CN104846004B - The method that mosaic disease resisting sugar cane breed is cultivated using artificial synthesized MV4 sequences - Google Patents
The method that mosaic disease resisting sugar cane breed is cultivated using artificial synthesized MV4 sequences Download PDFInfo
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Abstract
A kind of method that mosaic disease resisting sugar cane breed is cultivated using artificial synthesized MV4 sequences, including the artificial synthesized of MV4 sequences, RNAi interference carriers structure, the cultivation of mosaic disease resisting transgenic sugarcane material and the Disease Resistance Identification of disease-resistant transgenic sugarcane.The plant expression vector that the present invention obtains employs RNAi technology so that sugarcane Anti-virus Disease Breeding has broken away from the dependence of confrontation source gene, can effectively shorten sugarcane mosaic disease resisting breeding cycle.The acquired transfer-gen plant of the present invention, the features such as resistant wide spectrum, disease resistance are good, resistance is lasting and biological safety is high.
Description
Technical field
The present invention relates to a kind of method for cultivating sugarcane disease-resistant variety, and in particular to one kind utilizes artificial synthesized MV4 sequences
The method for cultivating mosaic disease resisting sugar cane breed, belongs to biological technical field.
Background technology
Mosaic of sugarcane also known as sugarcane mosaic, it is the viroid disease that each Chan Zhe states generally occur in the world, this disease exists
Many countries are listed in important disease, sugarcane it is susceptible it is latter as the underproduction 10%~40%, while such as crushing juice rate, reduced sugar, sucrose
Some process characters that grade are also influenced to different extents.At present, the cause of disease at least 3 of mosaic of sugarcane is clearly caused
Kind：Corn mosaic virus (Sugarcane Mosaic Virus, ScMV), sorghum mosaic virus (Sorghum Mosaic
Virus, SrMV) and sugarcane streak mosaic virus.Wherein corn mosaic virus and sorghum mosaic virus generally divide in China's sugarcane district
Cloth, both Tobamovirus Potyvirus (Potyvirus) members, it is single stranded positive-sense RNA virus, genome structure is simple,
Encode 10 maturation proteins, respectively P1, HC-pro, P3,6K1, CI, 6K2, VPg, NIa, Nib and CP.At present, world's sugarcane district
7 ScMV and SrMV virus strains are at least there are, including belongs to ScMV strain ScMV-A, B, D, E and belongs to SrMV strain
It is SrMV-H, I, M.Researcher is obtained to sugarcane successively by single CP and HC complete sequence channel genes into sugarcane
The transgenic sugarcane that the resistance of leaf disease significantly improves.But research shows that the dominant strain of mosaic virus ties up to natural conditions in sugarcane district
Under can also change, and produce new strain.Therefore, only turn using the term single gene of the mosaic virus of single strain as target
Improvement of genes obviously can not tackle the variation of sugarcane district cause of disease strain, complication and the change of advantage strain.
RNA interference (RNA interference, RNAi) is a kind of posttranscriptional gene expression silencing mechanism, and structure is transcribed
Plant expression vector for double-stranded RNA (dsRNA) simultaneously converts plant, and heritable gene that plant target gene can be achieved sinks
It is silent, moreover, triggering RNAi fragment and not needing complete gene order, it can also be multiple viral different genes fragments
Chimera, therefore, we are by the nucleotide sequence of each virus strains of the ScMV being collected into and the nucleotide sequence of each virus strains of SrMV
Multiple Sequence Alignment is carried out respectively, is therefrom respectively chosen 1 100% homologous sequence section, is connected on by the way of artificial synthesized
Together, as RNAi (RNA interference) fragment, inverted repeats is built, obtains a RNAi carrier.Pass through base
Because of rifle blast technique genetic transformation sugarcane, the transgenic sugarcane with silencing virus gene expression effect is obtained.
Patent No. ZL20071009226.8 patent of invention " cultivates mosaic disease resisting sugar cane breed using SrMV-P1 genes
Method ", describe and a kind of utilize SrMV-P1The method that gene cultivates mosaic disease resisting sugar cane breed, including SrMV-P1Gene
Clone, mosaic disease resisting sugar cane turn P1The building of gene plant expression vector, anti-mosaic of sugarcane turns P1The cultivation of genetic material and
The Screening and Identification of the high sugared transgenic sugarcane new material of disease-resistant high yield.
The content of the invention
It is an object of the invention to provide a kind of method that mosaic disease resisting sugar cane breed is cultivated using artificial synthesized sequence MV4.
For existing sugarcane district mosaic of sugarcane it is serious the problem of, it is artificial synthesized can simultaneously silence corn mosaic virus and sorghum mosaic virus
Novel nucleic acid sequence, build RNAi carrier, genetic transformation carried out by particle bombardment, obtain high anti-to mosaic disease turn
Gene sugarcane.
The purpose of the present invention is realized by the following method.
The method that mosaic disease resisting sugar cane breed is cultivated using artificial synthesized MV4 sequences of the present invention, include the people of MV4 sequences
Work synthesis, RNAi carrier structure, the cultivation of mosaic disease resisting transgenic sugarcane material and Disease Resistance Identification；It is characterized in that：
(1) MV4 sequences is artificial synthesized：
Respectively chosen together with one-stage serial from ScMV with SrMV nucleotide sequences, obtain MV4 sequences, while in positive-sense strand 5 '
End adds Xba I and Xho I restriction enzyme sites, and the end of antisense strand 5 ' adds Cla I and Kpn I restriction enzyme sites, using artificial synthesized
Mode, obtains purpose fragment A, and its sequence is as follows：
GATCTAGACT CGAGATGAGC GGACTAATGG TATGGTGCAT AGAAAATGGT TGCTCACCGA
ACATCAATGG TGTTTGGACC ATGATGGATG GAGAAGAACA AAGAACATTT CCTTTAAAGC
CAATAATTGA AAATGCTTCT CCAACATTTA GGCAGATTAT GCATCACTTT AGTGATGCAG
CTGAAGCGTA TATAGAATAC CGTAACTCAA CGGAACGCTA CATGCCAAGA TACGGACTTC
AGCGAAACTT GACCGACTGA GCGATACATG CCAAGATACG GTCTTCAGCG AAATCTCACC
GACTATAGCT TAGCGCGGTA TGCTTTCGAT TTCTATGAAA TGACTTCGCG CACACCAGCT
AGAGCTAAGG AAGCCCACAT GCAGATGAAA GCCGCAGCAG TTCGTGGTTC AAACACACGT
CTGTTCGGTC TGGACGGAAA TGTCGGCGAG ACTCAGGAGA ATACAGAGAG ACACACAGCT
GGCGACGTTA GTCGCAACAT GCACTCTCTG TTGGGAGTGC AGCAGCACCA CTAGGGTACC
ATCGATAG
Respectively chosen in the nucleotide sequence from ScMV with SrMV together with one-stage serial, refer to each viruses of the ScMV that will be collected into
The nucleotide sequence of each virus strain of nucleotide sequence and SrMV of strain carries out Multiple Sequence Alignment respectively, therefrom each to choose 1 100%
Homologous sequence section is cascaded, common 520bp, is target RNAi interference sequences.
(2) RNAi carrier is built：
(a) acquisition of purpose fragment I：Purpose fragment A is subjected to digestion using restriction enzyme Xho I and Kpn I, will
Digestion products are separated by agarose gel electrophoresis, reclaim purpose fragment I；The sequence of purpose fragment I is SEQ in sequence table
ID NO:Nucleotide sequence shown in 2；
(b) acquisition of purpose fragment II：By pHANNIBAL vector plasmid DNAs restriction enzyme Xho I and Kpn I
Digestion is carried out, digestion products are separated by agarose gel electrophoresis, reclaims purpose fragment II, the sequence of purpose fragment II
For SEQ ID NO in sequence table:Nucleotide sequence shown in 3；
(c) intermediate carrier B acquisition：The purpose fragment I of recovery and purpose fragment II are connected with T4-DNA ligases
Connect, and connection product is converted into bacillus coli DH 5 alpha, the checking of picking positive colony, obtain intermediate carrier B；Carried among described
Body B refers to for purpose fragment I to be inserted into the carrier obtained after pHANNIBAL carriers；
(d) acquisition of purpose fragment III：Extract intermediate carrier B DNA, and with restriction enzyme Cla I and Xba
I carries out digestion, and digestion products are separated by agarose gel electrophoresis, reclaims purpose fragment III；The sequence of purpose fragment III
It is classified as SEQ ID NO in sequence table:Nucleotide sequence shown in 4；
(e) acquisition of purpose fragment IV：Purpose fragment A is subjected to digestion with restriction enzyme Cla I and Xba I, will
Digestion products are separated by agarose gel electrophoresis, reclaim purpose fragment IV；The sequence of purpose fragment IV is in sequence table
SEQ ID NO:Nucleotide sequence shown in 5；
(f) intermediate carrier C acquisition：The purpose fragment III of recovery and purpose fragment IV are connected with T4-DNA ligases
Connect, and connection product is converted into bacillus coli DH 5 alpha, the checking of picking positive colony, obtain intermediate carrier C；Carried among described
Body C refers to for purpose fragment IV to be inserted into the carrier obtained after intermediate carrier B；
(g) acquisition of purpose fragment V：Intermediate carrier C DNA is extracted, and is carried out with restriction enzyme Not I
Digestion, digestion products are separated by agarose gel electrophoresis, reclaim purpose fragment V；The sequence of purpose fragment V is sequence
SEQ ID NO in list:Nucleotide sequence shown in 6；
(h) acquisition of purpose fragment VI：By plant expression vector pGreenII0229 DNA restriction enzymes
Not I carry out digestion, and digestion products are separated by agarose gel electrophoresis, reclaim purpose fragment VI；Purpose fragment VI
Sequence be sequence table in SEQ ID NO:Nucleotide sequence shown in 7；
(i) anti-sugarcane floral leaf RNAi carrier pG0229i-MV4 acquisition：By the purpose fragment V and purpose fragment VI of recovery
It is attached with T4-DNA ligases, and connection product is converted into bacillus coli DH 5 alpha, the checking of picking positive colony, is obtained
RNAi carrier pG0229i-MV4 available for genetic transformation sugarcane acceptor material；
(3) cultivation of mosaic disease resisting transgenic sugarcane material and Disease Resistance Identification：The RNAi carrier that extraction structure is completed
PG0229i-MV4 DNAs, its concentration and purity are determined with nucleic acid-protein analyzer, and quantified to 1 μ g/ μ l, particle gun
Blast technique genetic transformation sugarcane, Molecular Detection obtain positive transgenic plant, and carry out the disease resistance mirror of disease-resistant transgenic sugarcane
It is fixed.
The pHANNIBAL carriers, pGreenII0229 carriers, bacillus coli DH 5 alpha, by commercially available acquisition.
The method for cultivating mosaic disease resisting sugar cane breed using the artificial synthesized MV4 sequences of the present invention, in artificial inoculation conditions
Under, the ScMV-A for belonging to ScMV strains, E and the SrMV-H for belonging to SrMV strains are vaccinated with respectively, and fall ill by totally 4 strains, control by M
Rate has respectively reached 83.7%, 89.1%, 85.2% and 87.3%, and transgenic sugarcane illustrates to be transferred to artificial conjunction without morbidity
Into MV4 genes after improve the ability that sugarcane resists a variety of mosaic virus.The transgenosis obtained using the method screening of the present invention
Plant, all resistant wide spectrum, the characteristics of disease resistance is good, resistance is lasting and biological safety is high.
The advantages of the present invention：
1. the plant expression vector that the present invention obtains employs RNAi technology so that sugarcane Anti-virus Disease Breeding has broken away from confrontation
The dependence of source gene.
2. the artificial synthesized nucleotide sequence of the present invention contain with each virus strain's nucleotide sequences 100% of ScMV it is homologous and with
The homologous sequence section of each virus strain's nucleotide sequences 100% of SrMV, as RNAi sequences, the transfer-gen plant of acquisition, has
The features such as resistance wide spectrum, disease resistance are good, resistance is lasting and biological safety is high.
3. the RNAi carrier that the present invention obtains is used for transgenic sugarcane, it can effectively shorten sugarcane mosaic disease resisting breeding week
Phase.
It is the pG0229i-MV4 plasmid maps that structure is completed to illustrate Fig. 1.
Embodiment is said with reference to embodiments in order to which the present invention is furture elucidated rather than the limitation present invention
It is bright.Experimental method described in following embodiments, it is conventional method unless otherwise specified.The reagent and biomaterial for example without
Specified otherwise commercially obtains.
Embodiment one：Build the RNAi carrier of anti-mosaic of sugarcane
The method for building the RNAi carrier of anti-mosaic of sugarcane, comprises the following steps：
1st, MV4 sequences is artificial synthesized：
The nucleotide sequence of each virus strains of the ScMV being collected into and the nucleotide sequence of each virus strains of SrMV are carried out respectively
Multiple Sequence Alignment, 1 100% homologous sequence section is therefrom respectively chosen, is then cascaded by the way of artificial synthesized,
Common 520bp, as target RNAi interference sequences；Simultaneously Xba I and XhoI are added at the end of positive-sense strand 5 ' for intending artificial synthesized sequence
Restriction enzyme site, the end of antisense strand 5 ' add Cla I and Kpn I restriction enzyme sites, artificial synthesized acquisition purpose fragment A；The purpose piece
Section A refers to the nucleotide sequence after adding restriction enzyme site at the end of positive-sense strand 5 ' of target RNAi sequences and the end of antisense strand 5 '；Manually
After synthesis, purpose fragment A sequence is SEQ ID NO in sequence table:Nucleotide sequence shown in 1.
2nd, RNAi carrier is built：
The purpose fragment A obtained in step 1 is subjected to digestion using restriction enzyme Kpn I and Xho I, digestion is produced
Thing is separated by agarose gel electrophoresis, reclaims purpose fragment I, while by pHANNIBAL vector plasmid DNAs with restricted
Restriction endonuclease Kpn I and Xho I carry out digestion, and digestion products are separated by agarose gel electrophoresis, reclaim purpose fragment
II, the purpose fragment I of recovery and purpose fragment II are attached with T4-DNA ligases, and connection product is converted to large intestine
In bacillus DH5 α, the checking of picking positive colony, intermediate carrier B is obtained；Extract intermediate carrier B DNA, and with restricted interior
Enzyme cutting Xba I and Cla I carry out digestion, and digestion products are separated by agarose gel electrophoresis, reclaim purpose fragment III,
The purpose fragment A obtained in step 1 is also subjected to digestion with restriction enzyme Xba I and Cla I simultaneously, digestion products are led to
Cross agarose gel electrophoresis to be separated, reclaim purpose fragment IV, then use the purpose fragment III of recovery and purpose fragment IV
T4-DNA ligases are attached, and connection product is converted into bacillus coli DH 5 alpha, picking positive colony checking, in acquisition
Between support C；Intermediate carrier C DNA is extracted, and digestion is carried out with restriction enzyme Not I, digestion products are passed through into fine jade
Sepharose electrophoresis is separated, and reclaims purpose fragment V, while plant expression vector pGreenII0229 DNAs are limited
Property restriction endonuclease Not I processed carry out digestion, and digestion products are separated by agarose gel electrophoresis, reclaim purpose fragment VI,
The purpose fragment V of recovery and purpose fragment VI are attached with T4-DNA ligases, and connection product is converted to large intestine bar
In bacterium DH5 α, the checking of picking positive colony, RNAi carrier pG0229i-MV4 is obtained；The agarose gel electrophoresis, reference《Point
Son clone》The method of agarose gel electrophoresis in reference book；It is described to convert connection product into bacillus coli DH 5 alpha, conversion side
Method reference《Molecular cloning》In reference book the method with transformed competence colibacillus Escherichia coli is prepared with calcium chloride；The extraction plasmid
DNA method, with reference to plasmid extraction kit specification；The enzymatic cleavage methods, with reference to the specification of restriction enzyme；It is described
Recovery method, with reference to glue reclaim kit specification；It is described to be attached method with T4-DNA ligases, with reference to T4-DNA connections
Enzyme operational manual；It is as shown in Figure 1 to build the pG0229i-MV4 plasmid maps completed.
Embodiment two：A kind of cultivation of mosaic disease resisting transgenic sugarcane
A kind of cultivation of mosaic disease resisting transgenic sugarcane, comprises the following steps：
1st, material prepares：The RNAi carrier pG0229i-MV4 DNAs that extraction structure is completed, with nucleic acid-protein analyzer
Its concentration and purity are determined, and is quantified to 1 μ g/ μ l；Acceptor sugar cane breed is ROC22；
2nd, particle bombardment genetic transformation sugarcane：
The pretreatment of acceptor material：In sugarcane plant tip portion, the plump band of extracting waste lobus cardiacus more than growing point
Young tender lobus cardiacus in 10cm, with volumetric concentration be 75% ethanol disinfection after, and be cut into thickness be not more than 3mm disk,
Fiber differentiation 7d on MS+3.0mg/L 2.4-D+30g/L sucrose+6g/L agar powders pH 5.8 Fiber differentiation is seeded to, in base
4h goes to MS+2mg/L 2,4-D+0.2mol/L sorbierite+0.2mol/L mannitol+30g/L sucrose+6g/L fine jades before being bombarded because of rifle
In cosmetics pH 5.8 osmotic medium, pre-processed；
Biolistic bombardment converts：PDS-1000/He type particle gun operational manuals according to Bio-Rad companies prepare micro-
Bullet, adjustment target distance 6cm, air pressure 1,100psi, bombards the material of pretreatment, then the material after bombardment scatter, after
Continue and 20h is handled on osmotic medium；
Material culture and the acquisition of regeneration plant after conversion：Material after biolistic bombardment is converted goes to MS+2.0mg/L
2.4-D+30.0g/L sucrose+6g/L agar powders pH 5.8 recovery media, renewal cultivation 6d；Then by after renewal cultivation
Material moves to MS+2.0mg/L 2.4-D+30.0g/L sucrose+6.0g/L agar powder+4mg/L PPT pH 5.8 subculture screening
In culture medium, the screening and culturing 2-3 generations under 28 DEG C of dark conditions, 15d/ generations；Then material is gone into MS+1.0mg/L 6-BA+
In 0.5mg/L KT+30.0g/L sucrose+6.0g/L agar powder+4mg/L PPT pH 5.8 differentiation screening and culturing medium, at 28 DEG C
Under, 12h~14h 2000Lx illumination, screening and culturing 2-3 generations, 15d/ generations are given daily, until growing seedling；Treat that seedling grows tall
During to 3-4cm, 1/2MS+0.2mg/L 6-BA+3mg/L NAA+60g/L sucrose+6g/L agar powders pH 5.8 life is gone to
In root culture medium, at 28 DEG C, 12h~14h 2000Lx illumination is given daily, until seedling takes root；
3rd, Molecular Detection：By the seedling replanting after taking root into small flower (plant ash：Sand：Plantation soil=1:1:1), treat
After seedling survives, DNA is extracted respectively, is entered performing PCR detection, is obtained 56 plants of positive transgenic plant altogether；
4th, disease-resistant transgenic sugarcane Disease Resistance Identification
" mosaic disease resisting sugar cane product are cultivated with reference to the patent of invention of Patent No. 20071009226.8 using SrMV-P1 genes
Inoculation identification method in the method for kind ".
As a result：Under artificial inoculation conditions, the ScMV-A for belonging to ScMV strains is vaccinated with respectively, E and belong to SrMV strains
SrMV-H, M totally 4 strains, the control incidence of disease has respectively reached 83.7%, 89.1%, 85.2% and 87.3%, and transgenosis
Sugarcane without morbidity, illustrates to improve the ability that sugarcane resists a variety of mosaic virus after being transferred to artificial synthesized MV4 genes.
The plant expression vector of anti-mosaic of sugarcane is to build to obtain based on RNAi principles, and the technology can make antiviral
The dependence of confrontation source gene has been broken away from breeding, while artificial synthesized nucleotide sequence includes multistage and etiology nucleic acid sequence 100%
Homologous sequence section, as RNAi sequences, the transfer-gen plant of acquisition, resistant wide spectrum, disease resistance are good, resistance is lasting and
The features such as biological safety is high, while it is used for transgenic sugarcane, it can effectively shorten sugarcane mosaic disease resisting breeding cycle.
Claims (2)
1. a kind of method that mosaic disease resisting sugar cane breed is cultivated using artificial synthesized MV4 sequences, includes the artificial conjunction of MV4 sequences
Into, RNAi interference carriers structure, the cultivation of mosaic disease resisting transgenic sugarcane material and Disease Resistance Identification；It is characterized in that：
(1) MV4 sequences is artificial synthesized：
Respectively chosen together with one-stage serial from ScMV with SrMV nucleotide sequences, obtain MV4 sequences, while add at the end of positive-sense strand 5 '
Entering Xba I and Xho I restriction enzyme sites, the end of antisense strand 5 ' adds Cla I and Kpn I restriction enzyme sites, by the way of artificial synthesized,
Purpose fragment A is obtained, its sequence is as follows：
(2) RNAi carrier is built：
(a) acquisition of purpose fragment I：Purpose fragment A is subjected to digestion using restriction enzyme Xho I and Kpn I, by digestion
Product is separated by agarose gel electrophoresis, reclaims purpose fragment I；The sequence of purpose fragment I is SEQ ID in sequence table
NO:Nucleotide sequence shown in 2；
(b) acquisition of purpose fragment II：PHANNIBAL vector plasmid DNAs are carried out with restriction enzyme Xho I and Kpn I
Digestion, digestion products are separated by agarose gel electrophoresis, reclaim purpose fragment II, the sequence of purpose fragment II is sequence
SEQ ID NO in list:Nucleotide sequence shown in 3；
(c) intermediate carrier B acquisition：The purpose fragment I of recovery and purpose fragment II are attached with T4-DNA ligases, and
Connection product is converted into bacillus coli DH 5 alpha, the checking of picking positive colony, obtains intermediate carrier B；The intermediate carrier B is
Refer to the carrier that purpose fragment I is inserted into and obtained after pHANNIBAL carriers；
(d) acquisition of purpose fragment III：Intermediate carrier B DNA is extracted, and is entered with restriction enzyme Cla I and Xba I
Row digestion, digestion products are separated by agarose gel electrophoresis, reclaim purpose fragment III；The sequence of purpose fragment III is
SEQ ID NO in sequence table:Nucleotide sequence shown in 4；
(e) acquisition of purpose fragment IV：Purpose fragment A is subjected to digestion with restriction enzyme Cla I and Xba I, by digestion
Product is separated by agarose gel electrophoresis, reclaims purpose fragment IV；The sequence of purpose fragment IV is SEQ in sequence table
ID NO:Nucleotide sequence shown in 5；
(f) intermediate carrier C acquisition：The purpose fragment III of recovery and purpose fragment IV are attached with T4-DNA ligases,
And convert connection product into bacillus coli DH 5 alpha, the checking of picking positive colony, obtain intermediate carrier C；The intermediate carrier C
Refer to for purpose fragment IV to be inserted into the carrier obtained after intermediate carrier B；
(g) acquisition of purpose fragment V：Intermediate carrier C DNA is extracted, and digestion is carried out with restriction enzyme Not I,
Digestion products are separated by agarose gel electrophoresis, reclaim purpose fragment V；The sequence of purpose fragment V is sequence table
Middle SEQ ID NO:Nucleotide sequence shown in 6；
(h) acquisition of purpose fragment VI：By plant expression vector pGreenII0229 DNAs restriction enzyme Not I
Digestion is carried out, digestion products are separated by agarose gel electrophoresis, reclaims purpose fragment VI；The sequence of purpose fragment VI
For SEQ ID NO in sequence table:Nucleotide sequence shown in 7；
(i) anti-sugarcane floral leaf RNAi carrier pG0229i-MV4 acquisition：The purpose fragment V of recovery and purpose fragment VI are used
T4-DNA ligases are attached, and connection product is converted into bacillus coli DH 5 alpha, and the checking of picking positive colony, acquisition can
RNAi carrier pG0229i-MV4 for genetic transformation sugarcane acceptor material；
(3) cultivation of mosaic disease resisting transgenic sugarcane material and Disease Resistance Identification：The RNAi carrier that extraction structure is completed
PG0229i-MV4 DNAs, its concentration and purity are determined with nucleic acid-protein analyzer, and quantified to 1 μ g/ μ l, particle gun
Blast technique genetic transformation sugarcane, Molecular Detection obtain positive transgenic plant, and carry out the disease resistance mirror of disease-resistant transgenic sugarcane
It is fixed.
2. a kind of method that mosaic disease resisting sugar cane breed is cultivated using artificial synthesized MV4 sequences according to claim 1, its
It is characterised by each viruses of ScMV respectively chosen from ScMV with SrMV nucleotide sequences and together with one-stage serial, will be collected into
The nucleotide sequence of each virus strain of nucleotide sequence and SrMV of strain carries out Multiple Sequence Alignment respectively, therefrom each to choose 1 100%
Homologous sequence section is cascaded, common 520bp.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101126090A (en) * | 2007-07-19 | 2008-02-20 | 福建农林大学 | Method for cultivating mosaic disease resisting sugar cane breed by using SCMV-CP gene |
| CN101126089A (en) * | 2007-07-19 | 2008-02-20 | 福建农林大学 | Method for cultivating mosaic disease resisting sugar cane breed by using SrMV-P1 gene |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101126090A (en) * | 2007-07-19 | 2008-02-20 | 福建农林大学 | Method for cultivating mosaic disease resisting sugar cane breed by using SCMV-CP gene |
| CN101126089A (en) * | 2007-07-19 | 2008-02-20 | 福建农林大学 | Method for cultivating mosaic disease resisting sugar cane breed by using SrMV-P1 gene |
Non-Patent Citations (2)
| Title |
|---|
| Transgenic Sugarcane Resistant to Sorghum mosaic virus Based on Coat Protein Gene Silencing by RNA Interference;Jinlong Guo等;《BioMed Research International》;20150122;第2015卷;第35页第2段 * |
| 基于基因沉默的甘蔗三种主要病毒复合抗病基因构建及其转基因甘蔗研究;肖淑慧等;《2010年全国甘蔗保护技术研讨会论文集》;20110718;第2页右栏第1-2段、第4段，第3页左栏第1段至右栏第4段，第5页图2 * |
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