CN110184268A - A kind of rubber tree U6 gene promoter proHbU6.2 and its clone and application - Google Patents
A kind of rubber tree U6 gene promoter proHbU6.2 and its clone and application Download PDFInfo
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Abstract
The invention belongs to gene engineering technology fields, and in particular to a kind of rubber tree rna plymerase iii type promoter is more particularly to a kind of rubber tree U6 gene promoter proHbU6.2, and further discloses its cloning process and application.Clone obtains rubber tree rna plymerase iii type promoter -- the endogenous U6 promoter proHbU6.2 of rubber tree to the present invention in Para rubber tree for the first time, the promoter is rubber tree Endogenous RNA polymerase type III promoter, the promoter has efficient transcription activity, the expression of downstream sgRNA can be driven, and the activity of the promoter demonstrated by instantaneous conversion rubber tree protoplast and its applied to the feasibility of rubber tree CRISPR/Cas9 gene editing system, and realize the rubber tree genome targeting editor of CRISPR/Cas9 mediation for the first time.
Description
Technical field
The invention belongs to gene engineering technology fields, and in particular to a kind of rubber tree rna plymerase iii type promoter, more
More particularly to a kind of rubber tree U6 gene promoter proHbU6.2, and its cloning process and application are further disclosed.
Background technique
Natural rubber is always the important strategic materials and reserved resources in China, currently, the choosing of China's rubber tree new varieties
Educate is still based on traditional crossbreeding.Due to rubber tree slow growth, resulting in conventional breeding, there are low efficiencys, period
Long problem, this seriously hinders the process of Rubber Tree Breeding.With the development of biotechnology, molecular breeding technology is in rubber
Application in tree accelerates the cultivation of rubber tree new varieties, imports foreign gene by Agrobacterium or particle gun and loses as rubber tree
Pass the important way of improvement.However, genetic fragment is to be inserted randomly into rubber during above-mentioned exogenous genetic fragment imports
Genome is set, is inserted not only through site uncontrolled, while the problems such as there is also position benefits (Yutaka etc., 2018,
Chromosoma, doi:10.1007/s00412-018-0677-6.).
CRISPR/Cas9(clustered regularly interspaced short palindromic
Repeats/CRISPR-associated nuclease 9) genome editing technique appearance be genome it is accurate modify mention
New approach (Yongwei etc., 2016, Front Plant Sci, 7:1928 are supplied；Collonnier etc., 2017, Methods,
121-122:103-117；Yutaka etc., 2018, Chromosoma, doi:10.1007/s00412-018-0677-6), it is situated between
The basic principle for leading gene editing is to utilize nucleic acid by under single-stranded guide RNA (single guide RNA, sgRNA) guidance
Enzyme Cas9 fixed point combines and cutting upstream particular target site genome specificity site (PAM), generates DNA double chain fracture
(DSBs), and then cause the mutation of target site.Meanwhile it is raw by homologous recombination repair (HR) or non-homologous end joining (NHEJ)
Foreign donor DNA can also be inserted into sgRNA target site by the endogenous repair mechanism of object, to realize to specific site on genome
Mutation and specific DNA fragments (Lee etc., 2017, Elife, the 6:e25312. such as knockout, insertion and replacement；
Smirnikhina etc., 2018, Hum Genet, 138 (1): 1-19.), and the precision kind for being achieved in target gene changes
It is good.Currently, arabidopsis (Miki D etc., 2018, Nat Commun.9 (1): 1967.), rice (Li J etc., 2016, Nat
Plants, 2 (10): 16139.) and corn (Gil-Humanes J etc., 2017, Plant J, 89 (6): 1251-1262.) etc. is planted
The importing that foreign gene is realized based on plasmid CRISRP/Cas9 genome editing technique has been utilized in object.In addition, in jade
Rice, wheat have also also achieved the importing of foreign gene by CRISPR/Cas9-sgRNA RNP instantaneous conversion in rice
(Svitashev S etc., 2015, Plant Physiol, 169 (2): 931-945.；Liang Z etc., 2017, Nat Commun,
8:14261.；Sun Y W etc., 2016, Mol Plant, 9 (4): 628-631)；In addition, Liang etc. (2018, Nature
Protocols, 13 (3): 413-430.) also using particle gun by Cas9 and sgRNA expression vector plasmid, by instantaneous conversion by
Body cell realizes gene editing.
Studies have shown that is in CRISPR/Cas9 gene editing system, and the contents level of sgRNA is to influence in recipient cell
One of an important factor for editorial efficiency, therefore, the polymerase III type promoter that can accurately start sgRNA vivo transcription, obtain
Extensive concern (Jinek etc., 2016, science 337:816-821；Mali etc., 2013, science 339:823-826；
Cong etc., 2013, Science 339:819-823).U6RNA is a kind of non-coding RNA for participating in mRNA precursor montage, right
The U6 promoter answered is a kind of rna plymerase iii type promoter, and has been obtained in the CRISPR/Cas9 system of many species
Arrived a large amount of application (Kim and Nam, 2013, plant mol.Bio.Rep.31:581-593；Li et al., 2007,
J.integrate plant biol..49:222-229；H.Jia etc., 2014, pone, 9 (4): e93806.).
Although CRISPR/Cas9 genome editing technique has widely been applied in multiple species at present, needle
The gene editing technology of rubber tree is even had not been reported.Although this is mainly due in many species U6 promoter it is existing big
The report of amount, but external source U6 promoter usually and be not suitable for (X.Sun etc., 2015, Sci.Rep., 5p.10342).As it can be seen that
Lack the restriction factor that applicable U6 promoter has become current rubber tree CRISPR/Cas9 gene editing system, also limits
Application of the CRISPR/Cas9 genome editing technique in Rubber Tree Breeding.Therefore, the functional activity in rubber tree is filtered out
U6 promoter for rubber tree gene breeding technology development have positive meaning.
Summary of the invention
For this purpose, technical problem to be solved by the present invention lies in a kind of rubber tree U6 gene promoter proHbU6.2 is provided,
And its cloning process and application are further disclosed.
In order to solve the above technical problems, a kind of rubber tree U6 gene promoter proHbU6.2 of the present invention, described to open
Mover proHbU6.2 includes the DNA nucleotide sequence as shown in SEQ ID No:1.Above-mentioned rubber tree U6 gene promoter
ProHbU6.2 belongs to the rna plymerase iii type promoter of rubber tree U6snRNA gene, derives from Para rubber tree
(Heveabrasiliensis)。
Specifically, the DNA nucleotide sequence of the promoter proHbU6.2 is as shown in SEQ ID No:1.
The invention also discloses a kind of rubber tree instantaneous conversions to edit carrier, that is, contains the rubber tree U6 gene promoter
Sub- proHbU6.2.
Specifically, the instantaneous conversion editor carrier is recombinant plasmid proHbU6.2-sgRNA-163Cas9M.
The invention also discloses the methods of rubber tree U6 gene promoter proHbU6.2 described in clone a kind of, including such as
Lower step:
(1) 7-33-97 leaves genomic DNA is ground as template using Para rubber tree heat, designs following special primer:
ProHbU6.2-F:TCTAAGCTAAAGGAATAGCTTAGCAC；
ProHbU6.2-R:CAATTGCTACTGCCTATTC；
(2) PCR amplification is carried out in 20 μ l reaction systems using KOD FX enzyme；
(3) amplified production TA is cloned on pMD19-T carrier, converts in Escherichia coli Dh5 α and chooses recombinant monoclonal survey
Sequence is to get 345bp rubber tree U6 gene promoter DNA fragmentation proHbU6.2.
Specifically, in the step (2), the response procedures of the PCR amplification step are as follows: 95 DEG C of initial denaturation 2min, 98 DEG C
It is denaturalized 10s, 59 DEG C of annealing 30s, 72 DEG C of extension 1min, 35 circulations, 72 DEG C extend 5min eventually.
The invention also discloses a kind of methods of the editor's carrier of rubber tree instantaneous conversion described in building, including walk as follows
It is rapid:
(a) the promoter proHbU6.2 of clone is building up on intermediate vector SK-5G
With SalI and XhoI double digestion SK-5G carrier, 2913bp vector backbone segment is recycled, following primer is designed and exists respectively
ProHbU6.2 sequence and carrier gRNA sequence both ends introduce homologous sequence, and proHbU6.2 and gRNA are assembled into SK-5G carrier
On obtain proHbU6.2-SK-5G:
ProHbU6.2-lF:
GCGGCCGCAGATCTGCTAGCGTCGACTCTAAGCTAAAGGAATAGC；
ProHbU6.2-lR:
GTGTTGTGTTCACCTGCGAGCCAATTGCTACTGCCTATTC；
GRNA-sF:GCTCGCAGGTGAACACAACACC；
GRNA-sR:TTGGGTACCGAGGATCCTCTAGA；
(b) target site is building up on proHbU6.2-SK-5G carrier
Following target site is selected on rubber tree HbTFL1-3 gene:
GCCCAGCATAGGGATCCAC, with after AarI digestion proHbU6.2-SK-5G carrier both ends formed 3 '-TAAC and
5 '-GTTT, two cohesive ends；
Target site sequence is synthesized, and introduces two cohesive ends of 5 '-ATTG and 3 '-CAAA:
It is positive: ATTGGCCCAGCATAGGGATCCAC；
It is reversed: AAACGTGGATCCCTATGCTGGGC；
Above-mentioned forward and reverse target sequence DNA is mixed, carries out annealing at room temperature after 100 DEG C of processing, formed with
Then the segment is connected to load using T4 DNA ligase by the double-stranded DNA of proHbU6.2-SK-5G carrier complementary cohesive tennini
Body proHbU6.2 promoter downstream position obtains complete sgRNA expression cassette；
(c) sgRNA expression cassette is building up on instantaneous conversion editor carrier 163Cas9M
With KpnI and BglII double digestion proHbU6.2-sgRNA-SK-5G carrier, the small fragment sgRNA table of 494bp is recycled
Up to frame；
With KpnI and BamHI double digestion 163Cas9M carrier, 7919bp large fragment is recycled；
Small fragment sgRNA expression cassette is building up on 163Cas9M carrier to obtain rubber tree instantaneous using T4 DNA ligase
Conversion editor carrier proHbU6.2-sgRNA-163Cas9M to get.
The invention also discloses the methods that a kind of pair of rubber tree carries out genome editor, that is, include by the rubber tree wink
When conversion editor vector introduction rubber tree protoplast the step of.
The invention also discloses the rubber tree U6 gene promoter proHbU6.2 to lead in rubber tree molecular breeding technology
Application in domain.
The invention also discloses rubber tree instantaneous conversion editor's carriers in rubber tree molecular breeding technology field
Application.
Clone obtains rubber tree rna plymerase iii type promoter to the present invention in Para rubber tree for the first time -- in rubber tree
Source U6 promoter proHbU6.2, the promoter are rubber tree Endogenous RNA polymerase type III promoter, which has efficient
Transcriptional activity can drive the expression of downstream sgRNA, and by the target spot of the screening endogenous controlling gene HbTFL1-3 of blooming of rubber tree
After sequence, it is building up to proHbU6.2 promoter downstream in editor's carrier, rubber tree blade plasm wink is mediated by PEG
When conversion will edit and realize editor to HbTFL1-3 gene target site in vector introduction rubber tree cell, demonstrate
The activity of proHbU6.2 promoter also establishes effective CRISPR/Cas9 gene editing technology body in rubber tree for the first time
System.
The present invention is former by instantaneous conversion rubber tree for the first time by the rubber tree Endogenous RNA polymerase type III promoter of clone
Raw plastid demonstrates the activity of the promoter and its feasibility applied to rubber tree CRISPR/Cas9 gene editing system, and
The rubber tree genome targeting editor of CRISPR/Cas9 mediation is realized for the first time.It is verified, target site cloning and sequencing knot after editor
Fruit discovery mutation type is mostly the missing and insertion of single base, wherein the missing of polybase base and the insertion of small fragment also occurs.
Therefore, the promoter proHbU6.2 that the present invention clones can be applied to rubber tree CRISPR/Cas9 gene editing system, thus real
Now to rubber tree efficiently accurately breed improvement.
Detailed description of the invention
In order to make the content of the present invention more clearly understood, it below according to specific embodiments of the present invention and combines
Attached drawing, the present invention is described in further detail, wherein
Fig. 1 is that rubber tree HbU6.2 gene is compared with arabidopsis and cotton U6 gene order, wherein wire position is
The key element USE (upstream sequence element) and TATA box of U6snRNA transcription turn at arrow locations
Record initiation site；
Fig. 2 is clone's electrophoretogram of rubber tree HbU6.2 gene promoter, it is seen that has obtained 345bp's through PCR amplification
HbU6.2 promoter gene fragment；
Fig. 3 is that carrier proHbU6.2-sgRNA-163Cas9M structure diagram is edited in rubber tree instantaneous conversion, is in wire
Target sequence, underlined sequences are that BamHI identifies restriction enzyme site, and both ends are the cohesive end complementary with carrier；
Fig. 4 is rubber tree protoplast genome target fragment PCR product digestion (PCR/RE) qualification result again；Its
In, 1: rubber tree protoplast DNA target fragment PCR product；2: turning the protoplast DNA target base of control 163Cas9M carrier
Because of fragment PCR products BamHI digestion again；3: turning the protoplast DNA target gene of proHbU6.2-sgRNA-163Cas9M carrier
Fragment PCR products BamHI digestion again；Target fragment not to be edited is 500bp and 331bp small fragment, base by BamHI digestion
Because the target fragment after editor's mutation is the band that cannot be digested at arrow；
Fig. 5 is the digestion verification result of target fragment in monoclonal after TA is cloned, it is seen that obtains 13 target position point mutation
And it cannot be by the monoclonal of BamHI digestion；
Fig. 6 is target site edited result sequence alignment analysis；As it can be seen that after 13 edited target sites sequencings with open country
Raw type compares the missing and insertion for finding that main mutation type is single base, and there is also inserting for the missing of polybase base and small fragment
Enter.
Specific embodiment
It is conventional method unless otherwise instructed in the following embodiments of the present invention.Intermediate vector in following embodiments
SK-5G and instantaneous conversion carrier 163Cas9M give (Wang Kejian, Wang Chun, Shen Lan etc., plant by rice in China Suo Wangke sword seminar
The building and application of object polygenes knockout carrier), which only attaches most importance to used in the related experiment of duplicate invention, not can be used as
Other purposes use.
The acquisition of 1 Para rubber tree U6 gene promoter proHbU6.2 of embodiment
With arabidopsis AtU6-26 gene (Genebank accession number: X52528.1) and cotton GhU6-9 gene (Genebank
Accession number: XR_001680717.1) DNA sequence dna be reference, search for we foundation rubber tree genome database, by same
The mode that source compares finds a rubber tree HbU6 gene (Genebank accession number: XR_002491074.1), obtains this gene
Upstream reference sequence.
7-33-97 (Rubber Institute, Chinese Academy of Agricultural Science's cultivation) blade genome is ground with Para rubber tree heat
DNA is template, designs following proHbU6.2-F and proHbU6.2-R special primer, to clone promoter region 345bp DNA
Segment:
ProHbU6.2-F:TCTAAGCTAAAGGAATAGCTTAGCAC；
ProHbU6.2-R:CAATTGCTACTGCCTATTC；
PCR amplification, specific response procedures are carried out in 20 μ l reaction systems using KOD FX enzyme (TOYOBO) are as follows: 95 DEG C pre-
It is denaturalized 2min, 98 DEG C of denaturation 10s, 59 DEG C of annealing 30s, 72 DEG C of extension 1min, 35 circulations, 72 DEG C extend 5min eventually.
Amplified production TA is cloned on pMD19-T carrier, convert in Escherichia coli Dh5 α and chooses monoclonal sequencing, clone
Electrophoresis result is as shown in Figure 2.Finally, obtaining 345bp rubber tree U6 gene promoter DNA fragmentation as shown in SEQ ID No:1
proHbU6.2。
Using Vector NTI align X (Invitrogen) by the promoter sequence and arabidopsis AtU6-26 and cotton
GhU6-9 and its base sequence of promoter compare analysis, find key of the promoter sequence equally containing U6snRNA transcription
Site TATA box and USE (upstream sequence element) element (as shown in Figure 1), and the two element phases
Also almost the same with the promoter sequence of AtU6-26 and cotton GhU6-9 for the position of transcription initiation site, this is for geometry
The combination of symmetrical RNA polymerase plays a significant role (Kim and Nam, (2013) plant mol.Bio.Rep.31:581-
593)。
The building of 2 rubber tree gene editing carrier of embodiment
(1) proHbU6.2 is building up on intermediate vector SK-5G carrier
With SalI and XhoI double digestion SK-5G carrier and downstream gRNA segment, remove the rice U3 promoter on the carrier,
2913bp vector backbone segment is recycled, design primer:
ProHbU6.2-lF:
GCGGCCGCAGATCTGCTAGCGTCGACTCTAAGCTAAAGGAATAGC；
ProHbU6.2-lR:
GTGTTGTGTTCACCTGCGAGC(underscore show the homologous sequence of SK-5G carrier to CAATTGCTACTGCCTATTC
Column).
Using rubber tree genomic DNA as template, 95 DEG C of pre- changes in 20 μ l reaction systems using KOD FX enzyme (TOYOBO)
Property 2min, 98 DEG C of denaturation 10s, 60 DEG C of annealing 30s, 72 DEG C of extension 1min, 35 circulation, 72 DEG C eventually extend 5min expand to obtain
ProHbU6.2 promoter fragment, and SK-5G carrier homologous sequence is introduced at its both ends.Design primer:
GRNA-sF:GCTCGCAGGTGAACACAACACC；
GRNA-sR:TTGGGTACCGAGGATCCTCTAGA；
Using SK-5G vector plasmid as template, using KOD FX enzyme (TOYOBO) in 20 μ l reaction systems 95 DEG C of initial denaturations
2min, 98 DEG C of denaturation 10s, 60 DEG C of annealing 30s, 72 DEG C of extension 1min, 35 circulations, 72 DEG C extend 5min eventually and expand to obtain gRNA
Segment.
Using Gibson assembly Cloning Kit (NEB), the homologous sequence proHbU6.2 promoter that will be obtained
Segment and gRNA segment are assembled on SK-5G carrier and obtain proHbU6.2-SK-5G.
(2) target site is building up on proHbU6.2-SK-5G carrier
In order to verify the functional activity of proHbU6.2 promoter, select rubber tree bloom controlling gene HbTFL1-3 as
The target gene that CRISRP/Cas9 is edited selects suitable target point GCCCAGCATAG on the geneGGATCCAC, the site
Include BamHI recognition site GGATCC (underscore part) in the base positions of the site the PAM upstream AGG 2, can be used for target site quilt
Digestion identification after editor.
It is as follows to synthesize target site sequence:
It is positive:ATTGGCCCAGCATAGGGATCCAC；
It is reversed:AAACGTGGATCCCTATGCTGGGC, underscore show the two viscosity ends 5 '-ATTG and 3 '-CAAA
End.
3 '-TAAC and 5 '-are formed in proHbU6.2 promoter downstream with after AarI digestion proHbU6.2-SK-5G carrier
Two cohesive ends of GTTT.Meanwhile the forward and reverse target sequence that 10 μ l concentration are 100 μM being taken to mix respectively, at 100 DEG C
Annealing at room temperature is formed with the target site double chain DNA fragment with proHbU6.2-SK-5G carrier complementary cohesive tennini after managing 5min.
Then the segment is connected to proHbU6.2-SK-5G carrier proHbU6.2 promoter downstream using T4 DNA ligase (NEB)
Position obtains complete sgRNA expression cassette.
(3) sgRNA expression cassette is building up on instantaneous conversion CRISRP/Cas9 gene editing carrier 163Cas9M
With KpnI and BglII double digestion proHbU6.2-sgRNA-SK-5G carrier, the small fragment of 494bp is recycled
ProHbU6.2-sgRNA expression cassette；
With KpnI and BamHI double digestion 163Cas9M carrier, 7919bp large fragment is recycled；
It the characteristics of according to identical cohesive end is generated after BamHI and BglII digestion, will using T4 DNA ligase (NEB)
Small fragment sgRNA expression cassette, which is building up on 163Cas9M carrier, obtains rubber tree instantaneous conversion editor carrier proHbU6.2-
SgRNA-163Cas9M (building result is as shown in Figure 3).
3 rubber tree protoplast transformation of embodiment
The present embodiment mediates editor carrier proHbU6.2-sgRNA-163Cas9M to convert rubber tree protoplast with PEG,
And using the 163Cas9M carrier without sgRNA expression cassette as control.
It one month rubber tree heat will be cultivated grinds 7-33-97 tissue-cultured seedling and goes under 26-28 DEG C of dark condition and cultivate 5-7 days,
Protoplast is used to prepare after taking 2g discoloration phase blade to impregnate 10min in 0.6M mannitol solution immediately.The system of protoplast
The conversion process of standby sum is referring to (Yoo, S.D. etc., 2007, Nature Protocols, 2:1565-1575.).Original after conversion
Raw plastid cultivate the detection after 48 hours for target position point mutation under 26-28 DEG C of dark condition.
The detection in 4 rubber tree HbTFL1-3 target sequence mutational site of embodiment
Rubber tree protoplast genomic DNA is extracted using plant genome DNA extracts kit (Tiangen), is used
Genomic DNA PCR product carries out the method inspection of digestion (PCR/Restriction Enzyme digestion, PCR/RE) again
Survey mutation (Henao-Mejia J etc., 2016, Cold Spring Harbor on HbTFL1-3 gene target site sequence
Protocols,2016(2):pdb.prot090704.)。
Design the PCR amplification primer:
HbTFL1-3-F:CACCTAGGGCATAACTTCTAC；
HbTFL1-3-R:ACGGGATCTTAGTTGGATGG；
Using rubber tree protoplast genomic DNA as template, response procedures are as follows: 95 DEG C of initial denaturation 2min, 98 DEG C of denaturation
10s, 59 DEG C of annealing 30s, 72 DEG C of extension 1min, 35 circulations, 72 DEG C extend 5min eventually, and PCR amplification is obtained comprising editing sites
831bpHbTFL1-3 target fragment inside.
Because target gene editing sites include a BamHI restriction enzyme enzyme recognition site, by recycling
HbTFL1-3 genetic fragment carries out BamHI digestion to detect the editor of target site.As a result as shown in figure 4, part target fragment
Simultaneously digestion is no longer identified by BamHI because BamHI recognition site mutates, this tentatively shows that target gene has succeeded and is compiled
Volume, and target fragment not to be edited is then digested two small fragments for 500bp and 331bp.It is not thorough to exclude digestion
And remaining part does not edit the influence of target fragment, recycles the segment that cannot be digested in PCR product, its TA is cloned into
On pMD-19T (TAKARA) carrier, monoclonal is chosen after converting Escherichia coli Dh5 α, BamHI digestion is carried out to target site again and is tested
Card, as a result as shown in Figure 5；It takes the monoclonal that cannot be digested to carry out DNA sequencing, passes through sequence alignment analysis rubber tree
The mutation of HbTFL1-3 target site sequence, it was confirmed that the edited result in rubber tree protoplasm somatocyte.As a result such as Fig. 6 institute
Show.
As it can be seen that the present invention obtains rubber tree rna plymerase iii type promoter proHbU6.2 starting in Para rubber tree
Son has transcriptional activity, can drive the expression of downstream sgRNA, and realizes the rubber tree gene of CRISPR/Cas9 mediation for the first time
Group targeting editor；And target site cloning and sequencing is as a result, it has been found that mutation type is mostly the missing and insertion of single base after editing, wherein
Also there is the missing of polybase base and the insertion of small fragment.Therefore, promoter of the present invention can be applied to rubber tree CRISPR/
Cas9 gene editing system, to realize to rubber tree efficiently accurately breed improvement.
Obviously, the above embodiments are merely examples for clarifying the description, and does not limit the embodiments.It is right
For those of ordinary skill in the art, can also make on the basis of the above description it is other it is various forms of variation or
It changes.There is no necessity and possibility to exhaust all the enbodiments.And it is extended from this it is obvious variation or
It changes still within the protection scope of the invention.
Sequence table
<110>
<120>a kind of rubber tree U6 gene promoter proHbU6.2 and its clone and application
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 345
<212> DNA
<213> proHbU6.2
<400> 1
tctaagctaa aggaatagct tagcaccaac tttaatgttg gttattttaa gggtgtgaaa 60
aatgaaggtc tttataattg ttatagaagt ggaagtatct tgtaacttgt aagcttatgc 120
caacaccagg tgcccttagc gcatggcaag tagggtgtct tagcattggg ctttgaccaa 180
cttaggcctg atgtacaact cagaacccat ttgtgggcct aatgaaatta tttaacccat 240
cctggttgta aagtcatctg gttggttgga ttgaacttag tcccacatca tttagttaca 300
tatagttgca agccttcata agcaaagaat aggcagtagc aattg 345
Claims (10)
1. a kind of rubber tree U6 gene promoter proHbU6.2, which is characterized in that the promoter proHbU6.2 includes such as SEQ
DNA nucleotide sequence shown in ID No:1.
2. rubber tree U6 gene promoter proHbU6.2 according to claim 1, which is characterized in that the promoter
The DNA nucleotide sequence of proHbU6.2 is as shown in SEQ ID No:1.
3. carrier is edited in a kind of rubber tree instantaneous conversion, which is characterized in that contain rubber tree U6 base of any of claims 1 or 2
Because of promoter proHbU6.2.
4. carrier is edited in rubber tree instantaneous conversion according to claim 3, which is characterized in that the instantaneous conversion editor carries
Body is recombinant plasmid proHbU6.2-sgRNA-163Cas9M.
5. a kind of method for cloning rubber tree U6 gene promoter proHbU6.2 of any of claims 1 or 2, which is characterized in that
Include the following steps:
(1) 7-33-97 leaves genomic DNA is ground as template using Para rubber tree heat, designs following special primer:
ProHbU6.2-F:TCTAAGCTAAAGGAATAGCTTAGCAC；
ProHbU6.2-R:CAATTGCTACTGCCTATTC；
(2) PCR amplification is carried out in 20 μ l reaction systems using KOD FX enzyme；
(3) amplified production TA is cloned on pMD19-T carrier, converts in Escherichia coli Dh5 α and choose recombinant monoclonal sequencing,
Up to 345bp rubber tree U6 gene promoter DNA fragmentation proHbU6.2.
6. the method for rubber tree U6 gene promoter proHbU6.2, feature described in clone according to claim 5 exist
In, in the step (2), the response procedures of the PCR amplification step are as follows: 95 DEG C of initial denaturation 2min, 98 DEG C of denaturation 10s, 59 DEG C
Anneal 30s, and 72 DEG C of extension 1min, 35 circulations, 72 DEG C extend 5min eventually.
7. a kind of method of rubber tree instantaneous conversion editor's carrier described in building claim 3 or 4, which is characterized in that including
Following steps:
(a) the promoter proHbU6.2 of clone is building up on intermediate vector SK-5G
With SalI and XhoI double digestion SK-5G carrier, 2913bp vector backbone segment is recycled, following primer is designed and exists respectively
ProHbU6.2 sequence and carrier gRNA sequence both ends introduce homologous sequence, and proHbU6.2 and gRNA are assembled into SK-5G carrier
On obtain proHbU6.2-SK-5G:
ProHbU6.2-lF:
GCGGCCGCAGATCTGCTAGCGTCGACTCTAAGCTAAAGGAATAGC；
ProHbU6.2-lR:
GTGTTGTGTTCACCTGCGAGCCAATTGCTACTGCCTATTC；
GRNA-sF:GCTCGCAGGTGAACACAACACC；
GRNA-sR:TTGGGTACCGAGGATCCTCTAGA；
(b) target site is building up on proHbU6.2-SK-5G carrier
Following target site is selected on rubber tree HbTFL1-3 gene:
GCCCAGCATAGGGATCCAC forms 3 '-TAAC and 5 '-with both ends after AarI digestion proHbU6.2-SK-5G carrier
Two cohesive ends of GTTT；
Target site sequence is synthesized, and introduces two cohesive ends of 5 '-ATTG and 3 '-CAAA:
It is positive: ATTGGCCCAGCATAGGGATCCAC；
It is reversed: AAACGTGGATCCCTATGCTGGGC；
Above-mentioned forward and reverse target sequence DNA is mixed, carries out annealing at room temperature after 100 DEG C of processing, formed with
Then the segment is connected to load using T4DNA ligase by the double-stranded DNA of proHbU6.2-SK-5G carrier complementary cohesive tennini
Body proHbU6.2 promoter downstream position obtains complete sgRNA expression cassette；
(c) sgRNA expression cassette is building up on instantaneous conversion editor carrier 163Cas9M
With KpnI and BglII double digestion proHbU6.2-sgRNA-SK-5G carrier, the small fragment sgRNA expression of 494bp is recycled
Frame；
With KpnI and BamHI double digestion 163Cas9M carrier, 7919bp large fragment is recycled；
Small fragment sgRNA expression cassette is building up on 163Cas9M carrier using T4DNA ligase and obtains rubber tree instantaneous conversion
Edit carrier proHbU6.2-sgRNA-163Cas9M to get.
8. the method that a kind of pair of rubber tree carries out genome editor, which is characterized in that including by rubber described in claim 3 or 4
The step of vector introduction rubber tree protoplast, is edited in gum instantaneous conversion.
9. rubber tree U6 gene promoter proHbU6.2 of any of claims 1 or 2 is in rubber tree molecular breeding technology field
Application.
10. carrier answering in rubber tree molecular breeding technology field is edited in rubber tree instantaneous conversion described in claim 3 or 4
With.
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| CN117866966A (en) * | 2024-03-13 | 2024-04-12 | 中国热带农业科学院三亚研究院 | Betel nut U6 promoter and application thereof |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112980847A (en) * | 2021-04-21 | 2021-06-18 | 中国热带农业科学院橡胶研究所 | Rubber tree ubiquitin gene promoter proHbUBI3 and cloning and application thereof |
| CN112980847B (en) * | 2021-04-21 | 2023-05-02 | 中国热带农业科学院橡胶研究所 | Rubber tree ubiquitin gene promoter proHbUBI3 and cloning and application thereof |
| CN114774414A (en) * | 2022-03-31 | 2022-07-22 | 东北林业大学 | Fraxinus mandshurica U6 gene promoter proFmU6.5, and cloning and application thereof |
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| CN117487847A (en) * | 2023-10-31 | 2024-02-02 | 中国热带农业科学院橡胶研究所 | Method for obtaining homozygous gene editing plant of rubber tree |
| CN117866966A (en) * | 2024-03-13 | 2024-04-12 | 中国热带农业科学院三亚研究院 | Betel nut U6 promoter and application thereof |
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