CN103141498B - Compound for improving crop resistance and application of compound - Google Patents
Compound for improving crop resistance and application of compound Download PDFInfo
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- CN103141498B CN103141498B CN201310060015.2A CN201310060015A CN103141498B CN 103141498 B CN103141498 B CN 103141498B CN 201310060015 A CN201310060015 A CN 201310060015A CN 103141498 B CN103141498 B CN 103141498B
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Abstract
The invention provides a compound for improving wheat (especially winter wheat) resistance (especially cold resistance) and a preparation method of the compound. The compound comprises proline compounds and can be used for cold prevention of the wheat, such as the late spring coldness. Furthermore, the invention further provides a related cold-proof method, a cold-resistance application, a test method, a prepared intermediate and the like.
Description
Technical field
The invention belongs to plant cultivation field, particularly, the present invention relates to improve the proline compounds, preparation of crop resistance and in cold-resistant application of wheat etc.
Background technology
Wheat is one of most important crops in the world, has extensive plantation in northern China and high altitude localities, and wherein winter wheat need to survive the winter, and cold resistance be there are certain requirements.Therefore, the winter wheat of China's popularizing planting is substantially all the cold-resistant kind of artificially breeding at present.
For non-cold-resistant kind, cold-resistant by applying/to freeze agent, can improve the cold resistance of plant, wherein also can use the cold-resistant index of proline as plant endogenous property.For example, Chinese patent application 00128262 discloses cold-resistance agent for soybean, wherein contains the materials such as abscisic acid, can improve the proline content in soybean plant strain; And for example, Chinese patent application 200810010789 discloses plant stress-resistance (comprising freeze proof cold-resistant) derivant, wherein contains the materials such as chitosan oligosaccharide, can improve the proline content in soybean plant strain.In addition, proline also can directly use as the cold-resistant composition of external source.For example, Chinese patent 02100473 discloses degeneration-resistant (comprising the multiple adverse circumstances such as cold-resistant) the special foliage spraying agent of ball protection type cotton, comprises a small amount of proline in its Multiple components; Chinese patent 200610155114 discloses the coating agent for seed for ultra-sweet corn seeds, comprises a small amount of proline as antifreeze activity composition in its Multiple components; Further, Chinese patent 200910042200 discloses the cold-resistant seed soaking agent for rice direct-seeding, comprises a small amount of proline as anti-cryogen in its Multiple components.But, for cold-resistant kind, the especially cold-resistant kind of wheat, these are cold-resistant/freeze agent whether can have cold-resistant/freeze effectiveness, prior art does not provide enlightenment; In addition, because the current winter wheat of China's popularizing planting is substantially all cold-resistant kind, be also considered to there is no need to use extra cold-resistant/freeze agent, more there is no need to use separately active component only has the preparation of cold-resistant composition.
Study for a long period of time and test discovery through the inventor, the cold tolerance of existing cold-resistant wheat breed is mainly reflected in during Wintering Period at present, and after entering spring and turning green, cold tolerance will sharply decline, and will can cause freeze injury to (cold-resistant) wheat if now run into cold in the late spring weather; And, use in advance proline solution, although have certain effect, but cold in the late spring weather generally occurs in after rainfall in spring, proline easily runs off and subtracts effect, and comes just to use at cold in the late spring weather temporarily, and one carrys out weather is difficult to prediction, the 2nd, can bring burden to peasant, the 3rd, easily pollute if add stabilizing agent.So the experience that studies for a long period of time and put into practice in conjunction with the inventor, has obtained unexpectedly a kind of albumen, can more stably improve the cold tolerance of (cold-resistant) wheat, especially in there is cold in the late spring.
Summary of the invention
The technical problem to be solved in the present invention is the cold-resistant preparation of wheat that provides new, and it can improve the cold tolerance of (cold-resistant) wheat, especially in there is cold in the late spring.In addition, the present invention also provides associated Winter protection method, cold-resistant application, test method and has prepared intermediate etc.
Particularly, in first aspect, the invention provides the cold-resistant preparation of wheat, it comprises acceptable auxiliary material on proline compounds and agronomy.Have the wheat of cold-resistant demand mostly to be winter wheat, therefore in the present invention, preferably wheat is winter wheat.In this article, " acceptable auxiliary material on agronomy " refers to the agricultural auxiliary material that does not disturb, even can strengthen proline analogs onset, comprises excipient, thinner, emulsifier, pH adjusting agent etc.In the specific embodiment of the present invention, on agronomy, acceptable auxiliary material is water.Like this, can not use in the situation of stabilizing agent and use, no matter for cost and pollution control, be all very useful.
Preferably the preparation of first aspect present invention is made up of acceptable auxiliary material on proline compounds and agronomy.That is to say, except proline compounds, and except acceptable auxiliary material on agronomy, the preparation of first aspect present invention can not add other materials.For example, the preparation of first aspect present invention can be made up of acceptable auxiliary material on the proline compounds dissociating and agronomy.
In the preparation of first aspect present invention, can directly adopt free proline, under some irrigation method, this can obtain good cold-resistant effect.Preferably adopt at Coldproof protein of the present invention, the in the situation that of bound by theory not, this even can be in albumen proline amount strengthen cold-resistant effect in reducing, the proline that illustrate that the cold-resistant effect of protein should not only derive from (degraded generation), may also relate to other.Therefore, preferably in the preparation of first aspect present invention, proline compounds is that the Coldproof protein as shown in SEQ ID No:1 or its variant provide by amino acid sequence., the preparation of first aspect present invention comprises acceptable auxiliary material on the Coldproof protein of amino acid sequence as shown in SEQ ID No:1 or its variant and agronomy.
In this article, the amino acid sequence of the variant of Coldproof protein is to SEQ ID No:1 disappearance, adds and replace one or several and (be preferably no more than 7, more preferably no more than 5, as be no more than 3) amino acid residue and the sequence of the reservation SEQ ID No:1 antifreeze activity that obtains.Those skilled in the art, by recombinant DNA technology, can prepare the misfolded proteins obtaining through disappearance, interpolation and/or substituted amino acid residue.
Those skilled in the art can derive according to the amino acid sequence of albumen the nucleotide sequence of its coding, preferably codon optimized nucleotide sequence.In the specific embodiment of the present invention, Coldproof protein is nucleotide sequence coded by as shown in SEQ ID No:2.
More preferably in the preparation of first aspect present invention, Coldproof protein provide be by nucleotide sequence the gene as shown in SEQ ID No:2 in yeast, express and supernatant provide., the preparation of first aspect present invention comprise that by the nucleotide sequence gene as shown in SEQ ID No:2 is expressed in yeast and supernatant liquid and agronomy on acceptable auxiliary material.Like this, without purification, further saved cost.
Polynucleotides can be imported in bacterium by various modes well-known to those skilled in the art, make this bacterium express the albumen of described polynucleotide encoding.Described polynucleotides can directly be imported into, for example, utilize the transfered cell such as microsome, particle gun; Also can indirectly be imported into, for example can be by being structured in transfered cell on plasmid vector.The described polynucleotides that import can be incorporated on the genome of cell and express, and expression also can dissociate.Preferably in the present invention, the polynucleotides of encoding proteins can utilize shuttle plasmid to import in bacterium, import in secreting, expressing bacterium (as pichia yeast) as the polynucleotides of Coldproof protein can utilize E. coli-Yeast bacterium shuttle plasmid, in the supernatant of its expression, contain the Coldproof protein of amino acid sequence as shown in SEQ ID No:1.
In second aspect, the invention provides the cold-proof method of wheat, it is included in wheat during Wintering Period uses the preparation of first aspect present invention.In this article, cold-resistant be included in preventative cold-resistant (, cold-proof) before cold arrival and the remedial after cold arrives cold-resistant.For the main trend of cold in foreseeable winter, because current commercially available wheat (winter wheat) all has certain cold tolerance, therefore as long as selection is correctly suitable for wheat (winter wheat) kind of surviving the winter locality, general without extra antifreezing agent, therefore preferably prevent for cold in the late spring.Therefore the method that preferably the present invention second invents is the method for the anti-cold in the late spring of wheat.
Amount and the number of times of using the preparation of first aspect present invention can change to some extent along with the variation of wheat breed, and conventionally in the proline in preparation, amount of application is 1-100g/ mu, be preferably 5-50g/ mu, more preferably 10-20g/ mu, can divide and use for 1 ~ 5 time, preferably divides and uses for 1 ~ 3 time.
In the third aspect, the application of the preparation that the invention provides first aspect present invention in wheat is cold-resistant.For the convenience of using and normalization, preferably wherein cold-resistant is cold-proof; For the prediction difficulty to cold in the late spring, more preferably wherein cold-resistant is anti-cold in the late spring.
In fourth aspect, the invention provides the method for simulation wheat experience cold in the late spring, it comprises:
(1) before wheat is survived the winter, set up greenhouse support, not closed greenhouse of while, makes the temperature in greenhouse identical with environmental temperature;
(2), in wheat overwintering period closed greenhouse, make temperature in greenhouse higher than environmental temperature; With,
(3) closed greenhouse not again in the time that wheat is turned green in advance, makes the temperature in greenhouse identical with environmental temperature.
Aspect the 5th, the invention provides Coldproof protein or its variant, the amino acid sequence of described Coldproof protein is as shown in SEQ ID No:1, the amino acid sequence of the variant of described Coldproof protein is to SEQ ID No:2 disappearance, adds and replace one or several and (be preferably no more than 7, more preferably no more than 5, as be no more than 3) amino acid residue and the sequence of the reservation SEQ ID No:1 antifreeze activity that obtains.
Aspect the 6th, the invention provides the coding Coldproof protein of fifth aspect present invention or the gene of its variant, preferably its nucleotide sequence is as shown in SEQ ID No:2.
Aspect the 7th, the invention provides the carrier of the gene that comprises sixth aspect present invention, preferably E. coli-Yeast shuttle vector.
In eight aspect, the invention provides bacterial classification, especially secreting, expressing bacterium (as, yeast), the gene that it comprises the 6th aspect, or transform or transfection with the carrier of the 7th aspect.
Aspect the 9th, the invention provides the Coldproof protein that comprises fifth aspect present invention or the method for the supernatant of its variant of obtaining, it comprises the bacterial classification of under fermentation conditions cultivating eighth aspect present invention, after filtering, retains supernatant, optionally supernatant is concentrated and/or is dried.
Aspect the tenth, the invention provides ninth aspect present invention method obtain supernatant in the application of preparing in the cold-resistant preparation of wheat, preferably preparation first aspect present invention preparation in application.
The present invention has following beneficial effect: wheat that preparation of the present invention is ignored for people in agent plant cold resistance/freeze is always cold-resistant to be made up again, be particularly useful for the anti-fall cold spell in spring of wheat, particularly Coldproof protein of the present invention is not easy to run off and subtracts effect, has alleviated peasant's burden.In addition, Coldproof protein fermentation costs of the present invention is low, and process is simple, and supernatant directly (dilution) is used, and is convenient to promote.
For the ease of understanding, below will be described in detail the present invention by specific embodiment.It needs to be noted, these descriptions are only exemplary descriptions, do not form limitation of the scope of the invention.According to the discussion of this specification, many variations of the present invention, change are all apparent concerning one of ordinary skill in the art.
In addition, the present invention has quoted open source literature, and these documents are in order more clearly to describe the present invention, and their full text content is all included in and carried out reference herein, just looks like that repeated description is excessively the same in this article for their full text.
Embodiment
Further illustrate by the following examples content of the present invention.As do not specialize, the conventional means that in embodiment, technological means used is well known to those skilled in the art and commercially available common instrument, reagent, can be referring to references such as the manufacturers instructions of " molecular cloning experiment guide (the 3rd edition) " (Science Press), " Microbiology Experiment (the 4th edition) " (Higher Education Publishing House) and corresponding instrument and reagent.
the expression construct of the gene of embodiment 1 Coldproof protein
According to our design, the encoding gene of the synthetic Coldproof protein of student on commission's work biotechnology (Shanghai) limited company, its nucleotide sequence is as shown in the SEQ ID No:2 of sequence table, and the amino acid sequence of coded albumen is as shown in SEQ ID No:1.Then, build the yeast of secreting, expressing according to the operating guidance of " molecular cloning experiment guide " and commercialization reagent used, in brief, the gene that is about to this zytase of coding (has been introduced EcoR I restriction enzyme site) with forward primer as shown in the SEQ ID No:3 of sequence table, after amplification that reverse primer (has been introduced Not I restriction enzyme site) as shown in the SEQ ID No:4 of sequence table, use EcoR I and Not I double digestion, and be connected with T4 DNA ligase with pPIC3.5 plasmid through these two endonuclease digestions (can purchased from Invitrogen company), be transformed in bacillus coli DH 5 alpha.Choose positive colony, extract plasmid wherein, after sequence verification is correct, positive plasmid, with after Sal I linearization for enzyme restriction, is transformed into pichia yeast GS115 strain by electrotransformation, choosing positive Yeast transformant containing on the flat board of G418, return.
the expression of embodiment 2 Coldproof proteins
The positive Yeast transformant that embodiment 1 is obtained is inoculated into 50ml BMGY liquid nutrient medium, and (100mM kaliumphosphate buffer (pH6.0), wherein containing 1% yeast extract, 2% peptone, yeast nitrogenous base 1.34%, vitamin h 4*10
-5%, glucose 2%, glycerine 1%) in, be cultured to OD600 and reach 5.0 in 30 DEG C, 200rpm.
Above-mentioned culture is transferred in 1L BMGY liquid nutrient medium, cultivate 24 hours in 30 DEG C, 200rpm, add 5ml methyl alcohol, then continuing at 30 DEG C, 200rpm cultivates 8 days, within every 24 hours during this time, add 5ml methyl alcohol, ventilation is controlled dissolved oxygen (DO) and is greater than 20%, and to control pH be that 6.0(ammoniacal liquor regulates).After cultivation completes, with 0.22 μ m membrane filtration and retain supernatant, this supernatant detects the wherein Coldproof protein of corresponding 8kDa (wherein proline content accounts for 61%) through SDS-PAGE, finds that its table amount reaches 7g/L, directly freeze drying.When use, directly water is diluted to the use of the Coldproof protein aqueous solution after dissolving.
the anti-fall cold spell in spring test of embodiment 3 wheat
This natural phenomena of cold in the late spring be difficult to prediction, there is no at present suitable field modeling testing standard yet, for this reason, we are in the new new wheat of cold-resistant wheat breed of cultivating of October 19 sowing No. 19, and plot is randomly divided into 6 groups, 2 groups, positive control, negative control, proline 1, proline 2, albumen 1 and albumen etc.Wherein, the cultivation condition of positive controls (chemical fertilizer application etc.) all with reference to Dunhuang kind industry to the sell specification of " new wheat No. 19 ", i.e. field production, does not take green house, by the time wheat start to turn green after (February 27 next year) normally carry out water and fertilizer management; All take greenhouse support after planting for other 5 groups, but not plastic covering film, until December 6 plastic covering film, control temperature of shed is 8-16 DEG C, in the time that January 26 next year, wheat started to have the phenomenon of turning green to occur, just carry out in advance water and fertilizer management (wherein negative control, 1 group of proline and 1 group of employing flood irrigation of albumen, 2 groups of employing sprinkling irrigations of 2 groups of proline and albumen), and opened plastic foil (now outside canopy, temperature is-3-4 DEG C) January 29; In addition, 2 groups of 1 group of proline and proline respectively spray 1.5g/L proline solution once, every mu of 10L January 10 and January 20; 2 groups, 1 group, albumen and albumen also respectively spray the Coldproof protein aqueous solution (in Coldproof protein, 2g/L, amounts to proline content and be about 1.2g/L) prepared containing embodiment 2 once, every mu of 10L January 10 and January 20.Observe stem children's fringe cold mortality and gross yield with respect to positive control.
Result is as shown in table 1, with respect to the cultivation (positive control) of not experiencing cold in the late spring, the each group of generation of the phenomenon of freezing to death and the decline of output all having in various degree of experience cold in the late spring, wherein, do not use the cultivation (negative control) of any cold-resistance agent, cold mortality reaches more than 15%, production declining 40%; And other have used proline or Coldproof protein of the present invention, cold mortality and production declining degree all alleviate to some extent, have especially used Coldproof protein of the present invention, are better than directly using proline; In addition, the mode of irrigating also can affect the cold-resistant effect of proline or Coldproof protein of the present invention, adopt more easily impact (especially for directly use proline) of sprinkling irrigation, this may be to be not easy to be washed away to run off by water (rainwater, irrigation water) to cause because Coldproof protein of the present invention has better adhesive force.
The anti-fall cold spell in spring test data of table 1 wheat
| Group | Cold mortality (%) | Gross yield (kg/ mu) |
| Positive control | - | 506.7 |
| Negative control | 18.0 | 301.1 |
| Proline 1 | 8.3 | 436.0 |
| Proline 2 | 15.9 | 359.4 |
| Albumen 1 | 3.8 | 479.6 |
| Albumen 2 | 4.7 | 460.3 |
<110> Chengdu New Sun Crop Science Co., Ltd.
<120> improves compound and the application thereof of crop resistance
The application of <130> China
<160> 4
<170> PatentIn version 3.5
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<212> PRT
<213> is artificial
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Met Pro Thr Gly Pro Pro Pro Gly Pro Pro Glu Gly Pro Pro Pro Gly
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35 40 45
Pro Pro Lys Gly Pro Pro Pro Gly Pro Pro Glu Gly Pro Pro Pro Gly
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Pro Pro Arg Gly Pro Pro Pro Gly Pro Pro Thr Gly Pro Pro Pro Gly
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Pro Pro Thr Gly Pro
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Claims (9)
1. the cold-resistant preparation of wheat, it comprises acceptable auxiliary material on proline compounds and agronomy, wherein proline compounds is the Coldproof protein of amino acid sequence as shown in SEQ ID No:1.
2. preparation claimed in claim 1, it is made up of acceptable auxiliary material on proline compounds and agronomy.
3. preparation claimed in claim 1, wherein wheat is winter wheat.
4. the cold-proof method of wheat, it is included in wheat during Wintering Period uses the arbitrary described preparation of claim 1-3.
5. method claimed in claim 4, wherein wheat is winter wheat.
6. the application of arbitrary described preparation of claim 1-3 in wheat is cold-resistant.
7. application claimed in claim 6, wherein cold-resistant is cold-proof.
8. application claimed in claim 7, wherein cold-proof is anti-cold in the late spring.
9. application claimed in claim 6, wherein wheat is winter wheat.
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| CN104286026A (en) * | 2014-08-29 | 2015-01-21 | 徐州万国生物能源有限公司 | Plant antifreezing agent |
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| CN1194615C (en) * | 2002-02-05 | 2005-03-30 | 中国农业大学 | Anti-adversity cotton ball protection type foliage spraying agent for cotton |
| CN100397992C (en) * | 2006-12-08 | 2008-07-02 | 浙江大学 | Seed coating agent for raising cold resistance of super sweet corn seed |
| CN101642132B (en) * | 2009-08-27 | 2012-01-25 | 华南农业大学 | Cold-resistant seed soaking agent for direct sowing of rice as well as preparation method and applications thereof |
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Address after: 611630 No. five, No. 35 Industrial Road, Heshan Town, Pujiang County, Sichuan, Chengdu Patentee after: Chengdu new Chaoyang Crop Science Co., Ltd Address before: 611630 No. five, No. 35 Industrial Road, Heshan Town, Pujiang County, Sichuan, Chengdu Patentee before: CHENGDU NEWSUN CROPSCIENCE Co.,Ltd. |