CN104846005B - The method that mosaic disease resisting sugar cane breed is cultivated using artificial synthesized MV3 sequences - Google Patents
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Abstract
A kind of method that mosaic disease resisting sugar cane breed is cultivated using artificial synthesized MV3 sequences, including the artificial synthesized of MV3 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 features such as acquired transfer-gen plant of the present invention, 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 MV3 sequences
The method for cultivating mosaic disease resisting sugar cane breed, belongs to biological technical field.
Background technology
Mosaic of sugarcane is a kind of worldwide disease, and the influence to south, North America sugarcane yield is up to 30% ~ 40%, when serious
Up to 60% ~ 80%, the influence to South Africa sugarcane yield is up to 10% ~ 50%, and the influence to India's sugarcane yield is up to 10% ~ 15%, at me
State's sugarcane district also generally occurs, and the diseased plant rate of south China each department especially Upland of Guangxi mosaic of sugarcane reaches more than 30%, yield
Loss 3% ~ 50%.The disease cause of disease is mainly corn mosaic virus（Sugarcane Mosaic Virus, ScMV）With sorghum floral leaf
Viral (Sorghum Mosaic Virus, SrMV), belongs to Potyvirus (Potyvirus) member, for it is single-stranded just
Adopted RNA virus, genome structure is simple, encode 10 maturation proteins, be respectively P1, HC-pro, P3,6K1, CI, 6K2, VPg,
NIa, Nib and CP.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.At present, world's sugarcane district at least there are 7 ScMV and SrMV virus strains, bag
Include the strain ScMV-A, B, D, E that belong to ScMV and the strain SrMV-H, I, M that belong to SrMV.Therefore, only with single strain
Mosaic virus term single gene for target transgene improvement obviously can not tackle sugarcane district cause of disease strain variation, complicate with
And the change of advantage strain.
RNA is disturbed（RNA interference, RNAi）Refer to endogenous or exogenous double-stranded RNA（double-
Stranded RNA, dsRNA）Selective degradation occurs for the homologous mRNA of mediation, causes the expression silencing of target gene so as to produce
The deficient phenomena of corresponding function phenotype, belongs to the gene silencing phenomenon after transcription, moreover, triggering the fragment of RNAi and being not required
Complete gene order, can also be the chimera of multiple viral different genes fragments, for this reason, the ScMV that we will be collected into
The nucleotide sequence of each virus strain of nucleotide sequence and SrMV of each virus strain carries out Multiple Sequence Alignment respectively, therefrom each to choose 1
The homologous sequence section of bar 100%, is cascaded by the way of artificial synthesized, as RNAi (RNA interference)
Interference fragment, builds inverted repeats, obtains a RNAi carrier.By particle bombardment genetic transformation sugarcane, obtain
Transgenic sugarcane with silencing virus gene expression effect.
The patent of invention of Patent No. ZL20071009226.8 " 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
The object of the present invention is to provide a kind of method that mosaic disease resisting sugar cane breed is cultivated using artificial synthesized sequence MV3.
For existing sugarcane district mosaic of sugarcane it is serious the problem of, it is artificial synthesized can at the same time silence corn mosaic virus and sorghum mosaic virus
Novel nucleic acid sequence, build RNAi carrier, by particle bombardment carry out genetic transformation, obtain and mosaic disease highly resistance turned
Gene sugarcane.
The purpose of the present invention is what is be realized by the following method.
The method that mosaic disease resisting sugar cane breed is cultivated using artificial synthesized MV3 sequences of the present invention, includes the people of MV3 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）MV3 sequences it is artificial synthesized：
Respectively chosen together with one-stage serial from ScMV with SrMV nucleotide sequences, obtain MV3 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, its sequence is as follows：
GATCTAGACT CGAGATGGAA AAAAGTTACG TCGATCTCTT AAACCAAGCA TGGGCAGATT
TGCCATTACA TTCAAAATTA TATTCAATTT GGCGTGTGTA CGAAGTCAAG AAATATTACA
AGCCGTGCTT AGTCCTGAAA AGAGGCGTAG ATTTAGGCGC AATGTACAAT ATCTCAGCTA
CGCATCAAAT ATCAAGTTTA GTGCAGAAAA GTCGAGATCA AGTCAGCTCT ATTTCAACCA
AACTCCACCA CAGTTTATGT AATAATGGAA AAAAATTATG TAGATCAATT AAACCAGTCA
TGGGCAGAAT TATCATACTG TGGAAAATTT TCAGCAATAT GGCGTGTGTT CAGAGTCAAG
AAATACTACA AGCCATCTTT AACCGTGAGA AAAAGCGTAG ATTTAGGCGC TGTTTACAAT
ATATCAGCTA CGCATCTAAT ATCAGATTTA GTGCAGAGAA GTCGAGATCG AGTCAGCTCT
ACTTTAACCA AACTCCGCAA CGGTTTCTAG GTACCATCGA TAG；
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 495bp, is target RNAi interference sequences.
（2）RNAi carrier is built：
（a）The acquisition of purpose fragment I：Purpose fragment A is subjected to digestion using restriction enzyme Xho I and Kpn I,
Digestion products are separated by agarose gel electrophoresis, recycle purpose fragment I；The sequence of purpose fragment I is in sequence table
SEQ ID NO:Nucleotide sequence shown in 2；
（b）The 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, recycles purpose fragment II, the sequence of purpose fragment II
For SEQ ID NO in sequence table:Nucleotide sequence shown in 3；
（c）The acquisition of intermediate carrier B：The purpose fragment I of recycling and purpose fragment II are connected with T4-DNA ligases
Connect, and connection product is converted into bacillus coli DH 5 alpha, the verification of picking positive colony, obtains 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）The acquisition of purpose fragment III：Extract the Plasmid DNA of intermediate carrier B, and with restriction enzyme Cla I with
Xba I carry out digestion, and digestion products are separated by agarose gel electrophoresis, recycle purpose fragment III；Purpose fragment III
Sequence be sequence table in SEQ ID NO:Nucleotide sequence shown in 4；
（e）The 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, recycle purpose fragment IV；The sequence of purpose fragment IV is in sequence table
SEQ ID NO:Nucleotide sequence shown in 5；
（f）The acquisition of intermediate carrier C：The purpose fragment III of recycling and purpose fragment IV are carried out with T4-DNA ligases
Connection, and connection product is converted into bacillus coli DH 5 alpha, the verification of picking positive colony, obtains intermediate carrier C；The centre
Support C refers to for purpose fragment IV to be inserted into the carrier obtained after intermediate carrier B；
（g）The acquisition of purpose fragment V：The Plasmid DNA of intermediate carrier C is extracted, and uses restriction enzymeNotI is carried out
Digestion, digestion products are separated by agarose gel electrophoresis, recycle purpose fragment V；The sequence of purpose fragment V is sequence
SEQ ID NO in list:Nucleotide sequence shown in 6；
（h）The acquisition of purpose fragment VI：By plant expression vector pGreenII0229 Plasmid DNA restriction enzymesNot I carries out digestion, and digestion products are separated by agarose gel electrophoresis, recycles purpose fragment VI；Purpose fragment VI
Sequence be sequence table in SEQ ID NO:Nucleotide sequence shown in 7；
（i）The acquisition of anti-sugarcane floral leaf RNAi carrier pG0229i-MV3：By the purpose fragment V and purpose fragment VI of recycling
It is attached with T4-DNA ligases, and connection product is converted into bacillus coli DH 5 alpha, the verification of picking positive colony, obtains
RNAi carrier pG0229i-MV3 available for genetic transformation sugarcane acceptor material；
（3）The cultivation of mosaic disease resisting transgenic sugarcane material and Disease Resistance Identification：The RNAi carrier that extraction structure is completed
PG0229i-MV3 Plasmid DNA, its concentration and purity are measured with nucleic acid-protein analyzer, and are quantified to 1 μ g/ μ L, gene
Rifle blast technique genetic transformation sugarcane, Molecular Detection obtain regeneration positive transgenic plant, and carry out the anti-of disease-resistant transgenic sugarcane
Characteristic of disease is identified.
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 MV3 sequences of the present invention, in artificial inoculation conditions
Under, it is vaccinated with the ScMV-A for belonging to ScMV strains respectively, D, E and belongs to the SrMV-H of SrMV strains totally 4 strains, to approved for distribution
Sick rate has respectively reached 82.2%, 88.4%, 86.5% and 90.4%, and transgenic sugarcane illustrates to be transferred to artificial synthesized without morbidity
MV3 genes after improve the ability that sugarcane resists a variety of mosaic virus.The transgenosis that method screening using the present invention obtains is planted
Strain, 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 been broken away from pair
The dependence of anti-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 anti-
Property wide spectrum, the features such as disease resistance is 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
Cycle.
It is the pG0229i-MV3 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, 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, MV3 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, is therefrom respectively chosen 1 100% homologous sequence section, is then cascaded by the way of artificial synthesized,
Common 495bp, is target RNAi interference sequences；At the same time Xba I and Xho are added at the end of positive-sense strand 5 ' for intending artificial synthesized sequence
I restriction enzyme sites, 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, the sequence of purpose fragment A 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, by digestion
Product is separated by agarose gel electrophoresis, recycles purpose fragment I, while pHANNIBAL vector plasmid DNAs are limited
Property restriction endonuclease Kpn I and Xho I carry out digestion, digestion products are separated by agarose gel electrophoresis, recycle purpose piece
Section II, the purpose fragment I of recycling and purpose fragment II is attached with T4-DNA ligases, and connection product is converted to big
In enterobacteria DH5 α, the verification of picking positive colony, obtains intermediate carrier B；Extract the Plasmid DNA of intermediate carrier B, and with restricted
Restriction endonuclease Xba I and Cla I carry out digestion, and digestion products are separated by agarose gel electrophoresis, recycle purpose fragment
III, while the purpose fragment A obtained in step 1 is also subjected to digestion with restriction enzyme Xba I and Cla I, digestion is produced
Thing is separated by agarose gel electrophoresis, purpose fragment IV is recycled, then by the purpose fragment III and purpose fragment of recycling
IV is attached with T4-DNA ligases, and connection product is converted into bacillus coli DH 5 alpha, and the verification of picking positive colony, is obtained
Obtain intermediate carrier C；The Plasmid DNA of intermediate carrier C is extracted, and uses restriction enzymeNotI carries out digestion, and digestion products are led to
Cross agarose gel electrophoresis to be separated, recycle purpose fragment V, while by plant expression vector pGreenII0229 Plasmid DNA
Use restriction enzymeNot I carries out digestion, and digestion products are separated by agarose gel electrophoresis, recycles purpose fragment
VI, the purpose fragment V of recycling and purpose fragment VI are attached with T4-DNA ligases, and connection product is converted to big
In enterobacteria DH5 α, the verification of picking positive colony, obtains RNAi carrier pG0229i-MV3；The agarose gel electrophoresis, reference
《Molecular cloning》The method of agarose gel electrophoresis in reference book；It is described to convert connection product into bacillus coli DH 5 alpha, turn
Change method reference《Molecular cloning》In reference book the method with transformed competence colibacillus Escherichia coli is prepared with calcium chloride；The extraction matter
The method of grain DNA, with reference to plasmid extraction kit specification；The enzymatic cleavage methods, with reference to the specification of restriction enzyme；Institute
Recovery method is stated, with reference to plastic recovery kit specification；It is described to be attached method with T4-DNA ligases, connect with reference to T4-DNA
Connect enzyme operational manual；It is as shown in Figure 1 to build the pG0229i-MV3 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-MV3 Plasmid DNA that extraction structure is completed, with nucleic acid-protein analyzer
Its concentration and purity are measured, 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 10
Young tender lobus cardiacus in cm, with volumetric concentration be 75 % ethanol disinfection after, and be cut into thickness be not more than 3 mm disk, connect
Plant to Fiber differentiation 7 on the Fiber differentiation of+6 g/L agar powders pH 5.8 of MS+3.0 mg/L 2.4-D+30 g/L sucrose
D, 4h goes to MS+2 mg/L 2 ,+0.2 mol/L mannitol+30 of 4-D+0.2 mol/L sorbierites before biolistic bombardment
In the osmotic medium of+6 g/L agar powders pH 5.8 of g/L sucrose, pre-processed；
Biolistic bombardment converts：PDS-1000/He type particle gun operational manuals according to Bio-Rad companies prepare micro-
Bullet, 6 cm of adjustment target distance, 1,100 psi of air pressure, bombards the material of pretreatment, then the material after bombardment scatter,
Continuation handles 20 h on osmotic medium；
Material culture and the acquisition of regeneration plant after conversion：Material after biolistic bombardment is converted goes to MS+2.0
The recovery media of+6 g/L agar powders pH 5.8 of mg/L 2.4-D+30.0 g/L sucrose, 6 d of renewal cultivation；Then will be extensive
Material after multiple culture moves to+4 mg/L PPT of+6.0 g/L agar powders of MS+2.0 mg/L 2.4-D+30.0 g/L sucrose
In the subculture screening and culturing medium of pH 5.8, the screening and culturing 2-3 generations under 28 DEG C of dark conditions, 15d/ generations；Then material is gone to
+ 4 mg/L PPT pH 5.8 of+6.0 g/L agar powders of MS+1.0 mg/L 6-BA+0.5mg/L KT+30.0 g/L sucrose
Differentiation screening and culturing medium in, at 28 DEG C, be given once daily the illumination of 12 h~14 h, 2000 Lx, screening and culturing 2-3 generations,
15d/ generations, until growing seedling；When seedling grows up to 3-4cm, 1/2MS+0.2 mg/L 6-BA+3 mg/L are gone to
In the root media of+6 g/L agar powders pH 5.8 of NAA+60 g/L sucrose, at 28 DEG C, the h of 12 h~14 is given once daily
The illumination of 2000 Lx, 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, carries out PCR detections, obtains 65 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, D, E and belong to SrMV plants
The SrMV-H of system totally 4 strains, control incidence have respectively reached 82.2%, 88.4%, 86.5% and 90.4%, and transgenic sugarcane
Without morbidity, illustrate to improve the ability that sugarcane resists a variety of mosaic virus after being transferred to artificial synthesized MV3 genes.
The plant expression vector of anti-mosaic of sugarcane is to build to obtain based on RNAi principles, which 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% is same
The sequence section in source, as RNAi sequences, the transfer-gen plant of acquisition, resistant wide spectrum, disease resistance are good, resistance is lasting and raw
The features such as thing is safe, 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 MV3 sequences, includes the artificial conjunction of MV3 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）MV3 sequences it is artificial synthesized：
Respectively chosen together with one-stage serial from ScMV with SrMV nucleotide sequences, obtain MV3 sequences, while add at the end of positive-sense strand 5 '
Enter Xba I and Xho I restriction enzyme sites, the end of antisense strand 5 ' adds Cla I and Kpn I restriction enzyme sites, using artificial synthesized side
Formula, obtains purpose fragment A, its sequence is as follows：
GATCTAGACT CGAGATGGAA AAAAGTTACG TCGATCTCTT AAACCAAGCA TGGGCAGATT
TGCCATTACA TTCAAAATTA TATTCAATTT GGCGTGTGTA CGAAGTCAAG AAATATTACA
AGCCGTGCTT AGTCCTGAAA AGAGGCGTAG ATTTAGGCGC AATGTACAAT ATCTCAGCTA
CGCATCAAAT ATCAAGTTTA GTGCAGAAAA GTCGAGATCA AGTCAGCTCT ATTTCAACCA
AACTCCACCA CAGTTTATGT AATAATGGAA AAAAATTATG TAGATCAATT AAACCAGTCA
TGGGCAGAAT TATCATACTG TGGAAAATTT TCAGCAATAT GGCGTGTGTT CAGAGTCAAG
AAATACTACA AGCCATCTTT AACCGTGAGA AAAAGCGTAG ATTTAGGCGC TGTTTACAAT
ATATCAGCTA CGCATCTAAT ATCAGATTTA GTGCAGAGAA GTCGAGATCG AGTCAGCTCT
ACTTTAACCA AACTCCGCAA CGGTTTCTAG GTACCATCGA TAG；
（2）RNAi carrier is built：
（a）The acquisition of purpose fragment I：Purpose fragment A is subjected to digestion using restriction enzyme Xho I and Kpn I, by enzyme
Cut product to be separated by agarose gel electrophoresis, recycle purpose fragment I；The sequence of purpose fragment I is SEQ in sequence table
ID NO:Nucleotide sequence shown in 2；
（b）The 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, recycle purpose fragment II, the sequence of purpose fragment II is sequence
SEQ ID NO in list:Nucleotide sequence shown in 3；
（c）The acquisition of intermediate carrier B：The purpose fragment I of recycling and purpose fragment II are attached with T4-DNA ligases, and
Connection product is converted into bacillus coli DH 5 alpha, the verification 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 is obtained after pHANNIBAL carriers；
（d）The acquisition of purpose fragment III：Extract the Plasmid DNA of intermediate carrier B, and with restriction enzyme Cla I and Xba I
Digestion is carried out, digestion products are separated by agarose gel electrophoresis, recycles purpose fragment III；The sequence of purpose fragment III
For SEQ ID NO in sequence table:Nucleotide sequence shown in 4；
（e）The 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, recycles purpose fragment IV；The sequence of purpose fragment IV is SEQ in sequence table
ID NO:Nucleotide sequence shown in 5；
（f）The acquisition of intermediate carrier C：The purpose fragment III of recycling and purpose fragment IV are attached with T4-DNA ligases,
And convert connection product into bacillus coli DH 5 alpha, the verification of picking positive colony, obtains 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）The acquisition of purpose fragment V：The Plasmid DNA of intermediate carrier C is extracted, and uses restriction enzymeNotI carries out digestion,
Digestion products are separated by agarose gel electrophoresis, recycle purpose fragment V；The sequence of purpose fragment V is sequence table
Middle SEQ ID NO:Nucleotide sequence shown in 6；
（h）The acquisition of purpose fragment VI：By plant expression vector pGreenII0229 Plasmid DNA restriction enzymesNot I
Digestion is carried out, digestion products are separated by agarose gel electrophoresis, recycles purpose fragment VI；The sequence of purpose fragment VI
For SEQ ID NO in sequence table:Nucleotide sequence shown in 7；
（i）The acquisition of anti-sugarcane floral leaf RNAi carrier pG0229i-MV3：The purpose fragment V of recycling and purpose fragment VI are used
T4-DNA ligases are attached, and connection product is converted into bacillus coli DH 5 alpha, and the verification of picking positive colony, acquisition can
RNAi carrier pG0229i-MV3 for genetic transformation sugarcane acceptor material；
（3）The cultivation of mosaic disease resisting transgenic sugarcane material and Disease Resistance Identification：The RNAi carrier that extraction structure is completed
PG0229i-MV3 Plasmid DNA, its concentration and purity are measured with nucleic acid-protein analyzer, and are quantified to 1 μ g/ μ L, gene
Rifle blast technique genetic transformation sugarcane, Molecular Detection obtain regeneration positive transgenic plant, and carry out the anti-of disease-resistant transgenic sugarcane
Characteristic of disease is identified.
2. a kind of method that mosaic disease resisting sugar cane breed is cultivated using artificial synthesized MV3 sequences according to claim 1, its
It is characterized in that 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 495bp.
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