CN101987867B - Ethylene receptor NTHK1 interactive protein relevant to plant stress tolerance as well as coding gene and application thereof - Google Patents

Abstract

The invention discloses ethylene receptor NTHK1 interactive protein relevant to plant stress tolerance as well as a coding gene and application thereof. The protein is protein (1) comprising an amino acid sequence shown as a sequence 2 in a sequence table or protein (2) which is formed in such a way that an amino acid residual sequence of the sequence 2 in the sequence table is replaced and/or lost and/or added with one or some amino acid residues, is relevant to the plant stress tolerance and is derived by the protein (1). The method for culturing a transgenic plant with improved stress tolerance has important theoretical and practical significance and plays an important role in the genetic improvement of the plant.

Description

A kind of ethylene receptor NTHK1 interact protein relevant and encoding sox and application with plant stress tolerance

Technical field

The present invention relates to a kind of ethylene receptor NTHK1 interact protein relevant and encoding sox and application with plant stress tolerance.

Background technology

The variation of physics, chemical factor in the environment; For example arid, saline and alkaline, damage to plants caused by sudden drop in temperature, freeze injury, waterlogging etc. coerce factor and biotic factor; For example disease and pest has significant effects to growth and development of plant; Can cause the extensive underproduction of farm crop when serious, cultivating the resistance of reverse crop is one of major objective of plant husbandry.Improve the resistance of reverse of crop, except utilizing traditional breeding method, at present, one of field that the molecular genetic breeding has become the scientific worker to be paid close attention to.Under the coercing of abiotic and biological adverse circumstance; Higher plant cell has the number of ways impression and replys the variation of physico-chemical parameter in the external environment; The signal that born of the same parents are outer becomes intracellular signal, passes the signal along to nucleus through a series of phosphorylation cascade reactions, through the relevant functional gene of transcription factor regulation and control; Start the expression of adverse circumstance response gene, improve the resistance of reverse of plant.Verified at present, relevant with one of signal pipeline of response abiotic and biological environment stress in plant with ethene.The patent No. is the patent of ZL99 1 19096.3 and participated in above-mentioned signal conduction and relevant (Wanhong Cao with plant stress tolerance below with reference to the ethylene receptor NTHK1 that has all proved tobacco in the document; Jinsong Zhang&Shouyi Chen; Et al., Expression of tobacco ethylenereceptor NTHK1 alters plant responses to salt stress, Plant; Cell and Environment (2006) 29,1210-1219.).

NTHK1 is the II class ethylene receptor of tobacco; In the model plant tobacco, illustrate the function of NTHK1 interact protein; Signal transmission for the ethene relational approach of further understanding response abiotic and biological adverse circumstance; And be applied to the improvement of plant, both had important significance for theories and also had great practical value.

Summary of the invention

The purpose of this invention is to provide a kind of ethylene receptor NTHK1 interact protein and the encoding sox thereof relevant with plant stress tolerance.

The ethylene receptor NTHK1 interact protein name relevant with plant stress tolerance provided by the present invention is called Neip2, derives from tobacco, is following 1) or 2) protein:

1) protein of forming by the aminoacid sequence shown in the sequence in the sequence table 2;

2) with the aminoacid sequence of sequence in the sequence table 2 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and relevant with plant stress tolerance by 1) deutero-protein.

Sequence 2 is made up of 350 amino-acid residues in the sequence table, is the PEST structural domain from N-terminal 24-38 position, is three ankyrin structural domains from N-terminal 225-254 position, 258-287 position and 291-320 position.Existing report shows; The ankyrin structural domain is a conserved sequence that approximately comprises 33 amino acid lengths; Its center sequence can form the helix-loop-helix structure; The ankyrin structural domain is at mediation (the Becerra C that plays an important role in the protein-protein interaction; Jahrmann T, Puigdomenech P, Vicient CM (2004) Ankyrin repeat-containing proteins in Arabidopsis:characterization of a novel andabundant group of genes coding ankyrin-transmembrane proteins.Genes Dev 340:111-121).The PEST structural domain is the sequence of one section proline rich (P), L-glutamic acid (E), Serine (S) and Threonine (T) four seed amino acids; It mainly acts on is the phosphorylation site as protein kinase, and the phosphorylation in this site has often mediated the degraded of albumen through the 26S proteasome.The proteic structural representation of Neip2 is as shown in Figure 1.Its carbon teminal is the Tumor-necrosis factor glycoproteins of 3 Ankyrin structural domains, and the nitrogen end has a PEST structural domain.

In order to make 1) in Neip2 be convenient to purifying, label as shown in table 1 on proteinic N-terminal that can the aminoacid sequence shown in the sequence 2 is formed in by sequence table or C-terminal connect.

The sequence of table 1. label

Label	Residue	Sequence
			Poly-Arg	5-6 (being generally 5)	RRRRR
Poly-His	2-10 (being generally 6)	HHHHHH
			FLAG	8	DYKDDDDK
Strep-tag II	8	WSHPQFEK
			c-myc	10	EQKLISEEDL

Above-mentioned 2) but in the Neip2 synthetic, also can synthesize its encoding sox earlier, carry out biology again and express and to obtain.Above-mentioned 2) encoding sox of the Neip2 in can be through the codon that in the dna sequence dna shown in the 5 ' terminal 1-1050 bit base, lacks one or several amino-acid residue with sequence in the sequence table 1; And/or carry out the missense mutation of one or several base pair, and/or obtain at the encoding sequence that its 5 ' end and/or 3 ' end connects the label shown in the table 1.

The encoding sox of the above-mentioned ethylene receptor NTHK1 interact protein relevant with plant stress tolerance also belongs to protection scope of the present invention.

The encoding sox of the ethylene receptor NTHK1 interact protein relevant with plant stress tolerance specifically can be following 1)-4) in arbitrary described gene:

1) its encoding sequence is the deoxyribonucleotide from 5 ' terminal 1-1050 position of sequence 1 in the sequence table;

2) its nucleotide sequence is the sequence 1 in the sequence table;

The dna molecular of the dna sequence dna hybridization that 3) under stringent condition, can limit with sequence in the sequence table 1 and the above-mentioned ethylene receptor NTHK1 interact protein relevant of encoding with plant stress tolerance;

4) with 1) gene have the homology more than 90%, and the dna molecular of the above-mentioned ethylene receptor NTHK1 interact protein relevant of encoding with plant stress tolerance.

Gene in the said step 4) is with 1) gene the homology more than 95% is preferably arranged.

Sequence 1 in the sequence table is by 1053 based compositions, and its open reading frame (ORF) is from 5 ' terminal 1-1050 bit base, and encoding amino acid sequence is the Neip2 of sequence 2 in the sequence table.

Above-mentioned stringent condition can be at 6 * SSC, in the solution of 0.5%SDS, 65 ℃ of hybridization down, uses 2 * SSC then, and 0.1%SDS and 1 * SSC, 0.1%SDS respectively wash film once.

Increase above-mentioned Neip2 full length gene or arbitrary segmental primer to also belonging to protection scope of the present invention.

The recombinant vectors, expression cassette, transgenic cell line and the reorganization bacterium that contain the above-mentioned ethylene receptor NTHK1 interact protein encoding sox relevant with plant stress tolerance also belong to protection scope of the present invention.

Available existing plant expression vector construction contains the recombinant expression vector of Neip2 gene.Said plant expression vector comprises double base agrobacterium vector and the carrier etc. that can be used for the plant micropellet bombardment, like pCAMBIA3301, pCAMBIA1300, pBI121, pBin19, pCAMBIA2301, pCAMBIA1301-UbiN or other plant expression vector of deriving.Conventional biological methods such as the plant expression vector that carries the ethylene receptor NTHK1 interact protein encoding sox Neip2 relevant with plant stress tolerance of the present invention can lead through Ti-plasmids, Ri plasmid, plant viral vector, directly DNA conversion, microinjection, electricity, agriculture bacillus mediated are transformed in vegetable cell or the tissue.By the plant transformed host both can be monocotyledonss such as paddy rice, also can be dicotyledonss such as Arabidopis thaliana, soybean, tobacco.

When using the gene constructed recombinant plant expression vector of Neip2; Before its transcription initiation Nucleotide, can add any enhancement type, composing type, organizing specific type or inducible promoter; Like 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 said translation wave 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 in plant, express enzyme or the gene (gus gene, luciferase genes etc.) of luminophor, antibiotic marker thing (qingfengmeisu qiong affinity tag, kantlex affinity tag etc.) or the anti-chemical reagent marker gene (like anti-weedkiller gene) etc. that can produce colour-change with resistance as adding.From the security consideration of transgenic plant, can not add any selected marker, directly with adverse circumstance screening transformed plant.

Another object of the present invention provides a kind of method of cultivating the transgenic plant of resistance of reverse raising.

The method of the transgenic plant that cultivation resistance of reverse provided by the present invention improves is in the encoding sox Neip2 importing plant with the above-mentioned ethylene receptor NTHK1 interact protein relevant with plant stress tolerance, obtains the transgenic plant of resistance of reverse raising.

In the aforesaid method, said resistance of reverse is drought-resistant and/or salt tolerant and/or anti-injury.

Said plant can be monocotyledons or dicotyledons, like paddy rice, Arabidopis thaliana, soybean, tobacco etc.

Said plant specifically can be tobacco or Arabidopis thaliana.

The present invention has made up the expression vector of crossing of Neip2; And it is changed in the wild-type tobacco plant; Experiment showed, the resistance of reverse of the tobacco that changes Neip2 over to, particularly salt tolerance obviously shifts to an earlier date; Explain that Neip2 is the albumen relevant with plant stress tolerance, Neip2 albumen and encoding sox thereof can be used for improving the resistance of reverse of plant.The method of the transgenic plant that cultivation resistance of reverse of the present invention improves, for cultivating anti-abiotic and biological coercing, significant like plant variety and raising crop yield that drought stress, salt stress, disease and pest are coerced.

Below in conjunction with accompanying drawing and specific embodiment the present invention is further specified.

Description of drawings

Fig. 1 is the proteic structural representation of Neip2.

Fig. 2 is the two assorted principle schematic of CytoTrap yeast.

Fig. 3 is the synoptic diagram of recombinant vectors pSOS-NTHK1.

Fig. 4 is that two assorted system verification NTHK1 of CytoTrap yeast and Neip2 interact.

Fig. 5 is the expression of Neip2 gene when NaCl handles.

Fig. 6 is overexpression carrier and the RNAi vector construction process of Neip2.

Fig. 7 is the Molecular Identification result of transgene tobacco.

Fig. 8 is the phenotype analytical result of transgene tobacco.

Fig. 9 is the influence of condition of salt stress to the tobacco germination.

Figure 10 is the influence of condition of salt stress to the tobacco plant fresh weight.

Embodiment

Experimental technique described in the following embodiment like no specified otherwise, is ordinary method; Said reagent and biomaterial like no specified otherwise, all can obtain from commercial sources.

Embodiment 1, utilization yeast two-hybrid system obtain the mutual albumen Neip2 that does with ethylene receptor NTHK1

1, the principle of CytoTrap yeast two-hybrid system

The CytoTrap yeast two-hybrid system is to utilize a temperature sensitive yeast saccharomyces cerevisiae two mutants cdc25H to carry out the screening of interaction protein.The temperature sensitive of yeast saccharomyces cerevisiae two mutants cdc25H is because a point mutation with the cdc25 gene of human hSos dna homolog causes.Cdc25 genes encoding guanylic acid exchange factor; It can combine and activate the Ras signal path; Make yeast at 37 ℃ of (Petitjean et al.1990 that grow down; Comparisonof thermosensitive alleles of the CDC25 gene involved in the cAMP metabolism ofSaccharomyces cerecisiae, Genetics 124:797-806); The yeast saccharomyces cerevisiae two mutants cdc25H that has the cdc25 point mutation then can only be 22-25 ℃ of growth down.Since human hSos gene can complementary cdc25 gene function, therefore can be utilized in and express the hSos gene on the film and make yeast saccharomyces cerevisiae two mutants cdc25H at 37 ℃ of normal growths.

Be that encoding sequence with target protein (Neip2) is building up on the pMyr carrier in this experiment,, can target protein (Neip2) be anchored on the cytolemma owing to the sequence of coding myristylization is arranged at 5 ' end of this carrier.If can interact with the target protein (Neip2) that is positioned on the film with the bait protein (ethylene receptor NTHK1) of hSos gene fusion, then the hSos gene just can play a role, thereby makes yeast 37 ℃ of growths down.The two assorted principle schematic of CytoTrap yeast are as shown in Figure 2.Bait representes to contain the carrier of bait protein (in this experiment, being ethylene receptor NTHK1) encoding sox among Fig. 2.

2, the structure of tobacco cDNA expression library

We show in existing research; NTHK1 is a gene relevant with environment stress; What its expression received adverse circumstances such as salt, arid or injury induces the expression amount of its mRNA higher in floral organ (Zhang et al.2001 Atwo-component gene (NTHK1) encoding a putative ethylene-receptor homolog is bothdevelopmentally and stress-regulated in tobacco.Theor Appl Genet 102:815-824).Therefore, this experiment is cDNA with its reverse transcription with total RNA of tobacco floral organ with respectively through total RNA mixing of salt stress, drought stress and injury inductive tobacco again.Concrete experimentation is following:

Tobacco (Nicotiana tabacum var.Xanthi) seed (available from institute of microbiology of the Chinese Academy of Sciences) is seeded in the vermiculite,, cultivates illumination 16 hours, 25 ℃ of temperature in the greenhouse with the nutritive medium pouring that contains the 1/4MS inorganic salt.Treat that the tobacco seedling grew to about 4 weeks, be divided into four groups at random, handle as follows respectively: first group is carried out salt stress, handles 24 hours with 100mM NaCl; Second group is carried out drought stress, handles 24 hours with 20%PEG 6000; The 3rd group is injured processing, and detailed process is following: get the tobacco leaf of vegetative reproduction, remove middle arteries and veins, the blade of arteries and veins is cut into 1-2cm in will removing ²Fragment, fragment was placed in the 1/2MS liquid nutrient medium slowly wave and culture 3 hours; The 4th group of seedling is cultured to the adult plant, gets the floral organ of tobacco adult plant different development stage.Above four groups of materials are collected the back mix, liquid nitrogen flash freezer stores the extraction that is used for RNA in-80 ℃ of cryogenic refrigerators.

The extraction of total RNA adopts the method for guanidinium isothiocyanate-phenol-chloroform to extract (Zhang et al.2001 Atwo-component gene (NTHK1) encoding a putative ethylene-receptor homolog is bothdevelopmentally and stress-regulated in tobacco.Theor Appl Genet 102:815-824); The purifying of mRNA uses Promega test kit (available from Promega company) PolyAT tract mRNA isolationsystem IV to separate, and separates the mRNA that obtains and carries out quantitatively with ultraviolet spectrophotometer.The structure of cDNA carries out according to the method for CytoTrap cDNA library construction test kit (available from Stratagene company).5 μ g mRNA are used for the synthetic of first chain; Carry out the synthetic of second chain then; Mend flat end with pfu high-fidelity Taq enzyme, the synthetic double-stranded DNA carries out the connection of EcoR I joint, collecting precipitation after the extracting of phenol chloroform; Cut with Xho I enzyme; The double-stranded DNA that will contain EcoR I and Xho I directly is connected in the pMyr XR carrier (available from Stratagene company) (carrier is in advance with EcoR I and the two also dephosphorizations of cutting of Xho I), obtains recombinant vectors pMyr-Neip2, and recombinant vectors pMyr-Neip2 is changed in the XL-gold Kanr intestinal bacteria (available from Stratagene company).Confirming the titre in cDNA library according to the number of intestinal bacteria positive colony bacterium colony, is 2 * 10 through the titre that detects this cDNA library ⁵Cfu.10 mono-clonals of picking extract plasmid immediately, carry out pcr amplification with the universal primer on the pMyr carrier, insert pulsating size in the constructed cDNA library to detect.Primer sequence is pMyrF:5 '-ACTACTAGCAGCTGTAATAC-3 ' and pMyrR:5 '-CGTGAATGTAAGCGTGACAT-3 '.The result shows that the size of pcr amplification product is about 1.2kb.

3, the structure of recombinant vectors pSOS-NTHK1 (Δ TM) (Bait plasmid)

This experiment used yeast double cross test kit is the two assorted test kits (available from Stratagene company) of Cytotrap yeast.The structure of bait protein selects to remove the NTHK1 (145-762aa) of TMD; Be called NTHK1 (Δ TM); Itself and hSOS are formed fusion rotein; The concrete building process of recombinant vectors pSOS-NTHK1 (Δ TM) is following: the plasmid pBK-CMV (available from Stratagene company) to contain the NTHK1 encoding sox is a template, with (145-762aa) the sequences Design primer at two ends of the NTHK1 (GenBank Accession No:AF026267) that removes TMD, carries out pcr amplification.Forward primer: BaitK1F5 '-AGGGGATCCTTATGCTGAAAAAGAAAAC-3 ' (containing BamH I restriction enzyme site), reverse primer: BaitK1R:5 '-ACAGTCGACCGTGATTATGCTTGCCTG-3 ' (containing Sal I restriction enzyme site).Pcr amplification product is carried out double digestion with BamH I and Sal I; Enzyme is cut product and is connected to the pSOS carrier cut through same enzyme (available from Stratagene company; Contain the hSOS gene fragment in the pSOS carrier) in, with the recombinant vectors called after pSOS-NTHK1 (Δ TM) that obtains.The synoptic diagram of recombinant vectors pSOS-NTHK1 (Δ TM) is as shown in Figure 3.

4, yeast conversion

Used yeast saccharomyces cerevisiae two mutants cdc25H, plasmid etc. are and comprise in the test kit in this step experiment, and test kit is available from Stratagene company.

Yeast saccharomyces cerevisiae two mutants cdc25H is inoculated on the YPAD culture medium flat plate; Cultivated 4 days for 23-25 ℃, a plurality of single bacterium colonies of picking are inoculated in respectively in the YPAD liquid culture, and then on separate application and the YPAD solid medium flat board; Cultivated 4 days for 37 ℃; Picking is at 37 ℃ of mono-clonals that do not have the yeast colony growth, and the use final concentration is 25% glycerine preservation, and is subsequent use in-80 ℃ of preservations behind the liquid nitrogen flash freezer.

The yeast saccharomyces cerevisiae two mutants cdc25H that preserves is coated on the YPAD culture medium flat plate; The picking mono-clonal is inoculated in the centrifuge tube that 1.5ml YPAD liquid nutrient medium is housed; The thermal agitation mixing; Get bacterium liquid and be transferred in the 250ml triangular flask that the 50mlYPAD liquid nutrient medium is housed, 24 ℃ shaking culture 14-16 hour, guarantee the OD of nutrient solution ₆₀₀Value is greater than 1.With above-mentioned OD ₆₀₀Value joins in the 300ml YPAD liquid nutrient medium greater than 1 saccharomycetic nutrient solution, and 24 ℃ of shaking culture 3 hours guarantee the OD of nutrient solution ₆₀₀Value is greater than 0.7; 1000g is centrifugal 5 minutes under the room temperature condition, abandons supernatant, and bacterial sediment is resuspended with the aseptic ultrapure water of 50ml; Centrifugal 10 minutes of 1000g; Abandon supernatant, bacterial sediment is resuspended with 50ml LiSORB solution (contain the Tris-HCl of LiAc that final concentration is 100mM, PH 8.0 that final concentration is 10mM, EDTA and the final concentration that final concentration is 1mM is the sorbyl alcohol of 1M), and room temperature was placed 30 minutes.And then centrifugal 5 minutes of 1000g; Abandon supernatant; Collect bacterial sediment; Resuspended with 300 μ l LiSORB solution; And in 300 μ l LiSORB solution, adding salmon sperm dna (available from Sigma company), 5.4mlPEG/LiAc solution (LiAc and the quality percentage composition that contains final concentration and be the EDTA of the Tris-HCl of the PH 8.0 of 10mM, PH 8.0 that final concentration is 1mM, PH 7.5 that final concentration is 100mM is 40% PEG3350 (available from Sigma company)) and the 530 μ l DMSO of 600 μ l 8mg/ml, mixing is even, obtains the zymic competent cell.

5, the screening of yeast two-hybrid positive colony

In the yeast competent cell of above-mentioned steps 4 preparations, add the recombinant vectors pSOS-NTHK1 (Δ TM) of 40 μ g above-mentioned steps, 3 acquisitions and cDNA library plasmid (pMYR-Neip2) and the 200 μ l mercaptoethanols that 40 μ g above-mentioned steps 2 obtain; Mixing divides to install in 20 centrifuge tubes.Establish negative contrast simultaneously, promptly in the yeast competent cell of 500 μ l above-mentioned steps, 4 preparations, add cDNA library plasmid and 10 μ l mercaptoethanols that 2 μ g pSOS carriers and 2 μ g above-mentioned steps 2 obtain, room temperature was placed 30 minutes; Constantly shake during this time; 42 ℃ of heat shocks were handled 20 minutes, and ice bath is 3 minutes again, centrifugal collection yeast sedimentation; Sorbitol Solution USP with 0.5ml 1M is resuspended; It is 20mM that yeast after transforming is coated in the glucose final concentration, but lacks uridylic and leucic SD substratum (is on SD/glu (UL) substratum) (available from the Amresco company) flat board, cultivated 2 days for 24 ℃; Using the filter paper of sterilizing that saccharomycetic bacterium colony is copied to the semi-lactosi final concentration then is 0.2%; But lack uridylic and leucic SD substratum and (be on SD/gal (UL) substratum) (available from the Amresco company) flat board, cultivated 7 days for 37 ℃ that the clone who grows is candidate clone; Candidate's mono-clonal is transferred to SD/glu (UL) on the culture medium flat plate; Cultivated 2-3 days for 24 ℃, the mono-clonal that grows is washed with 25 μ l aqua sterilisas and thorough mixing, get 5 μ l bacteria suspensions and be inoculated in two SD/glu respectively (UL) and two SD/gal (UL) on the culture medium flat plate; (UL) (UL) culture medium flat plate is placed on 24 ℃ of cultivations 3-5 days, and (UL) (UL) culture medium flat plate is placed on 37 ℃ of cultivations 3-5 days to another SD/glu for culture medium flat plate and SD/gal for culture medium flat plate and SD/gal with one of them SD/glu.

The result shows; At the SD/glu of 24 ℃ of cultivations (UL) culture medium flat plate and the SD/gal (UL) normal growth all of the yeast on the culture medium flat plate; And on the flat board of 37 ℃ of cultivations; If (UL) clonal growth is arranged on the flat board, and corresponding be cloned in SD/glu (UL) dull and stereotyped going up can not be grown, and this clone is through the further positive colony of screening acquisition at SD/gal.

6, the checking of positive colony and analysis

The positive colony that above-mentioned steps 5 is screened be inoculated in SD/glu (UL) in the liquid nutrient medium, 24 ℃ shaking culture 2-3 days, guarantee OD ₆₀₀Value is greater than 1.Collect thalline, extract plasmid, with sharp being converted in the intestinal bacteria of plasmid electricity of extracting, coating LB culture medium flat plate, (LB contains for dull and stereotyped every liter: NaCl, 10g; Tryptone10g; Yeast extract 5g; 20g agar, adding distil water transfer to 7.0 with 5N NaOH with pH to 1000Ml.10 single bacterium colonies of picking on each flat board, each adds the granulated glass sphere (diameter 0.5mm) of 50 μ l pickling and the mixing solutions that 300 μ l are made up of isopyknic phenol and chloroform again with the resuspended thalline of 300 μ l yeast lysates (containing the Tris that final concentration is 50mM (PH 8.0), final concentration and be the LiCl of 2.5M, EDTA that final concentration is 62.5mM and 4% Trixon-X 100); Thermal agitation 1 minute; Centrifugal 5 minutes of 14000rpm room temperature moves to supernatant in another pipe, adds the absolute ethyl alcohol of precooling;-20 ℃ of placements spend the night or-80 ℃ placed 15 minutes; 14000rpm is centrifugal 10 minutes under 4 ℃ of conditions, and supernatant discarded is washed once with 75% ethanol; Dry up the back and melt, obtain saccharomycetic genomic dna with 10-20 μ l ultrapure water.

Saccharomycetic genomic dna with above-mentioned acquisition is a template, is that primer carries out pcr amplification with pMyrF:5 '-ACTACTAGCAGCTGTAATAC-3 ' and pMyrR:5 '-CGTGAATGTAAGCGTGACAT-3 '.Pcr amplification product is carried out agarose gel electrophoresis detect, see whether the amplified band of each single bacterium colony is consistent.If it is consistent; The recombinant vectors pSOS-NTHK1 (Δ TM) of above-mentioned steps 3 acquisitions and the cDNA library plasmid of above-mentioned steps 2 acquisitions are converted in the competent cell of yeast saccharomyces cerevisiae two mutants cdc25H once more; Carry out two assorted checkings according to the aforesaid operations step; If the yeast that finally obtains is (UL) clonal growth to be arranged on the flat board at SD/gal equally; And corresponding be cloned in SD/glu (can not grow on UL) dull and stereotyped, then this clone is final positive colony, and the plasmid that extracts this positive colony carries out sequencing analysis.

Sequencing result shows, has a gene to occur repeatedly, with this unnamed gene be Neip2 ( NTHK1- iNteractingproteins).The deoxyribonucleotide sequence of Neip2 gene is shown in sequence in the sequence table 1, and its amino acid sequence coded is shown in sequence in the sequence table 2.

The checking of embodiment 2, NTHK1 albumen and Neip2 protein-interacting

According to the resulting positive colony of the yeast two-hybrid of the foregoing description 1, need further verify just and can be sure of its interaction.

With the recombinant vectors pSOS-NTHK1 transformed yeast competent cell that the foregoing description 1 step 3 obtains, the result shows, in the time of 24 ℃, no matter be SD/glu (UL) on the substratum still at SD/gal (UL) the equal ability normal growth of all transformants on the substratum; And in the time of 37 ℃; Transformant (UL) all can not be grown on the substratum at SD/glu; And SD/gal (UL) on the substratum only positive control pSOS MAFB+pMYRMAFB (test kit carries; Proved that MAFB albumen can the oneself does mutually) with the yeast ability normal growth that changes recombinant vectors pSOS-NTHK1 and recombinant vectors pMYR-Neip2 simultaneously over to, show NTHK1 albumen can with the Neip2 protein-interacting.

NTHK1 albumen can be as shown in Figure 4 with Neip2 protein-interacting result.Wherein, pSOS+pMYR representes to change over to the yeast of empty carrier pSOS and pMYR, negative contrast; PSOS+HK1+pMYR representes to change over to the yeast of recombinant vectors pSOS-NTHK1 and empty carrier pMYR; PSOS+pMYRNeip2 representes to change over to the yeast of empty carrier pSOS and recombinant vectors pMYR-Neip2; PSOSHK1+pMYRNeip2 representes to change over to the yeast of recombinant vectors pSOS-NTHK1 and recombinant vectors pMYR-Neip2; PSOS MAFB+pMYRMAFB representes positive control.

Embodiment 3, Neip2 expression of gene pattern

Utilize the RT-PCR method to detect the expression pattern of Neip2 gene under the NaCl treatment condition.Tobacco (Nicotiana tabacum var.Xanthi) seed (available from Institute of Micro-biology of the Chinese Academy of Sciences) is directly sowed in containing the vermiculite of nutritive medium; Grow after 10 days; To the vermiculite that contains nutritive medium, 3-4 is after week in growth, with deionized water vermiculite is washed down gently with little transplantation of seedlings; The tobacco seedling is transferred in the NaCl solution that concentration is 300mM handled respectively 3,6,12 and 24 hours, after the processing tobacco seedling is put into the liquid nitrogen quick-frozen rapidly.Extract total RNA of tobacco, carry out the RT-PCR amplification.The RT-PCR amplification system is following: in the reverse transcription system of 25 μ l, add total RNA 2 μ g, Oligo (dT) 0.5 μ g, 5 μ l, 5 * reverse transcription damping fluid, 1.5 μ l dNTP (each 10mM) and 1 μ lMMLV ThermoScript II (available from promega company).37 ℃ of reactions after 1 hour 75 ℃ placed 15 minutes, make the ThermoScript II inactivation.With above-mentioned RT-PCR amplified production is template, is primer with pMyrF:5 '-ACTACTAGCAGCTGTAATAC-3 ' and pMyrR:5 '-CGTGAATGTAAGCGTGACAT-3 ', amplification Neip2 gene.Pcr amplification product is compared with Tublin A1 (tubulin A1) gene of tobacco; With this ratio is ordinate zou; With the NaCl treatment time is that X-coordinate is mapped, and three repetitions are established in experiment, and the expression of Neip2 gene when 300mM NaCl handles different time is as shown in Figure 5.The result shows, what the Neip2 expression of gene received that NaCl coerces induces.

The phenotype analytical of embodiment 4, Neip2 gene overexpression and RNAi transfer-gen plant

1, the structure of plant expression vector

In order to identify the function of Neip2 gene, made up the overexpression carrier and the RNAi carrier of this gene respectively.The carrier that sets out of overexpression is pROK2.Because the carrier pMYR that uses during the construction cDNA library is yeast and colibacillary shuttle plasmid; Therefore the positive colony that obtains of yeast two-hybrid screening extracts behind the plasmid directly transformed into escherichia coli; But the restriction enzyme site of pMYR is limited; Cut enzyme in the MCS of product insertion plasmid pBSK (available from Stratagene company) behind EcoRI and Xho I double digestion in the cDNA library that therefore can the foregoing description 1 be made up, and obtains recombinant vectors; And then this recombinant vectors imported in the intestinal bacteria; Behind BamHI and KpnI double digestion, obtain the Neip2 gene; The Neip2 gene that obtains is connected with the pROK2 plasmid of cutting through same enzyme (available from Stratagene company), obtains recombinant vectors pROK2-Neip2.Recombinant vectors pROK2-Neip2 imported to obtain transfer-gen plant in the tobacco.

The carrier that sets out of RNAi interference vector is pZH01 (available from a Stratagene company).The dna fragmentation (672bp) that does not contain the Neip2 of ankyrin structural domain encoding sox with 5 ' end is a template; With p1:5 '-TTCTAGAGCTCATGTCTGAGGGAGAGAAAGT-3 ' and P2:5 '-TGTCGACGGTACCTTCTGAGTCTTCTTCGTCTT-3 ' is that primer carries out pcr amplification; With pcr amplification product with before forward and the GUS fragment of oppositely inserting respectively among the plant expression vector pZH01 with afterwards; The insertion site of pcr amplification product is respectively SacI, KpnI and SalI, XbaI; Constituted the RNAi plant expression vector of Neip2, with the RNAi carrier called after pZH01-Neip2-RNA-interferance (RNAi) that obtains.The RNAi carrier that obtains through behind the leaf disc method transformation of tobacco, is obtained transfer-gen plant.Overexpression carrier and the RNAi vector construction process of Neip2 are as shown in Figure 6.Wherein, Fig. 6 A is the overexpression carrier pROK2-Neip2 of Neip2, and Fig. 6 B is RNAi carrier pZH01-Neip2-RNA-interferance (RNAi).

2, the Molecular Identification of transfer-gen plant

The expression amount of Neip2 gene in transgene tobacco detects respectively with Northern blot and RT-PCR method.Total RNA of the transgene tobacco that extraction step 1 obtains (being respectively transgene tobacco that changes recombinant vectors pROK2-Neip2 over to and the transgene tobacco that changes RNAi carrier pZH01-Neip2-RNA-interferance (RNAi) over to); The total RNA of 20 μ g forwards HybrondN to behind the sex change gel electrophoresis that contains 1% formaldehyde ⁺On the nylon membrane, UV-crosslinked after, the methylene blue with 0.1% carries out RNA dyeing, confirms the applied sample amount of each swimming lane RNA on the film.Hybridization is carried out at 65 ℃, and probe mark uses random primer labelling test kit (available from TaKaRa company), and the probe that Neip2 hybridization is used is the sequence of Neip2 gene 5 ' end 600bp.Hybridization signal shields record with phosphorus.Three repetitions are established in experiment, and the Molecular Identification result of transgene tobacco is as shown in Figure 7.Wherein, Fig. 7 A is a Neip2 expression of gene situation in the transgene tobacco (OE) of Neip2 gene overexpression, and Fig. 7 B changes Neip2 expression of gene situation in the transgene tobacco of RNAi carrier over to, and WT representes not genetically modified tobacco.Wherein, NtNeip2 representes that Northern blot detects the hybrid belt of Neip2 gene, and the band of rRNA when rRNA representes electrophoresis is as the contrast of applied sample amount.In the transgene tobacco, Neip2 expression of gene amount is the highest in No. 4 plant of tobacco and No. 19 plant, does not detect the Neip2 expression of gene and change in No. 12 plant of tobacco and No. 13 plant of RNAi.Therefore select 4,19,12 and No. 13 transgenic lines to do further to analyze.

3, the phenotype analytical of transgene tobacco

The phenotype analytical result of transgene tobacco is as shown in Figure 8.Wherein 12,13 expressions change the transgenic line of RNAi carrier over to; 16-4 representes the (Cao of strain system of NTHK1 gene overexpression; Et al.; Modulation of EthyleneResponses Affects Plant Salt-Stress Responses, Plant Physiol.2007 143:707-719.FirstPublished on December 22,2006; 10.1104/pp.106.094292); 4, the transgenic line of 19 expression Neip2 gene overexpressions.Fig. 8 A is the growing state of above-mentioned plant in solid medium; The growing state that Fig. 8 B and Fig. 8 E are respectively above-mentioned plant root and leaf when in vermiculite, growing relatively; Fig. 8 C and Fig. 8 D are respectively the statistic data of root length and plant fresh weight.As can be seen from Figure 8, overexpression Neip2 gene has promoted the growth of tobacco, and the growth of the RNAi transgene tobacco of this gene is then slow relatively, and though the difference in this growth be in medium and small seedling stage of plate or cultivation in the later stage all very obviously.Neip2 overexpression strain system and strain are that root length and the fresh weight of 16-4 is (WT) greater than the contrast strain obviously all, and the root length of the RNAi strain system of Neip2 and fresh weight all are significantly less than and contrast strain system; In vermiculite, Neip2 overexpression strain system and strain be the blade of 16-4 obviously than transgene tobacco is big with the strain system that changes the RNAi carrier over to, and it is littler than wild-type to change the blade that the strain of RNAi carrier is over to.Above result shows that the plant of Neip2 gene and NTHK1 gene overexpression has similar phenotype, explains that Neip2 is consistent with ethylene receptor NTHK1 aspect regulating plant growth, and Neip2 has regulated and control the reaction of plant to environment stress.

In order to detect the effect of Neip2 in plant reacts salt stress; The transgene tobacco of the Neip2 overexpression that wild-type tobacco, above-mentioned steps 1 obtained and the transgene tobacco seed that changes the RNAi carrier over to are planted in respectively on the MS flat board that contains different concns NaCl; In culturing room, cultivated for 4 weeks, observe the influence of condition of salt stress the tobacco germination.The result shows that under the normal cultured condition, the lotus throne of the transgene tobacco of overexpression Neip2 is a wild-type tobacco greater than contrast, and it is littler than transgene tobacco and the wild-type tobacco of overexpression Neip2 to change the lotus throne of transgene tobacco of RNAi over to.In adding the substratum of NaCl, above-mentioned difference is tending towards obviously, and is particularly more obvious when 300mMNaCl handles, but all death of all plant during 400mM NaCl.Fig. 9 A has shown contrast, Neip2 overexpression and has changed the germination of plant when normal, 0.2M, 0.3M and 0.4M NaCl handle of RNAi over to; Fig. 9 B has shown contrast, Neip2 overexpression and has changed the plant fresh weight of plant when normal, 0.2M, 0.3M and 0.4MNaCl handle of RNAi over to.The result shows; Though all plant percentage of germination under salt stress reduces; And fresh weight obviously descends; But the plant percentage of germination of overexpression Neip2 and plant significantly that decline scope obviously are lower than the plant that contrasts and change the RNAi carrier over to, and the plant percentage of germination and the plant fresh weight decline scope that wherein change the RNAi carrier over to are maximum.Therefore, the strain of Neip2 overexpression system shows the resistance to salt stress, and the strain system that changes the RNAi carrier over to then shows as the sensitivity to salt stress, shows that Neip2 has participated in the regulation and control of NTHK1 to the salt stress reaction.The transgene tobacco that changes the RNAi carrier over to shows as sensitivity to environment stress, and overexpression Neip2 has then increased the resistance of tobacco to environment stress, explains that Neip2 plays the positive regulation effect plant in the replying of high-salt stress.

Sequence table

< 110>Inst. of Genetics and Development Biology, CAS

< 120>a kind of ethylene receptor NTHK1 interact protein relevant and encoding sox and application with plant stress tolerance

<130>CGGNAZ92436

<160>2

<210>1

<211>1053

<212>DNA

< 213>tobacco (Nicotiana tabacum var.Xanthi)

<400>1

atgtctgagg gagagaaagt tttgcctact gcatcagcag atgagaagtc tggggcatct 60

gagaataaaa aatcttctga gtcttcctcc acagaagcac catcaggaga ggcgagaaca 120

acctctacgg ctgcggctgg agctgggctt caaaatccct ttgatttctc agccatgtct 180

ggactactta atgacccaag tatcaaagaa ctagcggagc agatagcgaa agatcctgca 240

tttaatcaga tggcggagca gcttcagaag acctttcaag gtgctgcagt cgaagagagc 300

gtccccaact ttgatagcca acaatactat tccacaatgc aacaggttat gcaaaatcct 360

caatttatga caatggctga gcggcttggt aatgcgttga tgcaggatcc atccatgtct 420

ggcatgcttg agagtttgtc aaaccctgct cagaaggagc aaattgaaga acgaatggca 480

cgcatcaaag aagacccatc gctgaaaccg attttggaag agatagagag cgggggacca 540

gctgcaatga tgaggtattg gaatgatcaa gaaacactga agaaaattgg tgaagcaatg 600

ggttttgctg ctgggggaga gggtgctacc tcttccgcaa tacctgggac cgatgaaacc 660

gaagaggcta atgaagatga atctgttgtt caccagtgtg ctagtgttgg tgatgcagag 720

ggcttgaagg ctgcactaac tgctggtgct gataaagacg aagaagactc agaaggaagg 780

acggcattgc attttgcttg tggatatggc gaggtgaagt gtgctcagat tcttctggaa 840

gctggggcaa aggttgatgc cttggacaag aataagaata ctgctcttca ctatgctgct 900

ggatatggta ggaaggagtg tgtcgcgctg ctgctagaga atggagccgc tgtaactctc 960

caaaacttgg atggtaagac accgatcgat gtggccaaac tcaacaacca gcaggaggtc 1020

ctgaagctgc tcgagaaaga tgtgtttctg tga 1053

<210>2

<211>350

<212>PRT

< 213>tobacco (Nicotiana tabacum var.Xanthi)

<400>2

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

1 5 10 15

Ser Gly Ala Ser Glu Asn Lys Lys Ser Ser Glu Ser Ser Ser Thr Glu

20 25 30

Ala Pro Ser Gly Glu Ala Arg Thr Thr Ser Thr Ala Ala Ala Gly Ala

35 40 45

Gly Leu Gln Asn Pro Phe Asp Phe Ser Ala Met Ser Gly Leu Leu Asn

50 55 60

Asp Pro Ser Ile Lys Glu Leu Ala Glu Gln Ile Ala Lys Asp Pro Ala

65 70 75 80

Phe Asn Gln Met Ala Glu Gln Leu Gln Lys Thr Phe Gln Gly Ala Ala

85 90 95

Val Glu Glu Ser Val Pro Asn Phe Asp Ser Gln Gln Tyr Tyr Ser Thr

100 105 110

Met Gln Gln Val Met Gln Asn Pro Gln Phe Met Thr Met Ala Glu Arg

115 120 125

Leu Gly Asn Ala Leu Met Gln Asp Pro Ser Met Ser Gly Met Leu Glu

130 135 140

Ser Leu Ser Asn Pro Ala Gln Lys Glu Gln Ile Glu Glu Arg Met Ala

145 150 155 160

Arg Ile Lys Glu Asp Pro Ser Leu Lys Pro Ile Leu Glu Glu Ile Glu

165 170 175

Ser Gly Gly Pro Ala Ala Met Met Arg Tyr Trp Asn Asp Gln Glu Thr

180 185 190

Leu Lys Lys Ile Gly Glu Ala Met Gly Phe Ala Ala Gly Gly Glu Gly

195 200 205

Ala Thr Ser Ser Ala Ile Pro Gly Thr Asp Glu Thr Glu Glu Ala Asn

210 215 220

Glu Asp Glu Ser Val Val His Gln Cys Ala Ser Val Gly Asp Ala Glu

225 230 235 240

Gly Leu Lys Ala Ala Leu Thr Ala Gly Ala Asp Lys Asp Glu Glu Asp

245 250 255

Ser Glu Gly Arg Thr Ala Leu His Phe Ala Cys Gly Tyr Gly Glu Val

260 265 270

Lys Cys Ala Gln Ile Leu Leu Glu Ala Gly Ala Lys Val Asp Ala Leu

275 280 285

Asp Lys Asn Lys Asn Thr Ala Leu His Tyr Ala Ala Gly Tyr Gly Arg

290 295 300

Lys Glu Cys Val Ala Leu Leu Leu Glu Asn Gly Ala Ala Val Thr Leu

305 310 315 320

Gln Asn Leu Asp Gly Lys Thr Pro Ile Asp Val Ala Lys Leu Asn Asn

325 330 335

Gln Gln Glu Val Leu Lys Leu Leu Glu Lys Asp Val Phe Leu

340 345 350

Claims (2)

1. a method of cultivating the transgenic plant of resistance of reverse raising is that the proteinic encoding sox of being made up of the aminoacid sequence shown in the sequence in the sequence table 2 is changed in the plant, obtains the transgenic plant that resistance of reverse improves;

Said resistance of reverse is a salt tolerant, and said plant is a tobacco.

2. method according to claim 1 is characterized in that: said encoding sox is following 1) or 2) described gene:

1) its encoding sequence is the deoxyribonucleotide from 5 ' terminal 1-1050 position of sequence 1 in the sequence table;

2) its nucleotide sequence is the sequence 1 in the sequence table.

Images