CN107267553A - A kind of transgenic watermelon new material initiative based on RNAi - Google Patents
A kind of transgenic watermelon new material initiative based on RNAi Download PDFInfo
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- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
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- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
- C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
- C12N15/8283—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for virus resistance
Abstract
The present invention relates to a kind of RNA interference carrier construction method and and its application in Watermelon Genetic Transformation, be related to plant virus controlling technical field, belong to biological and modern agricultural technology field application technology.By acting on host factor, destroy its mediate retroviral and infect function, watermelon is obtained antiviral activity, not yet have been reported that at present.The present invention builds RNA interference carrier, and genetic transformation watermelon, it is characterized in that clone watermelon eIF4E and its homology isomer eIF (iso) 4E genes, 4E iso justice and antisense fragments are respectively obtained after connection, connect again in intron sequences both sides, obtain 4E iso hairpin structure fragments；4E iso sense fragments, antisense fragments and hairpin structure fragment are inserted respectively by pCAMBIA1301 carriers by T4 connections and homologous recombination, RNA interference carrier is obtained；By agriculture bacillus mediated by target gene fragment conversion watermelon cotyledon explant, screening, which is cultivated, obtains the transgenic watermelon with target gene fragment.The present invention be mainly used for for watermelon virus disease evil preventing and treating new material and new technology are provided.
Description
Technical field
The invention belongs to technical field of molecular biology, more particularly to a kind of suitable dicotyledon genetic transformation, height are anti-
The structure of the RNA interference carrier of watermelon mosaic virus disease and its application in Watermelon Genetic Transformation.Further to watermelon
The expression vector and its genetic transformation watermelon of eIF4E genes and its homology isomer eIF (iso) 4E genes simultaneously obtain transgenosis plant
Strain.
Technical background
China's watermelon (Citrullus lanatus) cultivated area the first in the world, annual cultivated area is 1,200,000 hm2。
It is that the Curcurbitaceaes such as watermelon are made with the watermelon virus disease that watermelon mosaic virus (Watermelon mosaic virus, WMV) is representative
The main limiting factor of thing production, the long-term onset area of watermelon virus disease is up to 100,000 hm2, because its harm is serious, often result in huge
Economic loss, year, economic loss was up to 1.5 hundred million yuan.Watermelon mosaic virus is Potyvirus (Potyvirus) member.WMV
Viral genome is single stranded positive-sense RNA, and 5 ' ends are combined with albumen (the Viral protein of viral genome codes
Genome-linked, VPg), 3 ' ends are poly-A tail.The RNA first translates into a big polyprotein, then by certainly
Polyprotein is processed into functional albumen by the protease of body coding.
Traditional melon virus disease control method mainly has (1) control to pass virus mediator；(2) seed selection resistant variety；(3) it is viral
Cross protection.By controlling aphid transmission amboceptor to prevent and treat virosis, although had certain effect in production, but it is due to aphid
Worm easily breeds, and is also easy to produce the resistance to the action of a drug and limits its application.Seed selection resistant variety is most effective, most lasting virus disease control
Strategy.However, because it have been observed that virus resistance resource be difficult to transformation, significantly constrain breeding for disease resistance work progress.
Although cross protection has been reported that domestic there is presently no application low virulent strain system for preventing and treating the viruses such as ZYMV, CMV abroad
Prevent and treat the report of ground family crop virosis.
Recessive resistance genes generally existing in antivirus plant.Main mechanism is by lacking or being mutated some to disease
The host factor of malicious most important effect of having been survived in plant, so as to reach the work for making virus to breed in pin main body
With.At present, a considerable amount of recessive resistance genes have been cloned into from various crop, identification shows to belong to true these genes more
The eIF4E of nucleus translation initiation factor (eukaryotic translation initiation factors, eIF) and
EIF4G families.EIF4E is widely present in plant, mammal, drosophila and nematode.Be separated in plant 2 kinds it is different compound
Thing, eIF4E and its homology isomer eIF (iso) 4E.Melon middle eIF4E and eIF (iso) 4E is respectively by a gene code.
EIF4E is in plant with playing extremely critical effect in viral interaction.EIF4E has the ability that VPg is combined in plant, promotes it
Replicate and infect in host.There are some researches show potyvirus VPg albumen or its precursor and eIF4E or eIF
(iso) 4E direct interactions, this is the necessary condition of virus infection.
Due to important function of the eIF4E in virus infection, recessive resistance base is found by finding eIF4E mutant
Cause, it has also become improve the Critical policies of plant virus resistance.But, do not find corresponding mutant also so far in melon.
Therefore, passing through the interference to plant itself eIF4E, its gene expression of silence, the interaction destroyed between eIF4E and virus VPg, resistance
The duplication for virus of breaking and route of transmission, so that plant obtains corresponding virus resistance, have virus resistance watermelon as cultivating
The Critical policies of plant.
RNAi be by RNA mediate by specific interaction come the genetic intervention technology of inhibition of gene expression, it
Can specifically, effectively degrade mRNA, so as to cause the gene silencing of post-transcriptional level.In recent years, RNAi technology is planted in research
Thing known viral gene function is with there is extensive use in mechanism of causing a disease and plant disease-resistant mechanism, but most researchs are defined in virus
The silence of gene, and silence is carried out to host's gene by RNAi technology, it is lost the ability that mediate retroviral infects, so that
Plant obtains virus resistance, especially in terms of watermelon viral diseases research, yet there are no report.
The content of the invention
For above-mentioned deficiency, the present invention has cloned watermelon eIF4E genes and its homology isomer eIF (iso) 4E genes, and
Devise the dsRNA precursors of watermelon eIF4E genes and its homology isomer eIF (iso) 4E gene junction fragments 4E-iso mediations
Structure, is respectively adopted positive-sense strand and antisense strand site that 4E-iso fragments are inserted into interference vector pCAMBIA1301, recombination to construct
The RNAi interference vectors of suitable Watermelon Genetic Transformation.Recombinant RNA i carriers containing target gene fragment are converted into Agrobacterium
AGL-1 bacterial strains, then carry out genetic transformation operation to watermelon using Agrobacterium, target gene fragment are inserted into watermelon chromosome
In.In watermelon plant, by the recombinant vector loop-stem structure of structure, hairpin RNA is formed, is swashed as dsRNA existence form
PTGS reactions are sent out, so that the target gene fragment degraded with inserting has the watermelon eIF4E genes and its homologous isomery of homology
Body eIF (iso) 4E genes, destroy its mediation to virus infection, plant is obtained the virus resistance to WMV, final real
Key factor now is infected using RNAi technology silence pin main body inner virus, the expection of the watermelon new material with virus resistance is cultivated
Target.
The purpose of the present invention is achieved by the following technical solution：
The RNA interference vector construction side of one kind induction eIF4E genes and its homology isomer eIF (iso) 4E gene silencings
Method, it is characterised in that methods described comprises the following steps：
1) water melon leaf total serum IgE is extracted using Trizol methods；
2) according to watermelon eIF4E genes and its homology isomer eIF (iso) 4E gene orders the design clone being cloned into
The primer of eIF4E genes and eIF (iso) 4E genes：
4E-F：5′-ATGGTAGTTGAAGAKWCGATSAAAGC-3′
4E-R：5′-TCACACYRWATATTTRTTCTTYGCAT-3′
4E-iso-F：5′-ATGGCCGGTGAGGTAGCGGTGG-3′
4E-iso-R：5′-TCAAACACTRTATCGAGCTTTTGC-3′；
3) PCR primer of the design with restriction enzyme site, enters respectively to eIF4E genetic fragments and eIF (iso) 4E genetic fragments
Performing PCR is expanded, and is reclaimed and is obtained eIF4E gene sense fragments, eIF4E gene antisense fragments, eIF (iso) 4E gene sense fragments,
EIF (iso) 4E gene antisense fragments；4E-iso sense fragments and 4E-iso antisense fragments are obtained by over-lap PCR amplification again；
Specific primer is as follows, and the underscore part is restriction enzyme site：
4E-iso-1：5′-TTGACCATGGCCTGGGGTGCGTCTATCCG-3′
4E-iso-2：5′-TCTGCATTAGCCGGC GCCAGCCATTATCAG-3′
4E-iso-3：5′-CTGATAATGGCTGGC GCCGGCTAATGCAGA-3′
4E-iso-4：5′-GAGCTGGTCACCCGCACTGGCAAC-3′
4E-iso-5：5′-TTGACCATGGCGCACACTGGCAAC-3′
4E-iso-6：5′-GCCAGCCATTATCAGATTTGCCCCTTGAAA-3′
4E-iso-7：5′-TTTCAAGGGGCAAATCTGATAATGGCTGGC-3′
4E-iso-8：5′-GAGCTGGTCACCCCTGGGGTGCGTCTATC-3′；
4) primer of the design with restriction enzyme site, using cucumber gDNA as template, amplification includes sub-piece；By 4E-iso justice
Fragment, include sub-piece and 4E-iso antisense fragments are attached by homologous recombination, obtain containing 4E-iso sense fragments and
The hairpin structure fragment of 4E-iso antisense fragments, i.e. 4E-iso hairpin structures fragment；
Specific primer is as follows, and the underscore part is restriction enzyme site：
4E-iso-9：5′-GGTGACCAGCTCTATATACCTGCTGCC-3′
4E-iso-10：5′-GCCAGTGTGCGCTATAAACAAAGAAA-3′；
5) purpose fragment is connected on T- carriers, converts e. coli jm109, carries out cloning and sequencing identification, obtains positive
Plasmid；
6) double digestion is carried out to skeleton carrier pCAMBIA1301 using Nco I and BstE II, by 4E-iso sense fragments
PCAMBIA1301 carriers are inserted into by T4 ligases with 4E-iso antisense fragments, the carrier containing sense fragment is built respectively
PC4E-iso-S and antisense fragments carrier pC4E-iso-A；Constructed recombinant vector is again through determining nucleic acid sequence, and checking is inserted
Angle of striking and the correctness of Insert Fragment.
Using Nco I and BstE II double digestion pCAMBIA1301,4E-iso hair clip knots are contained using EcoR I single endonuclease digestions
The restructuring carrier T of tile section, inserts pCAMBIA1301 by purpose fragment by homologous recombination, obtains containing 4E-iso hair clip knots
The RNA interference carrier pC4E-iso-SA of tile section.Constructed recombinant vector verifies insertion point again through determining nucleic acid sequence
With the correctness of Insert Fragment.
Further, the present invention relates to a kind of carrier pC4E- containing sense fragment according to constructed by the above method
Iso-S and antisense fragments carrier pC4E-iso-A.
Further, the present invention relates to the application that watermelon cotyledon explant genetic transformation is applied to using above-mentioned carrier, its
It is characterised by, the application comprises the following steps：
1) plasmid containing target gene fragment, including pC4E-iso-S, pC4E-iso-A and pC4E-iso-SA are extracted,
Agrobacterium competent cell conversion is carried out using freeze-thaw method.Defrosting Agrobacterium AGL-1 competent cells, add ice after plasmid on ice
Bath 30 minutes, liquid nitrogen frozen after 1 minute 37 DEG C be incubated 5 minutes, add YEP 28 DEG C of fluid nutrient medium shaken cultivations 3 hours after from
The heart collects thalline, is coated on 28 DEG C of inversions on the YEP solid mediums containing antibiotic (50mg/L Kan, 100mg/L Rif)
Culture 48 hours；The laggard performing PCR identification of monoclonal is extracted, positive colony is further in the YEP fluid nutrient mediums containing antibiotic
Upper shaken cultivation 36 hours is to exponential phase, for the genetic transformation to watermelon explant；
2) kind is justified as material using Shandong, watermelon seed, which shells to induce on 1/2MS minimal mediums, to be sprouted；Culture 4-5 days
After cut cotyledon piece on MS inducing cultures preculture induce explant, the genetic transformation for recombinant vector；
3) tissue cultures obtain the watermelon transfer-gen plant containing target gene fragment 4E-iso positive-sense strands and antisense strand.West
Melon cotyledon piece explant is placed in total immersion in foregoing Agrobacterium bacterium solution and moistened, and is cultivated after being transferred to non-selective medium culture 1 week in selection
Squamous subculture is carried out on base, root media is transferred to after resistance adventitious bud is induced, root system development is well transplanted to greenhouse afterwards
Cellar culture.
4) transfer-gen plant is detected.
The antibiotic positive plant obtained through antibiotic-screening, is extracted after plant leaf DNA using CTAB methods, utilizes detection
Primer enters performing PCR amplification, screens the transgenic positive plant containing 4E-iso target gene fragments.
Specific primer is as follows：
Hyg-1：5′-CGGACGAGTGCTGGGGCGTCGG-3′
Hyg-2：5′-CACTGGCAAACTGTGATGGACGAC-3′
JD-1：5′-CTATACCTTCTCTACTGTTGAGG-3′
JD-2：5′-GGACTGTCACGAGCAGTAGAAAGG-3′
JD-3：5′-GCGCACTCAGGATCTTCCCATTT-3′
JD-4：5′-CGCGCGCGATAATTTATCCTAG-3′
Brief description of the drawings：
Fig. 1：Watermelon eIF4E and eIF (iso) 4E gene clonings；
(wherein M：DL2000,1：EIF4E, 2：eIF(iso)4E)
Fig. 2：Watermelon eIF4E and eIF (iso) 4E genes sense fragment is expanded
(wherein M：DL2000,1：EIF4E, 2：eIF(iso)4E)
Fig. 3：Watermelon eIF4E and eIF (iso) 4E gene antisense fragment amplifications
(wherein M：DL2000,1：EIF4E, 2：eIF(iso)4E)
Fig. 4：4E-iso sense fragments and antisense fragments connecting detection
(wherein M：DL2000,1-6：4E-iso sense fragments, 7,8：Negative control, 9-14：4E-iso antisense fragments, 15：
Negative control)
Fig. 5：4E-iso hairpin structure fragments PCR is detected
(wherein M：DL2000,1-5：4E-iso hairpin structure fragments, 6：Negative control)
Fig. 6：4E-iso sense fragments carrier and antisense fragments carrier digestion detection
(M1：1kb Marker, 1：Sense fragment carrier pC4E-iso-S：2：Antisense fragments carrier pC4E-iso-A, M2：
DL 5000)
Fig. 7：Interference vector digestion is detected
(wherein M1：DL 5000, M2：1kb Marker, Isosorbide-5-Nitrae, 7,10,13：1#, 2#, 3#, 4#, 5# hairpin structure fragment
Carrier pC4E-iso-AS, 2,5,8,11,14：1#, 2#, 3#, 4#, 5# hairpin structure fragment vector pC4E-iso-AS AsiS I/
Bste II double digestions, 3,6,9,12,15：The mono- enzymes of 1#, 2#, 3#, 4#, 5# hairpin structure fragment vector pC4E-iso-AS Sph I
Cut)
Fig. 8：Carrier pCAMBIA1301 collection of illustrative plates
Fig. 9：Exogenous sequences insertion vector pCAMBIA1301 schematic diagrames
Figure 10：Watermelon transgenosis flow chart
(wherein A：Watermelon cotyledon explant is induced, B：Agrobacterium cotransformation watermelon cotyledon explant, C：Explant after infiltration
Screening, D, E：Watermelon cotyledon explant resistance screening, F：Watermelon cotyledon explant squamous subculture, G：Watermelon vegetative seedling culture,
H：Watermelon vegetative seedling hot-house culture.)
Figure 11：Transfer-gen plant PCR is detected
(wherein M：DL 2000, P：Positive control, N：Negative control, 1-22：Transfer-gen plant)
Figure 12：Transfer-gen plant Dot blot are detected
(wherein P：Positive control, N：Negative control, 1-20：Transfer-gen plant)
Embodiment
The present invention is described in further details with reference to embodiment.Embodiments of the invention are only said for the present invention
Bright effect, without restriction effect.
It should be noted that in an embodiment of the present invention, although watermelon mosaic virus WMV interference viruses are described in detail
The construction method of expression vector, it is not intended that the interference vector combination of the present invention is only limited to convert WMV
With for cultivating the transfer-gen plant resistant to WMV.Therefore, the interference vector (pC4E-iso-SA) built using the present invention
And the control (pC4E-iso-S, pC4E-iso-A) of interference vector, with one of ordinary skill in the art grasped it is any
In a kind of any microorganism of method importing, plant or its tissue, cell, and thus obtain micro- with any anti-disease activity
Biological, plant, and such plant generations seed, hybridization and introgressive line, be included in right model of the presently claimed invention
Within enclosing.
Embodiment 1, PCR amplifications obtain watermelon plant eIF4E genes：
According to eIF4E gene orders, the present invention designs the primer of watermelon eIF4E genes first, as follows：
4E-F：5′-ATGGTAGTTGAAGAKWCGATSAAAGC-3′
4E-R：5′-TCACACYRWATATTTRTTCTTYGCAT-3′
Using the STb gene of watermelon plant leaf as template, total length eIF4E genes (708bp) (Fig. 1) are obtained through PCR amplifications.
Embodiment 2, PCR amplifications obtain watermelon plant eIF (iso) 4E genes：
According to eIF (iso) 4E gene orders, the present invention designs the primer of watermelon eIF (iso) 4E genes first, as follows：
4E-iso-F：5′-ATGGCCGGTGAGGTAGCGGTGG-3′
4E-iso-R：5′-TCAAACACTRTATCGAGCTTTTGC-3′
Using the STb gene of watermelon plant leaf as template, total length eIF (iso) 4E genes (612bp) (Fig. 1) are expanded through PCR.
Embodiment 3, PCR amplification eIF4E and eIF (iso) 4E gene sense fragments, connection form 4E-iso sense fragments
According to eIF4E and eIF (iso) 4E gene orders, the present invention designs amplification eIF4E and eIF (iso) 4E respectively first
The primer of genetic fragment, the primer contains restriction enzyme site, it may be connected in destination carrier.Using primer 4E-iso-1 and primer
4E-iso-2 expands eIF4E gene sense fragments；Using primer 4E-iso-3 and primer 4E-iso-4 amplification eIF (iso) 4E bases
Because of sense fragment；Using primer 4E-iso-1,4E-iso-2,4E-iso-3 and 4E-iso-4, over-lap PCR amplification obtains 4E-iso
Sense fragment.
Specific primer is following (underscore part is restriction enzyme site)：
4E-iso-1：5′-TTGACCATGGCCTGGGGTGCGTCTATCCG-3′
4E-iso-2：5′-TCTGCATTAGCCGGCGCCAGCCATTATCAG-3′
4E-iso-3：5′-CTGATAATGGCTGGCGCCGGCTAATGCAGA-3′
4E-iso-4：5′-GAGCTGGTCACCCGCACTGGCAAC-3′
EIF4E genes sense fragment (214bp), eIF (iso) 4E genes sense fragment (236bp) are obtained through PCR amplifications
(Fig. 2), over-lap PCR amplification obtains 4E-iso sense fragments (430bp) (Fig. 4).Purpose fragment is connected to pGEM T- after reclaiming
On easy carriers (being purchased from Promega companies), e. coli jm109 is converted, cloning and sequencing identification is carried out, obtains positive plasmid.
Embodiment 4, PCR amplification eIF4E and eIF (iso) 4E gene antisense fragments, connection form 4E-iso antisense fragments
According to eIF4E and eIF (iso) 4E gene orders, the present invention designs amplification eIF4E and eIF (iso) 4E respectively first
The primer of genetic fragment, the primer contains restriction enzyme site, it may be connected in destination carrier.Using primer 4E-iso-5 and primer
4E-iso-6 expands eIF (iso) 4E gene antisense fragments；Using primer 4E-iso-7 and primer 4E-iso-8 amplification eIF4E bases
Because of antisense fragments；Using primer 4E-iso-5,4E-iso-6,4E-iso-7 and 4E-iso-8, over-lap PCR amplification obtains 4E-iso
Antisense fragments.
Specific primer is following (underscore part is restriction enzyme site)：
4E-iso-5：5′-TTGACCATGGCGCACACTGGCAAC-3′
4E-iso-6：5′-GCCAGCCATTATCAGATTTGCCCCTTGAAA-3′
4E-iso-7：5′-TTTCAAGGGGCAAATCTGATAATGGCTGGC-3′
4E-iso-8：5′-GAGCTGGTCACCCCTGGGGTGCGTCTATC-3′
EIF4E gene antisenses fragment (214bp), eIF (iso) 4E gene antisenses fragment (236bp) are obtained through PCR amplifications
(Fig. 3), over-lap PCR amplification obtains 4E-iso antisense fragments (430bp) (Fig. 4).Purpose fragment is connected to pGEM T- after reclaiming
On easy carriers (being purchased from Promega companies), e. coli jm109 is converted, cloning and sequencing identification is carried out, obtains positive plasmid.
Embodiment 5, homologous recombination obtain 4E-iso hairpin structure fragments：
According to cucumber CSHSP70 gene intron sequences, primer of the design with restriction enzyme site, using cucumber gDNA as mould
Plate, amplification includes sub-piece.
Specific primer is following (underscore part is restriction enzyme site)：
4E-iso-9：5′-GGTGACCAGCTCTATATACCTGCTGCC-3′
4E-iso-10：5′-GCCAGTGTGCGCTATAAACAAAGAAA-3′
BstE II single endonuclease digestions contain the carrier T of 4E-iso sense fragments, will include sub-piece and 4E-iso antisense fragments,
It is attached by homologous recombination in carrier T, obtains the hair fastener knot containing 4E-iso sense fragments and 4E-iso antisense fragments
Tile section, i.e. 4E-iso hairpin structures fragment (966bp) (Fig. 5).
The RNA interference vector construction of embodiment 6, high anti-watermelon mosaic virus WMV
Using Nco I and BstE II double digestion pCAMBIA1301, while Nco I and BstE II double digestions contain 4E-
The restructuring carrier T of iso sense fragments, 4E-iso antisense fragments, is separately recovered 4E-iso sense fragments, 4E-iso antisense fragments,
Purpose fragment is inserted by pCAMBIA1301 by T4 ligases respectively, respectively obtained anti-containing 4E-iso sense fragments, 4E-iso
The just chain carrier pC4E-iso-S and antisense chain carrier pC4E-iso-A (Fig. 6) of adopted fragment.
Using Nco I and BstE II double digestion pCAMBIA1301,4E-iso hair clip knots are contained using EcoR I single endonuclease digestions
The restructuring carrier T of tile section, inserts pCAMBIA1301 by purpose fragment by homologous recombination, obtains containing 4E-iso hair clip knots
The RNA interference carrier pC4E-iso-SA (Fig. 7) of tile section.
The carrier pCAMBIA1301 used in the present invention applies to the interference vector of cucurbitaceous plant conversion, using agriculture
Bacillus infusion method carries out gene genetic conversion operation.PCAMBIA1301 contains CaMV 35S promoter sequences, under the promoter
Trip contains intron and GUS exon sequences.Nco I restriction enzyme sites are contained in intron and GUS exon sequences upstream, and downstream contains
There are BstE II restriction enzyme sites (Fig. 8).The present invention is by Nco I and BstE II double digestions by CaMV 35S promoter sequence downstreams
Intron and GUS exon sequences replace with target gene fragment.Build respectively anti-containing 4E-iso sense fragments, 4E-iso
The just chain carrier pC4E-iso-S and antisense chain carrier pC4E-iso-A of adopted fragment, contain 4E-iso hairpin structure fragments
RNA interference carrier pC4E-iso-SA (Fig. 9).
Embodiment 7, target plasmid conversion Agrobacterium
The plant expression plasmid built, converts JM109 competent cells, coated plate, 37 DEG C of overnight incubations.Picking single bacterium colony
Plasmid is extracted, after being the positive through PCR and digestion identification, Agrobacterium EHA105 competent cells are converted.Concrete operations are as follows：Take
Competent cell is prepared after the EHA105 strain culturings of preservation；Agrobacterium competent cell conversion is carried out using freeze-thaw method method, i.e.,
2 μ L plasmids are added into ice bath 30 minutes after 50 μ L Agrobacterium competent cells, then liquid nitrogen frozen is incubated 5 points for 37 DEG C after 1 minute
Clock, is collected by centrifugation thalline after adding YEP 28 DEG C of fluid nutrient medium shaken cultivations 3 hours, is coated on containing antibiotic (50mg/L
Kan, 100mg/L Rif) YEP solid mediums on 28 DEG C be inverted culture 48 hours；The laggard performing PCR identification of monoclonal is extracted,
Positive colony further on the YEP fluid nutrient mediums containing antibiotic shaken cultivation 36 hours to exponential phase.
Embodiment 8, Agrobacterium-mediated Transformation watermelon plant and transfer-gen plant are cultivated
Take the Agrobacterium containing target plasmid to be cultivated in 50mL YEP fluid nutrient mediums to exponential phase, be collected by centrifugation
Thalline is resuspended to OD with MS fluid nutrient mediums after thalline600Between 0.5 or so, for watermelon explant genetic transformation.
Watermelon seed is induced after shelling on 1/2MS minimal mediums to be sprouted；Culture cuts cotyledon piece after 4-5 days and made
Wound infects in favor of Agrobacterium, preculture 2 days on MS inducing cultures, and condition of culture is 28 DEG C, illumination in 12 hours；Take son
Leaf block is placed in total immersion in Agrobacterium bacterium solution and moistened 15 minutes, and filter paper, which is sucked, is transferred to non-selection inducing culture (MS+ after unnecessary bacterium solution
2mg·L-16-BA)；28 DEG C, low-light culture in 12 hours is transferred to Selective agar medium (MS+2mgL after one week-16-BA+80mg·
L-1Kan+100mg·L-1Hyg specific Antibiotics to be checked), 28 DEG C, the screening Fiber differentiation of illumination in 12 hours, every two weeks
Squamous subculture is once；Root media (MS+0.5mgL is transferred to after resistance adventitious bud is induced-1IBA+100mg·L- 1Hyg), 28 DEG C, 12 hours illumination cultivations；After root system development is good, practice seedling 3-5d days, be transplanted to advance autoclaved vermiculite：
In the nutritive cube that organic matter is 1: 1, sheltered from heat or light with plastic sheeting, after moisturizing 3d, transplant the Routine Management into greenhouse.(Figure 10)
The detection of embodiment 9, transfer-gen plant
Extracted using CTAB methods after rotaring gene plant blade DNA, using detection primer Hyg-1/Hyg-2 (531bp), JD-
1/JD-2 (629bp), JD-3/JD-4 (538bp) enter performing PCR amplification, screening transgenic positive plant respectively.Test positive
T0 harvests T1 for seed after breeding for transgenic watermelon by strain (single melon).
Specific primer is as follows：
Hyg-1：5′-CGGACGAGTGCTGGGGCGTCGG-3′
Hyg-2：5′-CACTGGCAAACTGTGATGGACGAC-3′
JD-1：5′-CTATACCTTCTCTACTGTTGAGG-3′
JD-2：5′-GGACTGTCACGAGCAGTAGAAAGG-3′
JD-3：5′-GCGCACTCAGGATCTTCCCATTT-3′
JD-4：5′-CGCGCGCGATAATTTATCCTAG-3′
Successful transgenic watermelon plant is converted present invention obtains hairpin structure.Through antibiotic-screening, converted altogether
157 plants of plant, detects through PCR, 33 plants of the transgenic watermelon plant containing target gene, and PCR positive transformants rate is 21.0%.
Plant (Figure 11) positive to PCR, using 4E-iso sense fragments as probe, carries out Dot-Southern blot detections.Through hybridization
Detect, 4 plants of PCR positive plants such as 8# are Southern blot positive, show that present invention obtains transfer-gen plant, conversion effect
Rate is 2.5% (Figure 12).
The present invention a kind of RNA interference carrier construction method and and its application in Watermelon Genetic Transformation pass through tool
The example of body is described, and those skilled in the art can use for reference present invention, the link such as appropriate feed change, process conditions
To realize corresponding other purposes, its correlation changes all without departing from present disclosure, all similar replacements and change pair
It is it will be apparent that being considered as being included within the scope of the present invention for those skilled in the art.
Claims (3)
1.RNA interference vectors are built and to watermelon cotyledon explant genetic transformation application.
It is characterized in that clone watermelon eIF4E genes and its homology isomer eIF (iso) 4E genes, respectively choose eIF4E and
EIF (iso) 4E Gene Partial fragments, over-lap PCR amplification obtains 4E-iso sense fragments and 4E-iso antisense fragments, inserts
PCAMBIA1301 carriers, build the carrier pC4E-iso-S containing sense fragment, the carrier pC4E-iso-A of antisense fragments respectively
With the interference vector pC4E-iso-SA containing hairpin structure fragment.Inducing watermelon cotyledon explant, will contain target gene fragment
Recombinant RNA i carriers conversion Agrobacterium AGL-1 bacterial strains.Heredity is carried out by agrobacterium-mediated transformation to watermelon cotyledon explant to turn
Change operation, recombinant vector pC4E-iso-S, pC4E-iso-A and pC4E-iso-SA are converted after watermelon respectively, make target gene
Fragment is inserted into watermelon chromosome.Insertion genetic fragment excites PTGS to react with dsRNA existence form, degrades and insertion
Target gene fragment has the watermelon eIF4E genes and its homology isomer eIF (iso) 4E genes of homology.Cultivate transgenosis
Watermelon plant, through antibiotic-screening and molecular biology identification, obtains the genes of eIF4E containing watermelon and its homology isomer eIF
(iso) 4E genetic fragments interfere the transfer-gen plant of expression vector.
2. according to the eIF4E genes and its homology isomer eIF (iso) 4E gene-specific RNA interference carriers of the watermelon of the requirement of right 1
Construction method, it is characterised in that including following steps：
1) watermelon total serum IgE, clone's watermelon eIF4E genes and its homology isomer eIF (iso) 4E genes are extracted, according to its sequence
Primer is designed, the two genetic fragments are connected as 4E-iso sense fragments and 4E-iso antisense fragments using overlapping pcr.
2) clone cucumber CSHSP70 gene introns, by homologous recombination by 4E-iso sense fragments, include sub-piece and 4E-
Iso antisense fragments are connected, and obtain 4E-iso hairpin structure fragments, and conversion Escherichia coli after being connected in carrier T, nucleic acid is surveyed
Sequence screening positive clone.
3) double digestion is carried out to skeleton carrier pCAMBIA1301 using Nco I and BstE II, by 4E-iso sense fragments and 4E-
Iso antisense fragments are inserted into pCAMBIA1301 carriers by T4 ligases, and the carrier pC4E- containing sense fragment is built respectively
Iso-S and antisense fragments carrier pC4E-iso-A.Constructed recombinant vector verifies insertion point again through determining nucleic acid sequence
With the correctness of Insert Fragment.
4) Nco I and BstE II double digestion pCAMBIA1301 are used, 4E-iso hairpin structures are contained using EcoR I single endonuclease digestions
The restructuring carrier T of fragment, pCAMBIA1301 is inserted by homologous recombination by purpose fragment, is obtained containing 4E-iso hairpin structures
The RNA interference carrier pC4E-iso-SA of fragment.Constructed recombinant vector again through determining nucleic acid sequence, checking insertion point and
The correctness of Insert Fragment.
3. according to the Watermelon Genetic Transformation method of the requirement of right 1, it is characterised in that including following steps：
1) plasmid containing 4E-iso target gene fragments is extracted, Agrobacterium competent cell conversion is carried out using freeze-thaw method, is turned
Positive colony after change is cultivated on the fluid nutrient medium containing antibiotic to exponential phase.
2) induce and sprout on 1/2MS minimal mediums after watermelon seed shells；Culture cuts cotyledon piece and induced in MS after 4-5 days
Preculture induces explant on culture medium.
3) watermelon cotyledon piece explant is placed in total immersion in the Agrobacterium bacterium solution containing 4E-iso target gene fragments and moistened, and carries out watermelon
Explant genetic transformation, the watermelon explant after conversion is transferred to non-selective medium renewal cultivation 1 week, is then transferred to selection culture
Base carries out resistance screening, induces and root media is transferred to after resistance adventitious bud, and root system development is well transplanted conventional to greenhouse afterwards
Culture.
4) the antibiotic positive plant obtained through antibiotic-screening, is extracted after plant leaf DNA using CTAB methods, is drawn using detection
Thing enters performing PCR amplification, screens the transgenic positive plant containing 4E-iso target gene fragments.
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| CN109136259A (en) * | 2018-10-09 | 2019-01-04 | 北京市农林科学院 | A kind of watermelon High-efficient Genetic Transformation and transgenic plant identification method |
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