CN105331622B - A method of improve that plant iron is biological reinforced and resistance - Google Patents
A method of improve that plant iron is biological reinforced and resistance Download PDFInfo
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- CN105331622B CN105331622B CN201410347908.XA CN201410347908A CN105331622B CN 105331622 B CN105331622 B CN 105331622B CN 201410347908 A CN201410347908 A CN 201410347908A CN 105331622 B CN105331622 B CN 105331622B
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Abstract
The present invention relates to a kind of biological reinforced methods with resistance of raising plant iron.A kind of new plant stress-resistance gene-sweet potato tonoplast pyrophosphorylation enzyme gene (IBAVP1) is disclosed, can be used for improveing the salt resistance ability of potato plant, drought-resistance ability, resistance to iron deficiency ability.Present invention can apply to stablize the yield of potato plant and expand planting area.
Description
Technical field
The present invention relates to phytobiology field, more particularly it relates to a kind of raising plant iron it is biological reinforced and
The method of resistance.
Background technology
Using sweet potato as the tuber crops of representative, the light, heat, water resource utilization efficiency with superelevation and unit area biology
Amount, content of starch are high, be suitble to plantation in drought and barren soil without with grain strive etc. characteristics, be most to have development prospect at present
Energy crop.
Sweet potato genus Convolvulaceae, sweet potato genus, sweet potato kind, for overgrowing property herbaceous plant.Sweet potato plant can be divided into root, stem, leaf,
The parts such as flower, fruit, seed.Sweet potato is the important source material of a kind of important cereal crops and food processing industry, nutritive value
It is abundant.Therefore, sweet potato variety optimization and cultivation, the research of regeneration techniques have great importance.
In view of sweet potato drought it is barren soil in survival ability, it is contemplated that in sweet potato genome there is with it is barren-resistant relevant excellent
Well-founded is because can help to the degeneration-resistant border of enhancing plant, the ability of impoverishment tolerant to the parsing of its related mechanism, cultivating rich iron, Gao Sheng
The crop of object effective iron content and resistance.
Therefore, this field is provided newly it is necessary to study the excellent genes with resistance in sweet potato with the improvement for plant
Approach.
Invention content
The purpose of the present invention is to provide a kind of biological reinforced methods with resistance of raising plant iron.
In the first aspect of the present invention, a kind of method improving potato plant stress-resistance ability is provided, the method includes：It carries
The expression of IbAVP1 albumen in high potato plant.
In a preference, the anti-adversity ability includes：Salt resistance ability, drought-resistance ability, resistance to iron deficiency ability.
In another preferred example, the IBAVP1 albumen is：
(a) such as SEQ ID NO:The albumen of amino acid sequence shown in 2；Or
(b) by SEQ ID NO:Amino acid sequence shown in 2 is by one or more (such as 1-20；Preferably 1-10；More
Good ground 1-5) replacing, missing or adding for amino acid residue and formed, and with improve plant stress-resistance ability function by
(a) albumen derived from；Or
(c) amino acid sequence that amino acid sequence and (a) are limited has 90% or more (preferably 95% or more；More preferably
98% or more；More preferably 99% or more) the phase same sex and with improve plant stress-resistance ability function polypeptide；Or
(d) the SEQ ID NO with (a) protein function:2 protein fragments.
In another preferred example, the method includes：The polynucleotides for encoding IbAVP1 albumen are transferred to potato plant
In.
In another preferred example, the polynucleotides of the coding IBAVP1 albumen are：
(i) there is SEQ ID NO:The polynucleotides of sequence shown in 1；
(ii) albumen that nucleotide sequence can hybridize and encode with (i) polynucleotide sequence limited under strict conditions
With the polynucleotides for improving plant stress-resistance ability function；Or
(iii) polynucleotide sequence that nucleotide sequence and (i) are limited has 90% or more (preferably 95% or more；More preferably
98% or more ground；More preferably 99% or more) albumen of the phase same sex and coding has the multinuclear glycosides for improving plant stress-resistance ability function
Acid.
In another preferred example, the method includes step：
(i) Agrobacterium for carrying expression vector, the polynucleotides of expression vector IBAVP1 albumen containing coding are provided；
(ii) polynucleotides of the coding IBAVP1 albumen are made to be transferred to potato plant using Agrobacterium.
In another preferred example, the potato plant is sweet potato.
In another aspect of this invention, the genetically modified plants or its filial generation obtained using the above method are provided, with open country
Raw type potato plant is compared, and the anti-adversity ability of the genetically modified plants or its filial generation improves.
In another aspect of this invention, the purposes of a kind of IbAVP1 albumen or the polynucleotides for encoding the albumen is provided, is used
In raising potato plant stress-resistance ability；
Improve H in potato plant+The PPi hydrolysing activities of-PPase (to promote auxin to be transported to root, promote potato
The well developed root system and raising Rhizosphere acidification ability of plant)；
Maintain potato plant chlorophyll content (chlorophyll is avoided to reduce) under drought stress conditions；
It reduces potato plant water content under drought stress conditions and declines degree；
Reduce the decline degree of potato plant chlorophyll content under condition of salt stress；
Improve Na of the potato plant under condition of salt stress+Content；Or
Promote root growth of potato plant under the conditions of iron deficiency.
In a preference, the anti-adversity ability includes：Salt resistance ability, drought-resistance ability, resistance to iron deficiency ability.
In another aspect of this invention, the purposes of a kind of IBAVP1 albumen or the polynucleotides for encoding it is provided, mirror is used as
It is colonized the molecular marked compound of the anti-adversity ability of object.
The other aspects of the present invention are apparent to those skilled in the art due to this disclosure
's.
Description of the drawings
The cross-film domain analysis of Fig. 1, IbAVP1.Blue lines represent the region being located in vacuole, and pink lines represent position
Region in cytoplasm, red boxes represent transmembrane domain.
IbAVP1 copy numbers in Fig. 2, Southern hybridization analysis sweet potato.Sweet Potato Leaf genomic DNA respectively with EcoR V,
Tri- kinds of endonuclease digestions of XbaI, XhoI, M：Marker.
The expression pattern of Fig. 3, IbAVP1 in different tissues.The RT-PCR and Real- that IbAVP1 is expressed in different tissues
Time PCR results.Leaf (L), stem (S), fibrous root (FR), root tuber (TR).
RT-PCR the and Real-time PCR results of IbAVP1 expressions when Fig. 4, NaCl processing.2 months sweet potato basins
Plant seedling 400mmol L-1NaCl handle 0,3,6,12,24,48 hours.
A, the expression in root；
B, the expression in leaf.
RT-PCR the and Real-time PCR results of IbAVP1 expressions under Fig. 5, treatment with mannitol.2 months sweet potatoes
Potted orchard 300mmol L-1Treatment with mannitol 0,3,6,12,24 and 48 hour.
A, the expression in root；
B, the expression in leaf.
The Real-time PCR results of Fig. 6, the lower IbAVP1 expressions of iron deficiency processing.
A, the expression in root；
B, the expression in leaf.
The Southern blotting testing results of Fig. 7, Transgenic Sweet Potato.Label probe is hygromix phosphotransferase
Gene, OE1-OE9：Transgenic line, WT：Wild type.M：marker.
Fig. 8, Transgenic Sweet Potato Real-time PCR testing results.OE1-OE9：Transgenic line, WT：Wild type.
actin：Internal reference
Fig. 9, Transgenic Sweet Potato V-ATPase and H+The active detections of-PPase.
A, Transgenic Sweet Potato V-ATPase activity；
B, Transgenic Sweet Potato tonoplast H+- PPase activity.OE3,4,8：Transgenic line, WT：Wild type.
Data as shown in the figure are the mean+SD of 3 repetitions, * and * * are illustrated respectively under identical treatment conditions
T-test significant differences (p compared with the control<0.05) and it is extremely aobvious
27 days phenotypes of Figure 10, IbAVP1 Transgenic Sweet Potato Osmotic treatment and physiology analysis.
A, the character mutation before and after Osmotic treatment；
B, the variation of chlorophyll content；
C, the variation of relative water content.
OE3,4,8：Transgenic line, WT：Wild type.
Data as shown in the figure are the mean+SD of 3 repetitions, * and * * are illustrated respectively under identical treatment conditions
T-test significant differences (p compared with the control<0.05) and extremely significantly (p<0.01).
Figure 11, IbAVP1 Transgenic Sweet Potato 200mmol L-1NaCl handles character mutation and physiology analysis in 24 days.
A, character mutation before and after salt treatment；
The variation of B, Fv/Fm；
C, the variation of chlorophyll content；
D, Na+The variation of content；
E, K+The variation of content.
OE3,4,8：Transgenic line, WT：Wild type.
Data as shown in the figure are the mean+SD of 3 repetitions, * and * * are illustrated respectively under identical treatment conditions
T-test significant differences (p compared with the control<0.05) and extremely significantly (p<0.01).
Figure 12, IbAVP1 Transgenic Sweet Potato iron deficiency treated character mutation and physiology analysis.
A, iron deficiency treated character mutation；
B, the variation of root system fresh weight；
C, the variation of weight of root system.
OE3,4,8：Transgenic line, WT：Wild type.
Data as shown in the figure are the mean+SD of 3 repetitions, * and * * are illustrated respectively under identical treatment conditions
T-test significant differences (p compared with the control<0.05) and extremely significantly (p<0.01).
Treated the iron content analysis of Figure 13, IbAVP1 Transgenic Sweet Potato iron deficiency.
A, the iron content variation of leaf after iron deficiency processing；
B, the variation of the iron content of root system after iron deficiency processing.
OE3,4,8：Transgenic line, WT：Wild type.
Data as shown in the figure are the mean+SD of 3 repetitions, * and * * are illustrated respectively under identical treatment conditions
T-test significant differences (p compared with the control<0.05) and extremely significantly (p<0.01).
Specific implementation mode
The present inventor passes through in-depth study, it was found that a kind of new plant stress-resistance gene-sweet potato tonoplast pyrophosphorylation
Enzyme gene (IBAVP1), and its coding albumen.The invention also discloses the purposes of this adversity gene, in particular for improveing potato
The salt resistance ability of class plant, drought-resistance ability, resistance to iron deficiency ability.Present invention can apply to stablize the yield of potato plant and expand kind
Growing area domain.
The IBAVP1 albumen (polypeptide) of the present invention can be recombinant polypeptide, natural polypeptides, synthesis polypeptide.The polypeptide of the present invention
Can be native purified product or chemically synthesized product, or using recombinant technique from protokaryon or eucaryon host (for example,
Bacterium, yeast, higher plant, insect and mammalian cell) in generate.
The invention also includes the segment of IBAVP1 albumen, derivative and analogue.As used herein, term " segment ", " spread out
Biology " and " analog " refer to the identical biological function of IBAVP1 albumen for being kept substantially the present invention or active polypeptide.
Polypeptide fragment, the derivative or the like of the present invention can be (i) there are one or multiple conservative or non-conservative amino acid residues
(preferably conservative amino acid) substituted polypeptide, and such substituted amino acid residue can be may not be by
Genetic code encoding, or (ii) polypeptide with substituent group in one or more amino acid residues, or (iii) additional
Amino acid sequence is fused to this polypeptide sequence and the polypeptide that is formed is (such as targeting sequencing or secretion sequence or for purifying this polypeptide
Sequence or proprotein sequence or fusion protein).Belong to this field according to the definition of this paper these segments, derivative and analogue
Range well known to those of skill in the art.
The bioactive fragment of any type IBAVP1 albumen can be applied in the present invention.Herein, IBAVP1 eggs
The meaning of white bioactive fragment refers to whole or the portion for the IBAVP1 albumen that overall length still can be kept as a kind of polypeptide
Divide function.Under normal conditions, the bioactive fragment at least keeps the activity of 50% overall length IBAVP1 albumen.More excellent
Under conditions of choosing, the active fragment can keep overall length IBAVP1 albumen 60%, 70%, 80%, 90%, 95%, 99%,
Or 100% activity.
In the present invention, term " IBAVP1 albumen " refers to the SEQ ID NO with IBAVP1 protein actives:2 sequences it is more
Peptide.The term further includes having and IBAVP1 albumen identical functions, SEQ ID NO:The variant form of 2 sequences.These variations
Form includes (but being not limited to)：Several (it is usually 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10
A, also more preferably such as 1-8,1-5) missing, insertion and/or the substitution of amino acid, and C-terminal and/or N-terminal (especially
N-terminal) addition or lack it is one or several (be usually 1-50, preferably 1-30, more preferably 1-20, most preferably 1-
10, also more preferably such as 1-8,1-5) amino acid.For example, in the art, being carried out with amino acid similar in performance
When substitution, the function of protein is not usually changed.For another example, in C-terminal and/or N-terminal (especially N-terminal) addition one
Or several amino acid will not generally also change the function of protein.The term further includes the active fragment and activity of IBAVP1 albumen
Derivative.
It is any it is high with the described IBAVP1 albumen homologies (such as with SEQ ID NO:The homology of sequence shown in 2 is
50% or higher；Preferably, homology is 60% or higher；Preferably, homology is 70% or higher；Preferably, homology
For 80% or higher；It is furthermore preferred that homology be 90% or higher, such as homology 95%, 98% or 99%) and have
The albumen of IBAVP1 albumen identical functions is also included in the present invention.
In the present invention, " IBAVP1 albumen conservative variation's polypeptides " refer to and SEQ ID NO:2 amino acid sequence is compared,
There are at most 20, preferably at most 10, more preferably at most 5, most preferably at most 3 amino acid are with similar or analogous properties
Amino acid is replaced and forms polypeptide.These conservative variation's polypeptides carry out amino acid substitution preferably based on table 1 and generate.
Table 1
| Amino acid residue | Representative substitution | Preferred substitution |
| 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 invention further relates to the polynucleotide sequences for encoding IBAVP1 albumen of the present invention or its conservative variation's polypeptides.It is described
Polynucleotides can be DNA form or rna form.DNA form includes cDNA, genomic DNA or artificial synthesized DNA.DNA
It can be single-stranded or double-strand.DNA can be coding strand or noncoding strand.The coding region sequence of encoding mature polypeptide can be with
With SEQ ID NO:Coding region sequence shown in 1 is identical or the variant of degeneracy.As used herein, " variant of degeneracy "
Referring to coding in the present invention has SEQ ID NO:2 protein, but with SEQ ID NO:Coding region sequence shown in 1 has difference
Other nucleic acid sequence.Preferably, selection IBAVP1 genome sequences (SEQ ID NO:1, while including exon and introne)
Or the variant (variant containing degeneracy) of the sequence for improve stress resistance of plant (including：Salt resistance ability, drought-resistance ability are resistance to
Iron deficiency ability).
Encode SEQ ID NO:The polynucleotides of 2 mature polypeptide include：The coded sequence of encoding mature polypeptide；It is ripe
The coded sequence of polypeptide and various additional coding sequences；The coded sequence (and optional additional coding sequence) of mature polypeptide and
Non-coding sequence.
Term " polynucleotides of coding polypeptide " can be the polynucleotides for including coding said polypeptide, can also be also to wrap
Include the polynucleotides of additional code and/or non-coding sequence.
The invention further relates to the variant of above-mentioned polynucleotides, coding has the more of identical amino acid sequence with the present invention
The segment of peptide or polypeptide, analogs and derivatives.The variant of this polynucleotides can be the allelic variant naturally occurred or
The variant that non-natural occurs.These nucleotide variants include substitution variants, Deletion variants and insert variation.Such as this
Known to field, allelic variant is the alternative forms of a polynucleotides, it may be one or more nucleotide substitution,
Missing or be inserted into, but not from substantially change its encode polypeptide function.
The invention further relates to the multinuclear glycosides for hybridizing and having between two sequences at least 80% phase same sex with above-mentioned sequence
Acid.The present invention is more particularly directed under strict conditions with the interfertile polynucleotides of polynucleotides of the present invention.In the present invention,
" stringent condition " refers to:(1) compared with the hybridization and elution under low ionic strength and higher temperature, such as 0.2 × SSC, 0.1%SDS,
60℃；Or (2) when hybridizing added with denaturant, such as 50% (v/v) formamide, 0.1% calf serum/0.1%Ficoll, 42 DEG C
Deng；Or the phase same sex of (3) only between two sequences at least just hybridizes at 90% or more, more preferably 95% or more.And
And the polypeptide of interfertile polynucleotide encoding and SEQ ID NO:Mature polypeptide shown in 2 have identical biological function and
Activity.
The present invention IBAVP1 protein nucleotides full length sequence or its segment usually can use PCR amplification method, recombination method or
Artificial synthesized method obtains.For PCR amplification method, can especially be opened according to related nucleotide sequence disclosed in this invention
It puts reading frame sequence and carrys out design primer, the commercially available libraries cDNA are used in combination or by prepared by conventional method well known by persons skilled in the art
The libraries cDNA as template, amplification and related sequence.
The present invention also relates to the carriers for including the polynucleotides, and with the carrier or IBAVP1 encoding histones
The genetically engineered host cell of sequence.
In the present invention, IBAVP1 protein polynucleotides can be plugged into recombinant expression carrier." recombinant expression carries term
Body " refer to bacterial plasmid well known in the art, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus or other
Carrier.As long as in short, can replicate and stablize in host, any plasmid and carrier can be used.One weight of expression vector
It is characterized in usually containing replication orgin, promoter, marker gene and translation control element.
The carrier for including above-mentioned appropriate DNA sequence dna and appropriate promoter or control sequence can be used for converting suitable
When host cell, allow it to expression protein.Host cell can be prokaryotic cell, such as bacterial cell；Or it is low
Eukaryocyte, such as yeast cells；Or higher eucaryotic cells, such as plant cell.Representative example has：Escherichia coli, streptomycete
Belong to, Agrobacterium；Fungal cell's such as yeast；Plant cell etc..
When the polynucleotides are expressed in higher eucaryotic cells, if will when being inserted into enhancer sequence in the carrier
Transcription is set to be enhanced.Enhancer is the cis-acting factors of DNA, generally about there is 10 to 300 base-pairs, acts on startup
Son is to enhance the transcription of gene.
Persons skilled in the art are aware that how to select carrier, promoter, enhancer and host cell appropriate.
It can be carried out with routine techniques well known to those skilled in the art with recombinant DNA conversion host cell.Converting plant can
Use the methods of Agrobacterium-mediated Transformation or via Particle Bombardment Transformation, such as spraying, leaf disk method, Rice Young Embryo conversion method etc..
Research shows that overexpression arabidopsis AVP1 genes significantly improve the salt tolerance of transgenic Arabidopsis plants and resistance to
Drought, and in sweet potato AVP1 and its function also not studies have reported that, and the present invention has inquired into AVP1 and has efficiently been utilized with iron for the first time
Between relationship, it is further clear and improve the mechanism such as absorption, transhipment, metabolism, storage of iron in plant, to solve global lack
The bad disease of melt quality provides new strategy and product, cultivates the crop of rich iron, high biologically effective iron content and resistance.
The present invention provides the purposes of the IBAVP1 albumen or its encoding gene, the degeneration-resistant energy for improving plant
Power；Also, the IBAVP1 albumen can be additionally used in：Improve H in potato plant+The PPi hydrolysing activities of-PPase are (to promote
Auxin is transported to root, is promoted the well developed root system of potato plant and is improved Rhizosphere acidification ability)；Maintain potato plant in arid
Chlorophyll content (chlorophyll is avoided to reduce) under stress conditions；It reduces potato plant water content under drought stress conditions and declines journey
Degree；Reduce the decline degree of potato plant chlorophyll content under condition of salt stress；Potato plant is improved under condition of salt stress
Na+Content；Promote root growth of potato plant under the conditions of iron deficiency.
The invention further relates to a kind of methods of improvement plant, and this method includes improving IBAVP1 albumen in the potato plant
Expression.After the purposes for knowing the IBAVP1 albumen, a variety of methods well known in the art may be used to carry
The expression of IBAVP1 albumen described in height.For example the expression of IBAVP1 genes can will be carried by approach known to those skilled in the art
Unit (such as expression vector or virus etc.) is delivered on target spot, and is allowed to the IBAVP1 albumen of expression activity.
Preferably, a kind of method of prepare transgenosis plant is provided, including：
(1) coded polynucleotide of the IBAVP1 albumen of external source is transferred to plant tissue, organ or tissue, is transformed into
Plant tissue, organ or the seed of the coded polynucleotide of IBAVP1 albumen；With
(2) plant tissue of the coded polynucleotide for being transferred to external source IBAVP1 albumen that obtains step (1), organ or
Seed regenerates plant.
Other IBAVP1 genes or the method for its homologous gene expression of increasing are well known in the art.For example, use can be passed through
Strong promoter drives to enhance the expression of IBAVP1 genes or its homologous gene.Or pass through enhancer (such as rice waxy bases
Because of First Intron, Actin gene First Introns etc.) enhance the expression of the IBAVP1 genes.Suitable for the method for the present invention
Strong promoter include but not limited to：35s promoters, rice, corn Ubi promoters etc..
Any conventional means appropriate, including reagent, temperature, pressure condition etc. can be used to implement the method.
Moreover, it relates to using IBAVP1 albumen or its encoding gene as a kind of genetic transformation progeny of plants
Tracking label.The invention further relates to using IBAVP1 albumen or its encoding gene as a kind of molecular labeling, pass through detection plant
The expression of middle IBAVP1 albumen, anti-adversity, yield height, stomatal frequency of plant identification etc..And utilize IBAVP1
The screening that albumen or its encoding gene are improved the breed.
In the specific implementation mode of the present invention, the present inventor is cloned into IbAVP1 genes from sweet potato, and converts potato
Plant inquires into IbAVP1 Transgenic Sweet Potatos with high salt, dry with the methods of plant physiology, science of heredity, molecular biology means
Tolerance under the conditions of drought and iron deficiency, improves the drought-enduring and resistance to iron deficiency ability of sweet potato salt tolerant, to cultivate rich iron and high biological effectiveness
The crop of iron content and resistance theorizes foundation and application foundation, and promotes Sweet Potato Breeding process, pulls Sweet Potato Industry
The effective means of the development of change；To promote Sweet Potato Industry to play the work of bigger in national food security and sustainable development
With.
Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention
Rather than it limits the scope of the invention.In the following examples, the experimental methods for specific conditions are not specified, usually according to conventional strip
Part such as J. Pehanorm Brookers etc. is write, Molecular Cloning:A Laboratory guide, the third edition, Science Press, the condition described in 2002, or
According to the normal condition proposed by manufacturer.
The Cloning and sequence analysis of embodiment 1, IbAVP1
It is cloned into H+-PPase genes IbAVP1 from sweet potato (Xu-shu No.22, the kind are obtained from Xuzhou sweet potato center) blade
(SEQ ID NO:1；GenBank：JN688962.1), the total 2644bp of cDNA overall lengths.The long 2304bp of sequence analysis open reading frame,
Predictive coding albumen (SEQ ID NO:2；GenBank：AFQ00710) contain 737 amino acid residues, molecular weight 77.7kDa,
Isoelectric point 5.84.With TMHMM softwares (http://www.cbs.dtu.dk/services/TMHMM/) IbAVP1 albumen is carried out
Hydrophobicity analysis finds that IbAVP1 has 14 hydrophobic transmembranes (Fig. 1).
There are one inorganic pyrophosphate catalyzed combination site (DVGADLVGKVE) and an I types H for IbAVP1 albumen+-PPase
The conservative sequence (EYYTS) of albumen, so speculating that IbAVP1 belongs to the I types H for relying on potassium ion activation+- PPase albumen.
For the copy number of IbAVP1 in analysis sweet potato genome, disappeared respectively with EcoR V, XbaI and tri- kinds of restriction endonucleases of XhoI
Change sweet potato genome, Southern hybridization is carried out using the 3 '-non-conservative area's segments in end as probe.Occurs 1 hybrid belt after hybridization
(Fig. 2), therefore speculate that IbAVP1 is likely to single copy.
The expression pattern analysis of embodiment 2, IbAVP1
The tissue expression pattern for studying gene is significant for the function of annotating gene.Exist for research IbAVP1 genes
Expression pattern in different tissues, the tissue for taking sweet potato (Xu-shu No.22, the kind are obtained from Xuzhou sweet potato center) different, including leaf
(L), stem (S), fibrous root (FR), root tuber (TR) etc..The Real-time PCR carried out with IbAVP1 specific primers.The results show that
The gene is expressed relatively by force in root tuber, and weaker (Fig. 3) is expressed in stem.
The IbAVP1 specific primer sequences of Real-time PCR applications are as follows：
FP：5'-GAGTCGCTTATTGAGGAGGAGG-3'(SEQ ID NO:3)；
RP：5'-AGAGCCCAGGAAAACAAAGATC-3'(SEQ ID NO:4).
The expression regulation of IbAVP1 under embodiment 3, Different stress
For expression regulations of the research IbAVP1 under stress, 400mmol L are used respectively-1NaCl and 300mmol L-1Sweet dew
Alcohol handles 2 months big sweet potato (Xu-shu No.22, the kind are obtained from Xuzhou sweet potato center) Potted orchards 0,3,6,12,24 and 48 hour.
RT-PCR with Real-time PCR results are consistent, show that the expression of IbAVP1 is regulated and controled by various abiotic stress processing.
400mmol L-1When NaCl processing, the expression quantity of IbAVP1 reaches peak after 3 hours in root, then gradually drops
It is low, it is preferably minimized after 24 hours, then rises (Fig. 4 A).The expression quantity of IbAVP1 reaches highest after handling 6 hours in leaf
Value then declines (Fig. 4 B).
300mmol L-1When treatment with mannitol, the expression quantity of IbAVP1 reaches peak after 3 hours in root, then gradually
It reduces, is preferably minimized after 24 hours, rise (Fig. 5 A) after 48 hours.In leaf the expression quantity of IbAVP1 processing 3 hours after on
Tune reaches peak, then rapid to decline, and minimum, then gradually up-regulation (Fig. 5 B) was dropped at 12 hours.
When iron deficiency processing, in root and leaf the expression of IbAVP1 induced, reach peak after 12h, it is then small 24
When relatively low (Fig. 6).
The IbAVP1 specific primer sequences of RT-PCR applications are as follows：
FP：5'-GAGTCGCTTATTGAGGAGGAGG-3'(SEQ ID NO:5)；
RP：5'-AGAGCCCAGGAAAACAAAGATC-3'(SEQ ID NO:6).
Embodiment 4, IbAVP1 are overexpressed the acquisition of Transgenic Sweet Potato
Build IbAVP1 over-express vectors：Using sweet potato cDNA as template, PCR amplification IbAVP1 genes, primer is as follows：
IbAVP1FP：5'-ATAGGATCCATGGTTGCGGCGACGAT-3'(SEQ ID NO:7)；
IbAVP1RP：5'-CAGCCATGGTCAAAAGATCTTGAAGAG-3'(SEQ ID NO:8)；
PCR product is connected in pMD18-T, and after sequence verification, gene connects sequence through I/Pst of BamH, I restriction enzyme sites
I sites I/Pst of BamH onto p1301 (pCAMBIA1301) plant vector form whole expression vector p1301-35S::
IbAVP1。
By expression vector (pCAMBIA1301-35S::IbAVP1) (Tai'an academy of agricultural sciences) No. 6 in Transformation of sweet potato Thailand,
Southern blotting verification displays：Eight strains are single copies (OE1, OE3-OE9), and one is double copy (OE2) (figures
7).The expression quantity of Real-time PCR detections IbAVP1 shows that the expression quantity of OE1, OE2 reduce, and OE5's is almost unchanged, other
Strain expression quantity increases (Fig. 8).The OE3 that single copy and expression quantity increase is chosen, OE4, OE8 do follow-up further research.
Embodiment 5, V-ATPase and H+The Activity determination of-PPase
To further confirm that overexpression IbAVP1 gene pairs the sweet potatoes ATPase and H of the preparation of embodiment 4+- PPase is active
It influences, the tonoplast of extraction Transgenic Sweet Potato strain OE3, OE4 and OE8 detect ATPase and H+- PPase activity.
V-H+- ATPase and V-H+The Phos amount that the hydrolysing activity of-PPase is discharged with their hydrolysising ATPs and PPi respectively
To indicate.V-H+- ATPase activity is that with nothing in reaction system and have 50mmol L-1NO3 –When V-H+The difference table of-ATPase activity
Show, because of NO3 –Inhibit tonoplast ATPase hydrolysing activity.V-H+- PPase activity be in reaction system with and without 50mmol L- 1V-H when KCl+The difference of-PPase activity indicates, because tonoplast pyrophosphatase hydrolysing activity is K+Dependence.
Measuring H+Contain 25mmol L in 500 μ L reaction solutions of-ATPase hydrolysing activities-1HEPES/Tris (pH7.5),
50mmol L-1KCl, 2mmol L-1MgSO4, 0.1mmol L-1(NH4)2MoO4, 0.1mmol L-1Na3VO4, 0.5mmol L- 1NaN3, 0.01% (V/V) Triton X-100 and 3mmol L-1ATPNa2, 10 μ g memebrane proteins are added and start reaction.Reaction temperature
It it is 37 DEG C, 30 minutes reaction time was added 0.1mL20%TCA and terminates reaction.After reaction be added phosphorus molybdenum orchid developing solution according to
Lin and Morales[40]Method measure reaction solution in Phos content.
H+Contain 25mmol L in the reaction solution that-PPase hydrolysing activities measure-1HEPES/Tris (pH7.5), 50mmol
L-1NaNO3, 2mmol L-1MgSO4, 0.1mmol L-1(NH4)2MoO4, 0.1mmol L-1Na3VO4, 0.5mmol L-1NaN3,
0.01% (V/V) Triton X-100 and 3mmol L-1Na4PPi, reaction process and coloration method and H+The active surveys of-ATPase
It is fixed identical.
By the way that the active detections of the special V-ATPase of tonoplast, tonoplast purity is 52-63%.Sweet Potato Leaf
ATPase and H+- PPase activity is as shown in figure 9, in wild type and Transgenic Sweet Potato, and the activity of V-ATPase is without significant difference
(Fig. 9 A), and the H of Transgenic Sweet Potato+The PPi hydrolysing activities of-PPase are significantly higher than wild type, in the higher strain of expression
In OE4 and OE8, H+The PPi hydrolysing activities of-PPase are higher by 132%, 79.6% (Fig. 9 B) than control wild type respectively.
On tonoplast, H+- PPase is a kind of proton pump of electricity production, can play hydrolysis as substrate using PPi and generate
Free energy is H+Across tonoplast film transhipment provides energy, while can be acidified vacuole.V-H+- PPase can also promote auxin
(mainly IAA) to the polar translocation of root, to promote the development of transfer-gen plant root system.It is overexpressed AVP1 and changes growth
The expression and distribution of plain transportation power P-ATPase contents and auxin Transport Carriers albumen PINl, to promote growth
Transport of the element to root, causes the root system of transfer-gen plant more flourishing and Rhizosphere acidification ability improves.
Embodiment 6, IbAVP1 are overexpressed the analysis of Transgenic Sweet Potato resistance
6.1IbAVP1 is overexpressed Transgenic Sweet Potato Drought Stress Tolerance Analysis of A
Osmotic treatment：The soil of phase homogenous quantities, Osmotic treatment incipient stage, the consistent potting of growth selection are poured into before plantation
Plant measures soil relative water content, and soil moisture in each basin is kept unanimously to stop watering afterwards (Osmotic treatment starts).Arid
Experiment carries out in glasshouse environment, selects 7-9 months, records daily weather condition, timing photograph observation phenotype.
Before Osmotic treatment, wild type and transfer-gen plant growing way are normal (Figure 10 A)；At 2 months big sweet potato seedling arids
Reason 27 days, as a result, it has been found that wild type growth, by serious inhibition, blade wilting falls off, and lobe numbers are considerably less than transgenosis plant
Strain (Figure 10 A).
It measures the chlorophyll content of wild type and transgenic line under non-drought stress and drought stress to change, method：It takes
Sample to be tested blade, distilled water flushing is clean, and blotting paper suck dry moisture takes 0.5g blades to be put into centrifuge tube, is added 80% the third
Ketone extracts chlorophyll.After blade bleaches, leaching liquor is taken, using the light absorption at spectrophotometric determination 663nm and 645nm,
The concentration of chlorophyll a (Chl a), chlorophyll b (Chl b) and Chlorophyll is calculated separately with following equation：
Chl a=12.7OD663-2.69OD645；
Chl b=22.9OD645-4.68OD663；
Chl a+Chl b=8.02OD663+20.21OD645。
Under non-drought stress, the chlorophyll content of all strains does not have significant difference, under drought stress, transgenic line
System keeps higher chlorophyll content, and wild type chlorophyll significantly reduces (Figure 10 B).
The water content difference of each strain before and after measurement Stress treatment：After blade is removed from plant, fresh weight is weighed immediately
(FW), then blade is immersed in deionized water and is no longer changed to weight for 4 hours, taking-up sucks surface moisture with filter paper, weighs
It is saturated fresh weight (TW), blade is then put into baking oven, and drying to constant weight in 70 DEG C, weighs dry weight (DW).Relative water content
(Relative water content, RWC) (%)=(FW-DW)/(TW-DW) × 100%.
As a result, it has been found that before Stress treatment, the relative water content of each strain is not significantly different, but after Stress treatment, wild
The relative water content of type is significantly lower than transfer-gen plant (Figure 10 C).
6.2IbAVP1 is overexpressed Transgenic Sweet Potato Salt Tolerance Analysis
With 200mmol L-1NaCl handled 2 months big sweet potato Potted orchards every 2 days, and every plant per treatment is given 200mL
NaCl, coprocessing 24 days.
Fv/Fm is measured：With with data gathering system fluorimeter (model PAM-101, H.Walz, Effeltrich,
Germany Fv/Fm) is measured.Before measurement, blade adapts to 30min in the dark, analyzes photosystem according to the method for Genty etc. later
The index Fv/Fm of II.
Fv/Fm=(Fm-Fo)/Fm；
Wherein Fo is the minimum chlorophyll fluorescence of dark adaptation blade, and Fm is its maximum chlorophyll fluorescence.
As a result, it has been found that before processing, WT and OE growing ways are normal (Figure 11 A), but after processing, and WT grows heavily suppressed, leaf
Piece flavescence falls off (Figure 11 A).Fv/Fm does not have marked difference, but after salt treatment under non-salt stress between all strains, turn
12-23% only drops in gene strain, and 34% (Figure 11 B) has then dropped in wild type.
It is investigated from chlorophyll content, transgenic line keeps higher chlorophyll content only to reduce about 17.4%-24.5%,
And wild type reduces 46.9% (Figure 11 C).
Detection such as preceding 200mmol L-1Na in wild type and Transgenic Sweet Potato blade before and after NaCl Stress treatments+And K+Contain
Amount, assay method：Sweet Potato Leaf is rinsed well with distilled water, it is 48 hours dry in 70 DEG C of baking ovens, claim its dry weight.With 1N salt
Acid was in 60 DEG C of warm bath 1 hour, then constant volume to 25mL.Take its supernatant, using thunder magnetic ion analyzer (PXSJ-216, Shanghai,
China) and configuration Na+And K+Ion-selective electrode measures the Na in Sweet Potato Leaf+、K+The content of ion.
Measurement result such as Figure 11 D, shown in E, NaCl Stress treatments cause different strain blade cell Na+Content increases and K+
Content reduces.Before salt stress processing, Na between each strain+Content is not significantly different, Transgenic Sweet Potato blade after processing
Na+Content respectively higher than wild type 30.8%, 24.7% and 30.9%, difference reaches the level of signifiance (Figure 11 D).For K+Contain
It measures, before NaCl Stress treatments, the content of each strain is not much different, and significant difference is not presented, and after NaCl stress, transgenosis
The K of strain+Though significant difference (Figure 11 E) is not present in content slightly above wild type.
6.3IbAVP1 is overexpressed the Transgenic Sweet Potato capability analysis of resistance to iron deficiency
The stem section of 3 expansion blades of transgenosis and WT lines top band is inserted into respectively contains 0mmol L-1、20mmol
L-1With 100mmol L-1FeEDTA SBM culture mediums (Ca (NO3)2300, MgSO450, NaH2PO430, K2SO450, H3BO33,
ZnSO40.4, CuSO40.2, MnCl20.5, (NH4)6Mo7O241 (unit：μM)) on, it was grown by 3 weeks, finds 100mmol L- 1OE and WT keeps normal growth on FeEDTA SBM culture mediums, but the root system ratio WT of OE is flourishing (Figure 12 A), in 0mmol L-1With
20mmol L-1On the culture medium of FeEDTA, WT growths are suppressed, and growth is slower, and the elongation of root is also suppressed, and OE roots
System grows impacted smaller (Figure 12 A), measures root system fresh weight and dry weight is also significantly greater than WT (Figure 12 B, C).
Water planting iron deficiency handled WT and OE sweet potatoes material after 25 days, takes new expansion blade and root system measured ion content, finds OE
Iron content is significantly higher than WT (Figure 13), illustrates that IbAVP1 can promote absorption of the Transgenic Sweet Potato to iron ion.
All references mentioned in the present invention is incorporated herein by reference, independent just as each document
It is incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can
To be made various changes or modifications to the present invention, such equivalent forms equally fall within model defined by the application the appended claims
It encloses.
Claims (7)
1. a kind of method improving the resistance to iron deficiency ability under the conditions of sweet potato iron deficiency, the method includes：Improve IbAVP1 in sweet potato
The expression of albumen；Wherein, the IbAVP1 albumen is such as SEQ ID NO:The albumen of amino acid sequence shown in 2.
2. the method as described in claim 1, which is characterized in that the method includes：The multinuclear of IbAVP1 albumen will be encoded
Thuja acid is transferred in sweet potato.
3. method as claimed in claim 2, which is characterized in that the polynucleotides of the coding IbAVP1 albumen are SEQ ID
NO:The polynucleotides of sequence shown in 1.
4. method as claimed in claim 3, which is characterized in that the method includes step：
(i) Agrobacterium for carrying expression vector, the polynucleotides of expression vector IbAVP1 albumen containing coding are provided；
(ii) polynucleotides of the coding IbAVP1 albumen are made to be transferred to sweet potato using Agrobacterium.
5. the purposes of a kind of IbAVP1 albumen or the polynucleotides for encoding the albumen, resistance under the conditions of iron deficiency for improving sweet potato
Iron deficiency ability；Wherein, the IbAVP1 albumen is such as SEQ ID NO:The albumen of amino acid sequence shown in 2.
6. the purposes of a kind of IbAVP1 albumen or the polynucleotides for encoding the albumen, for promoting root of sweet potato under the conditions of iron deficiency
System's growth；Wherein, the IbAVP1 albumen is such as SEQ ID NO:The albumen of amino acid sequence shown in 2.
7. the purposes of a kind of IbAVP1 albumen or the polynucleotides for encoding it, as resistance to scarce under the conditions of the iron deficiency for identifying sweet potato
The molecular marked compound of iron ability；Wherein, the IbAVP1 albumen is such as SEQ ID NO:The albumen of amino acid sequence shown in 2.
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