNHX albumen is typical ionic channel translocator.Rice grass proteic full length cDNA sequence of sodium hydrogen pump and coded protein thereof are carried out Nucleotide and protein homology retrieval with blast program in ncbi database, found that it and Arabidopis thaliana sodium hydrogen pump albumen have 77% homogeny on nucleotide level; On amino acid levels, it and Arabidopis thaliana sodium hydrogen pump protein have 73% homogeny.This shows that all there are higher homology in rice grass sodium hydrogen pump protein gene and Arabidopis thaliana sodium hydrogen pump protein gene on nucleic acid still is protein level, can think that both also have very high similarity on function.
Embodiment 2: the Southern hybridization analysis of rice grass SaNHX gene
The extraction of rice grass genomic dna is undertaken by the method for Paterson et al. (1993).Get 1 μ g genomic dna, cut with EcoRI and HindIII enzyme respectively.Enzyme is cut product, and (30-50V, O/N) separation, treatment gel on shaking table then: 0.125N HCl embathes 15min through 0.7% agarose gel electrophoresis, sex change liquid (1.5M NaCl, 0.5N NaOH) embathe 30min, neutralizer (1.5MNaCl, 0.5M Tris-HCl pH7.2) embathes 30min.Use 20 * SSC that southern blotting technique is arrived HybondN at last
+On the nylon membrane.
(Jin Dongyan etc., 1996 are all carried out in prehybridization, hybridization according to a conventional method; Hybond N
+Mannual, Amersham).
Probe and mark thereof: hybridize the dna fragmentation that used probe derives from one section about 800bp length in the SaNHX gene cDNA.Carrying out mark with the Primer-a-Gene test kit of Promega company spends the night.
Wash film and carry out (Hybond N with high tight method
+Mannual, Amersham): 2 * SSC, 0.1%SDS, 15min; 1 * SSC, 0.1%SDS, 65 ℃, 15min; 0.2 * SSC, 0.1%SDS, 65 ℃, 15min.
Preservative film parcel Hybond membrane exposes to the X-ray sheet under-70 ℃.
The result as shown in Figure 2, as can be seen from the figure, the enzyme that this gene is cut swimming lane and HindIII at the enzyme of EcoRI is cut and is all hybridized to two to three bands in the swimming lane, but each enzyme cut/have a band to seem brighter in the probe combinations.So tentatively conclude in the rice grass genome this gene and may have 2～3 with the copy number of the gene of this dna homolog.
Embodiment 3: the Northern hybridization analysis of rice grass SaNHX gene
The Hoagland nutrient solution culture was brought the rice grass up after 5 days, and adding NaCl is 400mmol/L to final concentration, and the time of coercing was respectively 0,1,3,6,10 and 24 hour, extracted the total RNA of blade and carried out the Northern analysis, and the result as shown in Figure 3.As can be seen from the figure, SaNHX expresses and to be subjected to Salt Stress-induced, and promptly reaches a higher level after coercing 6 hours, and it is still higher to coerce after 10 hours expression amount, and expression amount is higher after 24 hours, but descends to some extent.The expression amount of untreated control also has, but lower.This result and temporal sxemiquantitative RT-PCR be basically identical as a result.
Embodiment 4: rice grass sodium hydrogen pump albumen or polypeptide carry out the salt tolerance of eukaryotic cell expression and transfer-gen plant in rice cell identifies
1. the structure that contains the expression vector of goal gene SaNHX
According to the rice grass proteic full length sequence of sodium hydrogen pump (sequence 1 in the sequence table), design amplifies the primer that complete coding is read frame, and introduces restriction endonuclease sites (this is decided by the carrier of selecting for use) respectively on positive anti-primer, so that construction of expression vector.Amplified production with acquisition among the embodiment 1 is a template, behind pcr amplification, the cDNA of SaNHX is cloned into intermediate carrier (as T-simple), further be cloned into binary expression vector, under the prerequisite that guarantees reading frame, identify good expression vector, again it is changed in the Agrobacterium, utilize callus infestation method technical transform model plant paddy rice.
2. utilize and infect callus method rice transformation Japan fine (Nipponbare)
Rice paddy seed is shelled, sterile-processed after, be inoculated on the inducing culture NB, cultivate down for 27 ℃.Select good callus through 2～3 times subculture, select growth rapidly and the fine and close callus pre-5d of cultivation on subculture medium of quality.Agrobacterium after solid medium (YEB+50mg/L Rifampin+50mg/L kantlex+1.5% agar) is gone up cultivation 48h, is flushed to bacterium colony in the aseptic triangular flask OD with the AAM liquid nutrient medium
600Adjust to 1.5～2.0.After leaving standstill 1h, soak, callus is put on the aseptic filter paper, and blown 20～30min, receive then and contain on the NB-AS substratum, put the dark 3d of cultivation under 25 ℃ of conditions at Bechtop through pre-incubated callus 20min.With sterile distilled water flushing 2～3 times, use callus the Pyocianil (or cephamycin of 500mg/L) of 250mg/L to soak 30min again, be inoculated in upward cultivation of resistance screening substratum NBS1 with blotting on the rearmounted filter paper., be transferred to new NBS2 and continue screening after 2 weeks at the callus culture on the NBIS1.Every transforms the new callus that grows on the callus and is designated as a clone.
To be transferred to respectively on the division culture medium NBD through the callus of conversion and corresponding unconverted.Change the increment that root media 1/2MSR goes up increases root behind regeneration plant length to 3～4cm over to.
3. utilize PCR to detect the integration of SaNHX in transgenic rice plant
Method is extracted the total genomic dna of resistant plant in a small amount.Special primer with SaNHX carries out pcr amplification to carrying DNA, with the fine negative contrast of Japan.
4. the salt tolerance that contains the transfer-gen plant of SaNHX is identified
In view of the gene of the AtNHX1 of coding sodium hydrogen pump albumen such as Arabidopis thaliana has been proved to be salt stress had resistance, and the AtNHX1 of rice grass sodium hydrogen pump albumen (SaNHX) and Arabidopis thaliana has higher homology, and we further carry out salt tolerance to rice grass sodium hydrogen pump protein transgene plant and identify.The kanamycin-resistant callus tissue that screens is produced regeneration plant after through pre-differentiation and differentiation culture, behind experienced seedling, be transplanted to the glasshouse continued growth.Treat T
0For the solid back of paddy rice positive plant heading results seed.T
1For being sowed at after seed-soaking, the vernalization among the basin alms bowl, treat long after 20 day length of time rice seedlings grow with the pouring of 150mmol/LNaCl water.Use the clear water pouring after 10 days instead, observe phenotype after 15 days and change; After again with 150mmol/L NaCl water pouring 10 days, after use the clear water pouring instead, observe phenotype after one month and change.The result as shown in Figure 4, paddy rice tissue cultured seedling adjoining tree began to wither at 15 days, and is dead after one month, the paddy rice transfer-gen plant is because salt tolerant and normal growth.
The result proves that rice grass sodium hydrogen pump albumen truly has resistance to salt stress, can tolerate the NaCl solution of 150mmol/L.Rice grass sodium hydrogen pump albumen will can be used for utilizing in the research and industrialization of transgenic technology improvement plant stress-resistance (salt tolerant alkali).
Sequence table
SEQUENCE?LISTING
＜110〉University Of Agriculture and Forestry In Fujian
＜120〉spartina anglica sodium hydrogen pump protein gene SaNHX and application thereof
<130>
<160>2
<170>PatentIn?version3.1
<210>1
<211>2089
<212>DNA
<213>Spartina?anglica
<400>1
<210>2
<211>533
<212>PRT
<213>Spartina?anglica
<400>2
Claims (5)
1. spartina anglica sodium hydrogen pump protein gene SaNHX, its sequence is the base sequence shown in the sequence table SEQ ID NO:1.
2. rice grass sodium hydrogen pump Protein S aNHX, its sequence is the amino acid residue sequence shown in the sequence table SEQ ID NO:2.
3. contain the described expression carrier of claim 1.
4. the paddy rice transgenic plant cells system of containing the described expression carrier of claim 1.
5. the application of the described gene of claim 1 in cultivating the salt tolerant new rice variety.
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| CN103087159B (en) * | 2011-10-31 | 2014-08-20 | 中国科学院微生物研究所 | Sodium hydrogen pump protein, and coding gene and applications thereof |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2000157287A (en) * | 1998-09-24 | 2000-06-13 | Shokubutsu Kogaku Kenkyusho:Kk | Na+/H+ ANTIPORTER PROTEIN AND GENE CODING FOR THE SAME |
| JP2001223770A (en) * | 2000-02-09 | 2001-08-17 | Nec Shizuoka Ltd | Portable telephone terminal device |
| WO2002016423A2 (en) * | 2000-08-25 | 2002-02-28 | Basf Plant Science Gmbh | PLANT POLYNUCLEOTIDES ENCODING NOVEL Na+/H+ ANTIPORTERS |
| CN1454997A (en) * | 2003-01-09 | 2003-11-12 | 复旦大学 | Rape sodium-hydrogen pump transport protein coding sequence and application thereof |
| CN1651459A (en) * | 2004-12-01 | 2005-08-10 | 中山大学 | Small salt mustard sodium hydrogen pump protein gene TNHX1 and its anti salt application |
-
2006
- 2006-09-20 CN CN200610069038XA patent/CN101148671B/en not_active Expired - Fee Related
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2000157287A (en) * | 1998-09-24 | 2000-06-13 | Shokubutsu Kogaku Kenkyusho:Kk | Na+/H+ ANTIPORTER PROTEIN AND GENE CODING FOR THE SAME |
| JP2001223770A (en) * | 2000-02-09 | 2001-08-17 | Nec Shizuoka Ltd | Portable telephone terminal device |
| WO2002016423A2 (en) * | 2000-08-25 | 2002-02-28 | Basf Plant Science Gmbh | PLANT POLYNUCLEOTIDES ENCODING NOVEL Na+/H+ ANTIPORTERS |
| CN1454997A (en) * | 2003-01-09 | 2003-11-12 | 复旦大学 | Rape sodium-hydrogen pump transport protein coding sequence and application thereof |
| CN1651459A (en) * | 2004-12-01 | 2005-08-10 | 中山大学 | Small salt mustard sodium hydrogen pump protein gene TNHX1 and its anti salt application |
Non-Patent Citations (6)
| Title |
|---|
| BLUMWALD E..Sodium transport and salt tolerance in plants.CurrOp in Cell Biol12.2000,12431 - 434. |
| BLUMWALD E..Sodium transport and salt tolerance in plants.CurrOp in Cell Biol12.2000,12431- 434. * |
| YANG Q ET-AL.Cloning and exp ression analysis of a vacuolarNa + /H +antiporter gene from alfalfa.DNA Sequence16 5.2005,16(5),352 - 357. |
| YANG Q ET-AL.Cloning and exp ression analysis of a vacuolarNa + /H +antiporter gene from alfalfa.DNA Sequence16 5.2005,16(5),352- 357. * |
| 兰涛等.大米草Na + /H+ 逆转运蛋白基因片段的克隆.福建农林大学学报(自然科学版)35 1.2006,35(1),52-56. |
| 兰涛等.大米草Na + /H+ 逆转运蛋白基因片段的克隆.福建农林大学学报(自然科学版)35 1.2006,35(1),52-56. * |
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