CN107119071A - A kind of method for reducing plant amylose content and application - Google Patents
A kind of method for reducing plant amylose content and application Download PDFInfo
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Abstract
The present invention relates to biotechnology and technical field of plant transgene, and in particular to a kind of method of reduction plant amylose content and application, step one：Utilize polynary sgRNA CRISPR/Cas9 system editor's potato class plant GBSSI gene orders；Step 2：CRISPR/Cas9 polycomponent systems described in step one include：In system containing two and more than two sgRNA sequences CRISPR/Cas9 systems；Step 3：The sgRNA nucleotide sequences of CRISPR/Cas9 polycomponent systems in step 2 are selected from the group；(a) such as SEQ ID NO:Nucleotide sequence in 1 nucleotide sequence；(b) by SEQ ID NO:1 nucleotides sequence sequence is by formed by the substitution of one or more nucleotides, missing or addition, and with (a) functional nucleotide as nucleotide sequence derived from (a)；It can significantly adjust the development character of the storage root of potato class plant, can significantly adjust the amylose content of potato class plant, be had a good application prospect on the genetic improvement of plant amylum quality.
Description
【Technical field】
The present invention relates to biotechnology and technical field of plant transgene, and in particular to one kind reduction plant amylose contains
The method of amount and application.
【Background technology】
Potato class plant, which refers mainly to have, is available for edible roots or rhizomatous class terrestrial crop.There are root tuber, tubers, such as
Sweet potato (sweet potato, sweet potato), cassava, potato, Chinese yam (Chinese yam), sole potato etc., multirow vegetative propagation only stays potato wedge to plant, and
It can be bred with liana.This kind of general cold-hartliness of plant is weaker, is cultivated more in frostless season, and low temperature can suppress tuber crops
Growth, cause the underproduction of root tuber or stem tuber, therefore plantation tuber crops are tried one's best and avoid prolonged hypothermic phase；In addition, loose,
Fertile, deep soil and volume potash fertilizer is conducive to improving tuber crops yield and quality.
Starch has great importance as important grain and industrial raw materials in national product.The composition bag of starch
Containing amylose and amylopectin two large divisions.Amylose is the polysaccharide chain that D-Glucose base is connected with α-(Isosorbide-5-Nitrae) glycosidic bond,
There are 200 or so glucosyl groups, molecular weight 1~2 × 10 in molecule5, the degree of polymerization 990, space conformation crimps spirality, each
Turn round as 6 glucosyl groups.In branched amylopectin molecules in addition to the sugar chain for having α-(Isosorbide-5-Nitrae) glycosidic bond, also α-(1,6) glycosidic bond connects
Containing 300~400 glucosyl groups, molecular weight in the branch connect, molecule>2×107, the degree of polymerization 7200, each branch is also all curling
Spirality.The starch of different amylose contents has greatly difference in industrial applicability and food processing.
Therefore, the composition and property of research potato class plant amylum, find the key factor of regulation storage root character, are these
The emphasis of area research.
【The content of the invention】
In view of the defects and deficiencies of the prior art, the present invention intends to provide one kind reduces plant amylose content
With the method for improving amylopectin content.
A kind of method for reducing plant amylose content and improving amylopectin content of the present invention, it is using such as
Under method and step:
Step one：Utilize polynary sgRNA CRISPR/Cas9 system editor's potato class plant GBSSI gene orders；
Step 2：CRISPR/Cas9 polycomponent systems described in step one include：Contain two and two or more in system
SgRNA sequences CRISPR/Cas9 systems；
Step 3：The sgRNA nucleotide sequences of CRISPR/Cas9 polycomponent systems in step 2 are selected from the group：
(a) such as SEQ ID NO:Nucleotide sequence in 1 nucleotide sequence；
(b) by SEQ ID NO:1 nucleotides sequence sequence shape by the substitution of one or more nucleotides, missing or addition
Into, and with (a) functional nucleotide as nucleotide sequence derived from (a)；
(c) there is more than 70% homology with (a) nucleotide sequence limited and derivative by (a) with (a) nucleic acid function
Multinuclear acid sequence；
Step 4：In above-mentioned steps one, following aspect is included to the editor of GBSSI genes：
(a) editor to genome area where GBSSI genes or the volume to including genome area where GBSSI genes
Volume；
(b) editor in (a) is included：Substitution, missing or the addition of single or multiple nucleic acid sequences；
Step 5：Terminating the expression or termination of GBSSI polypeptides in plant has the formation of GBSSI polypeptides of biological function, from
And amylose content in potato class plant amylum can be improved, amylopectin and regulation potato class plant in reduction potato class plant amylum
Storage root weight, diameter or number；
Step 6：The expression or termination of GBSSI polypeptides have the GBSSI of biological function in termination plant in above-mentioned steps five
The formation of polypeptide includes：The polynary CRISPR/Cas9 carrier molecules of sgRNA nucleotide sequences with step 3 are transferred to plant
Thing, so as to edit in plant GBSSI gene nucleotide series in genome, and then terminate in plant the expression of GBSSI polypeptides or
Terminate the expression of the formation for the GBSSI polypeptides for having biological function；
Step 7：Reduce plant amylose content and improve the method for amylopectin content also including subsequent step：
From terminate GBSSI polypeptides expression or termination have biological function GBSSI polypeptides formation after plant in, selection
Go out to compare the plant of character acquisition change for plant before regulation, including：
Character obtains the plant of change for before the relatively regulation directly produced；
What hybridization or selfing were produced has the plant for comparing character acquisition change for plant before regulation of foreign gene；
Character obtains the plant of change to the comparing before regulation without foreign gene that hybridization or selfing are produced for plant.
Further, described potato class plant includes：Cassava, sweet potato, potato, Chinese yam, taro, the root of kudzu vine, konjaku, Jerusalem artichoke
And yacon.
Further, the sgRNA targeting GBSSI genome nucleotide sequences of editor's GBSSI nucleotide sequences；It is preferred that targetting
477-496 and 1722-1741 of GBSSI genomes.
Further, the material of its expression is adjusted using polynary CRISPR/Cas9 systems editor GBSSI genome sequences
Purposes, the starch for adjusting potato class plant is constituted and property.
After said structure, the present invention has the beneficial effect that：A kind of reduction plant amylose content of the present invention
With the method for improving amylopectin content, it finds to pass through in CRISPR/Cas9 polycomponent system editor's potato class Plant Genomes
GBSSI genes, expression of the regulation GBSSI polypeptides in potato class plant, can significantly adjust the development of the storage root of potato class plant
Character can significantly adjust the amylose content of potato class plant, have good answer on the genetic improvement of plant amylum quality
Use prospect.
【Brief description of the drawings】
Accompanying drawing described herein be for providing a further understanding of the present invention, constituting the part of the application, but
Inappropriate limitation of the present invention is not constituted, in the accompanying drawings：
Fig. 1 is sgRNA in the present invention position view in GBSSI genome sequences.Grey square frame area is extron
Area, black line area is to include sub-district.
Fig. 2 is the CRISPR/Cas9 multiple vectors schematic diagrames in the present invention.Wherein sgRNA-cas9 is that middle transition is carried
Body；PCAMBIA 1301S are whole expression vector.SgRNA1, sgRNA2 are started using arabidopsis U6 promoters；Cas9 genes are by intending
Southern mustard uBQ promoters start, with flag labels.Hygromycin gene is carried in whole carrier：Hyg II, are opened with CaMV35S
Mover starts.
Fig. 3 is the transfer-gen plant Southern blot identifications in the present invention.Mark：λ-HindIII Marker, gene
DNA is by HindIII digestions for group, is hybridized after agarose gel separation, transferring film with hygromycin probe, and red font is planted for single copy
Strain.It is greenhouse pot culture seedling leaf that DNA, which extracts material,.
Fig. 4 is the CRISPR/Cas9 transfer-gen plant PCR testing results in the present invention.M:DL2000plus marker；
WT:Wild type control group；L series：Transgenosis series strain.It is greenhouse pot culture seedling leaf, fine jade after PCR amplifications that DNA, which extracts material,
Lipolysaccharide glue gel is separated by electrophoresis, and red-label is strain to be selected.
Fig. 5 is the double base CRISR/Cas9 gene editing effect detections in the present invention.It is greenhouse pot culture seedling that DNA, which extracts material,
Blade, agarose gel gel electrophoresis is separated after PCR amplifications, sequencing.
A:MeGBSSI-sg-1 is represented in PCR primer in large fragment sequencing result in MeGBSSI-cas9 double element systems
The gene editing result in sgRNA1 sites；MeGBSSI-sg-2 represents the base in sgRNA2 sites in MeGBSSI-cas9 double element systems
Because of edited result
B:MeGBSSI-sg-out represents the sequencing result of small fragment in PCR, that is, knocks out sequencing result after target gene.Its
It is middle red for sgRNA sequences；Blueness is PAM sequences；Purple is insetion sequence；" -- " represents that nucleic acid is deleted；Grey is original
Sequence.
Fig. 6 be the polynary sgRNA mediations of use in the present invention CRISR-Cas9 technological transformation cassavas after amylose content.
The outdoor grown in field wild type of six months of plantation and transfer-gen plant are taken, starch is extracted, determines amylose and amylopectin
Content.WT:Wild type tapioca；MeGI series：Transgenosis after the CRISR-Cas9 system editors of polynary sgRNA mediations is planted
Strain.
【Embodiment】
Describe the present invention in detail below in conjunction with accompanying drawing and specific embodiment, illustrative examples therein and say
It is bright to be only used for explaining the present invention but not as a limitation of the invention.
As shown in figs 1 to 6, a kind of method of reduction plant amylose content described in present embodiment and should
With it is presented below：
The present invention " potato class plant " be also referred to as " tuber crops ", refer mainly to have be available for edible roots or rhizomatous one
The terrestrial crop of class.Including but not limited to：The root crops such as sweet potato of the root crops of Euphorbiaceae such as cassava, Convolvulaceae, Solanaceae
Tuberous plant such as potato, the root crops such as Chinese yam of Dioscoreaceae, the tuberous plant such as taro, konjaku, pulse family of Araeceae
The root crops such as root of kudzu vine, composite family tuberous plant such as Jerusalem artichoke, yacon etc..
The present invention includes CRISPR/Cas9 multiple vectors systems；Wherein：
1) term " polynary " in the present invention refers to be kept substantially GBSSI cores in the present invention with two or unnecessary two
The nucleotide sequence of sgRNA sequence identical biological functions or activity in nucleotide sequence.
Polynary can be two or more sgRNA both from SEQ ID NO:1 region；
Or two or more sgRNA at least one come from SEQ ID NO:1 region；
Or two or more sgRNA are all from SEQ ID NO:Beyond 1 region, but knock out or editing area
Include SEQ ID NO:1.
The sgRNA of any GBSSI genomic sources bioactive nucleotides fragment can be applied to the present invention
In.
In the present invention, the implication of sgRNA bioactive fragment refers to that, as a kind of nucleotide sequence, it still can keep
Normal sgRNA all or part of function.Under normal circumstances, bioactive fragment at least keeps 50% normal sgRNA
Activity.Under still more preferential conditions, active fragment can keep total length SRD polypeptides 60%, 70%, 80%, 90%,
95%th, 99% or 100% activity.
2) sgRNA sequences of the invention can be following statement：
The nucleotide sequence in subregion is included in GBSSI genomes or have one or more nucleotides it is substituted but
Still there is the nucleotide sequence of sgRNA functions in the present invention；
Or nucleotide sequence in GBSSI genome Exons region or have one or more nucleotides substituted
But still with the nucleotide sequence of sgRNA functions in the present invention
Or mutation or region is knocked out comprising GBSSI genomes in the present invention are whole or nucleotide sequence of subregion
Or have one or more nucleotides it is substituted but still with the nucleotide sequence of sgRNA functions in the present invention.According to herein
Definition these polynary, sgRNA belong to scope known to those skilled in the art.
3) in the present invention, term " GBSSI genomes " refers to the SEQ ID NO with bioactivity:Many nucleosides of 1 sequence
Acid.The term is also included with the SEQ ID NO with GBSSI nucleotides identical functions:The variant form of 1 sequence.These variations
Form includes (but being not limited to)：Several (it is usually 1-150, preferably 1-90, more preferably 1-60, most preferably 1-
30, also more preferably such as 1-24,1-15) missing of nucleotides, insertion and/or replace.For example, in the art, different product
There is polymorphism in the cassava GBSSI genome sequences for planting source, will not generally change the function of nucleotide sequence.Again such as, in C
End and/or N-terminal, which add one or several sequence labels, will not generally also change the function of nucleic acid sequence.
4) variant form of GBSSI nucleotide sequences includes：It is homologous sequence, conservative variant, allelic variant, natural
Mutant, induced mutants, DNA sequence dna that can be with GBSSI nucleic acid array hybridizings under the conditions of high or low stringency.
5) it is any high (such as with SEQ ID NO with GBSSI gene homologies:The homology of sequence shown in 1 is
70% or higher；It is preferred that, homology is 80% or higher；It is furthermore preferred that homology is 90% or higher, such as homology
95%, 98% or 99%) and be also included in the present invention with the nucleotide sequences of GBSSI gene order identical functions.
Although 6) it should be understood that the GBSSI gene orders of the present invention are preferably obtained from cassava, obtained from other plants and wood
Potato GBSSI gene orders very high homology (as having more than 70%, such as 80%, 90%, 95%, even 98% sequence thereto)
Other polynucleotides are also within the scope of the present invention contemplates.The Method and kit for of the aligned sequences phase same sex is also well known in the art
, such as BLAST.
7) the invention further relates to encode, the polynucleotides sequence of GBSSI polypeptides or its conservative variation's polypeptide in the present invention
Row.
Polynucleotides can be DNA form or rna form.
DNA form includes cDNA, genomic DNA or artificial synthesized DNA.
DNA 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 SEQ ID NO:Coding region sequence shown in 1 is identical or letter
And variant.As used herein, " variant of degeneracy " refers to that coding has SEQ ID NO in the present invention:2 albumen
Matter, but with SEQ ID NO:The differentiated nucleotide sequence of coding region sequence shown in 1.
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 coded polypeptide " can be included the polynucleotides of coding said polypeptide or also wrap
Include the polynucleotides of additional code and/or non-coding sequence.
8) 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 the 80% phase same sex.
9) GBSSI gene orders full length sequence or its fragment of the invention can generally use PCR TRAPs, recombination method or people
The method of work synthesis is obtained.
, can be according to relevant nucleotide sequence, especially open reading frame sequence disclosed in this invention for PCR TRAPs
To design primer, and with commercially available cDNA storehouses or the cDNA storehouses conduct as prepared by conventional method well known by persons skilled in the art
Template, expands and obtains relevant sequence.
10) the present invention also relates to include the carrier of described polynucleotides, and with described carrier or GBSSI gene sequences
Arrange the host cell produced through genetic engineering.
11) present invention also offers a kind of starch composition for adjusting potato class plant and the method for property, this method includes adjusting
Save the expression of GBSSI polypeptides in the potato class plant.
It is highly preferred that described method includes：GBSSI genome nucleotide sequences in the potato class plant are edited, are adjusted
GBSSI polypeptides expression (including make termination GBSSI polypeptides expression or termination have biological function GBSSI polypeptides shape
Into), so that the amylose of potato class plant amylum and the content of amylopectin are adjusted, including：Improve straight in potato class plant amylum
Chain content of starch；Reduce the content of amylopectin in potato class plant amylum；Adjust potato class plant storage root weight, diameter or number
Mesh.
Expression or termination that can be using the gene editing system being related in the present invention to terminate GBSSI polypeptides have biology
The formation of the GBSSI polypeptides of function.
12) CRISPR/Cas9 polycomponent system editor's potato class is passed through there is provided one kind as one embodiment of the present invention
GBSSI genes in Plant Genome, expression of the regulation GBSSI polypeptides in potato class plant, so as to improve potato class plant straight chain shallow lake
Powder content reduces the method for amylopectin content simultaneously.
Its method includes：Step one：The polynary CRISPR/Cas9 carrier molecules for carrying GBSSI genomes sgRNA are transferred to
Plant tissue, organ or seed, obtain the plant tissue, organ or seed for being transformed into the editting function；Step 2：By step
Plant tissue, the device of the one polynary CRISPR/Cas9 carrier molecules for being transferred to the carrying GBSSI genomes sgRNA obtained
Official or seed regeneration plant.
As a kind of preferred example, its method comprises the following steps：
(i) Agrobacterium for the carrier for carrying editable GBSSI genomes is provided, described carrier is selected from the group：
(a) encoding gene or the carrier of genetic fragment containing sgRNA the and Cas9 polypeptides started；
(b) carrier of GBSSI genomes and sweet potato sequence can be edited in plant by containing；
(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 its method 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 GBSSI genes, the characteristic identification GBSSI targets after plant is imported are can be designed that
The polynucleotides of point.The efficiency of specificity and editor is considered during design.The present invention does not have to the preparation method of sgRNA sequences
There is special limitation, include but is not limited to：Chemical synthesis, in-vitro transcription method etc..It should be understood that those skilled in the art are learning
The composition of CRISPR/Cas9 polycomponent systems, sgRNA sequences with after the correlation of plant trait, can with various approach systems
For described expression system is gone out, so that for adjusting plant trait.Described polycomponent system can be conveyed by transgenic technology
It can be transported into plant, or also using multiple technologies known in the art in plant.
13), can specific editor GBSSI as a preference of the present invention, there is provided a kind of sgRNA molecules of excellent effect
Genome sequence；And empirical tests, it has the effect of good regulation GBSSI polypeptide biological functions.SgRNA molecules are to contain
There are SEQ ID NO:The molecule of nucleotide sequence in 1 shown in 477-496 and 1722-1741, constitutes double element system.
14) double element system is constituted present invention also offers one kind, the double element system contains two sgRNA sequences, can be with
Orientation knocks out larger sequence fragment in genome.Described double element system, can be two in genome after importeding into plant
Individual special target spot is edited simultaneously can also be by between two target spots while two target spot mutation are caused after genome, editor
Nucleotide sequence fragment is knocked out.Generally, described double element system is located on expression vector.
15) present invention additionally comprises the plant obtained using foregoing any method, described plant include but is not limited to from
The expression or termination of termination GBSSI polypeptides, which have to select in the plant after the formation of the GBSSI polypeptides of biological function, compares regulation
Character obtains the plant of change for preceding plant, for example：Character obtains the plant of change for before the relatively regulation directly produced；It is miscellaneous
Hand over or selfing is produced have a foreign gene compare before regulation that character obtains the plant of change for plant；Hybridization or selfing
Character obtains the plant of change to the comparing before regulation without foreign gene produced for plant.Can be using any appropriate conventional hand
Section, including reagent, temperature, pressure condition etc. implement described method.
With reference to specific embodiment, the present invention is expanded on further.It should be understood that these embodiments are merely to illustrate the present invention
Rather than limitation the scope of the present invention.The experimental method of unreceipted actual conditions in the following example, generally according to conventional strip
Part such as J. Pehanorm Brookers etc. are write, Molecular Cloning:A Laboratory guide, the third edition, Science Press, the condition described in 2002, or
According to the condition proposed by manufacturer.
Specific embodiment one：Cassava MeGBSSI genes sgRNA selection and synthesis：
In the present invention, to obtain cassava MeGBSSI gene sgRNA sequences, this department utilizes ncbi database (https://
Www.ncbi.nlm.nih.gov/ the MeGBSSI genome nucleotide sequences of cassava) be have found.Total length 2097bp；By this three
Gene order is by Blastp and Blastn in RIKEN cassava cDNA databases (http://www.brc.riken.jp/
Inf/en/index.shtml its genome sequence is found in search in), and total length 3397bp, comprising 13 extrons, encodes 699
Amino acid.
Special sgRNA sequences MeGBSSI-sgRNA1 is filtered out from genome sequence:5’-
TTGGGATACCTCTGTATCGG-3’；MeGBSSI-sgRNA2：5 '-CGGGATATTGGAATCAGACA-3 ', synthesize sgRNA sequences
Row.It is as follows：
sgRNA1：
LP:5’-GATTGTTGGGATACCTCTGTATCGG-3’；
RP:5’-AAACCCGATACAGAGGTATCCCAAC-3’；
sgRNA2:
LP:5’-GATTGCGGGATATTGGAATCAGACA-3’
RP:5’AAACTGTCTGATTCCAATATCCCGC-3’；
Specific embodiment two：The structure of cassava MeGBSSI gene C RISPR/Cas9 binary vectors and obtaining for transgenosis cassava
：
Two sgRNA fragments are imported into the middle of two sgRNA-cas9 carry first by this department respectively by BbsI enzymatic cleavage methods
In body, then any one sgRNA is being imported in sgRNA-cas9 intermediate carriers formation double base using KpnI and XbaI double digestions
Between transition vector：sgRNA1-cas9-sgRNA2.The double base middle transition carrier built is transferred to expression vector pCAMBIA again
In 1301S.Then sgRNA1-cas9-sgRNA2-P1301s is transferred to Agrobacterium LBA4404, then cassava is infected by Agrobacterium
Fragility suspension callus, the callus after infecting obtains positive plant by processes such as regeneration, screenings, respectively by sgRNA1-
Cas9-sgRNA2-P1301s transgenosis cassavas are denoted as MeGBSSI-cas9, and MeGI is abbreviated as respectively.As shown in Figure 1 and Figure 2.
Specific embodiment three：The Molecular Identification of MeGBSSI-Cas9 transgenosis cassavas：
The sgRNA1-cas9-sgRNA2-P1301s positives are obtained altogether by agriculture bacillus mediated cassava suspension callus conversion to turn
Totally 20 strains such as gene cassava MeGI-1,5,12,16,17, then pass through that Southern blot screenings are last to obtain single copy
Transfer-gen plant is totally 4 strains such as MeGI L1/L2/L4/L9.
" the gene editing detection of MeGBSSI-Cas9 transgenosis cassavas of specific embodiment four
In order to verify CRISPR/Cas9 gene editing effect, the present inventor is extracted the gene of transgenosis cassava respectively
Group, designs amplimer：FP:5’-GTGATGTTCTTGGAGGACTCCCC-3’；RP:5’-
CCTGGACGTCCATGCCATTTATA-3 ' expands purpose editor's fragment of MeGBSSI in single copy plant by PCR.As a result show
Show in the serial transgenic lines of MeGBSSI-Cas9, there is small fragment (Fig. 3) occur in L2/L4/L9/L12, sequencing result shows
Show small fragment for the sequence after theoretical knock out, the target spot cut in sgRNA1 and sgRNA2 goes out to exist insertion mutation (Fig. 4)；To sheet
Section sequencing result is shown, prominent including insertion mutation, deletion there is also jumping phenomenon in sgRNA1 and sgRNA2 editing sites
Become, replace (Fig. 4).Illustrate our double element system with very high gene knockout efficiency and editorial efficiency.
Specific embodiment five：Influence of the MeGBSSI-Cas9 transgenosis cassava to starch constituent
In big Tanaka, growth, development and the yield of observation wild type cassava and MeGBSSI-Cas9 transgenosis cassavas
Character.
The seedling stem of MeGBSSI-Cas9 transgenosis cassavas is harvested from the seedling storehouse of Sanya in mid-April, in the village, often used in village names of Shanghai five
Have a try and test Tanaka's ridging, the high 50cm in ridge, ridge spacing is 100cm, by seedling stem cuttage on ridge, then spreads mulch film, at first three
Monthly average pours weekly a water, keeps soil moisture, after three months, pours within 20 days or so a water, keeps soil not non-irrigated,
And applied once-combined fertilizer in five month, until harvest.
The starch in wild type and the ripe storage root of transgenosis cassava is extracted, its amylose content is determined.As a result show
Wild type amylose tapioca content is 26%, is remarkably decreased in MeGBSSI-Cas9 amylose contents, between 5%-23%
Between.
All documents referred in the present invention are all incorporated as reference in this application, independent just as each document
It is incorporated as with reference to such.In addition, it is to be understood that after the above-mentioned instruction content of the present invention has been read, those skilled in the art can
To be made various changes or modifications to the present invention, these equivalent form of values equally fall within the model that the application appended claims are limited
Enclose.
SEQUENCE LISTING
<110>The broomcorn millet bio tech ltd of Jiangsu three
<120>A kind of method for reducing plant amylose content and application
<130> 20170607
<160> 10
<170> PatentIn version 3.5
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<211> 3397
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aatggtttaa ggtccctcaa cactatggat aaactccaaa tgaagacaca atcaaaagca 180
gtgaaaaagg tctctgccac cggcaatggt aggcctgctg ccaaaattat ttgtggtcat 240
ggaatgaatt taatctttgt tggagctgaa gttggtccct ggagcaaaac tggtggactt 300
ggtgatgttc ttggaggact cccccctgcc atggccgtaa gtagaggacc cttcttttgt 360
tctttctgcc tcgccttggt ttaattaata tgctgaattc aactgttact cctctttcag 420
gcaagagggc accgcgtcat gacagtgtct ccccgctatg accagtacaa ggatgcttgg 480
gatacctctg tatcggtgga ggtatctttt cttattttta agaggcattt tctttctttg 540
ttatgtatct ccaattgtag gatttttttt aaaaattttt tttctccttc tagctatgtg 600
catctcgatt ctcatttgca gattaaaatt ggagatagaa ttgaaactgt ccgcttcttc 660
cactcctaca aaagaggagt tgatcgggtc ttcgtggatc atccaatgtt ccttgagaag 720
gttggaaaga acttgcaata attatagaaa cttgtatagg aaaaaacatt tcgattgctt 780
ctttttcctg attccaaact tgttttaatc ttaaggtatg gggcaaaact ggatctaaaa 840
tatatggccc aagagcaggt ttggattacc aggacaacca actgcgattt agcttgttat 900
gccttgtaag tcttgcattt gtttcttttt tccccaaatt gaacaaagga taatgttttt 960
tccaagtaaa cggcgtttct tcttaaaaag acactcttta ttgagattca tgcatcgtct 1020
agtgctgctt tacagtttgt agctagcagg cggttattca aaaagttttc cctctaatac 1080
ctgcaggctg ctctggaggc accgagagtt ctgaacttga acagcagcaa aaatttctca 1140
ggaccctacg gtgtgtactt tttaccaaaa tcttaattgg gattttcaat ttctctgttg 1200
aaaagtaact atttttaaac caatgcattt tgctttctta aattcttaat gcaggagaag 1260
aagttgcctt cattgccaac gactggcaca ctgctctgct tccatgttat ctaaaagcca 1320
tttaccaacc tatggggatt tacaaacacg ccaaggtttt tacccctctt aattgggact 1380
gaatagttaa tgattctgga ttacttgaaa gtgtcatttt cggggttggt gacttgtttg 1440
tttttttaat cgatgatagg ttgccttttg catccacaac attgcatatc agggaagatt 1500
tgccttctca gacttcccac gacttaatct gccagataaa ttcaaaagct cttttgactt 1560
tatcgatggg tatgattatt agggcagttg gtcttgccaa tgtgactccc gttgtttatg 1620
gtttccatga attacttctc ttgcctagtg ttgaaaggct ttgctcttct tgtcaggtat 1680
gagaagcccg tgaagggaag gaaaatcaat tggatgaagg ccgggatatt ggaatcagac 1740
agggttttga ctgtgagccc atactatgcc caagaagtca tctctggagt tgaaagaggc 1800
gtcgagctgg ataacttcat tcgtaaaact ggcattgctg gtattataaa tggcatggac 1860
gtccaggagt ggaatcctgt tacagataaa tacattgaca tccactacga tgccacaact 1920
gtgagtgcct cgcagtaatt tgaagtttgt aagcagtaca taatcaatag caaatgatgg 1980
tgatcgttgg gatttttctt gttcaggtta tggacgcaaa acctttgttg aaggaagccc 2040
ttcaagcaga agtcggattg cctgttgata ggaatgttcc tttgataggc ttcattggta 2100
gattagaaga gcagaagggt tcagatattt ttgttgcagc tatttcccaa ttggttgaac 2160
acaatgtgca gatagtaatc cttgtaagta tcagaattaa ggatgtcctt ggttgtgttg 2220
tgagacatgc cgtgtctgaa gatatgactt gttctttcag ggaactggca aaaagaaatt 2280
tgagaagcag attgagcatc tggaggtttt gtaccctgac aaggcaagag gagttgcaaa 2340
attcaatgtg ccgttggcgc acatgatcac agctggtgca gactttatgc tggttccaag 2400
tagatttgag ccctgtggtc tcattcagtt gcatgctatg cgatatggaa cagtaatggc 2460
gctaagccct tttcttctcc tttctatggc tgctatcctg attctaatat ccatggctta 2520
tggcatggct tttcccgtca gttgccgcaa tggcagtaca tggcagttaa caacatctaa 2580
tctttgcgca ggttcccatt gttgcttcta ctggtggtct tgttgatact gttaaagaag 2640
gttacacagg attccaaatg ggggccttgc gcgttgaagt aagtaaagag gcgattgctg 2700
caattttatt cttttcgctc taaatattga aacgatgcga ctcaccagtg ggatattttc 2760
cagtgtgaca aaattgattc agcagatgta gctgcgatag ttaaaactgt ggcaagagct 2820
cttggcactt atgctaccgc tgcattaaga gaaatgatcc tgaattgcat ggcccaagac 2880
ttgtcatgga aggtttgtat atgactaagt atctaggagg ctcatctcta ttgttaacta 2940
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ctggaagttg ctggcagcga acctggcact gaaggggagg agatcgctcc tcttgctaag 3120
gagaacgttc ccacgccttg agcagcaagg ataatattaa tcttgacggt gtagaaatat 3180
tgacatttat ggatatacgt taacgcgcag ataaatatcc attccagtag gctagtctgc 3240
tgggatcaaa ggcacccttt gtttttctat tccaacggcc atgatttctt tgtgaagggt 3300
gaatggtgtc tgaatcagtg ttaaagagaa tttacaacta acgtattaca gcctttgcct 3360
ggcgttatgt gcgtaaataa aggttcatct tctgaca 3397
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355 360 365
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385 390 395 400
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Claims (4)
1. a kind of method for reducing plant amylose content and application, it is characterised in that：It uses following method and step:
Step one：Utilize polynary sgRNA CRISPR/Cas9 system editor's potato class plant GBSSI gene orders；
Step 2：CRISPR/Cas9 polycomponent systems described in step one include：It is containing two and more than two in system
The CRISPR/Cas9 systems of sgRNA sequences；
Step 3：The sgRNA nucleotide sequences of CRISPR/Cas9 polycomponent systems in step 2 are selected from the group：
(a) such as SEQ ID NO:Nucleotide sequence in 1 nucleotide sequence；
(b) by SEQ ID NO:1 nucleotides sequence sequence is formed by the substitution of one or more nucleotides, missing or addition
, and with (a) functional nucleotide as nucleotide sequence derived from (a)；
(c) there is more than 70% homology and with (a) nucleic acid function as multinuclear derived from (a) with (a) nucleotide sequence limited
Acid sequence；
Step 4：In above-mentioned steps one, following aspect is included to the editor of GBSSI genes：
(a) editor to genome area where GBSSI genes or the editor to including genome area where GBSSI genes；
(b) editor in (a) is included：Substitution, missing or the addition of single or multiple nucleic acid sequences；
Step 5：Terminating the expression or termination of GBSSI polypeptides in plant has the formation of GBSSI polypeptides of biological function, so as to
Improve amylopectin and regulation potato class plant storage in amylose content in potato class plant amylum, reduction potato class plant amylum
Root weight, diameter or number；
Step 6：The expression or termination of GBSSI polypeptides have the GBSSI polypeptides of biological function in termination plant in above-mentioned steps five
Formation include：The polynary CRISPR/Cas9 carrier molecules of sgRNA nucleotide sequences with step 3 are transferred to plant, from
And GBSSI gene nucleotide series in genome are edited in plant, and then terminate the expression or termination of GBSSI polypeptides in plant and have
The expression of the formation of the GBSSI polypeptides of biological function；
Step 7：Reduce plant amylose content and improve the method for amylopectin content also including subsequent step：
From terminate GBSSI polypeptides expression or termination have biological function GBSSI polypeptides formation after plant in, select phase
Character obtains the plant of change for plant before relatively adjusting, including：
Character obtains the plant of change for before the relatively regulation directly produced；
What hybridization or selfing were produced has the plant for comparing character acquisition change for plant before regulation of foreign gene；
Character obtains the plant of change to the comparing before regulation without foreign gene that hybridization or selfing are produced for plant.
2. a kind of method for reducing plant amylose content according to claim 1 and application, it is characterised in that：It is described
Potato class plant include：Cassava, sweet potato, potato, Chinese yam, taro, the root of kudzu vine, konjaku, Jerusalem artichoke and yacon.
3. a kind of method for reducing plant amylose content according to claim 1 and application, it is characterised in that：Editor
The sgRNA targeting GBSSI genome nucleotide sequences of GBSSI nucleotide sequences；It is preferred that the 477- of targeting GBSSI genomes
496 and 1722-1741.
4. a kind of method for reducing plant amylose content according to claim 1 and application, it is characterised in that：Utilize
Polynary CRISPR/Cas9 systems editor GBSSI genome sequences adjust the purposes of the material of its expression, for adjusting potato class plant
Starch composition and property.
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