CN102051364B

CN102051364B - Plant salt-resisting protein as well as encoding gene and application thereof

Info

Publication number: CN102051364B
Application number: CN2010105834001A
Authority: CN
Inventors: 曹树青; 吕申超; 江力; 袁怀波
Original assignee: Hefei University of Technology
Current assignee: Hefei University of Technology
Priority date: 2010-12-10
Filing date: 2010-12-10
Publication date: 2012-07-25
Anticipated expiration: 2030-12-10
Also published as: CN102051364A

Abstract

The invention discloses application of a plant protein encoded with a DNA sequence of a sequence table SEQ ID: 1 or an amino acid sequence of a sequence table SEQ ID: 2 in the aspect of controlling the salt resistance of a plant. In the invention, the encoding gene of the salt-resisting protein is used for enhancing the salt resistance of the plant, such as inhibition of the expression of the encoding genes of the protein related to the plant salt resistance in the plant, and a gene and a technology support are provided for breeding of salt-resisting crops.

Description

A kind of plant anti-salt albumen and encoding sox and application

Technical field

The present invention relates in the bioengineering field plant anti-salt GAP-associated protein GAP and encoding sox and application, particularly utilize the method for this gene enhancement of plant salt resistance.

Background technology

Food problem is one of very big difficult problem of facing of the world today.Through improve per unit area yield or enlarge cultivated area, the approach such as improve the low and medium-yield farmland of increasing investment in agriculture increases grain yield and all can run into and need overcome the adverse circumstance restriction or alleviate the problem of adverse circumstance harm.Therefore, the understanding plant is improved the resistance of plant to the reaction mechanism of adverse circumstance, has become the further emphasis of research of agriculture prodn and food safety, and extremely the concern of politics circle, countries in the world and academia also is the focus of current life science.

Salting of soil is two important adverse circumstance factors of influence restriction plant growth, also is the important factor in order of crop production reduction, therefore seeks new anti-salt functional gene and illustrate its function to have important theory and practice significance.Except traditional breeding method, utilize genetic engineering technique to improve the crop saline-alkaline tolerance and have important theory and economic implications.

Arabidopis thaliana is a kind of model plant, is widely used in research fields such as plant genetics, developmental biology and molecular biology.Most of genes of Arabidopis thaliana can both find in other plant, and any discovery of relevant Arabidopis thaliana can both be applied to other plant research.Therefore, the output that the research of the anti-molecules of salt biological mechanism of Arabidopis thaliana is improved crop to the specific region has important theory and economic implications.The arabidopsis gene group checks order fully; Seeking and find that according to Arabidopis thaliana sequencing data storehouse (www.arabidopsis.org) the new functional gene with independent intellectual property right is one of focus of international phytology research field, also is the focus of science and technology competition between the country variant.Arabidopis thaliana has about 1.3 hundred million base pairs, 2.9 ten thousand genes.The function of most gene is not clear at present, utilizes mutating technology research gene function to become a kind of effective means.Through research, found the function of some salt resistant genes, like SOS1, SOS2, SOS3, NHX1, HKT1 etc. to two mutants.

According to the genome sequence that the Arabidopis thaliana DB is announced, find that salt processing HIS1-3 gene knockout plant shows as tolerance, this shows that this shows that the HIS1-3 gene relates to the regulation and control of salt resistance.For this reason, we have studied this gene function, and find that this gene has the effect of regulation and control plant salt tolerance.

Summary of the invention

A kind of new plant anti-salt GAP-associated protein GAP and encoding sox thereof have been the objective of the invention is to find.Plant salt tolerance provided by the present invention and reduction plant salt absorb the encoding sox of GAP-associated protein GAP, are called HIS1-3 (AT2G18050), derive from the Arabidopis thaliana of Colombia's wild-type, and its albumen is the protein with following amino acid residue sequences:

SEQ ID No:2 in the sequence table, the sequence 2 in the sequence table is made up of 167 amino-acid residues.

The cDNA gene of HIS1-3 is selected from one of following nucleotide sequence;

(1) dna sequence dna of SEQ ID No:1 in the sequence table;

(2) polynucleotide of SEQ ID No:2 protein sequence in the code sequence tabulation;

The nucleotide sequence of the dna sequence dna hybridization that (3) under the rigorous condition of height, can limit with SEQ ID No:1 in the sequence table;

(4) with sequence table in the dna sequence dna that limits of SEQ ID No:1 have 90% above homology and encode identical function protein DNA sequence.

Sequence 1 in the sequence table is by 763 based compositions, and its open reading frame (ORF) is to hold the 46th to 549 bit bases, the protein of encoding sequence SEQ ID No:2 from 5 '.

The expression vector that contains HIS1-3 of the present invention, clone and host bacterium all belong to protection scope of the present invention.Arbitrary segmental primer is to also belonging to protection scope of the present invention among the amplification HIS1-3.

Second purpose of the present invention provides a kind of method of utilizing this gene enhancement of plant salt resistance.

The method of enhancement of plant salt resistance provided by the present invention is the expression that suppresses the above-mentioned salt resistance of plants GAP-associated protein GAP encoding sox in the plant.

Suppress the expression of the above-mentioned salt resistance of plants GAP-associated protein GAP encoding sox HIS1-3 in the plant and can pass through accomplished in many ways; Method like the plant viral vector mediated gene silencing; The method of antisense technology silencer, the gene silencing methods of siRNA mediation etc.The method of inhibition of gene expression of the present invention is not limited to above-mentioned several method, all can as long as can suppress the HIS1-3 expression.

Utilize any plant gene to knock out technology, behind this gene knockout (or reticent), plant shows as anti-salt.

HIS1-3 gene of the present invention or its antisense nucleic acid 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 (qingfengmeisu qiong, kantlex etc.) that adds the alternative mark (gus gene, luciferase genes etc.) of plant or have resistance.By the plant transformed host both can be monocotyledons, also can be dicotyledons, as: paddy rice, wheat, rape, corn, cucumber, tomato, willow, turfgrass or lucerne place etc.Carry that HIS1-3 expression carrier of the present invention 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 through using, and plant transformed is become plant through tissue cultivating.

Plant anti-salt GAP-associated protein GAP of the present invention and coding base thereof can be the anti-salt breeding of farm crop the support of gene with technology are provided.

Below in conjunction with embodiment technical scheme of the present invention is further described.

Description of drawings

Fig. 1-A-1-D is that his1-3, wild-type plant are erect cultivation, and directly 20 days comparison photo, 1-E, the quantizating index (1-AMS of 1-F for adding up are vertically cultivated in dibbling under the normal illumination condition on the substratum that contains or do not contain salt (NaCl); 1-B 50mM NaCl; 1-C 75mM NaCl; 1-D 100mM NaCl; The 1-E root is long; The 1-F fresh weight).

Fig. 2-A-2-D is that his1-3, wild-type plant are handled a week in 100mM NaCl condition, under the normal cultured condition, replys week back phenotype contrast photo (wild-type and his1-3 plant before 2-A, the 2-B processing; 2-C, 2-D reply back wild-type and his1-3 plant).

Fig. 3-A-3-D be his1-3, wild-type plant after handling with 75mM NaCl, the comparison (comparison of 3-A underground part sodium content of sodium, potassium content and sodium potassium ratio in the plant; The comparison of 3-B over-ground part sodium content; The comparison of 3-C underground part potassium content; The comparison of 3-D over-ground part potassium content; The comparison of 3-E underground part sodium-potassium ratio).

Fig. 4 is an Expression of Related Genes level under his1-3, the wild-type plant condition of salt stress.

Embodiment

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

The acquisition of embodiment 1, HIS1-3 and encoding sox thereof

The about 50-100mg of seedling with the environmental Arabidopis thaliana of wild-type Colombia is a material, extracts its total RNA with Trizol, with UV spectrophotometer measuring RNA concentration.Pressing RevertAidTM First Strand cDNA Synthesis Kit (Fermentas company) test kit specification sheets, is template with the total RNA that extracts, synthetic cDNA article one chain.Article one chain cDNA to extract is a template, and carry out following PCR reaction: 20 μ l reaction systems include 10 * PCR damping fluid, 2 μ l; DNTPs (10mM) mixture 0.4 μ l; Each 2 μ l of primer 1 and primer 2, Tag enzyme (5U/ μ l) 0.2 μ l, all the other add distilled water to 20 μ l.Wherein, primer 1:5 '-ACCACCACTCATCCTCCATACTTT-3 '; Primer 2: 5 '-TCTCGCCTTCTTCACTTTCCTCT-3 '.On Life Express gene-amplificative instrament, increase: first 94 ℃ of preparatory sex change 3min, 94 ℃ of 30sec again, 54 ℃ of 30sec, 72 ℃ of 60sec amount to 30 circulations, and last 72 ℃ are extended 10min, the PCR product that obtains are carried out electrophoresis at 1% (m/v) sepharose.The cDNA fragment that PCR is obtained is connected in the pGEM-T carrier; Obtain containing the segmental carrier pT-HIS1-3 of purpose, the evaluation of checking order, the result shows that cDNA fragment that PCR obtains has the dna sequence dna of sequence SEQ ID No:1 in the sequence table; CDNA gene for HIS1-3; By 763 based compositions, its encoding sequence is that coding has the protein of the amino acid residue sequence of sequence SEQ ID No:2 in the sequence table from 5 ' end the 46th bit base to the, 549 bit bases.

Embodiment 2, cultivation salt resistance enhanced Arabidopis thaliana

1, the acquisition of the homozygous mutation body his1-3 that knocked out of HIS1-3 gene

Obtain T-DNA from U.S. Arabidopis thaliana germ plasm resource center and inserted two mutants seed (SAIL 799A07; Http:// www.arabidopsis.org/servlets/TairObj ect? Type=germplasm&id=1006546836).Be the homozygous mutation body of identifying that the HIS1-3 gene is knocked out, sowing SAIL 799A07 seed also extracts individual plant plant DNA, as template, with 3 pairs of primers it is carried out pcr amplification.Wherein, primer 1LB1:5 '-GCCTTTTCAGAAATGGATAAATAGCCTTGCTTCC-3, primer 2: HIS 1-3-LP 5 '-GACCGAAAGGAGGAGCTAATG-3 ', primer 3:HIS1-3-RP 5 '-AAGATGATAACGAGGCAGCAG-3 '.According to the agarose gel electrophoresis result of PCR product, obtain the homozygous mutation body his1-3 that the HIS1-3 gene is knocked out.From phenotype; This two mutants and wild-type (WT) plant does not have significant difference (Fig. 1-A) under the normal growth condition; The expression vector excessively of the strong mover of cDNA sequence construct 35S of this gene of usefulness also transforms two mutants; Recovered the wild-type phenotype on this transfer-gen plant form, under condition of salt stress, also recovered wild-type plant symptom, confirmed that this gene controls the salt resistance shape really.

2, the salt resistance of his1-3 and wild-type plant relatively

Wild-type (WT) is seeded in the petridish that diameter is 90mm with his1-3 simultaneously; Substratum is respectively the solid medium that adds sodium and do not add sodium; Erectting the cultivation of cultivation or level places 22 ℃ of constant temperature illumination (photoperiod is illumination in 16 hours, 8 hours dark) to cultivate.Cultivate after 20 days, can observe: WT that on the normal cultured base, grows and his1-3 all do not have significant difference at phenotype, fresh weight, rectangular of root.On the substratum that is containing sodium after direct dibbling of plant or the transplanting, cultivate, his1-3 shows the proterties of tangible salt tolerance.At 75mM, 100mM NaCl coerces down, and the fresh weight of his1-3 and root are kept burning day and night to show higher than WT.The above results shows that his1-3 obviously tolerates salt stress than WT.

3, the sodium of his1-3 and wild-type plant accumulation relatively

WT and the intravital sodium-potassium content of his1-3 plant to salt is handled are measured, and find that the sodium accumulation is starkly lower than WT in the his1-3 plant body, and potassium content do not have significant difference; The Na of while wild-type ⁺/ K ⁺Apparently higher than his1-3, show that the his1-3 plant is than the difficult accumulation of WT Na ⁺, to Na ⁺Certain tolerance is arranged.

4, his1-3 and wild-type plant related gene expression compare under the salt treatment condition

WT and the intravital related gene expression level of his1-3 plant that salt is handled detect; Find that interior SOS family gene of his1-3 plant body and NHX1 gene are apparently higher than WT; And HKT1 genetic expression does not have notable difference, shows that the his1-3 plant maybe be relevant with the activation of SOS family gene to the salt ion response.

Claims

1. a regulate and control method that strengthens the Arabidopis thaliana salt resistance is characterized in that: insert mutating technology through T-DNA and suppress the vegetable-protein expression of gene of dna sequence dna shown in SEQ ID No:1 in the plant materials.