CN1273604A - Antifreeze proteins, DNA and expression system - Google Patents

Abstract

The winter rye, upon cold-induction or aclimation, produces a family of antifreeze proteins that are similar to pathogen-related proteins. Two of these proteins, both of which are chitinase-like proteins, are cloned using molecular biology techniques and are expressed in bacterial and yeast (Pichia) systems and Arabidopsis thaliana. The recombinant proteins showed both chitinase and antifreeze activities. The invention includes the DNA and protein sequences of the chitinase-like antifreeze proteins, any modifications of the said sequences, the expression of these proteins and their appplication in agriculture, food industry and medicine.

Description

Antifreeze protein, DNA and expression system thereof

In present patent application, intactly quote and adopted following patent: English Patent provisional application number 9112774.69 (on June 13rd, 1991 filed an application), U. S. application number 08/060,425 (on May 11st, 1993 filed an application), U. S. application number 08/419,061 (file an application April 10 nineteen ninety-five), U. S. application number 08/485,647 (file an application June 10 nineteen ninety-five), Canadian Patent 2,110,510 (on June 12nd, 1992 filed an application), European Patent Application No. 92911435.3 (on June 12nd, 1992 filed an application), PCT application number PCT/CA92/00255 (on June 12nd, 1992 filed an application), and Application No. 08/903,872.Present patent application requires the right of priority based on Application No. 08/903,872.

Scope of the present invention

The present invention relates to plant antifreeze protein, peptide and polypeptide (hereinafter being called " AFP " again), they are set on the crystal of ice, stop the ice recrystallization, and liposome, cytolemma and protein, cell and the organism of protection under the low temperature situation.

The invention still further relates to the dna sequence dna of giving protein coding with chitinase and freeze proof activity, and the expression method of the dna sequence dna in bacterium, yeast, plant and animal, so that produce freeze proof active chitinase is arranged.

Background of the present invention

For the production of farm crop, low temperature is a kind of main environmental restraint factor.For example, the frost in late spring has been incured loss through delay the rudiment of seed, and the frost of early autumn has reduced the quality and the output of farm crop harvests, and the low temperature in winter has reduced the chance of surviving of winter crop (such as grain and fruit tree).Yet some plant can be long-term, and subfreezing temperature is lived in tolerance.By recognizing and be separated in the protein that helping in these plants forms anti-frost ability, and corresponding gene, can be transformed into farm crop the farm crop of anti-frost, and prolong the production phase of crop frost-rib.

People such as GUY (I-5.1) analyze protein total content in the spinach tissue of cold domestication and the protein total content in warm regional growing plants tissue are compared analysis, find that protein is relevant to the tolerance of cold with plant.Find that in the extract of the leaf of cold domestication molecular weight is 110,82,66,55 and the protein of 13 KD, do not appear in the extract of leaf of warm area growth.People such as GUY (I-5.2) have also checked the protein total content in the leaf of spinach of cold domestication, and have observed gathering of high-molecular weight protein (110,90 and 79KD).For people such as GUY (I-5.1), at inside plants, it is unknown that these proteinic positions and function remain.

Yet we know that many biological organisms can be under survival under the subfreezing temperature condition by the formation of avoiding icing.This strategy requires synthetic antifreeze protein (AFP), and this protein is called " thermal hysteresis protein " again (THP).Various AFP are frozen in the surface of ice crystal, prevent the further generation of ice crystal.The existence of various AFP is determined by following: 1) in the process that ice crystal forms, investigate the shape (III-19, III-35, III-22) of ice crystal, 2) measure thermal hysteresis, this hysteresis quality is the ice crystal temperature of fusion in the solution and the difference (III-19) of freezing temperature, and 3) measure inhibition ability to the recrystallization of ice crystal.

In fish, recognize and four kinds of different AFP types (II-4), and in insect, recognize and some different THP (III-12).These previous discoveries have been pointed out, and this adaptation mechanism independently forms in different organisms, and this mechanism is preventing anyly to play effect aspect icing inner generation of organism.It is conventionally believed that various AFP are not present in the middle of the plant, because can be long-term under subfreezing temperature condition, in fact the most of plants that survive have ice to form in their tissue, normally in the intercellular space, and in xylem vessel.KURKELA and FRANCK (I-9.1) reported that a kind of plant gene that shows was given a kind of protein coding under cold condition, be similar to the situation (II-4) of encoding to the various antifreeze proteins of flatfish in amino acid that DAVUS and GEW reported.KURKELA and FRANCK (I-9.1) fail the coded protein of sufficient amount and determine whether to present freeze proof activity.People (I-1.1) such as people such as CUTLER (I-1.1) CUTLER point out that the plant tissue that has been injected into the flatfish antifreeze protein demonstrates its freeze proof activity slight raising.People such as GEORGES (I-3.1) use a kind of synthetic gene of flatfish antifreeze protein to transform the protoplastis of cereal, attempt to improve the freeze proof activity of plant.The result has generated a kind of protein, but it fails to secrete effectively, and fails to detect freeze proof activity.

Although the antifreeze protein of separating from fish and insect has been used to develop the food that is applicable under the temperature condition that is stored in low temperature and low freezing point and the new additive of biological substance, but these protein still come into the market, and are inappropriate because they are thought by the human consumer.For example, a kind of fish antifreeze protein added to be considered to unacceptable in the icecream, a kind of plant antifreeze protein is added to then be considered to acceptable in the icecream.The AF that separates from plant is expected and can application be widely arranged than those antifreeze proteins that has been named as from animal in market.

Brief description of drawings

First-selected embodiment of the present invention will add explanation in conjunction with the accompanying drawings.These accompanying drawings comprise: the cold tolerance of I. plant

Fig. 1 has described the proteinic concentration of iuntercellular of taking from the winter naked barley leaf that is grown under the condition of different temperatures.

Fig. 2 has described " SDS-PAGE " that separates from the polypeptide of the exoprotein that is grown in the winter naked barley leaf under the condition of different temperatures.First road: molecular weight marker; Second road: the outer polypeptide of born of the same parents that separates the naked barley crop under the temperature condition that is grown in 20/16 degree centigrade (daytime/night) (accounting for daytime 16 hours); The 3rd road: the outer polypeptide of born of the same parents that separates the naked barley crop under the temperature condition that is grown in 5/2 degree centigrade (daytime/night) (accounting for daytime 16 hours); The 4th road: the outer polypeptide of born of the same parents that separates the naked barley crop under the temperature condition that is grown in 5/2 degree centigrade (daytime/night) (accounting for daytime 8 hours).

Fig. 3 has described to separate from growth and had accounted for by day 8 hours, at " SDS-PAGE " of the polypeptide of the exoprotein of the cold domestication winter naked barley leaf of different growth phases.First road: molecular weight marker; Second road: separate outer polypeptide from the born of the same parents that are grown in 7 days naked barley crop of growth under 20/16 degree centigrade (daytime/night) (accounting for daytime 16 hours) temperature condition; The 3rd road: the temperature condition that separating confesses one's crime is grown in 20/16 degree centigrade earlier growth 7 days down, be transplanted to then 5/2 degree centigrade (daytime/night), account for the outer polypeptide of born of the same parents of 28 days naked barley crop of regrowth under 8 hours the illumination system daytime; The 4th road: the outer polypeptide of born of the same parents that separates the naked barley crop under the temperature condition that the temperature condition of being transplanted to 5/2 degree centigrade was grown 43 days down; The 5th road: the outer polypeptide of born of the same parents that separates the naked barley crop under the temperature condition that the temperature condition of being transplanted to 5/2 degree centigrade was grown 50 days down; The 6th road: the outer polypeptide of born of the same parents that separates the naked barley crop under the temperature condition that the temperature condition of being transplanted to 5/2 degree centigrade was grown 71 days down; The 7th road: the outer polypeptide of born of the same parents that separates the naked barley crop under the temperature condition that the temperature condition of being transplanted to 5/2 degree centigrade was grown 95 days down.

Fig. 4 has described " SDS-PAGE " that separates from the polypeptide of the exoprotein of taming winter naked barley leaf.First road: molecular weight marker; Second road: the temperature condition that separating confesses one's crime is grown in 20/16 degree centigrade earlier growth 7 days down, be transplanted to then 5/2 degree centigrade (daytime/night), account for the outer polypeptide of born of the same parents of 42 days naked barley crop of regrowth under 8 hours the illumination system daytime; Third and fourth and five roads: be respectively to separate certainly at first through the described process of growth in second road, and then be transplanted to 20/16 degree centigrade, the shared time on daytime is the outer polypeptide of born of the same parents of the naked barley crop of regrowth 4 days, 6 days and 8 days respectively under 16 hours the illumination system;

Fig. 5 has described and has separated from being grown in naked barley leaf under the various condition of different temperatures, forms ability through the ice crystal of the outer extract of the filtering born of the same parents of high-precision.

Fig. 6 has illustrated the freeze proof activity in the extract outside the born of the same parents of the winter of cold domestication naked barley leaf.Freeze proof activity is that (manufacturer: New York, United States Hart Ford city CLIFTON technology physics company, II-5) form of observation ice crystal is measured by utilizing a millimicron osmometer.Ice crystal orientation in C, D, E and F: the A axis represents that along basic planar growth, the C axis is then represented perpendicular to basic planar growth.

Fig. 7 has illustrated the component of the outer extract of born of the same parents of the naked barley leaf that utilizes column chromatography to separate self cooling domestication.

Fig. 8 has illustrated that the part of the leaf that separates self cooling domestication winter naked barley is purified and the ice crystal form of spissated antifreeze protein.A: crystalline is orientated as described in Figure 6.B, C and D: in the process that temperature descends, the succession of ice crystal.B: incomplete biconical; C: biconical; D: needle-like crystal.

Fig. 9 with at the relevant polypeptide " SDS-PAGE " of the column fractionation of each the 280 millimicrons of peak value shown in Fig. 8.

Figure 10 has described and has utilized trihydroxy-Wheat flavone damping fluid (TRIS-TRICNEBUFFERS) by " SDS-PAGE " polypeptide outside the born of the same parents that the winter naked barley leaf of cold domestication is separated.Polypeptide is come out by elution from gelinite, is used to check freeze proof activity.Molecular weight is 161,93 to 99,33,31,27,23,15 and the polypeptide of 11 KD all demonstrate freeze proof activity.

Figure 11 has illustrated and has separated from A: not through the winter naked barley leaf of domestication, and B: through the outer extract of born of the same parents of the naked barley leaf of cold domestication.Polypeptide is dissolved by " SDS-PAGE " and separates, and uses anti-chitinase antiserum(antisera) as main antibody it to be tested then.Molecular weight standard (is unit with kD) provides in the C row.

Figure 12 has illustrated a kind of immunoblotting assay of extract outside the born of the same parents that extract from the leaf of cold domestication winter naked barley.Polypeptide is separated by " SDS-PAGE ", and trace uses anti-chitinase antiserum(antisera) to test as main antibody to nitrocotton then.

Figure 13 has illustrated to use and has separated from the winter naked barley crop of cold domestication that extract suppresses recrystallization outside the born of the same parents that obtain.When being diluted to 1: 10,000 o'clock, proteinic concentration was approximately every liter 28 microgram.When being cooled to-20 degrees centigrade, at-8 degrees centigrade after following 6 hours, form slip then.(remarks: allow slip " annealing " 6 hours under-8 degrees centigrade temperature, to observe the recrystallization of ice crystal.The photo is here provided by American National atmospheric research center C HARLES doctor KNIGHT.)

Figure 14 illustrated in the leaf of non-domestication (σ), through (λ) in the leaf of cold domestication and in order to support exoprotein content before freezing by the leaf of the cold domestication of extractive process in the degree of freezing injury of (υ), measure to leak.These leaves are put in the water, are cooled to icing temperature takes place, and keep under this temperature 22 minutes then.After this, from freezing bath, leaf is taken out, be put on ice and slowly thaw.Ion leaks and may be calculated the total electric conductivity of the frozen solution conductivity rate afterwards of leaf divided by the solution after boiling.The data that obtain are used without the ion leakage of the sample that freezes and are corrected, and the form with mean value ± SE provides then, and N equals 3 here.Fatal harm occurs in tissue and appears at 50% or bigger ion when leaking.II. the herbal freeze proof activity of cold domestication

Figure 15 has illustrated the freeze proof activity in the leaf of plant of non-domestication (NA) and cold domestication (CA).Freeze proof activity in the outer extractives of the born of the same parents of every kind of plant leaf is to measure in the form of the ice crystal of growth from solution by observation.In the extractives of the leaf of all non-domestications, do not observe freeze proof activity.Through the collard of cold domestication and the extractives of winter canola leaf, and through cold domestication winter naked barley and spring naked barley, winter wheat and spring wheat, winter barley and spring the avenaceous leaf extractives all demonstrate freeze proof activity.All photos all are to take with identical magnification.Amplify bar and represent 17 microns.

Figure 16: following various crops through catching a cold between domestication. the exoprotein in its leaf gathers---A: the winter naked barley, winter barley and winter wheat; B: spring naked barley, spring wheat, spring oat and corn; C: spinach, winter canola and kale; D: spring canola and tobacco.Plant grows under the condition of non-domestication (0 time point), transfers to (5/2 degree centigrade) 1 to 7 week of experience under the condition of carrying out cold domestication then.Protein concn shows with the form of mean value ± SE, and N equals 3 here.

Figure 17: the outer polypeptide of the born of the same parents among the winter naked barley cv Voima gather immunodetection with antifreeze protein.A: from vegetative period is that the naked barley (first road) of the non-domestication (NA) in 3 weeks is isolated extractives outside the spissated born of the same parents, and each was respectively a week since vegetative period, (second road), fortnight, (the 3rd road), four stars phase, (the 4th road), seven-star phase, (the 5th road) and nine weeks, (the 6th road), temperature condition is 5/2 degree centigrade (daytime/night), isolate the outer extract of spissated born of the same parents through (CA) wheat of cold domestication, outside these concentrated born of the same parents, isolate the polypeptide (per pass 5 micrograms) of equivalent then by SDS-PAGE the extractives.The outer extract SDS-polyacrylamide gel body trace of the concentrated born of the same parents that are mounted with equivalent (per pass one microgram) is come out, use B to test then.B is the protein antiserum(antisera) (thinning ratio is 1: 10000) with reference to the anti-similar dextranase of the protein preparation of the similar dextranase of winter naked barley 32kD; C is the anti-class chitinase protein antiserum(antisera) (thinning ratio is 1: 10000) with reference to the anti-class chitinase protein preparation of winter naked barley 35kD; And D is the protein antiserum(antisera) (thinning ratio is 1: 10000) with reference to the anti-similar West Africa arrowroot essence of the protein preparation of the similar West Africa arrowroot essence of winter naked barley 25kD.The clear and definite detection of corresponding every peptide species indicates on the right.Left side numeral is meant Bio-Rad lower molecular weight sign.CLP, the class chitinase protein; GLP, class dextranase albumen; TLP, the protein of class West Africa arrowroot essence.

Figure 18 illustrated non-domestication (NA) and through the born of the same parents in the cereal of complete cold domestication (CA) outside the gathering of polypeptide.In 15% SDS-polyacrylamide gel body, isolate polypeptide (per pass 1 microgram) outside the born of the same parents of equivalent.A: a kind of gelinite of catching bio-Rad silver mark.This gelinite is used B, C and D comes trace and test.B is the protein antiserum(antisera) (thinning ratio is 1: 10000) of anti-similar dextranase; C is anti-class chitinase protein antiserum(antisera) (thinning ratio is 1: 10000); And D is anti-class West Africa arrowroot protamine antiserum(antisera) (thinning ratio is 1: 10000).The clear and definite detection of corresponding every peptide species indicates on the right.Left side numeral is meant Bio-Rad lower molecular weight marker.

Figure 19 illustrated non-domestication (NA) and through the born of the same parents in the cereal of complete cold domestication (CA) outside the gathering of polypeptide.In 15% SDS-polyacrylamide gel body, isolate polypeptide (per pass 1 microgram) outside the born of the same parents of equivalent.A: a kind of gelinite of catching Bio-Rad silver color.After shifting, use B, C and D to come immunoblotting is tested.B is the protein antiserum(antisera) (thinning ratio is 1: 10000) of anti-similar dextranase; C is anti-class chitinase protein antiserum(antisera) (thinning ratio is 1: 10000); And D is anti-class West Africa arrowroot protamine antiserum(antisera) (thinning ratio is 1: 10000).The clear and definite detection of corresponding every peptide species indicates on the right.Left side numeral is meant the separation of Bio-Rad lower molecular weight marker.

Figure 20 has illustrated and has separated from (the white tape) of non-domestication and the total reducing sugar amount of existence in through the monocotyledons (A) of (the hypographous tape) of cold domestication and dicotyledons (B).Utilize glucose to come the concentration of sugar is calculated as milligram/gram FW as standard, and be expressed as mean value+SE, wherein n equals 3.III. the DNA with freeze proof active class chitinase protein is separated and protein sequence

Figure 21 has described a kind of DNA and protein sequence with freeze proof active class chitinase protein.A:CH-9 full length DNA and aminoacid sequence; B:CH-9 DNA and aminoacid sequence (signal peptide coding region that lacks prediction); C:CH-9 comprises a preamble sequence of being made up of 20 seed amino acids, and this sequence is removed (purifying at last obtains, have chitinase and freeze proof active protein lacks this preamble sequence) from vegetable cell; The D:CH-9 aminoacid sequence lacks the signal sequence of a supposition of being made up of 20 seed amino acids.

Figure 22 has described a kind of DNA and protein sequence with freeze proof active class chitinase protein.A:CH-46 DNA and aminoacid sequence (the untranslated sequence that is removed and the preamble sequence of supposition); B:CH-46 DNA and aminoacid sequence (the untranslated sequence that is removed); C:CH-46 sequence (the untranslated sequence that is removed); D:CH-46 (the untranslated sequence that is removed).

Figure 23 is a multiple formation of being made up of from the aminoacid sequence of the chitinase of different plants separation.The 1st, separate aminoacid sequence (protein information source registration number JC 2071) from the chitinase-a of naked barley seed, rye (naked barley) separates from the chitinase-c of naked barley seed (protein information source registration number JN 0884, pCHT 9 and pCHT 46 are two kinds of aminoacid sequences with prediction of freeze proof active winter naked barley class chitinase protein).The representation of the dna sequence dna of coding class chitinase protein

Figure 24 has illustrated the dna sequence dna in intestinal bacteria and the representation of class chitinase protein.All cell extract is to use uninduced cell (the 1st road and the 31st road) and IPTG-inducing cell (the 2nd road and the 4th road) to prepare, and adopts SDS-PAGE to separate, and through immunoblotting.A kind ofly recognized by anti-class chitinase protein antiserum(antisera), molecular weight is the protein of 32 kD, clearly be present in (the 2nd road and the 4th road) in the inducing cell.The class chitinase protein of purifying and obtaining with through the winter naked barley (M) of cold domestication also has been described among the figure.

Figure 25 A has illustrated the dna sequence dna in yeast and the representation of class chitinase protein.Yeast cell in the YPD medium, grown respectively 24 hours (the 1st road) and 48 hours (the 2nd road).Cell is through centrifugal treating, and supernatant liquid is with after sample buffer mixes, and polypeptide is separated by SDS-PAGE.In twice, all having a kind of molecular weight is the polypeptide of 32kD.

Figure 25 B has illustrated and has carried out a kind of immunoblotting assay to having sero-fast supernatant liquid of anti-class chitinase protein and cell section.The protein of taking from supernatant liquid (the 3rd road and the 4th road) and cell section (the 1st road and the 2nd road) is dissolved, adopts SDS-PAGE to separate then, and through immunoblotting.Most protein is found and is present in the supernatant liquid (the 3rd road and the 4th road).The class chitinase protein of purifying and obtaining with through the winter naked barley of cold domestication also has been described among the figure.

Figure 26 has illustrated and has adopted SDS-PAGE to separate the heat-staple antifreeze protein that obtains.Extract is heated to 60 degrees centigrade respectively outside the born of the same parents that separate through the winter naked barley leaf of cold domestication, lasts 30 minutes in (A road) and be heated to 100 degrees centigrade, lasts 10 minutes.The C road demonstrates the molecular weight marker that BioRad haves a wide reach (207,121,81,51.2,33.6,28.6,21.1 and 7.5kD).GLP: class dextranase albumen; TLP: class West Africa arrowroot protamine.

Brief summary of the present invention

In the past, people it is generally acknowledged that the mechanism relevant with the freeze proof activity of plant is present in the inside of cell, thereby should protect cell, make it can not form ice crystal internally.Nobody once expected having at inside plants to have antifreeze protein and the proteinic possibility that forms the ice crystal nucleus ability is arranged.In addition, nobody once expected existing such possibility: these protein can be positioned at the outside of cell, for the evil that protects plants from frost is being carried out a kind of diverse mechanism.Making us very surprised is, we have found that have one group of polypeptide to accumulate in the outside of cell, and demonstrates ability and the freeze proof activity that forms ice crystal, thereby can be controlled at space between the cell and the ice-crystal growth in the xylem.In their natural form, these protein also present enzymic activity, for example hydrolysis of dextran and chitinase, and this hydrolytic action is divided the cell walls that changes plant, and/or suppress the growth of low temperature pathogenic agent.The outer polypeptide of these born of the same parents is positioned at the apoplastic part of extracting of plant, and this part comprises the test-tube baby and the conduit of zone, cell walls, cell middle level, intercellular space and xylem between outside surface, membrane plasmapheresis and the cell walls of membrane plasmapheresis.Should be pointed out that in the full content of patent specification term " born of the same parents are outer " all has above-mentioned implication.

Winter, naked barley was a kind ofly can be lower than under-20 degrees centigrade the temperature condition under the survival annual herb plant that survives the winter.When the leaf when freezing of winter naked barley, ice is growth (III-40) in the intercellular space of mesophyll inside and in xylem vessel.Can the plant tissue that freeze survive and depend on that can the growth that stop owing to intercellular ice crystal cause cell damage.Between cold domestication, accumulate in various antifreeze proteins (plant antifreeze protein, peptide and polypeptide) in the apoplast of winter naked barley leaf and have the ability to change the situation (III-35 and III-22) of growing in the tissue that ice crystal freezing, and be relevant to the ability (III-35) that stronger tolerance that winter naked barley leaf had is freezed.In addition, shown in immunolocalization, antifreeze protein is relevant with epidermis, and be with through cold domestication winter naked barley the intercellular space of leaf around mesophyll cell relevant (III-2).These positions are equivalent on leaf surface, and the place (III-40 and III-41) of the known generation ice in the intercellular space of mesophyll inside, and the prompting antifreeze protein can also stop the inoculation of cell to freeze (III-41).

The six kind antifreeze proteins of its molecular weight from 16 to 35kD from through domestication winter naked barley the mesophyll of leaf separate (III-22).Their (PR) proteinic members relevant with pathogeny with three classes are similar: it is the protein (CLPs) that is similar to chitinase that two kinds of antifreeze proteins are arranged, have two kinds of antifreeze proteins be the protein that is similar to beta-1,3-glucanase (TLPs, III-23).One kind chitinase protein is from being homogeneity through purifying the winter naked barley leaf of cold domestication, and demonstrates and have freeze proof activity and chitinase activity (III-23) simultaneously.Because this; A little low temperature naked barley protein demonstrate two kinds of activity, and they can work (chitinase activity) aspect the indefinite disease of opposing, also can work aspect the ability that strengthens tolerance frost (freeze proof activity, III-48).

If the ability of the situation of the growth of the ice crystal of change in apoplast is a freeze proof active important integral part, so, it may be a kind of very general mechanism in the middle of plant.Investigation result to various overwintering plants shows, in the middle of expressed resin from the tissue of many plants, has freeze proof activity, these plants comprise 21 kinds of dicotyledonss and 9 kinds of monocotyledonss (III-19, III-17, III-52, III-11, III-12).A kind of protein from bittersweet (Solanum dulcamara, a kind of bittersweet Solanum overgrow xylophyta) (III-10).The antifreeze protein of nightshade is the glycosylated protein of a kind of macromolecule (67kD), has higher glycine content (about 24%) usually, and has a kind of n terminal amino acid sequence (III-1) of uniqueness.

The antifreeze protein of separating from plant origin is that those relate to the upgrowth situation that changes ice crystal, the disease that minimizing is caused by microorganism, rotten and damage under cold condition, and improvement is exposed to the consequence that low temperature causes, comprise and producing and storing frozen food, and the low-temperature storage of biological substance.Is being crucial to the gene of antifreeze protein coding aspect the expression system of producing more antifreeze proteins as additive, for increase antifreeze protein synthetic with promote survival and support injury in cold environment and disease aspect also be crucial.According to an aspect of the present invention, we have prepared strong some the common antifreeze proteins of plant of freezing tolerance.These protein are positioned at the born of the same parents outside, to control ice crystal in the xylem of inside plants and the growth in the intercellular space.Having freeze proof active polypeptide is to choose from the one group of polypeptide that has following approximate apparent molecular weight respectively: about 5 to 9kD, about 9 to 11kD, about 11 to 15kD, about 21 to 23kD, about 24 to 27kD, about 30 to 31kD, about 31 to 33kD, about 32 to 36kD, about 60 to 68kD, about 89 to 100kD, and about 161kD

According to another aspect of the present invention, we also provide has above-mentioned molecular weight, and separates from frost being had the polypeptide of the plant of tolerance.In these polypeptide, some are arranged is nucleators of ice crystal, starts the process of ice-crystal growth in born of the same parents' external space of the tissue of their plants.It is enzymes that some polypeptide are arranged, and they can change the cell walls of plant and/or suppress the growth of the pathogenic agent in the plant intercellular space.

According to each different aspect of the present invention, the shown feature of polypeptide is their apparent molecular weight: their apparent molecular weight is and they relevant with respect to the migration of known molecular weight marker in the SDS-PAGE gelinite.Can be understood as, different migrations can take place in each peptide species of the present invention in dissimilar gelinites, particularly when gelinite acrylamide concentration not simultaneously, or reductive condition is not simultaneously, or employed operation damping fluid is not simultaneously, and situation especially like this.Therefore, molecular weight characteristic of the present invention plans to be used to replace the polypeptide of equivalent, because the molecular weight of the polypeptide of equivalent in different gelinites can be slightly variant.In addition, the outer polypeptide of born of the same parents is found and is present in separation from the tissue of plant, contains in the extract of all soluble protein.

According to another aspect of the present invention, we have prepared separation from unifacial leaf herbaceous plant and dicotyledonous herbal antifreeze protein.Freeze proof activity and all protein gather to be found in and separate in the outer extract of 8 kinds of herbal leaf cells that is grown in the low temperature environment, comprising the unifacial leaf herbaceous plant (winter naked barley and spring naked barley, winter wheat and spring wheat Triticum aestivum L., winter barley Hordeum vulgare l., spring oat Avena sativaL.) and dicotyledonous herbaceous plant (winter canola Brassica napul L. and kale Brassicaoleracea L.).The secretory product that can change the exoprotein of ice-crystal growth situation is can the survive the winter aspect of mechanism of survival of these unifacial leaf herbaceous plant and dicotyledonous herbaceous plant.In addition, the protein secreting thing enters in the apoplast, and is accompanied by freeze proof active enhancing, is all common responses that are grown in all plants gramineous in the cold environment.Therefore, in another embodiment, the present invention relates to a kind of freeze proof active polypeptide that is separated that has, choose free Gramineae member and Cruciferae member and constitute in one group of plant.In a top-priority embodiment, grass is selected from by in barley, wheat and naked barley, spring naked barley, winter naked barley, spring wheat, winter wheat, winter barley and the one group of plant that spring, oat constituted, and cress then is selected from kale and winter canola.

According to another aspect of the present invention, some monocotyledons through after inducing or taming, can produce a series of protein relevant antifreeze proteins relevant with pathogeny, shown in the N terminal sequence of the protein that passes through cold inductive and immunoblotting.We have prepared relevant plant from grass, comprise naked barley, wheat and barley, have gathered between cold domestication and have been similar to the protein relevant with pathogeny.Therefore, the invention still further relates to the cold inductive protein relevant with pathogeny, they are separated from a kind of unifacial leaf herbaceous plant, and be have freeze proof active.Unifacial leaf is preferentially chosen from Gramineae by herbaceous plant.In a kind of embodiment preferred, the unifacial leaf herbaceous plant is selected from by barley, oat and naked barley, spring naked barley, winter naked barley, spring wheat, winter wheat and winter barley.The present invention also comprises a kind of mimicry of every peptide species.In a kind of embodiment preferred, each peptide species of the present invention can be chosen the histone matter that free class chitinase protein, class West Africa arrowroot protamine and glucose enzyme constitute.These polypeptide can be used to produce antifreeze protein; in order to strengthen the freeze proof activity in plant and the microorganism; the recrystallization that suppresses the ice crystal in biological substance or the foodstuff products; the disease that minimizing causes in the activity under the cold condition owing to microorganism in biological substance or foodstuff products, rotten or damage; the very low temperature refrigeration of cell; the cryoprotection of cell, the cold protection of human body platelet, and kill tumor cell.

According to another aspect of the present invention, may form one or more antibody, for example selectedly in immunoassay be applicable to the level of measuring antifreeze protein, or be used for determining whether similar antifreeze protein can be produced by other plant aforementioned polypeptides.Having the outer antifreeze protein of three kinds of born of the same parents to separate from the winter naked barley now comes, they are equivalent to the protein of the West Africa arrowroot essence of the protein of similar chitinase of the protein of the similar dextranase of a kind of 32-kD, a kind of 32-kD and a kind of 25-kD, they are purified out, and turn out antiserum(antisera) with respect in the middle of them each.

According to another aspect of the present invention, in the preparation process of frozen product, can add one or more above-mentioned polypeptide.Specifically, one or more polypeptide in icecream and fruit product, have been added, so that obtain to have the superior product of small crystalline structure.In addition, aspect the refrigeration of the refrigeration of the refrigeration of organization of production protection, various tissues and germplasm, polypeptide also is of great use.

According to another aspect of the present invention, we have determined that multiple sugar is present in through in the outer extract of the born of the same parents of cold domestication, and have changed the upgrowth situation of the outer ice crystals of born of the same parents, have suppressed the recrystallization of ice crystal simultaneously.These sugar have strengthened the activity of antifreeze protein.

According to another aspect of the present invention, two kinds of separation are utilized molecular biotechnology and duplicate out from the class chitinase protein of winter naked barley, and express with bacterial system and Yeast system simultaneously.Demonstrated the active and freeze proof activity of chitinase simultaneously by expressed protein.The present invention includes the DNA of class chitinase protein and protein sequence, to the representation of any correction of above-mentioned sequence, these sequences and they agricultural, foodstuffs industry and medical aspect application.The present invention is a kind of nucleic acid molecule of separating from a kind of monocotyledons, gives a kind of antifreeze protein coding.In a preferred embodiment, molecule has the dna sequence dna in Figure 21 .a., Figure 21 .b or Figure 22 .a or Figure 22 .B.The present invention also comprises a kind of such molecule, and its sequence and dna sequence dna in Figure 21 .a., Figure 21 .b or Figure 22 .a or Figure 22 .b all or part of is that identical (below this identity is discussed with reference to the quantity of sequence signature) this molecule can be chosen from the one group of nucleic acid that is made of mRNA, cDNA, sense strand (sense) DNA, antisense strand (anti-sense) DNA, single stranded DNA and double-stranded DNA substantially.The invention still further relates to a peptide species by nucleic acid molecule encoding, and a kind of purification and the mimicry of separating the polypeptide that obtains.In a preferred embodiment, polypeptide has at the aminoacid sequence shown in Figure 21 .a., Figure 21 .b, Figure 21 .c, Figure 21 .d, Figure 22 .a, Figure 22 .B, Figure 22 .c or Figure 22 .d.In another embodiment, polypeptide can be substantially with the whole identical of the aminoacid sequence shown in Figure 21 .a., Figure 21 .b, Figure 21 .c, Figure 21 .d, Figure 22 .a, Figure 22 .B, Figure 22 .c or Figure 22 .d or part is identical.

The invention still further relates to the dna sequence dna to the polypeptide signal sequence encoding, this polypeptide signal sequence allows secretory product leave cell.The present invention includes a kind of molecule, its sequence is whole identical with at the dna sequence dna shown in Figure 21 .a., Figure 21 .c, Figure 21 .d, Figure 22 .B or Figure 22 .d substantially.This molecule can be chosen from the one group of nucleic acid that is made of mRNA, cDNA, sense strand DNA, antisence strand dna, single stranded DNA and double-stranded DNA.The invention still further relates to a peptide species by nucleic acid molecule encoding, and a kind of purification and the mimicry of separating the polypeptide that obtains.In a preferred embodiment, polypeptide has at the aminoacid sequence shown in Figure 21 .a., Figure 21 .c, Figure 21 .d, Figure 22 .B or Figure 22 .d.In another embodiment, polypeptide can be substantially with the whole identical of the aminoacid sequence shown in Figure 21 .a., Figure 21 .c, Figure 21 .d, Figure 22 .B or Figure 22 .d or part is identical.This target sequence can be used in transgenosis organism of protein secreting thing importing or in the expression system.The main body of expressing can be plant, vegetable cell, alginic cell, bacterium, yeast, Mycophyta, animal and zooblast.

The present invention also comprises a gene expression system of giving class chitinase protein coding, comprises that one is expressed vector and is embedded in the cDNA that this expresses the class chitinase protein in the vector.In a preferred embodiment, expressing vector is intestinal bacteria plastid pET 12a.In another embodiment, this system is made of Pichia vector pGAPZ α A.In a preferred embodiment, this system is included in the aminoacid sequence of the similar chitinase shown in Figure 21 .a., Figure 21 .b, Figure 22 .a or Figure 22 .b.The present invention also comprises a kind of plant, vegetable cell, animal, zooblast, bacterium, Mycophyta or the yeast by the system reform.Other aspect of the present invention comprises a kind of method of expressing the class chitinase protein, and this method constitutes by transforming a kind of expression main body and culture expression main body that has class chitinase protein DNA expression vector.This expression main body can be a kind of plant, vegetable cell, alginic cell, bacterium, yeast, Mycophyta, animal and zooblast.The product of expression system can be used to produce antifreeze protein; strengthen the cold-resistant ability of plant, animal, microbe and the ability of the disease that the opposing low temperature causes; the recrystallization that suppresses the ice crystal in biological substance or the foodstuff products; the very low temperature refrigeration of cell; the cryoprotection of cell; the cold protection of human body platelet, and kill tumor cell.

The invention still further relates to chitinase coded DNA sequence in the cold inductive of process, chitinase is that all members from monocotyledons and/or grass obtain in separation in this, and its usage is identical with the description to the usage of winter naked barley sequence in this patent application.

The invention still further relates to the organism of being transformed by the antifreeze protein dna sequence dna (plant, animal and microorganism), to promote survival ability, the survival ability of surviving the winter and the freeze proof activity in genetically modified organism during refrigerated storage.Organism can be used for exceedingly producing protein by transformation, and antifreeze protein also allow body this near zero degrees celsius or be lower than and store under the temperature condition of zero centigrade, and allow to continue they are carried out work.Because many bacterium 1 animals, plant and yeast cell pedigree are very responsive to frost and cold.

The present invention is a kind of nucleic acid molecule of separating from a kind of monocotyledons, gives a kind of antifreeze protein coding.In one embodiment, nucleic acid molecule has the dna sequence dna in Figure 21 .a., Figure 21 .b or Figure 22 .a or Figure 22 .b.Nucleic acid molecule can dispense at the target sequence shown in Figure 21 or Figure 22.This molecule preferably has 40% sequence with the whole identical of dna sequence dna in Figure 21 .a., Figure 21 .b or Figure 22 .a or Figure 22 .b or part is identical.This molecule is preferably chosen in one group of nucleic acid that free mRNA, cDNA, sense strand DNA, antisence strand dna, single stranded DNA and double-stranded DNA constitute and is chosen.The present invention also comprises some such nucleic acid molecule, and they give same amino acid sequence encode as the nucleic acid molecule among Figure 21 and Figure 22.The present invention also comprises so a kind of nucleic acid molecule, this molecule is given a kind of antifreeze polypeptide coding of similar chitinase, the coding strand making nucleic acid molecular hybridization of 340 to 744 each positions among this peptide species and Figure 22 (a), its process in a kind of strictness by 0.2 * SSC to 2 * SSC, in the eluant that 0.1% SDS (sodium laurylsulfonate) forms, be to carry out under 42 degrees centigrade the temperature condition in temperature.Another embodiment of the invention also comprises a kind of such nucleic acid molecule, this molecule is given a kind of antifreeze protein coding of similar chitinase, the coding strand making nucleic acid molecular hybridization of 541 to 745 each positions among this protein and Figure 21 (a), its process in a kind of strictness by 0.2 * SSC to 2 * SSC, in the eluant that 0.1% SDS forms, be to carry out under 42 degrees centigrade the temperature condition in temperature.

In another embodiment, the present invention includes a kind of polypeptide, as described in the last period by a kind of nucleic acid molecule encoding of the present invention.The present invention also comprises through purifying and a kind of mimicry of separating the polypeptide that obtains.This peptide species preferably includes at the aminoacid sequence shown in Figure 21 (a), Figure 21 (b), Figure 21 (c), Figure 21 (d), Figure 22 (a), Figure 22 (b), Figure 22 (c) or Figure 22 (d).In another embodiment, this peptide species include at least 40% sequence with in that the aminoacid sequence shown in Figure 21 (a), Figure 21 (b), Figure 21 (c), Figure 21 (d), Figure 22 (a), Figure 22 (b), Figure 22 (c) or Figure 22 (d) is all identical or part is identical.

The present invention comprises with a kind of variation and separating from a kind of monocotyledonous, through a kind of antifreeze polypeptide of cold inductive, or separates from a kind of monocotyledonous, through a kind of protein relevant with pathogeny of cold inductive.This monocotyledons preferably is selected from Gramineae.This monocotyledons preferably is selected from by barley, wheat and naked barley, spring naked barley, spring wheat, winter wheat, winter barley and one group of grass that spring, oat was formed.The present invention also comprises the mimicry of these polypeptide.

The present invention includes a kind of freeze proof active polypeptide that is separated that has, choose free barley, wheat and naked barley, spring naked barley, spring wheat, winter wheat and winter barley, winter canola, spring oat and one group of plant forming of kale.This peptide species is preferably chosen one group of enzyme that free chitinase, West Africa arrowroot essence and dextranase are formed.This peptide species is in plant, animal and microorganism survival ability and the output in the open air under the temperature condition that is lower than zero degrees celsius for producing tolerance, the enhancing to severe cold of antifreeze protein, enhancing plant and little organism; be suppressed at the recrystallization of the ice crystal in biological substance or the foodstuff products; the refrigeration of cell; the low-temperature protection of cell; the cold protection of human body platelet, and the kill tumor cell is very useful.

The present invention also comprises a kind of NDA recombinant chou, and this recombinant chou comprises a kind of dna molecular of the present invention and a promotor district, and they are mutual link on function, thereby can make promotor strengthen the transcribing of molecule of the above-mentioned DNA in host cell.The present invention also comprises a system expressing a kind of chitinase gene, and this system comprises a kind of vector and chitinase cDNA that is embedded in this expression vector of expressing.This expression vector preferably includes a plastid, for example intestinal bacteria vector (for example pET 12a).This expresses vector also can be Pichia vector (for example pGAPZ α A).In this expression system, chitinase dna is included in all or part of of dna sequence dna among Figure 21 (a), Figure 21 (b) or Figure 22 (a) or Figure 22 (b) at last.The present invention also comprises by a kind of plant, vegetable cell, animal, zooblast, bacterium and the yeast of this system reform.

In another embodiment, the present invention is a method of expressing chitinase.This method comprises: a kind of expressive host that has a chitinase dna vector is transformed, and cultivated this expressive host.In this method, expressive host is preferably chosen one group of biology that a free kind of plant, vegetable cell, bacterium, yeast, Mycophyta, protozoon, marine alga, animal and zooblast are formed.This expression system is useful especially for following work: production antifreeze protein, enhancing plant and microbe are in plant, animal and microorganism survival ability and the output in the open air under the temperature condition that is lower than zero degrees celsius to tolerance, the enhancing of severe cold; be suppressed at the recrystallization of the ice crystal in biological substance or the foodstuff products; the refrigeration of cell; the low-temperature protection of cell; the cold protection of human body platelet, and the kill tumor cell etc.

The present invention also comprises a kind of nucleotide sequences, and this sequence to be to be target by the protein secreting thing in the plant that target sequence or its complement were constituted of coding strand, as Figure 21 (a), shown in 1 to 60 of the position.This is that the nucleotide sequences of target preferably includes coding strand and complement thereof with the protein secreting thing in the plant, as Figure 22 (a), shown in 1 to 60 of the position.

The freeze proof active method of a kind of enhancing of the present invention, this method comprise antifreeze polypeptide combined, and improving the activity of antifreeze polypeptide, thereby suppresses the recrystallization of ice crystal, and change the normal growth situation of ice crystal.The present invention includes a kind of by the sugared a kind of component that is constituted that combines of one or more antifreeze proteins and one or more.These polypeptide of the present invention can combine use with a kind of sugar; for example can be used for producing antifreeze protein; strengthen the plant, animal, microbe survival ability and the output in the open air that are under the temperature condition that is lower than zero degrees celsius; the recrystallization that suppresses the ice crystal in biological substance or the foodstuff products; the very low temperature refrigeration of cell; the cryoprotection of cell, the cold protection of human body platelet, and kill tumor cell.

These polypeptide of the present invention can be used for being suppressed at certain kind of plant, certain frozen product or any refrigeration biological substance, the generation and the development of the disease that causes by certain low temperature pathogenic agent, and damage.The present invention also comprises a method of isolating antifreeze protein from vegetable material or recombinant chou expression system, this method comprises the extract of solubility to minimum about 60 degrees centigrade temperature, the protein and the solubility material of sex change are removed in centrifuge dehydration or filtration then.The invention still further relates to a kind of cDNA of a kind of separation that give from a kind of monocotyledonous antifreeze polypeptide coding.This monocotyledons is preferably chosen from Gramineae or Triticum plant (preferably choosing the winter naked barley).This cDNA preferably returns a signal sequence encoding of polypeptide transposition.The invention still further relates to separating chitinase nucleic acid molecules encoding in the cold inductive of certain monocotyledonous process of Gramineae or Triticum.The invention still further relates to the polypeptide that generates from nucleic acid molecule.The invention still further relates to the purposes of these nucleoproteins, its usage with in this patent application to the winter naked barley identical with the description of other antifreeze polypeptide.

The present invention includes a kind of separating, comprise a kind ofly from such one group of nucleotide sequences that material is chosen.This group material comprises: as Figure 21 or a kind of nucleic acid molecule shown in Figure 22, or its complement; A nucleotide sequences of under the condition of medium strictness or highly strictness, hybridizing with the nucleotide sequences of above-mentioned (a), for example condition described in this patent application, and this sequence is given a kind of antifreeze polypeptide coding; A kind of antifreeze nucleic acid molecule, have with the nucleotide sequences of above-mentioned (a) and to be at least 40% respectively, to be at least 60%, to be at least 80%, to be at least 90%, to be at least 95%, 97%, 98% or 99% sequence identity, and this antifreeze nucleic acid molecule is given a kind of antifreeze polypeptide coding; A kind ofly as the nucleotide sequences of (a), give same amino acid sequences encoded nucleotide sequences; And a kind ofly as the nucleotide sequences of (b), give same amino acid sequences encoded nucleotide sequences.

The invention still further relates to a kind of nucleic acid molecule of separating, this molecule can be under the condition of medium strictness or highly strictness, with a kind of nucleotide sequences hybridization in the monocotyledons, this sequence is given has freeze proof active certain peptide coding, and in the middle of this, dna molecular can be under the condition of medium strictness or highly strictness, with Figure 21 or a kind of nucleotide sequences hybridization shown in Figure 22.

The condition of " medium strictness " is used to example 25,25.1 and 25.2.6.And the condition of certain " highly strict " also can be used, and for example, in routine 25.2.2, by temperature being increased to 55 degrees centigrade, and uses the gene application specific probe that is used for CHT 46 described in example 26.1.Stringent condition (0.2 * the SSC of normal use, 0.1% SDS, 30 minutes, 42 degrees centigrade), just we propose the sort of condition of claim for our hybridization probe, at people such as F.Ausubel work, and the 25th page of " the concise and to the point agreement in the molecular biology " book that nineteen ninety-five is published in nineteen ninety-five by John Wiley and Sons publishing company, in the 4th chapter, be defined as medium stringent condition.

The invention still further relates to a kind of from winter the naked barley CH 9 or CH 46 nucleic acid molecule and a kind of expression vector that comprises nucleic acid molecule that duplicate out, and a kind of promotor of controlling developed by molecule.The present invention also comprises the expression product of nucleic acid molecule.The gene that the invention still further relates to a kind of antifreeze nucleic acid molecule or duplicate out from a kind of Gramineae monocotyledons (preferably adopting the winter naked barley).

The invention still further relates in a sample, detect CH 46, CH9 or a kind of a kind of method of giving the existence of antifreeze protein nucleic acid molecules encoding by following step: A) among Figure 21 or Figure 22 nucleotide sequence in select a kind of probe; B) this probe and biological sample hybridization; And C) detect by a kind of hybridization complex that antifreeze making nucleic acid molecular hybridization generated in probe and CH 46, CH 9 or the sample, in the middle of this, the existence of complex body indicates and have CH 46, CH 9 or antifreeze nucleic acid molecule in sample.This nucleic acid can be DNA (thymus nucleic acid) or RNA (Yeast Nucleic Acid).This probe is at least 75% nucleotide sequences supplying the molecule of 10 adjacency that exist in a kind of probe disclosed in this patent application preferably.The sequence that probe described in this patent application is supplied also is of great use.

The invention still further relates to a kind of plant that can tolerate cold, in its gene group, embedded a recombinant chou that comprises following content successively, double chain DNA molecule:

A) promotor district, its effect in plant is to cause producing a RNA sequence, this sequence on function with B) link;

B) can cause producing a kind of dna sequence dna of RNA sequence, this RNA sequence can be given a kind of antifreeze polypeptide coding of the sequence that has in Figure 21 or Figure 22, and this peptide species is a kind of variant, or the polypeptide of equivalence on biological function.

In another patented technology embodiment of the present invention, dna sequence dna on function with at least with a signal sequence and a untranslated sequence link.Promotor is preferably with respect to the dna sequence dna xenogeneic, and can cause the expression of enough antifreeze proteins in the cell or tissue of plant, to strengthen with certain kind of plant of this genetic modification tolerance to cold.

Nucleic acid molecule of the present invention can be used to transform a kind of host cell by a method, is transformed through the vector that adopts a kind of expression to comprise nucleic acid molecule, and this host cell can produce antifreeze polypeptide.The invention still further relates to one and can be used for by utilizing a kind of a kind of nucleic acid molecule of the present invention that comprises, and expression is transformed the content (overexpression antifreeze polypeptide) that certain host cell changes the antifreeze polypeptide in certain cell by the polypeptide expression vector of nucleic acid molecule.This expression vector preferably includes a kind of antifreeze nucleic acid molecule and a promotor district, and they are to link mutually on function, thereby make promotor can strengthen described dna molecular transcribing in host cell.The present invention also comprises a kind of method that is used to produce a kind of plant that process is transformed on genetics, this expression of plants antifreeze protein.This method comprises following several steps: 1) a recombinant chou, double chain DNA molecule embeds in a kind of genome of host plant cell, this double chain DNA molecule contains a kind of promotor, and its function is to strengthen transcribing of an adjacent dna encoding sequence in vegetable cell; 2) an antifreeze dna molecular of the present invention is linked to this promotor, from this host plant cell, bears a kind of plant that process is transformed on genetics more then.This dna molecular preferably includes an independently dna molecular, it is CH 9 or CH 46, or as Figure 21 or a kind of dna molecular with CH 9 or CH 46 nucleotide sequences shown in Figure 22, a kind of variant, or under medium stringent condition or height stringent condition, the molecule of equivalence on biological function.The present invention also comprises the plant of using this method to produce.

The invention still further relates to a kind of method that changes the content of the antifreeze polypeptide in certain kind of plant.This method comprises the steps:

DNA is delivered to a vegetable cell, plant regenerates from here on, wherein, DNA comprises an independently dna molecular, and it is CH 9 or CH 46, perhaps with shown in Figure 21 or 22, has the hybridization of CH 9 or CH 46 nucleotide sequences, a kind of variant, or the molecule of equivalence on biological function under the condition of medium strictness or highly strictness, in the middle of this, this DNA gives an antifreeze polypeptide coding;

Then from this vegetable cell this kind of plant of regenerating, so that allow this DNA of this expression of plants.

In one of this method preferential specific embodiments of selecting, this DNA is a kind of nucleic acid molecule, separate from a kind of monocotyledons gramineous, preferably the winter naked barley.This molecule also can separate from certain dicotyledons.This preferred DNA has changed the content of endogenous antifreeze polypeptide, thereby makes the ability of tolerance cold of this kind of plant with respect to the normal circumstances of this plant species change take place.

The present invention also comprises a kind of by utilizing nucleic acid molecule of the present invention and transforming certain cell or plant by expressing gene, allows cell or plant obtain the method for the ability (preferably resisting the ability of fungal disease) of opposing low temperature disease.This method also comprises uses a kind of component, and this component comprises polypeptide of the present invention and one can received carrier on agricultural.Various chitinases, dextranase and West Africa arrowroot essence all are antimycotic protein.Each peptide species of the present invention shows the activity of chitinase, dextranase and West Africa arrowroot essence.Even when polypeptide obtained freeze proof activity, this activity still existed.It is to be caused by microorganism that can normal growth in low temperature environment that numerous disease is arranged.Freezing can only delaying, and can not stop microbial growth, and any microorganism in food or biological substance can grow between frost free period fast.

The invention still further relates to a kind of for plant provide the component of the cold ability of tolerance, this component comprise antifreeze polypeptide and a kind of can received carrier on agricultural.The present invention also comprises a kind of component that is suitable for adding in the foodstuff products, comprises a kind of antifreeze polypeptide and a kind of carrier that is suitable for human consumption.Detailed description of the invention

Antifreeze protein has a lot of interesting property, comprises constraint and inhibition to ice-crystal growth, and (more detailed description is seen III-21, III-17) to the protection of liposome, protein, cytolemma and cell by the recrystallize that suppresses ice.Therefore, these protein will be widely used in foodstuffs industry, improve under freezing state grain edible or that store with freezing mode, vegetables, fruit, meat and a lot of its shelf-livess of quality, quality and increase of other food by the recrystallize that suppresses ice, reduce the loss that causes by microorganism.In addition, these protein can be used for the low temperature storage of cell, film, ovocyte, tissue (III-4), and the low-temperature protection of cell and tissue (III-44).Antifreeze protein (AFP) also has some up-to-date purposes, comprises the hematoblastic cold protections of the mankind (III-49).What more attract people's attention is that the characteristic that AFP regulates ice-crystal growth has other biomedical uses, can kill for example cancer cells of some cell.In transgenic plant, when expression of plants AFP, these AFP can improve the freezing tolerance of plant and strengthen the resistibility of plant to the low temperature pathogenic agent.Plant is to the tolerance of cold

The unusual protein relevant with plant winter hardiness that we find has three classes:

I) ice crystal becomes nucleoprotein

Ii) antifreeze protein (AFP)

Iii) plant and fungal cell wall are carried out the polypeptide of enzyme modification

In the process of moisture freezes, if allow water to freeze under in check condition, the moisture in the plant tissue just passes cell walls and enters the intercellular space, causes the formation of intercellular ice crystal in plant materials.The proteic effect of ice crystal nucleation is to cause ice crystal to form in extracellular plant tissue intercellular space.AFP is present in the extracellular equally, the growth in the intercellular space with constraint and regulation and control ice crystal, thus stop breaking of cytolemma.The enzyme that is present in the extracellular space works to modify cell wall substance, increases the elasticity of cell wall substance, is viability when freezing and thaw to strengthen plant tissue.These enzymes cell wall substance of low temperature pathogenic agent of can also degrading, thus some resistibility to the low temperature pathogenic agent is provided.

It is believed that plant is resulted from the body by vegetable cell with freeze proof proteins associated matter in the process that is exposed to the cryogenic freezing environment, see through plasmalemma and be secreted into generation effect in the intercellular space, the formation of regulation and control ice crystal.Anti-ly freeze proteic formation and can be understood as and comprise one or more specific polypeptide.Many in these polypeptide are bound up, and proteinic a kind of ad hoc structure is provided, and make it to have the ice crystal nucleation, one or more characteristics in the significant anti-frost characteristic such as anti-freeze or enzyme effect.

We find, warm environment as 20 ℃ under, vegetable cell produces and anti-ability drop of freezing relevant polypeptide.When plant is in following time of frost weather condition in early spring or late fall, the generation of these polypeptide just significantly increases.As far as we know, the initial discovery in plant tissue of Here it is frost resistance induces polypeptide.Found in the intercellular space of plant that by us this angle of these polypeptide sets out, we have extracted these polypeptide (I-16 and II-12) in these positions.In general, adopt two following step extraction methods:

I) leaf with plant shreds or shreds, and carries out vacuum extraction with the buffer reagent that preferably contains 20mM calcium chloride and 10mM ascorbate salt

Ii) extract extracts from plant tissue, and cell is not damaged

The extracting solution that obtains has ice crystal and becomes nuclear activity, dextranase activity and chitinase activity and anti-freeze activity.Ice crystal becomes nuclear activity to be more suitable for freezing to test with droplet measuring.In the example of discussing, when adding resembled the such sulfydryl reductive agent of dithiothreitol (DTT) and mercaptoethanol, ice crystal became nuclear activity just to reduce.The freeze proof activity of extracting solution is determined by the ice crystal forming process that observation is installed in the freezing stage on the opticmicroscope.Such structure is similar with the antifreeze protein matter of other type of extracting (II-4) from other source as extra large snakeheaded fish.Experiment finds that when adding proteolytic enzyme, the freeze proof activity of extracting solution disappears, thereby has confirmed the existence of AFP.Dextranase activity is by the measurement of enzyme release glucose Equivalent from solubility laminarin (poly β-1,3-glucose) is measured.Dextranase is more active in the presence of calcium.The chitinase activity is by (I-10.1) measured in (external source chitinase) and enzymatic discharges glucosamine in (endogenous chitinase) from the chitin oligomer measurement from tobacco brown spot pathogen.

Be appreciated that in order from the intercellular space, to extract extract and do not destroy vegetable cell, can take different isolation technique.These technology are included in the centrifuge tube places a funnel or cone-shaped body makes the leaf vertical orientation to avoid the bending of blade.Then that blade is centrifugal, thus under the situation of not destroying cell, obtain extract.Other technology can be used for the extraction of polypeptide.For example, can come the extracting blade by pouring into suitable extracted solution.When plant tissue homogenized, extracellular polypeptide was water miscible, will be distributed in the whole soluble component.

When freezing generation, cause that all be useful to the polypeptide of frost tolerance to the plant of any kind.Any plant can be used or itself contains these polypeptide by different modes, and these polypeptide can be regulated the growth of ice crystal.If plant contains these albumen, just can be in cold or freezing temperature the exposure of tolerance longer time, just can in harsher weather, survive, just can be arranged the longer season of growth because the frost of spring and autumn to they to influence meeting littler.

In addition, indivedual proteic uses are that the plant that is exposed in the sub-zero zero temperature has been selected another kind of survival mechanism.For example, some plant avoids frostbite by stoping the formation of ice fully.This supercooled strategy requires not exist ice crystal nucleus former, perhaps has the former material of passivation ice crystal nucleus.AFP shows and suppresses the characteristic (II-13) that ice crystal nucleus forms.Therefore, any plant tissue can be tamed generation or give AFP from the outside by different modes, forms and promote coldization of mistake of plant sap to stop ice crystal nucleus.

Just as described, tolerance proteins associated matter provided by the invention and freezing serves many purposes.Important purposes is exactly to be used to detect the characteristic of plant aspect freeze proof.At these polypeptide, prepared multiple antibody.Rely on these antibody, can determine the existence of similar polypeptide in other plant, produce the ability of the cold AFP of inducing, ice crystal nucleus and enzyme to judge them, and measure the power of their anti-freezing abilities by the method for immunoassay.Be appreciated that equally can the be encoded genetic information of aforementioned polypeptides of plant transforms, thereby in the plant of other type, improve or provide anti-frost ability.Polypeptide among the present invention can be applicable to the batch production operation equally, but necessary practical appropriate carriers is to quicken the absorption to these polypeptide of plant, fruit, grain or other biomaterial.AFP must be present in the aqueous solution, with the formation of influence ice.For promoting to absorb, we use some physical methods, as vacuum extraction, perfusion, mixing, stirring, and the immersion under grinding or non-grinding situation.In the plantation of vegetables, fruit, when surrounding temperature is plummeted to sub-zero zero, can use these polypeptide with the mode of spraying, with the destruction that prevents that frost from causing.In addition, these polypeptide are applied in the low temperature storage of biological tissue.These polypeptide also are widely used in the quality of all frozen product, particularly ice-creams and other food edible under freezing state.The application of these polypeptide makes the structure of ice crystal become small and reduce recrystallize, thereby produces higher-quality product.In principle, as long as where need to suppress the recrystallize of ice crystal, the polypeptide among the present invention just can be applied.This inhibition to recrystallize has kept the small of crystal ice granule, has prevented the damage of pair cell wall and film, thereby has kept the existence of refrigeration cell and tissue.By following going through and illustration, the present invention is further understood.

Embodiment 1: the extraction of extracellular protein and quantification

With winter naked barley (Secale cereale L.cv.Musketeer) planting seed in the 15cm plastics pot that thick vermiculite is housed, 20: 16 ℃ (daytime: night) germinate a week down, during the length in daytime of every day be 16 hours.Plant is transferred to 5: 2 ℃, and (daytime: environment night), illumination system are 16: 8 hours (daytimes: night), be called cold domestication naked barley (RH) through the plant of this processing.(daytime: night) under the environment, but the illumination system is 8: 16 hours (daytimes: night), be called short type in daytime cold domestication naked barley (RH-SD) through the plant of this processing in 5: 2 ℃ in plant-growth.Remaining plastics pot is stayed three weeks of regrowth in 20 ℃ the growth room, in contrast, or claims non-domestication naked barley (RNH).(daytime: night) (8 hour daytime: 16 hour night) growth four days are transferred in 20 ℃ of growth rooms in growth whole seven weeks to naked barley then under the condition, and this process is called takes off domestication (Deacc) at 5: 2 ℃.All plant are watered with modified Hoagland nutritive medium (Huner and Macdowall prescription are seen 1-6).

From RNH, RH extracts extracellular protein in RH-SD and the Deacc plant respectively.In each the extraction, all use 5mM EDTA, the 10mM xitix, the 10mM mercaptoethanol, 1mM phenyl methyl sulfonic acid fluoride, 2mM caproic acid and 2mM benzenyl amidine are equipped with the outer extracting solution of born of the same parents with the vacuum extraction legal system.The program of this vacuum extraction is to carry out according to the description of Mauch and Staehelin (I-16).Treated blade is put into centrifuge tube after vertically filling in a funnel, to avoid the bending of blade.Centrifugal with the outer extract of separation born of the same parents, and be collected in the centrifuge tube.This extracting solution is to obtain under the situation of not destroying leaf cell.

From RNH, RH extracts extracellular protein in RH-SD and the Deacc blade respectively, as outlining in the preamble " proteins extraction ".The protein concn of extracting solution is standard test with the Bio-Rad method with BSA, does four times during mensuration at least and independently repeats.The proteinic amount of extracting from the intercellular space is different because of the difference of plant growing condition.0.034 milligram of albumen of extracellular protein content average out to/gram fresh weight in the non-naturalized plant blade.Under 5 ℃ of conditions respectively at 16 hours and 8 hour daytime long under the blade of growth, its extracellular protein content is 0.149 milligram of albumen of average out to/gram fresh weight and 0.307 milligram of albumen/gram fresh weight respectively.As seen, the naked barley that low temperature and short daytime grow under the condition, its extracellular protein content has increased by nine times.And when they are transferred to when taming under 20 ℃ of conditions, these protein levels descend (Fig. 1).Embodiment 2: the electrophoresis of extracellular protein

For as Fig. 2, Fig. 3 and Fig. 4 result displayed, be electrophoretic needs, add the methanol solution of 1% acetate of 1.5 times of amounts in the outer extracting solution of born of the same parents ,-20 ℃ of following incubated overnight, so that extracellular protein precipitates the extracting solution outside born of the same parents.Protein precipitation washs down at 5 ℃ with 100% ethanol and 70% ethanol respectively, and is dry in moisture eliminator then.Protein Laemmli (I-10) sample buffer [60mM Tris-HCl, pH6.8; 10% glycerine; 2% sodium laurylsulfonate (SDS); 5% mercaptoethanol] resuspending, carry out electrophoretic separation with Biorad dye-free standard, adopt 10% acrylamide gel, to spacer gel 90V voltage, to separation gel 110V voltage (I-10).With Coomassie blue to gel-colored.For the result that Fig. 9 shows, the protein that is present in the post component of the outer extract of born of the same parents directly is dissolved in Laemmli sample buffer (I-10), carries out electrophoretic separation with Biorad pre-staining standard, adopts 13.5% acrylamide gel, 200V voltage.Gel dyes with ammonia silver.

Between dissimilar leaf, the protein form in the outer extracting solution of born of the same parents shows significant difference.SDS-PAGE (10% acrylamide gel) is presented in the outer extracting solution of born of the same parents and has 12 peptide species at least.Wherein the molecular weight of two peptide species is respectively 77 and 73kD, is dyed redness by Coomassie blue, and they only find (Fig. 2 in the leaf of cold domestication; The the 3rd and the 4th road).In the leaf of domestication, molecular weight be respectively 36,33,30,25,21,15,14 and the polypeptide of 13kD be detected the increase (Fig. 2) of content.Molecular weight be respectively 23 and two peptide species of 20kD only this just detects increase at the leaf of short cold domestication in daytime.Embodiment 3: polypeptide gathers between cold domestication

For further studying the characteristic of polypeptide in the intercellular space, carried out the time-histories experiment, its objective is the appearance of these polypeptide of understanding and the relation between the freezing tolerance development.Detect and find, in non-domestication leaf, the content of most of intercellular polypeptide very low (Fig. 3, the 2nd road).At the 35th, 50,57 and 78 day of cold domestication, be measured to the lasting accumulation (Fig. 3, the 3rd, 4,5 and the 6th road) of polypeptide.At the 78th day, the freezing tolerance of cold domestication in short daytime naked barley leaf reached maximum (LT50=-30 ℃), showed the outer content of peptides of the highest born of the same parents simultaneously and the novel polypeptide that a kind of molecular weight is 109kD occurred.After 102 days, the polypeptide of 109kD, 77kD and 73kD has not existed in cold domestication, and the freezing tolerance of leaf reduces simultaneously, and supposition may be because the vernalized cause of plant (Fig. 3, the 7th road).This discovery shows, most existence in the outer polypeptide of these 13 kinds of born of the same parents is relevant with freezing tolerance and the vernalization of plant.In taking off the domestication process, the plant that is about to cold domestication is transferred to the time period of experience different lengths in 20 ℃ the environment from the harshest environment, also detects the form of extracellular protein simultaneously.As shown in Figure 4, the content of most of polypeptide in 12 peptide species (the 2nd road, the cold domestication of top) reduces after this 4 days take off in the domestication (the 3rd road) greatly, and takes off after this 6 days and 8 days that (the 4th and the 5th road) continues reduction in the domestication.A hurdle of left hand is the molecular weight graduated scale among the figure, can estimate the molecular weight of polypeptide in the 1st road by this.Molecular weight graduated scale between the 1st road and the 2nd road believes it is more accurate.Embodiment 4: the ice crystal of the outer polypeptide of born of the same parents becomes nuclear activity

With the method by Amicon corpusculum (I-13) ultrafiltration the extracellular protein of leaf is concentrated ten times.In given temperature range, concentrated solution is determined the spectrum of active ice crystal nucleus with the droplet freezing technology.Between pool period (Fig. 5), the outer extracting solution of born of the same parents of cold domestication naked barley leaf is at-9 ℃ of short photoperiodic nucleation ice crystals of growing down, at 10 ℃ of long photoperiodic nucleation ice crystals of next growth.Non-domestication and take off the domestication leaf extracting solution then under minimum temperature (13 ℃) just begin to freeze.The leaf extract ice crystal nucleation of non-domestication and cold domestication active different (Fig. 5) may be because kept higher protein content (Fig. 1) in the intercellular space of domestication leaf.Method with ultrafiltration obtains the non-domestication of same protein content and the influence that cold domestication leaf extract is measured protein concn.When becoming nuclear activity to measure and calculate with the droplet freezing method to the ice crystal of two kinds of extracting solutions, the ice crystal nucleus cumulative number of finding every gram fresh weight in cold domestication leaf is the remarkable increase (p=0.01) on the statistics.Under-15 ℃, (mean standard deviation is 2269 ± 292 n=4) to the ice crystal check figure of every gram fresh weight, and the ice crystal check figure of every gram fresh weight is 7048 ± 917 in the outer extracting solution of born of the same parents of cold domestication in long daytime naked barley leaf in the outer extracting solution of the born of the same parents of non-domestication leaf.Low ice crystal nucleus forms starting temperature and shows, the ice crystal releaser that is present in the extracting solution is not that complete ice crystal forms point.Embodiment 5: the freeze proof activity of the outer extracting solution of born of the same parents

The outer extract of born of the same parents that the technology of mentioning with preamble prepares is estimated in the activity of freeze proof invention.Freeze proof active mensuration, be by with receive that the form that rises the ice crystal that forms in the osmometer observation extracting solution realizes (Clifton Technical Physics, Hartford, N.Y., U.S.A, II-3, II-5).In pure water, the normal growth of ice crystal parallel with the basal plane of lattice (a-direction of principal axis) and seldom perpendicular to basal plane (c-direction of principal axis) growth, so the outward appearance of ice crystal is flat and justify (II-5) (Fig. 6 A).On the contrary, the AFP of lower concentration (nM) preferentially suppresses ice crystal along the axial growth of a-, thereby ice crystal presents six prismatic planes (II-6) (Fig. 6 G).In the AFP of greater concn, ice crystal mainly along the growth of c-direction of principal axis, forms double hexcone shape (II-6) (Fig. 8 C).

In this experiment, the freezing of non-domestication naked barley leaf extract as distilled water, that is, only observe the ice crystal (Fig. 6 B) of Bao Eryuan.On the contrary, all from cold domestication winter naked barley leaf the intercellular space the crude extract that extracts when freezing, all produce hexagonal ice crystal (Fig. 6 C to 6G).When temperature reduced, ice crystal at first along the expansion of c-axle, formed incomplete double hexcone shape (Fig. 6 D), along the growth of a-axle, formed six prisms (Fig. 6 D) and bigger hexagon ice crystal (Fig. 6 E to 6G) then.The formation of six prismatic ice crystals and ice crystal be along the growth of c-axle, shows in the crude extract of these naked barley to have freeze proof activity (II-3,5) in winter.In addition, extracting solution adds 5% (w/v) Streptomyces griseus proteolytic enzyme (Sigma Chemical Co. outside cold domestication naked barley leaf born of the same parents, St.Louis, MO, U.S.A.) hatched one hour down at 22 ℃, extracting solution just disappears to the influence of ice crystal form, and the freeze proof activity of this statement of facts is relevant with protein.Embodiment 6: extracellular protein is to the inhibition of recrystallize

One of AFP effect in frost resistance plant and organism is the recrystallize (II-10) that stops ice.Although ice is to form little ice crystal at first, yet if there is not the existence of AFP, As time goes on, these ice crystals can merge and form big ice crystal, thereby biological tissue is caused physical abuse.With " strip experiment " recrystallize is measured.The outer extracting solution of a small amount of born of the same parents is added drop-wise on-20 ℃ the surface, forms the little ice crystal of skim.Then this strip was annealed 6 hours down at-8 ℃.The size of observing annealing back ice crystal with opticmicroscope and polarized light, and the outer extract of born of the same parents of the present invention contrasted with distilled water, the effect (Figure 13) of the recrystallize of icing in the inhibition water whether had to determine extract.Ice crystal in all dilution extracting solutions is all obviously little than the observed ice crystal in pure water annealing back.Extracting solution is by 1: 10 outside born of the same parents, and 000 dilution promptly is diluted to every liter when containing about 28 micrograms of protein, still shows significant inhibition recrystallization.Embodiment 7: proteinic fractional separation in the outer extracting solution of born of the same parents

The outer extracting solution of born of the same parents is concentrated five times, exchange to the NH of 50mM with ultrafiltration ₄HCO ₃(Centriprep-10, Amicon Canada Ltd., Oakville, ON, Canada) in, and with 50mM NH ₄HCO ₃Be medium, (Sweden) post (0.5 * 32cm) for Pharmacial LKBBiotechnology, Uppsala to adopt Sephacryi 200.Elutant is monitored down at 280nm (O--O) and 230nm (v-v) with ultraviolet absorption method.Standard protein is washed out respectively to estimate proteinic size.Ferritin, 440kD washes out at 9.5ml; Zymohexase, 158kD washes out at 11.5ml; Bovine serum albumin, 67kD washes out at 13.5ml; And trypsinogen, 24kD washes out at 16.5ml.As shown in Figure 7, monitor four tangible macromole peak: 305kD (peak 1), 5kD (peak 2), 2kD (peak 3) and＜1kD (peak 4).When the anti-freeze characteristic of each component of test, have only component formation hexagon and the bipyramid shape ice crystal relevant with peak 2.

As shown in Figure 7, with SDS-PAGE (13.5% acrylamide, silver dyeing gel, as shown in Figure 9) to 280nm under the post at each peak divide relevant polypeptide to estimate.The 1st road comprises prestained standard macromole; The 2nd road comprises the outer extract of primitive unit cell; The 3rd road is included in the polypeptide (peak 1) that 8ml washes out; The 4th road is included in the polypeptide (shoulder at peak 2) that 18ml washes out; The 5th road is included in the polypeptide (peak 2) that 22ml washes out; The 6th road is included in the polypeptide (shoulder at peak 2) that 26ml washes out; The 7th road is included in the polypeptide (peak 3) that 31ml washes out; The 8th road is included in the polypeptide (peak 4) that 35ml washes out.The 2 post branches (Fig. 7) that obtain comprise several main polypeptide from the peak, and its size is (Fig. 9, the 4th road, and Table I and Table II) from 5kD to 36kD.This post branch has the anti-freeze activity.Embodiment 8: the ice crystal of the outer polypeptide of classified isolating born of the same parents becomes nuclear activity

Protein is after the Sephacryl post washes out, and 1 monitors out low ice crystal with peak 4 and becomes nuclear activity from the peak, as shown in Figure 4.Peak 3 then demonstrates higher ice crystal and becomes nuclear activity.SDS-PAGE isolates two kinds of molecular weight and is respectively 60 and the polypeptide of 68kD.Ice crystal become nucleoprotein can be both one of, also can be both combinations.This two peptide species is identified as the polypeptide of 77kD and 73kD respectively in Fig. 2, Fig. 3 and Fig. 4 because 60 and the 68kD polypeptide by Coomassie blue stain.Embodiment 9: the thermal hysteresis of the outer polypeptide of classified isolating born of the same parents

The growth non-colligative property ground that AFP passes through to stop the formation of ice crystal and suppress ice crystal reduces the freezing point of solution, and these protein then only depend on according to counting effect (II-5) the change of fusing point.This thermal hysteresis (phenomenon that freezing point and fusing point are inequality) is measured the influence of single ice-crystal growth by observing temperature.Being dissolved in ice crystal change bowlder begins to take place; Be frozen in ice crystal (II-5) takes place when the c-axle extends.

Be the existence of proof thermal hysteresis, we absorb 280nm and have freeze proof active post branch (peak 2) and merge, and dissolving again in distilled water is used to observe thermal hysteresis after the freeze-drying.Under this higher protein concn, be suppressed (Fig. 8 B to 8D) along the axial ice-crystal growth of a-.In addition, five ice crystals are arranged along the c-direction of principal axis, and its average freezing temperature is-1.10 ℃.Their average melting temperature (Tm) is-0.78 ℃, thus the thermal hysteresis temperature be measured as 0.33 ± 0.06 ℃ (average ± standard deviation, n=5).Therefore, the naked barley leaf has produced AFP the winter.This (these) AFP has the normal mode of modification ice-crystal growth and the ability that non-colligative property ground reduces freezing point of solution.The thermal hysteresis effect that winter naked barley AFP shows than the AFP that from the fish of polar region, finds (about 0.6 ℃, II-7) with insect this find AFP (5 ℃, II-7, II-8) little.This may be because winter naked barley AFP does not have complete purifying, or because the difference on structure and the function.Embodiment 10: the freeze proof activity of at least 11 peptide species in the outer polypeptide of born of the same parents

Use 20mM CaCl ₂From winter naked barley leaf, extract polypeptide outside the born of the same parents with the 10mM xitix, separate with the SDS-polyacrylamide gel electrophoresis, use the Tris-tricine buffer system and big (16 * 18 * 0.15cm) 12% acrylamide gels of no reductive agent (not containing dithiothreitol (DTT)) during electrophoretic separation.Insulation is after 10 minutes in the 0.25M KCl of ice temperature, and polypeptide develops the color in gel.Gel cuts each wave band after the distillation washing, with 0.1%SDS and 50mM Tris-HCl polypeptide is washed out from gel.Polypeptide is separated out from elution buffer at-20 ℃ with 80% acetone then, precipitation is also air-dry.With polypeptide 0.1M NH ₄HCO ₃Dissolve again, measure the freeze proof activity of each peptide species respectively with the method for observing the ice crystal metamorphosis.As shown in figure 10,8 peptide species are arranged, molecular weight can change the ice-crystal growth of normal type and forms the hexagon ice crystal from 111kD to 161kD.In fact the polypeptide of 93kD represents one group to have freeze proof active polypeptide, and they are separated into one group that molecular weight does not wait from 93kD to 99kD in the Tris-tricine gel systems.These polypeptide are dyed red-violet colour according to it by Xylene Brilliant Cyanine G and are discerned.In the experiment, polypeptide separates with 12.5% glue or gradient glue with the Tris-glycine buffer system in early days, so compares different in the performance of the size of these polypeptide in native system and in early days the gel.Embodiment 11: the analysis amino acid contained to AFP

To having the aminoacid component analysis revealed of freeze proof active polypeptide, they are rich in glycine, l-asparagine or aspartic acid, L-Ala, glutamine or L-glutamic acid relatively, and Serine (seeing Table the displaying of III), but do not contain hexosamine (monitoring condition: amino acid analysis is carried out in every peptide species 15 picomole hydrolysis after 4 hours) to all polypeptide.The high lactamine content characteristic (II-10) of showing antifreeze glycoprotein and I type AFP without any polypeptide.On the contrary, the naked barley polypeptide demonstrates high hydrophile amino acid content, as detected in extra large snakeheaded fish and Atlantic salmon (II-4); Simultaneously also has high-load glycine, as detected in some insect AFP (II-7, Table III).Embodiment 12: with immunoabsorption the class chitinase protein in the outer extracting solution of born of the same parents is identified

Separate the outer polypeptide of born of the same parents with SDS-PAGE, and with its electro-adsorption to nitrocotton.The primary antibody probe of trace to chitinase.Primary antibody source: Dr.Michel Legrand, Laboratoire de Virologie, Institut de Biologie Moleculaire et Celluire de laRecherche Scientifique, 15, rue Descartes, 67000 Strasbourg, France (I-10.1).Needs for developing carry out probe with secondary antibodies (anti-rabbit 1gG antibody combines with alkaline phosphatase) to trace.The result shows, two kinds of molecular weight be respectively 27 and the polypeptide of 32kD have and the similar epitope of chitinase (Figure 11).The content of 27kD polypeptide in cold domestication leaf is higher than non-domestication leaf, and the 32kD polypeptide is then produced by low temperature induction.Secondary immunosorption as shown in figure 12.Each road among the figure is represented respectively with polypeptide outside the winter naked barley born of the same parents after 5 ℃ were grown for 2,5,6,7,8 and 9 weeks down of chitinase antibody probe.The 27kD chitinase exists not obvious in the leaf in 2 ages in week, but continues accumulation in the time in whole 9 weeks.The 32kD chitinase is only when plant just obviously existence after 7 weeks of growth under 5 ℃.To the 9th week, two kinds of chitinases all (32 and 31kD, 27 and 26kD) occur with dual road.Embodiment 13: the outer polypeptide role in the freezing tolerance of naked barley of born of the same parents

5 ℃ and 8 hour daytime long under the winter naked barley leaf 20mM CaCl of cold domestication ₂With the extracting of 10mM xitix, to reduce the protein concn in the apoplast.Non-domestication leaf, cold domestication leaf and be cut into the long fragment of 2.5cm respectively through extractive cold domestication leaf, and with the abundant rinsing of distilled water.Each part in three duplicate samples all put into broken blade 50 test tubes that the 4mL hplc grade water is housed.These test tubes are placed ice bath, temperature is reduced by 1 ℃ every 22 minutes.Under each temperature, all freezing sample is moved and places on ice and slowly thaw.Then sample is placed the conductivity of room temperature and working sample.Sample is boiled to remove all interior ions, be cooled to room temperature, measure the conductivity of solution once more.The result as shown in figure 14.Extracting to extracellular protein causes cold domestication leaf fatal frostbite to occur at-11 ℃.If extracellular protein is not by extracting, ice crystal is when-1 ℃ of following nucleation, and cold domestication leaf arrives still can normal existence under-30 ℃ of low like this temperature.Even allow without extractive cold domestication leaf naturally frozen, they can not freezed to death under the temperature more than-13 ℃.This shows that the existence of extracellular protein has alleviated the freezing injury that brings really.The n terminal amino acid sequential analysis of embodiment 14:AFP proves that AFP and pathogenesis-related protein are similar

To three kinds in seven kinds of main polypeptide shown in Fig. 9 the 4th road mensuration of having carried out the partial amino-acid order.The N-that wherein has freeze proof active 9kD polypeptide holds preceding 20 amino acid to be checked order:

NH ₂-ALA-ILE-PHE-CYS-GLY-GLN-VAL-ASN-PRO-ALA-LEU-GLY-

PRO-PRO-ILE-TYR-PRO-ALA-PHE-GLY-

Preceding 16 amino acid whose orders of 11kD polypeptide are as follows:

NH ₂-ARG-SER-PHE-SER-ILE-THR-ASN-ARG-CYS-TRP-SER-PHE-

THR-VAL-PRO-GLY-

Wherein preceding 11 amino acid show that the transfer protein (list in the filename MUSHCK and TVMSHC of Protein Information Resource under) relevant with a kind of kinases has 55% homology.

The proteinic N-of 30kD holds the sequence of preceding 30 residues as follows:

NH ₂-ILE-GLY-VAL-CYS-TYR-GLY-VAL-ILE-GLY-ASN-ASN-LEU-

PRO-SER-ARG-SER-ASP-VAL-VAL-GLN-LEU-TYR-ARG-SER-GLY-

X-ILE-ASN-X-MET-

Wherein X represents unknown amino-acid residue.

The protein listed in this order and the supercomputer database of listing in National Cancer Institute is carried out homology relatively.This sequence is with in the past from barley in the dextran of purifying-1, and 3-beta-glucosidase (EC3.2.1.39) has 63% homology.Show as one 31 to 33kD band when 30kD has, be speculated as the precursor of endo-dextranase.These results show, one of cold domestication institute inductive process is to produce the ability of modifying cell walls.The increase of cell walls flowability, for freeze-thawing cycle in the contraction and the expansion after this of cell may be important.This dextranase activity perhaps can also suppress the growth of hypha,hyphae, thereby the resistibility to the low temperature disease is provided.

In addition six kinds show the outer amino acid sequence of polypeptide of freeze proof active born of the same parents and have also obtained determining.These polypeptide separate with SDS-PAGE, adopt the Tris-tricine damping fluid, wash out from gel, carry out freeze proof active mensuration, as shown in figure 10, are used for sequential analysis then.Their N-terminal sequence is as follows:

The 11kD polypeptide:

NH ₂-ALA-ILE-SER-X-GLY-GLU-GLN-VAL-ASN-

SER-ALA-LEU-[GLY]-PRO-X-ILE-[SER]-TYR-

ALA-[ARG]-[GLY]

To disclose a kind of molecular weight in this sequence and the barley be that 20 amino acid overlap of the lipid transfer protein of 9kD has 80% homology (II-14.2) in retrieval to the FASTA of Protein Information Resource.Mentioned through the polypeptide of order-checking corresponding among Figure 10 in the polypeptide of this 11kD and Fig. 9 preamble.Just as mentioned before, the difference of this molecular weight is that mutability by gel electrophoresis causes.

The 15kD polypeptide:

NH ₂-ARG-SER-PHE-SER-ILE-THR-ASN-ARG-X-

ALA-PHE-THR-VAL-X-PRO-ALA-ALA-THR-PRO-

VAL-GLY-GLY-GLY-GLY-GLN

FASTA retrieval to state-run biological medicine research foundation protein information resources bank finds that there is 75% homology one section 24 amino acid whose overlap that this sequence and a kind of class West Africa arrowroot protamine that extracts are reported from Oryza sativa.The polypeptide of 15kD is perhaps corresponding with the 11kD polypeptide among preamble Fig. 9 among this Figure 10.

The 25kD polypeptide:

NH ₂-ALA-THR-ILE-THR-VAL-VAL-ASN-[LYS]-

PHE-SER-TYR-THR-VAL-X-PRO-GLY-ALA-LEU-

PRO-PHE-GLY-GLY-VAL-GLY-LEU-GLY-PRO-GLY

-GLN-

Find that through the FASTA retrieval there is 79% homology one section 29 amino acid whose overlap in the smart homologous protein 1 of this sequence and barley West Africa arrowroot.It simultaneously also with oat in one section 22 amino acid whose overlap among the isolating avematin 77% homology is arranged, with one section 22 amino acid whose overlap among the isolating trimatin in the wheat 82% homology is arranged.Be proved to be with the smart similarly albumen of West Africa arrowroot and have a series of activity, comprise alpha-amylase activity, protease activity and membrane permeation activity (II-8.2).

31kD polypeptide among Figure 10 is corresponding with the 30kD polypeptide among preamble Fig. 9, just has more one section aminoacid sequence:

NH ₂-ILE-GLY-VAL-X-TYR-GLY-VAL-ILE

The 32kD polypeptide:

NH ₂-ILE-GLY-VAL-X-TYR-GLY-VAL-ILE-GLY-

ASN-ASN-LEU-PRO-[SER]-ARG-[SER]-ASP-VAL-

VAL-GLU

The 33kD polypeptide:

NH ₂-ILE-GLY-VAL-X-TYR-GLY-VAL-ILE-GLY-

ASN-ASN-LEU-PRO-SER

Three sequences listed above all with dextran in-1,3-glucuroide (EC3.2.1.39) has significant homology.

The 33kD polypeptide:

NH ₂-GLU-GLN-X-GLY-SER-GLN-ALA-GLY-GLY-

ALA-THR-X-PRO-ASN-ASN-LEU-LEU-

The FASTA retrieval shows that one section 16 amino acid whose overlap has 81% homology in the hevein of this sequence and class rubber tree.This sequence simultaneously also with the endogenous chitinase of from paddy rice and tobacco, separating (EC3.2.1.14) in one section 16 amino acid whose overlap have 88% homology, also with isolating lectin from wheat (claiming different hemagglutinin 11 again) in one section 14 amino acid whose overlap have 92% homology.

Resemble class West Africa arrowroot protamine (TLP), the such protein of dextranase and chitinase all is known and pathogenesis proteins associated matter, because accumulate in their large quantities of plants that grow that are pathogenic agent.We can prove now, class West Africa arrowroot protamine (TLP), and class dextranase albumen (GLP) and class chitinase protein (CLP) produce between the cold domestication of freezing tolerant plants, and represent freeze proof activity.Except that our work, other scientific literature is not seen has report low temperature can induce the protein relevant with pathogenesis to produce, the pathogenesis associated protein of the low temperature induction of not appearing in the newspapers shows freeze proof activity, and the product of the cold temperature induced protein matter of also not appearing in the newspapers or low temperature induction gene represents freeze proof activity.Embodiment 15: the dextranase and the chitinase activity of the outer extracting solution of born of the same parents

The extraction agent extracting winter naked barley leaf of the pH3.5 that forms with 20mM calcium chloride and 10mM xitix obtains the outer extracting solution of born of the same parents.Measure outside the born of the same parents beta-1,3-glucanase activity (II-1.0) in the extracting solution by measure the glucose from laminarin, discharge with edlefsen's reagent.In our experiment, dextranase is measured ideal under the following conditions: pH3.5,1% laminarin, CaCl ₂(with MgCl ₂Or MnCl ₂Opposed), 5 ℃, and the light absorption ratio of measuring 470nm.In the extracting solution, the beta-1,3-glucanase activity per hour is approximately every milligram of total protein of 312 milligrams of glucose equivalents outside thick born of the same parents.When adopting carboxymethyl cellulose to make substrate, extracting solution does not show β-1, the 4-dextranase activity.

5 ℃ short daytimes long under outside the thick born of the same parents of winter naked barley leaf of growth in the extracting solution, endogenous chitinase activity (I-10.1) per hour discharges 115 nmole glucosamines for every gram fresh weight, and external source chitinase activity then per hour discharges 9 nmole glucosamines for every gram fresh weight.20 ℃ down the naked barley plant of growth then show every gram fresh weight and per hour discharge the active and every gram fresh weight of the endogenous chitinase of 34 nmole glucosamines and per hour discharge external source chitinase activity less than 3 nmole glucosamines.Embodiment 16: the active sign of pair cell suspension ice crystal nucleation

Winter naked barley leaf texture with 20 ℃ and 5 ℃ degrades with polygalacturonase respectively, uses the density gradient method purifying then, to obtain the suspension of single mesophyll cell.For former the carrying out of the ice crystal nucleus that exists in the winter naked barley quantitatively carried out the determination of activity of ice crystal nucleation with the droplet technology to the single-cell suspension liquid through serial dilution.Concerning the mesophyll cell of separating from 20 ℃ of leaves with from 5 ℃ of leaves, average ice crystal nucleation is initial not to have marked difference, is-7.3 ℃ (Table IV and Table V).

Ice crystal nucleus ultimate constituent in the growing plants leaf under different condition is by the method for individual cells insulation in the presence of known compound that can influence protein, sulfydryl and the disulfide linkage that links to each other with protein, carbohydrate and phosphatide (Table IV) and enzyme is determined.With cell handle and be heated to 3M urea 90 ℃ make protein denaturation after, ice crystal becomes nuclear activity obviously to descend.Nonspecific protease (pronase e and Proteinase K) also makes ice crystal become nuclear activity to reduce.This shows that the ice crystal nucleus relevant with winter naked barley mesophyll cell be former to have proteinic composition.Dithiothreitol (DTT) also reduces ice crystal with the N-ethyl maleimide with the reaction of free sulfhydryl groups to the reduction of disulfide linkage and becomes nuclear activity, shows that active generation is important to proteinic structure to the ice crystal nucleation.Boric acid and Periodic acid can both react with carbohydrate, and two kinds of compounds can both make ice crystal become nuclear activity to reduce, and this also proves the former carbohydrate components that contains equally of ice crystal nucleus.At last, Phospholipase C equally also can reduce ice crystal and become nuclear activity owing to can discharge the head of phosphate and phosphatide.Show that based on the above results the ice crystal nucleus relevant with winter naked barley mesophyll cell be former to have protein, carbohydrate and phospholipid composition.Because the ice crystal of free polypeptide becomes nuclear activity to occur in the intercellular space of plant tissue, its performance explanation ice crystal in the cell suspending liquid experiment becomes nucleoprotein to be tied on the cell walls, and is reduced reagent place's release of disulfide linkage.Embodiment 17: the antibody with freeze proof active polypeptide

Polypeptide in the outer extracting solution of the winter naked barley leaf born of the same parents that are present in cold reinforcement is prepared mono-clonal and polyclonal antibody.Independent polypeptide with SDS-PAGE separation and electric elution from gel (using through biology radiation preliminary cell) is used as antigen.With antibody purification, be used for the immune purifying of polypeptide, become their dextranases of nuclear activity, frost resistance and rhizoma nelumbinis or chitinase activity with the ice crystal of determining each polypeptide.In addition, these antibody are used for the immunoassay of polypeptide.The preparation of antibody and purifying, antigenic immune purifying, and the operation steps of immunoassay is pressed the detailed description (II-8.1) of Harlow and Lane.The detection of these polypeptide and quantitatively can be used for is chosen as and reduces plant frostbite and the output that low temperature causes and reduce and the program of design.Selection to program in the past depends on winter crop existence in the winter time, and to increase the tolerance to frost, these programs are unsuccessful.Immunoassay are because have a high susceptibility, thereby provide a kind of non-nocuous method for the plant of selecting to contain high density relevant with antifreeze (existence of AFP and former not the existing of ice crystal nucleus) or anti-freezing (ice crystal becomes the proteic existence of nucleoprotein, antifreeze protein and dextranase).The evaluation that antibody also can be used for antifreeze protein matter in other plant with separate.In addition, these antibody also can increase its freeze proof activity by combining with AFP.Embodiment 18: the anti-freeze activity in crude extract and the different plant organs

Leaf, hat and the root of cold domestication winter naked barley are separated.These organs of plant are packed in the plastics bag, freezing and at room temperature thaw in liquid nitrogen.Then with these plant tissues through squeezing, filtration and centrifugal treating, to obtain soluble component.Measure the ability of these component regulation and control ice-crystal growths.In freeze proof determination of activity, the soluble component of leaf, hat and root all shows the formation of hexagon bipyramid.These results show that freeze proof activity not only is present in the crude extract, and this activity is present in all sites of plant.Embodiment 19: have freeze proof active different plant speciess and mutation

16 different plant speciess or mutation had not only comprised monocotyledons but also had comprised that dicotyledons (seeing Table IV) longly grew down in 5/2 ℃ (order temperature/night temperature) and 16 hour daytime.The leaf of all these 16 kind of plant extracts with the vacuum extraction method respectively, and centrifuging is separated then, adopts 20mMCaCl ₂With the 10mM xitix, the solution of pH3.5.Although degree is different between mutation and kind, all these 16 kinds of intercellular extracting solutions all show the ability of the normal mode of regulating ice-crystal growth.In the plant of experiment, winter naked barley (Secale cereale cv.Musketeer), periwinkle (Vinca minor), winter wheat (Triticum aestivum cv.Karatand Ruby) and winter barley (Hordeum vulgrare cv.Acton) extract show maximum effect to ice-crystal growth by forming hexagon bipyramid ice crystal, and winter canola (Brassica napus cv.Ceres) extracting solution only forms hexagonal dish shape ice crystal to the minimum that influences of ice-crystal growth.

As these polypeptide are further described proved like that, at least 11 peptide species are synthetic under non-freezing low temperature, the those polypeptides of Fig. 9 the 4th road and Figure 10 just, molecular weight from 5kD to 36kD, and 60,68kD, 93 arrive the additional polypeptide of 99kD and 16kD.We are verified, and six peptide species wherein comprise two kinds of dextranases, and two kinds of chitinases and two kind West Africa arrowroot protamine matter have beat all frost resistance.These polypeptide are also the same with other polypeptide of the present invention simultaneously, present the activity that suppresses recrystallize under very low concentration.

Importantly pay close attention to these results of Fig. 3, particularly the variation of naked barley leaf intercellular albumen in cold domestication process in the time-histories experiment.Between the increase of the seriousness of frost and intercellular polypeptide, there is a kind of dependency.The content of the outer polypeptide of born of the same parents reached the climax at the 78th day, to enter the harshest stage corresponding with the cold domestication of naked barley plant under growing 8 hour daytime for this.After rudiment the 102nd day, the outer polypeptide of most born of the same parents has reduced concentration, remaining then again detect less than.This result shows lose its freezing tolerance when plant vernalization.Ice crystal becomes nuclear activity also to manifest in Table V, and its level is up to-7 ℃.This believes it is reported first to character relevant with ice crystal nucleus in the higher plant protein properties.Also there is sign as if to show, needs some ice crystals to become core molecule in the template procedure of ice-crystal growth in assembling.Be appreciated that it is important for freezing tolerance development in the cold domestication leaf that ice crystal in the intercellular space becomes nucleoprotein.

About separating as mentioned above and having established clear conclusions with the polypeptide of the description that meets invention, promptly plant is to this advantage of frost tolerance, be rely on the ice crystal nucleogenesis and to the anti-freeze mechanism of this two aspect of adjusting of ice crystal in conjunction with realizing.These extracellular proteins have anti-mycotic activity equally and resistibility to low temperature pathogenic agent such as fungi and bacterium are provided.What obtained proving is also can not come to harm because of freezing when reaching-12 ℃ even the winter naked barley leaf of cold domestication is lowered the temperature when first, but not domestication winter naked barley leaf to be as long as come to harm once freezing.Simultaneously, according to the present invention, the formation of icing in the intercellular space shows that the existence of AFP is not unique factor of the low temperature limit of cells survival under the freezing condition of decision.Along with the reduction of temperature, intracellular moisture is lost because of the formation of iuntercellular ice cube, thereby causes the dehydration of cell itself.Therefore, the survival in low temperature limit of frost tolerance type plant also with cell to the exsiccant tolerance degree relevant (II-16, II-18).

Traditional cultivation program can't be improved the frost resistance of crop, is in default of specific physiology mark (II-2).As the present invention confirmed, the discovery that the former and freezing tolerant plants of ice crystal nucleus produces intrinsic AFP indicated the important breakthrough on the agricultural.This mainly contains two reasons.At first, the former and AFP of ice crystal nucleus is proved to be to participate in the polypeptide that influence forming process of ice in the plant materials directly for the first time, thereby anti-freeze and ice crystal nucleation polypeptide can be proved to be the selective marker into increase winter crop surviving rate.Secondly, separate and description with the further of AFP along with ice crystal nucleus is former, they will be used to produce the more transgenic plant of high-survival rate and higher output yield.To in the future, crop will become possibility in the area that can not plant because of the restriction of cryogenic freezing and seasonal growth.

As having explained, also can be used for producing the low temperature storage of frozen product and biological tissue.Handle frozen product with these polypeptide, can guarantee that food keeps good quality in the back that thaws.Equally, no matter be to produce such product or the cryopreservation biological tissue of ice-creams, all need littler ice crystal structure.The characteristic of the size of AFP restriction ice crystal and prevention ice recrystallize all is very useful for superior prod being provided and reducing the storage loss.Being proved in embodiment, the consumption of polypeptide in these biomaterials and food composition is very little.For example, adding the polypeptide that resembles the such a small amount of of 25 micrograms in every liter of water content in biomaterial and food will tell on.This freeze proof active embodiment 20 of cold domestication herbaceous plant: the freeze proof activity in the different plants

Winter naked barley (Secale cereale L.cv.Voima), spring naked barley (S.Cereale L.cv.Jo02), winter wheat (Triticum aestivum L.cv.Ruby) and spring barley (T.Aestivum L.cv.Katepwa) are planted in thick vermiculite, grow under the length in 20/16 ℃ (daytime/night), 16 hour daytime, use through improved Hoagland solution (III-34) and water once weekly.After 7 days, or with these plants results so that the plant tissue of non-domestication to be provided, or transfer to low temperature (5/2 ℃) and 8 hour daytime and carry out the cold domestication in 7 weeks long time.Spring oats (Avena sativa L.cv.Ogle), winter canola (Brassica napus cv.Ceres), kale (the blue look of B.Oleracea var.acephala cv.Dwarf curls) and tobacco (Nicotiana tabacum L.) planted in Pro-Mix and 23 ℃ and grow 16 hour daytime under grow, arrive enough greatly up to long, so that provide abundant leaf to be used for experiment.Then, or with these plants results so that the plant tissue of non-domestication to be provided, or under above-described condition cold 7 weeks of domestication.

Use winter barley, winter wheat and spring wheat, winter naked barley and spring naked barley, spring oats, spinach, winter canola and spring canola respectively, and the outer extracting solution of the born of the same parents of the leaf of kale is done freeze proof determination of activity (Figure 15).In all non-naturalized plants, all detect, only form flat and ice crystal (Figure 15) circle in the outer extracting solution of their born of the same parents less than freeze proof activity.Through after the cold domestication, the leaf of all winter in spring cereals all tolerates low temperature, and the temperature range of tolerance is from-10 ℃ to-27 ℃, and all shows anti-freezing activity.Freeze proof activity is detecting in the winter of cold domestication naked barley, winter wheat, winter barley, spring naked barley, spring wheat and spring oats, winter canola and kale.Therefore, freeze proof activity is present in the outer extracting solution of these born of the same parents that test all anti-monocotyledons leaves that freeze that detected, all grasses that they comprise in the experiment being detected (winter naked barley, winter wheat, winter barley, spring naked barley, spring wheat) (Figure 15, table 1).

In the dicotyledons winter of cold domestication canola and kale, also detect low-level freeze proof activity (Figure 15) .Duman and Olsen (III-11) have also detected high-caliber thermal hysteresis in the full extracting solution of collard, and we just detect the outer extracting solution of born of the same parents.The freeze proof activity of some of kale might be present in the cell.In the skin that the interior AFP of born of the same parents that obtains identifying at first is a flatfish in the winter time (III-15), it is believed to prevent that extraneous ice is inoculated on the epidermic cell.We are detected low freeze proof active another kind of the explanation in kale, be relevant with etap or environmental factors.Urrutia etc. (III-52) are other member of Cruciferae, comprise in the seedling bud of the subspecies of Brassica oleracea such as Caulis et Folium Brassicae capitatae and Brussel detecting the thermal hysteresis activity, 5 ℃ of of cold of but must be domestications, 4 months down.In addition, also find Brassica napus only after under being exposed to slight freezing condition (III-27) just show stronger frost resistance.Therefore, the dicotyledons that survives the winter produces the condition of AFP, may be must be exposed in the slight frost.Detect less than freeze proof activity in the tobacco, 5/2 ℃ of and of even if the cold domestication of process also is like this.Table 1.12 kind of herbaceous plant, 8 hour daytime long under frost resistance (LT50, LT50) after the cold domestication.LT50 (℃) determine according to conductivity, be the mean value ± standard deviation that is taken to few three cold domestication samples of independence. monocotyledon LT50 (℃) dicotyledon LT50 (℃) winter naked barley-27 ± 1.0 spinach-17.3 ± 1.3 winter wheat-19.5 ± 1.7 winter canola-16.0 ± 1.2 spring wheat-15.0 ± 0.6 collard-14.7 ± 0.7 winter barley-14.7 ± 1.3 spring canola-14.0 ± 0.0 spring naked barley-14.0 ± 1.6 tobacco＞1 spring oats-10.3 ± 0.5 corn＞-2 embodiment 21: the accumulation of extracellular protein in different plants

We have carried out quantitatively the extracellular protein accumulation at low temperatures of different plants.The leaf of cereal grass is cut into the segment of 3cm, carries out the extracting of extracellular protein, according to the operation steps of (III-22) such as Hon.The leaf of broad leaved plant then is cut into specification and equates that (other operation of small pieces of 3cm * 1cm) is according to carrying out with the similar step of cereal grass.Method by vacuum extraction was extracted 30 minutes with 20mM xitix and 20mM calcium chloride (pH3), and is centrifugal then.With BSA is standard, uses the Ltd. through Bio-Rad Laboratoties, Mississauga, and ON, the improved Bradford of Canada (1976) protein determination carries out the mensuration of protein concn, and each sample is done independent the repetition three times.These result of experiment show that the accumulation of protein in apoplast generally betides monocotyledons (Figure 16) under the low temperature.The accumulation (Figure 16) of extracellular protein is in a small amount arranged in the anti-dicotyledons of freezing equally.Embodiment 22: the immunoassay of AFP in different cold naturalized plants

Cumulative extracellular protein between cold domestication is concentrated, and detect with SDS-PAGE.For AFP being carried out the needs of immunodetection, adopt the previously prepared trace of three class Musketeer naked barley AFP (III-2) antiserum(antisera)s being surveyed the outer polypeptide of born of the same parents.These three kinds of antiserum(antisera)s act on 35 and 32kD GLP, 35 and 28kD CLP respectively specifically, and 25 and 16kD TLP.This three peptide species all shows freeze proof activity in the extracting solution outside the Musketeer of cold domestication naked barley leaf (III-2) born of the same parents.

With the method for the extracellular protein of equivalent through extracting, in the 15%SDS polyacrylamide gel, separate according to Laemmli (1970).With ultrafiltration process rare extracting solution is concentrated, and will carry out electrophoresis and dyeing, dye with Xylene Brilliant Cyanine G or silver staining (Bio-Rad) to these polypeptide.Be the needs of western blot determination, use miniature transfer-trace groove (Bio-Rad) that protein transduction is moved on on the 0.45-m Nitrocellulose film according to the indication of manufacturers.Trace is at 25mM Tris-HCl (pH7.6), and 140mM NaCl becomes piece in the damping fluid of 0.01% (v/v) Tween 20 and 5% skim-milk.The antiserum(antisera) of anti-winter naked barley GLP and TLP uses under 1: 10000 extent of dilution, and the antiserum(antisera) of anti-winter naked barley CLP is by dilution in 1: 1000 and incubated overnight, as (1996 years) as described in the Antikainen etc.(MO is USA) with 5-bromo-4-chloro-3-indoles phosphoric acid-toluene amine salt (BCIP for Sigma Chemical Co., St.Louis by alkaline phosphatase bonded goat anti-rabbit igg in immune response; Sigma) and nitroblue tetrazolium (NBT; Sigma) detect for substrate.The extracellular protein of winter naked barley cv.Musketeer is through separating and shift, as all immunoblottings over against photograph.As long as clear and legible over against impinging upon under the background, trace just can continue to launch.

All three kinds of AFP (being GLP, GLP and TLP) detect with immunization method in the extracting solution outside the born of the same parents of Musketeer winter naked barley, Voima winter naked barley, spring naked barley, winter wheat, spring wheat and spring barley, but detect less than (Figure 17,18) in the oat.These AFP find (III-6) in all members of after testing Gramineae Triticum plant.In dicotyledons, anti--TLP serum is unique serum (Figure 19 D) that detects polypeptide outside the born of the same parents (molecular weight is 25kD) from the spinach of cold domestication.A kind of possibility is arranged, and is because do not produce cross reaction from the antiserum(antisera) of winter naked barley polypeptide generation and the polypeptide of dicotyledons.

The distribution of cold specificity wheat protein WCS 120 and the cold AFP similar but incomplete same (III-24) that induces.WCS120 winter wheat, winter naked barley and winter barley in find, but in the corn and the paddy rice of oat or low-temperature sensitive, or in the dicotyledons of cold tolerance such as winter canola, all detect less than (III-24).Embodiment 23: the effect that the outer carbohydrate of born of the same parents is exchanged ice-crystal growth between the ganglion cell

Plant can be regulated the growth of iuntercellular ice crystal equally with the excretory carbohydrate.We detect the concentration (Figure 20) of sugar in all non-domestications and the outer extracting solution of complete cold naturalized plant born of the same parents with the anthrone experiment.Because the outer extract of born of the same parents will be subjected to the hydrolysis of the vitriol oil, so this experiment may both measure the monose in the extracting solution, measured the monose that discharges because of polysaccharide and glycoprotein hydrolysis again.By the t under 5% significance check, we find, winter naked barley, spring oats, winter barley and winter wheat cold domestication afterwards outside the born of the same parents carbohydrate significantly increase (Figure 20 A).Observe then in spring wheat or spring naked barley that less variation has taken place carbohydrate outside the born of the same parents.And in corn, the outer increase of carbohydrate level under cold inducing of born of the same parents then do not present remarkable on the statistical significance.Our result proves that the same plant of accumulation carbohydrate also accumulates AFP (Figure 16, Figure 17, Figure 18 and Figure 20) usually in apoplast.These results show that ice can not replace AFP in the formation that is accumulated in adjusting ice of the outer carbohydrate of born of the same parents.On the contrary, situation more likely is that these carbohydrates have strengthened the freeze proof activity of cold naturalized plant tissue.Winter outer carbohydrate being accumulated in the former study between cold domestication of born of the same parents of naked barley was found once also that (III-39), but the carbohydrate that early stage institute is detected is bigger saccharan, is identified as pectinose sill glycan (III-29) for III-37, III-38.

Be the required consumption of minimizing antifreeze protein generation effectiveness, or the freeze proof activity for strengthening or supplying antifreeze protein, it is useful adding carbohydrate in antifreeze protein.Use the same purpose that one or more antifreeze proteins reached for reaching, it also is effective using the carbohydrate of one or more types and the prescription of one or more antifreeze proteins.Suitable carbohydrate comprises fructose, glucose, sucrose or Polylevulosan (polyfructosan).For example, be to strengthen the activity of antifreeze protein, can be before one or more antifreeze proteins be added ice-creamss, afterwards or when adding antifreeze protein fructose or other sugar are added in the ice-creams.Embodiment 24: with the method purifying winter naked barley antifreeze protein of heating

For simplifying the purge process of AFP, the thermostability of naked barley AFP is measured to the winter.Winter naked barley and winter wheat at low temperatures the growth, as described in embodiment 1.The acquisition of full leaf texture extracting solution is with containing 20mM xitix and 20mM CaCl ₂Aqueous buffer solution, or with the damping fluid that contains the 20mM Ammonium Bicarbonate, Food Grade and leaf and/or hat tissue homogenate, the outer extracting solution of born of the same parents then obtains according to embodiment 1 described program.The outer extracting solution of full leaf texture and born of the same parents is heated to 60 ℃ continues 30 minutes, or be heated to 100 ℃ and continue 10 minutes, centrifugal denatured protein and the particulate matter removed.No matter before heating or after the heating, freeze proof activity is measured in the adjusting of ice crystal with observation, complete all find winter wheat and the winter naked barley leaf texture's extracting solution and born of the same parents outward the supernatant liquid of extracting solution in the scope of pH8, have freeze proof activity at pH3.With the extracting solution of SDS-PAGE detection, show class dextranase albumen and class West Africa arrowroot protamine after heating still solvable (Figure 26) through heating.The technology of this simplification purge process is also effective to recombinant protein.This technology is useful equally to described other polypeptide of the application's book or polypeptide fragment.

The present invention separates the method for antifreeze protein from vegetable material or recombinant expression system, this method comprises soluble extract is heated to about 60 ℃ at least, and is then will be through the extracting solution of heating centrifugal or filter, with protein and the insoluble substance of removing sex change.The DNA of class chitinase A FP separates and protein sequence (CH-9 and CH-46) embodiment 25: the cDNA of class chitinase A FP separates

The purpose of this research is the cDNA that separates chitinase, and the class chitinase A FP that determined those dna encodings.The people of those skilled these technology believes that it is difficult that the clone has freeze proof active chitinase, because Plant Genome itself contains the gene cluster of chitinase encoding.The someone thinks equally, under some occasion, only needs a protein is done very little modification, just can give freeze proof activity to this albumen, therefore is difficult to not possess freeze proof active chitinase and makes a distinction with possessing freeze proof active chitinase.For example, KVEwart etc. (1998, Biochemistry 37:4080-4085) show that wherein one section Glu-Pro-Asn changes into Glu-Pro-Asp in the C-of fish type Sugar receptors, has just given freeze proof activity with this albumen.By from making only to possess under the condition that freeze proof active chitinase can express, that is, the method of separating mRNA in the naked barley plant that grows under the condition of low temperature and pathogen-free domestic or other pressure, we are cloned has freeze proof active chitinase.The separated classification then of the cold cDNA that induces chitinase has been encoded and ice crystal has been had the albumen of regulating power to determine which section cDNA.The colleague who is skilled at this technology will find, also available other method is come the gene of clones coding naked barley AFP.The Southern engram analysis of 25 winter naked barley genomic dnas

The naked barley genomic dna is carried out the Southern engram analysis, estimating there are what chitinase genes in the naked barley genome, thereby know that what may be isolated clones.Genomic dna extracts (III-42) according to the method after improving.From the naked barley plant of 8 ages in days, gather leaf and hat tissue, freezing in liquid nitrogen, use the mortar grinding powder.To join extraction buffer (100mM Tris, 1.4M NaCl, the 20mM EDTA of 60 ℃ of 5mL through the plant tissue of above-mentioned processing, pH 8.0,2% (w/v) CTAB adds 0.3% (v/v) 2 mercapto ethanol before using) in, and add 50mg PVP (polyvinylpyrrolidone).This solution is incubated under 60 ℃ and vibrated 1 hour, is cooled to room temperature, uses the 6mL chloroform: octanol (24: 1) extraction.After centrifugal 20 minutes, upper strata liquid is used the 6mL chloroform once more through 3000rpm: octanol (24: 1) extraction.The 5M NaCl of 0.5 times of volume of adding and the 95%EtOH of 2 times of volumes are incubated 30 minutes down at 4 ℃ earlier, are incubated 10 minutes down at-20 ℃ then, with prep dna.After centrifugal, throw out 70%EtOH washed twice, dry back suspends once more with TE damping fluid (10mMTris-HCl, pH 8.0,0.1mM EDTA).DNA usefulness ribonuclease A and Proteinase K are handled, and use phenol: chloroform: IAA (25: 24: 1) extracting twice, and in 20 ℃ of following 3M NaOAc with 0.1 times of volume, the 100%EtOH sedimentation of pH 5.2 and 2 times of volumes.After centrifugal, throw out 70%EtOH washed twice, dry and suspension once more in 200 μ L TE.

The all DNA Bam HI, Eco RI, Hind III, Xba I or the Xho I digestion with restriction enzyme that from 8 ages in days non-domestication winter naked barley leaf and hat tissue, extract.Part separates with agarose gel electrophoresis, and transfers on the nylon membrane.Adopt a kind of known can be by the basic barley chitinase cDNA of pathogen-inducible, promptly pHvch2a is used to differentiate chitinase gene.Film is hybridized under two kinds of different strict control conditions with pHvcht2a: 30% or 50% methane amide, 42 ℃, 6 * SSC, and under 42 ℃, be washed till 0.2 * SSC and 0.1% SDS, use radioautograph exposure (Fig. 3 B) then.Under high stringent condition (50% methane amide), there are three genes at least,, three DNA parts all arranged with the pHvch2a probe hybridization on Hind III and the Xba I road because at Eco RI.There are two bands to appear at the BamHI road under the high stringent condition.In low stringency condition hybridization, obviously also have more two genes, because five bands occur in Eco RI road.Five DNA parts hybridize on the probe, wherein three very fuzzy.Similarly, at Xba I and Hind III road two extra bands appear at least.25.1 cold domestication winter naked barley cDNA library screening

With known can be by the basic barley chitinase cDNA of pathogen-inducible, i.e. pHvch2a, the cDNA library that is used to screen cold domestication winter naked barley.The sequence of PHvch2a is the one section 1028bp that inserts in the Eco RI site of pBluescript SK+.Eco RI is used to separate the part as probe.The size that is used as the part of probe is 915bp, because insertion person is having an Eco RI restriction site near 3 of cDNA ' end.The PHvcn2a chitinase of encoding, this chitinase have the chitinase catalysis district of height preservation, but disappearance chitin bundle feature district.Because barley also is monocotyledons, and with naked barley be close relative (III-6), can imagine that therefore the chitinase of barley and naked barley perhaps has closely similar nucleosides order.

Before the screening library, determine with Northern engram analysis method whether pHvcht2a is suitable for screening the library and detects all naked barley chitinases.Full RNA separates from non-domestication and cold domestication naked barley leaf, and carries out gel electrophoresis and separate in sex change formaldehyde agarose gel, transfers on the nylon membrane then.Film is hybridized down at high stringent condition (50% methane amide, 6 * SSC, 42 ℃) with pHvcht2a, and under 42 ℃, be washed till 0.2 * SSC and 0.1% SDS.Trace exposes to the radioautograph film, demonstrates the mRNA that hybridizes on the probe.Two kinds of different chitinase mRNA that size is respectively 1.25kb and 1.00kb obtain identification.The transcript of another about 3.7kb size also hybridizes on the pHvcht2a, but this may be because with the non-specific binding of probe.1.25 and the transcript of 1.00kb is consistent with the transcript size that foundation 35 and 26kD class chitinase A FP are predicted respectively.The pHvcht2a probe is used to screen the λ Zap-cDNA library that obtains from the isolating poly A+RNA of cold domestication naked barley leaf, and is with identification overall length chitinase cDNA, as mentioned below.

From tissue, extract full RNA with preamble described (III-33) through improved method.The plant tissue of liquid nitrogen freezing is ground in mortar.Z6 damping fluid (8M Guanidinium hydrochloride, 20MmMES pH 7.0,20mM EDTA) and the 2 mercapto ethanol of 200 μ L are joined in the plant tissue that under freezing conditions further homogenizes.Phenol with this soup compound and 1 times of volume: chloroform: primary isoamyl alcohol (25: 24: 1) is mixed to extract protein.Through 8000 * g centrifugal 15 minutes, supernatant liquid was used the phenol of 1 times of volume once more: chloroform: isoamyl alcohol extracting, mix with the Virahol of 1 times of volume and the 3M NaOAc pH 5.2 of 0.1 times of volume, and-20 ℃ of insulations 2 hours with sedimentation RNA.With solution under 8000 * g centrifugal 45 minutes, make the RNA sedimentation.Throw out with 70%EtOH washing 2 times, is suspended in the water of handling through DEPC after the drying once more.RNA separates out 20 ℃ of following sedimentations with 8M LiCl.Under 8000rpm centrifugal 45 minutes, make the RNA precipitation, and use the 70%EtOH washed twice.With drying precipitate, in the water of handling through DEPC, suspend once more.(WI USA) separates poly A+mRNA from full RNA for Promega, Madison with PolyATtract mRNA separation system.

(CA USA) makes the cDNA library with poly A+RNA for Stratagene, La Jolla with λ Zap-cDNAGigapack clone test kit.Film is being contained 6 * SSC, 0.5% (w/v) SDS, 50% methane amide, 5 * DenhardtShi solution (1 * DenhardtShi solution is 0.02% (w/v) Ficoll, 0.02% (w/v) PVP, 0.02% (w/v) BSA) and 100mg mL ^-1Prehybridization when under 42 ℃, proceeding to when young in 50% formamide soln of the salmon sperm dna of shearing sex change.Spend the night with the hybridization under 42 ℃ of barley chitinase cDNA probe.(Sweden agricultural science and technology university, Svalov Sweden) provides probe, uses by Tomas doctor Bryngelsson ³²P-dCTP marks, and is filled to about 1 * 10 at random ⁸To 2 * 10 ⁸Cpm μ g ^-1DNA.The composition of hybridization solution is the same during with prehybridization, just without salmon sperm, and adds 1 * 10 to fresh sex change label probe ⁶Cpm mL ^-1Concentration.After 42 ℃ of incubated overnight, under 42 ℃, film is washed from the different stringent conditions of 1 * SSC and 0.1 (v/v) SDS to 0.2 * SSC and 0.1 (v/v) SDS, last 30 minutes.About 92700 clones obtain screening, and 89 hot spots are identified.Take turns in the screening second, reclaim phage and be divided into 17 groups from hot spot.The E.coli cell uses the phage through grouping to infect again, recoats flat board, transfers on the Nitrocellulose film, screens with pHvcht2a again.Identify 48 clones that infer, and by excising on the pBluescript SK-plasmid of from phage, recombinating in the body.These clones represent with recombinant plasmid pCHT-1 to pCHT-48.25.2 infer clone's digestion of grouping 25.2.1 Pvu II Restriction Enzyme and G-trajectory analysis

The digestion of Pvu II Restriction Enzyme can allow the pBluescript plasmid that rebuilds discharge the insertion fragment, inserts segmental size with decision.The pBluescriptr plasmid has 2 Pvu II restriction enzyme sites, and at a distance of 445bp, flank has a plurality of cloning sites.Enzymic digestion discharges the insertion fragment, and 450bp is combined in the insertion fragment of actual size.The plasmid size is 2500bp on agar gel, inserts fragment and is shown as another band.Contain any Pvu II restriction site if insert fragment itself, should have two or more take out of now so generally, promptly 2500bp represent plasmid, and other band is the total size of insertion fragment.Digestion clone's agargel electrophoresis shows that different insertion clip size is between the 350-2655bp.Some clones show identical banding pattern, can be at first with these clone's groupings according to Pvu II restriction enzyme digestion pattern.There are 17 classifications to have similar band after the digestion.Clearly, greater than 950bp, and 7 classifications that contain 17 clones are inserted fragments less than 950bp by the insertion fragment of 10 classifications that contain 31 clones.Estimating to be used for 36KD and 26KD class chitinase A FP total length transcribes the insertion fragment length minimum of the Northern blot hybridization data of using pHvcht2a and is 950bp.The G-trajectory analysis can determine those clones to be identical thereby to be clone more than needed.Is the G reaction that primer carries out the sequential analysis of mulberry lattice to each clone with T3 and T7 sequence, carries out polyacrylamide gel electrophoresis again and separates, and carries out the banding pattern contrast with genomic each clone of primer, and it is identical which is determined.The G-trajectory analysis can obtain some sequence datas and indicate identical clone.8 classifications that experiment shows 9 clones are cloned mutually and are not matched.25.2.2 pHvcht2a hybridization

The enzymic digestion thing is separated through gel electrophoresis, transfer DNA to nylon membrane, (6 * SSC under strict control condition, 50% methane amide, 42 ℃) be high strict control condition (0.2 * SSC, 0.1% SDS behind the probe hybridization with pHvcht2a, 55 ℃) flushing, decision false positive dna clone (Fig. 6 B).Experiment shows 9 clones and does not hybridize again with pHvcht2a.These clones are PCHT-10 ,-13 ,-17 ,-18 ,-19 ,-28 ,-32 ,-38 and-39, and these genes may not be chitinase cDNA.25.2.3 the sequential analysis of the chitinase cDNA of purifying.

At first the sequential analysis of carrying out is those clones that have full length sequence in pvuII restriction enzyme digestion data, promptly selects the clone of those insertion sequences greater than 950bp.These are cloned the hybridization analysis that at first carries out following T3 and T7 universal primer, can take some sequential analysis strategies on this basis to obtain more detailed sequence data.Some sequences with restriction enzyme site allow the segmental part of insertion to delete from plasmid, then, are that primer links the plasmid order-checking again to obtain more sequence information with T3 or T7.Can not also need design primer by above method order-checking in some cases, proceed order-checking with sequence data.It is the-terminal amino acid sequence that PCHT9 inserts fragment, identical with the N-end sequence (III-23) of 35KD class chitinase A TP.The insertion segment of PCHT46 also has full length sequence.The class chitinase A FP of gene translation product and 26KD is identical, and other classification shows 5 ' the longest non-translational region relatively.The-terminal amino acid sequence of estimation gene translation product and the aminoacid sequence of pHvcht2a are closely similar.Two kinds of genes all have ATG initiation codon and open reading-frame (ORF) (ORF), and are consistent with known chitinase (in proper order) to guarantee its amino-acid sequence.25.2.4 CHT9 signature analysis

Children's medical treatment biotech research center in University of Toronto, according to multiple structure plasmid with by initial and ever-increasing order data design primer, with the two-way mensuration CHT9 order of ALFexpressDNA sequenator.The poly-A tail of cDNA band, length is 1193bp.Open reading-frame (ORF) (ORF) comprises 318 amino acid of coding, begins to finish to the 955bp terminator codon of ATG startup codon by being positioned at the 48bp codon methionine that inserts fragment 5 ' terminal downstream.First amino acid of estimating gene product is L-glutamic acid, and is in 21 sites of open reading-frame (ORF) (ORF), identical with the terminal nucleotide sequence of N-of coding 35KD class chitinase protein.Estimate that gene product contains 301 amino acid, molecular weight is 31647Da, and iso-electric point is 6.96.The non-coding region that 190bp is arranged at 3 ' end.The psort analysis of cDNA shows one 20 amino acid whose signal sequences and shifts relevant (Figure 21 (c) with protein to the extracellular.The present invention also comprises some mutant, and these mutant are and identical peptide, polypeptide and the protein of signal sequence (or coding nucleic acid molecule) biological function, have shown similar or identical signal targeting vigor.The mutant discussion of relevant antifreeze peptide will be in the following difference that relates to signal sequence itself.This signal sequence can be readily incorporated on the polypeptide except that antifreeze peptide to activate them to extracellular transfer.The present invention also includes by recombinant DNA peptide, polypeptide or protein transduction is moved on to extracellular method, adopt protein analysis or other technology that polypeptide, peptide or the proteinaceous solid that produces fixed on the signal sequence (or identical biological function sequence), polypeptide, peptide or protein transduction are moved on to the extracellular.25.2.5 CHT46 signature analysis

Known have can the encode class chitinase A FP of 26KD of a lot of available clones.By pharmacia T7 kit test kit to possible cloning and sequencing, to determine those to have total length and the clone of the 26KD class chitinase A FP that can encode.Utilize the information of sequential arrangement of T3 and the effect of T7 primer generation, determine the chitinase and the 26KD class chitinase A FP of CHT46 coding total length, and its whole orders (Figure 22).CHT46 contains the insertion fragment of 998bp size, and it has the 78bp downstream that begins from the ATG that starts 252 amino acid whose open reading-frame (ORF)s (ORF).The molecular weight of gene product is 26835Da, and iso-electric point is 8.25.Terminator codon is positioned at from ATG and starts the 757bp site that codon begins, and containing 163bp is 3 ' non-translational region.Psort analysis predicts that also this albumen has one 22 amino acid whose sequences [figure (d)], and is transferred to the extracellular.25.2.6 the sequence of CHT9 and CHT46 contrast

Studies show that by the GAP that uses GCG sequence analysis software package, PCHT9 and PCHT46 (Figure 23) nucleotide level have 62.1% identical, amino acid levels have 58.6% identical with 63.3% similar.Simultaneously, CHT46 Nucleotide and amino acid levels and PHvcht2a than PCH9 more similar (two levels all greater than 90% similar).ABLAST research can be used for every kind of clone, to disclose and to concern the similarity analysis of close other chitinase of kind.CHT9 and Chinese spring chitinase height homology (similar up to more than 90% of Nucleotide and amino acid levels).Contrast CHT9; CHT46 and naked barley seed chitinase, they have 82% identical at nucleotide level, have 86.6% similar at amino acid levels.And CHT46 and naked barley seed chitinase only are 57.4% identical in nucleic acid level, amino acid levels be 62.2% similar.CHT9, CHT46, Hvcht2a, tobacco chitinase (x16939), paddy chitinase (D16223); The aminoacid sequence of 2 kinds of chitinases of spring wheat chitinase (x76041) and naked barley seed is conservative (conserved) very.Our result in the past shows that tobacco does not accumulate extracellular AFP enzyme or chitinase at cold condition (Figure 16 and 19).Although tobacco and naked barley chitinase sequence are high conservatives, the fact is that naked barley albumen is subjected to low temperature induction, shows freeze proof vigor and tobacco protein is non-low temperature induction, does not show freeze proof vigor.

Order design cDNA probe according to Figure 21 and 22 is identified class chitinase A FP.A kind of is the probe (CH46bp) of 404bp, forms [Figure 22 (b)] by one section gene order of mending mutually with the gene section of the 340th Nucleotide to 774 Nucleotide of CHT46.Second kind is 404bp probe (ch9hp), its sequence and CHT9 sequence begin the homologous region complementation [Figure 21 (a)] that finishes to the 945th Nucleotide with 541 Nucleotide.The nucleotide sequence mutant of these probes (identical biological function probe) is useful equally.These probes are of value in order to following method identifies similar DNA, cDNA or RNA sequence.Doubt DNA or RNA are transferred on the film, preferably then with 50% methane amide, 6 * SSC, 0.5%SDS, (a * 1 solution is the 0.02%W/V ficoll to 5 * Denhardt solution, 0.02%W/V PVP, 0.02%W/V BSA) prehybridization and under the 100kg/ml cutting denatured salmon sperm dna condition, temperature is 42 ℃, and doubt DNA or RNA can replace salmon sperm dna to hybridize with the sex change ch46p of mark or the sex change ch9hp of mark under similarity condition.Film is better than 42 ℃ most and washed 30 minutes with 0.2 * SSC and 0.1%SPS down.

Will go through as following, invent and not only comprise naked barley class chitinase A FP (by the coding of the gene order shown in Figure 21 and 22), but also contain the peptide of identical biological function vigor, and polypeptide and protein, they all demonstrate the same or analogous freeze proof vigor with naked barley ATPs.The present invention also comprises some and CHT9, CHT46, and PCHT9 or PCHT46 have the nucleotide sequence of identical biological function.Embodiment 26: the expression analysis 26.1 of chitinase CHT9 and CHT46 is in the expression of low temperature and non-domestication by low temperature leaf

The total rna content that separates non-domestication by low temperature and domestication by low temperature leaf.By denaturing formaldehyde agarose gel electrophoresis (Figure 11 A); Isolate the RNA total amount; Be shown as the ribosome-RNA (rRNA) band.RNA is transferred on the nylon membrane back and CHT9; CHT46 gene specific probe hybridization.Design two kinds of probes respectively according to both 3 ' different non-translational regions.CHT9 gene specific probe is for containing 30 Nucleotide oligonucleotide: CGAATAATGGTGCAATCCATCGCAAGATGC.CHT46292bpcDNA,A ( TCGAGTGCGGCATGGGCCGGAACGACGCCAACGTCGACCGCATCGGCTACTACACACGCTACTGCGGCATGCTTGGCACGGCCAGGGGGGGCAACTCGACTGCATCACCCAGCGAAACTTCGCTAGCTAGACAGTGTATCTCACGTGTTATAAATAAATGGCAATGCATATGCCATCCCCGAATAAATAATTCAACATGTGACAGTTGATTTGTATGGTAATACGAGTAAGTTGTTGCAACAAATTATGAATATTGAATAAAATCAAATTTTATCAAAAAAAAAAAAAAAAA ) 。

CHT9 gene specific probe hybridization product is a 1.25kb transcript and CHT46 gene specific probe hybridization thing is the 1.0kb transcript.For CHT46 gene specific probe, should be in down flushing of the strict control condition of height (temperature is 65 ℃ for 0.2 * SSC, 0.1%SDS), the non-specific combination that causes with the poly-A tail that reduces probe.The relative intensity of non-domestication and cold domestication RNA is weighed by optical density(OD).For CHT9 and CHT46, the intensity of the more non-respectively cold domestication leaf of the intensity of cold domestication leaf has increased by 2.5 and 1.3 times.26.2 the expression of different naked barley tissues between cold domestication

Between cold domestication to the naked barley sample of tissue.In a week between cold domestication, in three weeks, in five weeks, respectively to hat (mitogenetic or meristem), young leaves, Lao Ye and root tissue are taken a sample after seven weeks, and the plant tissue around having grown with non-cold domestication condition is contrast.The leaf that grows after plant is transferred under the cold domestication condition is the young leaves sample, and under non-cold domestication condition, transferring to the leaf that cold domestication condition stretched in the past is the Lao Ye sample.

Extracting goes out total rna content of sample tissue and analyzes with sex change formaldehyde gel electrophoresis (Figure 13), after transfer on the nylon membrane, carry out the Northern engram analysis with CHT9 described above and CHT46 gene specific probe.The hybridization product of CHT9 is the transcript (except the root tissue) of 1.25kb, and the strength of signal of this transcript is lower after cold one week of domestication, but constantly strengthens subsequently, reaches the highest after seven weeks of domestication; The hybridization product of CHT46 gene specific probe is the transcript of 1.0kb, and the change in signal strength of transcript is the same, and is promptly lower after cold one week of domestication, but constantly strengthens subsequently.CHT46 probe hybridization transcript is present in all tissue samples that comprise root tissue.26.3 the expression under non-domestication, cold domestication and the domestication minimizing condition in leaf

For confirming that low temperature induction is reversible, we put non-next week of domestication condition with the naked barley plant, and the back moves into cold seven weeks of domestication condition, rotates back into non-cold domestication condition again.With under the non-cold domestication condition growth two weeks plant in contrast.Be behind 6,12,36 hours and 1,3,5,7 weeks, after 7 weeks plant to be transferred under the non-cold domestication condition sample time between cold domestication, respectively at sampling after 6,12,30 hours and 9 days.Extracting RNA total amount from these sample tissues is analyzed banding pattern with the denaturing formaldehyde agarose gel electrophoresis respectively, then hybridizes with gene specific probe (seeing embodiment 26.1).For CHT9 gene specific probe, the transcript of 1.25kb just obtained in 6,12 hours after seven weeks of cold domestication and after transferring to non-domestication condition, later signal strength weakening, and in other sample tissue no signal intensity; And PCHT46 gene specific probe hybridization thing (transcript that is kb in 1 under low control condition comes across all sample tissues, and its strength of signal improves constantly under the low-temperature reinforcement condition, and slowly disappearance after plant is transferred to non-domestication condition.These results confirm that the expression low temperature adjusting of CHT9 and CHT46 can reverse.26.4 the expression in the acid-treated leaf of bigcatkin willow

Whitfield's ointment can be induced the genetic expression of the antimycotic chitinase of coding.Under cold domestication condition, the naked barley plant in two weeks of growing is carried out Whitfield's ointment (the being dissolved in 0.5%Tween liquid) spraying of 20ppm, once a day, continuous 8 days.With the plant of not spraying is contrast, every day Whitfield's ointment handle back one hour to the leaf sample of tissue separating total rna content, analyze expression with the Northern blotting (under low control condition, washing) of CHT46 gene specific probe.The transcript of 1kb and 3.7kb is present in (Whitfield's ointment is handled or non-processing sample) in all samples, and the transcript of 3.7kb may be because the non-specific combination that low control condition flushing causes.Beginning, the strength of signal of transcript is similar, and Whitfield's ointment was handled after 5 and 6 days, and the transcript strength of signal of check sample is higher than the acid-treated plant sample transcript of bigcatkin willow intensity.Use CHT9 gene specific probe not produce any positive findings.Above result shows that the expression of CHT46 and CHT9 is not to resemble the antimycotic chitinase gene by identical signal induction.Embodiment 27: the super expression of CHT46 and CHT9 in the Arabidopis thaliana (Arabidopsis thaliana)

CHT9 and CHT46 to be cloned into pkylx7.1-35s2 Agrobacterinmtemefaciecs consistency carrier, carrier to be transformed in the A.tumefacicn bacterial strain (C58PGV3850) by electroporation. vacuum filtration will obtain seed after making bacterial strain import Arabidopis thaliana.These seeds are carried out the kalamycin resistance screening, the T1 plant is inserted the number of fragments analysis.Containing among two CHT9 or the CHT46 any one inserts segmental plant and represents homozygosity at T3.Northern blotting (embodiment 25) by the gene specific probe and freeze proof vitality test confirm the accumulation of class chitinase A FP.Freeze proof vigor can be measured in the extracellular extract of embodiment 18 described thick extracts or embodiment 5 descriptions.

The overexpression of dna encoding class chitinase A FP

" coding DNA " is meant winter naked barley AFP in the code book invention or the proteinic chromosomal DNA of other AFP example class chitinase A FP, plasmid DNA, cDNA or synthetic DNA.Coding DNA of the present invention can import microorganism, plant or animal host.The microorganism host comprises algae, bacterium, fungi and protozoon.The coding DNA that imports the host can be embedded in the plasmid of microorganism cells nuclear ability organoid, the karyomit(e).Super expression is that the accumulation or the more general native gene of non-existent recombinant protein under normal conditions has higher expression level.The secretion 28.1 clone CHT9 of the expression of CHT9 in embodiment 28 intestinal bacteria (E.coli) and class chitinase A FP are to bacterial expression vector

The CHT9 gene is inserted into intestinal bacteria ((E coli) expression vector pET12a (Novagen, Madson, WI) BamHI site, as primer CHT9 is carried out pcr amplification with synthetic oligonucleotide 5 '-TTAAGGATCCGGAGCAGTGCGGCTCGCAGGC and 5 '-GGTTGGATCCTGCGAACGGCCTCTGGTTGTA, at the beginning and the ending generation BamHI restriction enzyme site of gene.With the about 800bp pcr amplified fragment of BamHI digestion, connect the single BamHI site of pET12a.To connect mixture and be transformed into DH52, coating LB-phenalgin penicillin (150kg/ml) flat board, 37 ℃ of cultivations.Analyze the plasmid of single bacterium colony, insert the segmental clone of BamHI to find to contain by correct direction, for example the CHT9 reading direction is identical with secretion signal direction on the carrier.Identify bacterium colony and called after cht9/12a.For confirming the dna sequence dna of the chitinase cDNA that PCR-increases, (Quebec Canada) uses the T7 dna sequencing kit, carries out the double-stranded DNA order-checking according to the dideoxy nucleotide chain cessation method for Pharmacia, Montreal according to business directory.28.2 the expression of ch9/12a recombinant plasmid

Plasmid DNA with QIAGEN DNA purification kit (QIAGEN Germany) purifying worm ch9/12a from the DH52 cell, after be transformed into and derive from Novagen (Madison, WI) host BL-21 (DE3) cell, these cells contain the T7 polysaccharase of plasmid embedding to instruct plasmid-encoded expression of gene.For expressing class chitinase A FP, from the single bacterium colony of the dull and stereotyped picking of fresh line LB; Be inoculated in the 2ml LB/ penbritin nutrient solution in 30 ℃ of shaking culture, spend the night, after the overnight culture of 1ml is inoculated in shaking culture in the 250ml flask that contains 50ml LB/ penbritin nutrient solution, temperature is 30 ℃, reaches 0.4 up to the OD600 of nutrient solution.Adding IPTG (sigma) again in nutrient solution is 0.4mm up to its ultimate density, continue to cultivate 3 hours, and then centrifugal down (rotating speed is 5000 * g) 5 minutes, harvested cell in 4 ℃ with nutrient solution.28.3 the purifying of expressing protein and analysis

Be the purifying periplasm protein, bacterial precipitation is suspended in ice-cold 20% sucrose again, 2.5mmEDTA in the 20mm tris-HCL PH8.0 liquid, reaches the concentration of 50D550 unit/ml and cultivated 10 minutes on ice up to it.With suspension centrifugal (rotating speed is 1500 * g) 5 minutes, the above again operation of bacterial precipitation, after (changeed 15000 * g) 10 minutes suspension is centrifugal again.Supernatant liquor is the periplasm protein fragment, uses SDS-PAGE and further analyzes (seeing embodiment 17) by the antiserum(antisera) immunoblotting that naked barley class chitinase A FP produces.

Bacterial expression vector pET12a contains the proteic leader sequence of ompT (outer membrane protein T).In some cases, target protein can be transferred to extracellular space.Leader sequence is essential for transferring to pericentral siphon, but is not sufficient.Proteic transhipment also depends on the maturation zone of target protein.A simple method that obtains the pericentral siphon part from the BL-21 cell is the osmotic shock method.Figure 24 has shown the expression of using pEL12a/cha cell transformed chitinase in the cell.The albumen extract that all cells obtains has shown that about 32Kda (row 2.4) mainly is with, and lacks (row 1,3) in cellular control unit.Although pericentral siphon partly contains expressing protein, the great majority of the class chitinase A FP that expresses are present in the cell (data is not seen).For whether the class chitinase A FP in the assessment pericentral siphon part has freeze proof vigor, these part concentration are brought up to 1000 times, its freeze proof vigor is determined by observing the ice crystal form that forms in the refrigerating process in the back.The result shows that these fragments have freeze proof vigor, because ice crystal is with vertical six two pyramid form growths.Recombinant protein shows chitinase activity equally, and this can be measured by embodiment 15 described methods.Embodiment 29: the expression of class chitinase A FP and secrete the CHT gene of the terminal His/MYC mark of 29.1 tool C-in Pichia pastoris

From Ivitrogen (San Diego, CA) buy the Pichia expression vector, CHT9 cDNA is inserted into EcoRI and the Notl site of expression vector PGAPZA, with synthetic oligonucleotide 5 '-AATTGAATTCGAGCAGTGCGGCTCGCAGGCC and 5 '-AATTGCGGCCGCTGCGAACGGCCTCTGGTTGTA is that primer carries out the PCR spread spectrum with CHT9, and 5 ' and 3 ' end at cDNA produces EcoRI and Notl site respectively.With Ecoli and Notl digest amplification fragment, link to each other with the PGAPZ α A carrier that disappears colored with the same restrictions enzyme.To connect mixture and be transformed into DH52, be coated on less salt LB-Zeocin (25ug/ml) flat board, 37 ℃ of cultivations.Analyze bacterium colony and called after pGAPZ/cht9 that recombinant plasmid is differentiated to be had in EcoRT/Notl fragment embedding yeast-factor sequence.Determine the dna sequence dna of recombinant plasmid with above-mentioned dna sequence analysis method.The strategy that serves as a mark with His/MYC helps to produce active chitinase and other antifreeze protein.Therefore the present invention also comprises a kind of method of purifying chitinase and other antifreeze protein.The mark similar with the His/MYC mark also can be used for purifying.Do not make up the clone 29.2 there is the end-labelled CHT9 of C-

With the new oligonucleotide 5 ' of synthetic-TCTGGAGACTATGCGAACGGCCTCTTGGTT-3 ', 5 '-oligonucleotide of having described more than reaching carries out the PCR reaction as primer to CHT9, produces CHT9 5 ' and 3 ' end EcoRI and Xbal site respectively.Link to each other with EcoRI and xbal digest amplification fragment and with PGAP2A with the same restrictions enzymic digestion.This strategy has guaranteed can be attached to the C-end of CHT9 by the sequence of the band His/MYC mark of vector encoded.Select and the amplification recombinant plasmid as above method.Positive bacteria called after pGAPZ α A/chtgc-tag), it helps to improve the freeze proof vigor of recombinant protein.29.3 pGAPZ α A/cht9 and pGAPZ α A/chtg (tag) are transformed into Pichiax-33

Use a large amount of DNA purification kits (QIAGEN Germany) purifying pGAPZ α A/chtg and pGAPZ α A/chtg (plasmid DNA tag).The parameter of recommending according to user manual is carried out electroporation (Bio-Rad gene pulse) plasmid DNA of about 10ug is transformed into yeast cell strain x-33 (Invitrogen), with transformant in YPDS flat board (1% yeast water that contains 100ug/ml Zeocin, 2%D-glucose, the 2M Sorbitol Powder, 2% agar) cultivated 3 days.At fresh YPS/Zeocin plate streaking to obtain single bacterium colony, more single step purification.Analyze 8 kinds of anti-Zeocin Picha transformants to know existing of CHT9 cDNA.Isolate the gene DNA of single bacterium colony and express handbook (invitrogen) according to Pichia and carry out pcr amplification and insert segmental the existence determining.Be chosen in the clone that karyomit(e) is inserted with CHT9cDNA and carry out expression analysis.29.4 the expression of recombinant C HT and class chitinase A FP analyze in Pichia

The single colony inoculation of picking is in the YPD of 10ml substratum (the female juice of 1% enzyme from a reorganization bacterium colony, 2% peptone, 2% glucose), 28-30 ℃ of shaking culture spent the night, and the nutrient solution at night of about 0.1ml is inoculated in continues in the 250ml flask that contains the 50mlYPD substratum to cultivate 2-3 days.When finish every day, collect the 1ml nutrient solution, centrifugal, with SDS-PAGE and immunoblotting analytically clear liquid and precipitation.

PGAPZ α A (2.9kb) carrier is with the recombinant protein among the GAP promotor constructive expression Pichia pastoris.GAP is the promotor of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, constructive expression all in comprising Pichta pastoris 5 a lot of organisms.In addition, the N-terminal peptide of this carrier one coding cereuisiae fermentum (Saccharomyces cererisisiae) alpha factor secretion signal in some cases, can be transferred to foreign protein extracellular space (supernatant liquor).Figure 25 a has shown the super expression and the secretion of the recombinant plasmid of yeast cell.The protein sample of yeast culture supernatant liquor in SDS-PAGE analyzes is-band of Yue 32KD ( row 1,2).Figure 25 b has shown the immunoblotting assay of same sample.Yet in any case, different with bacterial expression system described above is, the most of chitin AFP that express in yeast are present in ( contrast row 1,2 and 3,4) in the supernatant liquor, and this explanation secretion process hexyl is originally finished.Expression and secretion level are that every liter of nutrient solution contains 50 to 100mg as calculated.Pichiapastoris seldom secretes oneself protein, and the successful secretion of recombination classes chitinase A FP is more prone to purifying and characterized thus.Embodiment 30.PGAPZ α A/chtg (expression tag) and secretion

For the albumen that makes acquisition meets its natural form more, can select to make up a gene and eliminate the His/MYC mark and be connected with natural chitinase C-is terminal.Expression and the secretion level of contrast and PGAPZ α A/chtg, SDS-PAGE of both expressing proteins and immunoblotting assay show that recombination classes chitinase A FP enzyme has freeze proof vigor, because of formed hexasomic bodily form ice crystal in AFP liquid.Recombination classes chitinase A FP also shows chitinase activity, and this is described in embodiment 15.Embodiment 31: the expression of CHT46 in the intestinal bacteria (E.coli)

CHT46 by carrier (seeing embodiment 27) in intestinal bacteria (E.coli) with fusion protein form expression, its fusion rotein has a leading strand that contains 6 Histidines.With the preparation of embodiment 27 described methods and with Ni inner complex post purification of recombinant proteins from lysate, through refolding (refolding) and the excision of His mark, ripe recombinant protein shows certain freeze proof vigor, because of formed six body ice crystals in recombinant AFP liquid.Embodiment 32: the enhancing of freeze proof vigor

The freeze proof vigor of site selectivity sudden change can further enhancing class chitinase A FP.For example can improve the vigor of Atlantic salmon (Ocean Pout) by this approach (III32).Use albumen model analysis and some other Forecasting Methodology, we determine, and the ice land of AFP and other key amino acid residue can suddenly change, chimeric or deletion.By U.S.E (cancellation of unique site) sudden change test kit (pharmacia biotechnology) or other similar commercialization sudden change test kit the DNA plasmid expression vector that contains class chitinase A FP gene is studied.In case dna sequence analysis is confirmed to undergo mutation, promptly available any expression system carries out the expression and the antifreeze protein vitality test thereof of mutain.This approach can not only improve the freeze proof vigor of albumen, and helps the operation of the functional zone of other proterties, for example interpolation of the application of protein purification or its biological function or the like.Make synthetic dna fragmentation become possibility simultaneously, less albumen of these fragment codings and expression more easily based on chitinase sample ATPs sequence.This also may modify the expression of cDNA in addition, so that its expression can be induced by envrionment conditions, and for example low temperature, different chemical inducer or hormone etc.Moreover, making protein can locate different distributions by modified dna sequence, Figure 21 and 22 are seen in the modification of all dna sequence dnas, and the present invention has also comprised by these modification sequence expressed protein.Embodiment 33: the heredity of antifreeze protein in winter wheat and the spring wheat

Cheyenne winter wheat (Triticum aesticum aestivum Lcv Cheyenne), the growing state of Chinese spring (Triticum aesticum Lcv Chinese Spring) and 21 karyomit(e) replacement strains is seen embodiment 1.Replace in the strain at every kind of karyomit(e), the dyad of Chinese spring is replaced by the homologous chromosomes of Cheyenn winter wheat, and the stronger low temperature tolerance of Cheyene winter wheat tool.From the leaf of Chinese spring, Cheyene winter wheat and 21 karyomit(e) replacement kinds, obtain the apoplast extract as what describe in embodiment 20 and 21, every kind of extract is carried out freeze proof vitality test (seeing embodiment 5), quantize albumen (seeing embodiment 21) in the extract, the SDS-PAGE method is separated, use the antiserum(antisera) immunoblotting assay that produces by naked barley AFP, these AFP and dextranase, chitinase and West Africa arrowroot essence similar (seeing embodiment 22).

After the cold domestication, the food value of leaf ectosome extract of all kinds all shows freeze proof vigor, dextranase is all arranged, the smart AFP of chitinase and class West Africa arrowroot exists that (chun et al 1998, Euphytica102:219-226) to be positive coorperativity relevant the survival time in winter of the apoplast albumen of every leaf and freeze proof activity level and 23 kinds of kinds.Karyomit(e) 5B has carried with more apoplast albumen accumulation with 5D and has reached the more relevant oligogene of high anti-freezing vigor.Embodiment 34: the expression product of AFP in eukaryotic cell and the prokaryotic cell prokaryocyte

From the cDNA library, can obtain dna sequence dna of the present invention (also claiming nucleic acid molecule), method as described in Example 25 (gene DNA library).Also can obtain nucleic acid molecule from other source, for example the expression analysis of flag sequence or external (ln vitro) are synthetic.Here the AFP coding DNA of Ying Yonging (containing identical biological function mutant) can import multiple eucaryon and protokaryon host cell and be expressed.The recombinant nucleotide molecule of AFP contains transcribing of the suitable connection operated and translates regulatory factor, can obtain suitable regulatory factor by multiple source, and they can optionally be read [Sambrook, J., Fritsch, E.E; Maniatis, T (1989) molecular cloning laboratory manual, press of cold spring harbor laboratory, New York; Ausubel et al (1989) Current Protocols in Molecular Biology, John Wiley﹠amp; Sons Inc.].For example, if will improve expression of gene, can be by being inserted with adopted sequence and suitable promotor in carrier.Plant promoter can be inducibility, composition, environment and growth modulability or cell and tissue specificity.Transcription also can for example use 35s promotor [Shah et al. (1986) Science 233:478-481] promotor of super promotor (superpromoter) [Niet al (1995) Plant Journal 7:661-676] or cauliflower embedded virus to strengthen by some known skills.

Evoked promoter comprises: a) arid and ABA evoked promoter contain ABA-response factor [Ono et al. (1996) Plant physiol.112:483-491; Abe et al. (1997) Plant Cell9:1859-1868]; B) the heat shock induction promotor comprises the HSES (heat shock factor) and the CCAAT box sequence factor [Raghitgama et al., (1997) Plant Mol Biol 34:393-402]; D) copper inducible promotor comprises ACE1 binding site [Mett et al., (1996) Transgenic Res5:105-113.]; E) steroid evoked promoter comprises the expression vector [Schena et al., PNAS 88:10421-10425] of glucocorticoid response factor and encoding mammalian steroid receptor.

In addition, utilization tissue-specific promoter also can obtain specifically expressing, for example, regulate Fd (Ferredorin) promotor [Vorst et al., (1990) Plant Mol Biol14:491-499.] of greenery high level expression and the peroxidase promoter [Wanapu﹠amp that root-specific is expressed; Shinmyo (1996) Ann.NY Acad.Sci.782:107-114]. pollen, flower, fruit and seed specific promoters are suitable for too.These promotors can change the ratio and the efficient of transcribing.

If will turn down expression of gene, can in carrier, embed antisense sequences and suitable promotor; These technology are existing to be published.Nucleic acid molecule or gene fragment can be separated, be synthesized by nature gene source (in that justice or antisense orientation are arranged), also can obtain by suddenling change source or composition sequence or both combinations.

Regulatory factor includes for example transcripting promoter and enhanser or RNA polymerase binding sequence, ribosome binding sequence, ribosome binding sequence (containing the translation initiating signal) etc.In addition, according to different carriers, some other gene example selective marker can be integrated into recombinant molecule.Other regulatory region such as promotor and termination zone also can be used as regulatory factor.Above regulatory factor can be obtained by animal, plant, yeast, bacterium, fungi, virus, bird, insect or other source, comprises synthetic and mutation factor.

Except that the used expression vector of embodiment 28, polypeptide can be inserted in the recombinant nucleic acid molecules of expression system of known bacterium, virus, Mammals, insect, fungi or birds.Utilization can be inserted recombinant molecule in the cell such as the technology such as Agrobacterium tumefaciens mediated conversion, particle-bombardment mediated transformation, directly absorption, microinjection, co precipitation, transfection and electroporation based on cell type.Carrier has retriever vector, adenovirus carrier, dna viral vector and liposome.An amount of structure sequence embeds expression vector, and it comprises the selected marker of transformant.Making up sequence can be embedded on the restriction enzyme site that is obtained by Restriction Enzyme.

A core of the present invention is that the nucleic acid fragment that has nucleic acid molecule of the present invention or be embedded into carrier can be transformed with the cell of expressing AFP in cell and can breed and the identical secretion characteristic of tool.Genome or gene are separated by natural source, and be synthetic or obtained by the combination of sudden change source, composition sequence and these several respects.

Another core of the present invention is to transform nucleic acid molecule of the present invention or the pulsating insertion plasmid of nucleic acid molecule, and the method that makes the method for cell expressing AFP and express polypeptide of the present invention in cell.

Vegetable cell after transcribing can further carry out tissue culture, seed or whole plants.Produce the combination and the existing article report of method of sudden change.

Conversion and plant regeneration in monocotyledons and dicotyledons, have successfully been carried out.Can transform the AFP nucleic acid molecule or be contained the plant (or vegetable cell) that the component of AFP handles and comprise that asparagus, arabidopsis, potato, tobacco, rape belong to (for example canola), cotton, sunflower, strawberry, spinach, lettuce, rice, soybean, wheat, garden grass, highland barley, barley, atriplex, saliconia oat, Chinese sorghum, clover, beet, pepper, lemon, pumpkin, pea, cocoa tree, hemp, coffee tree, sugarcane, cucumber and grape.Tree also can be transformed, and comprises maple, birch, pine tree, eucalyptus, apple tree, pear tree, Japanese plum, apricot, olive, Fructus Fici, lemon, linden, tangerine, red tangerine and natsudaidai here.Ornamental flowering plant for example carnation and rose also can transform gene of the present invention.The pod plant is Semen Caryae Cathayensis for example, English walnut and as the tree etc. can transcribe out the antifreeze protein nucleic acid molecule.The plant of some spices and herbal medicine, for example genseng, ginger, ginkgo, lemonweed, Chinese cassia tree, Beans are cool, pepper and Cuminum cyminum L can be transformed by the antifreeze protein nucleic acid molecule equally.

Outstanding feature of the present invention is that we select that those have transformed and show that the plant of freeze proof active plant tissue cell or culture and expression antifreeze protein and low thirsty tolerance is the regeneration of culture.These plants for example can breed by to the tolerance to low temperature plant being arranged and not having mutual pollination of tolerance to low temperature plant etc.If self pollinating of plant, the not low temperature resistant filial generation gene of homologous can be identified in seed.For example with these plant growings in low temperature environment, use the activity detection of tolerance of genetic marker or low temperature or antifreeze protein etc.Can cultivate through the maturation plant that mutual pollination obtains, grow into that sexual maturity is further pollinated mutually or self pollinates.

Also can insert nucleic acid molecule at some floristics by the cultivating method of backcrossing produces and the low temperature resistant nucleic acid molecule homologous of AFP plant.Can be used as plant hybridization that male parent or maternal and disappearance AFP stand nucleic acid molecule for the plant of AFP nucleic acid molecule autozygote and can produce and hybridize plant, normally its nucleic acid molecule is a heterozygote.Two plant hybridization of AFP resistance nucleic acid molecule autozygote can produce heterozygote, and it is a resistance nucleic acid autozygote.

Nucleic acid molecule of the present invention can be used as mark in the transformation experiment of plant.The low-temperature sensitive plant can transform and have the antifreeze protein nucleic acid molecule, has linked useful nucleic acid molecule, and the plant that has been transformed useful nucleic acid molecule like this can grow under low temperature environment, and on the contrary, unconverted plant can not grow.

Nucleic acid molecule of the present invention also can be used for the only expression of some part of plant.For example, nucleic acid molecule can be only in fruit or the results part express, with improve its under low temperature or freezing environment the storage quality and improve shell or shelf life.Nucleic acid molecule of the present invention can only be expressed at the temperature sensitive position of plant, for example spends, and it is bloomed in spring but frost may take place; Perhaps rhizome, it in the plant that passes the winter to low-temperature sensitive.

Also may command nucleic acid molecule low expression level of the present invention, mainly be nucleic acid molecule to be handled by antisense technology or common the inhibition, to reduce the expression level of wild-type and importing nucleic acid molecule, so just increased plant to cryogenic sensitivity, for example can be by weeds are handled, make it low-temperature sensitive is lowered into motility rate, reach the purpose of removing weeds.Embodiment 35: identical biological function peptide, polypeptide and protein

At present, the present invention not only refers to the protein described by among the class chitinase A FP of Figure 21,22 nucleic acid sequence encoding and the embodiment, and comprise peptide, polypeptide and the protein of the identical plant function of those tools, they and use in AFP have same or analogous freeze proof activity.Identical biological function bioactive peptide, polypeptide and protein refer to that those have one or more peptide, polypeptide and the protein of the same or similar AFP vigor of AFP that detects with the present invention." same or similar AFP vigor " means the ability that has with the same or similar function of AFP of the present invention.Some zones of these peptides, polypeptide and protein inclusion or part with use in identical corresponding zone or the part of AFP, but this is not certain requirement, as long as both show same or analogous AFP activity.Identical being meant with the line up similarity of the matching degree that obtains both of two peptide species (or nucleotide sequence).According to the article of having delivered, for example following GAP or the BestFit software of introducing can calculate identical degree.For example, (claim " sequence A ") if a polypeptide with Figure 21 bright or 22 have 90% identical, so, sequence A is just identical with the corresponding section of polypeptide in Figure 21 or bright or 22, unless sequence A contains up to 10 site mutation, for example deleted or replace per 100 amino acid in polypeptide portion zone in Figure 21 or bright or 22 by other amino acid.Peptide, polypeptide and the protein identical with the AFP biological function can following various mutations bodily form formula occur.

As described in Example 18, the AFP in the application also can be used for the antigen that antibody is prepared, and is used for purifying or indication AFP or other class AFP protein.The existing article of the preparation techniques of mono-clonal and polyclonal antibody is delivered.For example United States Patent (USP) is seen in the preparation of monoclonal antibody and application: 5688681,5688657,5683693,5667781,5665356,5591628,5510241,5503987,5501988,5500345 and 5496705.United States Patent (USP) is seen in the preparation and the application of polyclonal antibody: 5512282,4828985,5225331 and 5124147.The antibody capable of identification AFP is used for screening and contains AFP or the proteic organism of class AFP.Simultaneously, antibody is of value to purifying AFP and class AFP albumen from thick extract.

A) conserved amino acid in the .AFP sequence changes

The active identical peptide of the proteic biological function of AFP, polypeptide and protein contain aminoacid sequence identical with the AFP sequence but that also have part to change.Identical biological function bioactive peptide, polypeptide and protein and spontaneous polypeptide or corresponding section have 40% sequence identical, or higher, and be identical at least about 60%, 80% or 90%.In most cases, at least 97%, 98% or 99% is identical." sequence is identical " determined by GAP or BestFit software package.The BestFit software arrangement goes out the suitableeest part of two similar sequences.The portion homologous algorithm of available Smith and Waterman determines to arrange (1981 Adv.Appl.Math.2:482-489).What GAP software used is the algorithm (1970 J Mol.Biol.48:443-453) of Needlemen and Wunsch.

B). antifreeze protein fragment and mutant

The present invention has comprised AFP fragment and the mutant that has similar or identical AFP vigor with AFP.

C) .AFP fragment

Polypeptide fragment of the present invention has similar or identical vigor with polypeptide.This peptide great majority are made up of at least 5 amino acid, under the best circumstances, are made up of the the 6th to 10,16 to 25,26 to 50 amino acids of polypeptide chain of the present invention.As long as fragment and AFP keep similar or identical AFP enzyme activity, by from the C-end, the terminal or proteinic interior region (or above combination) of N-is removed one or more amino acid and is obtained the AFP fragment.These fragments can be the spontaneous mutation of AFP or the product that the restriction nuclease enzyme of coding nucleotide sequence is handled.Polypeptide fragment can be used for measuring in conjunction with the same compound thing of polypeptide.The method that some articles have been delivered can be identified these segmental agonist and antagonists.

AFP fragment among the present invention and modification must be mostly and generation albumen or respective regions partly have 40% identical sequence, preferably at least 60%, 80%, 90% or 95% naturally.In most of the cases, fragment is with generation albumen or respective regions partly have 97%, 98% or 99% identical sequence, any mensuration that all is fit to the fragment homogeny of GAP or BestFit software naturally.

The present invention also comprises the polypeptide fragment of the similar or identical vigor of some no polypeptide, but they can be used as the research tool that peptide characteristic is analyzed.Embodiment 36:AFP mutant

Sudden change (embodiment 32) or the experimental technique of shearing can be created the mutant of AFP.AFP spontaneous mutation of the present invention also can produce the modification mutant.Aminoacid replacement as a lot of articles have been delivered points to sudden change by the site in the protein engineering and can produce protein modification mutant of the present invention.The combination of these known technologies can be used for amino acid whose replacement, removal and interpolation.For example, the hydrophobic residue of methionine(Met) can be replaced by other hydrophobic residue example L-Ala; Alanine residue can be replaced by more hydrophobic residue example leucine, propylhomoserin short of money or Isoleucine; One aromatic residue example phenylalanine can be replaced by Threonine; One acid negative charge amino acid for example aspartic acid can be replaced by L-glutamic acid; One positively charged amino acid for example Methionin can be by for example arginine replacement of positively charged amino acid.The polypeptide that the mass treatment of use change side chain group is invented can be modified albumen, for example, a hydrogen base is converted into other routine hydroxyl or amino acid group.

The present invention has noticed the amino acid whose peptide of one or more D-and the terminated acetylated amino acid whose peptide of one or more N-has been arranged.The data of having delivered shows that these can use multiple technologies that polypeptide is handled and be beneficial to make up modified peptides (for example modified peptides or polypeptide or protein), they not only have the same or similar biologos of corresponding peptides binding substances of the present invention, and have than the more favourable vigor of peptide in solvability, stability or to aspects such as hydrolysis and proteolysis susceptibilities.Specific embodiment is seen Morgan and Gainor, Ann.Rep.Med.Chem., 24:243-252 (1989).

The present invention also comprises hybrid gene and polypeptide.For example, when the nucleotide sequence of a gene and other nucleotide sequence in conjunction with forming fusogenic peptide.For example the AFP nucleic acid molecule of the AFP that describes in the nucleic acid molecule of useful AFP or other nucleic acid molecule and the code book invention can be connected.Utilize chemosynthesis or other known technology also can produce fusion gene and peptide.

The same with the AFP that generates naturally, mutant maintains similar or identical AFP vigor preferably.Here can carry out the AFP vitality test to this type of modification.

Above-mentioned have the mutant (with generating naturally) of similar or identical AFP vigor to comprise in the present invention (for example: aminoacid addition, removal, replacement or triplicity) by the biotechnology bonded.Embodiment 37: identical biologically active nucleic acid molecule

The present invention includes those and have the nucleotide sequence of identical biological function vigor with sequence shown in Figure 21 or 22 or other sequence.The nucleotide sequence of identical biological function vigor is meant DNA and RNA, and (for example gene DNA, cDNA, synthetic DNA and mRNA nucleotide sequence, encoded peptide, polypeptide and protein have and Figure 21 or 22 or the contained same or analogous AFP vigor of AFP of the present invention.The nucleotide sequence of identical biological function vigor can encode with the present invention in AFP have the identical peptide of part, polypeptide and protein.

A) nucleic acid molecule that conserved amino acid changes among the coding AFP the present invention includes the nucleotide sequence of the identical biological function vigor of the conserved amino acid variation in the coding AFP aminoacid sequence, and these sequences produce silent amino acid to the AFP sequence simultaneously and change.B) nucleotide sequence of replacement, interpolation or the removal of non-conserved amino acid among the coding AFP

The present invention includes those and have the nucleotide sequence of identical biological function vigor with sequence shown in Figure 21 or 22 or other sequence.The nucleotide sequence of identical biological function vigor is meant DNA and RNA (for example gene DNA, cDNA, synthetic DNA and mRNA nucleotide sequence, the replacement of the non-conserved amino acid of encoding, interpolation or removal but have and Figure 21 or 22 or the contained same or analogous AFP vigor of AFP peptide, polypeptide and the protein of the present invention.Fragment or the modification of AFP in DNA and the invention of RNA energy code book.Determine the AFP or the class AFP vigor of these fragments and modification with the above detection method.Nucleotide sequence, fragment and the modification of the identical AFP biological function vigor that the present invention includes should have 40% identical sequence at least with the polypeptide or the matching area that generate naturally, and better at least 60%, 80%, 95%.Best is that fragment has 97%, 98% or 99% identical sequence at least with the polypeptide or the matching area that generate naturally.The present invention includes those and have the nucleotide sequence of identical biological function vigor with AFP shown in Figure 21 or 22.The sequence homogeny adopts Gap or BestFit software to measure.(1) derives from winter naked barley and the DNA of other antifreeze protein.For example: as Figure 21 (a)

Shown in sequence for example had part Nucleotide to insert by nature or artificial mutation or remove,

Thereby changed sequence length.If the encoding sequence length with Figure 21 (b) is decided to be 100%,

The nucleotide sequence length that has identical biological function vigor so only is 60-120%,

Be well about 80-110%.(2) contain part nature or artificial mutation nucleotide sequence (be generally total length 80% or

Few, better 60% or few, preferably 40% or still less), the different amino of they codings

Acid, but its product has and the same or analogous AFP enzyme of antifreeze protein of generation is naturally lived

Power.Mutant DNA encoded protein matter sequence should with the AFP enzyme preface of Figure 21 or 22

Show at least 40%, better 60%, 80%, 90% and 95%, best 97%, 98%

Or 99% identical.The sequence homogeny adopts Gap or BestFit software to measure.

C) hereditary degenerate core nucleotide sequence

Along with the degeneracy of gene codon, we will recognize Figure 21 or 22 and other nucleotide sequence no longer be the unique sequence of freeze proof enzyme, dextranase, chitinase or the smart active polypeptide of West Africa arrowroot of encoding.The present invention includes the nucleotide sequence that the nucleic acid molecule described among those and Figure 21 or 22 has main identical information.Compare with the nucleotide sequence of describing in the research and as the sequence of Figure 21 or 22 coded polypeptides that show, the nucleic acid molecule (containing RNA) that has one or more nucleic acid to change has also been included scope of the present invention in.All nucleotide sequence degeneracy forms of discussing in application form also are contained among the present invention.

The present invention also comprise those have nucleic acid molecule that one or more nucleic acid changes and and Figure 21 or 22 chemofacies is arranged like the amino acid whose but polypeptide of existing one or more amino acid replacement.

D) the several nucleotides sequence of the identical biological function nucleotide sequence coding AFP of hybridization indication is seen Figure 21 or 22 or use and should can determine at an easy rate with described method.Adopt traditional DNA-DNA or DNA-RNA hybridization technique can isolate the identical biological function form of the nucleic acid of coding AFP at an easy rate.Therefore, the present invention also comprises the sequence of hybridizing with one or more chain of Figure 21 or 22, and their complementary sequence and those codings and AFP are that (thing of these sequence expression is peptide, polypeptide and protein to the sequence that same or similar vigor is arranged.These nucleotides sequences are listed under the medium paramount control condition (sees Sambrook etal. (1989) Molecular Cloning:Alaboratory Manual than one or more sequence hybridization in easy and Figure 21 or bright or 22, Second Edition, ColdSpring Harbor, N.Y.).Optimum condition sees that embodiment 25 describes.

The present invention comprises that also the synthetic dna molecule of aminoacid sequence of those and gene DNA, cDNA or code book invention AFP or genetic devolution form (under with the salt and humidity condition described in should using, genetic code is degenerated and caused) and coding are peptide, polypeptide and the proteinic sequence of identical biological function vigor with AFP of the present invention.If still can observe the restraining effect that recrystallize or ice-crystal growth are modified, so above-described nucleotide sequence is considered to have the relative biological function that is equal to AFP gene of the present invention.When the concentration of encoded polypeptides is 0.1g/l or littler, better 1g/l or still less, or better 100mg/l or still less.Embodiment 38: probe

The present invention also comprises the oligonucleotide probe that is obtained by the clone APF nucleic acid molecule among the embodiment 25 or other nucleic acid molecule.Probe is 15 to 30 Nucleotide on length, preferably at least 30 or above Nucleotide.Embodiment 25 has provided the probe of be satisfied with (superior).These probes are that than other superior part it helps distinguishing encode in the plant antifreeze peptide, polypeptide and nucleic acid sequences to proteins, also comprise using polypeptide, peptide and the nucleic acid sequences to proteins of describing identical with the ATPs biological function.Under the control hybridization conditions, oligonucleotide probe can with Figure 21 or 22 or other gene order hybridization of the present invention.Employing just can be invented a nucleic acid molecule of contained peptide by isolating code book in other organism under high control condition and label probe screening by hybridization gene library.Peptide, polypeptide and protein vigor by nucleic acid molecule encoding adopt the dna clone of embodiment 25 descriptions and expression to detect.Expression product is carried out the smart determination of activity of dextranase, chitinase or West Africa arrowroot.

Adopt the amplification of PCR aspect can from other plant, isolate identical AFP biological function gene, identical AFP code cDNA s and synthetic DNA s.According to the aminoacid sequence of Figure 21 or 22, can prepare Oligonucleolide primers, comprise degenerated primer, these primers are in conjunction with the round pcr with reversed transcriptive enzyme, with the identical biological function DNAs that increases and obtain from other organic gene cDNA.

What can select in addition is oligonucleotide, comprises that degeneracy nucleic acid also can be used as probe and screens the cDNA library.The super expression of the allos of embodiment 39:AFP

Use the expression vector among the embodiment 25 can obtain high-caliber protein expression.The plant and the cell culture that have transformed nucleic acid molecule of the present invention are the excellent research instruments.According to the multiple technologies of announcing in the article, adopt plant and cell cultures under study for action.By the carrier transformation cell lines (immobilized cell culture or initiating cell culture) that comprises AFP nucleic acid molecule (or mutant), the expression level that can nucleic acid molecule and the vigor of nucleic acid molecule.A polypeptide of the present invention is used to distinguish the mixture in conjunction with polypeptide.Method in the article can be used for distinguishing the activator and the antagonist of polypeptide.The plant that you can not express AFP in experiment detects the AFP expression of gene.The interior zygote that employing contains the dissimilar genes (or similar AFP, AFP fragment nucleic acid molecule) of AFP gene is transformed into the plant of experimental group, to measure the protein level that produces and the phenotype (phenotype) of function and plant.Polypeptide is of value to the in-vivo analysis of AFP vigor equally.For example, performance albumen can be used for microscope or the research of X ray crystallization topology.Other expression system also is used in the super expression of AFP in the recombinant plant.Embodiment 40: the cloned nucleic acid molecule of the unnecessary AFP of coding in the winter highland barley

The inventive method that adopts embodiment 25 to describe, DNA and the RNA of coding class dextranase and the smart AFP of class West Africa arrowroot in clone's winter highland barley.Adopt the allos monocotyledons, or the probe (embodiment 25) that preferably obtains in the grass, cDNA library can be screened by isolating mRNA generation from the health tissues of low temperature instructionization winter highland barley.This probe (embodiment 25.1) under medium control condition utilizes the Northern trace and the transcript hybridization of low temperature induction highland barley AFP shows its coding dextran or class West Africa arrowroot protamine, and hybridizing definite positive gene by these probes can carry out feature and sequential analysis as what embodiment 25 described.Other expression system as embodiment 27,39 or embodiment 28,29,30,31,34 is described, and cDNAs can be expressed by transforming plant.Identical biological function peptide, polypeptide and protein have been the present invention includes, these AFP modification and class dextranase, the smart identical biological function nucleic acid of AFP tool of class West Africa arrowroot and their nucleotide sequence.The present invention also comprises the probe that is applicable to coding dextranase sample and the smart AFP nucleic acid of class West Africa arrowroot.Embodiment 41: in food or biomaterial are stored as the production of the AFP of additive

Can be with thick extract, part sheet extract or purified peptide, polypeptide, proteinaceous product form are produced peptide, polypeptide or the protein at protokaryon or eukaryotic expression system synthetic coding AFP or identical biological function AFP, are used for food and biomaterial.For example, the either overt or covert 2 invention DNA of Figure 21 can express in plant, obtain total dissolving extract (embodiment 18,24) or extracellular extract ( embodiment 1,15,19 and 20) then from plant, and these extracts can be used for food or biomaterial.By Nidcel Chelation, tobacco brown spot pathogen affinity chromatography, immunoaffinity chromatography, standard liposomal chromatographic technique purified peptide, polypeptide or protein.

If protokaryon or eukaryotic cell lines with the peptide, polypeptide or the protein secreting that produce at growth medium or be kept in the cell, we just can obtain always to dissolve extract so, partial purification extract or purified peptide, polypeptide or protein are to be used for the additive of food or biomaterial.

Here it is particularly particularly detailed to the description of core to have described content of the present invention in detail.In any case, as long as have the people of general understanding all can understand the reason of changing ten thousand times without leaving the original aim or stand, for example, talk about polypeptide, so very clearly be bound to relate to peptide and protein the technical ability in the article.

This specification sheets integrates all publications, patent and patent application at this, and keeps its integrity; Each independent publication, patent or patent application all specifically or individually indicate it to integrate, and keep its integrity.

Reference is for the ease of discussing to known all respects and the invention of this main body about the ability of plant tolerance frost, here according to the call number of following I group, II group and III group reference with reference to several magazine articles.I group: 1.0 authors: Andersson JA, Ashworth EN (1986); Title of article: Streptomycin sulphate, drying and ultraviolet irradiation are to the influence of the ice crystal nucleation that caused by pseudomonas viridiflava; Magazine name: plant physiology; 80 phases, 956 to 960 pages.1.1 author: Cuter AJ, Saleem M, Kendall E, Gusta LV, Georges F, Fletcher GL (1989); Title of article: the winter flounder antifreeze protein can improve the resisting power of plant tissue to cold; Magazine name: plant physiology magazine; 135 phases, 351 to 354 pages.2. author: Duman JG, Morris JP, Castellino FJ (1984); Title of article: take from the rheum officinale honeybee royal, proteinic purification of ice crystal nucleation and the composition of Vespula maculata; 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Title of article: extract proteinic purification of thermal hysteresis and representation feature from a kind of bittersweet nightshade Solanum dulcamara; Magazine name: Biochim.Biophys.Acta, 1206 phases, 129 to 135 pages.11. author: Duman JG, Olsen TM (1993); Title of article: the thermal hysteresis activity of proteins in the plant of bacterium, Mycophyta and various different sorts histories; Magazine name: cryobiology; 30 phases, 322 to 328 pages.12. author: Duman JG, Wu DW, Olsen TM, Urrutia M, Tursman D (1993); Title of article: thermal hysteresis protein; Magazine name: senior cryobiology; 2 phases, 131 to 182 pages, book number: ISBN 1-55938-536-7.13. author: Feinberg AP, Vogelstein B (nineteen eighty-three); Title of article: a kind of is DNA constraint endonuclease fragment label the technology of high specific acitivity with radio isotope; Magazine name: analytical biochemistry; 132 phases, 6 to 13 pages.14. author: Gatschet MJ, Taliaferro CM, Porter DR, Anderson MP, Anderson JA, Jackson KW (1996); 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Title of article: the growth of winter wheat and its are to the mutual relationship between the tolerance of frost; Magazine name: plant physiology; 87 phases, 335 to 344 pages.19. author: Griffith M, Ala P, Yang DSC, Hon W-C, Moffatt BA (1992); Title of article: give birth to the antifreeze protein that forms in winter naked barley leaf; Magazine name: plant physiology; 100 phases, 593 to 596 pages.20. author: Harlow E, Lane D (1988); Title of article: immunoblotting.See: antibody: laboratory manual (E.Harlow and D Lane, eds) 471 to 510 pages, ColdSpring Harbor laboratory, New York.21. author: Hew Cl, Yang DSC (1992); Title of article: the interaction of protein and ice; Magazine name: European biology magazine; 203 phases, 33 to 42 pages.22. author: Hon W-C, Griffith M, Chong P, Yang DSC (nineteen forty-four); Title of article: from winter naked barley (Secale cereale L.) leaf, extract and separate antifreeze protein; Magazine name: plant physiology; 104 phases, 971 to 980 pages.23. author: Hon W-C Griffith M, Mlynarz A, Kwok YA, Yang DSC (nineteen ninety-five); 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Claims (37)

1. from monocotyledonous isolated nucleic acid molecule, a kind of antifreeze protein of this molecule encoding.

2. the molecule in the claim 1 is included in the dna sequence dna among Figure 21 (A), Figure 21 (B), Figure 22 (A) or Figure 22 (B).

3. the molecule in the claim 1, wherein, it is all identical with dna sequence dna in Figure 21 (A), Figure 21 (B), Figure 22 (A) or Figure 22 (B) or part is identical that this sequence comprises at least 40% sequence.

4. the molecule in the claim 1, this molecule is to choose from the one group of nucleic acid that is made of mRNA, cDNA, sense strand DNA, antisence strand dna, single stranded DNA and double-stranded DNA.

5. as the nucleic acid molecule in the claim 2, give same amino acid sequences encoded sequence of nucleic acid molecules.

6. antifreeze polypeptide nucleic acid molecules encoding of giving similar chitinase, this peptide species is the making nucleic acid molecular hybridization from 340 positions to 744 positions and coding strand in Figure 22 (A), its process is by 0.2 * SSC to 2 * SSC in a kind of strictness, in the eluant that 0.1% SDS forms, be to carry out under 42 degrees centigrade the temperature condition in temperature.

7. nucleic acid of giving the antifreeze protein coding of similar chitinase, this antifreeze protein is hybridized at the nucleotide sequences from 541 positions to 745 positions and coding strand from Figure 21 (A), its process is by 0.2 * SSC to 2 * SSC in a kind of strictness, in the eluant that 0.1% SDS forms, be to carry out under 42 degrees centigrade the temperature condition in temperature.

8. in claim 1, with a peptide species of nucleic acid molecule encoding.

9. a kind of process in claim 8 is purified and isolating mimicry.

10. the polypeptide in claim 8, this peptide species has at the aminoacid sequence shown in Figure 21 (a), Figure 21 (b), Figure 21 (c), Figure 21 (d), Figure 22 (a), Figure 22 (b), Figure 22 (c) or Figure 22 (d).

11. it is all or part of identical with the aminoacid sequence shown in Figure 21 (a), Figure 21 (b), Figure 21 (c), Figure 21 (d), Figure 22 (a), Figure 22 (b), Figure 22 (c) or Figure 22 (d) that the polypeptide in claim 8, this peptide species have 40% sequence at least.

12. the cold inductive antifreeze polypeptide of separating from a kind of monocotyledons of a kind of process.

13. separate from a kind of monocotyledons, relevant with pathogenic agent, have a cold inductive antifreeze polypeptide of freeze proof active a kind of process.

14. the polypeptide in claim 12, wherein employed monocotyledons is chosen from Gramineae.

15. the polypeptide in claim 14 wherein uses and chooses free barley, wheat and naked barley, spring naked barley, winter wheat, winter barley and one group of plant that spring, oat was formed.

A 16. mimicry of the polypeptide in claim 12 or claim 13.

17. one have freeze proof active, the polypeptide that is separated, this peptide species choose free barley, wheat and naked barley, spring naked barley, winter wheat, winter barley, winter canola and spring oat and one group of plant forming of kale.

18. the polypeptide in claim 12 or claim 13, this peptide species are chosen one group of enzyme that free chitinase, West Africa arrowroot essence and dextranase are formed.

19. the polypeptide in claim 12 or claim 13, its purposes are choose from following one group of purposes a kind of: produce antifreeze protein, strengthen plant and microorganism to the tolerance of frost, strengthen ability and output, the recrystallization that is suppressed at the ice crystal in biological substance or the foodstuff products, the very low temperature refrigeration of cell, the low-temperature protection of cell, human hematoblastic cold protection and kill tumor cell that the plant, animal and the microorganism that are exposed to the temperature that is lower than zero degrees celsius survives in the open air.

20. a DNA recombinant chou is included in Accessory Right requirement 1 any dna molecular and promotor district among the claim 7, links mutually on function, makes promotor strengthen this dna molecular transcribing in certain host cell.

21. the expression system of a chitinase gene comprises a kind of vector and chitinase cDNA that is embedded in this expression vector of expressing.

22. the expression system in claim 21, expression vector wherein comprises a kind of plastid.

23. the expression system in claim 22, plastid wherein comprise a kind of intestinal bacteria vector.

24. the expression system in claim 23, intestinal bacteria vector wherein comprises pET12a.

25. the expression system in claim 21, expression vector wherein comprises a kind of Pichia vector.

26. the expression system in claim 25, Pichia vector wherein comprises pGAPZ α A.

27. the expression system in claim 21, chitinase dna wherein are included in all or part dna sequence dna among Figure 21 (a), Figure 21 (b), Figure 22 (a), Figure 22 (b).

28. by certain kind of plant, vegetable cell, animal, zooblast, bacterium or yeast in any system reform of claim 1 in the claim 27.

29. a method of expressing chitinase, this method comprises: use a chitinase dna to express vector and transform an expressive host; And cultivation host.

30. the method in claim 29, expressive host wherein are to choose from one group of biology being made up of certain kind of plant, vegetable cell, bacterium, yeast, Mycophyta, protozoon, marine alga, animal and zooblast.

31. at the product of any one expression system of claim 21 in the claim 27, its purposes is choose from following one group of purposes a kind of: produce antifreeze protein, strengthen plant and microorganism to the tolerance of frost, strengthen ability and output, the recrystallization that is suppressed at the ice crystal in biological substance or the foodstuff products, the very low temperature refrigeration of cell, the low-temperature protection of cell, human hematoblastic cold protection and kill tumor cell that the plant, animal and the microorganism that are exposed to the temperature that is lower than zero degrees celsius survives in the open air.

32. the nucleotide sequences of the protein secreting thing in following plant as target, these plants are by forming at coding strand and the complement thereof 60 from position 1 to the position shown in Figure 21 (A).

33. the nucleotide sequences of the protein secreting thing in following plant as target, these plants are by forming at coding strand and the complement thereof 60 from position 1 to the position shown in Figure 22 (A).

34. one kind strengthens freeze proof active method, this method comprises: antifreeze polypeptide and carbohydrate are combined, and with the activity of enhancing antifreeze polypeptide, thus the recrystallization of inhibition ice crystal, and the normal growth situation of adjusting ice crystal.

35. a component comprises that one or more antifreeze proteins and one or more carbohydrates combine.

36. Accessory Right require 8,10 to 15 or claim 17 to 19 in any in the middle of polypeptide and a kind of sugar combine, its purposes is choose from following one group of purposes a kind of: produce antifreeze protein, strengthen plant and microorganism to the tolerance of frost, strengthen ability and the output that the plant, animal and the microorganism that are exposed to the temperature that is lower than zero degrees celsius survive, the recrystallization that is suppressed at the ice crystal in biological substance or the foodstuff products, the very low temperature refrigeration of cell, low-temperature protection, the hematoblastic cold protection of the mankind and the kill tumor cell of cell in the open air.

37. Accessory Right require 8,10 to 15 or claim 17 to 19 in any in the middle of polypeptide, be used for suppressing the generation of certain disease that causes at certain kind of plant, frozen product or any very low temperature biological substance by the low temperature pathogenic agent or damage or spread.

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