CN106148293A - A kind of method improving agronomic characters of cassava and application thereof - Google Patents
A kind of method improving agronomic characters of cassava and application thereof Download PDFInfo
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Abstract
The invention provides the purposes of the polynucleotide of ROS related polypeptide and coding thereof, specifically, described ROS related polypeptide and the polynucleotide of coding may be used for improving the economical character of Maninot esculenta crantz., and the physiological especially extending Maninot esculenta crantz. goes bad, drought resisting and/or cold-resistant etc..Present invention also offers a kind of method improving agronomic characters of cassava and application thereof.
Description
The application is the Application No. 201110369895.2 submitted on November 18th, 2011, and invention entitled " one changes
The method of kind agronomic characters of cassava and application thereof " divisional application.
Technical field
The present invention relates to botany field, in particular it relates to a kind of method improving agronomic characters of cassava and
Application.
Background technology
Along with the increase day by day of whole world fossil energy demand, energy scarcity is the universal difficult problem faced of countries in the world, profit
Carry out biomass liquid fuel research and development with industrial biotechnology and have become as one of whole world focus.
Maninot esculenta crantz. is Euphorbiaceae (Euphorbiaceae) cassava (Manihot) plant.Maninot esculenta crantz. because of its have extraordinary light,
Heat, water utilization rate, the bioenergy yield of unit are is almost higher than other all of raise crop, and has subject range
Extensively, the high characteristic of tuber starch rate, the development and utilization of bioenergy occupies very important position.Utilize deserted mountain,
Maninot esculenta crantz. plantation is carried out in the edge zones such as wasteland, sandy soil ground, can accomplish not strive ground with grain, have again environmental benefit simultaneously, meet life
The thing energy and the long-range strategy of grain-production harmonious development, be conducive to ensureing the grain security of country.Therefore, Maninot esculenta crantz. is as China
Important energy source plant, be responsible for produce per year 500,000 tons of alcohol fuels task, become the head of China's non-grain starch type energy-source plant
One of choosing.
But, the maximum bottleneck that Cassava industry faces is the physiology decay adopting rear tuber.Maninot esculenta crantz. is necessary after gathering in the crops
Processed in 3 days, post-harvest physiology otherwise can be occurred to decay, cause tuber brownization and rot, affect its processing and properties of product, with
Time also bring pressure for starch processing enterprise.What the annual physiology decay owing to adopting rear tuber caused is lost in harvest yield
More than 5%, direct economic loss reaches more than 200,000,000 yuan.
For a small amount of storage root, after harvesting can be by sealing wax immediately, bagging or be processed into dry tablet and suppress physiological
Rotten, but add the cost of raw material, therefore it is not suitable for scale Maninot esculenta crantz. processing enterprise.Gather the last fortnight cane to Maninot esculenta crantz.
Carry out pruning and the post-harvest physiology of storage root can be delayed to a certain extent to go bad, but edible quality and the shallow lake of storage root can be caused
Opaque amount is substantially reduced, and limits the application of the method.
Additionally, Maninot esculenta crantz. can stand certain low temperature, but at a certain temperature, as less than 10 DEG C can occur poor growth,
Cell and cell membrane damage, organ senescence etc. changes.Under extreme drought condition, there is leaves water loss, wilting, even death
Deng.
So far, not yet have a kind of for improving such as: the post-harvest physiology of suppression Maninot esculenta crantz. storage root is rotten, drought resisting, cold-resistant
Deng the method for economical character, therefore agronomic characters of cassava can well be improved in the urgent need to exploitation one is effective in this area
Method.
Summary of the invention
It is an object of the invention to provide a kind of method improving agronomic characters of cassava and application thereof.
Another object of the present invention is to provide a kind of have improve the plant that physiological is rotten.
Another object of the present invention is to provide a kind of and there is drought resisting and/or cold-resistant plant.
Another object of the present invention is to be provided in polypeptide relevant for ROS or polynucleotide for improving agronomic characters of cassava
Purposes.
In a first aspect of the present invention, it is provided that the use of the polynucleotide of the ROS related polypeptide of a kind of separation or its coding
On the way, wherein, described ROS related polypeptide or its coded polynucleotide are SOD, CAT, APX, or a combination thereof, described polypeptide or many
Nucleotide is for improving the economical character of plant.
In another preference, described in be combined as the combination of SOD and CAT, or the combination of SOD and APX；Or SOD, CAT and
The combination of APX three.
In another preference, described ROS related polypeptide has the feature that
Described SOD polypeptide is selected from lower group: (i-a) has the polypeptide of aminoacid sequence shown in SEQ ID NO:2；Or (ii-
A) by aminoacid sequence shown in SEQ ID NO:2 through one or several amino acid residue replacement, lack or add and formed
And the polypeptide derivative by (i-a) with SOD function；
Described CAT polypeptide is selected from lower group: (i-b) has the polypeptide of aminoacid sequence shown in SEQ ID NO:6；Or (ii-
B) by aminoacid sequence shown in SEQ ID NO:6 through one or several amino acid residue replacement, lack or add and formed
And the polypeptide derivative by (i-b) with CAT function；
Described APX polypeptide is selected from lower group: (i-c) has the polypeptide of aminoacid sequence shown in SEQ ID NO:10；Or
(ii-c) by aminoacid sequence shown in SEQ ID NO:10 through one or several amino acid residue replacement, lack or add and
That formed and that the there is APX function polypeptide derivative by (i-c).
In another preference, the polynucleotide of coding ROS related polypeptide have the feature that
The coded polynucleotide of described SOD polypeptide is selected from lower group: (i) has many nucleoside of sequence shown in SEQ ID NO:1
Acid；Or (ii) has and the polynucleotide of complementary shown in SEQ ID NO:1；
The coded polynucleotide of described CAT polypeptide is selected from lower group: (i) has the multinuclear of sequence shown in SEQ ID NO:5
Thuja acid；Or (ii) has the polynucleotide of the sequence with complementary shown in SEQ ID NO:5；
The coded polynucleotide of described APX polypeptide is selected from lower group: (i) has the multinuclear of sequence shown in SEQ ID NO:9
Thuja acid；Or (ii) has the polynucleotide of the sequence with complementary shown in SEQ ID NO:9.
In another preference, the polynucleotide of described ROS related polypeptide or its coding are derived from Euphorbiaceae
(Euphorbiaceae) cassava (Manihot) plant, more preferably derives from Maninot esculenta crantz..
In another preference, described plant is Euphorbiaceae (Euphorbiaceae) cassava (Manihot) plant, more
Goodly for Maninot esculenta crantz..
In another preference, described economical character is selected from lower group: extend the shelf life；Physiological is delayed to go bad；Improve anti-
Drought ability；Improve tolerance to cold；Or a combination thereof.
In a second aspect of the present invention, it is provided that a kind of method improving agronomic characters of cassava, described method includes: improve
The expression of ROS related polypeptide or activity in Maninot esculenta crantz., described ROS related polypeptide is SOD, CAT, APX, or a combination thereof.
In another preference, the described combination being combined as SOD and CAT, or the combination of SOD and APX, or SOD, CAT
With the combination of APX three, and:
Described SOD polypeptide is selected from lower group: (i-a) has the polypeptide of aminoacid sequence shown in SEQ ID NO:2；Or (ii-
A) by aminoacid sequence shown in SEQ ID NO:2 through one or several amino acid residue replacement, lack or add and formed
And the polypeptide derivative by (i-a) with SOD function；
Described CAT polypeptide is selected from lower group: (i-b) has the polypeptide of aminoacid sequence shown in SEQ ID NO:6；Or (ii-
B) by aminoacid sequence shown in SEQ ID NO:6 through one or several amino acid residue replacement, lack or add and formed
And the polypeptide derivative by (i-b) with CAT function；
Described APX polypeptide is selected from lower group: (i-c) has the polypeptide of aminoacid sequence shown in SEQ ID NO:10；Or
(ii-c) by aminoacid sequence shown in SEQ ID NO:10 through one or several amino acid residue replacement, lack or add and
That formed and that the there is APX function polypeptide derivative by (i-c).
In another preference, described economical character is selected from lower group: extend the shelf life；Physiological is delayed to go bad；Improve
Drought-resistant ability；Improve tolerance to cold；Or a combination thereof.
In another preference, described method includes step:
(1) Agrobacterium of expression vector is carried in offer, and described expression vector contains the coded sequence of ROS related polypeptide；
(2) plant cell of Maninot esculenta crantz. or tissue or organ are contacted with the Agrobacterium in step (1), so that described ROS
The coded sequence of related polypeptide proceeds to plant cell, and is incorporated on the chromosome of plant cell；
(3) select to have proceeded to the plant cell of the coded sequence of described ROS related polypeptide or tissue or organ；
(4) plant cell in step (3) or tissue or neomorph are become plant.
In another preference, described SOD polypeptide is selected from lower group: (i-a) has aminoacid sequence shown in SEQ ID NO:2
The polypeptide of row；Or (ii-a) is passed through the replacement of one or several amino acid residue by aminoacid sequence shown in SEQ ID NO:2, is lacked
Lose or add and formed and the polypeptide derivative by (i-a) with SOD function；
Described CAT polypeptide is selected from lower group: (i-b) has the polypeptide of aminoacid sequence shown in SEQ ID NO:6；Or (ii-
B) by aminoacid sequence shown in SEQ ID NO:6 through one or several amino acid residue replacement, lack or add and formed
And the polypeptide derivative by (i-b) with CAT function；
Described APX polypeptide is selected from lower group: (i-c) has the polypeptide of aminoacid sequence shown in SEQ ID NO:10；Or
(ii-c) by aminoacid sequence shown in SEQ ID NO:10 through one or several amino acid residue replacement, lack or add and
That formed and that the there is APX function polypeptide derivative by (i-c).
In should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the present invention and having in below (eg embodiment)
Can be combined with each other between each technical characteristic that body describes, thus constitute new or preferred technical scheme.As space is limited, exist
This tires out the most one by one states.
Accompanying drawing explanation
Drawings below is used for illustrating specific embodiments of the present invention, rather than limits and to be defined by the claims
The scope of the invention.
Fig. 1 shows tri-gene expression doses of MeCu/ZnSOD, MeAPX2, MeCAT1 in PPD generating process.
Fig. 2 shows the expression casette constructed by the present invention: Fig. 2 a shows carrier pCAMBIA1301-P54-MeCu/
The structure of ZnSOD, Fig. 2 b shows that the structure of carrier pCAMBIA1301s-35s-CAT1, Fig. 2 c show carrier
The structure of pCAMBIA1301s-35s-APX2；Fig. 2 d and Fig. 2 e shows the structure of bivalent carrier.
Fig. 3 shows that the expression of transgenic Maninot esculenta crantz., left figure are transgene APX2 and MeCu/ZnSOD transgenic Maninot esculenta crantz.
Relative expression levels；Right figure is transgene CAT and the relative expression levels of MeCu/ZnSOD transgenic Maninot esculenta crantz..
Fig. 4 shows that each plant (before process, WT, SA1, SC2) is through H2O2Coloration result.
Fig. 5 shows the H of the Manihot Esculenta C3 of non-transgenic2O2Content significantly raises, and the Manihot Esculenta of transgenic is (left
Figure) SA1, SA2, SA4, SA6 and (right figure) SC3, SC4, SC11, SC23 are not changed in substantially.
Fig. 6 shows transgenic line (SA1, SA2) and C3 wild-type plant methyl viologen (MV) result.
Fig. 7 shows that plant methyl viologen (MV) processes the chlorophyll content testing result of rear blade.
Fig. 8 shows tapioca root post-harvest physiology, and rotten a situation arises.
Fig. 9 shows use active oxygen probe dyeing tapioca root fluorogram, and left column is 0h testing result, and the right side is classified as 24h detection
As a result, first row is fluorescence picture, and second row is transmission picture, and the 3rd row is synthesising picture.
Figure 10 shows WT lines (WT) and transfer-gen plant (SC4) overall diagram before and after cold treatment and Local map.
Figure 11 shows WT lines (WT) and transfer-gen plant (SC2, SC4, SC11) MDA content before and after cold treatment,
The enzyme of SOD and CAT is lived.
Figure 12 shows before WT lines (WT) and transfer-gen plant (SC2) Osmotic treatment and processes 16 days, 30 days, and multiple
The water growth conditions of 7 days.
Figure 13 shows the leaves water loss after WT lines (WT) and transfer-gen plant (SC2, SC4, SC11) Osmotic treatment
Result.
Figure 14 shows genetic transformation route.
Detailed description of the invention
The present inventor, through extensively in-depth study, establishes a kind of method improving agronomic characters of cassava first, meaning
Other places finds, by the important gene relevant with ROS metabolism process LAN in plant, the successful plant of gene transformation has good
The excellent agronomic characteristics that antibiosis rationality is rotten and drought resisting is cold-resistant.
Specifically, APX2, CAT, and MeCu/ZnSOD gene is proceeded to Maninot esculenta crantz. fragility callus by the present inventor, it is thus achieved that
Regeneration plant significantly improves oxidation resistance, and Maninot esculenta crantz. is hidden rotten (PPD) process of physiological of root and significantly delays, and significantly improves
The anti-cold drought-resistant ability of plant, the important enzyme relevant with ROS metabolism, as CAT, SOD significantly improve, relevant with membrane protective
Mda content be remarkably decreased.
Term
Reactive oxygen species (ROS)
Aerobic cell can produce in metabolic process a series of reactive oxygen species (reactive oxygen species,
ROS), including O2-, H2O2, OH etc..The ROS of middle and high concentration is by cellular oxidation stress inducing cell apoptosis even
Causing it downright bad, the free radical of low concentration can affect a series of signal transduction pathway.Additionally, ROS also can double regulation control some
The apoptosis of tumor cell and propagation, and and free radical and cell signalling between exist internal association, the freedom in body
Base is balanced by the life and death of its concentration change regulation body cell, and except causing apoptosis, downright bad function, ROS is all right
Activating transcription factor, promotes cell proliferation and differentiation.In animal body, ROS is relevant with aging, in plant, and ROS and fruit
Ripe, corrupt and physiological goes bad closely related.
SOD(Super Oxide Dimutase)
SOD i.e. superoxide dismutase, is antioxidase important in organism, is distributed widely in various organism,
Such as animal, plant, microorganism etc..SOD has special physiologically active, is the interior primary material removing free radical of organism.SOD
Superoxide dismutase by multiple common presented in, they are using copper and zinc or manganese, ferrum or nickel as cofactor.Substantially
The superoxide dismutase (Cu/Zn SOD) with copper and zinc is all contained in upper all of eukaryotic cell.Cu/Zn SOD is one
Dimer, molecular weight is 32,500, two subunits are mainly combined by hydrophobic and electrostatic interaction, copper and zinc then with
Histidine side chains on avtive spot forms coordinate bond.Almost all of mitochondrion and many antibacterials (such as escherichia coli) are containing knot
Close the superoxide dismutase (Mn SOD) of manganese.Manganese ion and the side chain of three histidine, the side chain of an aspartic acid and
Individual hydrone or hydroxyl (depending on the oxidation state of manganese) coordination combine.Escherichia coli and some other antibacterial are possibly together with combining ferrum
Superoxide dismutase (Fe SOD), some antibacterials are containing only Fe SOD, and other are containing only Mn SOD, also have some then both of which contain
Having, Fe SOD is also found to exist in the chromatoplast of what plant, and the avtive spot of Mn SOD and Fe SOD has same type of
Amino acid and metallic ion coordination.The structure in the mankind 2 type superoxide dismutase activity site in higher plant, multi-form
The superoxide dismutase location different cellular compartment of what in.Mn SOD is present in mitochondrion and peroxisome；FeSOD
It is predominantly located at chloroplast, but also is able to be detected in peroxisome；Cu/Zn SOD is then positioned protoplasm, Ye Lv
In body, peroxisome and apoplast.
Those skilled in the art can use general method to be measured the activity of SOD.In a preference,
The activity measuring SOD utilizes the reaction of riboflavin and NBT and methionine thereof to be measured, and its reactant mixture can be found in table 2.
CAT (catalase)
Catalase is the marker enzyme of peroxisome, accounts for the 40% of peroxisome enzyme total amount.Peroxidating
Hydrogen enzyme is a kind of enzyme scavenger, is also called catalase, is the desmoenzyme with iron porphyrin as prothetic group.Catalase is prevalent in
In the organism that can breathe, it is primarily present in the chloroplast of plant, mitochondrion, endoplasmic reticulum, the liver of animal and erythrocyte.It can
Promote H2O2Being decomposed into molecular oxygen and water, the hydrogen peroxide in purged body, so that cell protects against H2O2Murder by poisoning, be raw
One of key enzyme of thing defense system.CAT acts on the mechanism substantially H of hydrogen peroxide2O2Disproportionation, it is necessary to have two
H2O2Successively meet with CAT and collide on active center, just can react.H2O2Concentration is the highest, and decomposition rate is the fastest.
Those skilled in the art can use general method to be measured the activity of SOD.In a preference,
Reaction system includes enzyme extraction buffer, and H2O2, under 240nm, measure absorbance, with A in 1min240The enzyme amount of minimizing 0.1 is
One enzyme unit (U) alive.
APX (ascorbate peroxidase enzyme)
Ascorbate peroxidase enzyme (ascorbate peroxidase) is single-minded with ascorbic acid microelectronics donor
The peroxidase that property is strong, is primarily present in plant chloroplast and endochylema it is considered to be remove general H in chloroplast2O2Pass
Key enzyme.The method measuring Specific peroxidase (PPOD) as electron donor with guaiacol can not measure most work
Property, it and superoxide dismutase SOD, hydrogen peroxide CAT, monodehydroascorbate reductase MDARD, double hydroascorbic acids
Reductase HDARD and glutathion reductase GR constitutes the enzyme system in SCAVENGING SYSTEM OF ACTIVATED OXYGEN together.
In the present invention, term " SOD albumen ", " SOD polypeptide ", " SOD enzyme " are used interchangeably, and all refer to have superoxides
The albumen of dismutase activity or polypeptide.In the present invention, term " CAT albumen ", " CAT polypeptide ", " CAT enzyme " are used interchangeably,
All refer to albumen or the polypeptide with catalase activity.In the present invention, term " APX albumen ", " APX polypeptide ", " APX enzyme "
It is used interchangeably, all refers to albumen or the polypeptide with ascorbate peroxidase enzymatic activity.When unspecified, term " SOD
Albumen ", " CAT albumen " and " APX albumen " include wild type and mutein.
As used herein, it is (if crude, former that " separation " refers to that material is separated from its primal environment
Beginning environment is i.e. natural surroundings).If the polynucleotide under the native state in active somatic cell and polypeptide are the most isolated and purified
, but same polynucleotide or polypeptide are as separated with in other materials existed from native state, then be isolated and purified
's.
As used herein, " SOD albumen ", " CAT albumen " and " APX albumen " that term separates refers to described albumen substantially
Without natural other albumen relative, lipid, saccharide or other material.Those skilled in the art can be with the albumen of standard
Albumen described in matter purification techniques.The purest polypeptide can produce single master on non-reducing polyacrylamide gel
Band.
The polypeptide of the present invention can be recombinant polypeptide, natural polypeptides, synthesis polypeptide, preferably recombinant polypeptide.The present invention is many
Peptide can be native purified product, or the product of chemosynthesis, or uses recombinant technique from protokaryon or eucaryon host (example
As, antibacterial, yeast, higher plant, insecticide and mammalian cell) middle generation.According to the host used by recombinant production scheme, this
The polypeptide of invention can be glycosylated, can be maybe nonglycosylated.The polypeptide of the present invention may also include or not include initiateing
Methionine residues.
Present invention additionally comprises the fragment of described albumen, derivant and analog.As used herein, term " fragment ", " derivative
Thing " refer to be kept substantially the identical biological function of the native protein of the present invention or the polypeptide of activity with " analog ".This
Bright polypeptide fragment, derivant or the like can be that (i) has one or more conservative or non-conservative amino acid residue (preferably
Conservative amino acid) polypeptide that is replaced, and such substituted amino acid residue can be to may not be by heredity
Cryptography, or (ii) have the polypeptide of substituted radical in one or more amino acid residues, or (iii) mature polypeptide with
Another compound (such as extending the compound of polypeptide half-life, such as Polyethylene Glycol) merges the polypeptide formed, or (iv)
Polypeptide that additional aminoacid sequence is fused to this peptide sequence and is formed (as targeting sequencing or secretion sequence or be used for purification this
The sequence of polypeptide or proprotein sequence, or fusion protein).According to teaching herein, these fragments, derivant and analog belong to
Scope known to those skilled in the art.In the present invention, this term also includes having and described albumen identical function
Variant form.These variant forms include (but being not limited to): one or more (usually 1-50, preferably 1-30, more
Good ground 1-20, most preferably 1-10) amino acid whose disappearance, insert and/or replace, and add at C-terminal and/or N-terminal
One or several (usually 20 within, within preferably 10, within being more preferably 5) aminoacid.Such as, in ability
In territory, when replacing with similar nature or similar aminoacid, generally will not change the function of protein.The most such as, at C end
End and/or N-terminal interpolation one or several aminoacid generally also will not change the function of protein.This term also includes described egg
White active fragment and reactive derivative.
The variant form of this polypeptide includes: homologous sequence, conservative variant, allelic variant, natural mutation, induction
Mutant, under conditions of high or low rigor can with the albumen coded by the DNA of the DNA encoding sequence hybridization of described albumen,
And utilize polypeptide or the albumen of the antiserum acquisition of anti-described albumen.In addition to the polypeptide of almost total length, present invention additionally comprises
The soluble fragments of described albumen.Generally, this fragment has at least about 10 continuous amino acids of wild type protein sequence, logical
Often at least about 30 continuous amino acids, the most at least about 50 continuous amino acids, the most at least about 80 continuous amino acids,
The most at least about 100 continuous amino acids.
(the most the not changing primary structure) form of modification includes: the chemically derived form such as acetyl of inner or in vitro polypeptide
Change or carboxylated.Modify and also include glycosylation.Modified forms also include having phosphorylated amino acid residue (such as phosphotyrosine,
Phosphoserine, phosphothreonine) sequence.Also include being modified thus improve its anti-Proteolytic enzyme performance or optimize molten
Solve the polypeptide of performance.
In the present invention, " albumen conservative variation's polypeptide " refers to compared with the aminoacid sequence of wild-type protein, have to
Many 10, the most at most 8, the most at most 5, the most at most 3 aminoacid is by the similar or close amino of character
Acid is replaced and is formed polypeptide.These conservative variation's polypeptide carry out aminoacid replacement preferably based on table 1 and produce.
Table 1
| Initial residue | Representational replacement | Preferably replace |
| Ala(A) | Val；Leu；Ile | Val |
| Arg(R) | Lys；Gln；Asn | Lys |
| Asn(N) | Gln；His；Lys；Arg | Gln |
| Asp(D) | Glu | Glu |
| Cys(C) | Ser | Ser |
| Gln(Q) | Asn | Asn |
| Glu(E) | Asp | Asp |
| Gly(G) | Pro；Ala | Ala |
| His(H) | Asn；Gln；Lys；Arg | Arg |
| Ile(I) | Leu；Val；Met；Ala；Phe | Leu |
| Leu(L) | Ile；Val；Met；Ala；Phe | Ile |
| Lys(K) | Arg；Gln；Asn | Arg |
| Met(M) | Leu；Phe；Ile | Leu |
| Phe(F) | Leu；Val；Ile；Ala；Tyr | Leu |
| Pro(P) | Ala | Ala |
| Ser(S) | Thr | Thr |
| Thr(T) | Ser | Ser |
| Trp(W) | Tyr；Phe | Tyr |
| Tyr(Y) | Trp；Phe；Thr；Ser | Phe |
| Val(V) | Ile；Leu；Met；Phe；Ala | Leu |
The polynucleotide of the present invention can be DNA form or rna form.DNA form includes cDNA, genomic DNA or people
The DNA of work synthesis.DNA can be strand or double-strand.DNA can be coding strand or noncoding strand.Encoding mature polypeptide
Polynucleotide include: the coded sequence of an encoding mature polypeptide；The coded sequence of mature polypeptide and various additional coding sequence；
The coded sequence (with optional additional coding sequence) of mature polypeptide and non-coding sequence.
Term " polynucleotide of coded polypeptide " can be the polynucleotide including encoding such peptides, it is also possible to is also to include
Additional code and/or the polynucleotide of non-coding sequence.The invention still further relates to the variant of above-mentioned polynucleotide, it encodes and this
Invention has polypeptide or the fragment of polypeptide, the sum analogous to general Dedekind sum of identical aminoacid sequence.The variant of these polynucleotide can
To be allelic variant or the variant of non-natural generation of natural generation.These nucleotide variants include replace variant,
Deletion variants and insertion variant.As known in the art, allelic variant is the alternative forms of polynucleotide, and it can
Can be the replacement of one or more nucleotide, lack or insert, but will not be from the function of the polypeptide substantially changing its coding.
The invention still further relates to have at least 50%, the most at least between above-mentioned sequence hybridization and two sequences
70%, the polynucleotide of more preferably at least 80% homogeny.The present invention be more particularly directed to many with of the present invention under strict conditions
The interfertile polynucleotide of nucleotide.In the present invention, " stringent condition " refers to: (1) is at relatively low ionic strength and higher temperature
Under hybridization and eluting, such as 0.2 × SSC, 0.1%SDS, 60 DEG C；Or added with denaturant during (2) hybridization, such as 50% (v/v) formyl
Amine, 0.1% calf serum/0.1%Ficoll, 42 DEG C etc.；Or (3) only homogeny between two sequences at least 90% with
On, just hybridize when more preferably more than 95%.
The nucleotide full length sequence of the encoding said proteins of the present invention or its fragment generally can use PCR TRAP, restructuring
The method of method or synthetic obtains.For PCR TRAP, can be according to relevant nucleotide sequence disclosed in this invention, especially
Be open reading frame sequence to design primer, and with commercially available cDNA storehouse or by conventional method institute well known by persons skilled in the art
The cDNA storehouse of preparation, as template, expands and obtains relevant sequence.When sequence is longer, it is often necessary to carry out twice or repeatedly PCR
Amplification, is stitched together the fragment that each time amplifies by proper order the most again.
Once obtain relevant sequence, it is possible to obtain relevant sequence in large quantity with recombination method.This is typically will
It is cloned into carrier, then proceeds to cell, then by conventional method relevant sequence of isolated from the host cell after propagation.
Additionally, can also be used with the method for synthetic to synthesize relevant sequence, when especially fragment length is shorter.Generally, by first synthesizing
Multiple small fragments, are attached obtaining the fragment that sequence is the longest the most again.At present, it is already possible to come by chemosynthesis completely
Obtain the DNA sequence of code book invention albumen (or its fragment, or derivatives thereof).Then this DNA sequence can be introduced this area
In various existing DNA moleculars (or such as carrier) known in and cell.Additionally, sudden change is introduced this also by chemosynthesis
In invention protein sequence.
The present invention also relates to the carrier of the polynucleotide comprising the present invention, and produce through genetic engineering with the carrier of the present invention
Raw host cell, and the method producing polypeptide of the present invention through recombinant technique.
By conventional recombinant DNA technology (Science, 1984；224:1431), the polynucleotide of the available present invention
Sequence can be used to express or produce recombiant protein.In general there are following steps: (1). with the multinuclear of the encoding proteins of the present invention
Thuja acid (or variant), or convert or suitable host cell of transduceing with the recombinant expression carrier containing these polynucleotide；(2).
The host cell cultivated in suitable culture medium；(3). separation, protein purification from culture medium or cell.
In the present invention, polynucleotide sequence can be plugged in recombinant expression carrier.Term " recombinant expression carrier " refers to ability
Bacterial plasmid, phage, yeast plasmid, plant cell virus, mammalian cell virus or other carriers known to territory.Always
It, as long as can replicate in host and stablize, any plasmid and carrier can be used.One key character of expression vector is
Usually contain origin of replication, promoter, marker gene and translation and control element.
Method well-known to those having ordinary skill in the art can be used for building containing protein coding DNA sequence and suitably transcribing/turn over
Translate the expression vector of control signal.These methods include recombinant DNA technology in vi, DNA synthetic technology, In vivo recombination technology etc..
Described DNA sequence can be effectively connected in the suitable promoter in expression vector, to instruct mRNA to synthesize.Expression vector also wraps
Include ribosome binding site and the transcription terminator of translation initiation.
Additionally, expression vector preferably comprises one or more selected marker, to provide for selecting conversion
The phenotypic character of host cell, dihydrofolate reductase, neomycin resistance and the green fluorescence egg cultivated such as eukaryotic cell
In vain (GFP), or for colibacillary tetracycline or amicillin resistance.
Comprise above-mentioned suitable DNA sequence and suitable promoter or control the carrier of sequence, may be used for converting and fit
When host cell, allow it to marking protein.Host cell can be prokaryotic cell, such as bacterial cell；Or low etc.
Eukaryotic cell, such as yeast cells；Or higher eucaryotic cells, such as plant cell.Representative example has: escherichia coli, streptomycete
Genus, Agrobacterium；Fungal cell's such as yeast；Plant cell etc..When the polynucleotide of the present invention are expressed in higher eucaryotic cells, as
Fruit will make to transcribe to be strengthened when inserting enhancer sequence in the carrier.Enhancer is the cis-acting factors of DNA, the most greatly
There are about 10 to 300 base pairs, act on promoter transcribing with enhancing gene.How persons skilled in the art are aware that
Select suitable carrier, promoter, enhancer and host cell.
Can carry out with routine techniques well known to those skilled in the art with recombinant DNA transformed host cell.When host is former
When core biology is such as escherichia coli, the competent cell that can absorb DNA can be gathered in the crops at exponential growth after date, uses CaCl2Method processes, institute
Step generally well-known in the art.Another kind of method is to use MgCl2.Also can be with the side of electroporation if it is required, convert
Method is carried out.When host is eukaryote, can be selected for following DNA transfection method: calcium phosphate precipitation, conventional mechanical methods is such as
Microinjection, electroporation, liposome packaging etc..Convert plant and the method such as Agrobacterium-mediated Transformation or via Particle Bombardment Transformation, example are preferably used
Such as leaf disk method.Plant cell, tissue or organ for converting can use conventional method regeneration plant, thus obtain transgenic
Plant.
The transformant obtained can be cultivated by conventional method, expresses the polypeptide of the coded by said gene of the present invention.According to used
Host cell, culture medium used in cultivation is selected from various conventional medium.Under conditions of being suitable to host cell growth
Cultivate.When after host cell growth to suitable cell density, with suitable method (such as temperature transition or chemical induction)
The promoter that induction selects, is further cultured for a period of time by cell.
Albumen or the polypeptide of restructuring are of use in many ways.Such as with the SOD containing process LAN, and/or CAT, and/or
The Maninot esculenta crantz. of APX albumen delays physiological to go bad, and removes the ROS of fruit, strengthens the ability etc. that the drought resisting of plant is cold-resistant.
The invention still further relates to a kind of method improveing Maninot esculenta crantz., as a kind of optimal way of the present invention, described method includes
Step: (1) provides the Agrobacterium carrying expression vector, and described expression vector contains selected from SOD, CAT, APX any one or more
The DNA encoding sequence of albumen；(2) plant cell or tissue or organ are contacted with the Agrobacterium in step (1), so that this egg
White DNA encoding sequence proceeds to plant cell, and is incorporated on the chromosome of plant cell；(3) select and proceed to described albumen
The plant cell of DNA encoding sequence or tissue；(4) plant cell in step (3) or tissue regeneration are become plant.Wherein,
Any suitable conventional means can be used, including reagent, temperature, pressure condition etc., implement the method.
Below in conjunction with specific embodiment, the present invention is expanded on further.Should be understood that these embodiments are merely to illustrate the present invention
Rather than restriction the scope of the present invention.The experimental technique of unreceipted actual conditions in the following example, generally according to conventional strip
Part such as Sambrook et al., molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory
Press, 1989) condition described in, or according to the condition proposed by manufacturer.
General experimental technique
1. vector construction
With Maninot esculenta crantz. leaf cDNA template, PCR amplification obtains genes of interest SOD, APX, CAT.This gene is inserted
PCAMBI1301 carrier builds bivalent carrier.
2. genetic transformation route
Genetic transformation route sees Figure 14.
3.Real time RT-PCR detects
Extracting total serum IgE with Trizol reagent, description is shown in operation in detail, and the total serum IgE of extraction digests through DNase, then makes
Reverse transcription is carried out with oligo dT (17-19).Reverse transcription gained cDNA dilution 50 times is carried out PCR amplification for template.Real
Time RT-PCR reaction system: 2 × SYBR Green Master Mix (TOYOBO, Code:QPK-201) 10 μ l, 50ng
CDNA, 400nM forward primer, 400nM reverse primer, instrument is Bio-Rad CFX96.Pcr amplification reaction condition is: 95 DEG C,
1min, 95 DEG C of 15sec, 60 DEG C of for 15s, 72 DEG C of 30s, circulate 40 times, with actin as internal reference.
4.CTAB method extracts Maninot esculenta crantz. genome DNA
1) in extract with CTAB buffer, 2% beta-mercaptoethanol is added before experiment；
2) take fresh leaf tissue, be fully ground in liquid nitrogen, in the blade powder ground, add 400 μ L CTAB
In Extraction buffer, reverse mixing gently；
3) 65 DEG C of water-bath 30min, period is shaken gently for several times, so that Extraction buffer is fully contacted with sample；
4) add isopyknic chloroform, mix gently, 12000rpm, centrifugal 10min；
5) draw supernatant, add the dehydrated alcohol of 2 times of volumes, after mixing ,-20 DEG C of precipitation 20min；
6) 12000rpm, 4 DEG C of centrifugal 10min, abandon supernatant；
7) precipitation 70% washing with alcohol 2 times；
8) precipitation is dried up, be dissolved in appropriate amounts of sterilized water, add 1 μ L 10mg/L RNaseA, place 20min for 37 DEG C；
9) DNA obtained now with or-20 DEG C save backup.
5.H2O2The mensuration of content
1) the TCA solution of 1g blade+10ml 0.1% pre-cooling, ice bath grinds, homogenate 12000g, 15min；
2) 1ml supernatant, adds 1ml 100mM phosphate buffer (PBS) (pH7.0), 2ml KI (1mol/L), shakes up,
Standing a moment (10min), 390nm surveys OD value (ultraviolet spectrophotometer)；
3) H is tried to achieve according to standard curve2O2Content.
6.CAT, APX, SOD enzyme activity determination method
1) sample addition 5ml enzyme extraction buffer (50mM phosphate buffer, 1%PVP, 1mM EDTA) of 1g grinds, and 4
DEG C, 10000rpm is centrifuged 20min, takes supernatant, is transferred in EP pipe (about 3-4ml) by enzyme liquid, puts on ice；
2) CAT measures: 2ml reaction system=1.6ml enzyme extraction buffer+0.2ml 0.1mol/L, enzyme liquid+0.2ml
The H2O2 of 0.1mol/L.Adding H2O2 to clock immediately, test tube turns upside down mixing, measures absorbance, read every 1min under 240nm
Several 1 time, survey 5min altogether.The enzyme amount reducing 0.1 with A240 in 1min is 1 enzyme unit (u) alive；
3) APX measures: 2ml reaction mixture=1.8ml enzyme extraction buffer+0.2ml enzyme liquid+0.5mmol/L AsA+
The H of 20ul 0.01mol/L2O2, record 10s and the numerical value of 40s.AsA decrement and enzymatic activity in the unit of account time；
4) SOD measures: chromogenic reaction: take transparency, 5, quality same glass test tube, 3 for measuring, 2 be comparison,
Reagent is added by table 1.
Table 2
After mixing according to table 1, to double-deck black cardboard set shading longer than test tube on 1 comparison shroud, each with other
Pipe is concurrently placed under 4000lx daylight lamp reaction 10min, and (requiring that each pipe irradiation situation is consistent, reaction temperature controls at 25～35 DEG C
Between, suitably adjust the response time depending on enzymatic activity height).When control tube becomes indigo plant, and when sample cell is still yellow, terminate reaction,
Measure the absorbance of each pipe.
SOD activity=(A0-AS) × VT/A0 × 0.5 × FW × V1
The absorbance value of A0: irradiation comparison light pipe；The absorbance value of AS: sample cell；VT: sample liquid cumulative volume (ml)；V1: survey
Timed samples consumption (ml)；FW: sample fresh weight (g)
7.Dab colouring method
Adding 10ml Dab dyeing liquor in 50ml centrifuge tube, take in a certain amount of addition blade, evacuation 10min, by leaf
Sheet is immersed in Dab liquid, is placed under light, observes color change at any time, and dye 6-8h.Outwell dyeing liquor during colour developing, add
95% ethanol, 80 DEG C of decolouring 10min.
8.MV dyes
Glass dish puts 2 filter paper, adds the MV working solution of 25ml 10uM, is placed in a liquid by blade, lasting light
Place according to condition, observe blade change.
9.MDA assay method
1) weigh the examination material 1g shredded, add 2ml 10%TCA and a small amount of quartz sand, be ground to homogenate, then add 8ml TCA
Grinding further, centrifugal (4000 × g) 10min of homogenate, supernatant is sample extracting solution；
2) chromogenic reaction and mensuration draw centrifugal supernatant 2ml (comparison adds 2ml distilled water), add 2ml0.6%TBA
Solution, homomixture reacts 15min on boiling water bath, is centrifuged after cooling rapidly again, take supernatant measure 532nm, 600nm and
Dullness under 450nm wavelength；
3) computing formula: MDA=6.45 × (A532-A600)-0.56 × A450.
10. measuring chlorophyll content
Blade soaks 95% ethanol 12-24h in the dark and extracts pigment, and to blade white, spectrophotometer method is surveyed
Determine OD663, OD646, OD470；
Computing formula is as follows:
Chl a=12.21*A663 2.81A646；
Chl b=20.13*A646 5.03*A663；
Chl a+Chl b=8.02*A663+20.2A646.
11. mitochondrial oxidations react PPD the real time measure
1) tapioca root section, is immersed in 50uM fluorescent probe Dihydrorhodamine123 lucifuge and hatches 10min；2)
Laser confocal microscope is at exciting light: 520nm, launches light: 529nm and observes staining conditions.
Embodiment 1 Maninot esculenta crantz. physiological rotten (PPD) and the relation of ROS
It is a kind of distinctive biological phenomenon of Maninot esculenta crantz. having oxidation to excite that Maninot esculenta crantz. post-harvest physiology goes bad, and relates to a series of life
Reason Biochemical changes, the present embodiment is studied for three genes, investigates Maninot esculenta crantz. physiological rotten (PPD) and APX, CAT, Cu/
The relation of Zn-SOD.
Fig. 1 shows Maninot esculenta crantz. MeCu/ZnSOD, MeAPX2, MeCAT1 gene expression water in PPD generating process after harvesting
Flat, result shows, APX, CAT, Cu/Zn-SOD these three gene raises the most aobvious in Maninot esculenta crantz. post-harvest physiology metamorphic process
Write.
Embodiment 2 cDNA clone and binary vector con-struction
According to MeCu/ZnSOD, MeAPX2, MeCAT1 gene order announced, (NCBI Accession No is respectively as follows:
AY642137, AY973622, AF170272), Cloning of full length sequence is inserted into CaMV35S (constitutive promoter) or P54 (dimension pipe
Bundle specifically expressing) promoters driven binary vector in, convert Maninot esculenta crantz..
1. extracting Maninot esculenta crantz. blade RNA, reverse transcription is cDNA, with cDNA as template, amplifying target genes.
The cDNA sequence of 2.SOD gene is as shown in SEQ ID NO:1；The peptide sequence such as SEQ ID NO:2 institute of its coding
Show；SOD amplimer is as follows:
SEQ ID NO:3 5 '-GCGTCTAGAATGGTGAAGGCCGTTGCTG-3 '
SEQ ID NO:4 5 '-GCGGAGCTCCTATCCTTGCAAACCAAT-3 '
3.CAT1 gene cDNA sequence is as shown in SEQ ID NO:5: the peptide sequence such as SEQ ID NO:6 institute of its coding
Show；CAT1 amplimer is as follows:
SEQ ID NO:7 5 '-GCGGTACCTTCACTTTCTTTGTCATGG-3 '
SEQ ID NO:8 5 '-ATACTGCAGCCCATAGCCTCATCTTCA-3 '
4.APX2 gene order as shown in SEQ ID NO:9, its coding peptide sequence as shown in SEQ ID NO:10,
APX2 amplimer is as follows:
SEQ ID NO:11 P 5 '-CAGGTACCGCTCAGAATCGCAGAA-3 '
SEQ ID NO:12 P 5 '-CTACTGCAGTCCGACCATCATCACA-3 '
5. vector construction: build pCAMBIA1301-P54-MeCu/ZnSOD carrier and pCAMBIA1301s-35s-respectively
CAT1, pCAMBIA1301s-35s-APX2 carrier, its structure is as shown in figs. 2 a-2 c.By EcoRI single endonuclease digestion P54-SOD-Nos
This fragment, then carry by EcoRI single endonuclease digestion pCAMBIA1301s-35s-CAT1 and pCAMBIA1301s-35s-APX2 the two
Body (EcoRI is positioned at before 35S promoter), reconnects fragment and carrier, obtains bivalent carrier, its structure such as Fig. 2 d-2e institute
Show.
The preparation of embodiment 3 transgenic Maninot esculenta crantz. and plant regeneration
Will in embodiment 2 preparation the Agrobacterium-mediated Transformation Maninot esculenta crantz. fragility wound healing containing binary vector, utilize embryo occur and
Adventitious organogenesis regeneration obtain transfer-gen plant and in containing Antibiotic medium screening obtain positive colony.
Embodiment 4 transfer-gen plant gene expression dose detects
Utilize qRT-PCR MeCu/ZnSOD, MeAPX2 and MeCAT1 gene table from molecular level detection transgenic Maninot esculenta crantz.
Reach situation.
Fig. 3 is the expression of transgenic Maninot esculenta crantz., and left figure is transgene APX2 and MeCu/ZnSOD transgenic Maninot esculenta crantz.
Relative expression levels；Right figure is transgene CAT and the relative expression levels of MeCu/ZnSOD transgenic Maninot esculenta crantz..Result shows,
These gene expression doses obtain rise in various degree,
Embodiment 5 transgenic Maninot esculenta crantz. oxidation resistance detects
1. transfer-gen plant and C3 WT lines are through the H of 50mM2O2After processing 24h, Dab dyeing is utilized to observe in blade
H2O2Accumulation degree.
Fig. 4 shows that each plant (before process, WT, SA1, SC2) is through H2O2Coloration result, as we can see from the figure wild type
The blade of plant substantially browning color, illustrates to have accumulated a large amount of H2O2；And rotaring gene plant blade brown is considerably lighter, explanation
H2O2Accumulation is little.
2.H2O2H is detected before and after process2O2Content, Fig. 5 shows the H of the Manihot Esculenta C3 of non-transgenic2O2Content is notable
Raise, and the Manihot Esculenta of transgenic (left figure) SA1, SA2, SA4, SA6 and (right figure) SC3, SC4, SC11, SC23 do not have substantially
Change, i.e. WT content significantly raises, and transfer-gen plant changes not quite substantially.Result illustrates, the antioxidation energy of transfer-gen plant
Power is obviously improved relative to wild type.
3. pair transgenic line and C3 WT lines process at the MV (methyl viologen, ROS derivant) of 100 μMs, 48h
After, transgenic line blade still keeps green, and the blade of wild type the most substantially turns yellow (Fig. 6), and WT is the Maninot esculenta crantz. of non-transgenic
Result after the vanes MV process of plant C3；SA1, SC2 are respectively rotaring gene plant blade result after MV processes.Experiment
Result illustrates, oxidation resistance is significantly increased by transfer-gen plant relative to wild type.
4. measure the content of blade Determination of Chlorophyll before and after MV processes.Fig. 7 shows that plant methyl viologen (MV) processes posterior lobe
The chlorophyll content testing result of sheet, WT chlorophyll content in leaf blades reduces clearly, and transfer-gen plant still maintains higher containing
Amount.
Embodiment 6 transgenic Maninot esculenta crantz. PPD delays ability to observe
1. observe Maninot esculenta crantz. PPD generating process according to CIAT method: cut at the two ends of its tuber immediately, far after Maninot esculenta crantz. results
Axle head preservative film seals, and proximal ends is exposed in air, and tuber places the room of room temperature 21-28 DEG C humidity 70-80%, and one
Sampling of fixing time is observed.From Fig. 8 it is observed that the tuber of transgenic Maninot esculenta crantz. (SA1, SC2) preserves 96h, and PPD does not occurs yet, and
Wild type (WT) substantially occurs rotten, it was demonstrated that transgenic line storage root PPD occurs substantially to delay.
2. simultaneously, the present inventor utilizes fluorescent probe Dihydrorhodamine123 that mitochondrial oxidation reacts to Maninot esculenta crantz.
PPD carries out the real time measure.Fig. 9 shows use active oxygen probe dyeing tapioca root fluorogram, and left column is 0h testing result, and the right side is classified as
24h testing result, first row is fluorescence picture, and second row is projection picture, and the 3rd row is synthesising picture.
Result shows, just the tapioca root of results internal ROS accumulation is considerably less, at the PPD of WT, 24h, tuber thin-walled occurs
Histiocyte have accumulated substantial amounts of ROS, thus produces bright fluorescence signal.And 24h tuber fluorescence signal is more weak in transgenic,
Illustrate not yet to occur PPD, ROS accumulation to be still within normal level.
The anti-cold-working of embodiment 7 is used
Wild type Maninot esculenta crantz. and transfer-gen plant P54::MeCu/ZnSOD-35S::MeCAT1 (SC) grow in the controlled environment chamber
About 1 month, 4 DEG C of cold treatments 4 days.
Figure 10 shows WT lines (WT) and transfer-gen plant (SC4) overall diagram before and after cold treatment and Local map,
Observe wild type Maninot esculenta crantz. after 4 DEG C of cold treatments, the obvious shrinkage of lower blade, damaged to plants caused by sudden drop in temperature phenotype serious；And transfer-gen plant leaf
Sheet still stretches, and the most significantly damages to plants caused by sudden drop in temperature phenotype.
Figure 11 shows WT lines (WT) and transfer-gen plant (SC2, SC4, SC11) MDA content before and after cold treatment,
The enzyme of SOD and CAT is lived, and shows that this transfer-gen plant improves cold toleration.
Plant organ is old and feeble or sustains an injury under adverse circumstance, tends to occur peroxidation of membrane lipids, and malonaldehyde (MDA) is film
The final catabolite of lipid peroxidation, after the position produced from film discharges, reacting with protein, nucleic acid, it is special to modify it
Levy；Make the bridged bond between cellulosic molecule loose, or the synthesis of suppression protein.Film and cell may be caused one by the accumulation of MDA
Fixed injury, MDA content is the highest, then the extent of injury of explanation film and cell is big.
CAT enzyme work is in WT, and after process, specific activity is low before processing, serious owing to being damaged to plants caused by sudden drop in temperature injury in WT, is damaging to plants caused by sudden drop in temperature
Early stage CAT enzyme is lived has rising to remove ROS, processes the injury of later stage whole young plant serious, and the albumen of organism inner cell is badly damaged,
Reduce so enzyme is lived.But in transfer-gen plant, after process, Enzyme activity is higher than before processing, and this is due under cold adverse circumstance environment,
The ROS that the work of CAT enzyme produces in significantly improving to remove organism, keeps normal plants.
Embodiment 8 drought resistance function
Wild type Maninot esculenta crantz. and transfer-gen plant P54::MeCu/ZnSOD-35S::MeCAT1 (SC) grow in the controlled environment chamber
About 1 month, Osmotic treatment 1 month, rehydration 7 days, observe character mutation.
Figure 12 shows before WT lines (WT) and transfer-gen plant (SC2) Osmotic treatment and processes 16 days, 30 days, and multiple
The water growth conditions of 7 days, figure is observed wild type Maninot esculenta crantz. arid 16 days, all leaves water loss are wilted, and transfer-gen plant
Blade is simply wilted by lower blade, and upper blade is opened up the most entirely.Arid 30 days, wild type Maninot esculenta crantz. major part blade withered and yellow fall
Falling, rotaring gene plant blade upper part blade maintains normal condition, and observation rehydration situation of then watering, wild type Maninot esculenta crantz. dehydration is withered
Extremely, transfer-gen plant rehydration reply normal growth.Simultaneously in arid early stage, measure wild type Maninot esculenta crantz. and rotaring gene plant blade
Percentage of water loss relatively.Figure 13 shows that the blade after WT lines (WT) and transfer-gen plant (SC2, SC4, SC11) Osmotic treatment loses
Water result.Result shows, wild type Maninot esculenta crantz. blade percentage of water loss is significantly higher than rotaring gene plant blade.
The all documents mentioned in the present invention are incorporated as reference the most in this application, just as each document by individually
It is incorporated as with reference to like that.In addition, it is to be understood that after the above-mentioned teachings having read the present invention, those skilled in the art can
To make various changes or modifications the present invention, these equivalent form of values fall within the model that the application appended claims is limited equally
Enclose.
Claims (10)
1. the ROS related polypeptide separated or the purposes of polynucleotide for its coding, wherein, described ROS related polypeptide or
Its coded polynucleotide is SOD, CAT, APX, or a combination thereof, it is characterised in that described polypeptide or polynucleotide are planted for improvement
The economical character of thing.
2. purposes as claimed in claim 1, it is characterised in that described in be combined as the combination of SOD and CAT, or SOD and APX
Combination.
3. purposes as claimed in claim 1 or 2, it is characterised in that: described ROS related polypeptide has the feature that
Described SOD polypeptide is selected from lower group: (i-a) has the polypeptide of aminoacid sequence shown in SEQ ID NO:2；Or (ii-a) by
Aminoacid sequence shown in SEQ ID NO:2 through one or several amino acid residue replacement, lack or add and formed and
There is the polypeptide derivative by (i-a) of SOD function；
Described CAT polypeptide is selected from lower group: (i-b) has the polypeptide of aminoacid sequence shown in SEQ ID NO:6；Or (ii-b) by
Aminoacid sequence shown in SEQ ID NO:6 through one or several amino acid residue replacement, lack or add and formed and
There is the polypeptide derivative by (i-b) of CAT function；
Described APX polypeptide is selected from lower group: (i-c) has the polypeptide of aminoacid sequence shown in SEQ ID NO:10；Or (ii-c)
By aminoacid sequence shown in SEQ ID NO:10 through one or several amino acid residue replacement, lack or add and formed
And there is the polypeptide derivative by (i-c) of APX function.
4. purposes as claimed in claim 3, it is characterised in that the polynucleotide of coding ROS related polypeptide have the feature that
The coded polynucleotide of described SOD polypeptide is selected from lower group: (i) has the polynucleotide of sequence shown in SEQ ID NO:1；Or
(ii) have and the polynucleotide of complementary shown in SEQ ID NO:1；
The coded polynucleotide of described CAT polypeptide is selected from lower group: (i) has the polynucleotide of sequence shown in SEQ ID NO:5；
Or (ii) has the polynucleotide of the sequence with complementary shown in SEQ ID NO:5；
The coded polynucleotide of described APX polypeptide is selected from lower group: (i) has the polynucleotide of sequence shown in SEQ ID NO:9；
Or (ii) has the polynucleotide of the sequence with complementary shown in SEQ ID NO:9.
5. purposes as claimed in claim 1, it is characterised in that described plant is Maninot esculenta crantz..
6. purposes as claimed in claim 1, it is characterised in that described economical character is selected from lower group: extend the shelf life；Delay raw
Rationality goes bad；Improve drought-resistant ability；Improve tolerance to cold；Or a combination thereof.
7. the method improving agronomic characters of cassava, it is characterised in that improve expression or the work of ROS related polypeptide in Maninot esculenta crantz.
Property, described ROS related polypeptide is SOD, CAT, APX, or a combination thereof.
8. method as claimed in claim 7, it is characterised in that the described combination being combined as SOD and CAT, or SOD and APX
Combination,
Described SOD polypeptide is selected from lower group: (i-a) has the polypeptide of aminoacid sequence shown in SEQ ID NO:2；Or (ii-a) by
Aminoacid sequence shown in SEQ ID NO:2 through one or several amino acid residue replacement, lack or add and formed and
There is the polypeptide derivative by (i-a) of SOD function；
Described CAT polypeptide is selected from lower group: (i-b) has the polypeptide of aminoacid sequence shown in SEQ ID NO:6；Or (ii-b) by
Aminoacid sequence shown in SEQ ID NO:6 through one or several amino acid residue replacement, lack or add and formed and
There is the polypeptide derivative by (i-b) of CAT function；
Described APX polypeptide is selected from lower group: (i-c) has the polypeptide of aminoacid sequence shown in SEQ ID NO:10；Or (ii-c)
By aminoacid sequence shown in SEQ ID NO:10 through one or several amino acid residue replacement, lack or add and formed
And there is the polypeptide derivative by (i-c) of APX function.
9. method as claimed in claim 7, it is characterised in that described economical character is selected from lower group: extend the shelf life；Delay
Physiological goes bad；Improve drought-resistant ability；Improve tolerance to cold；Or a combination thereof.
10. the method as described in claim 7-9 is arbitrary, it is characterised in that include step:
(1) Agrobacterium of expression vector is carried in offer, and described expression vector contains the coded sequence of ROS related polypeptide；
(2) plant cell of Maninot esculenta crantz. or tissue or organ are contacted with the Agrobacterium in step (1), so that described ROS is correlated with
The coded sequence of polypeptide proceeds to plant cell, and is incorporated on the chromosome of plant cell；
(3) select to have proceeded to the plant cell of the coded sequence of described ROS related polypeptide or tissue or organ；
(4) plant cell in step (3) or tissue or neomorph are become plant.
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| CN2011103698952A CN103122340A (en) | 2011-11-18 | 2011-11-18 | Method for improving agronomic characters of cassava and application of method |
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| CN104094852B (en) * | 2014-07-24 | 2016-04-06 | 广西壮族自治区农业科学院经济作物研究所 | A kind of method of cassava plantlet in vitro strengthening seedling and rooting |
| CN108085333B (en) * | 2016-11-14 | 2021-06-29 | 中国科学院分子植物科学卓越创新中心 | Method for delaying physiological deterioration of potato plants |
| CN109929946B (en) * | 2019-03-07 | 2022-02-11 | 广东出入境检验检疫局检验检疫技术中心 | Kit for quantitatively detecting cassava components based on micro-drop digital PCR and application thereof |
| CN110669744A (en) * | 2019-11-05 | 2020-01-10 | 海南大学 | Cassava ascorbic acid peroxidase gene and construction and application of prokaryotic expression vector thereof |
| CN114703154B (en) * | 2022-03-30 | 2024-01-09 | 云南大学 | Polypeptide, protein containing same and application |
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| WO2006054815A1 (en) * | 2004-11-17 | 2006-05-26 | Korea Research Institute Of Bioscience And Biotechnology | Recombinant expression vector for production of plants having multiple stress tolerances, and method for preparing multiple stress-tolerant plants using the same |
| CN102161996A (en) * | 2011-03-01 | 2011-08-24 | 洪洞县维民生生物科技有限公司 | Jujube tree ascorbate peroxidase gene and application thereof in improving stress resistance of plants |
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| WO2006054815A1 (en) * | 2004-11-17 | 2006-05-26 | Korea Research Institute Of Bioscience And Biotechnology | Recombinant expression vector for production of plants having multiple stress tolerances, and method for preparing multiple stress-tolerant plants using the same |
| CN101061227A (en) * | 2004-11-17 | 2007-10-24 | 韩国生命工学研究院 | Recombinant expression vector for production of plants having multiple stress tolerances, and method for preparing multiple stress-tolerant plants using the same |
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