CN110093349A - SgRNA and application using CRISPR/Cas9 systemic characteristic shearing rice xal3 gene promoter - Google Patents
SgRNA and application using CRISPR/Cas9 systemic characteristic shearing rice xal3 gene promoter Download PDFInfo
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Abstract
The sgRNA and its application that the present invention provides one group using CRISPR/Cas9 systemic characteristic shearing rice xal3 gene promoter, belong to field of plant genetic project technology.SgRNA of the present invention includes g1RNA and g2RNA；The nucleotide sequence of the g1RNA is as shown in SEQ ID NO.1；The nucleotide sequence of the g2RNA is as shown in SEQ ID NO.2.Rice plant is transfected using the recombinant expression carrier that sgRNA provided by the invention combination building obtains, specifically the promoter region 149nt of xal3 gene can be lacked, the transgenic paddy rice for lacking the promoter fragment is set to show resistance to leaf spot bacteria by the ability of leaf spot bacteria inducing expression to make in rice body xal3 gene lose.In addition, method provided by the invention does not influence other characters of transgenic paddy rice, transgenic paddy rice fertility is good.
Description
Technical field
The invention belongs to field of plant genetic project technology, and in particular to one group utilizes CRISPR/Cas9 systemic characteristic
Shear sgRNA and its application of rice xal3 gene promoter.
Background technique
Resistance breeding to pests is one of the main target of breeding in agricultural production, and bacterial blight of rice is by leaf spot bacteria
(Xanthomonas oryzae pv.Oryzae, Xoo) causes, and is in the world to the maximum bacterial disease of rice hazard.Mesh
It is preceding to have identified a Resistant To Xanthomonas Oryzae major gene resistance more than 30 in rice, wherein it is recessive for having 5.Xa13 is in recent years gram
The very special rice recessive Bacterial blight resistance gene of grand one (Chu et al.2006), xa13 and its dominant allele
Xa13 does not have difference in the code area of gene, but the sequence variations of xa13 starting subdivision result in its table in rice leaf
Up to being remarkably decreased, so that rice plant obtains the resistance to leaf spot bacteria bacterial strain PXO99 specificity.Composing type inhibits
Rice can be improved to bacterial strain PXO99 resistance in the expression of xa13 equipotential dominant gene Xa13, but will lead to pollen fertility simultaneously
It is remarkably decreased, shows that the gene not only controls disease resistance also related (the Bart et al.2006 with the fertility of rice of rice；Chu
et al.2006)。
It is especially in crossbreeding in breeding, the use and inconvenience due to recessive resistance genes (need to improve simultaneously
Two parents of Hybrid), therefore breeder more payes attention to the application, such as Xa21, Xa23 or Xa7 etc. of dominant resistance gene.
Due to the evolution of pathogenic bacteria, a kind of disease-resistant gene often gradually loses its resistance using several years or more than ten years in production.
The function of recessive disease-resistant gene xa13 is related with disease-resistant and fertility simultaneously, and the function of the disease-resistant gene found in the past has larger difference
Different, theoretically, it is the weight to existing disease-resistant gene resource that anti-disease mechanism and other disease-resistant genes, which should also have biggish difference,
It supplements.
It is generally acknowledged that CRISPR-Cas9 technology is to can be used for constructing base after the technologies such as Zinc finger nuclease (ZFN) and TALEN
Because fixed point knocks out third generation gene editing technology, and the characteristics of efficient high, speed is fast and simple economy.The work of this system is former
Reason is that crRNA (CRISPR-derived RNA) is combined by base pairing and tracrRNA (trans-activating RNA)
TracrRNA/crRNA compound is formed, this compound guides nuclease Cas9 albumen in the sequence target site matched with crRNA
Shear double-stranded DNA.And by both RNA of engineer, it may sgRNA (single of the transformation formation with guiding function
Guide RNA), guidance Cas9 cuts the fixed point of DNA.
Genome fixed point editor was successfully completed using Cas9 in model plant arabidopsis for the first time since 2014
(Jiang et al., 2013), rice, wheat, corn there are also the crops such as mushroom also have successfully report (Sun et al.,
2016；Liu et al.,2017；Hussain et al.,2018).Gene editing is carried out using Cas9 within 2014 to successfully obtain
The wheat of mildew-resistance, the Cas9 of anti-browning in 2016 is to the mushroom of polyphenol oxidase (PPO) gene editing by United States Department of Agriculture
Ruling does not need to need additional supervision as traditional genetically modified crops, obtains anti-weeding also with the technology within 2016
The rice of agent.It is bigger that this shows that the crop of the gene editing obtained using cas9 technology is had compared with traditional genetically modified crops
Degree of recognition has big advantage in terms of application.
Summary of the invention
The problem of in view of background technique, utilizes CRISPR/Cas9 system the purpose of the present invention is to provide a kind of
The sgRNA of system specific cleavage rice xal3 gene promoter and its application, are obtained using transgenic approach without transgenosis trace
High bacterial leaf spot resistant and the normal rice of fertility.
The present invention provides one group to utilize CRISPR/Cas9 systemic characteristic shearing rice xal3 gene promoter
SgRNA, the sgRNA include g1RNA and g2RNA；The nucleotide sequence of the g1RNA is as shown in SEQ ID NO.1；It is described
The nucleotide sequence of g2RNA is as shown in SEQ ID NO.2.
The present invention provides a kind of recombinant expression carrier, the recombinant expression carrier is using pYLCRISPR/Cas9-MH as base
Plinth, including Cas9 gene, U3 promoter, g1RNA, U6a promoter and the g2RNA being sequentially connected in series.
Preferably, the nucleotide sequence of the Cas9 gene is as shown in SEQ ID NO.3；The nucleotide of the U3 promoter
Sequence is as shown in SEQ ID NO.4；The nucleotide sequence of the U6a promoter is as shown in SEQ ID NO.5.
Preferably, the U3 promoter is connect by target spot connector T1 with g1RNA；The target spot connector T1 is by Pxal3U3-
F and Pxal3U3-R denaturation annealing is made；The nucleotide sequence of the Pxal3U3-F is as shown in SEQ ID NO.6；It is described
The nucleotide sequence of Pxal3U3-R is as shown in SEQ ID NO.7.
Preferably, the U6a promoter is connect by target spot connector T2 with g2RNA；The target spot connector T2 by
Pxal3U6a-F and Pxal3U6a-R denaturation annealing is made；The nucleotide sequence of the Pxal3U6a-F such as SEQ ID NO.8 institute
Show；The nucleotide sequence of the Pxal3U6a-R is as shown in SEQ ID NO.9.
The present invention provides a kind of for producing turning for the normal rice of high bacterial leaf spot resistant and fertility without transgenosis trace
Trans-genetic hybrid rice cell, which is characterized in that contain the simultaneously energy above-mentioned recombinant expression carrier of positive expression in the Transgenic rice cells.
The present invention also provides above-mentioned g1RNA and g2RNA, recombinant expression carrier or Transgenic rice cells to turn in production nothing
Application in the normal rice of high bacterial leaf spot resistant and fertility of gene trace.
The present invention provides a kind of high bacterial leaf spot resistant of no transgenosis trace and the normal Rice Production methods of fertility, including
Following steps:
(1) above-mentioned recombinant expression carrier is transformed into rice cell, obtains transformed cells；
(2) using the transformed cells as raw material, culture obtains aftergrowth；
(3) positive-selecting is carried out to the aftergrowth using PCR detection technique, the highly resistance for obtaining no transgenosis trace is white
Leaf is withered and the normal rice of fertility.
Preferably, step (1) rice cell is the cell of japonica rice ZH11.
Preferably, the target gene of step (3) the PCR detection is the marker gene that the recombinant expression carrier carries.
The utility model has the advantages that being opened the present invention provides one group using CRISPR/Cas9 systemic characteristic shearing rice xal3 gene
The sgRNA of mover and its application.The sgRNA includes g1RNA and g2RNA；The nucleotide sequence of the g1RNA such as SEQ ID
Shown in NO.1；The nucleotide sequence of the g2RNA is as shown in SEQ ID NO.2.It is combined and is constructed using sgRNA provided by the invention
Obtained recombinant expression carrier transfection rice plant, can specifically lack the promoter region 149nt of xal3 gene,
To making xal3 gene in rice body lose by the ability of leaf spot bacteria inducing expression, make to lack the promoter fragment turns base
Because rice shows resistance to leaf spot bacteria.In addition, method provided by the invention does not influence other characters of transgenic paddy rice,
Transgenic paddy rice is while obtaining high bacterial leaf spot resistant character, no transgenosis trace, and fertility is good.
Detailed description of the invention
Fig. 1 is the structural schematic diagram of recombinant expression carrier described in the embodiment of the present invention 1；
Fig. 2 is the carrier is carrier used in building process of recombinant expression carrier described in the embodiment of the present invention 1, wherein scheming
2-a is the structural schematic diagram of pYLcrispr/Cas9-MH expression vector；Fig. 2-b is sgRNA donor vehicle pYLgRNA-OsU6a
~c；The structural schematic diagram of-OsU3；
Fig. 3 be the embodiment of the present invention 2 described in transgenic plant blade PCR identification, Sourthern copy number detection and
PD1-6 sequencing result；
Fig. 4 is the phase of Xa13 gene in PD1-6 T0 generation homozygous shearing transgenic line blade described in the embodiment of the present invention 4
To the testing result of expression quantity；
Fig. 5 is the economical character and resistance result of rice described in the embodiment of the present invention 5.
Fig. 6 is PCR testing result described in the embodiment of the present invention 6.
Specific embodiment
The present invention reported in the promoter sequence of xa13 gene and the sequence by pathogen controlling element sequence,
A cas9 cutting target site is respectively selected in the upstream and downstream of this element sequences, engineer obtains one according to the two target sites
Group using CRISPR/Cas9 systemic characteristic shearing rice xal3 gene promoter sgRNA, the sgRNA include g1RNA and
g2RNA；The nucleotide sequence of the g1RNA is as shown in SEQ ID NO.1；The nucleotide sequence of the g2RNA such as SEQ ID
Shown in NO.2.
Rice plant is transfected using the recombinant expression carrier that sgRNA provided by the invention combination building obtains, does not influence to turn
Other characters of trans-genetic hybrid rice can specifically lack the promoter region 149nt of xal3 gene, to make rice body
Interior xal3 gene loses the transgenic paddy rice dialogue leaf for being made to lack the promoter fragment by the ability of leaf spot bacteria inducing expression
Blight bacterium shows resistance.
The present invention provides a kind of recombinant expression carrier, the recombinant expression carrier is connected preferably by PCR with digestion
Method constructs to obtain.In the present invention, the recombinant expression carrier preferably wraps preferably based on pYLCRISPR/Cas9-MH
Include the Cas9 gene being sequentially connected in series, U3 promoter, g1RNA, U6a promoter and g2RNA.In the present invention, the Cas9 gene
Nucleotide sequence preferably as shown in SEQ ID NO.3；The nucleotide sequence of the U3 promoter is preferably such as SEQ ID NO.4 institute
Show；The nucleotide sequence of the U6a promoter is preferably as shown in SEQ ID NO.5.The U3 promoter is preferably connect by target spot
Head T1 is connect with g1RNA；The target spot connector T1 is made by Pxal3U3-F and Pxal3U3-R denaturation annealing；The Pxal3U3-
The nucleotide sequence of F is preferably as shown in SEQ ID NO.6；The nucleotide sequence of the Pxal3U3-R is preferably such as SEQ ID NO.7
It is shown.The U6a promoter preferably passes through target spot connector T2 and connect with g2RNA；The target spot connector T2 by Pxal3U6a-F and
Pxal3U6a-R denaturation annealing is made；The nucleotide sequence of the Pxal3U6a-F is preferably as shown in SEQ ID NO.8；It is described
The nucleotide sequence of Pxal3U6a-R is preferably as shown in SEQ ID NO.9.In the present invention, the polymerization in the denaturation annealing is excellent
Choosing uses archaeal dna polymerase, and the denaturation temperature in the denaturation annealing is preferably 95 DEG C.
The present invention provides a kind of for producing turning for the normal rice of high bacterial leaf spot resistant and fertility without transgenosis trace
Trans-genetic hybrid rice cell, in the Transgenic rice cells containing and can the above-mentioned recombinant expression carrier of positive expression.The present invention is to institute
The construction method for stating Transgenic rice cells is not particularly limited, conventional method in that art.
The present invention also provides above-mentioned sgRNA combination, recombinant expression carrier or transgenic cell in production without transgenosis trace
Application in the normal rice of high bacterial leaf spot resistant and fertility of mark.
Specifically, the present invention provides a kind of high bacterial leaf spot resistant of no transgenosis trace and the normal Rice Production sides of fertility
Method includes the following steps:
(1) above-mentioned recombinant expression carrier is transformed into rice cell, obtains transformed cells；
(2) using the transformed cells as raw material, culture obtains aftergrowth；
(3) positive-selecting is carried out to the aftergrowth using PCR detection technique, the highly resistance for obtaining no transgenosis trace is white
Leaf is withered and the normal rice of fertility.
In the present invention, step (1) described rice cell is preferably the cell of japonica rice ZH11.Step (3) the PCR detection
Target gene be preferably the included marker gene of the recombinant expression carrier, more preferably hygromycin gene.The tide
The PCR primer of mycin resistant gene is preferably Hpt-F/R.The present invention it is described to step (1) conversion, step (2) it is described culture with
And the specific method of step (3) described screening is not particularly limited, this field routine operation.
Technical solution provided by the invention is described in detail below with reference to embodiment, but they cannot be understood
For limiting the scope of the present invention.
Primer information involved in various embodiments of the present invention is shown in Table 1.
1 primer information of table
Embodiment 1
The selection of Cas9 target spot and the building of Cas9+P13 (g1RNA+g2RNA) expression vector
According to the document that Chu et al. (2006) is delivered, sequence number of the Xa13 gene in Genebank is obtained
"DQ421395".It is obtained in the Genebank of the website NCBI (www.ncbi.nlm.nih.gov) according to the sequence number
The sequence of DQ421395.According to the sequence of DQ421395 the website TIGR Rice (http: //
Rice.plantbiology.msu.edu/LocusNameSearch.shtml blastn) is carried out, obtains it in TIGR Rice
In locus identifier:Os08g42350.1.2000bp is chosen forward as its promoter sequence in gene 5 ' end.With reference to
The article that Romer (2010) and Yuan (2011) are delivered obtains the element sequences regulated and controled in promoter by pathogenic bacterium inducing.At this
The upstream and downstream of a element sequences respectively selects a cas9 cutting target site.
With reference to the method (Ma et al., 2015) of Liu Yaoguang seminar, U3 is constructed using the method that PCR is connected with digestion
The two segments are finally connected into through BsaI by the expression vector of the g2RNA and Cas9 albumen of the g1RNA of driving, U6a driving simultaneously
In the pYLCRISPR/Cas9-MH of digestion.Detailed process is as follows:
(1) Pxa13U3-F and Pxa13U3-R, Pxa13U6a-F and Pxa13U6a-R are mixed respectively, after 95 DEG C of denaturation
The cooling completion annealing of room temperature is moved to complete the preparation of target spot connector.
(2) BsaI digestion carrier pYLgRNA-OsU3/LacZ and pYLgRNA-OsU6 are utilized, after digging glue recovery purifying,
It connect to form intermediate vector g1RNA-OsU3/LacZ and g2RNA-OsU6a with target spot connector.
(3) using intermediate vector g1RNA-OsU3/LacZ and g2RNA-OsU6a as template, it is utilized respectively primer pair U-F/
Pxa13U3-R, Pxa13U3-F/gRNA-R and primer pair U-F/Pxa13U6a-R, Pxa13U6a-F/gRNA-R are carried out for the first time
PCR reaction.
(4) will PCR reaction product dilute 10 times after respectively mix after be used as template, using primer pair Uctga-B2 '/
GRcggt-BL and Uctcg-B1 '/gRctga-B2 carries out second of PCR reaction.By reaction product and after BsaI digestion
PYLCRISPR/Cas9-MH carrier connects to form whole support C as9+P13 (g1RNA+g2RNA).The plasmid that building is completed carries out
Sequencing analysis, confirmation construct successfully.
Embodiment 2
(1) conversion of Cas9+P13 (g1RNA+g2RNA) carrier
With reference to the long-grained nonglutinous rice method for transformation of Hiei etc. (1994) mediated by agriculture bacillus delivered, by the Cas9+P13 (g1RNA of building
+ g2RNA) expression vector is transformed into common japonica rice conversion kind ZH11.Utilize the PCR primer Hpt-F/ of hygromycin gene
R carries out the PCR positive-selecting of transgenic plant, obtains 40 independent T0 for positive transformants plant；Again with PCX-F/R to 40
Strain positive transformants plant is detected, and discovery wherein has 6 plants of target sites to realize shearing completely.
(2) the target site PCR sequence verification of homozygous shearing transgenic plant
In order to prove target gene group sequence deletion in this research really due to Cas9 and two sgRNA mediate it is special
Property cutting caused by, the present embodiment expands the obtained PCR product of transgenic plant with PCX-F/R, after T-easy carrier cloning
Sequencing.All operations carry out all in accordance with the specification that producer provides.The present invention to 6 conversions individual (PD1, PD2, PD3,
PD4, PD5, PD6) target site sequence amplification after the TA cloning vector that obtains be sequenced.
The blade PCR of transgenic plant identifies that the detection of Sourthern copy number and PD1-6 sequencing result are shown in Fig. 3.Fig. 3
In, Fig. 3-a is the blade PCR qualification result of PD1-6 transgenic plant；Fig. 3-b is PD1-6 sourthern copy number detection knot
Fruit；Fig. 3-c is PD1-6 target site sequencing result sequencing result.Fig. 3 the result shows that:
1. having 13 plants of target sequences for finding that 6 plants of realization target sequences shear completely in transgenic plant altogether in 40 plants of T0
Not exclusively shearing.
2. it is single copy that there are two single plants through sourthern detection in the plant of 6 plants of homozygous shearings.
3. being sequenced after carrying out PCR amplification to plant (PD1-6) target site of 6 plants of homozygous shearings, 6 all transgenosis men
It is the position that target site DNA is broken all is between the 3rd and 4 base in the site PAM.Wherein PD1, PD3, PD4, PD5 and
Five transgenic lines such as PD6 have lacked the institute between two broken sites including pathogen specificity inducing expression element orderly
Column, and then there are two the radom insertions of base between two broken sites by PD2.The sequencing result meet Cas9 albumen and
The general specificity rule for the genome cutting that sgRNA is mediated shows that the missing of target gene genome is because of Cas9 albumen
And caused by the cutting of the mRNA specificity of two sgRNA of g1, g2 mediation.
Embodiment 3
The bacterial leaf spot Resistance Identification of target site homozygosis shearing transgenic paddy rice
In order to detect whether genetically modified plants obtain expected disease resistance, the present embodiment is in tillering regularity, to all
T0 transgenic plant is vaccinated with leaf spot bacteria biological strain PXO99 and (using turbidimetry, connects bacteria concentration control in 9~1,200,000,000/ml
Between).Each transgenosis single plant is inoculated with 5~6 leaves, 14 days investigation scab length and lesion area after inoculation.Inoculation result is shown in
Table 2.In table 2: spending 11 (ZH11) morbidity obvious in wild type, mean leaf scab length is 16.82cm, lesion area
65.82%.And transgenic plant (PD1-PD6) shows the disease resistance significantly improved.The mark used by Chu Zhaohui (2006)
Quasi- (scab length < 3cm is that highly resistance is horizontal after inoculation 14 days) calculates, the transgenosis T0 of 6 target site homozygosis shearing for plant all
The resistance of high resistance to hoja blanca bacterium PXO99 is shown, scab length is in 3cm or less.
Table 2 is inoculated with PXO99 experimental result
Table 2 the result shows that: utilize the starting that is induced by cause of disease of CRISPR/Cas9 systemic characteristic shearing rice xa13 gene
The disease resistance of transgenic plant can be improved in subsequence, and does not influence its fertility.
Embodiment 4
The expression quantity of the Xa13 gene of transgenic plant detects
The case where in order to detect the expression quantity of Xa13 gene in transgenic resistance plant leaf, the present embodiment utilize real-
The method of time PCR have detected in rotaring gene plant blade be inoculated with leaf spot bacteria before and inoculation 3 days after Xa13 gene expression
It measures (X13-Qt1-F/R).The detection knot of the relative expression quantity of Xa13 gene in PD1-6T0 generation homozygous shearing transgenic line blade
Fruit sees Fig. 4.Wherein, Fig. 4-a is the RT-PCR testing result of Xa13 gene in blade and anther in PD1；Fig. 4-b is in PD1-6
The expression pattern qualification result of Xa13 gene.
The RT-PCR of Xa13 gene is as the result is shown: expression is not apparent from PD1 blade after 3 days after being inoculated with leaf spot bacteria
Variation, and raised in wild type obvious；But in the expression quantity and wild type in PD1 anther, expression quantity is all very high；qRT-
PCR the results show that PD1-6T0 generation homozygous shearing transgene vaccination the result shows that after inoculation leaf spot bacteria PXO99, in blade
The expression quantity of Xa13 gene does not show obviously to raise compared with before inoculation；And Xa13 gene after inoculation in wild type ZH11
Expression quantity rises as many as 36 times.Show in transgenic plant Xa13 by pathogenic bacterium inducing ability with the missing of the element
And lose, to obtain the bacterial leaf spot resistance of height.
Embodiment 5
The fertility of transgenic plant is investigated
In the research of (2006) Chu et al., although the expression using the hpRNA interference Xa13 gene of constitutive expression can
To improve transgenic plant to the disease resistance of PXO99 bacterial strain, but the pollen potassium iodide rate of dyeing of transgenic plant is abnormal, fertility
Be generally reduced, should the result shows that Xa13 in addition to having outside the Pass with Resistant To Xanthomonas Oryzae, it is closely related with the pollen development of rice.
The present embodiment investigates the economical character and resistance of transgenic paddy rice, as a result sees Fig. 5.Wherein, Fig. 5-a
For the appearance Agronomic of transgenic paddy rice；Fig. 5-b is the disease-resistant sex expression of transgenic paddy rice；Fig. 5-c is transgenosis water
The fertility performance of rice.
It is as shown in Figure 5: to spend 11 controls and part that there is high disease resistance transgenic paddy rice strain to have carried out pollen in wild type
The detection of potassium iodide dyeing (0.67% potassium iodide and 0.33% iodine), the results showed that high disease-resistant plant pollen development is just
Often, with compare no significant difference, setting percentage is also normal.
The setting percentage of transgenic plant after maturation is investigated, spent in wild type 11 setting percentage be 82.65 ±
3.22%；6 transgenic lines are 78.95%, 78.68%, 57.15%, 72.37%, 85.94% and 85.94% respectively；Two
Group numerical value P=0.215, and there was no significant difference.
Under normal conditions, transgenic protocol will lead to some transgenic plants fertility decline or infertility, this often due to
Somatic mutation or foreign gene insertion cause the factors such as endogenous important gene inactivation to cause in tissue culture procedures.This research
In, the fertility of resistant plant shows transgenosis relative to the fertility of disease plant without statistical difference in transgenic plant
Plant Sterility variation is unrelated with disease resistance.
Embodiment 6
The separation of single copy rice family of the plant PD1 offspring without transgenosis trace
T1 generation 30 familys of the present invention in singly copy PD1 have carried out PCR detection (cas9-F/R) to external source Cas9 albumen,
Testing result is shown in Fig. 6.It will be appreciated from fig. 6 that the present invention has obtained 8 plants of external source introgressed segments separated family PD1-2,4,12,
16,21,26,27,30, and to this 30 T1 for family promoter gene type and Resistance Identification as the result is shown with T0 generation
PD1 is consistent.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Sequence table
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<400> 3
atggctccta agaagaagcg gaaggttggt attcacgggg tgcctgcggc tgacaagaag 60
tactccatcg gcctcgacat cggcaccaac agcgtcggct gggcggtgat caccgacgag 120
tacaaggtcc cgtccaagaa gttcaaggtc ctgggcaaca ccgaccgcca ctccatcaag 180
aagaacctca tcggcgccct cctcttcgac tccggcgaga cggcggaggc gacccgcctc 240
aagcgcaccg cccgccgccg ctacacccgc cgcaagaacc gcatctgcta cctccaggag 300
atcttctcca acgagatggc gaaggtcgac gactccttct tccaccgcct cgaggagtcc 360
ttcctcgtgg aggaggacaa gaagcacgag cgccacccca tcttcggcaa catcgtcgac 420
gaggtcgcct accacgagaa gtaccccact atctaccacc ttcgtaagaa gcttgttgac 480
tctactgata aggctgatct tcgtctcatc taccttgctc tcgctcacat gatcaagttc 540
cgtggtcact tccttatcga gggtgacctt aaccctgata actccgacgt ggacaagctc 600
ttcatccagc tcgtccagac ctacaaccag ctcttcgagg agaaccctat caacgcttcc 660
ggtgtcgacg ctaaggcgat cctttccgct aggctctcca agtccaggcg tctcgagaac 720
ctcatcgccc agctccctgg tgagaagaag aacggtcttt tcggtaacct catcgctctc 780
tccctcggtc tgacccctaa cttcaagtcc aacttcgacc tcgctgagga cgctaagctt 840
cagctctcca aggataccta cgacgatgat ctcgacaacc tcctcgctca gattggagat 900
cagtacgctg atctcttcct tgctgctaag aacctctccg atgctatcct cctttcggat 960
atccttaggg ttaacactga gatcactaag gctcctcttt ctgcttccat gatcaagcgc 1020
tacgacgagc accaccagga cctcaccctc ctcaaggctc ttgttcgtca gcagctcccc 1080
gagaagtaca aggagatctt cttcgaccag tccaagaacg gctacgccgg ttacattgac 1140
ggtggagcta gccaggagga gttctacaag ttcatcaagc caatccttga gaagatggat 1200
ggtactgagg agcttctcgt taagcttaac cgtgaggacc tccttaggaa gcagaggact 1260
ttcgataacg gctctatccc tcaccagatc caccttggtg agcttcacgc catccttcgt 1320
aggcaggagg acttctaccc tttcctcaag gacaaccgtg agaagatcga gaagatcctt 1380
actttccgta ttccttacta cgttggtcct cttgctcgtg gtaactcccg tttcgcttgg 1440
atgactagga agtccgagga gactatcacc ccttggaact tcgaggaggt tgttgacaag 1500
ggtgcttccg cccagtcctt catcgagcgc atgaccaact tcgacaagaa cctccccaac 1560
gagaaggtcc tccccaagca ctccctcctc tacgagtact tcacggtcta caacgagctc 1620
accaaggtca agtacgtcac cgagggtatg cgcaagcctg ccttcctctc cggcgagcag 1680
aagaaggcta tcgttgacct cctcttcaag accaaccgca aggtcaccgt caagcagctc 1740
aaggaggact acttcaagaa gatcgagtgc ttcgactccg tcgagatcag cggcgttgag 1800
gaccgtttca acgcttctct cggtacctac cacgatctcc tcaagatcat caaggacaag 1860
gacttcctcg acaacgagga gaacgaggac atcctcgagg acatcgtcct cactcttact 1920
ctcttcgagg atagggagat gatcgaggag aggctcaaga cttacgctca tctcttcgat 1980
gacaaggtta tgaagcagct caagcgtcgc cgttacaccg gttggggtag gctctcccgc 2040
aagctcatca acggtatcag ggataagcag agcggcaaga ctatcctcga cttcctcaag 2100
tctgatggtt tcgctaacag gaacttcatg cagctcatcc acgatgactc tcttaccttc 2160
aaggaggata ttcagaaggc tcaggtgtcc ggtcagggcg actctctcca cgagcacatt 2220
gctaaccttg ctggttcccc tgctatcaag aagggcatcc ttcagactgt taaggttgtc 2280
gatgagcttg tcaaggttat gggtcgtcac aagcctgaga acatcgtcat cgagatggct 2340
cgtgagaacc agactaccca gaagggtcag aagaactcga gggagcgcat gaagaggatt 2400
gaggagggta tcaaggagct tggttctcag atccttaagg agcaccctgt cgagaacacc 2460
cagctccaga acgagaagct ctacctctac tacctccaga acggtaggga tatgtacgtt 2520
gaccaggagc tcgacatcaa caggctttct gactacgacg tcgaccacat tgttcctcag 2580
tctttcctta aggatgactc catcgacaac aaggtcctca cgaggtccga caagaacagg 2640
ggtaagtcgg acaacgtccc ttccgaggag gttgtcaaga agatgaagaa ctactggagg 2700
cagcttctca acgctaagct cattacccag aggaagttcg acaacctcac gaaggctgag 2760
aggggtggcc tttccgagct tgacaaggct ggtttcatca agaggcagct tgttgagacg 2820
aggcagatta ccaagcacgt tgctcagatc ctcgattcta ggatgaacac caagtacgac 2880
gagaacgaca agctcatccg cgaggtcaag gtgatcaccc tcaagtccaa gctcgtctcc 2940
gacttccgca aggacttcca gttctacaag gtccgcgaga tcaacaacta ccaccacgct 3000
cacgatgctt accttaacgc tgtcgttggt accgctctta tcaagaagta ccctaagctt 3060
gagtccgagt tcgtctacgg tgactacaag gtctacgacg ttcgtaagat gatcgccaag 3120
tccgagcagg agatcggcaa ggccaccgcc aagtacttct tctactccaa catcatgaac 3180
ttcttcaaga ccgagatcac cctcgccaac ggcgagatcc gcaagcgccc tcttatcgag 3240
acgaacggtg agactggtga gatcgtttgg gacaagggtc gcgacttcgc tactgttcgc 3300
aaggtccttt ctatgcctca ggttaacatc gtcaagaaga ccgaggtcca gaccggtggc 3360
ttctccaagg agtctatcct tccaaagaga aactcggaca agctcatcgc taggaagaag 3420
gattgggacc ctaagaagta cggtggtttc gactccccta ctgtcgccta ctccgtcctc 3480
gtggtcgcca aggtggagaa gggtaagtcg aagaagctca agtccgtcaa ggagctcctc 3540
ggcatcacca tcatggagcg ctcctccttc gagaagaacc cgatcgactt cctcgaggcc 3600
aagggctaca aggaggtcaa gaaggacctc atcatcaagc tccccaagta ctctcttttc 3660
gagctcgaga acggtcgtaa gaggatgctg gcttccgctg gtgagctcca gaagggtaac 3720
gagcttgctc ttccttccaa gtacgtgaac ttcctctacc tcgcctccca ctacgagaag 3780
ctcaagggtt cccctgagga taacgagcag aagcagctct tcgtggagca gcacaagcac 3840
tacctcgacg agatcatcga gcagatctcc gagttctcca agcgcgtcat cctcgctgac 3900
gctaacctcg acaaggtcct ctccgcctac aacaagcacc gcgacaagcc catccgcgag 3960
caggccgaga acatcatcca cctcttcacg ctcacgaacc tcggcgcccc tgctgctttc 4020
aagtacttcg acaccaccat cgacaggaag cgttacacgt ccaccaagga ggttctcgac 4080
gctactctca tccaccagtc catcaccggt ctttacgaga ctcgtatcga cctttcccag 4140
cttggtggtg ataagcgtcc tgctgccacc aaaaaggccg gacaggctaa gaaaaagaag 4200
tag 4203
<210> 4
<211> 383
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
aaggaatctt taaacatacg aacagatcac ttaaagttct tctgaagcaa cttaaagtta 60
tcaggcatgc atggatcttg gaggaatcag atgtgcagtc agggaccata gcacaagaca 120
ggcgtcttct actggtgcta ccagcaaatg ctggaagccg ggaacactgg gtacgttgga 180
aaccacgtgt gatgtgaagg agtaagataa actgtaggag aaaagcattt cgtagtgggc 240
catgaagcct ttcaggacat gtattgcagt atgggccggc ccattacgca attggacgac 300
aacaaagact agtattagta ccacctcggc tatccacata gatcaaagct ggtttaaaag 360
agttgtgcag atgatccgtg gca 383
<210> 5
<211> 414
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
accatccgaa tgataggata ggaaaaatat ccaagtgaac agtattccta taaaattccc 60
gtaaaaagcc tgcaatccga atgagccctg aagtctgaac tagccggtca cctgtacagg 120
ctatcgagat gccatacaag agacggtagt aggaactagg aagacgatgg ttgattcgtc 180
aggcgaaatc gtcgtcctgc agtcgcatct atgggcctgg acggaatagg ggaaaaagtt 240
ggccggatag gagggaaagg cccaggtgct tacgtgcgag gtaggcctgg gctctcagca 300
cttcgattcg ttggcaccgg ggtaggatgc aatagagagc aacgtttagt accacctcgc 360
ttagctagag caaactggac tgccttatat gcgcgggtgc tggcttggct gccg 414
<210> 6
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 6
ggcactatat aaacactgag cca 23
<210> 7
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 7
aaactggctc agtgtttata tag 23
<210> 8
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 8
gccggagaga gggacagatc tag 23
<210> 9
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 9
aaacctagat ctgtccctct ctc 23
<210> 10
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 10
ctccgtttta cctgtggaat cg 22
<210> 11
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 11
cggaggaaaa ttccatccac 20
<210> 12
<211> 40
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 12
ttcagaggtc tctctcgact agtggaatcg gcagcaaagg 40
<210> 13
<211> 37
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 13
agcgtgggtc tcgtcagggt ccatccactc caagctc 37
<210> 14
<211> 38
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 14
ttcagaggtc tctctgacac tggaatcggc agcaaagg 38
<210> 15
<211> 40
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 15
agcgtgggtc tcgaccgacg cgtccatcca ctccaagctc 40
<210> 16
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 16
gcatcattgt ccatggttg 19
<210> 17
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 17
gctagagagg aaggcttaag 20
<210> 18
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 18
ctgcaactca gaagaccgtt g 21
<210> 19
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 19
cctgttgtca ccctggaagt c 21
<210> 20
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 20
agtcgacggg agggtacag 19
<210> 21
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 21
gacgaggtag agggtggtga 20
<210> 22
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 22
agaatctcgt gctttcagct tcga 24
<210> 23
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 23
tcaagaccaa tgcggagcat atac 24
<210> 24
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 24
atgactctct taccttca 18
<210> 25
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 25
tagttcttca tcttcttg 18
Claims (10)
1. one group of sgRNA using CRISPR/Cas9 systemic characteristic shearing rice xal3 gene promoter, which is characterized in that
The sgRNA includes g1RNA and g2RNA；The nucleotide sequence of the g1RNA is as shown in SEQ ID NO.1；The g2RNA's
Nucleotide sequence is as shown in SEQ ID NO.2.
2. a kind of recombinant expression carrier, which is characterized in that the recombinant expression carrier based on pYLCRISPR/Cas9-MH,
Including be sequentially connected in series Cas9 gene, U3 promoter, described in g1RNA, U6a promoter and claim 1 described in claim 1
g2RNA。
3. recombinant expression carrier according to claim 2, which is characterized in that the nucleotide sequence of the Cas9 gene is such as
Shown in SEQ ID NO.3；The nucleotide sequence of the U3 promoter is as shown in SEQ ID NO.4；The nucleosides of the U6a promoter
Acid sequence is as shown in SEQ ID NO.5.
4. recombinant expression carrier according to claim 2 or 3, which is characterized in that the U3 promoter passes through target spot connector
T1 is connect with g1RNA；The target spot connector T1 is made by Pxal3U3-F and Pxal3U3-R denaturation annealing；The Pxal3U3-F
Nucleotide sequence as shown in SEQ ID NO.6；The nucleotide sequence of the Pxal3U3-R is as shown in SEQ ID NO.7.
5. recombinant expression carrier according to claim 2 or 3, which is characterized in that the U6a promoter passes through target spot connector
T2 is connect with g2RNA；The target spot connector T2 is made by Pxal3U6a-F and Pxal3U6a-R denaturation annealing；It is described
The nucleotide sequence of Pxal3U6a-F is as shown in SEQ ID NO.8；The nucleotide sequence of the Pxal3U6a-R such as SEQ ID
Shown in NO.9.
6. it is a kind of for producing the Transgenic rice cells of the normal rice of high bacterial leaf spot resistant and fertility without transgenosis trace,
It is characterized in that, recombinant expression described in simultaneously energy positive expression claim 2~5 any one is contained in the Transgenic rice cells
Carrier.
7. g1RNA and g2RNA described in claim 1, recombinant expression carrier or right described in claim 2~5 any one are wanted
Ask application of 6 Transgenic rice cells in high bacterial leaf spot resistant and fertility normal rice of the production without transgenosis trace.
8. the high bacterial leaf spot resistant and the normal Rice Production method of fertility of a kind of no transgenosis trace, which is characterized in that including such as
Lower step:
(1) recombinant expression carrier described in claim 2~5 any one is transformed into rice cell, obtains transformed cells；
(2) using the transformed cells as raw material, culture obtains aftergrowth；
(3) positive-selecting is carried out to the aftergrowth using PCR detection technique, obtains the high bacterial leaf spot resistant of no transgenosis trace
And the normal rice of fertility.
9. according to the method described in claim 8, it is characterized in that, step (1) rice cell is the cell of japonica rice ZH11.
10. according to the method described in claim 9, it is characterized in that, the target gene of step (3) the PCR detection is described
The included marker gene of recombinant expression carrier.
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| CN201910375501.0A CN110093349A (en) | 2019-05-07 | 2019-05-07 | SgRNA and application using CRISPR/Cas9 systemic characteristic shearing rice xal3 gene promoter |
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| CN201910375501.0A CN110093349A (en) | 2019-05-07 | 2019-05-07 | SgRNA and application using CRISPR/Cas9 systemic characteristic shearing rice xal3 gene promoter |
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Cited By (13)
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| CN111411123A (en) * | 2020-04-08 | 2020-07-14 | 上海市农业生物基因中心 | Method for simultaneously improving rice fragrance and bacterial leaf blight resistance by using CRISPR/Cas9 system and expression vector |
| CN111621515A (en) * | 2020-05-14 | 2020-09-04 | 中国计量大学 | Method for enhancing gene editing efficiency of CRISPR/Cas9 system |
| CN111849969A (en) * | 2020-01-21 | 2020-10-30 | 扬州大学 | Promoter Wx of rice Wx geneb1And use thereof |
| CN111849973A (en) * | 2020-01-21 | 2020-10-30 | 扬州大学 | Promoter Wx of rice Wx geneb5And use thereof |
| CN111849974A (en) * | 2020-01-21 | 2020-10-30 | 扬州大学 | Promoter Wx of rice Wx geneb7And use thereof |
| CN111849971A (en) * | 2020-01-21 | 2020-10-30 | 扬州大学 | Promoter Wx of rice Wx geneb4And use thereof |
| CN111849976A (en) * | 2020-01-21 | 2020-10-30 | 扬州大学 | Promoter Wx of rice Wx geneb6And use thereof |
| CN111849975A (en) * | 2020-01-21 | 2020-10-30 | 扬州大学 | Promoter Wx of rice Wx geneb2And use thereof |
| CN111849972A (en) * | 2020-01-21 | 2020-10-30 | 扬州大学 | Promoter Wx of rice Wx geneb3And use thereof |
| CN112175969A (en) * | 2020-10-13 | 2021-01-05 | 广东省农业科学院果树研究所 | Preparation and application of corn ZmFKF1 gene and gene editing mutant thereof |
| CN112609019A (en) * | 2020-12-21 | 2021-04-06 | 华智生物技术有限公司 | Screening method of gene editing site homozygote without transgene |
| CN114686498A (en) * | 2022-04-22 | 2022-07-01 | 中佳源物种(深圳)生物科技有限公司 | Apple mutant gene, encoding protein and preparation method of apple mutant |
| CN116262927A (en) * | 2021-12-13 | 2023-06-16 | 中国科学院微生物研究所 | Method for regulating gene expression based on CRISPR/Cas system and application thereof |
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| CN111849974A (en) * | 2020-01-21 | 2020-10-30 | 扬州大学 | Promoter Wx of rice Wx geneb7And use thereof |
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| CN111621515A (en) * | 2020-05-14 | 2020-09-04 | 中国计量大学 | Method for enhancing gene editing efficiency of CRISPR/Cas9 system |
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| CN112609019A (en) * | 2020-12-21 | 2021-04-06 | 华智生物技术有限公司 | Screening method of gene editing site homozygote without transgene |
| CN116262927A (en) * | 2021-12-13 | 2023-06-16 | 中国科学院微生物研究所 | Method for regulating gene expression based on CRISPR/Cas system and application thereof |
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| CN114686498A (en) * | 2022-04-22 | 2022-07-01 | 中佳源物种(深圳)生物科技有限公司 | Apple mutant gene, encoding protein and preparation method of apple mutant |
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