CN102373187A

CN102373187A - Plant stress tolerance-associated protein and encoding gene and application thereof

Info

Publication number: CN102373187A
Application number: CN2011100255543A
Authority: CN
Inventors: 刘公社; 苏蔓; 齐冬梅; 李晓峰; 陈双燕
Original assignee: Institute of Botany of CAS
Current assignee: Institute of Botany of CAS
Priority date: 2010-08-11
Filing date: 2011-01-24
Publication date: 2012-03-14
Anticipated expiration: 2031-01-24
Also published as: CN102373187B

Abstract

The invention discloses a plant delta1-pyrroline-5-carboxylate synthetase and an encoding gene and application thereof. The protein provided by the invention is a protein shown as (a) or (b), wherein (a) is a protein constituted by an amino acid sequence shown as a sequence 1 in a sequence table, or (b) a protein which is obtained by performing substitution and/or deletion and/or addition of one or more amino acid residues on an amino acid residue sequence shown as a sequence 1, participates in proline synthesis and is derived from the sequence 1. The protein and the encoding gene thereof provided by the invention can be used for adjusting and controlling the synthesis of plant proline, and participating in responding to the salt and drought stress of guinea grass under the inducing action of salt and drought stress. The gene provided by the invention is introduced into a plant, so that the stress tolerance of the plant can be improved. The protein and the encoding gene thereof provided by the invention have important practical values on the culturing of guinea grass with higher stress tolerance and other novel plant varieties.

Description

One kind of plant conversion-resisting resisting related protein and encoding sox and application

Technical field

The present invention relates to a kind of plant conversion-resisting resisting related protein and encoding sox and application.

Background technology

Arid and salt stress are to influence the most serious environment stress factor of plant-growth, are the primary limiting factors of China's crop yield and sown area.Proline(Pro) is the osmotic stress material that plant accumulates under environment stress, plays and regulates Premeabilisation of cells pressure, protected protein matter molecule, as the effects such as scavenging agent of active oxygen, to keep the normal growth of plant.Plant accumulates the height of proline(Pro) ability, has determined the power of stress resistance of plant.

In plant materials, synthetic ornithine/l-arginine and two approach of L-glutamic acid of passing through of proline(Pro), under environment stress, the L-glutamic acid approach is the main path of proline biosynthesis.Δ ¹-pyrroles beautiful jade-5-carboxylic acid synthetase (P5CS) is the rate-limiting enzyme in the L-glutamic acid approach; It is a bifunctional enzyme; Have Glutamate kinase and paddy ammonia enzyme-γ-semialdehyde deaminase active, the preceding 2 steps reaction of catalysis proline(Pro) synthetic, it again can be by product proline(Pro) feedback inhibition simultaneously.

Sheep's hay has another name called the alkali grass, is one of important edificato of Eurasia grassland region east meadow steppe.Sheep's hay is the herbage of a kind of high yield, good palatability, and salt tolerant alkali, but intolerant to waterlogging or severe drought are cultivated the high resistance to cold and diseases sheep's hay, can bring huge economic benefit and ecological benefits undoubtedly.The drought resisting of plant, salt tolerant alkalescence shape are the quantitative characters of controlled by multiple genes, give through the resistance of cross-breeding raising crop and bring very big difficulty, therefore can improve the resistance of crop through genetically engineered.Increasing plant P5CS expression of gene amount through transgenic, thereby improve the ability of plant accumulation proline(Pro), is an effective way of cultivating drought resisting, saline alkali tolerant plant.

Summary of the invention

The purpose of this invention is to provide anti-contrary (drought resisting, salt tolerant) the GAP-associated protein GAP Δ of a kind of plant ¹-pyrroles beautiful jade-5-carboxylic acid synthetase and encoding sox and application.

Plant conversion-resisting resisting related protein provided by the invention (LcP5CS1) is a kind of Δ ¹-pyrroles beautiful jade-5-carboxylic acid synthetase derives from sheep's hay (Leymus chinensis (Trin.) Tzvel.), is (a) or protein (b) as follows:

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

(b) with the aminoacid sequence of sequence 1 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and participate in the proline(Pro) synthetic by sequence 1 deutero-protein.

Sequence 1 is made up of 730 amino-acid residues in the sequence table; From the 21st the-the 295th amino acids residue of N-terminal is the conserved domain of AAK_super family; From 310-710 of N-terminals is the conserved domain of ALDH-SF super family, is P5CS Multi-domain sequence from the 22nd the-the 730th amino acids residue of N-terminal.

For the ease of the purifying of LcP5CS1, can connect label as shown in table 1 at proteinic aminoterminal of forming by the amino acid residue sequence of sequence 1 or carboxyl terminal.

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 (b) but in the LcP5CS1 synthetic, also can synthesize its encoding sox earlier, carry out biology again and express and to obtain.The encoding sox of LcP5CS1 in above-mentioned (b) can be through the codon with one or several amino-acid residue of disappearance in the dna sequence dna shown in the sequence in the sequence table 2; 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 gene of plant conversion-resisting resisting related protein (LcP5CS1) also belongs to protection scope of the present invention on the coding.

Said gene can be following 1) or 2) or 3) or 4) dna molecular:

1) in the sequence table sequence 2 from the dna molecular shown in the Nucleotide of 5 ' terminal 3-2195 position;

2) dna molecular shown in the sequence in the sequence table 2;

3) can be with 1 under stringent condition) or 2) gene recombination that limits and the gene of encoding said proteins;

4) with 1) or 2) gene that limits has homology and the gene of encoding said proteins more than 90%.

Sequence 2 in the sequence table is by 2197 based compositions, and its open reading frame (ORF) is Nucleotide from 5 ' terminal 3-2195 position.

Above-mentioned stringent condition can be 0.1 * SSPE (or 0.1 * SSC), in the solution of 0.1%SDS, hybridization and wash film under 65 ℃ of conditions.

The recombinant vectors that contains above arbitrary said gene also belongs to protection scope of the present invention, like recombinant expression vector.

Available existing plant expression vector construction contains the recombinant expression vector of said gene.

Said plant expression vector comprises double base agrobacterium vector (like pBI121, pBin19, pCAMBIA2301, pCAMBIA3301, pCAMBIA1301-UbiN, pCAMBIA1300 etc.) and can be used for carrier of plant micropellet bombardment etc.Said plant expression vector also can comprise 3 ' end untranslated zone of foreign gene, promptly comprises the dna fragmentation of polyadenylic acid signal and any other participation mRNA processing or genetic expression.Said polyadenylic acid signal can guide polyadenylic acid to join 3 ' end of mRNA precursor, and the non-translational region of inducing (Ti) plasmid gene (like kermes synthetic enzyme Nos gene), plant gene (like soybean storage protein gene) 3 ' end to transcribe like the Agrobacterium crown-gall nodule all has similar functions.

When using said gene constructed recombinant plant expression vector; Before its transcription initiation Nucleotide, can add any enhancement type promotor, constitutive promoter or inducible promoter; Like the ubiquitin promoter (Ubiquitin) of cauliflower mosaic virus (CAMV) 35S promoter, corn, stress induced promoter Rd29A 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 produce the enzyme of colour-change or the gene of luminophor (gus gene, luciferase genes etc.) as adding the coding that in plant, to express, have antibiotic marker thing (qingfengmeisu qiong affinity tag, kantlex affinity tag etc.) or the anti-chemical reagent marker gene (like anti-weedkiller gene) of resistance etc.From the security consideration of transgenic plant, can not add any selected marker, directly with adverse circumstance screening transformed plant.

The expression cassette, transgenic cell line and the reorganization bacterium that contain above arbitrary said gene (LcP5CS1) all belong to protection scope of the present invention.

The total length of amplification said gene or arbitrary segmental primer are to also belonging within protection scope of the present invention.

In said albumen, said gene, said recombinant expression vector, expression cassette, transgenic cell line or the reorganization bacterium any one all can be applicable to cultivate plant with adverse resistance.

Another object of the present invention provides a kind of method of cultivating anti-salt, drought-enduring transgenic plant.

The method of the anti-salt of cultivation provided by the present invention, drought-enduring transgenic plant can be with the above-mentioned Δ of coding ¹The gene (LcP5CS1) of-pyrroles beautiful jade-5-carboxylic acid synthetase imports in the purpose plant (like vegetable cell or tissue), obtains the transgenic plant that resistance of reverse is higher than said purpose plant.

Utilize any carrier that can guide foreign gene in plant, to express,, can obtain anti-salt, drought tolerance enhanced transgenic cell line and transfer-gen plant the gene transfered plant cell of encoding said proteins.Carry that said expression carrier 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 the plant transformed tissue cultivating is become plant.By the plant transformed host both can be monocotyledons, also can be dicotyledons, as: tobacco, lucerne place, Arabidopis thaliana, paddy rice, wheat, corn, cucumber, tomato, willow, turfgrass etc.

Albumen provided by the invention and encoding sox thereof are the rate-limiting enzymes of plant proline biosynthesis under environment stress.With gene transfered plant of the present invention, thereby the accumulation volume that improves the proline(Pro) of plant improves the resistance of plant, but particularly arid and this expression of gene of high-salt stress induced strong improve the crop Δ through the transgenic means ¹-pyrroles beautiful jade-5-carboxylic acid synthetase expression amount can improve the resistance of crop and grass type, helps to improve crop yield, expand sown area and realize the healthy and sustainable development of grassland ecosystem.Albumen provided by the invention and encoding sox thereof have important practical value for the sheep's hay of cultivating the resistance raising and other neies variety of plant.The present invention helps to understand plant accumulates proline(Pro) under osmotic stress the mechanism of action, plant is kept normal growth grows with the regulatory function of resistance, with the genetically engineered research that improves quality the genetics foundation is provided for improving crop yield for disclosing the proline(Pro) Accumulation Mechanism.

Description of drawings

Fig. 1 is the agarose gel electrophoresis detected result of the total RNA of chinense seedlings;

Fig. 2 is the agarose gel electrophoresis detected result of pcr amplification LcP5CS1 full-length cDNA;

Fig. 3 is the systematic evolution tree of LcP5CS1;

Fig. 4 is the response modes (sxemiquantitative RT-PCR) of LcP5CS1 gene under the salt stress treatment condition;

Fig. 5 is the response modes (sxemiquantitative RT-PCR) of LcP5CS1 gene under arid treatment condition;

Fig. 6 is the accumulation volume of sheep's hay proline(Pro) under salt stress;

Fig. 7 is the accumulation volume of the proline(Pro) of sheep's hay under drought stress;

Fig. 8 is that the PCR of recombinant vectors pSN1301-LcP5CS1 identifies;

Fig. 9 is that the double digestion of recombinant vectors pSN1301-LcP5CS1 is identified;

Figure 10 is an Agrobacterium positive transformant PCR qualification result behind the conversion Agrobacterium;

Figure 11 is a transgenic arabidopsis PCR qualification result;

Figure 12 is that high salt (250mM NaCl) is handled the Arabidopis thaliana of conversion LcP5CS1 after 3 days and the phenotype of wild-type Arabidopis thaliana;

Embodiment

Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique among the following embodiment like no specified otherwise, is ordinary method.Used test materials among the following embodiment like no specified otherwise, is to buy from routine biochemistry reagent shop and obtains.

Sheep's hay (lucky giving birth to No. one): in September, 2005 is available from Jisheng Improved Variety Station of Aneurolepidium Chinense, Jilin Province

Embodiment 1, sheep's hay plant conversion-resisting resisting related protein (Δ ¹-pyrroles beautiful jade-5-carboxylic acid synthetase) and the acquisition of encoding sox

One, the clone of the sheep's hay plant gene of anti-retrocorrelation LcP5CS1

1, the extraction of vegetable material processing and total RNA

Handle 12h sheep's hay (lucky giving birth to No. one) seedling with 250mM NaCl, concrete grammar is:

The normal growth sheep's hay in 8 weeks (lucky giving birth to No. one) seedling is carried out salt stress and handles.

Soak root with the 250mM NaCl aqueous solution, incubated at room temperature, getting and handling 12h sheep's hay (lucky giving birth to No. one) seedling is material, extracts total RNA, carries out 1.2% agarose gel electrophoresis and detects, the result is as shown in Figure 1.The RNA that is extracted has two tangible electrophoretic bands, is followed successively by 28S RNA and 18S RNA from top to bottom, shows to have obtained higher, the more complete total RNA of purity.

2, the clone of the plant gene of anti-the retrocorrelation

According to this laboratory high-flux sequence result, in conjunction with disclosed plant Δ ¹The amino acid residue sequence of-pyrroles beautiful jade-5-carboxylic acid synthetase is sought conservative region, and according to conservative region sequences Design primer, concrete primer sequence is following:

S1?5′-GAATGGGCAGGGGTGGCAT-3′；

S2?5′-CCTCATTGCAAAGGAAGATCC-3′。

The total RNA that extracts chinense seedlings with step 1 is a template, uses PrimeScript ^TM1st Strand cDNASynthesized Kit test kit (Takara company) and reference reagent box specification sheets, the synthetic first chain cDNA of counter-rotating.Reaction system and reaction conditions are following: Oligo-dT (10pmol/ μ l) 1.0 μ l, dNTP Mixture (10mmol/leach) 1.0 μ l, Total RNA (≤1 μ g) 1.0 μ l, RNase-free water7.0 μ l, 65 ℃ of 5min; Add 5 * Buffer, 4.0 μ l, RNase Inhibitor (40U/ μ l) 0.5 μ l, PrimeScript RTase (200U/ μ l) 0.5 μ l then, 42 ℃ of 45min, 70 ℃ of 15min, with the synthetic first chain cDNA be stored in-20 ℃ subsequent use.

The first chain cDNA to obtain is a template, with the primer of S1 and S2 composition to carrying out pcr amplification; The PCR reaction system is: each 1 μ l (10umol/l) of cDNA template, S1 and S2,10 * Buffer, 2.5 μ l, dNTP Mixture (10mmol/leach) 2 μ l, Taq enzyme 0.25 μ l, ddH ₂O 12.25 μ l; Reaction conditions is: 94 ℃ of preparatory sex change 5min of elder generation; 94 ℃ of 30s then, 55 ℃ of 30s, 72 ℃ of 2min 30s, totally 35 circulations; Last 72 ℃ are extended 10min.

After reaction finishes, pcr amplification product is carried out 1.2% agarose gel electrophoresis detect, the result is as shown in Figure 2.Among Fig. 2, swimming lane M is a DL2000Plus dna molecular amount standard (Beijing Quanshijin Biotechnology Co., Ltd), and swimming lane 1 is a pcr amplification product.The result shows, obtained the purpose fragment that length is about 2200bp through pcr amplification.Reclaim and purified pcr product; Be connected on the PMD-18T carrier (Takara company); Connect product transformed into escherichia coli DH5 α competent cell (Beijing Quanshijin Biotechnology Co., Ltd); Screening positive clone carries out bacterium liquid PCR identifies that the plasmid that extracts positive colony checks order, and sequencing result is carried out BLAST analyze.

Sequencing result shown in sequence 2, through analysis revealed, this sheet segment length 2197bp, the protein shown in the sequence 1 of code sequence tabulation.With the protein called after LcP5CS1 shown in the sequence 1 (being made up of 730 amino-acid residues), with the encoding sox called after LcP5CS1 of LcP5CS1, its full-length cDNA is shown in the sequence 2 of sequence table.

The bioinformatic analysis of embodiment 2, LcP5CS1 and LcP5CS1

One, the structure function of LcP5CS1 Gene Sequence Analysis and proteins encoded thereof prediction

Utilize DNAMAN and OMIGA software that the full length cDNA sequence of LcP5CS1 is carried out bioinformatic analysis, this sequence total length 2197bp is ORF from the 3rd the-the 2195th at 5 ' end.Infer that its molecular weight is 78.887kDa, iso-electric point pI value 6.69.With the structural domain of online B1AST instrument (http://blast.ncbi.nlm.nih.gov/Blast.cgi) analysis LcP5CS1, the result shows that this albumen belongs to P5CS Multi-domain, shows that this albumen is a member in the P5CS family.

Two, the homology and the systematic evolution tree analysis of LcP5CS1 and other P5CS proteinoid sequence

(the GeneBank number of landing is: TaP5CS is respectively AAX35536, SbP5CS1ACU65226, SbP5CS2ACU65227 to other P5CS aminoacid sequence of having cloned in LcP5CS1 and the plant to utilize online Blast instrument; 0sP5CS1 BAA19916, ZmP5CS ABI21839, AtP5CS1NP181510; AtP5CS2NP191120, BnP5CSAAAK01360, BnP5CSB AAK01361; TomPR01 AAB67877; TomPR02 Q96480, EcGPK YP539318 carry out the homology analysis comparison, see Fig. 3.

Result such as Fig. 3 show that the homology of LcP5CS1 and monocotyledons Chinese sorghum SbP5CS1 is respectively 87%; And it is lower with the dicots P5CS homology of other unifacial leaf P5CS.The evolutionary tree analysis revealed; Bigger difference has appearred in P5CS proteinoid during evolution; It is one type that monocotyledonous P5CS gathers, and except that the TomPR01 of tomato and the colibacillary EcGPK homology of prokaryotic organism were nearer, it was one type that other dicotyledonous P5CS also gathers in the dicotyledons.

Embodiment 3, the LcP5CS1 expression pattern analysis under arid and condition of salt stress

The normal growth sheep's hay in 8 weeks (lucky giving birth to No. one) seedling is carried out different treatment.

First group (salt stress processing): soak root, incubated at room temperature with the 250mM NaCl aqueous solution;

Get respectively and handle 0,1,2,4,8,12,24, the processing material blade of 48h is stored in the liquid nitrogen rapidly.

Second group (drought stress processing): stop to supply water incubated at room temperature;

Getting respectively cuts off the water supply handles 0,1,2,3,4,5, and the chinense seedlings blade of 6d freezes in liquid nitrogen rapidly.

Extract total RNA of above-mentioned two groups of chinense seedlings of handling, carry out RT-PCR.

LcP5CS1 primer: 1:5 '-GTCGGCACCGCTATTGTC-3 ';

2：5′-GCCAAACTGTCGTTATCCC-3′；

Confidential reference items Actin primer: 1:5 '-TGGACTCTGGTGATGGTGTGAG-3 ';

2：5′-GTGCTAAGGGAGGCAAGGATG-3′。

Confidential reference items reaction conditions: 94 ℃ of 5min; 94 ℃ of 30s, 55 ℃ of 30s, 72 ℃ of 30s, 26 circulations.

LcP5CS1 reaction conditions: 94 ℃ of 5min; 94 ℃ of 30s, 55 ℃ of 30s, 72 ℃ of 40s, 30 circulations.

The result sees Fig. 4, Fig. 5.The result shows that the LcP5CS1 transcriptional level obviously receives arid and Salt Stress-induced, and under inductive condition, the relative expression quantity of LcP5CS1 gene increases sharply.

The functional verification of embodiment 4, LcP5CS1 (measuring the content of its product proline(Pro))

Arid, proline content is measured under the salt stress, and confirming the relation with proline content that increases of under arid and salt stress LcP5CS1 expression amount, thereby whether the content of verifying proline(Pro) is relevant with the expression amount of LcP5CS1.The concrete grammar of arid, salt stress is with embodiment 3.

1. proline(Pro) extracts: get 0.05-0.5g and coerce processing back different time points sheep's hay blade, with 3% sulphosalicylic acid solution milling and extracting, the final volume of sulphosalicylic acid is 5ml.Homogenate changes in the glass centrifuge tube, in boiling water bath, extracts 10min.After the cooling, with the centrifugal 10min of 3000r/min, it is to be measured to get supernatant.

2. assay

1) standard curve making: at 1-10ug/ml concentration of proline scope production standard curve. get each 2ml of standardized solution; Add 2ml 3% sulphosalicylic acid; 2ml glacial acetic acid and 4ml 2.5% ninhydrin solution, put in the boiling water bath colour developing 60min. cooling after, add 4ml extracted in toluene red material. after leaving standstill; Get toluene and measure the absorption value of 520nm wavelength mutually, according to proline(Pro) amount and respective absorption value drawing standard curve.

2) sample determination: get the 2ml supernatant, add 2ml water, add colouring reagentss such as glacial acetic acid again, develop the color, extraction and colorimetric with the typical curve program.

Result such as Fig. 6,7 show; After coercing processing; Proline content raises in the plant materials; And increasing after the P5CS expression amount increases of proline content proves that improving the LcP5CS1 expression amount can increase proline content in the plant materials, explains that LcP5CS1 is a proline(Pro) synthetic key enzyme under the environment stress.

The acquisition of embodiment 5, LcP5CS1 transgenic arabidopsis

The LcP5CS1 gene that the foregoing description 1 amplification is obtained is gone into carrier PMD18-T (TaKaRa company) with the primer LcP5CSSN1301-s that contains KpnI and BamHI (5 '-CGGGATCCATGGGCAGGGGTGGCAT-3 ') and LcP5CSSN1301-r (5 '-GGGGTACCTCATTGCAAAGGAAGATCCT-3 ') rear clone that increases, and the pcr amplification condition is: 94 ℃ of preparatory sex change 5min; 94 ℃ of 30s then, 58 ℃ of 30s, 72 ℃ of 2min 30s, totally 35 circulations; Last 72 ℃ are extended 10min.Identify back positive bacteria liquid upgrading grain,, be inserted into through between the KpnI and BamHI restriction enzyme site of the plant expression vector pSN1301 (available from CAMBIA company) that same enzyme is cut, the recombinant vectors called after pSN1301-LcP5CS1 that obtains with KpnI and BamHI double digestion.With primer LcP5CSSN1301-s and LcP5CSSN1301-r pSN1301-LcP5CS1 being carried out PCR identifies; Reaction conditions is the same; The result is as shown in Figure 8; Swimming lane M is for being DL2000Plus dna molecular amount standard (Beijing Quanshijin Biotechnology Co., Ltd), and swimming lane 1,3,4,5,7,8,9,10 is for identifying correct recombinant plasmid.With KpnI and BamHI pSN1301-LcP5CS1 being carried out double digestion identifies; The result is as shown in Figure 9; Wherein, Swimming lane M is a DL2000Plus dna molecular amount standard (Beijing Quanshijin Biotechnology Co., Ltd), cuts the band that obtains about a 2000bp through enzyme, proves that the purpose fragment correctly connects in the plant expression vector.

Recombinant vectors pSN1301-LcP5CS1 imports to Agrobacterium EHA105 strain system (available from U.S. Clontech company; Cat. no K1612-1) in, PCR screens positive recombinant, and primer and reaction conditions are the same; The result is shown in figure 10; Among Figure 10, swimming lane M is a DL2000Plus dna molecular amount standard (Beijing Quanshijin Biotechnology Co., Ltd), swimming lane 1,2,3,5 positive recon PCR results.Arabidopis thaliana seed (Columbia is environmental, derives from the world, Fan Ersai research centre, French academy of agricultural sciences Arabidopis thaliana seed bank) is with 75% ethanol disinfection 2min, behind the 2%NaClO sterilization 15min; Be seeded in the MS substratum, illumination cultivation moved to nutrition soil with seedling after 3 days: in the soil of vermiculite=1: 1; Treat that most of plant is all after the bolting; Cut whole main tongue at the inflorescence base portion, remove its apical dominance, about 1 all backs grow 4-6 newborn side tongue at the axillalry bud position; Treat that side tongue inflorescence forms bud and part and blooms or forms 1-2 angle really the time, just can be used for conversion.The 10mL pipe adds YEB liquid nutrient medium 2mL, and adds each 50mg/L of the gentle kantlex of screening rifamycin antibiotic.The single colony inoculation of picking Agrobacterium is in pipe, and 28 ℃ of 200rpm shake bacterium.Transferred night on the same day in the triangular flask that contains the antibiotic YEB liquid nutrient medium of same concentration 50ml, 28 ℃ are shaken bacterium and spend the night.Changed in second day 250ml contain shake in the antibiotic YEB liquid nutrient medium bacterium to 0D 600 about 1.2, change in the centrifuge tube the centrifugal 5min of room temperature 5000rmp over to.Abandon supernatant, deposition is changed in the zero(ppm) water that contains 3% sucrose and 0.1%Selwert (Sigma company), make its 0D600 about 0.8.Wild-type plant inflorescence was soaked in the transformed bacteria liquid 20 seconds, and the preservative film parcel keeps in Dark Place and takes off film, normal maintenance after spending the night.Obtain sterilizing behind the seed, be seeded on the MS screening culture medium of the Totomycin that contains 20mg/L, screen positive transgenic seedling; These seedlings are moved in the soil; Grow and after 10 days transfer-gen plant is carried out the PCR detection, reaction conditions and primer are the same, and the result is shown in figure 11; Swimming lane M is a DL2000Plus dna molecular amount standard (Beijing Quanshijin Biotechnology Co., Ltd), and swimming lane 2,5,6,7,8,9,10,12 is the PCR qualification result for 8 positive transgenic plant strains that obtain.The positive transgenic plant selfing that obtains obtains T2 for seed.

The resistance experiment of embodiment 6, LcP5CS transgenic arabidopsis

((Columbia is environmental in contrast with wild-type Arabidopis thaliana seed; Derive from the world, Fan Ersai research centre, French academy of agricultural sciences Arabidopis thaliana seed bank)) and the LcP5CS1 transgenic arabidopsis T2 that obtains of embodiment 5 be seeded in respectively in the MS substratum for seed; After three days seedling is moved in the soil; After one week of hot-house culture, carry out salt stress and handle (250mM NaCl), observe phenotype after three days.

Transgenic arabidopsis does not have death, the left figure of result such as Figure 12 after salt is handled three days as a result; And wild-type wilting death fully, the right figure of result such as Figure 12.Above-mentioned experimental result shows the anti-adversity ability that LcP5CS1 of the present invention can enhancement of plant, thereby the candidate gene that can be used as plant genetic engineering is applied, and is used for improveing the degeneration-resistant proterties of plant.

Claims

1. protein is (a) or protein (b) as follows:

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

2. coding claim 1 said proteic gene.

3. gene according to claim 2 is characterized in that: said gene is following 1) or 2) or 3) or 4) dna molecular:

1) dna molecular shown in the sequence 2 in the sequence table;

2) under stringent condition with 1) the dna sequence dna hybridization and the coded delta that limit ¹The dna molecular of-pyrroles beautiful jade-5-carboxylic acid synthetase;

3) with 1) or 2) dna sequence dna that limits has 90% above homology, and coded delta ¹The dna molecular of-pyrroles beautiful jade-5-carboxylic acid synthetase.

4. the recombinant expression vector, expression cassette, transgenic cell line or the reorganization bacterium that contain claim 2 or 3 said genes.

5. total length or its any segmental primer of amplification claim 2 or 3 said genes are right.

6. any one application in cultivating plant with adverse resistance in the said albumen of claim 1, claim 2 or 3 said genes, the said recombinant expression vector of claim 4, expression cassette, transgenic cell line or the reorganization bacterium.

7. application according to claim 6 is characterized in that: the said anti-contrary drought-enduring and/or salt tolerant that is.

8. a method of cultivating transgenic plant is that claim 2 or 3 said genes are imported in the purpose plant, obtains the transgenic plant that resistance of reverse is higher than said purpose plant; Said purpose plant is Arabidopis thaliana, tobacco, Root or stem of Littleleaf Indianmulberry, paddy rice, wheat, corn, cucumber, tomato, willow, turfgrass, lucerne place, soybean, cotton or sheep's hay.

9. method according to claim 6 is characterized in that: said resistance of reverse is drought tolerance and/or salt tolerance.