CN1821405A

CN1821405A - Xinjiang Saussurea involucrata cold regulation protein and its code gene and use

Info

Publication number: CN1821405A
Application number: CN 200510053240
Authority: CN
Inventors: 王志方; 王博; 艾秀莲
Original assignee: INSTITUTE OF MICROBIOLOGY XINJIANG ACADEMY OF AGRICULTURAL SCIENCES
Current assignee: INSTITUTE OF MICROBIOLOGY XINJIANG ACADEMY OF AGRICULTURAL SCIENCES
Priority date: 2005-02-17
Filing date: 2005-02-17
Publication date: 2006-08-23

Abstract

The present invention discloses cold regulating protein originated from Xinjiang Saussurea involucrata and its coding gene and application, and aims at provides cold regulating protein with high cold resisting performance and its coding gene. The cold regulating protein, named as XLCOR, is protein with the amino acid residue sequence of SEQ ID No. 2 in the sequence list, or derivative protein with the amino acid residue sequence of SEQ ID No. 2 in the sequence list through substitution, deletion or addition of one or several amino acid residues and possessing the same activity. The XLCOR coding gene is nucleotide with one of the following DNA sequences: 1) SEQ ID No. 1 in the sequence list; and 2. the sequence with over 90% homology to SEQ ID No. 1, and coding the protein of the same functions. The gene of the present invention is significant in breeding cold tolerant plant variety and raising the yield of crops.

Description

Xinjiang Saussurea involucrata cold regulation protein and encoding gene thereof and application

Technical field

The present invention relates to plant cold-regulated proteins and encoding gene thereof and application, particularly come from cold-regulated proteins encoding gene, cold-regulated proteins and application thereof in the Herba Saussureae Involueratae.

Background technology

Low temperature is a kind of main environmental restraint factor in the agriculture production, the low temperature disaster cause farm crop significantly the underproduction be ubiquitous problem in the world wide, the annual whole world only is used to alleviate the harm of frost in requisition for more than 100,000,000 dollar (Pearce, 1999) of cost.Yet some plant can withstand long term exposure live subfreezing temperature.At Weiser in 1970 imagination that cold acclimation may cause that genetic expression changes is proposed.Find that subsequently most of plants all have the existence of cool tone joint material in the cold acclimation process.Guy etc. discover that cold instructionization can induce and increase some expression of gene, several genes is expressed changed.Mohapatra etc., Houde etc. have been separated to the special cDNA of multiple cold domestication in clover, cf to dormin induce, dehydration or heat-shocked handle inductive and transcribe accumulation and the obvious positive correlation of freezing tolerance.Artus N.N. etc. change in the Arabidopis thaliana after the great expression the freeze proof genes involved cor15am of Arabidopis thaliana over to, compare the winter hardiness that transgenic plant are improved chloroplast(id) and protoplastis respectively with wild-type, and strengthen plasmalemma stability.

Herba Saussureae Involueratae (Sasussured involucrata Kar.et Kir.) belongs to the composite family hieracioides and belongs to, exist only in Chinese Xinjiang, be the perennial disposable high mountain herbaceous plant that blossoms and bears fruit, generally be grown on the high mountain of height above sea level 2400-4100 rice, the cold performance that studies show that saussurea involucrata is very outstanding, winter-30 ℃ even lower severe cold can be survived the winter smoothly, exist in its body in abundant cold-resistant resource.By separate and clone's Herba Saussureae Involueratae in freeze proof relevant gene, utilize genetic engineering technique can be transformed into farm crop the farm crop of anti-frost, and prolong the vegetative period of crop frost-rib, can bring huge income to agriculture production.

Summary of the invention

The cold-regulated proteins XLCOR and the encoding gene thereof that the purpose of this invention is to provide Herba Saussureae Involueratae, this albumen has regulating effect to saussurea involucrata cold-resistant.

A kind of Xinjiang Saussurea involucrata cold regulation protein XLCOR encoding gene provided by the invention is one of following nucleotide sequences:

1), the dna sequence dna of SEQ ID NO:1; Or with

2), the dna sequence dna that limits of SEQ ID NO:1 has 90% above homology, and the identical function protein DNA sequence of encoding.

The cold-regulated proteins XLCOR of Herba Saussureae Involueratae provided by the present invention has the gene of SEQ ID NO:1 coding Xinjiang Saussurea involucrata cold regulation protein by 650 based compositions, and the reading frame of this gene is from 5 ' end the 65th to the 571st bit base.

The cold-regulated proteins XLCOR that the invention provides Herba Saussureae Involueratae has the amino acid residue sequence of SEQ ID NO:2.

Xinjiang Saussurea involucrata cold regulation protein XLCOR sequence SEQ ID NO:2 provided by the present invention is made up of 170 amino-acid residues.

The present invention provides the cold-regulated proteins XLCOR of Herba Saussureae Involueratae by SEQ ID NO:2 deutero-protein simultaneously, it is characterized in that with the amino acid residue sequence among the SEQ ID NO:2 through the replacement of one or several amino-acid residue have with SEQ ID NO:2 in the identical activity of aminoacid sequence.

The present invention provides the cold-regulated proteins XLCOR of Herba Saussureae Involueratae by SEQ ID NO:2 deutero-protein simultaneously, it is characterized in that with the amino acid residue sequence among the SEQ ID NO:2 through the disappearance of one or several amino-acid residue have with SEQ ID NO:2 in the identical activity of aminoacid sequence.

The present invention provides the cold-regulated proteins XLCOR of Herba Saussureae Involueratae by SEQ ID NO:2 deutero-protein simultaneously, it is characterized in that with the amino acid residue sequence among the SEQ ID NO:2 through the interpolation of one or several amino-acid residue have with SEQ ID NO:2 in the identical activity of aminoacid sequence.

The present invention also provides Xinjiang Saussurea involucrata cold regulation protein XLCOR the expression vector of encoding gene, it is characterized in that the expression vector transformed plant that can use any foreign gene of can transduceing in plant, to express all can obtaining the transfer-gen plant that the low temperature stress tolerance is enhanced.

The present invention also provides the expression vector of Xinjiang Saussurea involucrata cold regulation protein XLCOR encoding gene can comprise double base agrobacterium vector and the carrier that is used for the monocotyledons micropellet bombardment.

The present invention also provides the expression vector of Xinjiang Saussurea involucrata cold regulation protein XLCOR encoding gene also can comprise 3 ' untranslated zone, but promptly comprise the polyadenylic acid signal and any other effect mRNA processing or the dna segment of genetic expression.

Xinjiang Saussurea involucrata cold regulation protein XLCOR encoding gene provided by the invention is in being building up to plant expression vector, before its transcription initiation Nucleotide, can add any strong promoter or inducible promoter, it is characterized in that to use separately or being used in combination with other plant promoter.

Xinjiang Saussurea involucrata cold regulation protein XLCOR encoding gene provided by the invention is in being building up to plant expression vector, before its transcription initiation Nucleotide, also can use enhanser, comprise translational enhancer or transcriptional enhancer, it is characterized in that the enhanser zone can be ATG initiator codon and neighboring region initiator codon, but must be identical with the reading frame of encoding sequence, to guarantee the translation of sequence.

Xinjiang Saussurea involucrata cold regulation protein XLCOR encoding gene provided by the present invention is in being applied in transgenic plant, transgenic plant or plant are identified and when screening as needs, can process employed carrier, comprise adding the alternative mark of plant, also can any selection markers.

Xinjiang Saussurea involucrata cold regulation protein XLCOR encoding gene provided by the present invention has application widely in the cold resistant plant kind, it is characterized in that comprising that the plant expression vector conversion plant host of cold-regulated proteins XLCOR encoding gene of the present invention both can be a monocotyledons, also can be dicotyledons, can cultivate cold-resistant plant variety.

The gene of coding cold-regulated proteins XLCOR provided by the present invention, expression vector (these plant expression vectors comprise double base agrobacterium vector and the carrier that the is used for the monocotyledons micropellet bombardment) transformed plant that can use any foreign gene of can transduceing to express in plant all can obtain the transfer-gen plant that low temperature side of body tolerance is enhanced.Carrier also can comprise 3 ' untranslated zone, but comprise the polyadenylic acid signal and any other effect mRNA processing or the dna segment of genetic expression.Polyadenylic acid signal guidance polyadenylic acid joins 3 ' end of mRNA precursor.The example in 3 ' zone has the Agrobacterium crown-gall nodule to induce (Ti) plasmid gene (as kermes synthetic enzyme Nos gene), plant gene (as the major issue storage protein gene) 3 ' non-translational region of transcribing etc.3 ' untranslated zone of XLCOR gene of the present invention can be used to express in plant.Gene of the present invention can add any strong promoter or inducible promoter in being building up to plant expression vector the time before its transcription initiation Nucleotide.These promotors are a lot, and as cauliflower mosaic virus (CAMV 35S) and Ubiqutin promotor etc., they can use separately or be used in combination with other plant promoter.1 gene of the present invention also can use enhanser in being building up to plant expression vector the time, comprises translational enhancer or transcriptional enhancer.These enhanser zones can be ATG initiator codon and neighboring region initiator codon etc., but must be identical with the reading frame of encoding sequence, to guarantee the translation of trimming sequence.Translation control signal and initiator codon can be multiple different sourcess such as natural or synthetic.Translation initiation region can be from initiation region or structure gene.

For the ease of transgenic plant or plant being identified and screening, can process employed carrier, comprise adding the alternative mark of plant.The selected marker of using can be the gene of coding to the antibiotics resistance enzyme, and microbiotic comprises gentamicin, Totomycin, kantlex etc.Also can be to produce the enzyme of colour-change or the mattress of luminophor, as GUS, luciferase, can also be the gene of anti-chemical reagent (for example weedkiller).Certainly, also can any selection markers.

Carry XLCOR expression carrier of the present invention and can import plant (Weissbach by using conventional biotechnological meanss such as Ti-plasmids, Ri plasmid, plant viral vector, directly DNA conversion, microinjection, electroporation, 1998, method for Plant Molecular Biology VIII, Academy Press, Now York, pp 421-463; Geiserson and Corey, 1998, plant MolecularBiolory (2nd Editon).

Can use the plant expression vector that comprises cold-regulated proteins gene of the present invention to transform plant host, it both can be monocotyledons, it also can be dicotyledons, as: paddy rice, wheat, cotton, corn, cucumber, tomato, willow, turfgrass, clover etc., can cultivate cold-resistant plant variety.

Description of drawings

Fig. 1: gene library results of hybridization.

Previous figure is a forward subtracted library results of hybridization among the figure; The figure in back is reverse subtracted library results of hybridization.

Fig. 2: gene library results of hybridization.

The A point is the results of hybridization of total RNA of the Herba Saussureae Involueratae tissue culture seedling of probe and 20 ℃ of cultivations among the figure, and the B point is positive contrast, and the C point is the results of hybridization of total RNA of the Herba Saussureae Involueratae tissue culture seedling of probe and 4 ℃ of cultivations.

Specific embodiment

The structure in embodiment 1, Herba Saussureae Involueratae cDNA library

Collection grows in the Herba Saussureae Involueratae blade of 4000 meters of Tianshan Mountains height above sea level, take by weighing 0.1g and in liquid nitrogen, grind, add 1mlTRIZOL reagent, fully mixing, add the 200ul chloroform, mixing, the centrifuging and taking supernatant adds the 250ul Virahol, the centrifugal supernatant that goes, after 75% washing with alcohol, in drying at room temperature, precipitation 15ulDEPC water dissolution.In strict accordance with BD Biosciences company " SMART cDNA Library Contruction Kit " service manual, get the total RNA of 1ug, synthetic double chain cDNA under the effect of PowerScript ThermoScript II, after Proteinase K and restriction enzyme Sfi I processing, carry out fractional separation with CHROMA SPIN-400 pillar, be connected, connect product and pack with the lambda particles phage packaging protein with carrier λ Trip1EX2, the library that obtains behind titer determination, be stored in-70 ℃.

The structure of embodiment 2, Herba Saussureae Involueratae cDNA subtracted library

Herba Saussureae Involueratae tissue culturing seedling is handled fortnight at 0 ℃-2 ℃, use in contrast without the Herba Saussureae Involueratae tissue culturing seedling of subzero treatment, " Clontech PCR-Select cDNASubtraction Kit " according to BDBioxciences company uses service manual, extract two kinds of total RNA of tissue culturing seedling respectively, separate its PolyA+RNA, synthetic double chain cDNA.Tissue culturing seedling to subzero treatment adds different joints respectively, as " test cDNA " (Tester cDNA), tissue culturing seedling's conduct without subzero treatment " drives cDNA " (DrivercDNA), after twice hybridization, the hybridization product is carried out the PCR reaction, obtain the Herba Saussureae Involueratae cDNA of specifically expressing at low temperatures, these cDNA are connected with the T-carrier, make up and obtain Herba Saussureae Involueratae cDNA low temperature forward subtracted library; The cDNA of tissue culturing seedling to normal temperature growth adds different joints respectively simultaneously, as " test cDNA " (Tester cDNA), the tissue culturing seedling of low temperature induction as " driving cDNA " (DrivercDNA), after twice hybridization, the hybridization product is carried out the PCR reaction,, these cDNA are connected with the T-carrier, make up and obtain the reverse subtracted library of Herba Saussureae Involueratae cDNA low temperature.

The clone of embodiment 3, Herba Saussureae Involueratae cool tone joint gene XLCOR

From Herba Saussureae Involueratae cDNA low temperature forward subtracted library and reverse 300 clones of each picking the subtracted library, extract plasmid after, carry out the PCR reaction, obtain full-length cDNA, with its point on nylon membrane; The low temperature forward difference of preparation is subtracted cDNA carries out behind the mark hybridizing with the cDNA that point is got well as probe with digoxin (can buy by market).Results of hybridization is referring to shown in Figure 1.A is a forward subtracted library results of hybridization among the figure, and B is reverse subtracted library results of hybridization among the figure.Two films are compared, and selecting in the forward subtracted library has hybridization signal, and in reverse hybridized storehouse, do not have hybridization signal cDNA carry out sequencing.Sequencing result in GENEBANK, carry out sequence relatively after, obtain Herba Saussureae Involueratae cool tone joint gene XLCOR.

The relation of embodiment 4, Herba Saussureae Involueratae cool tone joint gene XLCOR and low temperature stress

Herba Saussureae Involueratae tissue culture seedling was cultivated 14 days at 2 ℃, taken by weighing the 0.1g blade and in liquid nitrogen, grind, add 1mlTRIZOL reagent, abundant mixing, add the 200ul chloroform, mixing, centrifuging and taking supernatant, add the 250ul Virahol, the centrifugal supernatant that goes is after 75% washing with alcohol, in drying at room temperature, precipitation obtains its total RNA with 15ul DEPC water dissolution.Extract total RNA of the Herba Saussureae Involueratae tissue culture seedling of 20 ℃ of cultivations in the same way, XLCOR is a probe with cool tone joint gene, carries out Northern hybridization with above-mentioned two RNA samples respectively, and results of hybridization is referring to shown in Figure 2.The A point is the results of hybridization of total RNA of the Herba Saussureae Involueratae tissue culture seedling of probe and 20 ℃ of cultivations among the figure, the positive contrast of B point, and the C point is the results of hybridization of total RNA of the Herba Saussureae Involueratae tissue culture seedling of probe and 4 ℃ of cultivations.The result shows that this cool tone joint gene is subjected to low temperature induction, does not express under normal temperature is cultivated.

Sequence table

＜110〉Microorgan Application Inst., Xinjiang Agricultural Academy

＜120〉Xinjiang Saussurea involucrata cold regulation protein and encoding gene thereof and application

<130>111

<160>2

<170>Patent?In?version?3.3

<210>1

<211>650

<212>DNA

＜213〉Herba Saussureae Involueratae Sasussured involucrata Kar.et Kir.

<220>

<221>5′UTR

<222>(1)..(64)

<220>

<221>CDS

<222>(65)..(571)

<220>

<221>3′UTR

<222>(572)..(650)

<400>1

atctccaaca?acccaaatta?aaaagaagaa?taaaaacaag?attgtgaaag?ttgaaggtct 60

aaca?atg?gcg?aat?gca?agt?gat?gag?ttc?agg?gtg?gca?tct?tcc?ggc?atc 109

Met?Ala?Asn?Ala?Ser?Asp?Glu?Phe?Arg?Val?Ala?Ser?Ser?Gly?Ile

1 5 10 15

gat?cat?gaa?ggc?cgt?ctt?cca?cga?aaa?tac?acc?gcc?gaa?ggt?caa?ggt 157

Asp?His?Glu?Gly?Arg?Leu?Pro?Arg?Lys?Tyr?Thr?Ala?Glu?Gly?Gln?Gly

20 25 30

aca?aag?aaa?gac?ata?tcg?cca?cca?ata?gaa?tgg?cac?aat?gtt?ccc?gat 205

Thr?Lys?Lys?Asp?Ile?Ser?Pro?Pro?Ile?Glu?Trp?His?Asn?Val?Pro?Asp

35 40 45

gag?acg?aaa?aca?tta?gca?cta?gtg?gtc?gag?gat?atc?aac?gaa?gca?gac 253

Glu?Thr?Lys?Thr?Leu?Ala?Leu?Val?Val?Glu?Asp?Ile?Asn?Glu?Ala?Asp

50 55 60

ccc?agt?ggt?atg?ctc?gtt?cct?tgg?aca?ttg?tgg?gtg?gtg?atc?aat?ata 301

Pro?Ser?Gly?Met?Leu?Val?Pro?Trp?Thr?Leu?Trp?Val?Val?Ile?Asn?Ile

65 70 75

cca?ccc?acc?tta?aaa?ggg?ttg?ccg?gag?ggg?ttt?tcc?ggc?aaa?gaa?gag 349

Pro?Pro?Thr?Leu?Lys?Gly?Leu?Pro?Glu?Gly?Phe?Ser?Gly?Lys?Glu?Glu

80 85 90 95

gac?aac?ggt?gag?gct?tcc?gct?aac?att?aaa?gaa?ggg?aac?aat?gat?ttt 397

Asp?Asn?Gly?Glu?Ala?Ser?Ala?Asn?Ile?Lys?Glu?Gly?Asn?Asn?Asp?Phe

100 105 110

aag?gtg?ccg?gga?tgg?agc?ggc?cca?aag?atg?ccg?tca?ccg?gga?cat?aga 445

Lys?Val?Pro?Gly?Trp?Ser?Gly?Pro?Lys?Met?Pro?Ser?Pro?Gly?His?Arg

115 120 125

ttt?gag?ttc?aag?ctt?tat?gcg?ttg?gat?act?gaa?att?gat?ttg?gga?aat 493

Phe?Glu?Phe?Lys?Leu?Tyr?Ala?Leu?Asp?Thr?Glu?Ile?Asp?Leu?Gly?Asn

130 135 140

aag?gtg?aca?aag?gaa?aag?cta?cta?gat?gca?att?gat?ggg?cat?gtg?ctt 541

Lys?Val?Thr?Lys?Glu?Lys?Leu?Leu?Asp?Ala?Ile?Asp?Gly?His?Val?Leu

145 150 155

gag?gaa?gcc?gtt?cta?acg?gca?tta?tat?gaa?tagcgttgtt?ttggttgggt 591

Glu?Glu?Ala?Val?Leu?Thr?Ala?Leu?Tyr?Glu

160 165

cgagaatagt?gcgttagtgc?tatacttcta?tgtaaggttt?aattttgatt?ttgtactcg 650

<210>2

<211>169

<212>PRT

＜213〉Herba Saussureae Involueratae Sasussured involucrata Kar.et Kir.

<400>2

Met?Ala?Asn?Ala?Ser?Asp?Glu?Phe?Arg?Val?Ala?Ser?Ser?Gly?Ile?Asp

1 5 10 15

His?Glu?Gly?Arg?Leu?Pro?Arg?Lys?Tyr?Thr?Ala?Glu?Gly?Gln?Gly?Thr

20 25 30

Lys?Lys?Asp?Ile?Ser?Pro?Pro?Ile?Glu?Trp?His?Asn?Val?Pro?Asp?Glu

35 40 45

Thr?Lys?Thr?Leu?Ala?Leu?Val?Val?Glu?Asp?Ile?Asn?Glu?Ala?Asp?Pro

50 55 60

Ser?Gly?Met?Leu?Val?Pro?Trp?Thr?Leu?Trp?Val?Val?Ile?Asn?Ile?Pro

65 70 75 80

Pro?Thr?Leu?Lys?Gly?Leu?Pro?Glu?Gly?Phe?Ser?Gly?Lys?Glu?Glu?Asp

85 90 95

Asn?Gly?Glu?Ala?Ser?Ala?Asn?Ile?Lys?Glu?Gly?Asn?Asn?Asp?Phe?Lys

100 105 110

Val?Pro?Gly?Trp?Ser?Gly?Pro?Lys?Met?Pro?Ser?Pro?Gly?His?Arg?Phe

115 120 125

Glu?Phe?Lys?Leu?Tyr?Ala?Leu?Asp?Thr?Glu?Ile?Asp?Leu?Gly?Asn?Lys

130 135 140

Val?Thr?Lys?Glu?Lys?Leu?Leu?Asp?Ala?Ile?Asp?Gly?His?Val?Leu?Glu

145 150 155 160

Glu?Ala?Val?Leu?Thr?Ala?Leu?Tyr?Glu

165

Claims

1, a kind of Saussurea involucrata cold regulation protein XLCOR encoding gene, this protein gene comprises the nucleotide sequence shown in the SEQ ID NO:2;

2, a kind of nucleotide sequence of the aminoacid sequence shown in the SEQ ID NO:2 of encoding;

3, nucleotide sequence as claimed in claim 2, this sequence is shown in SEQ ID NO:1;

4, a kind of SEQ ID NO:2 sequence Xing as claimed in claim 2 protein of giving birth to, it is characterized in that with the amino acid residue sequence among the SEQ ID NO:2 through the replacement of one or several amino-acid residue have with SEQ ID NO:2 in the identical activity of aminoacid sequence;

5, a kind of SEQ ID NO:2 sequence Xing as claimed in claim 2 protein of giving birth to, it is characterized in that with the amino acid residue sequence among the SEQ ID NO:2 through the disappearance of one or several amino-acid residue have with SEQ ID NO:2 in the identical activity of aminoacid sequence;

6, a kind of SEQ ID NO:2 Xing as claimed in claim 2 protein of giving birth to, it is characterized in that with the amino acid residue sequence among the SEQ ID NO:2 through the interpolation of one or several amino-acid residue have with SEQ ID NO:2 in the identical activity of aminoacid sequence;

7, a kind of SEQ ID NO:1 sequence as claimed in claim 3 is characterized in that the dna sequence dna that limits with SEQ IDNO:1 has 90% above homology, and coding identical function protein DNA sequence;

8, a kind of expression vector, it contains the expression vector just like each described Xinjiang Saussurea involucrata cold regulation protein XLCOR encoding gene of claim 2 to 7;

9, expression vector as claimed in claim 8 is characterized in that the expression vector transformed plant that can use any foreign gene of can transduceing to express in plant, all can obtain the transfer-gen plant that low temperature side of body tolerance is enhanced;

10, the application of any Xinjiang Saussurea involucrata cold regulation protein of a kind of claim 2 to 7 XLCOR encoding gene in the cold resistant plant kind, it is characterized in that, the plant expression vector conversion plant host that comprises cold-regulated proteins XLCOR encoding gene of the present invention both can be a monocotyledons, also can be dicotyledons, can cultivate cold-resistant plant variety.