CN105039356B - The method for cultivating anti-stripe mosaic disease sugarcane using RNAi silencing SceIF4E1 gene - Google Patents

Abstract

RNAi silencing is utilized the present invention relates to a kind ofSceIF4E1The method that gene cultivates anti-stripe mosaic viral transgene sugarcane, includingSceIF4E1Clone, the building of RNAi interference carrier, genetic transformation, the cultivation of mosaic disease resisting transgenic sugarcane material and the Disease Resistance Identification of gene.WithSceIF4E1Target sequence is interfered as RNAi, the transgenic plant obtained after genetic transformation, produce the resistance of wide spectrum to the different strains of sugarcane stripe mosaic virus, have the characteristics that disease resistance is good, resistance is lasting and biological safety is high, the cultivation of render transgenic sugarcane gets rid of the dependence of confrontation source gene, and can effectively shorten sugarcane mosaic disease resisting breeding cycle.Mosaic disease resisting transgenic sugarcane is cultivated using method of the invention, on GMO bio-safety, better than the transgenic sugarcane for being transferred to virus sequence.

Description

The method for cultivating anti-stripe mosaic disease sugarcane using RNAi silencing SceIF4E1 gene

Technical field

The present invention relates to a kind of methods for cultivating sugarcane (Saccharum sp.hybrid) disease-resistant variety, and in particular to one Kind cultivates anti-sugarcane stripe mosaic virus (Sugarcane streak mosaic using RNAi silencing SceIF4E1 gene Virus, SCSMV) sugar cane breed method, i.e., using RNAi technology interference sugarcane in eukaryotic translation initiation factor gene The method that SceIF4E1 cultivates anti-SCSMV transgenic sugarcane, belongs to field of biotechnology.

Background technique

Sugarcane (Saccharum sp.hybrid) is the most important sugar crop in China or even the whole world and energy crop, Cane suger accounts for the 92% of China's sugar total amount, accounts for the 74% of world's sugar total amount, and sugarcane ethyl alcohol accounts for world's biomass fuel ethyl alcohol 60%.Mosaic of sugarcane is one of the Disease for seriously endangering sugarcane production, in China Guangxi, Yunnan, Guangdong, Hainan Etc. ground sugarcane district generally occur (Li Wenfeng etc., 2007；Xu Donglin etc., 2008).After sugarcane infects mosaic disease, there is yellowish green phase in blade Between, striped not of uniform size, seedling germination rate decline, tiller reduce, plant is short and small, and slow growth, quality deteriorates, crystallization of sucrose Rate decline (Joyce et al., 1998；Wu et al.,2012；Li et al.,2013).The cause of disease of mosaic of sugarcane is main There are 3 kinds, i.e. SCSMV, corn mosaic virus (Sugarcane Mosaic Virus, SCMV) and sorghum mosaic virus (Sorghum Mosaic virus, SrMV), belong to marmor upsilon section (Potyviridae) (Li Wenfeng etc., 2007；Xu Donglin etc., 2008；Xu et al.,2008；Black bearberry such as, 2011).SCMV and SrMV belongs to Potyvirus (Potyvirus). SCSMV had found and reported for the first time in the U.S. in 1978, determination in 1998 its belong to Potyviridae, international virus in 2012 The classification committee (International Committee on Taxonomy of Viruses, ICTV) stood for the section it is new Belong to, i.e. standing grain Tobamovirus (Poacevirus) (http://www.ictvonline.org).SCSMV is mainly distributed on China, Meng Jia Explosion type prevalence is presented in Chinese sugarcane main producing region Guangxi, Yunnan in the ground such as drawing, India, Japan, Indonesia, Pakistan, Generally occur and wide-scale distribution (Li et al., 2011) on Guangdong, Zhejiang and Fujian and other places, when Field diseases reach 50%, Production loss is up to 20% (Putra et al., 2013).

SCSMV is single stranded positive-sense RNA virus, and genome structure is simple, and length is about 10Kb, encodes a big poly egg White (ployprotein), after digesting formed 10 maturation proteins, from N-terminal to C-terminal successively are as follows: the first protein (P1), auxiliary at Point-protease (helper component proteinase, HC-Pro), third albumen (P3), first 6K albumen (6K1), Cylindrical inclusion albumen (cylindrical inclusion protein, CI), second 6K albumen (6K2), viral genome Terminal binding protein (Viral Protein Genome-linked, VPg), nuclear inclusion a albumen (Nuclear Inclusion A protein, NIa), nuclear inclusion b albumen (Nuclear Inclusion b protein, NIb) and coat protein (Coat Protein,CP)(Riechmann et al.,1992；Urcuqui-Inchima et al.,2001).In addition, in P3 gene Inside, in G₂A₇Frameshit occurs for conserved domain, encodes a new albumen, be P3NPIPO (Chung et al., 2008；Wei et al.,2010；Vijayapalani et al.,2012).

Plant virus is as a kind of intracellular obligate parasite, it is necessary to by certain specific factors of host, could post It establishes in main body and infects, complete the basic vital movement such as translation, duplication, movement (Charron et al., 2008).Plant virus It is the key link that virus completes that systematicness infects in the intracorporal translation of host.The study found that being turned in marmor upsilon coe virus During translating, VPg plays a key effect.Since 5 ends ˊ of the coe virus genome do not have cap sequence m7GpppN, (N is to appoint Meaning nucleic acid), virus must serve as cap sequence and eukaryotic translation initiation factor (Eukaryotic by the VPg of its coding Translation initiation factor, eIF) interaction, it forms the tetramer and recruits other factors, virus could be started The translation (Murphy et al., 1991,1996) of RNA.

Translation initiation factor eIF4E is the necessary host factor that marmor upsilon coe virus starts translation in pin main body, For a long time by viral Selection utilization, it is virus and plant that the function in viral life activity, which has opposite conservative and stability, The product (Charron et al., 2008) of object coevolution, the recessive disease-resistant gene that this kind of host factor gene mutation generates With broad spectrum activity and persistence (Pavan et al., 2010).Recessive disease-resistant gene is generally existing in antivirus plant, because of it To the resistance of wide spectrum of virus, it is widely used in crop disease-resistant breeding.Nearest 10 years, with virus and the interaction research of host Deeply, it is found that these recessive disease-resistant genes in biology are inherently the mutant with the host factor gene of viral interaction, As resistant to PVY (Potato virus Y, PVY) in capsicum and tobacco etch virus (Tobacco etch virus, TEV the pvr6 of pvr2, anti-capsicum vein mottle virus (Pepper vein mottle virus, PVMY)), anti-pea in pea Beans kind passes the sbm1 etc. of mosaic virus (Pea seed-born mosaic virus, PSbMV), is all the host with viral interaction The mutant of translation initiation factor gene eIF4E1 or eIF4E2.This resistance is very lasting, and the pvr6 of the anti-PVMY of capsicum makes With more than 50 years, still effectively.It has been reported that 12 recessive disease-resistant genes resistant to marmor upsilon, wherein 7 What a natural mutation generated, 5 are generated by the methods of T-DNA gene knockout, are all the prominent of translation initiation factor gene Variant (Robaglia&Caranta, 2006；Maule et al.,2007；Truniger et al.,2009).

Host's recessiveness disease-resistant gene it is this because losing and generating the function to viral resistance of wide spectrum due to viral interaction ability, Basic of Biology has been established to carry out broad-spectrum disease resistance transgenic research using RNAi technology.Using RNAi technology, successfully The translation initiation factor gene of the plants such as silencing arabidopsis, tomato, obtains the mutant with resistance of wide spectrum.Arabidopsis When AteIF4E1 gene mutation, produce to the anti-of clover yellow vein virus (Clover yellow vein virus, ClYVV) Property；When AteIF4E2 gene mutation, mutant strain is produced to Brassica 2 et 4 (Turnip mosaic virus, TuMV), lettuce The resistance of lettuce mosaic virus (Lettuce mosaic virus, LMV) and plumpox virus (Plum pox virus, PPV) (Duprat et al.,2002；Sato,2005；Decroocq et al.,2006).In tomato, silencing SleIF4E1 base Marmor upsilon (Potato virus Y, PVY) and capsicum mottle virus (Pepper mottle is immunized in cause, mutant strain simultaneously Virus, PepMoV) two kinds of viruses (Piron et al., 2010), while SleIF4E1 the and SleIF4E2 base of silencing tomato Cause, mutant strain generate viral to PVY, TEV, capsicum mottle virus (Pepper mottle virus, PepMoV), color leaf green pepper (Ecuadorian rocotto virus, ERV), capsicum heavy type mosaic virus (Pepper severe mosaic virus, PepSMV), capsicum yellow mosaic virus (Pepper yellow mosaic virus, PepYMV) and potato V virus (Potato Virus V, PVV) totally 7 kinds of viral resistances (Mazier et al., 2011).It is especially noted that having at these wide In the mutant for composing resistance, phenotype all occur, or substantially there is no variations, being capable of normal growth and development, completion life Period strain (Duprat et al., 2002；Sato,2005；Decroocq et al.,2006；Piron et al., 2010；Mazier et al.,2011).

Virus Associated With Sugarcane Mosaic Disease Occurred is mainly propagated in a manner of non-standing by aphid, is difficult to prevent by conventional means.Anti- flower Leaf disease is always one of cane breeding target, by traditional Crossing system, can select the sugar cane breed of mosaic disease resisting.But Be, because of reasons such as flowering asynchronism, superior genotypes are difficult to polymerize, the period is long, be difficult to obtain the economical characters such as high yield and high sugar with The genotype of mosaic disease resisting polymerization.Transgenic plant disease resistance (the pathogen-derived mediated by viral autogene Resistance, PDR) discovery of (Abel et al., 1986) and the development of transgenic technology, make to utilize biological engineering means Mosaic disease resisting transgenic sugarcane is cultivated to be possibly realized.The principle of PDR is RNAi, and RNAi technology is occurred in eucaryote body , a kind of gene expression regulation mechanism based on nucleic acid sequence homology.I.e. double-stranded RNA (double-stranded RNA, DsRNA the siRNA of 21-26nt) is degraded to by Dicer, and the latter instructs cognate rna to identify and cut, and leads to target The degradation for marking mRNA, to cause gene silencing.RNAi technology has applied the cultivation with mosaic disease resisting transgenic sugarcane material, But its objects interfered is mainly the CP gene of virus, since CP gene pleiomorphism is very high, the transgenic line of acquisition is only right CP gene source virus strain is immune, to same other viral strain resistances deficiencies or loses resistance.Joyce (1998) will The coat protein gene (CP) of SCMV-E is transferred to sugarcane, take at random field morbidity Sugarcane Leaves juice (not yet explicitly viral species and Strain) to mutant frictional inoculation, mutant is generated by resisting a series of types of sense.Ingelbrecht (1999) is by SrMV-H The CP channel genes sugarcane of strain is cooked virus infection experiment to mutant with SrMV-H, SrMV-I, SrMV-M and SCMV-D, hair Existing mutant is positively correlated to the resistance of virus with CP gene order similarity: to SrMV-H highly resistance, same virus strain CP base Because homology is 100%；Resist in SrMV-I and SrMV-M, the CP gene of the CP gene and SrMV-H of the two virus strains Homology is 95%；To SCMV-D high sense, the CP genetic homology of the virus strain and SrMV-H are 75%.

Translation initiation factor gene is the endogenous gene of plant, is the key gene that control virus is translated in pin main body, The transgenic line or strain that a certain all strains of virus are generated with resistance will be obtained by interfering it to express or knock out the gene.

Summary of the invention

The anti-stripe mosaic disease product of sugarcane are cultivated using RNAi silencing SceIF4E1 gene the object of the present invention is to provide a kind of The method of kind.Using RNAi technology, using the conserved region of the SceIF4E1 gene of sugarcane as RNAi interference fragment, reversed weight is constructed Complex sequences obtains a RNAi carrier.By particle bombardment genetic transformation sugarcane, to obtain the different strains to SCSMV Virus has the transgenic sugarcane of resistance of wide spectrum.

The purpose of the present invention is what is be realized by the following method.

A kind of gene SceIF4E1 with the sugarcane translation initiation factor of the VPg interaction of SCSMV, it is characterised in that the base The nucleotide sequence of cause is as follows:

ATGGCCGACGAGATCGACACGAGGCCGGCCTCCGCGGGCTCCCGGGGCCGCCCCGCGCACGCTACGGA GGAGGACGACAGGGAGGAGGGCGAGATCGCCGATGACGCCCCCGCGCCCGCCCTCCCCGCCACGCACCCGCTCGAG CACTCGTGGACCTTCTGGTTCGACAACCAGCAGGGCAAGAGCAAGCAGGCCGCCTGGGGGAGCTCCATCCGACCCA TCCACACCTTCTCCACCGTCGAGGACTTCTGGGGCCTTTACAATAATATTCATCACCCTAGCAAGTTGGCCATGGG AGCTGACTTCCATTGCTTCAAGAACAAGATTGAACCAAAATGGGAAGACCCTATTTGTGCTAATGGCGGTAAATGG ACCATCAGCTGTGGAAGAGGAAAATCTGACACGCTGTGGCTGCACACCCTGTTGGCTATGATTGGCGAACAATTCG ACTATGGTGATGAAATTTGTGGAGCAGTCGTCAGCGTGCGTGGCAAGCAGGAAAGAATAGCTATCTGGACAAAAAA TGCTGCTAATGAAGCTGCTCAGATAAGCATTGGGAAGCAGTGGAAGGAATTCCTGGATTACAAGGACTCCATTGGA TTCATTGTTCATGACGATGCAAAGAAGGCGGACAAGGGACCGAGGAACCGTTACACGGTTTGA。

SceIF4E1 amino acid sequence is as follows:

MADEIDTRPASAGSRGRPAHATEEDDREEGEIADDAPAPALPATHPLEHSWTFWFDNQQGKSKQAAWG SSIRPIHTFSTVEDFWGLYNNIHHPSKLAMGADFHCFKNKIEPKWEDPICANGGKWTISCGRGKSDTLWLHTLLAM IGEQFDYGDEICGAVVSVRGKQERIAIWTKNAANEAAQISIGKQWKEFLDYKDSIGFIVHDDAKKADKGPRNRYTV。

A kind of a pair of of specific primer Sc4E1-F and Sc4E1-R for the design of SceIF4E1 gene, it is characterised in that institute The sequence for stating primer is as follows:

Sc4E1-F:TCTAGAGAATTCATCCACACCTTCTCCACCGTC

Sc4E1-R:AAGCTTGGTACCGTCCCTTGTCCGCCTTCTTTG

A method of the anti-SCSMV kind of sugarcane, including SceIF4E1 base are cultivated using RNAi silencing SceIF4E1 gene Clone, the building of RNAi interference carrier, genetic transformation, the cultivation of mosaic disease resisting transgenic sugarcane material and the Disease Resistance Identification of cause； It is characterized by:

(1) clone of SceIF4E1 gene:

Using the SceIF4E1 gene of Homology-based cloning clone sugarcane, with the noble cane sugarcane Badila of susceptible SCSMV Blade be experimental material, using Trizol method extract RNA, reverse transcription is at cDNA；Compare sorghum, corn, rice, arabidopsis EIF4E1 gene conservative section, design PCR primer, using cDNA as template, through PCR amplification and RACE (rapid Amplification of cDNA ends, RACE) reaction, obtain the opening code-reading frame (open of sugarcane SceIF4E1 gene Reading frame, ORF), gene ORF length is 663bp, encodes 220 amino acid；The core of the SceIF4E1 gene Nucleotide sequence is nucleotide sequence shown in SEQ ID NO:1 in sequence table；Its encoding amino acid sequence is SEQ in sequence table Amino acid sequence shown in ID NO:2；

(2) RNAi interferes the acquisition of target sequence:

For the conservative section of SceIF4E1 gene, a pair of of specific primer Sc4E1-F and Sc4E1-R are designed, in upstream Xba I and EcoR I restriction enzyme site are introduced on primer Sc4E1-F, introduce Hind III and Kpn I enzyme on downstream primer Sc4E1-R Enzyme site；5 ' ends are obtained by PCR reaction and have Xba I and EcoR I restriction enzyme site, and 3 ' have Hind III and Kpn I digestion The PCR product in site；Digestion products are carried out separation and recycling by agarose gel electrophoresis, RNAi is obtained and interferes target sequence； The nucleotides sequence of the upstream primer Sc4E1-F is classified as nucleotide sequence shown in SEQ ID NO:9 in sequence table；Downstream primer The nucleotides sequence of Sc4E1-R is classified as nucleotide sequence shown in SEQ ID NO:10 in sequence table；The core of RNAi interference target sequence Nucleotide sequence is nucleotide sequence shown in SEQ ID NO:11 in sequence table；

(3) RNAi interference carrier constructs:

(a) acquisition of positive segment S1: double digestion is carried out to RNAi interference target sequence with EcoR I and Kpn I, by digestion Product is separated by agarose gel electrophoresis, recycles positive segment S1；The sequence of positive segment S1 is SEQ in sequence table Nucleotide sequence shown in ID NO:3；

(b) acquisition of intermediate vector pS1: EcoR I and Kpn I double digestion pHANNIBAL carrier are used, digestion products are led to It crosses agarose gel electrophoresis to be separated and recovered from, is then connect digestion products with forward direction segment S1 with T4 DNA ligase, And convert connection product by state Escherichia coli (Escherichia coli) DH5 α, the verifying of picking positive colony obtains Intermediate vector pS1；

(c) acquisition of reverse complemental segment S2: target sequence is interfered with III double digestion RNAi of Xba I and Hind, digestion is produced Object is separated by agarose gel electrophoresis, recycles reverse complemental segment S2；The sequence of the reverse complemental segment S2 is sequence Nucleotide sequence shown in SEQ ID NO:4 in list；

(d) acquisition of intermediate vector pS1S2: III double digestion carrier pS1 of Xba I and Hind is used, digestion products are passed through into fine jade Sepharose electrophoresis is separated and recovered from, and is connect recovery product with reverse complemental segment S2 with T4 DNA ligase, and will Connection product is converted by state Escherichia coli (Escherichia coli) DH5 α, picking positive colony verifying, obtain with Introne intron on pHANNIBAL carrier is as spacer region, the intermediate vector with hairpin structure reverse complementary sequence pS1S2；

(e) acquisition of hairpin structure segment: I single endonuclease digestion intermediate vector pS1S2 of Not is used, digestion products are passed through into agarose Gel electrophoresis is separated, and hairpin structure segment is recycled；The sequence of the hairpin structure segment is SEQ ID NO in sequence table: Nucleotide sequence shown in 12；

(g) acquisition of RNAi interference carrier pG0229-4E1: I single endonuclease digestion pGreenII0229 carrier of Not produces digestion Object is separated and recovered from by agarose gel electrophoresis, then uses T4 DNA ligase by recovery product and hairpin structure segment Connection, and connection product is converted by state Escherichia coli (Escherichia coli) DH5 α, the verifying of picking positive colony, Obtain the RNAi interference carrier pG0229-4E1 that can be used for genetic transformation of sugarcane；

(4) genetic transformation, the cultivation of mosaic disease resisting transgenic sugarcane material and Disease Resistance Identification: extract what building was completed RNAi interference carrier pG0229-4E1 Plasmid DNA, measures its concentration and purity with nucleic acid-protein analyzer, and is quantified to 1 μ G/ μ L, particle bombardment genetic transformation sugarcane, Molecular Detection obtains positive transgenic plant, and carries out disease-resistant transgenic sugarcane Disease Resistance Identification.

The pHANNIBAL carrier, pGreenII0229 carrier, bacillus coli DH 5 alpha, restriction enzyme is by business Purchase obtains.

Mosaic disease resisting sugar cane material is obtained to using RNAi carrier Transformation of Sugarcane of the invention, by artificial infection, respectively It is vaccinated with the JP1 (Yunnan isolate) for belonging to SCSMV, JP2 strain (Yunnan isolate), FJ strain (Fujian isolate) control is invaded Dye rate has respectively reached 90.0%, 95.0% and 90%, and 13 plants of transgenic sugarcanes are not fallen ill, but wherein has 2 plants to turn base Because PCR detected the CP gene amplification product of SCSMV on the superior leaf of the inoculation leaf of sugarcane.Illustrate silencing and the VPg of SCSMV The sugarcane SceIF4E1 gene of interaction, has blocked virus genomic translation, and render transgenic sugarcane obtains the wide spectrum to SCSMV Resistance.The 11 plants of transgenic sugarcane plant obtained using method screening of the invention, identification, all resistant wide spectrum, disease resistance The feature 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 uses RNAi technology, so that sugarcane Anti-virus Disease Breeding avoids virus The problem of strain complexity, the cultivation of render transgenic sugarcane get rid of the dependence of confrontation source gene.

2. the present invention selects the transgenosis obtained with the sugarcane factor gene of the VPg interaction of SCSMV as RNAi sequence to plant Strain, has the characteristics that the resistance of wide spectrum to SCSMV difference strain, disease resistance is good, resistance is lasting and biological safety is high.

3. the RNAi interference carrier that the present invention obtains is used for transgenic sugarcane, excellent to other economical characters but do not resist The sugar cane breed of SCSMV carries out genetic improvement, can effectively shorten sugarcane mosaic disease resisting breeding cycle.

4, silencing of the present invention is sugarcane endogenous gene, therefore, on GMO bio-safety, better than being transferred to virus sequence Transgenic sugarcane.

Detailed description of the invention

Fig. 1 is pG0229-4E1 plasmid map.

Specific embodiment

In order to which the present invention is furture elucidated rather than the limitation present invention, it is illustrated with reference to embodiments.Following implementations Experimental method described in example is unless otherwise specified conventional method.The reagent and biomaterial are unless otherwise specified It obtains from commercial channels.

Embodiment one: the clone of sugarcane translation initiation factor gene SceIF4E1, SceIF4E2, SceIF4E3 gene and its With SCSMV-, SCMV-, the verifying of SrMV-VPg interaction:

Utilize SceIF4E1, SceIF4E2 and the SceIF4E3 gene of Homology-based cloning clone sugarcane.With susceptible SCSMV Noble cane sugarcane Badila blade be experimental material, using Trizol method extract RNA, reverse transcription is at cDNA.It is relatively high Fine strain of millet, corn eIF4E1, eIF4E2, opening code-reading frame (open reading frame, ORF) sequence of eIF4E3 gene, if Count 3 pairs of specific PCR primers, using cDNA as template, through PCR amplification and RACE, obtain sugarcane SceIF4E1, SceIF4E2 and The ORF of SceIF4E3 gene.The nucleotides sequence of SceIF4E1 gene is classified as nucleotides sequence shown in SEQ ID NO:1 in sequence table Column, overall length 663bp, encoding amino acid sequence are amino acid sequence shown in SEQ ID NO:2 in sequence table, encode 220 Amino acid；The nucleotides sequence of SceIF4E2 gene is classified as nucleotide sequence shown in SEQ ID NO:5, overall length in sequence table 630bp；Its encoding amino acid sequence is amino acid sequence shown in SEQ ID NO:6 in sequence table, encodes 209 amino acid； The nucleotides sequence of SceIF4E3 gene is classified as nucleotide sequence shown in SEQ ID NO:7, overall length 690bp in sequence table；It is compiled Code amino acid sequence is amino acid sequence shown in SEQ ID NO:8 in sequence table, encodes 229 amino acid.

Using yeast-two hybrid technique and bimolecular fluorescence complementary technology, specify SceIF4E1, SceIF4E2, The interaction of SceIF4E3 and SCSMV-, SCMV-, SrMV-VPg, the results are shown in Table 1, shows SrMV-VPg and SCMV- VPg with SceIF4E1, SceIF4E2 interaction, but SCSMV-VPg only with SceIF4E1 interaction, it was demonstrated that SceIF4E1 be participate in The host factor of SCSMV translation, i.e. SCSMV selective use SceIF4E1 complete the translation of genome.The VPg is viral base Because of group end binding protein.

1. yeast two-hybrid of table and bimolecular fluorescence complementary verifying SCSMV-, SCMV-, SrMV-VPg and SceIF4E1, The interaction of SceIF4E2, SceIF4E3

Embodiment two: the RNAi interference carrier of anti-sugarcane stripe mosaic virus is constructed

The method for constructing the RNAi interference carrier of anti-sugarcane stripe mosaic virus, comprising the following steps:

(1) RNAi interferes the acquisition of target sequence: using the RNAi carrier design library in GenBank to SceIF4E1 gene Target position section is screened, and target position section of the 220th to the 640 bit base segment of SceIF4E1 gene as RNAi has been selected. A pair of specific primer Sc4E1-F (upstream primer) and Sc4E1-R (downstream primer) for introducing restriction enzyme site of design, to target position area Section is transformed, and is made it easier to orientation and is connected to intermediate vector pHANNIBAL.Xba is introduced at the 5 ' ends of upstream primer Sc4E1-F 5 ' ends of I and EcoR I restriction enzyme site, downstream primer Sc4E1-R introduce Hind III and Kpn I restriction enzyme site.The core of Sc4E1-F Nucleotide sequence is that nucleotide sequence shown in SEQ ID NO:9, the nucleotides sequence of Sc4E1-R are classified as in sequence table in sequence table Nucleotide sequence shown in SEQ ID NO:10.Using this to primer, SceIF4E1 gene is expanded, PCR reaction system For 25 μ L:10 × PCR Buffer, 2.5 2.0 μ L of μ L, dNTPs, upstream primer Sc4E1-F (10 μm of ol/L) 1.0 μ L, downstream is drawn Object Sc4E1-R (10 μm of ol/L) 1.0 μ L, Taq enzyme (5U/ μ L) 0.125 μ L, ddH₂17.375 μ L of O, 1.0 μ L of template (cDNA), 25 μ L of total volume.PCR response procedures: 94 DEG C of initial denaturation 4min；Then it runs 35 to recycle, 94 DEG C of 30s, 60 DEG C of 30s, 72 DEG C 1min；Last 72 DEG C of 10min.Electrophoresis detection PCR product simultaneously returns target fragment using OMIGA gel extraction kit It receives and purifies, obtain RNAi interference target sequence, nucleotide sequence nucleotides sequence as shown in SEQ ID NO:11 in sequence table Column；

(2) building of RNAi interference carrier: target sequence is interfered with EcoR I and Kpn I double digestion vector rna i, by digestion Product is separated by agarose gel electrophoresis, recycles positive segment S1, SEQ ID in nucleotide sequence such as sequence table Nucleotide sequence shown in NO:3；EcoR I and Kpn I double digestion pHANNIBAL carrier are used simultaneously, and digestion products are passed through into fine jade Sepharose electrophoresis is separated and recovered from, and is connect recovery product with forward direction segment S1 with T4-DNA ligase, and will connection Product is converted by state bacillus coli DH 5 alpha, and the verifying of picking positive colony obtains recombinant plasmid pS1.Extract intermediate vector pS1 Plasmid DNA, with III double digestion carrier pMD19-T-4E1 of Xba I and Hind, by digestion products by agarose gel electrophoresis into Row separation, recycles reverse complemental segment S2, nucleotide sequence nucleotide sequence as shown in SEQ ID NO:4 in sequence table； With III double digestion intermediate vector pS1 of Xba I and Hind, digestion products are separated and recovered from by agarose gel electrophoresis, Recovery product is connect with reverse complemental segment S2 with T4-DNA ligase, and connection product is converted to by state Escherichia coli In DH5 α, the verifying of picking positive colony obtains using pHANNIBAL carrier introne (intron) as spacer region, has hair fastener The intermediate vector pS1S2 of structure inverted repeats.Intermediate pS1S2 Plasmid DNA is extracted, with Not I single endonuclease digestion, by digestion products It is separated by agarose gel electrophoresis, recycles hairpin structure segment, SEQ ID NO in nucleotide sequence such as sequence table: Nucleotide sequence shown in 12；With Not I single endonuclease digestion pGreenII0229 carrier, digestion products are passed through into agarose gel electrophoresis It is separated and recovered from, is connect recovery product with S1-intron-S2 segment with T4-DNA ligase, and connection product is turned Change to by state bacillus coli DH 5 alpha, the verifying of picking positive colony obtains RNAi interference carrier pG0229-4E1.The agarose Gel electrophoresis, referring to the method for agarose gel electrophoresis in " Molecular Cloning:A Laboratory guide " (second edition) chapter 6 first segment；Institute It states and converts connection product into bacillus coli DH 5 alpha, method for transformation is referring to " Molecular Cloning:A Laboratory guide " (second edition) chapter 1 Section five, with the method for calcium chloride preparation and transformed competence colibacillus Escherichia coli in；The picking of the bacterium colony containing positive colony, Referring to the identification method of the bacterial clump containing recombinant plasmid in Section six of " Molecular Cloning:A Laboratory guide " (second edition) chapter 1；Institute The method for extracting Plasmid DNA is stated, referring to plasmid extraction kit specification；The enzymatic cleavage methods, referring to restriction enzyme Specification；The recovery method, referring to plastic recovery kit specification；It is described to be attached method with T4-DNA ligase, ginseng According to T4-DNA ligase operational manual；It is as shown in Figure 1 to construct the pG0229-4E1 plasmid map completed.

Embodiment three: there is the different strains of a kind of pair of sugarcane stripe mosaic virus the transgenic sugarcane of resistance of wide spectrum to cultivate

A kind of sugarcane stripe mosaic virus difference strain has the transgenic sugarcane cultivation of resistance of wide spectrum, including following step It is rapid:

1, material prepares: extracting the RNAi interference carrier pG0229-4E1 Plasmid DNA that building is completed, is measured with nucleic acid-protein Instrument measures its concentration and purity, and is quantified to 1 μ g/ μ L；Receptor sugar cane breed is ROC22；

2, 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 The tender lobus cardiacus of children in 10cm after the ethanol disinfection for being 75% with volumetric concentration, and is cut into the disk that thickness is not more than 3mm, It is seeded to MS+3.0mg/L 2, Fiber differentiation 7d on the Fiber differentiation of 4-D+30g/L sucrose+6g/L agar powder pH5.8, in gene 4h goes to MS+2mg/L 2,4-D+0.2mol/L sorbierite+0.2mol/L mannitol+30g/L sucrose+6g/L agar before rifle bombards In the osmotic medium of powder pH 5.8, pre-processed；

Biolistic bombardment conversion: it is prepared according to the PDS-1000/He type particle gun operational manual of Bio-Rad company micro- Bullet adjusts target distance 6cm, air pressure 1,100psi, bombards pretreated material, the material after bombardment is scatter, and continues 20h is handled on osmotic medium；

The acquisition of material culture and regeneration plant after conversion: the material after biolistic bombardment is converted goes to MS+2.0mg/L The recovery media of 2.4-D+30g/L sucrose+6g/L agar powder pH 5.8, renewal cultivation 6d；It then will material after renewal cultivation Material moves to the subculture screening training of MS+2.0mg/L 2.4-D+30.0g/L sucrose+6.0g/L agar powder+4mg/L PPT pH 5.8 It supports in base, the screening and culturing 2-3 generation under 28 DEG C of dark conditions, 15d/ generation；Then material is gone into MS+1.0mg/L 6-BA+ In the differentiation screening and culturing medium of 0.5mg/L KT+30.0g/L sucrose+6.0g/L agar powder+4mg/L PPT pH 5.8, at 28 DEG C Under, 12~14h 2, the illumination of 000Lx, screening and culturing 2-3 generation, 15d/ generation is given once daily, until growing seedling；It grows tall to seedling When to 3-4cm, the life of 1/2MS+0.2mg/L 6-BA+3mg/L NAA+60g/L sucrose+6g/L agar powder pH 5.8 is gone to In root culture medium, at 28 DEG C, the illumination of 12~14h 2000Lx is given once daily, until seedling takes root；

3, Molecular Detection: by the seedling replanting after taking root into small flower (plant ash: sand: plantation soil=1: 1: 1), to After seedling survives, clip blade extracts RNA respectively, and reverse transcription utilizes quantitative PCR technique detection SceIF4E1 gene at cDNA Expression.The transgenic plant of 19 plants of complete silencings of SceIF4E1 gene is obtained altogether, wherein 13 plants of transgenic sugarcane plant exist The economical characters such as plant height, Ye Zi, stem diameter aspect no notable difference compared with acceptor material, being numbered is FJT1, FJT2 ... ..., FJT13；

4, transgenic sugarcane Disease Resistance Identification

First by vegetative propagation, expand numerous transgenic sugarcane.Turn base for 13 plants similar with acceptor material of biological character Because sugarcane plant is planted in greenhouse, each basin alms bowl plants 1 plant of transgenic sugarcane, normal water and fertilizer management, and the maturity period takes cane stalk In in greenhouse, each basin alms bowl plants 2 simple bud stems for simple bud plantation.Acceptor material ROC22 is planted in the same way in greenhouse It is interior.When plant enters fast growing period, " SrMV-P1 is utilized referring to the patent of invention of Patent No. ZL200710009226.8 Inoculation identification method in the method for gene cultivation mosaic disease resisting sugar cane breed ", carries out challenge viral dosage to transgenic sugarcane plant.Often A transgenic line takes 3 plants, is inoculated with the JP1 strain, JP2 strain and FJ strain virus of SCSMV respectively.Acceptor material ROC22 60 plants, it is divided into 3 groups, every group 20 plants, is inoculated with the JP1 strain, JP2 strain and FJ strain virus of SCSMV respectively.Meanwhile for The conserved region of the CP gene of SCSMV separately designs special primer, using round pcr, carries out PCR inspection to the superior leaf of inoculation blade It surveys, whether clear virus, which establishes systematicness, is infected.

As a result: being control with acceptor material ROC22, to JP1, JP2 and FJ of 13 plants of transgenic sugarcane artificial infection SCSMV Strain carries out challenge test, the results showed that this 13 plants of transgenic sugarcanes are not fallen ill.The transgenosis for being inoculated with JP1 strain virus is sweet In sugarcane plant, the superior leaf of the inoculation leaf of FJT-5 and FJT-7 detects the CP gene amplification product of SCSMV；In 20 plants of controls, 18 plants show typical floral leaf disease symptoms, do not detect that CP gene expands on the superior leaf of the inoculation leaf of 2 plants of non-disease plants Increase production object, infection rate 90.0%.It is inoculated in the transgenic sugarcane plant of JP2 strain virus, FJT-7 is inoculated with the superior leaf inspection of leaf Measure CP gene amplification product；In 20 plants of controls, 19 plants show typical floral leaf disease symptoms, the inoculation of 1 plant of non-disease plant The superior leaf of leaf does not detect CP gene amplification product, infection rate 95.0%.The transgenic sugarcane for being inoculated with FJ strain virus is planted CP gene amplification product is not detected on the superior leaf of the inoculation leaf of strain；In 20 plants of controls, 17 plants show typical floral leaf Disease symptoms have the superior leaf of 1 plant of inoculation leaf to detect CP gene amplification product in 3 plants of aobvious disease plant, share 18 plants it is right According to being infected, infection rate 90.0%.See Table 2 for details for virus inoculation experimental result.In 19 plants of transgenic sugarcanes, there are 13 plants of growths Resistance normally and to SCSMV is shown, rejects FJT-5 and FJT-7 that superior leaf detects CP gene, 11 plants of acquisition is right altogether The transgenic sugarcane material of the virus immunity of 3 strains of SCSMV illustrates that silencing SceIF4E1 gene render transgenic sugarcane obtains To the resistance of wide spectrum of SCSMV.

2. transgenic sugarcane virus inoculation qualification result of table

The plant expression vector of anti-mosaic of sugarcane is to construct to obtain based on RNAi principle, and method of the invention can make Anti-virus Disease Breeding gets rid of the dependence of confrontation source gene, using the sugarcane endogenous gene with SCSMV interaction as RNAi interference sequence Transgenic plant obtained has resistance of wide spectrum to all strains of SCSMV.Meanwhile because silencing is the endogenous base of sugarcane Cause, on GMO bio-safety, better than the transgenic sugarcane for being transferred to virus sequence.

The foregoing is merely presently preferred embodiments of the present invention, all equivalent changes done according to scope of the present invention patent with Modification, is all covered by the present invention.

SEQUENCE LISTING

Sequence table

<110>University Of Agriculture and Forestry In Fujian

<120>method for cultivating anti-stripe mosaic disease sugarcane using RNAi silencing SceIF4E1 gene

<130>

<160> 10

<170> PatentIn version 3.5

<210> 1

<211> 663

<212> DNA

<213>sugarcane

<400> 1

atggccgacg agatcgacac gaggccggcc tccgcgggct cccggggccg ccccgcgcac 60

gctacggagg aggacgacag ggaggagggc gagatcgccg atgacgcccc cgcgcccgcc 120

ctccccgcca cgcacccgct cgagcactcg tggaccttct ggttcgacaa ccagcagggc 180

aagagcaagc aggccgcctg ggggagctcc atccgaccca tccacacctt ctccaccgtc 240

gaggacttct ggggccttta caataatatt catcacccta gcaagttggc catgggagct 300

gacttccatt gcttcaagaa caagattgaa ccaaaatggg aagaccctat ttgtgctaat 360

ggcggtaaat ggaccatcag ctgtggaaga ggaaaatctg acacgctgtg gctgcacacc 420

ctgttggcta tgattggcga acaattcgac tatggtgatg aaatttgtgg agcagtcgtc 480

agcgtgcgtg gcaagcagga aagaatagct atctggacaa aaaatgctgc taatgaagct 540

gctcagataa gcattgggaa gcagtggaag gaattcctgg attacaagga ctccattgga 600

ttcattgttc atgacgatgc aaagaaggcg gacaagggac cgaggaaccg ttacacggtt 660

tga 663

<210> 2

<211> 220

<212> PRT

<213>sugarcane

<400> 2

Met Ala Asp Glu Ile Asp Thr Arg Pro Ala Ser Ala Gly Ser Arg Gly

1 5 10 15

Arg Pro Ala His Ala Thr Glu Glu Asp Asp Arg Glu Glu Gly Glu Ile

20 25 30

Ala Asp Asp Ala Pro Ala Pro Ala Leu Pro Ala Thr His Pro Leu Glu

35 40 45

His Ser Trp Thr Phe Trp Phe Asp Asn Gln Gln Gly Lys Ser Lys Gln

50 55 60

Ala Ala Trp Gly Ser Ser Ile Arg Pro Ile His Thr Phe Ser Thr Val

65 70 75 80

Glu Asp Phe Trp Gly Leu Tyr Asn Asn Ile His His Pro Ser Lys Leu

85 90 95

Ala Met Gly Ala Asp Phe His Cys Phe Lys Asn Lys Ile Glu Pro Lys

100 105 110

Trp Glu Asp Pro Ile Cys Ala Asn Gly Gly Lys Trp Thr Ile Ser Cys

115 120 125

Gly Arg Gly Lys Ser Asp Thr Leu Trp Leu His Thr Leu Leu Ala Met

130 135 140

Ile Gly Glu Gln Phe Asp Tyr Gly Asp Glu Ile Cys Gly Ala Val Val

145 150 155 160

Ser Val Arg Gly Lys Gln Glu Arg Ile Ala Ile Trp Thr Lys Asn Ala

165 170 175

Ala Asn Glu Ala Ala Gln Ile Ser Ile Gly Lys Gln Trp Lys Glu Phe

180 185 190

Leu Asp Tyr Lys Asp Ser Ile Gly Phe Ile Val His Asp Asp Ala Lys

195 200 205

Lys Ala Asp Lys Gly Pro Arg Asn Arg Tyr Thr Val

210 215 220

<210> 3

<211> 431

<212> DNA

<213>artificial sequence

<400> 3

aattcatcca caccttctcc accgtcgagg acttctgggg cctttacaat aatattcatc 60

accctagcaa gttggccatg ggagctgact tccattgctt caagaacaag attgaaccaa 120

aatgggaaga ccctatttgt gctaatggcg gtaaatggac catcagctgt ggaagaggaa 180

aatctgacac gctgtggctg cacaccctgt tggctatgat tggcgaacaa ttcgactatg 240

gtgatgaaat ttgtggagca gtcgtcagcg tgcgtggcaa gcaggaaaga atagctatct 300

ggacaaaaaa tgctgctaat gaagctgctc agataagcat tgggaagcag tggaaggaat 360

tcctggatta caaggactcc attggattca ttgttcatga cgatgcaaag aaggcggaca 420

agggacggta c 431

<210> 4

<211> 439

<212> DNA

<213>artificial sequence

<400> 4

agcttggtac cgtcccttgt ccgccttctt tgcatcgtca tgaacaatga atccaatgga 60

gtccttgtaa tccaggaatt ccttccactg cttcccaatg cttatctgag cagcttcatt 120

agcagcattt tttgtccaga tagctattct ttcctgcttg ccacgcacgc tgacgactgc 180

tccacaaatt tcatcaccat agtcgaattg ttcgccaatc atagccaaca gggtgtgcag 240

ccacagcgtg tcagattttc ctcttccaca gctgatggtc catttaccgc cattagcaca 300

aatagggtct tcccattttg gttcaatctt gttcttgaag caatggaagt cagctcccat 360

ggccaacttg ctagggtgat gaatattatt gtaaaggccc cagaagtcct cgacggtgga 420

gaaggtgtgg atgaattct 439

<210> 5

<211> 630

<212> DNA

<213>sugarcane

<400> 5

atggcggagg tcgaggttcc agctgctgcg gttgcgacga cgacccctga ggcggcggcg 60

accgagggcg gagccgcgac ggaggcgaag ggtccgcaca agctgcaccg gcagtggacc 120

ttctggtacg acatccagtc gaagcccaag ccgggcgctg cgtggggcac ctccctcaaa 180

aaggcctaca ccttcgacac cgtcgaggat ttttggagct tgtatgatca gatttttcgt 240

ccaagcaagc tgtctgggaa tgctgatttt catctgttca aggctggagt agagccaaaa 300

tgggaagacc cagagtgtgc aaatggtggc aaatggactg tcccatgcaa cagaaaggca 360

acctttgaga acatgtggct tgaaacgttg atggcactta ttggggagca gtttgatgaa 420

acagaggaca tttgtggaat tgttgctagt gtccgtgcga gaggagataa gcttgcatta 480

tggactagga ctgccagcaa tgaagctgtc caggtaaaca ttggcaagaa atggaaggat 540

gtcatcgact acaatgacaa gatcacctac actttccatg acgactcgag aagagaggag 600

ccgagcagag gcggacggta caccgtgtaa 630

<210> 6

<211> 209

<212> PRT

<213>sugarcane

<400> 6

Met Ala Glu Val Glu Val Pro Ala Ala Ala Val Ala Thr Thr Thr Pro

1 5 10 15

Glu Ala Ala Ala Thr Glu Gly Gly Ala Ala Thr Glu Ala Lys Gly Pro

20 25 30

His Lys Leu His Arg Gln Trp Thr Phe Trp Tyr Asp Ile Gln Ser Lys

35 40 45

Pro Lys Pro Gly Ala Ala Trp Gly Thr Ser Leu Lys Lys Ala Tyr Thr

50 55 60

Phe Asp Thr Val Glu Asp Phe Trp Ser Leu Tyr Asp Gln Ile Phe Arg

65 70 75 80

Pro Ser Lys Leu Ser Gly Asn Ala Asp Phe His Leu Phe Lys Ala Gly

85 90 95

Val Glu Pro Lys Trp Glu Asp Pro Glu Cys Ala Asn Gly Gly Lys Trp

100 105 110

Thr Val Pro Cys Asn Arg Lys Ala Thr Phe Glu Asn Met Trp Leu Glu

115 120 125

Thr Leu Met Ala Leu Ile Gly Glu Gln Phe Asp Glu Thr Glu Asp Ile

130 135 140

Cys Gly Ile Val Ala Ser Val Arg Ala Arg Gly Asp Lys Leu Ala Leu

145 150 155 160

Trp Thr Arg Thr Ala Ser Asn Glu Ala Val Gln Val Asn Ile Gly Lys

165 170 175

Lys Trp Lys Asp Val Ile Asp Tyr Asn Asp Lys Ile Thr Tyr Thr Phe

180 185 190

His Asp Asp Ser Arg Arg Glu Glu Pro Ser Arg Gly Gly Arg Tyr Thr

195 200 205

Val

<210> 7

<211> 690

<212> DNA

<213>sugarcane

<400> 7

atggaggcgg cggtggagaa gaaggagacc gagcaggagg agcagcagct acctcacgcg 60

cggaaggatg acgcgcccgc cgccgccgag gaagacgaag cggattcgga ggagaccgag 120

cgccgcaacc gcgacctcaa ggctggccac caccccctta ggcgcaagct cgtgctctgg 180

tacacgcgcc ggacgcctgg tgcgaggtcg caggcgtacg aggacaacat caagaagatc 240

atcgatttca gcacagtcga atcgttctgg gtttgctact gccaccttgc gcgcccttct 300

tccctgccaa gccccactga ccttcatctc ttcaaggatg gcatccgtcc cctctgggag 360

gatcctgcaa atcagaatgg tggcaagtgg ataattagat tcaaaaaggc agtttcaggt 420

cgtttctggg aggatttggt gctggtgcta gttggtgacc aacttgaata tagtgatgat 480

gtttgcggtg ttgtgcttag tgtccgtttc aatgaagaca ttctgagcgt ctggaaccgg 540

aacgcatcag accatcaggc ggtgatggca ttgagggatt ctatcaagag gcacctcaag 600

ctgccacaca gctatctgat ggagtacaaa ccacatgatg cttcgcggcg tgataactcg 660

tcctacagga acacttggct gagaggataa 690

<210> 8

<211> 229

<212> PRT

<213>sugarcane

<400> 8

Met Glu Ala Ala Val Glu Lys Lys Glu Thr Glu Gln Glu Glu Gln Gln

1 5 10 15

Leu Pro His Ala Arg Lys Asp Asp Ala Pro Ala Ala Ala Glu Glu Asp

20 25 30

Glu Ala Asp Ser Glu Glu Thr Glu Arg Arg Asn Arg Asp Leu Lys Ala

35 40 45

Gly His His Pro Leu Arg Arg Lys Leu Val Leu Trp Tyr Thr Arg Arg

50 55 60

Thr Pro Gly Ala Arg Ser Gln Ala Tyr Glu Asp Asn Ile Lys Lys Ile

65 70 75 80

Ile Asp Phe Ser Thr Val Glu Ser Phe Trp Val Cys Tyr Cys His Leu

85 90 95

Ala Arg Pro Ser Ser Leu Pro Ser Pro Thr Asp Leu His Leu Phe Lys

100 105 110

Asp Gly Ile Arg Pro Leu Trp Glu Asp Pro Ala Asn Gln Asn Gly Gly

115 120 125

Lys Trp Ile Ile Arg Phe Lys Lys Ala Val Ser Gly Arg Phe Trp Glu

130 135 140

Asp Leu Val Leu Val Leu Val Gly Asp Gln Leu Glu Tyr Ser Asp Asp

145 150 155 160

Val Cys Gly Val Val Leu Ser Val Arg Phe Asn Glu Asp Ile Leu Ser

165 170 175

Val Trp Asn Arg Asn Ala Ser Asp His Gln Ala Val Met Ala Leu Arg

180 185 190

Asp Ser Ile Lys Arg His Leu Lys Leu Pro His Ser Tyr Leu Met Glu

195 200 205

Tyr Lys Pro His Asp Ala Ser Arg Arg Asp Asn Ser Ser Tyr Arg Asn

210 215 220

Thr Trp Leu Arg Gly

225

<210> 9

<211> 33

<212> DNA

<213>artificial sequence

<400> 9

tctagagaat tcatccacac cttctccacc gtc 33

<210> 10

<211> 33

<212> DNA

<213>artificial sequence

<400> 10

aagcttggta ccgtcccttg tccgccttct ttg 33

<210> 11

<211> 445

<212> DNA

<213>artificial sequence

<400> 11

tctagagaat tcatccacac cttctccacc gtcgaggact tctggggcct ttacaataat 60

attcatcacc ctagcaagtt ggccatggga gctgacttcc attgcttcaa gaacaagatt 120

gaaccaaaat gggaagaccc tatttgtgct aatggcggta aatggaccat cagctgtgga 180

agaggaaaat ctgacacgct gtggctgcac accctgttgg ctatgattgg cgaacaattc 240

gactatggtg atgaaatttg tggagcagtc gtcagcgtgc gtggcaagca ggaaagaata 300

gctatctgga caaaaaatgc tgctaatgaa gctgctcaga taagcattgg gaagcagtgg 360

aaggaattcc tggattacaa ggactccatt ggattcattg ttcatgacga tgcaaagaag 420

gcggacaagg gacggtacca agctt 445

<210> 12

<211> 3816

<212> DNA

<213>artificial sequence

<400> 12

gcggccgctc gacgaattaa ttccaatccc acaaaaatct gagcttaaca gcacagttgc 60

tcctctcaga gcagaatcgg gtattcaaca ccctcatatc aactactacg ttgtgtataa 120

cggtccacat gccggtatat acgatgactg gggttgtaca aaggcggcaa caaacggcgt 180

tcccggagtt gcacacaaga aatttgccac tattacagag gcaagagcag cagctgacgc 240

gtacacaaca agtcagcaaa cagacaggtt gaacttcatc cccaaaggag aagctcaact 300

caagcccaag agctttgcta aggccctaac aagcccacca aagcaaaaag cccactggct 360

cacgctagga accaaaaggc ccagcagtga tccagcccca aaagagatct cctttgcccc 420

ggagattaca atggacgatt tcctctatct ttacgatcta ggaaggaagt tcgaaggtga 480

aggtgacgac actatgttca ccactgataa tgagaaggtt agcctcttca atttcagaaa 540

gaatgctgac ccacagatgg ttagagaggc ctacgcagca ggtctcatca agacgatcta 600

cccgagtaac aatctccagg agatcaaata ccttcccaag aaggttaaag atgcagtcaa 660

aagattcagg actaattgca tcaagaacac agagaaagac atatttctca agatcagaag 720

tactattcca gtatggacga ttcaaggctt gcttcataaa ccaaggcaag taatagagat 780

tggagtctct aaaaaggtag ttcctactga atctaaggcc atgcatggag tctaagattc 840

aaatcgagga tctaacagaa ctcgccgtga agactggcga acagttcata cagagtcttt 900

tacgactcaa tgacaagaag aaaatcttcg tcaacatggt ggagcacgac actctggtct 960

actccaaaaa tgtcaaagat acagtctcag aagaccaaag ggctattgag acttttcaac 1020

aaaggataat ttcgggaaac ctcctcggat tccattgccc agctatctgt cacttcatcg 1080

aaaggacagt agaaaaggaa ggtggctcct acaaatgcca tcattgcgat aaaggaaagg 1140

ctatcattca agatctctct gccgacagtg gtcccaaaga tggaccccca cccacgagga 1200

gcatcgtgga aaaagaagac gttccaacca cgtcttcaaa gcaagtggat tgatgtgaca 1260

tctccactga cgtaagggat gacgcacaat cccactatcc ttcgcaagac ccttcctcta 1320

tataaggaag ttcatttcat ttggagagga cacgctcgag gaattcatcc acaccttctc 1380

caccgtcgag gacttctggg gcctttacaa taatattcat caccctagca agttggccat 1440

gggagctgac ttccattgct tcaagaacaa gattgaacca aaatgggaag accctatttg 1500

tgctaatggc ggtaaatgga ccatcagctg tggaagagga aaatctgaca cgctgtggct 1560

gcacaccctg ttggctatga ttggcgaaca attcgactat ggtgatgaaa tttgtggagc 1620

agtcgtcagc gtgcgtggca agcaggaaag aatagctatc tggacaaaaa atgctgctaa 1680

tgaagctgct cagataagca ttgggaagca gtggaaggaa ttcctggatt acaaggactc 1740

cattggattc attgttcatg acgatgcaaa gaaggcggac aagggacggt accccaattg 1800

gtaaggaaat aattattttc ttttttcctt ttagtataaa atagttaagt gatgttaatt 1860

agtatgatta taataatata gttgttataa ttgtgaaaaa ataatttata aatatattgt 1920

ttacataaac aacatagtaa tgtaaaaaaa tatgacaagt gatgtgtaag acgaagaaga 1980

taaaagttga gagtaagtat attattttta atgaatttga tcgaacatgt aagatgatat 2040

actagcatta atatttgttt taatcataat agtaattcta gctggtttga tgaattaaat 2100

atcaatgata aaatactata gtaaaaataa gaataaataa attaaaataa tattttttta 2160

tgattaatag tttattatat aattaaatat ctataccatt actaaatatt ttagtttaaa 2220

agttaataaa tattttgtta gaaattccaa tctgcttgta atttatcaat aaacaaaata 2280

ttaaataaca agctaaagta acaaataata tcaaactaat agaaacagta atctaatgta 2340

acaaaacata atctaatgct aatataacaa agcgcaagat ctatcatttt atatagtatt 2400

attttcaatc aacattctta ttaatttcta aataatactt gtagttttat taacttctaa 2460

atggattgac tattaattaa atgaattagt cgaacatgaa taaacaaggt aacatgatag 2520

atcatgtcat tgtgttatca ttgatcttac atttggattg attacagttg ggaaattggg 2580

ttcgaaatcg ataagcttgg taccgtccct tgtccgcctt ctttgcatcg tcatgaacaa 2640

tgaatccaat ggagtccttg taatccagga attccttcca ctgcttccca atgcttatct 2700

gagcagcttc attagcagca ttttttgtcc agatagctat tctttcctgc ttgccacgca 2760

cgctgacgac tgctccacaa atttcatcac catagtcgaa ttgttcgcca atcatagcca 2820

acagggtgtg cagccacagc gtgtcagatt ttcctcttcc acagctgatg gtccatttac 2880

cgccattagc acaaataggg tcttcccatt ttggttcaat cttgttcttg aagcaatgga 2940

agtcagctcc catggccaac ttgctagggt gatgaatatt attgtaaagg ccccagaagt 3000

cctcgacggt ggagaaggtg tggatgaatt ctctagagtc ctgctttaat gagatatgcg 3060

agacgcctat gatcgcatga tatttgcttt caattctgtt gtgcacgttg taaaaaacct 3120

gagcatgtgt agctcagatc cttaccgccg gtttcggttc attctaatga atatatcacc 3180

cgttactatc gtatttttat gaataatatt ctccgttcaa tttactgatt gtaccctact 3240

acttatatgt acaatattaa aatgaaaaca atatattgtg ctgaataggt ttatagcgac 3300

atctatgata gagcgccaca ataacaaaca attgcgtttt attattacaa atccaatttt 3360

aaaaaaagcg gcagaaccgg tcaaacctaa aagactgatt acataaatct tattcaaatt 3420

tcaaaaggcc ccaggggcta gtatctacga cacaccgagc ggcgaactaa taacgttcac 3480

tgaagggaac tccggttccc cgccggcgcg catgggtgag attccttgaa gttgagtatt 3540

ggccgtccgc tctaccgaaa gttacgggca ccattcaacc cggtccagca cggcggccgg 3600

gtaaccgact tgctgccccg agaattatgc agcatttttt tggtgtatgt gggccccaaa 3660

tgaagtgcag gtcaaacctt gacagtgacg acaaatcgtt gggcgggtcc agggcgaatt 3720

ttgcgacaac atgtcgaggc tcagcaggac ctgcaggcat gcaagctagc ttactagtga 3780

tgcatattct atagtgtcac ctaaatctgc ggccgc 3816

Claims (2)

1. a kind of sugarcane translation initiation factor gene SceIF4E1 with the VPg interaction of SCSMV, it is characterised in that the gene Nucleotides sequence is classified as nucleotide sequence shown in SEQ ID NO:1 in sequence table.

2. a kind of method for cultivating the anti-SCSMV kind of sugarcane using RNAi silencing SceIF4E1 gene, it is characterised in that:

(1) RNAi interferes the acquisition of target sequence:

For the conservative section of SceIF4E1 gene, a pair of of specific primer Sc4E1-F and Sc4E1-R are designed, in upstream primer Xba I and EcoR I restriction enzyme site are introduced on Sc4E1-F, introduce Hind III and Kpn I digestion position on downstream primer Sc4E1-R Point；5 ' ends are obtained by PCR reaction having Xba I and EcoR an I restriction enzyme site, 3 ' with Hind III and Kpn I restriction enzyme site PCR product；Digestion products are carried out separation and recycling by agarose gel electrophoresis, RNAi is obtained and interferes target sequence；On described The nucleotides sequence of trip primer Sc4E1-F is classified as nucleotide sequence shown in SEQ ID NO:9 in sequence table；Downstream primer Sc4E1- The nucleotides sequence of R is classified as nucleotide sequence shown in SEQ ID NO:10 in sequence table；The nucleotides sequence of RNAi interference target sequence It is classified as nucleotide sequence shown in SEQ ID NO:11 in sequence table；The nucleotides sequence of the SceIF4E1 gene is classified as sequence table Nucleotide sequence shown in middle SEQ ID NO:1；

(2) RNAi interference carrier constructs:

(a) acquisition of positive segment S1: double digestion is carried out to RNAi interference target sequence with EcoR I and Kpn I, by digestion products It is separated by agarose gel electrophoresis, recycles positive segment S1；The sequence of positive segment S1 is SEQ ID in sequence table Nucleotide sequence shown in NO:3；

(b) acquisition of intermediate vector pS1: EcoR I and Kpn I double digestion pHANNIBAL carrier are used, digestion products are passed through into fine jade Sepharose electrophoresis is separated and recovered from, and is then connect digestion products with forward direction segment S1 with T4 DNA ligase, and will Connection product is converted by state Escherichia coli (Escherichia coli) DH5 α, and the verifying of picking positive colony obtains centre Carrier pS1；

(c) acquisition of reverse complemental segment S2: target sequence is interfered with III double digestion RNAi of Xba I and Hind, digestion products are led to It crosses agarose gel electrophoresis to be separated, recycles reverse complemental segment S2；The sequence of the reverse complemental segment S2 is sequence table Nucleotide sequence shown in middle SEQ ID NO:4；

(d) acquisition of intermediate vector pS1S2: III double digestion carrier pS1 of Xba I and Hind is used, digestion products are passed through into agarose Gel electrophoresis is separated and recovered from, and is connect recovery product with reverse complemental segment S2 with T4 DNA ligase, and will connection Product is converted by state Escherichia coli (Escherichia coli) DH5 α, picking positive colony verifying, obtain with Introne intron on pHANNIBAL carrier is as spacer region, the intermediate vector with hairpin structure reverse complementary sequence pS1S2；

(e) acquisition of hairpin structure segment: I single endonuclease digestion intermediate vector pS1S2 of Not is used, digestion products are passed through into Ago-Gel Electrophoresis is separated, and hairpin structure segment is recycled；The sequence of the hairpin structure segment is SEQ ID NO:12 institute in sequence table The nucleotide sequence shown；

(f) acquisition of RNAi interference carrier pG0229-4E1: I single endonuclease digestion pGreenII0229 carrier of Not passes through digestion products Agarose gel electrophoresis is separated and recovered from, and is then connect recovery product with hairpin structure segment with T4 DNA ligase, And convert connection product by state Escherichia coli (Escherichia coli) DH5 α, the verifying of picking positive colony obtains It can be used for the RNAi interference carrier pG0229-4E1 of genetic transformation of sugarcane；

(3) it is dry genetic transformation, the cultivation of mosaic disease resisting transgenic sugarcane material and Disease Resistance Identification: to extract the RNAi that building is completed Carrier pG0229-4E1 Plasmid DNA is disturbed, measures its concentration and purity with nucleic acid-protein analyzer, and is quantified to 1 μ g/ μ L, Particle bombardment genetic transformation sugarcane, Molecular Detection obtain positive transgenic plant, and carry out the anti-of disease-resistant transgenic sugarcane Characteristic of disease identification.