CN106701814A - Method for adjusting content of starch in tuber leaves, and application thereof - Google Patents
Method for adjusting content of starch in tuber leaves, and application thereof Download PDFInfo
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- CN106701814A CN106701814A CN201510469270.1A CN201510469270A CN106701814A CN 106701814 A CN106701814 A CN 106701814A CN 201510469270 A CN201510469270 A CN 201510469270A CN 106701814 A CN106701814 A CN 106701814A
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Abstract
The invention relates to a method for adjusting the content of starch in tuber leaves, and an application thereof. Substantial adjustment of the starch properties of the tuber plants through changing the expression of SRD gene in tuber plants is disclosed for the first time, and the method has good application prospect in the genetic improvement of the quality of the plants.
Description
Technical field
The invention belongs to biotechnology and botany field, more particularly it relates to adjust potato class leaf
The method of content of starch and application in piece.
Background technology
Potato class plant refers to the terrestrial crop for being available for edible roots or a rhizomatous class.There are root tuber, block
Stem class, such as sweet potato (Ipomoea batatas, sweet potato), cassava, potato, Chinese yam (Chinese yam), sole potato, multirow without
Sexual reproduction, only stays potato wedges to plant, it is possible to be bred with liana.This kind of general cold-hartliness of plant is weaker,
It is many that in the cultivation of frostless season, low temperature can suppress the growth of tuber crops, cause the underproduction of root tuber or stem tuber,
Therefore plantation tuber crops are tried one's best and avoid prolonged hypothermic phase；Additionally, loose, fertile, deep soil
Earth and volume potash fertilizer are conducive to improving tuber crops yield and quality.
Starch, current potato class plant all can be largely enriched with the blade and storage root of most potato class plants
Storage root be the primary raw material for producing starch, but blade fixes carbon dioxide as photosynthesis is carried out
Major organs, it may have the potential of production novel starch.By regulating and controlling the accumulation of starch in blade and changing
The property of starch in good blade, gives full play to the advantage production novel starch of potato class blade, is always ability
The emphasis of domain research.
The content of the invention
It is an object of the invention to provide a kind of method for adjusting content of starch in potato class blade and application.
In the first aspect of the present invention, there is provided a kind of method of the starch property of the blade of regulation potato class plant,
Methods described includes：The expression of SRD polypeptides in regulation potato class plant.
In a preference, described potato class plant includes：Cassava, sweet potato, potato, Chinese yam, taro
Head, the root of kudzu vine, konjaku, Jerusalem artichoke, Smallantus sonchifolium etc..
In another preference, described SRD polypeptides are selected from the group：
(a) such as SEQ ID NO:The polypeptide of 2 amino acid sequences；
B () is by SEQ ID NO:2 amino acid sequences are by one or more (such as 1-20；Preferably 1-10；
More preferably 1-5) substitution of amino acid residue, missing or addition and formed, and with (a) polypeptide function
As polypeptide derived from (a)；Or
C polypeptide sequence that () and (a) are limited have more than 70% (preferably more than 80%, more than 85%, more preferably
Ground more than 90%, such as more than 95%, more than 98%, more than 99%) homology and with (a) polypeptide function
As polypeptide derived from (a).
In another preference, methods described includes：The expression of SRD polypeptides in plant is reduced, so that：
Improve the content of starch in potato class plant leaf blade；
Improve the diameter of starch granules in potato class plant leaf blade；
Reduce the phosphorylation degree of starch in potato class plant leaf blade；
Increase the content of amylose in potato class plant leaf blade；And/or
Increase starch in short chain (preferably DP6-14) content, reduce starch in long-chain moiety (preferably
Be DP20-37) content.
In another preference, the expression for reducing SRD polypeptides in plant includes：The SRD will be disturbed
The disturbing molecule of expression of polypeptides is transferred to plant (such as cell of plant, tissue, organ or seed), so as to lower
The expression of SRD polypeptides in plant.
In another preference, the volume of the disturbing molecule targeting SRD polypeptides of the SRD expression of polypeptides is disturbed
Code gene or its transcript；It is preferred that targetting 741-1193 or its transcript of the encoding gene.
In another preference, described disturbing molecule contains the structure shown in formula (I)：
SeqIt is positive-X-SeqReverselyFormula (I),
In formula (I),
SeqIt is positiveIt is the fragment of the encoding gene of SRD polypeptides, SeqReverselyIt is and SeqIt is positiveComplementary polynucleotides；
It is preferred that SeqIt is positiveIt is 741-1193 of the encoding gene of SRD polypeptides；
X is positioned at SeqIt is positiveAnd SeqReverselyBetween intervening sequence, and the intervening sequence and SeqIt is positive
And SeqReverselyIt is not complementary.
In another preference, the structure shown in formula (I) forms two grades shown in formula (II) after plant is transferred to
Structure：
In formula (II), SeqIt is positive、SeqReverselyIt is as defined above with X and is stated,
| | represent in SeqIt is positiveAnd SeqReverselyBetween the relation being substantially complementary.
In another preference, methods described also includes subsequent step：From after the expression of regulation SRD polypeptides
Plant in select and compare before regulation that proterties obtains the plant of change for plant.
In another aspect of this invention, there is provided one kind lowers the use of the material of (as disturbed) SRD expression of polypeptides
On the way, for adjusting the starch property of the blade of potato class plant.
In a preference, the material of described downward SRD expression of polypeptides is used for：
Improve the content of starch in potato class plant leaf blade；
Improve the diameter of starch granules in potato class plant leaf blade；
Reduce the phosphorylation degree of starch in potato class plant leaf blade；
Increase the content of amylose in the provisional starch of potato class plant leaf blade；And/or
Increase the content of short chain in starch, reduce the content of long-chain moiety in starch.
In another preference, described potato class plant includes：Cassava, sweet potato, potato, Chinese yam, taro
Head, the root of kudzu vine, konjaku, Jerusalem artichoke, Smallantus sonchifolium etc..
In another aspect of this invention, there is provided a kind of downward SRD expression of polypeptides is so as to adjust potato class plant
The disturbing molecule of the starch property of blade, it contains the structure shown in formula (I)：
SeqIt is positive-X-SeqReverselyFormula (I),
In formula (I),
SeqIt is positiveIt is the encoding gene segment of SRD polypeptides, SeqReverselyIt is and SeqIt is positiveComplementary polynucleotides；
It is preferred that SeqIt is positiveIt is 741-1193 of the encoding gene of SRD polypeptides；
X is positioned at SeqIt is positiveAnd SeqReverselyBetween intervening sequence, and the intervening sequence and SeqIt is positive
And SeqReverselyIt is not complementary.
In another aspect of this invention, there is provided a kind of carrier, described carrier contains described disturbing molecule.
In another aspect of this invention, there is provided the purposes of a kind of SRD polypeptides or its encoding gene, for making
To identify the molecular labeling of the starch property of the blade of potato class plant；The shallow lake of the blade of described potato class plant
Mealiness shape includes：
Content of starch in potato class plant leaf blade；
The diameter of starch granules in potato class plant leaf blade；
The phosphorylation degree of starch in potato class plant leaf blade；
The content of amylose in the provisional starch of potato class plant leaf blade；And/or
Increase the content of short chain in starch, reduce the content of long-chain moiety in starch.
Other side of the invention, due to this disclosure, is to those skilled in the art aobvious
And be clear to.
Brief description of the drawings
Fig. 1, the RNAi binary expression vector schematic diagrames containing hairpin structure.
Fig. 2, transfer-gen plant Southern blot are identified.
The change of cassava SRD expressions in Fig. 3, SRDRNAi transfer-gen plant.
The Western blot analysis results of MeSRD protein expressions in Fig. 4, transfer-gen plant.
Antibody：MeSRD rabbits source polyclonal antibody；
Internal reference：It is albumen applied sample amount internal reference with Rubisco；
Control：Wild type cassava TMS60444；
Protein extraction material is the mature leaf of greenhouse Manihot Esculenta.
The content of provisional starch in Fig. 5, wild type cassava TMS60444 and transgenosis cassava blade.Leaf
Piece takes from the Manihot Esculenta grown under the village, often used in village names pilot plant test field natural conditions of Shanghai five, and data shown in figure are 3
Secondary experiment is repeated.
Fig. 6, dark week end of term wild type cassava TMS60444 and transgenosis different development stage cassava blade iodine
Dye result.
The ESEM result of starch in Fig. 7, wild type cassava TMS60444 and transgenosis cassava blade.
Amylum body in blade is extracted rear by scanning electron microscopic observation, and A-D is respectively WT, transgenic line
It is the starch in G1i-12, transgenic line G1i-17, transgenic line G1i-31 blades.
The phosphorylation level collection of illustrative plates of starch in Fig. 8, wild type cassava TMS60444 and transgenosis cassava blade.
A：G-6-P standard sample；
B：Glucose -3- phosphate standard samples；
C：G-6-P and glucose -3- phosphorus acid contents in wild type cassava TMS60444 blades；
D：G-6-P and glucose -3- phosphorus acid contents in SRDRNAi transgenosis cassava blades.
The phosphorylation degree of starch in Fig. 9, wild type cassava TMS60444 and transgenosis cassava blade.
Ordinate is the amount of the G-6-P contained in every milligram of starch, starch isolation material crop field
The blade of Manihot Esculenta.* represents t-test inspections difference extremely significantly (p<0.01).
The amylose content of starch in Figure 10, wild type cassava TMS60444 and transgenosis cassava blade.
Data shown in figure are three reproducible results.* t-test inspection significant differences (p is represented<0.05), * *
Represent t-test inspections difference extremely significantly (p<0.01).
The XRD diffracting spectrums of starch in Figure 11, wild type cassava TMS60444 and transgenosis cassava blade.
The diffracting spectrum of the provisional starch of blade；Starch is extracted from the blade and root tuber of crop field cassava respectively.
The chain length distribution of starch in Figure 12, wild type cassava TMS60444 and transgenosis cassava blade.
A：The chain length distribution of wild type cassava TMS60444 blade amylopectin；
B-F：Respectively wild type cassava TMS60444 root tuber storage characteristics starch, transfer-gen plant
The provisional starch of the blade of G1i-12,17,28,31 and the provisional shallow lake of wild type cassava TMS60444 blades
The difference of powder chain length distribution.Data above is that three experiments are repeated.
Specific embodiment
, by in-depth study, discovery, can by changing expression of the SRD in potato class plant for the present inventor
Significantly to adjust the starch property of potato class plant, there is good application on the genetic improvement of plant quality
Prospect.
As used herein, " potato class plant " of the invention is also referred to as " tuber crops ", refers mainly to have
It is available for the terrestrial crop of edible roots or a rhizomatous class.Including but not limited to：The root tuber of Euphorbiaceae is planted
The root crops such as sweet potato of thing such as cassava, Convolvulaceae, the tuberous plant such as potato of Solanaceae, Dioscoreaceae
Root crops such as Chinese yam, the tuberous plant such as taro, konjaku of Araeceae, the pulse family root crops such as root of kudzu vine,
Composite family tuberous plant such as Jerusalem artichoke, Smallantus sonchifolium etc..
Fragment, derivative and analog present invention additionally comprises SRD polypeptides.As used herein, term " piece
Section ", " derivative " and " analog " refer to be kept substantially SRD polypeptides identical life of the invention
The polypeptide of thing function or activity.Polypeptide fragment of the invention, derivative or the like can be that (i) has one
Or multiple conservative or substituted polypeptides of non-conservative amino acid residue (preferably conservative amino acid), and
Such substituted amino acid residue can may not be by genetic code encoding, or (ii) is at one
Or the polypeptide with substituted radical in more amino acid, or (iii) additional amino acid sequence is fused to this
Polypeptide sequence and the polypeptide (such as targeting sequencing or secretion sequence or sequence or egg for purifying this polypeptide that are formed
White original sequence, or fusion protein).Definition these fragments, derivative and analog according to this paper belong to this
Scope known to skilled practitioner.
The bioactive fragment of any SRD polypeptides can be applied in the present invention.Herein, SRD
The implication of the bioactive fragment of polypeptide refers to that, used as a kind of polypeptide, it still can keep the SRD of total length many
All or part of function of peptide.Under normal circumstances, described bioactive fragment at least keep 50% it is complete
The activity of SRD polypeptides long.Under still more preferential conditions, the active fragment can keep total length SRD many
The activity of 60%, 70%, 80%, 90%, 95%, 99% or the 100% of peptide.
In the present invention, term " SRD polypeptides " refers to the SEQ ID NO with SRD polypeptide actives:2
The polypeptide of sequence.The term also includes having and SRD polypeptide identical functions, SEQ ID NO:2 sequences
Variant form.These variant forms include (but being not limited to)：Several (usually 1-50, preferably
1-30, ground, more preferably 1-20, most preferably 1-10, also more preferably such as 1-8,1-5) amino acid
Missing, insertion and/or replace, it is and (logical in C-terminal and/or N-terminal addition or missing one or several
Often within 20, within preferably 10, more preferably within 5) amino acid.For example,
In this area, when being replaced with similar nature or similar amino acid, will not generally change protein
Function.Again such as, C-terminal and/or N-terminal addition or missing one or several amino acid generally also not
The function of protein can be changed.The term also active fragment and reactive derivative including SRD polypeptides.
The variant form of polypeptide includes：Homologous sequence, conservative variant, allelic variant, natural mutation
Body, induced mutants, the DNA that can hybridize with SRD polypeptid DNAs under the conditions of high or low stringency
Coded albumen.Present invention also offers other polypeptides, the fusion such as comprising SRD polypeptides or its fragment
Albumen.
It is any high with described SRD peptides homologous (such as with SEQ ID NO:Sequence shown in 2 it is same
Source property is 70% or higher；Preferably, homology is 80% or higher；It is furthermore preferred that homology is 90%
Or it is higher, such as homology 95%, 98% or 99%) and with the albumen of SRD polypeptide identical functions
It is included in the present invention.
Invention also provides the analog of SRD polypeptides or polypeptide.The difference of these analogs and natural SRD polypeptides
Can not be difference on amino acid sequence, or do not influence the difference on the modified forms of sequence,
Or have both at the same time.These polypeptides include natural or induction genetic variant.Induction variant can lead to
Cross various technologies to obtain, such as produce random mutagenesis by radiation or exposed to mutagens, can also be by fixed
The technology of point mutagenesis or other known molecular biology.Analog also includes having being different from natural L- ammonia
The analog of the residue (such as D- amino acid) of base acid, and with non-naturally occurring or synthesis amino acid
The analog of (such as β, gamma-amino acid).It should be understood that polypeptide of the invention is not limited to the above-mentioned generation for enumerating
The polypeptide of table.
It should be understood that although SRD polypeptides of the invention are preferably obtained from cassava, available from other plants with
Cassava SRD polypeptides very high homology is (as having more than 70%, such as 80%, 90%, 95%, even 98% sequence
Row homogeny) other polypeptides also within the scope of the present invention contemplates.The method of aligned sequences homogeny and
Instrument is also well known in the art, such as BLAST.
Polynucleotides sequence the invention further relates to encode SRD polypeptides of the present invention or its conservative variation's polypeptide
Row.Described polynucleotides can be DNA form or rna form.DNA form include cDNA,
Genomic DNA or artificial synthesized DNA.DNA can be single-stranded or double-strand.DNA can be with
It is coding strand or noncoding strand.The coding region sequence of encoding mature polypeptide can be with SEQ ID NO:Shown in 1
Coding region sequence is identical or variant of degeneracy.As used herein, " variant of degeneracy " exists
Refer to that coding has SEQ ID NO in the present invention:2 protein, but with SEQ ID NO:Volume shown in 1
The code differentiated nucleotide sequence of region sequence.
Coding SEQ ID NO:The polynucleotides of 2 mature polypeptide include：The coding of encoding mature polypeptide
Sequence；The coded sequence of mature polypeptide and various additional coding sequences；Mature polypeptide coded sequence (and appoint
The additional coding sequence of choosing) and non-coding sequence.
Term " polynucleotides of coded polypeptide " can include the polynucleotides of coding said polypeptide, also may be used
Being also to include the polynucleotides of additional code and/or non-coding sequence.
The invention further relates to have at least 50% between above-mentioned sequence hybridization and two sequences, preferably
At least 70%, more preferably at least polynucleotides of 80% homogeny.
The nucleotides full length sequence or its fragment of SRD genes of the invention generally can with PCR TRAPs,
Recombination method or artificial synthesized method are obtained.For PCR TRAPs, can be had according to disclosed in this invention
Nucleotide sequence, especially open reading frame sequence is closed to design primer, and with commercially available cDNA storehouses or
CDNA storehouses as prepared by conventional method well known by persons skilled in the art must have as template, amplification
Close sequence.
The present invention also relates to the carrier comprising described polynucleotides, and carrier or SRD bases described in
Because of the host cell that sequence is produced through genetic engineering.
New discovery based on the present inventor, present invention also offers a kind of method for improveing potato class plant, the party
Method includes the expression of SRD polypeptides in the regulation potato class plant.
It is highly preferred that described method includes：The expression for reducing SRD polypeptides in the plant (including makes
SRD polypeptides are not expressed or low expression) so that：Improve the content of starch in potato class plant leaf blade；Improve potato
The diameter of starch granules in class plant leaf blade；Reduce the phosphorylation degree of starch in potato class plant leaf blade；With/
Or increase the content of amylose in the provisional starch of potato class plant leaf blade；Increase the content of short chain in starch,
Reduce the content of long-chain moiety in starch.
The expression of SRD polypeptides can be reduced using various methods well known in the art or is allowed to lack
, such as be delivered to for the ceneme (such as expression vector or virus etc.) of carrying antisense SRD genes by expression
On target spot so that expression SRD polypeptides are not expressed or reduced to cell or plant tissue.
As one embodiment of the present invention, there is provided the expression of SRD polypeptides in a kind of reduction plant
Method, described method includes：
(1) disturbing molecule of SRD gene expressions will be disturbed to be transferred to plant tissue, organ or seed, is obtained
It is transformed into plant tissue, organ or the seed of the disturbing molecule；With
(2) plant tissue for being transferred to the disturbing molecule, organ or the seed regeneration for obtaining step (1)
Plant.
Used as a kind of preferred example, described method includes step：
I () provides the Agrobacterium for carrying the carrier that may interfere with gene expression, described carrier is selected from the group：
The encoding gene of a SRD polypeptides that () starts containing opposite direction or the load of genetic fragment (antisense molecule)
Body；
B () contains the encoding gene expression (or transcription) that specificity interference SRD polypeptides can be formed in plant
Composition disturbing molecule carrier；
(ii) tissue or organ of plant are contacted with the Agrobacterium in step (i), so that the carrier is transferred to
Plant tissue or organ.
It is preferred that methods described also includes：
(iii) select and be transferred to the plant of the carrier and knit or organ；With
(iv) by the plant tissue in step (iii) or neomorph into plant.
Based on the nucleotide sequence of SRD genes, to can be designed that and can form specificity after plant is imported
Disturb the polynucleotides of the molecule of SRD gene expressions.The effect of specificity and interference is considered during design
Rate.The present invention has no particular limits to the preparation method of disturbing molecule, including but not limited to：Chemistry is closed
Cheng Fa, in-vitro transcription method etc..It should be understood that those skilled in the art are knowing SRD genes and vegetalitas
After the correlation of shape, described disturbing molecule can be prepared with various approach, so as to be planted for adjusting
Physical property shape.Described disturbing molecule can be transported in plant by transgenic technology, or can also be used
Multiple technologies known in the art are transported in plant.
It is described as particularly preferred mode of the invention, there is provided a kind of disturbing molecule of excellent effect
Disturbing molecule can specific interference SRD genes expression；And empirical tests, it has good interference
The effect of SRD gene expressions.Described disturbing molecule is to contain SEQ ID NO:741-1193 in 1
The molecule of shown nucleotide sequence, constitutes hairpin structure.
Present invention also offers a kind of disturbing molecule, the disturbing molecule contains following structure：SeqIt is positiveFor
SRD genetic fragments (preferably SEQ ID NO:Nucleotide sequence in 1 shown in 741-1193),
SeqReverselyIt is and SeqIt is positiveComplementary polynucleotides；X is positioned at SeqIt is positiveAnd SeqReverselyBetween intervening sequence,
And the intervening sequence and SeqIt is positiveAnd SeqReverselyIt is not complementary.
Described disturbing molecule can be folded into the loop-stem structure of stabilization, stem ring knot after importeding into plant
The stem both sides of structure include the two sequences being substantially complementary.That is, forming secondary structure as follows：
Wherein, | | represent in SeqIt is positiveAnd SeqReverselyBetween the relation being substantially complementary.Described stem ring knot
Structure can further be acted on, process or sheared by the various materials in plant, and form double-strand
RNA(dsRNA)。
Generally, described disturbing molecule is located on expression vector.
Present invention additionally comprises the plant obtained using foregoing any one method, described plant includes：It is transferred to
The genetically modified plants of SRD genes or its homologous gene；Or SRD expression of polypeptides amount (including low expression or
Do not express) plant etc. that reduces.
Any appropriate conventional meanses can be used, implements described including reagent, temperature, pressure condition etc.
Method.
Moreover, it relates to by the use of SRD polypeptides or its encoding gene as a kind of genetic transformation plant
The tracking mark of offspring.The invention further relates to by the use of SRD polypeptides or its encoding gene as a kind of molecule mark
Note, by detecting the expression of SRD polypeptides in plant, the proterties of plant identification.Also using SRD
The plant characteristic of gene-correlation as true hybrid during hybrid seeding cue mark.
The potato that the content of starch that the method for the present invention can be prepared in blade is improved and starch granules increases
Class plant, so as to a large amount of techniques for extracting starch from the blade of potato class plant can be realized.
Also, the method for the present invention can improve the diameter (3-10 μm) of starch granules in potato class plant leaf blade,
So that starch granules therein more connects with the starch granules from beans (such as mung bean) or cereals (such as rice)
Closely, this granular starch has good application value as starch granule (being less than 10 μm).
With reference to specific embodiment, the present invention is expanded on further.It should be understood that these embodiments are only used for
Illustrate the present invention rather than limitation the scope of the present invention.The reality of unreceipted actual conditions in the following example
Proved recipe method, generally writes, Molecular Cloning:A Laboratory guide, the 3rd according to normal condition such as J. Pehanorm Brookers etc.
Version, Science Press, the condition described in 2002, or according to the condition proposed by manufacturer.
The Cloning and sequence analysis of embodiment 1, MeSRD
The present inventor is by Blastp and Blastn in RIKEN cassava cDNA databases
(http://www.brc.riken.jp/inf/en/index.shtml) two est sequences of middle search discovery, it is defined as wood
The MeSRD genes of potato, its total length 4230bp encodes 1410 amino acid.
MeSRD nucleotide sequences (SEQ ID NO:1)：
ATGAGTAATAGCATAGGGCATAATTTATTCCAACAGAGTTTGATTCGTCCCGCGAGTT
TTAAACATGGAAGCAATCTCAATTCTTCTGGCATTCCTGCAAGCTATTTATTCCAATCTGCC
TCTGTGAGTCGAGGACTGCAGATAAGCAGGTCGCCAATATCCTCTAGTTTTTATGGAAAAA
ATTTGAGGGTGCGGAAATCAAAATTAGCCGTTGTAAATCCTCGTCCAGCTATAACAATTCC
ACGGGCTATATTGGCGATGGATCCGGCATCCCAGCTCCTAGGAAAATTCAACCTTGATGGA
AATGTTGAATTGCAGGTGTTTGTTAGCAGTCACACTTCTGCTTCTACTGTGCAAGTACACA
TTCAGATAACATGCACTAGTGATTCTTTGCTCCTACACTGGGGTGGGAAACATGATAGAAA
GGAAAACTGGGTACTTCCTAGTCGTTATCCAGATGGAACCAAAAATTATAAGAGCAGAGC
CCTTAGAAGTCCTTTTGTCAAGTCTGGTTCAAGTTCTTACCTGAAAATAGAGATTGATGAT
CCTGCAATACAAGCCTTAGAATTTCTTATACTTGATGAAGCGCATAATAAATGGTTTAAAA
ATAATGGTGACAACTTTCATGTTAAATTACCTGCACGAGAGAAGCTGATAATTCCAAATAT
CTCAGTTCCTGAAGAGCTTGTACAAGTTCAAGCATATCTGAGGTGGGAAAGAAATGGTAA
ACAAATGTATACCCCAGAACAAGAAAAGAAAGAATATGAAGCCGCTCGTATTGAACTATT GGAGGAAGTAGCTAAGGGTACTTCCATTGAGGGCCTCCGAGCAAGGCTGACAAACAAAAA TGAAATTAAGGAGTCATCTGTCTCTAAAACACAAAGCAAGATACACGCTCAAGCTCATAG AAGATGGGAAAAATCTACTACTAGTAATGAAAGGTTTCAGCGCAATCAGAGGGACTTAGC ACAGCTTGTTACCAAATCTGCTACTAAAAAATCTGCAGAGGAAGCTGTTTCAGTAGAACCA AAACCAAAAGCATTGAAGGCAGTTGAACTTTTTGCTAAAGAAAAGGAAGAACGGGTTGGG GGTGCTGTTCTGAACAAGAAGATCTTTAAGCTCCAAGATGCGGAACTTCTGGTGCTTGTGA CCAAGCCTGCTGATAAGATGAAGGTTTATGTTGCCACTGATTTCAAAGAACCAGTCACTCT
TCACTGGGCATTATCTAGGAAGGGTAAAGAGTGGTTGGCGCCACCACCAAGTGTGTTGCCT
CCTGGTTCAGTTTCTTTGAACGAGGCTGCTGAAACACAACTTAAAAGCATTTCTTCAACTG
AACTTTCTTATCAGGTCCAATACTTTGAAACGGAGATCGAAGAGAATTTTGTAGGGATGCC
CTTTGTGCTTTTTTCTAATGAAAAATGGATAAAGAATAAGGGCTCTGACTTTTATGTTGAA
CTTAGTGGCGGACCTAGGCCAGTCCAAAAGGATGCTGGTGATGGAAGAGGTACAGCAAAA
GTTTTATTGGACACAATTGCAGAGCTGGAGAGTGAAGCACAGAAATCCTTCATGCACCGA
TTTAATATTGCAGCTGATTTGATGGAGGATGCAAAGGATGCTGGTGAGTTGGGTTTTGCAG
GGATCTTGGTGTGGATGAGATTTATGGCCACGAGGCAACTTATTTGGAACAAAAACTACA
ATGTGAAACCACGTGAGATCAGCAAGGCACAGGATAGGCTCACAGACTTGCTCCAGAATA
CTTATACAAGTCATCCTCAATATCGGGAGCTTTTGCGGATGATTATGTCTACTGTCGGTCG
AGGTGGTGAAGGTGATGTGGGGCAGCGAATTCGGGATGAAATTTTAGTTATCCAGAGAAA
CAATGATTGCAAAGGTGGTATGATGGAGGAATGGCATCAGAAGCTGCATAATAACACAAG
CCCTGATGATGTTGTTATCTGCCAGGCATTAATGGATTACATTAAAAGTGACCTTGACATC
AGTGTGTACTGGAAAACTTTGAATGAAAATGGAATAACAAAAGAACGACTTTTAAGCTAT
GATCGTGCAATCCATTCTGAACCAAGCTTCAGGAGAGATCAAAAGGACGGTCTTTTGCGTG
ATCTCGGCAACTATATGAGAAGTTTGAAGGCAGTTCATTCTGGTGCAGATCTTGAGTCTGC
TATTGCAAATTGTATGGGCTATAAAGATGAGGGTCAAGGTTTCATGGTTGGAGTGCAAATA
AATCCCATTTCAGGCTTGCCATCTGGATTTCCAGAGTTGCTTCGATTTGTTCTCAAACATGT
TGAAGATAGAAATGTAGAAGCACTTCTTGAGGGTTTGCTGGAGGCTCGTCAGGAGCTGAG
GCCATTGCTGTTTAAGTCTAATAATCGTCTGAAAGATCTTCTATTTTTGGATATTGCCCTTG
ATTCTACTGTTAGGACAGCCATTGAGAGAGGATATGAGGAATTAAATGATGCTGGACCAG
AGAAAATTATGTATTTCATCACCCTGGTTCTTGAAAATCTTGCGCTTTCATCAGATGATAAT
GAAGAGTTTGTCTATTGCTTGAAGGGATGGAATTATGCCCTAAGCATGTCCAAAAGTAAA
AGCAATCACTGGGCATTATATGCAAAATCAGTCCTTGACAGAACTCGCCTTGCCCTGGCCA
GCAAGGCTGAATGGTATCAGCAAGTTTTGCAACCATCAGCAGAGTATCTTGGATCACTGCT
TGGAGTGGATCAGTGGGCTGTGAACATATTCACTGAAGAAATAGTTCGTGCTGGATCAGCT
GCAGCTGTATCCTTGCTTCTTAATCGACTTGATCCAGTTCTTCGGAAGACTGCTCATCTTGG
AAGTTGGCAGGTTATTAGCCCAGTTGAAGCTGCTGGGTATGTTGTTGTTGTGGATGAGTTG
CTCACAGTACAGAATTTATCTTACGACCGCCCTACAATTTTAGTGGCAAGAAGAGTAAGTG
GAGAAGAAGAAATTCCTGATGGTACAGTTGCTGTGCTGACATCTGACATGCCAGATGTCCT
ATCCCATGTTTCTGTACGAGCAAGAAATAGCAAGGTTTGCTTTGCCACATGTTTTGATCAC
AACATTCTGGACAATCTCCGAGCAAATGAAGGGAAATTATTGAATTTGAAACCTACATCA
GCAGATATAGTCTATAGCGTGATCGAGGGTGAATTAGCAGATTTAAGTTCAAATAAGCTG
AAAGAAGTTGGTCCTTCACCTATAAAGTTGATAAGAAAGCAGTTCAGTGGTAGATATGCC
ATATCATCGGAGGAGTTCACCGGTGAAATGGTTGGTGCCAAATCACGCAATATCGCGCAT
CTAAAAGGAAAAGTACCATCCTGGATTGGGATTCCTACATCGGTTGCCTTACCATTTGGAG
TTTTTGAGAAGGTTCTTTCAGATGGTTCAAATCAAGAAGTGGCTAAGAAGTTGGAAGTTTT
GAAGAAACAGTTGGAAGGAGGAGAGTCTAGTGTCCTCAGGAGAATTCGTGAGACAGTTTT
ACAGCTGGCAGCACCACCACAGCTGGTGCAAGAGCTGAAGACAAAGATGAAAAGTTCTGG
GATGCCTTGGCCTGGCGATGAAGGTGAACAGCGATGGGAGCAAGCATGGATGGCTATAAA
GAAGGTCTGGGCTTCAAAATGGAATGAGAGAGCATACTTCAGCACAAGGAAAGTGAAGTT
GGACCATGATTACCTCTGCATGGCTGTCCTGGTTCAGGAGATAATAAATGCCGATTATGCA
TTTGTTATCCACACGACCAATCCATCTTCTGGGGATTCATCAGAGATATATGCTGAGGTAG
TGAAGGGACTTGGAGAAACTCTTGTTGGAGCCTATCCCGGCCGTGCTTTGAGTTTTATCTG
CAAGAAAAAAGATCTGAATTCTCCTCAGGTGTTGGGTTACCCAAGCAAACCCATTGGCCTT
TTTATAAGACGTTCTATAATCTTCAGATCTGACTCCAATGGTGAAGATCTGGAAGGTTATG
CTGGTGCTGGTCTTTATGATAGTGTTCCAATGGATGAGGAAGAGAAAGTTGTGCTTGATTA
CTCATATGATCCATTGATCACCGATGAAAGCTTCCGAAAATCAATTCTCTCTAACATAGCT
CGTGCTGGAAGTGCCATTGAAGAGCTCTATGGATCTCCACAAGACATTGAAGGAGTAATA
AGGGACGGTAAACTCTATGTGGTTCAGACAAGGCCTCAGATGTAA
MeSRD amino acid sequences (SEQ ID NO:2)：
MSNSIGHNLFQQSLIRPASFKHGSNLNSSGIPASYLFQSASVSRGLQISRSPISSSFYGKNLR
VRKSKLAVVNPRPAITIPRAILAMDPASQLLGKFNLDGNVELQVFVSSHTSASTVQVHIQITCTS
DSLLLHWGGKHDRKENWVLPSRYPDGTKNYKSRALRSPFVKSGSSSYLKIEIDDPAIQALEFLI
LDEAHNKWFKNNGDNFHVKLPAREKLIIPNISVPEELVQVQAYLRWERNGKQMYTPEQEKKE
YEAARIELLEEVAKGTSIEGLRARLTNKNEIKESSVSKTQSKIHAQAHRRWEKSTTSNERFQRN
QRDLAQLVTKSATKKSAEEAVSVEPKPKALKAVELFAKEKEERVGGAVLNKKIFKLQDAELL
VLVTKPADKMKVYVATDFKEPVTLHWALSRKGKEWLAPPPSVLPPGSVSLNEAAETQLKSISS
TELSYQVQYFETEIEENFVGMPFVLFSNEKWIKNKGSDFYVELSGGPRPVQKDAGDGRGTAKV
LLDTIAELESEAQKSFMHRFNIAADLMEDAKDAGELGFAGILVWMRFMATRQLIWNKNYNVK
PREISKAQDRLTDLLQNTYTSHPQYRELLRMIMSTVGRGGEGDVGQRIRDEILVIQRNNDCKG
GMMEEWHQKLHNNTSPDDVVICQALMDYIKSDLDISVYWKTLNENGITKERLLSYDRAIHSEP
SFRRDQKDGLLRDLGNYMRSLKAVHSGADLESAIANCMGYKDEGQGFMVGVQINPISGLPSG
FPELLRFVLKHVEDRNVEALLEGLLEARQELRPLLFKSNNRLKDLLFLDIALDSTVRTAIERGY
EELNDAGPEKIMYFITLVLENLALSSDDNEEFVYCLKGWNYALSMSKSKSNHWALYAKSVLD
RTRLALASKAEWYQQVLQPSAEYLGSLLGVDQWAVNIFTEEIVRAGSAAAVSLLLNRLDPVL
RKTAHLGSWQVISPVEAAGYVVVVDELLTVQNLSYDRPTILVARRVSGEEEIPDGTVAVLTSD
MPDVLSHVSVRARNSKVCFATCFDHNILDNLRANEGKLLNLKPTSADIVYSVIEGELADLSSN
KLKEVGPSPIKLIRKQFSGRYAISSEEFTGEMVGAKSRNIAHLKGKVPSWIGIPTSVALPFGVFE
KVLSDGSNQEVAKKLEVLKKQLEGGESSVLRRIRETVLQLAAPPQLVQELKTKMKSSGMPWP
GDEGEQRWEQAWMAIKKVWASKWNERAYFSTRKVKLDHDYLCMAVLVQEIINADYAFVIH
TTNPSSGDSSEIYAEVVKGLGETLVGAYPGRALSFICKKKDLNSPQVLGYPSKPIGLFIRRSIIFR
SDSNGEDLEGYAGAGLYDSVPMDEEEKVVLDYSYDPLITDESFRKSILSNIARAGSAIEELYGS
PQDIEGVIRDGKLYVVQTRPQM*
The acquisition of embodiment 2, the structure of MeSRDRNAi carriers and transgenosis cassava
The present inventor first by selecting MeSRD (pdm02348) gene-specific fragment (741-1193bp),
Construct RNA interference (RNAi) binary vector that specificity suppresses MeSRD expression：
pPCaMV35S::SRDRNAi。
The present inventor first by selecting MeSRD (pdm02348) gene-specific fragment (741-1193bp),
Construct RNA interference (RNAi) binary vector that specificity suppresses MeSRD expression：
pPCaMV35S::SRDRNAi。
With cassava (manihot utilissima) genome as template, with 5 '-
ATGGTACCCCCAGAACAAGAAAAGAAAGAA-3’(SEQ ID NO:And 5 ' 3)-
CTATCGATAA ATCAGTGGCAACATAAACCT-3’(SEQ ID NO:4) amplification is obtained
MeSRD (pdm02348) gene-specific fragment (741-1193bp), by what is expanded
MeSRD (741-1193bp) forward direction fragment be inserted into pRNAi-dsAC1 binary vectors (referring to
Biotechnology and Bioengineering, Vol.108, No.8, August, 2011,1925-1935)
In KpnI/ClaI restriction enzyme sites；With 5 '-
ATGGATCCCCCAGAACAAGAAAAGAAAGAA-3’(SEQ ID NO:And 5 ' 5)-
CTCTCGAGAAATCAGTGGCAACATAAACCT-3’(SEQ ID NO:6) it is primer,
Amplification obtains MeSRD (pdm02348) gene-specific fragment (741-1193bp), by what is expanded
MeSRD (741-1193bp) inverted repeat is inserted into and foregoing has been already inserted into positive fragment
In the XhoI/BamHI restriction enzyme sites of pRNAi-dsAC1 binary vectors, acquisition includes hairpin structure
RNAi recombinant vectors.
The RNAi recombinant vectors that will be built are transferred to Agrobacterium LBA4404, then infect wood by Agrobacterium
Potato fragility suspension callus, the callus after infecting obtains positive plant by processes such as regeneration, screenings,
By pPCaMV35S::SRDRNAi transgenosis cassavas are denoted as SRDRNAi, are abbreviated as G1i.
Embodiment 3, the Molecular Identification of MeSRDRNAi transgenosis cassavas
Converted by agriculture bacillus mediated cassava suspension callus and obtain pPCaMV35S altogether::SRDRNAi is positive
Totally 20 strains such as transgenosis cassava G1i-1,5,12,16,17, then pass through Southern blot
The last list copy transfer-gen plant that obtains of screening is totally ten one strains such as G1i-12,17,18,22,23.
(1) gene expression dose identification
In order to verify RNA interference effect, the present inventor with
5’-ACCTCTGCATGGCTGTCCTGGTT-3’(SEQ ID NO:7) with 5 '-GCACGGC
CGGGATAGGCTCC-3’(SEQ ID NO:8) it is primer, by Real-time RT-PCR couple
The expression of MeSRD is analyzed in single copy plant.
Result shows, in most of pPCaMV35S::In the mono- copy transgenosis cassavas of SRDRNAi,
The expression quantity of MeSRD is all substantially lowered, wherein being lowered with G1i-2,12,18,28,31 the brightest
It is aobvious, relative to wild type cassava TMS60444, the expression quantity of MeSRD in G1i-12 transgenic lines
90% is reduced, such as Fig. 3.
(2) protein expression identification
Although target gene receives strong suppression on expression, whether also received on protein level
To suppression, it is necessary to further prove.The present inventor is anti-for the specific protein fragments designs of MeSRD
Former polypeptide simultaneously have successfully been obtained the rabbit source polyclonal antibody of cassava MeSRD.Harvest wild type cassava and
Three single copies transgenosis cassava (G1i-12, G1i-17, G1i-28) that MeSRD gene expressions are substantially lowered
Blade of the same size, after extracting total protein, further detects the egg of MeSRD by Western blot
Bai Liang.It is albumen applied sample amount internal reference with Rubisco, is control (WT) with wild type cassava TMS60444；
Protein extraction material is the mature leaf of greenhouse Manihot Esculenta.
Result shows there is the purpose band of MeSRD in wild type, size about in 140kD, with prediction
MeSRD it is in the same size；Do not have the purpose of MeSRD in transfer-gen plant G1i-12,17,18
Band, illustrates that the present inventor not only reduces the expression of genes of interest by the means of RNAi, while
Also the protein content of genes of interest, such as Fig. 4 are successfully reduced.
The above results show, have successfully been obtained the MeSRDRNAi that the expression of MeSRD effectively disturbed and turn
Gene cassava.
Regulation of the embodiment 4, MeSRD in the provisional content of starch of blade
Respectively under the village, often used in village names pilot plant test field natural conditions of Shanghai five cultivate wild type cassava TMS60444 and
SRDRNAi transgenosis cassavas.Take respectively 6 in the photoperiod:00、12:00、18:00、0:00 climax leaves
Piece, first removes soluble polysaccharide with methyl alcohol, and Total Starch (K-TSTA, Megazyme) are passed through after drying
Content of starch in kit measurement blade.Result such as Fig. 5, the blade of wild type cassava starts in the photoperiod
When, content of starch is gradually increasing, and 6:00 to 12:Starch is accumulated rapidly between 00, and 12:00 to 18:00 it
Between, starch accumulation speed becomes relatively slow, reaches highest to content of starch at the end of the photoperiod, about
2%；When starting in the dark cycle, starch starts degraded, and content of starch is gradually reduced, in the whole dark cycle
The degradation rate of starch is held essentially constant, to dark all end of term (6:00) starch is dropped completely substantially when.And
In the blade of SRDRNAi transgenosis cassavas, content of starch is always held at level higher in the photoperiod,
About 11%, the fluctuation in change of its content of starch in the photoperiod is also without rule.
The wild type and transgenosis cassava blade of different development stage after dark end cycle are taken, it is de- by alcohol
After color, with 0.2% iodine staining, it is found that wild-type leaves can not be coloured, and during transgenosis cassava difference
The blade of phase all substantially becomes blue, illustrates that wild type cassava blade no longer accumulates starch in the dark end of term in week, and turns
Gene cassava blade still contains substantial amounts of starch, such as Fig. 6.
Influence of the embodiment 5, MeSRD to the provisional starch form of blade
Respectively under the village, often used in village names pilot plant test field natural conditions of Shanghai five cultivate wild type cassava TMS60444 and
SRDRNAi transgenosis cassavas, culture to harvest time.Respectively extract wild type cassava TMS60444 and
Starch in SRDRNAi transgenosis cassava blades, by ESEM (SEM) observation blade and root tuber
Starch form, as a result finds that the starch in blade is presented round pie, starch diameter in wild type cassava blade
About 2-3 μm, and amylum body generally becomes big in SRDRNAi transfer-gen plants, most of starch diameter can
Up to more than 5 μm, starch is straight in transfer-gen plant G1i-12,17 that MeSRD expression in part is lowered
Footpath reaches as high as 10 μm or so, such as Fig. 7.
The increase of amylum body can not only reduce the cost of starch isolation technique, while also closer to mung bean, paddy
The small starch granules in thing source.
Regulation of the embodiment 6, MeSRD to the provisional starch phosphorylation degree of blade
Respectively under the village, often used in village names pilot plant test field natural conditions of Shanghai five cultivate wild type cassava TMS60444 and
SRDRNAi transgenosis cassavas, culture to harvest time.Photoperiod Mo maturation cassava blade is collected, leaf is extracted
Starch in piece, containing for the G-6-P in its hydrolyzate is analyzed after sour water solution by HPAEC-PAD
Amount.
Chromatogram shows that the standard items of G-6-P and G-3-P went out at 22.7min and 24.2min minutes respectively
Existing eluting peak, obvious G-6-P is also detected that in the provisional starch hydrolyzate of blade of wild type cassava
And the eluting peak of G-3-P.Similar with other species, the content of G-3-P is significantly less than the content of G-6-P,
And in the provisional starch of blade of RNAi transfer-gen plants G-6-P eluting peaks become it is very faint, about
The 0.5% of wild type cassava, MeSRD also has glucose residue C-6 in catalytic starch in illustrating cassava
The function of position phosphorylation, such as Fig. 8.With G1i-12,17,18 in MeSRDRNAi transgenosis Manihot Esculentas
The phosphorylation degree of middle starch is lowered the most substantially, about 1ng/mg starch, G1i-28,31 secondly,
Respectively 7ng/mg starch and 15ng/mg starch, and starch phosphorylation level is 20 in wild-type leaves
Ng/mg starch, its phosphorylation level substantially with the expression of MeSRD genes into positive correlation, such as Fig. 9
It is shown.
Regulation of the embodiment 7, MeSRD to amylose content in the provisional starch of blade
With the change of starch form in blade, its amylose content also there occurs obvious change.
Wild type and SRDRNAi transgenosis are cultivated under the village, often used in village names pilot plant test field natural conditions of Shanghai five respectively
Cassava, culture to harvest time.Determine amylose content in the provisional starch of blade.
Assay method：The measure of amylose content is with reference to country of People's Republic of China (PRC) mark in tapioca
Quasi- GB/T 15683-2008/ISO 6647-1：2007.Mainly comprise the following steps：Weigh 50mg ± 0.5mg
Tapioca, amylose standard specimen (Sigma, St.Louis, MO, USA) and amylopectin standard specimen (Sigma,
St.Louis, MO, USA) in test tube, add the ethanol of 500 μ L 95% to be bonded on inboard wall of test tube
Sample sweeps away, boiling water bath 10min after gently shaking up and add 4.5mL 1.0M sodium hydroxide solutions to mix
Above until amyloid is completely dispersed, constant volume in 50mL volumetric flasks is transferred to after being cooled to room temperature；Simultaneously
Prepared by same steps and be not added with any amyloid blank；Formed sediment using the amylose and side chain of constant volume
Powder standard specimen prepare amylose content be 0,10%, 20%, 30%, 40%, 50%, 60%, 70%,
80%th, 90% and 100% each 1mL of series standard content solution is standby；Add after the dilution of the iodine solutions of 8mL 2%
4mL 1.0M HAC solution is simultaneously settled to 200mL as starch coloration liquid, now with the current, with a collection of
Sample uses identical nitrite ion；Blank, tapioca sample, standard content solution respectively take 100 μ L
Mixing colour developing in 1mL starch coloration liquid is added, blank takes 4 times, and other samples take 3 times, colour developing
The measurement of 720nm wavelength OD values is carried out on spectrophotometer Libra S22 (Biochrom Ltd., UK) afterwards；
One-variable linear regression is carried out using absorbance of the standard content solution at 720nm and ask for phase relation
Number, sets up the equation of amylose content and absorbance, by tapioca sample absorbance substitution side to be measured
Formula asks for its amylose content.
Result such as Figure 10, relative to the wild type provisional starch of cassava blade, in rotaring gene plant blade
The content of amylose has and obviously rises, and amylose content there are about 9% in wild type cassava blade,
The content of amylose rises to 22-37% in the provisional starch of SRDRNAi rotaring gene plant blades, with block
The content of root storage characteristics starch kind amylose is close.
Regulation of the embodiment 8, MeSRD to the provisional starch macroscopic property of blade
Differential calorific value scanning (Differential Scanning Calorimetry, DSC) is determined in heating process
A kind of thermodynamics means of the heat absorption capacity of middle starch, the heat absorption capacity of starch is mainly formed sediment by starch straight chain
The influence of the aspects such as powder content, starch crystals degree, amylopectin chain length distribution.Had a try in the village, often used in village names of Shanghai five
Culture wild type and SRDRNAi transgenosis cassavas under the natural conditions of field are tested, is cultivated to harvest time, carry out heat
Mechanical property is determined.
Macroscopic property assay method is：Using Q2000 differential scanning calorimeters (TA Instruments,
Norwalk, CT, USA) make starch sample gelatinization thermodynamic analysis.By sample, (10mg starch adds 30 μ L
Water, using blank panel as blank) it is sealed in alumina crucible, after equilibrium at room temperature 24h, with 10 DEG C/min
Speed, be warming up to 95 DEG C by 30 DEG C, scan thermal change.Data are soft using Universal Analysis
Part is analyzed.
Result such as table 1, in SRDRNAi transfer-gen plants, the provisional starch To (initial gelatinization temperature) of blade,
Tp (peak value gelatinization point) does not have notable difference with the provisional starch of wild-type leaves, but Tc (is represented and terminated paste
Change temperature), Δ H (gelatinization enthalpy) significantly improve, wherein four times higher than wild type or so of Δ H.
The macroscopic property parameter of starch in the wild type of table 1 and transgenosis cassava blade
Note：Data shown in figure are the mean+SD that 3 experiments are repeated.Indicated in every group of data
Difference is not notable between the value of same letter (a, b, c, d, e) represents each measured value with this understanding
(p<0.05)。
Regulation of the embodiment 9, MeSRD to the provisional starch structure of blade
Wild type and SRDRNAi transgenosis cassavas are cultivated under the village, often used in village names pilot plant test field natural conditions of Shanghai five,
Culture collects cassava blade to harvest time, and provisional starch is extracted from blade, is spread out using X-ray
Penetrate the diffracting spectrum that instrument determines the provisional starch of blade.
Starch XRD diffracting spectrums can be divided into A, B, C, V-type starch, it is considered that the XRD of starch
Diffracting spectrum is made up of the diffraction maximum of crystal region and the background peaks of noncrystalline domain.In wild type cassava blade
Starch XRD spectrum shows that its diffracting spectrum has multiple crystal region characteristic peaks, but noncrystalline domain background peaks ratio
Weaker, the provisional starch noncrystalline domain in indication wild-type leaves is fewer, and crystallinity is higher；And
Provisional starch in SRDRNAi rotaring gene plant blades has crystal region and noncrystalline as common starch
District's groups into, its diffraction maximum is mainly shown as 15 °, and 17 °, 23 ° of diffraction maximums are the c-type starch of standard, with
The diffracting spectrum of storage characteristics starch is close, such as Figure 11.
Additionally, the provisional starch of blade is utilized after isoamylase (Isoamylase) is hydrolyzed
HPAEC-PAD analyzes the glucan composition in its hydrolyzate.
Such as Figure 12, analysis result shows, most short dextran chain in the provisional starch of wild type cassava blade
It is DP6, there are two peaks at DP11-12 and DP45, there is an acromion at DP18-20.Different from wood
The chain length distribution of potato leaf starch, short chain content increases (2.2%), middle chain content drop in root tuber storage characteristics starch
Low (- 0.8%).Rotaring gene plant blade starch compared with wild type, its chain length changes in distribution trend and root tuber
Starch change is similar：Its short chain (DP6-14) has obvious rising, and highest can improve 0.4%；Middle chain part
(DP20-37) significantly decrease, about -0.2% or so.
The all documents referred in the present invention are all incorporated as reference in this application, just as each text
Offer and be individually recited as with reference to such.In addition, it is to be understood that reading above-mentioned instruction content of the invention
Afterwards, those skilled in the art can make various changes or modifications to the present invention, and these equivalent form of values are same
Fall within the application appended claims limited range.
Claims (14)
1. it is a kind of adjust potato class plant blade starch property method, it is characterised in that methods described
Including：The expression of SRD polypeptides in regulation potato class plant.
2. the method for claim 1, it is characterised in that described potato class plant includes：Cassava,
Sweet potato, potato, Chinese yam, taro, the root of kudzu vine, konjaku, Jerusalem artichoke, Smallantus sonchifolium.
3. the method for claim 1, it is characterised in that described SRD polypeptides are selected from the group：
(a) such as SEQ ID NO:The polypeptide of 2 amino acid sequences；
B () is by SEQ ID NO:2 amino acid sequences pass through the substitution of one or more amino acid residues, lack
Lose or addition and formed, and with (a) polypeptide function as polypeptide derived from (a)；Or
C polypeptide sequence that () limits with (a) has more than 70% homology and being spread out by (a) with (a) polypeptide function
Raw polypeptide.
4. the method for claim 1, it is characterised in that methods described includes：In reduction plant
The expression of SRD polypeptides, so that：
Improve the content of starch in potato class plant leaf blade；
Improve the diameter of starch granules in potato class plant leaf blade；
Reduce the phosphorylation degree of starch in potato class plant leaf blade；
Increase the content of amylose in potato class plant leaf blade；And/or
Increase the content of short chain in starch, reduce the content of long-chain moiety in starch.
5. method as claimed in claim 4, it is characterised in that SRD polypeptides in the reduction plant
Expression includes：The disturbing molecule of the SRD expression of polypeptides will be disturbed to be transferred to plant, so that in lowering plant
The expression of SRD polypeptides.
6. method as claimed in claim 5, it is characterised in that the interference SRD expression of polypeptides it is dry
Disturb the encoding gene or its transcript of molecular targeted SRD polypeptides；It is preferred that targetting the of the encoding gene
741-1193 or its transcript.
7. method as claimed in claim 6, it is characterised in that described disturbing molecule contains formula (I) institute
The structure shown：
SeqIt is positive-X-SeqReverselyFormula (I),
In formula (I),
SeqIt is positiveIt is the fragment of the encoding gene of SRD polypeptides, SeqReverselyIt is and SeqIt is positiveComplementary polynucleotides；
It is preferred that SeqIt is positiveIt is 741-1193 of the encoding gene of SRD polypeptides；
X is positioned at SeqIt is positiveAnd SeqReverselyBetween intervening sequence, and the intervening sequence and SeqIt is positive
And SeqReverselyIt is not complementary.
8. the method for claim 1, it is characterised in that methods described also includes subsequent step：
Proterties for comparing the preceding plant of regulation is selected from the plant after the expression of regulation SRD polypeptides to be changed
Plant.
9. a kind of purposes of the material of downward SRD expression of polypeptides, the shallow lake of the blade for adjusting potato class plant
Mealiness shape.
10. purposes as claimed in claim 9, it is characterised in that described downward SRD expression of polypeptides
Material be used for：
Improve the content of starch in potato class plant leaf blade；
Improve the diameter of starch granules in potato class plant leaf blade；
Reduce the phosphorylation degree of starch in potato class plant leaf blade；
Increase the content of amylose in the provisional starch of potato class plant leaf blade；And/or
Increase the content of short chain in starch, reduce the content of long-chain moiety in starch.
11. purposes as described in claim 9 or 10, it is characterised in that described potato class plant includes：
Cassava, sweet potato, potato, Chinese yam, taro, the root of kudzu vine, konjaku, Jerusalem artichoke, Smallantus sonchifolium etc..
A kind of 12. downward SRD expression of polypeptides are so as to adjust the interference of the starch property of the blade of potato class plant
Molecule, it contains the structure shown in formula (I)：
SeqIt is positive-X-SeqReverselyFormula (I),
In formula (I),
SeqIt is positiveIt is the encoding gene segment of SRD polypeptides, SeqReverselyIt is and SeqIt is positiveComplementary polynucleotides；
It is preferred that SeqIt is positiveIt is 741-1193 of the encoding gene of SRD polypeptides；
X is positioned at SeqIt is positiveAnd SeqReverselyBetween intervening sequence, and the intervening sequence and SeqIt is positive
And SeqReverselyIt is not complementary.
13. a kind of carriers, it is characterised in that described carrier contains the interference point described in claim 12
Son.
The purposes of a kind of 14. SRD polypeptides or its encoding gene, it is characterised in that for as identification potato
The molecular labeling of the starch property of the blade of class plant；The starch property bag of the blade of described potato class plant
Include：
Content of starch in potato class plant leaf blade；
The diameter of starch granules in potato class plant leaf blade；
The phosphorylation degree of starch in potato class plant leaf blade；
The content of amylose in the provisional starch of potato class plant leaf blade；And/or
Increase the content of short chain in starch, reduce the content of long-chain moiety in starch.
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| CN113293170A (en) * | 2021-05-27 | 2021-08-24 | 中国热带农业科学院热带生物技术研究所 | Gene MeTIR1 for regulating cassava starch content and application thereof |
| CN116143891A (en) * | 2023-03-07 | 2023-05-23 | 江苏省农业科学院 | Application of transcription factor CeMyb108 in regulation and control of taro starch synthesis |
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