CN109392702A - A kind of method of the normal wild rice stem of artificially breeding - Google Patents

Abstract

A kind of method of the normal wild rice stem of artificially breeding, belongs to wild rice stem breeding technical field.It the following steps are included: be mixed for wild rice smut haploid strains UET1 △ Itd1 and wild rice smut haploid strains bacterium solution to connect bacterium in equal volume；Select the wild hay seedling of 3-5 leaf phase as object of inoculation；Mixed bacteria liquid injects seedling base portion with syringe, until bacterium solution is overflowed above leaf sheath；By treated, inoculation seedling is trimmed；Inoculation is completed in transition culture after two weeks, can transplant outdoor outdoor cultivation；The outdoor outdoor cultivation of transplanting.The wild rice smut for wild rice smut haploid strains UET1 △ Itd1 and wild rice smut haploid strains UET2 △ Itd1 a pair of the combination that the present invention is screened using wild rice smut is inoculated in wild hay, makes the pregnant normal wild rice stem out of its success.This method is easy to operate, effective, provides a new direction for wild rice stem breeding.

Description

A kind of method of the normal wild rice stem of artificially breeding

Technical field

The invention belongs to wild rice stem breeding technical fields, and in particular to a kind of method of the normal wild rice stem of artificially breeding.

Background technique

Wild rice Ustilago is a kind of typical dichotype fungi, with corn tumor smut in Basidiomycotina Ustilago Nearly source.So far, wild rice stem is unique host known to it, and the pregnant hay of wild rice stem is wild rice stem plant and this colonizes in body in specific manner The interior coefficient result of wild rice smut.So far, the plantation of wild rice stem and conservation breeding still use the side of hay pier separation screening Formula, manpower and material resources investment are larger.It therefore is the developing direction of wild rice stem breeding using artificial infection mode.And it is existing at present artificial Breeding technique can only make the pregnant ash discharge hay of wild hay.

Summary of the invention

In view of the problems of the existing technology, it is an object of the invention to design to provide a kind of normal wild rice stem of artificially breeding The technical solution of method.

A kind of method of the normal wild rice stem of artificially breeding, it is characterised in that the following steps are included:

1) by wild rice smut (Ustilago esculenta) haploid strains UET1 △ Itd1 and wild rice smut (Ustilago esculenta) haploid strains UET2 △ Itd1 respectively 25-30 DEG C in YEPS fluid nutrient medium under the conditions of shake bacterium to OD₆₀₀For 2.0, then bacterium is shaken to OD under the conditions of 25-30 DEG C into YEPS fluid nutrient medium with the dilution proportion of 1:100₆₀₀For 0.8-1.0, from The heart collects thallus, is diluted to final concentration of OD with pure water₆₀₀For 2.0 bacterium solution, by wild rice smut haploid strains UET1 △ Itd1 It is mixed for connecing bacterium in equal volume with wild rice smut haploid strains bacterium solution, the wild rice smut (Ustilago esculenta) Haploid strains UET1 △ Itd1 deposit number be CGMCC No.16723, the wild rice smut (Ustilago esculenta) Haploid strains UET2 △ Itd1 deposit number is CGMCC No.16724；

2) the wild hay tubulose root progress nursery of selection, greenhouse cultivation 20-30 days, and transplant to small basin, Nutrition Soil slightly covers root, And continue to cultivate 7-10 days in the greenhouse of the same terms, select the wild hay seedling of 3-5 leaf phase as object of inoculation；

3) seedling base portion is injected to the mixed bacteria liquid syringe that step 1) obtains, until bacterium solution is overflowed above leaf sheath；

4) by treated, inoculation seedling is trimmed, and placing excessive transpiration leads to here plant withers；

5) seedling after inoculation is continued to be placed on 22-25 DEG C of greenhouse transition culture, completes inoculation after two weeks, outdoor dew can be transplanted Its cultivation；

6) time of the outdoor outdoor cultivation of transplanting should control in the 3-4 month and 9-10 month, prevent outdoor temperature too high or too low It is unfavorable for the growth of bacterium, fertilising plants standard with reference to normal wild rice stem.

A kind of method of the normal wild rice stem of artificially breeding, it is characterised in that YEPS Liquid Culture in the step 1) Base contains following components in percentage by weight: peptone 2%, sucrose 2%, yeast powder 1% and water surplus.

A kind of method of the normal wild rice stem of artificially breeding, it is characterised in that step 2) the medium temperature chamber breeding condition At 22-25 DEG C, light application time is 8-12 hours for temperature control.

The method of the normal wild rice stem of a kind of artificially breeding, it is characterised in that the medium and small basin size of the step 2) is 10 ×10cm。

A kind of method of the normal wild rice stem of artificially breeding, it is characterised in that injection seedling base portion children in the step 3) The above position of seedling growing point.

Wild rice smut that the present invention is screened using wild rice smut (Ustilago esculenta) haploid strains UET1 △ Itd1 and wild rice smut (Ustilago esculenta) haploid strains UET2 △ Itd1 a pair of combination the black powder of wild rice Bacterium is inoculated in wild hay, makes the pregnant normal wild rice stem out of its success.This method is easy to operate, effective, provides one for wild rice stem breeding New direction.

Detailed description of the invention

Fig. 1 is gene cloning electrophoretogram；

Fig. 2 is upstream and downstream knockout carrier schematic diagram；

Fig. 3 be bacterial strain UET1 △ Itd1 and UET2 △ Itd1 regular-PCR verifying andItd1The verifying of gene expression amount；

Fig. 4 be wild rice smut (Ustilago esculenta) haploid strains UET1 △ Itd1 and wild rice smut (Ustilago esculenta) after haploid strains UET2 △ Itd1 infects wild hay, the pregnant hay phenotypic map of wild rice stem and the copolymerization of wild rice stem histotomy are burnt aobvious Micro mirror observation figure.

Specific embodiment

Further illustrate the present invention with reference to embodiments.

Embodiment 1: wild rice smut (Ustilago esculenta)Itd1The clone of gene

1. the clone of Itd1 gene: by the analysis of the transcript profile data of normal wild rice stem and grey hay, and combining wild rice smut gene Group three generations's sequencing data, prediction have obtained forming relevant wild rice smut gene loci to teleutospore being g1449.Utilize NCBI's ORF Finder function predicts g1449 gene open reading frame (ORF) sequence, it is found that its open reading frame length is 2670bp.With wild rice smut (Ustilago esculenta) haploid strains UET1 and wild rice smut (Ustilago esculenta) haploid strains UET2 genomic DNA be template, and design primer Itd1-F/R expand Itd1 gene sequence Column, primer I td1-UF/UR amplificationItd1Upstream region of gene promoter sequence, primer I td1-DF/DR amplification Itd1 downstream of gene open Promoter sequences.The total serum IgE for extracting two bacterial strains simultaneously, passes through PrimeScript II 1st strand cDNA Synthesis Kit reverse transcription obtains the first chain of cDNA and as template, with primer I td1-cF/cR amplification Itd1 gene CDNA sequence.

Pcr amplification reaction system (50 μ l) are as follows: 10 × PCR buffer, 54 μ L of μ l, dNTP, 1 μ l of upstream primer, downstream is drawn 1 μ l of object, 1 μ l of DNA profiling, 0.5 μ L of Taq enzyme add ddH₂O to 50 μ L.PCR amplification program is 94 DEG C of 4 min；94 ℃ 30 s, 58 DEG C of 30 s, 72 DEG C of 2 min, 30 circulations；72 ℃ 10 min；4 DEG C of terminations, primer sequence are as follows:

Itd1-F:5 '-ATGGTTTCCCACCAAGTCGC-3 ' (as shown in SEQ ID NO.5)；

Itd1-R:5 '-TCACGCCGCGGGTCTTGTCAA-3 ' (as shown in SEQ ID NO.6)；

Itd1-UF:5 '-GGTGCACGTCATTTGCCCAC-3 ' (as shown in SEQ ID NO.7)；

Itd1-UR:5 '-GTCGAGTGGCTGTGAGAAAC-3 ' (as shown in SEQ ID NO.8)；

Itd1-DF:5 '-TGCAGACGAATCAGGGCTGG-3 ' (as shown in SEQ ID NO.9)；

Itd1-DR:5 '-ACGTTTGCGGACCCAACCAT-3 ' (as shown in SEQ ID NO.10)；

Itd1-cF:5 '-ATGGTTTCCCACCAAGTCGCC-3 ' (as shown in SEQ ID NO.11)；

Itd1-cR:5 '-TCACGCCGCGGGTCTTGTCA-3 ' (as shown in SEQ ID NO.12).

By above-mentioned PCR product after 1.0% agarose electrophoresis, specific fragment is recycled, and be connected to PMD19-T carrier On, it is cloned.The positive transformant that clone obtains is sequenced, obtains g1449 genome sequence, the gene nucleosides respectively Acid sequence is named as shown in SEQ ID No.1 and its upstream and downstream and CDS sequence (CDS sequence is as shown in SEQ ID No.2) Itd1 gene.Sequence and cDNA sequence on its genome are compared by Clone Manager software, finds have in Itd1 gene The introne that one length is 111bp, carries out protein translation to Itd1cDNA sequence with EditSeq software, it is found that the gene is compiled 852 amino acid of code, amino acid sequence is as shown in SEQ ID No.3.As Fig. 1 be wild rice smut (Ustilago esculenta) Itd1 gene cloning electrophoretogram.

Embodiment 2: wild rice smut (Ustilago esculenta) haploid strains UET1 △ Itd1 and wild rice smut (Ustilago esculenta) haploid strains UET2 △ Itd1 acquisition

1. the clone of upstream and downstream knockout carrier: using the principle of homologous recombination, using bacterial strain UET1 as template, with primer I td1- U2/U3 expands upstream and knocks out segment, segment is knocked out with primer I td1-D1/D2 amplification downstream, using plasmid pUM1507 plasmid as mould Plate, expands hygromycin fragment upstream with primer hyg-F/3, hygromycin segments downstream is expanded with primer hyg-4/R, by upstream region of gene The method of segment and hygromycin fragment upstream fusion DNA vaccine, and obtain upstream with primer I td1-U2/hyg3 and knock out long segment, and Carrier T is connected, Escherichia coli are converted；By the method for downstream of gene segment and hygromycin segments downstream fusion DNA vaccine, and use primer Hyg4/Itd1-D2 obtains downstream and knocks out long segment (Fig. 2 is upstream and downstream knockout carrier schematic diagram), and connects carrier T, and conversion is big Enterobacteria, for replacingItd1Hygromycin (Hyg) gene order of gene order is as shown in SEQ ID No.4.Primer sequence is such as Under:

Itd1-U2:5 '-CGCGTTCTCGACTCGATTG-3 ' (as shown in SEQ ID No.13)；

Itd1-U3:5 '-GTAGTTACCACGTTCGGCCAATCTAGTGAATGCGGCGAGAG-3 ' (such as SEQ ID No.14 It is shown)；

Itd1-D1:5 '-TGTCAAACATGAGGCCTGAGTTGAAGATTGCGTGGCTCG-3 ' is (such as SEQ ID No.15 institute Show)；

Itd1-D2:5 '-TTGCGGACCCAACCATTTC-3 ' (as shown in SEQ ID No.16)；

Hyg3:5 '-GGATGCCTCCGCTCGAAGTA-3 ' (as shown in SEQ ID No.17)；

Hyg4:5 '-CGTTGCAAGACCTGCCTGAA-3 ' (as shown in SEQ ID No.18).

2. the preparation and conversion of protoplast: from -80 DEG C of preservation refrigerators take out the wild rice smut monoploid UET1 frozen and UET2 is simultaneously activated in YEPS solid medium, is placed in 28 DEG C incubator 2-3 days, black from picking wild rice on YEPS solid medium The single colonie of powder bacterium monoploid UET1 and UET2 are inoculated into 5-10 mL YEPS fluid nutrient medium, 28 DEG C of 180 r/min training It supports to OD_600nmBetween 0.8-1.5,1 mL renewed vaccination of bacterium solution is taken to continue into 50 mL YEPS fluid nutrient mediums It cultivates to OD_600nmFor 0.6-0.8.After microexamination confirmation is pollution-free, thalline were collected by centrifugation, and 10 mL are added into thallus SCS buffer, piping and druming mix, and 3000 rpm of room temperature is centrifuged 2min, discard supernatant；900 μ L lywallzyme (15 mg/ are added ML), 100 μ L driselase (15 mg/mL), gently blows and beats suspension thalline, and 30 DEG C of 110 r/min digests 15-20 min, show It is microcosmic to examine enzymatic hydrolysis situation, it can be carried out after occurring 30%-40% Pellet form Strain in the visual field in next step.It is added into enzymolysis product The SCS buffer of 10 mL pre-cooling, gently piping and druming mixes, and 4 DEG C of 2400 rpm is centrifuged 5 min, discards supernatant, weighs on ice Multiple previous step；In the STC liquid that 6 mL pre-cooling is added into precipitating on ice, gently piping and druming is mixed, 4 DEG C of 2400rpm 5 min are centrifuged, are discarded supernatant；In the STC solution that 1 mL pre-cooling is added into precipitating on ice, gently piping and druming is mixed, according to Every 100 μ L of pipe is dispensed, and is marked, -80 DEG C of preservations.The protoplast for taking out and preparing is filled from -80 DEG C of refrigerators to be placed in 10 min on ice；It falls the regeneration culture medium containing 100 μ g/mL hygromycin when waiting, falls 10-15mL on each plate；Xiang Xie 1 μ L Heparin solution (15 mg/mL) is added in the protoplast that frozen piece is carved, then is separately added into the upper and lower of 1-5 μ g equivalent Knockout carrier linear DNA is swum, piping and druming mixes, 10 min of ice-water bath；500 μ L STC-PEG solution are added, piping and druming mixes, 15 min of ice-water bath；When waiting in the resistant panel solidified fall regeneration culture medium the second layer, each plate 10-15 mL；It takes 200 μ L protoplast transformation liquid to be spread evenly across on regeneration culture medium, 28 DEG C of constant incubators is inverted in after drying Middle culture 5 days or so, picking single colonie.

3. the screening and verifying of transformant: picking transformant on the YEPS solid medium containing hygromycin after being commissioned to train Support secondary, then picking single colonie expands culture on the YEPS solid medium containing hygromycin again.And it is verified using gene Primer, Itd1-YZ-F/ Itd1-YZ-R, hygromycin verify primer, hyg-YZ-/FR, and primer I td1-YZ-U/ is verified in upstream Primer hyg-YZ-F/ Itd1-YZ-D is verified in hyg-YZ-R and downstream；Pcr amplification reaction system (15 μ l) are as follows: rTaq Nasemix 7.5 μ l, primer-F(10 μM) 0.6 μ l, primer-R(10 μM) 0.6 μ l, 1 μ L of template adds ddH₂O to 15 μL.PCR response procedures: 94 DEG C of 4 min of initial denaturation；94 DEG C of 15 s, anneal 15 s of Tm, 72 DEG C of extension 15 sec/kb, and 35 A circulation；72℃ 5 min；4 DEG C of terminations.Primer sequence is as follows:

Itd1-YZ-F:5 '-ACCGACGGCATGATCTGGAG-3 ' (as shown in SEQ ID No.19)；

Itd1-YZ-R:5 '-CGTTGCCGCTACTGACATGC-3 ' (as shown in SEQ ID No.20)；

Hyg-YZ-F:5 '-TCGTTATGTTTATCGGCACT-3 ' (as shown in SEQ ID No.21)；

Hyg-YZ-R:5 '-TCGGCGAGTACTTCTACACA-3 ' (as shown in SEQ ID No.22)；

Itd1-YZ-U:5 '-TCTAGCACTTGCACAGCGTC-3 ' (as shown in SEQ ID No.23)；

Itd1-YZ-D:5 '-TGCGACGAGTTGACAACACG-3 ' (as shown in SEQ ID No.24).

It screens to obtain the wild rice smut for knocking out wild rice brand spores and forming related gene UeItd1 by the above method (Ustilago esculenta) haploid strains UET1 △ Itd1 and wild rice smut (Ustilago esculenta) monoploid Bacterial strain UET2 △ Itd1.The regular-PCR of bacterial strain UET1 △ Itd1 and UET2 △ Itd1 verify andItd1The verifying of gene expression amount, As shown in Figure 3.

Wild rice smut (Ustilago esculenta) haploid strains UET1 △ Itd1 and wild rice smut (Ustilago esculenta) haploid strains UET2 △ Itd1 has the property that

Morphological feature: rod-short, for mature spore in 15-20 microns, monokaryon carries out budding without diaphragm, like saccharomycete, but It is bigger than saccharomycete.Cultural character: solid culture bacterium colony is in milk yellow, and surface is smooth, relatively wet, bigger thicker than bacterial clump, It is more sticky than bacterium when picking, similar yeast colony.Liquid medium such as bacterial solution.

Physiological metabolism characteristic: the carbon nitrogen source of the bacterial strain preference reducing sugar and organic nitrogen source as its growth.

Cultural method: can in vitro culture, be suitable on PDA or YEPS solid medium carry out single colonie scribing line culture, Culture is easily reduced bacterium activity in Liquid Culture.In addition the bacterium is not suitable for subculture more than three times, is not suitable for 7 days or more continuous Culture or low temperature are placed, and are otherwise caused activity reduction or thallus degradation, are suitable for spending 20% glycerol stocks -80.

And by wild rice smut (Ustilago esculenta) haploid strains UET1 △ Itd1 and wild rice smut (Ustilago esculenta) haploid strains UET2 △ Itd1 carries out preservation, preservation information is as follows:

Wild rice smut (Ustilago esculenta) haploid strains UET1 △ Itd1 on November 09th, 2018 China it is micro- The common micro-organisms center preservation of biological inoculum preservation administration committee, deposit number are CGMCC No.16723, depositary institution address Are as follows: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, it is proposed that name ustilago esculentaUstilago esculenta；Wild rice smut (Ustilago esculenta) haploid strains UET2 △ Itd1 on November 09th, 2018 in Chinese microorganism strain preservation Administration committee's common micro-organisms center preservation, deposit number are CGMCC No.16724, depositary institution address are as follows: Beijing's southern exposure The institute 3 of area North Star West Road 1, it is proposed that name ustilago esculentaUstilago esculenta。

Embodiment 3: make the Inoculation Method of the pregnant hay of wild rice stem plant

1) by wild rice smut (Ustilago esculenta) haploid strains UET1 △ Itd1 and wild rice smut (Ustilago esculenta) haploid strains UET2 △ Itd1 respectively in YEPS fluid nutrient medium 25-30 DEG C shake bacterium to OD₆₀₀For 0.8- 1.0, thalline were collected by centrifugation, is diluted to final concentration of OD with 0.5 × YEPS fluid nutrient medium₆₀₀For the bacterium solution of 2.0-3.0, by this Bacterium solution is mixed for connecing bacterium two-by-two；

2) by the wild hay tubulose root progress nursery with 3 or more complete internodes, hot-house culture 15-20 days, with sprouting have 3 with The tubulose root of upper small seedling stage plant is object of inoculation, and carries out pricking hole processing to seedling base portion；

3) mixed bacteria liquid for obtaining step 1) syringe syringe-like root, until having bacterium solution spilling；

4) by treated, inoculation seedling is trimmed, and prevents excessive transpiration from plant being caused here to wither, then leaching is placed in residue Mixed bacteria liquid in, 22-25 DEG C of greenhouse dark places 12-24 h；

5) the wild hay seedling after leaching bacterium is transferred to progress transition inoculation in the small basin with Nutrition Soil, it will mixing after soil is sufficiently humidified so as to Bacterium solution is poured into soil, 22-25 DEG C of 7 d of greenhouse dark culturing, completes inoculation；

6) seedling after inoculation is transplanted and carries out outdoor cultivation in outdoor or incubator, the control of inoculation planting time is in March, fertilising Standard is planted with reference to normal wild rice stem.

With Inoculation Method in embodiment 3, the pregnant normal wild rice stem out of artificial infection is realized for the first time, is inoculated within ten days after inoculation Shoot survival percent is 15 ± 4.1%, is 87 ± 14.1% based on the pregnant hay rate counted on the basis of survival rate.The inoculation method survival rate It is too low, it is not suitable for applying to field production, for the Inoculation Method for obtaining efficient stable, we have carried out inoculation system excellent Change.

Embodiment 4: the selection of inoculation position

By wild rice smut (Ustilago esculenta) haploid strains UET1 △ Itd1 and wild rice smut (Ustilago esculenta) haploid strains UET2 △ Itd1 respectively in YEPS fluid nutrient medium 25-30 DEG C shake bacterium to OD₆₀₀It is 2.0, with The dilution proportion of 1:100 shakes bacterium to OD for 25-30 DEG C into YEPS fluid nutrient medium₆₀₀For 0.8-1.0, thalline were collected by centrifugation, and use is pure Water is diluted to final concentration of OD₆₀₀For 2.0 bacterium solution, UET1 △ Itd1 and UET2 △ Itd1 bacterium solution is mixed in equal volume Combined bacteria liquid.

To pick up from the wild hay tubulose root in Wujiang as object of inoculation, according to the difference of inoculation position, be divided into miaoye, leaf sheath, Tubulose root；It is required that the leaf of selection 3-5 leaf phase open country hay seedling, is divided into two sections and is cleaned twice with ultrapure water, it is layered on equipped with wet filter In the 15 cm glass culture dish of diameter of paper, and both ends of the blade wound is covered with wet filter paper or cotton.Processing one: needle is used Point slightly causes scratch with syringe needle on blade, draws 6 μ l bacterium solutions and is covered on the intrusion that wound surface is convenient for pathogen；Processing Two: the filter paper or cotton covering that bacterium solution bedews in the wound of both ends of the blade impregnate；Processing three: bacterium solution is directly covered on leaf On piece；Control: cotton covers after the filter paper bedewed with both ends of the blade wound pure water.Sample is placed in 28 DEG C of trainings after infecting It supports in case, sampling observation copolymerization is burnt after being protected from light culture 3 days, and observation discovery cannot make the black powder of wild rice using the method to Infectikon Bacterium successfully infects wild rice stem blade.Selection has 3 leaf phase open country hay seedling of the complete tubulose root of one to two sections, and bacterium solution is injected inlet pipe Until bacterium solution overflows and 12 hr of processing are set in mixed bacteria liquid leaching in treatment site, sampling observation copolymerization is burnt after being inoculated with 3 days sends out shape root It is now invaded without wild rice smut mycelia, shows that wild rice smut cannot infect wild hay from tubulose root；Select the tri-leaf period open country hay children of single plant Seedling injects mixed bacteria liquid to seedling base portion, and 12 hr of processing is set in leaching in treatment site and mixed bacteria liquid, and sampling in 3 days is seen after inoculation The intrusion it can be observed that wild rice smut is examined, and along with great-hearted mycelia is enriched, shows to be inoculated with successfully.Same sight in October Examine, discovery only can make the pregnant ash discharge hay of wild hay to the method that seedling base portion be inoculated with, after the inoculation of remaining vaccination ways with compare nothing obviously Difference.Statistics discovery, the method survival rate of plant that basal part of stem is impregnated in injection only has 13.3 ± 2.2%, pregnant in the wild rice stem seedling survived Hay rate reaches 91.6 ± 7.8%.

Embodiment 5: the optimization to the method for basal part of stem processing

The single plant open country hay seedling of 3 leaf phases of same selection carries out the optimization processing of 3 kinds of inoculation methods respectively, processing one: uses Mixed Microbes Liquid injects seedling basal part of stem, until bacterium solution is overflowed from top leaf sheath；Processing two: seedling seedling base portion is soaked in mixed bacteria liquid Set 12 hr；Processing three: with mixed bacteria liquid live seedling basal part of stem until bacterium solution from top leaf sheath overflow, and by treatment site in 12 hr are set in leaching in mixed bacteria liquid；October is unified it has been observed that processing one, processing three can pregnant hay, processing two cannot pregnant hay, In addition, the inoculation method survival rate of plant (78.3 ± 6.2%) of processing one is higher than three (20 ± 4.08%) of processing, in the hay survived The pregnant hay rate of Bai Miaozhong processing one is 79.5 ± 9.6%, and the pregnant hay rate for handling three is 91.7 ± 11.8%.Thus we filter out note The method of launched field hay seedling basal part of stem can preferably realize high-survival rate and high pregnant hay rate.

Embodiment 6: optimization of the method based on injection basal part of stem to bacterial concentration

Using the acquisition methods of 4 mixed bacteria liquid of embodiment, OD is obtained₆₀₀Respectively 2.0,1.5,1.0,0.5,0 mixing Bacterium solution infects wild hay seedling using the method for the injection basal part of stem optimized in embodiment 4, and sampling in 3 days is observed after inoculation Laser confocal microscope discovery, uses OD₆₀₀It is that 2.0,1.5,1.0 mixed bacteria liquids are equal it is observed that invasive bacterium at the initial stage of infecting Silk, remaining bacterial concentration is not it is observed that infect mycelia, and October, unified observation was found: OD₆₀₀Be 2.0,1.5,1.0 it is mixed The wild hay seedling percent of combined bacteria liquid inoculation is similar, is respectively 79.4 ± 4.1%, 11.6 based on the pregnant hay rate on the basis of survival rate ± 4.5%, 0.Thus we, which can determine that out, injects OD₆₀₀It is really effective inoculation bacterial concentration for 2.0 mixed bacteria liquid.

Embodiment 7: based on using OD₆₀₀Wild hay seedling of the method to different seedling ages of basal part of stem is injected for 2.0 mixed bacteria liquid It optimizes

According to the difference of seedling age, 2 leaf phases, 3 leaf phases, 5 leaf phases, the seedling of difference leaf phase 8 leaf phases four, using embodiment 4 are selected The middle method for screening the optimization obtained carries out injection bacterium solution inoculation, three days slice confocal laser scanning microscope hairs after inoculation Existing, 2 leaf phases, 3 leaf phases, 5 leaf phases, 8 leaf phases had mycelia to be formed, but in the sample of 8 leaf phases, it is several that hyphae length is obviously weaker than remaining Ye Qi, unification in October hay it has been observed that the wild hay Miao Jun of all leaf phases is pregnant, survival rate is respectively 51.6 ± 4.7%, 85 ± 4.1%, 91.6 ± 2.3%, 96.6 ± 2.3%, show that the survival rate as seedling age is bigger, after inoculation is also higher, is based on survival rate On the basis of pregnant hay rate be respectively 72.05 ± 11.9%, 82.9 ± 12.2%, 90.8 ± 5.3%, 17.2 ± 4.7%.Thus it obtains The wild rice stem seedling of selection 3-5 leaf phase infect can preferable seedling age selection.

Embodiment 8: the application of the artificially breeding of normal wild rice stem

In conjunction with the method for the efficient artificial infection wild rice smut of optimization, by the wild rice smut monoploid UET1 △ to mutation Itd1 and UET2 △ Itd1 mixed bacteria liquid is inoculated with wild hay seedling.

1) by the wild rice smut haploid strains UET1 △ Itd1 and UET2 △ Itd1 25- in YEPS fluid nutrient medium respectively 30 DEG C are shaken bacterium to OD₆₀₀It is 2.0, bacterium is shaken to OD for 25-30 DEG C into YEPS fluid nutrient medium with the dilution proportion of 1:100₆₀₀For 0.8-1.0, thalline were collected by centrifugation, is diluted to final concentration of OD with pure water₆₀₀For 2.0 bacterium solution, by UET1 △ Itd1 and UET2 △ Itd1 bacterium solution is mixed for connecing bacterium in equal volume；

2) the wild hay tubulose root progress nursery of selection, greenhouse cultivation 20-30 days, and transplant to the small basin of 10 × 10cm, Nutrition Soil is light Micro- covering root, and continue to cultivate 7-10 days in the greenhouse of the same terms, select the wild hay seedling of 3-5 leaf phase as inoculation pair As；

3) seedling base portion is injected to the mixed bacteria liquid syringe that step 1) obtains, until bacterium solution is overflowed above leaf sheath；

4) by treated, inoculation seedling is trimmed, and prevents excessive transpiration from leading to here plant withers；

5) seedling after inoculation is continued to be placed on 22-25 DEG C of greenhouse transition culture, completes inoculation after two weeks, outdoor dew can be transplanted Its cultivation；

6) time of the outdoor outdoor cultivation of transplanting should control in the 3-4 month and 9-10 month, prevent outdoor temperature too high or too low It is unfavorable for the growth of bacterium, fertilising plants standard with reference to normal wild rice stem；

To October in autumn, it is inoculated with the pregnant hay of seedling success, pregnant hay rate reaches 87.3 ± 6.7%, and is normal wild rice stem, the pregnant hay of the wild rice stem Phenotypic map and wild rice stem histotomy confocal microscopy figure, as shown in Figure 4.

Sequence table

<110>China's metering university

<120>method of the normal wild rice stem of a kind of artificially breeding

<160> 24

<170> SIPOSequenceListing 1.0

<210> 1

<211> 2670

<212> DNA

<213>wild rice smut (Ustilago esculenta)

<400> 1

atggtttccc accaagtcgc cacgtttcag ggctatgtgt atgttgattc ggctcggcac 60

catccgccct tctactgtcc agctttgtgc tgattggctg acaacttgaa tattcgacct 120

tctctatcgt cttccgttgt ttctcacagc cactcgactc gagatgcttt gcttatcttt 180

gaagcggtac ggcgcggaca actgcccaag atcacacgac gactccgcga cgatgagcgc 240

aagatgatcc aaagcggaac catctttgtg tttgatgagg ctgaatccag catcaagaga 300

tggaccgacg gcatgatctg gagtccaagt cgaatcctga acaactttct agtctatcgt 360

gaagtggaga aaaaggatcc gaagcctgcg accgaccaac ctgccttcgc cacatcgcac 420

tctagcttcg cgatgcaggg cgcagctctc aacagcagcc atccctcgca gcattcgtct 480

gtccgctctg tgcccctcac gctgatgccc aatgctatgg acgcggatgc acaaatgacg 540

cactacaagc aagagcacag tggctaccat tcggtcgagg gcgtctcttc ctcttccaat 600

cctggctact cggaacacga tggctttttt ggcggccatg ctcatcacca tcattccgcg 660

cacgcacaag cgcatgcgca agcagccggg ctggtaggtc tgttggacga aggcgccgca 720

tcttcgtcgg cggtcaaacg cgaagtagag ctggatagga cgatcgttgg aagcttgaca 780

agcagctacc catttgtgcg tgatggactg tgcaagaaga ccatctcgat ccaggtggaa 840

ggctccacac agcacctcat ttcgtactac aagatcgacg atgtgcacca tggacgtctg 900

acgattccat ccaaccttcc cgagctcttc tgttttagca tctcgcccat cttcctcaac 960

aagagcaact ttcgctaccc ccccatcgtc gagatgggtc ccgacggctt gccgaggtac 1020

gtgggcgaat caaccgacaa cgccatgcgt gcctcggggc atgtcagtag cggcaacgag 1080

agctactcgt ctgggtccga ggcgaggcac gaagggcttg gaagagccgc tagtcggtcg 1140

ctgtccatct actcgccagc tcttccctcc gatcctacgc acaacgagtt ctacagtagc 1200

agatacccgc accagatcgg cggggatggt atacttccga actcggctaa tttaccctcg 1260

tcatcgacct cttcgttgcc gtaccggagc aggcgctcat ccgacgctcc acgcagacga 1320

gctaattcga ggtacgagcc ataccagcag gcacctggct ttggccatgg gatggtgccc 1380

actcaacttt actcgaatag cccggcgatg gatcatggcc ccgtctcgcc atcggggcgc 1440

cgagcctttt tcccacctcc gcgtggctac agcgacgcgg aggcagcaca cgcatcagaa 1500

catacatcgt ttggaatgca tcgtcaagat gctgggttct tcgcgatcgg acaggtagat 1560

cacgcaagcg gtcagaatgg cgggcatctg aaccagcccc acgcttcgat ggaccaggtt 1620

gatcagcacg gcgatctaca gcagtcgttt cagccagggc ccgtgattcc ttttgtgcct 1680

agtcagccta gccgcagtca cacgagctac tcgacagggg ccgacttgat gcacggcagg 1740

tacgacgctg ttgggatgaa gcaggaacag gcggatccat ttcacttttc atcgcggctt 1800

gcgagaggaa gctgggactc gaaccagccg agccatccga atgttgtgca ccacgtgcag 1860

caacctcctg cgacgagcca gggcttgacg gcgatatcga tccacagtca gtccaacttt 1920

ggtgggcccg tgtatggccg tctggttggg tcgcctccca acacgagctc gtcgcacgac 1980

agccgccact cttacatggg cagcggaccc acacacgcga cgggtcgcat cggagagttg 2040

gtggatggcg tgcttccgac gacagcggct cggctcgagg aagcaaacta ctcgtcgcgc 2100

cccatgtctc gtgctggaga aggaggatac gtcggcgatc tggactcggc aggtgtggta 2160

caaacagggc cgttggaggc gctgcaagtt gattctgacc caaccagccc ctatgcgagg 2220

tctcaccagc atccattgca tgcatcgcat gcgcagcatc aaatactggg gaatgacgct 2280

gccggagtcg acggctacgg tcggcctgcc accgagatgg atgcacagcc gcctttctat 2340

ccgccaccgc agacgtcgta tccgccacag gaacatgacg gcgtccagct ggaccagcaa 2400

gctttcggtc gtcatgctca agacggctat gcatcgacgc aggcctacgc gtcgaacgga 2460

atcgtgcctg gatcggcgca taacgagcaa tcctggtcgg atccggtcca agagggcaac 2520

ggcgctacca cggcggatca atcatacccg tcgcggccgg ggacatccca cgatcaggct 2580

taccaccacc agtaccgcaa cgaaggcggt gacgccaatg cagacgaatc agggctggaa 2640

aaggtcatgt tgacaagacc cgcggcgtga 2670

<210> 2

<211> 2559

<212> DNA

<213>wild rice smut (Ustilago esculenta)

<400> 2

atggtttccc accaagtcgc cacgtttcag ggctatgtcc actcgactcg agatgctttg 60

cttatctttg aagcggtacg gcgcggacaa ctgcccaaga tcacacgacg actccgcgac 120

gatgagcgca agatgatcca aagcggaacc atctttgtgt ttgatgaggc tgaatccagc 180

atcaagagat ggaccgacgg catgatctgg agtccaagtc gaatcctgaa caactttcta 240

gtctatcgtg aagtggagaa aaaggatccg aagcctgcga ccgaccaacc tgccttcgcc 300

acatcgcact ctagcttcgc gatgcagggc gcagctctca acagcagcca tccctcgcag 360

cattcgtctg tccgctctgt gcccctcacg ctgatgccca atgctatgga cgcggatgca 420

caaatgacgc actacaagca agagcacagt ggctaccatt cggtcgaggg cgtctcttcc 480

tcttccaatc ctggctactc ggaacacgat ggcttttttg gcggccatgc tcatcaccat 540

cattccgcgc acgcacaagc gcatgcgcaa gcagccgggc tggtaggtct gttggacgaa 600

ggcgccgcat cttcgtcggc ggtcaaacgc gaagtagagc tggataggac gatcgttgga 660

agcttgacaa gcagctaccc atttgtgcgt gatggactgt gcaagaagac catctcgatc 720

caggtggaag gctccacaca gcacctcatt tcgtactaca agatcgacga tgtgcaccat 780

ggacgtctga cgattccatc caaccttccc gagctcttct gttttagcat ctcgcccatc 840

ttcctcaaca agagcaactt tcgctacccc cccatcgtcg agatgggtcc cgacggcttg 900

ccgaggtacg tgggcgaatc aaccgacaac gccatgcgtg cctcggggca tgtcagtagc 960

ggcaacgaga gctactcgtc tgggtccgag gcgaggcacg aagggcttgg aagagccgct 1020

agtcggtcgc tgtccatcta ctcgccagct cttccctccg atcctacgca caacgagttc 1080

tacagtagca gatacccgca ccagatcggc ggggatggta tacttccgaa ctcggctaat 1140

ttaccctcgt catcgacctc ttcgttgccg taccggagca ggcgctcatc cgacgctcca 1200

cgcagacgag ctaattcgag gtacgagcca taccagcagg cacctggctt tggccatggg 1260

atggtgccca ctcaacttta ctcgaatagc ccggcgatgg atcatggccc cgtctcgcca 1320

tcggggcgcc gagccttttt cccacctccg cgtggctaca gcgacgcgga ggcagcacac 1380

gcatcagaac atacatcgtt tggaatgcat cgtcaagatg ctgggttctt cgcgatcgga 1440

caggtagatc acgcaagcgg tcagaatggc gggcatctga accagcccca cgcttcgatg 1500

gaccaggttg atcagcacgg cgatctacag cagtcgtttc agccagggcc cgtgattcct 1560

tttgtgccta gtcagcctag ccgcagtcac acgagctact cgacaggggc cgacttgatg 1620

cacggcaggt acgacgctgt tgggatgaag caggaacagg cggatccatt tcacttttca 1680

tcgcggcttg cgagaggaag ctgggactcg aaccagccga gccatccgaa tgttgtgcac 1740

cacgtgcagc aacctcctgc gacgagccag ggcttgacgg cgatatcgat ccacagtcag 1800

tccaactttg gtgggcccgt gtatggccgt ctggttgggt cgcctcccaa cacgagctcg 1860

tcgcacgaca gccgccactc ttacatgggc agcggaccca cacacgcgac gggtcgcatc 1920

ggagagttgg tggatggcgt gcttccgacg acagcggctc ggctcgagga agcaaactac 1980

tcgtcgcgcc ccatgtctcg tgctggagaa ggaggatacg tcggcgatct ggactcggca 2040

ggtgtggtac aaacagggcc gttggaggcg ctgcaagttg attctgaccc aaccagcccc 2100

tatgcgaggt ctcaccagca tccattgcat gcatcgcatg cgcagcatca aatactgggg 2160

aatgacgctg ccggagtcga cggctacggt cggcctgcca ccgagatgga tgcacagccg 2220

cctttctatc cgccaccgca gacgtcgtat ccgccacagg aacatgacgg cgtccagctg 2280

gaccagcaag ctttcggtcg tcatgctcaa gacggctatg catcgacgca ggcctacgcg 2340

tcgaacggaa tcgtgcctgg atcggcgcat aacgagcaat cctggtcgga tccggtccaa 2400

gagggcaacg gcgctaccac ggcggatcaa tcatacccgt cgcggccggg gacatcccac 2460

gatcaggctt accaccacca gtaccgcaac gaaggcggtg acgccaatgc agacgaatca 2520

gggctggaaa aggtcatgtt gacaagaccc gcggcgtga 2559

<210> 3

<211> 852

<212> PRT

<213>wild rice smut (Ustilago esculenta)

<400> 3

Met Val Ser His Gln Val Ala Thr Phe Gln Gly Tyr Val His Ser Thr

1 5 10 15

Arg Asp Ala Leu Leu Ile Phe Glu Ala Val Arg Arg Gly Gln Leu Pro

20 25 30

Lys Ile Thr Arg Arg Leu Arg Asp Asp Glu Arg Lys Met Ile Gln Ser

35 40 45

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

50 55 60

Thr Asp Gly Met Ile Trp Ser Pro Ser Arg Ile Leu Asn Asn Phe Leu

65 70 75 80

Val Tyr Arg Glu Val Glu Lys Lys Asp Pro Lys Pro Ala Thr Asp Gln

85 90 95

Pro Ala Phe Ala Thr Ser His Ser Ser Phe Ala Met Gln Gly Ala Ala

100 105 110

Leu Asn Ser Ser His Pro Ser Gln His Ser Ser Val Arg Ser Val Pro

115 120 125

Leu Thr Leu Met Pro Asn Ala Met Asp Ala Asp Ala Gln Met Thr His

130 135 140

Tyr Lys Gln Glu His Ser Gly Tyr His Ser Val Glu Gly Val Ser Ser

145 150 155 160

Ser Ser Asn Pro Gly Tyr Ser Glu His Asp Gly Phe Phe Gly Gly His

165 170 175

Ala His His His His Ser Ala His Ala Gln Ala His Ala Gln Ala Ala

180 185 190

Gly Leu Val Gly Leu Leu Asp Glu Gly Ala Ala Ser Ser Ser Ala Val

195 200 205

Lys Arg Glu Val Glu Leu Asp Arg Thr Ile Val Gly Ser Leu Thr Ser

210 215 220

Ser Tyr Pro Phe Val Arg Asp Gly Leu Cys Lys Lys Thr Ile Ser Ile

225 230 235 240

Gln Val Glu Gly Ser Thr Gln His Leu Ile Ser Tyr Tyr Lys Ile Asp

245 250 255

Asp Val His His Gly Arg Leu Thr Ile Pro Ser Asn Leu Pro Glu Leu

260 265 270

Phe Cys Phe Ser Ile Ser Pro Ile Phe Leu Asn Lys Ser Asn Phe Arg

275 280 285

Tyr Pro Pro Ile Val Glu Met Gly Pro Asp Gly Leu Pro Arg Tyr Val

290 295 300

Gly Glu Ser Thr Asp Asn Ala Met Arg Ala Ser Gly His Val Ser Ser

305 310 315 320

Gly Asn Glu Ser Tyr Ser Ser Gly Ser Glu Ala Arg His Glu Gly Leu

325 330 335

Gly Arg Ala Ala Ser Arg Ser Leu Ser Ile Tyr Ser Pro Ala Leu Pro

340 345 350

Ser Asp Pro Thr His Asn Glu Phe Tyr Ser Ser Arg Tyr Pro His Gln

355 360 365

Ile Gly Gly Asp Gly Ile Leu Pro Asn Ser Ala Asn Leu Pro Ser Ser

370 375 380

Ser Thr Ser Ser Leu Pro Tyr Arg Ser Arg Arg Ser Ser Asp Ala Pro

385 390 395 400

Arg Arg Arg Ala Asn Ser Arg Tyr Glu Pro Tyr Gln Gln Ala Pro Gly

405 410 415

Phe Gly His Gly Met Val Pro Thr Gln Leu Tyr Ser Asn Ser Pro Ala

420 425 430

Met Asp His Gly Pro Val Ser Pro Ser Gly Arg Arg Ala Phe Phe Pro

435 440 445

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

450 455 460

Thr Ser Phe Gly Met His Arg Gln Asp Ala Gly Phe Phe Ala Ile Gly

465 470 475 480

Gln Val Asp His Ala Ser Gly Gln Asn Gly Gly His Leu Asn Gln Pro

485 490 495

His Ala Ser Met Asp Gln Val Asp Gln His Gly Asp Leu Gln Gln Ser

500 505 510

Phe Gln Pro Gly Pro Val Ile Pro Phe Val Pro Ser Gln Pro Ser Arg

515 520 525

Ser His Thr Ser Tyr Ser Thr Gly Ala Asp Leu Met His Gly Arg Tyr

530 535 540

Asp Ala Val Gly Met Lys Gln Glu Gln Ala Asp Pro Phe His Phe Ser

545 550 555 560

Ser Arg Leu Ala Arg Gly Ser Trp Asp Ser Asn Gln Pro Ser His Pro

565 570 575

Asn Val Val His His Val Gln Gln Pro Pro Ala Thr Ser Gln Gly Leu

580 585 590

Thr Ala Ile Ser Ile His Ser Gln Ser Asn Phe Gly Gly Pro Val Tyr

595 600 605

Gly Arg Leu Val Gly Ser Pro Pro Asn Thr Ser Ser Ser His Asp Ser

610 615 620

Arg His Ser Tyr Met Gly Ser Gly Pro Thr His Ala Thr Gly Arg Ile

625 630 635 640

Gly Glu Leu Val Asp Gly Val Leu Pro Thr Thr Ala Ala Arg Leu Glu

645 650 655

Glu Ala Asn Tyr Ser Ser Arg Pro Met Ser Arg Ala Gly Glu Gly Gly

660 665 670

Tyr Val Gly Asp Leu Asp Ser Ala Gly Val Val Gln Thr Gly Pro Leu

675 680 685

Glu Ala Leu Gln Val Asp Ser Asp Pro Thr Ser Pro Tyr Ala Arg Ser

690 695 700

His Gln His Pro Leu His Ala Ser His Ala Gln His Gln Ile Leu Gly

705 710 715 720

Asn Asp Ala Ala Gly Val Asp Gly Tyr Gly Arg Pro Ala Thr Glu Met

725 730 735

Asp Ala Gln Pro Pro Phe Tyr Pro Pro Pro Gln Thr Ser Tyr Pro Pro

740 745 750

Gln Glu His Asp Gly Val Gln Leu Asp Gln Gln Ala Phe Gly Arg His

755 760 765

Ala Gln Asp Gly Tyr Ala Ser Thr Gln Ala Tyr Ala Ser Asn Gly Ile

770 775 780

Val Pro Gly Ser Ala His Asn Glu Gln Ser Trp Ser Asp Pro Val Gln

785 790 795 800

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

805 810 815

Gly Thr Ser His Asp Gln Ala Tyr His His Gln Tyr Arg Asn Glu Gly

820 825 830

Gly Asp Ala Asn Ala Asp Glu Ser Gly Leu Glu Lys Val Met Leu Thr

835 840 845

Arg Pro Ala Ala

850

<210> 4

<211> 1884

<212> DNA

<213>hygromycin gene (Hyg)

<400> 4

tggccgaacg tggtaactac cagcgagttc tgcaaacttc aaaaaaaaaa tctgggcacg 60

atgaaagttg agctaacgct gacgctcaca aatggcgtgg ctaaaggaag cgagacaatc 120

ggaaaattgt tctctcgggc accacaaagc tgttgttagt cgctgaagaa caattccaac 180

tgattccgcc gccttcctat tgcgtcagcc ttgtacctaa gctgccgagt aacgtcactc 240

aacctctctt ttcagactgc tttgctccgc gaatactttt cttctatgcg ctcaagaaaa 300

tgacacagca caccaagctc tgcaaacttt cttcgctaat ctgacgcgaa atgtgagcca 360

tttcttctcg cctgcaatgg caatgcgtct gtgcggcgat gagaatcacg atgcggaatg 420

ggtggctgga agttcataga gatgctgagt tgttggagcg acatggtaca taagcatgag 480

tctgtcctga tttccaccct cccgtctttc atcaactttc tcgtctgacc cttccgttgc 540

cagatcccgg ggggccatgg aaaagcctga actcaccgcg acgtctgtcg agaagtttct 600

gatcgaaaag ttcgacagcg tctccgacct gatgcagctc tcggagggcg aagaatctcg 660

tgctttcagc ttcgatgtag gagggcgtgg atatgtcctg cgggtaaata gctgcgccga 720

tggtttctac aaagatcgtt atgtttatcg gcactttgca tcggccgcgc tcccgattcc 780

ggaagtgctt gacattgggg aattcagcga gagcctgacc tattgcatct cccgccgtgc 840

acagggtgtc acgttgcaag acctgcctga aaccgaactg cccgctgttc tgcagccggt 900

cgcggaggcc atggatgcga tcgctgcggc cgatcttagc cagacgagcg ggttcggccc 960

attcggaccg caaggaatcg gtcaatacac tacatggcgt gatttcatat gcgcgattgc 1020

tgatccccat gtgtatcact ggcaaactgt gatggacgac accgtcagtg cgtccgtcgc 1080

gcaggctctc gatgagctga tgctttgggc cgaggactgc cccgaagtcc ggcacctcgt 1140

gcacgcggat ttcggctcca acaatgtcct gacggacaat ggccgcataa cagcggtcat 1200

tgactggagc gaggcgatgt tcggggattc ccaatacgag gtcgccaaca tcttcttctg 1260

gaggccgtgg ttggcttgta tggagcagca gacgcgctac ttcgagcgga ggcatccgga 1320

gcttgcagga tcgccgcggc tccgggcgta tatgctccgc attggtcttg accaactcta 1380

tcagagcttg gttgacggca atttcgatga tgcagcttgg gcgcagggtc gatgcgacgc 1440

aatcgtccga tccggagccg ggactgtcgg gcgtacacaa atcgcccgca gaagcgcggc 1500

cgtctggacc gatggctgtg tagaagtact cgccgatagt ggaaaccgac gccccagcac 1560

tcgtccgagg gcaaaggaat agagcggccg cccggctgca gatcgttcaa acatttggca 1620

ataaagtttc ttaagattga atcctgttgc cggtcttgcg atgattatca tataatttct 1680

gttgaattac gttaagcatg taataattaa catgtaatgc atgacgttat ttatgagatg 1740

ggtttttatg attagagtcc cgcaattata catttaatac gcgatagaaa acaaaatata 1800

gcgcgcaaac taggataaat tatcgcgcgc ggtgtcatct atgttactag atccgatgat 1860

aagctgtcaa acatgaggcc tgag 1884

<210> 5

<211> 20

<212> DNA

<213>primer (primer)

<400> 5

atggtttccc accaagtcgc 20

<210> 6

<211> 21

<212> DNA

<213>primer (primer)

<400> 6

tcacgccgcg ggtcttgtca a 21

<210> 7

<211> 20

<212> DNA

<213>primer (primer)

<400> 7

ggtgcacgtc atttgcccac 20

<210> 8

<211> 20

<212> DNA

<213>primer (primer)

<400> 8

gtcgagtggc tgtgagaaac 20

<210> 9

<211> 20

<212> DNA

<213>primer (primer)

<400> 9

tgcagacgaa tcagggctgg 20

<210> 10

<211> 20

<212> DNA

<213>primer (primer)

<400> 10

acgtttgcgg acccaaccat 20

<210> 11

<211> 21

<212> DNA

<213>primer (primer)

<400> 11

atggtttccc accaagtcgc c 21

<210> 12

<211> 20

<212> DNA

<213>primer (primer)

<400> 12

tcacgccgcg ggtcttgtca 20

<210> 13

<211> 19

<212> DNA

<213>primer (primer)

<400> 13

cgcgttctcg actcgattg 19

<210> 14

<211> 41

<212> DNA

<213>primer (primer)

<400> 14

gtagttacca cgttcggcca atctagtgaa tgcggcgaga g 41

<210> 15

<211> 39

<212> DNA

<213>primer (primer)

<400> 15

tgtcaaacat gaggcctgag ttgaagattg cgtggctcg 39

<210> 16

<211> 19

<212> DNA

<213>primer (primer)

<400> 16

ttgcggaccc aaccatttc 19

<210> 17

<211> 20

<212> DNA

<213>primer (primer)

<400> 17

ggatgcctcc gctcgaagta 20

<210> 18

<211> 20

<212> DNA

<213>primer (primer)

<400> 18

cgttgcaaga cctgcctgaa 20

<210> 19

<211> 20

<212> DNA

<213>primer (primer)

<400> 19

accgacggca tgatctggag 20

<210> 20

<211> 20

<212> DNA

<213>primer (primer)

<400> 20

cgttgccgct actgacatgc 20

<210> 21

<211> 20

<212> DNA

<213>primer (primer)

<400> 21

tcgttatgtt tatcggcact 20

<210> 22

<211> 20

<212> DNA

<213>primer (primer)

<400> 22

tcggcgagta cttctacaca 20

<210> 23

<211> 20

<212> DNA

<213>primer (primer)

<400> 23

tctagcactt gcacagcgtc 20

<210> 24

<211> 20

<212> DNA

<213>primer (primer)

<400> 24

tgcgacgagt tgacaacacg 20

Claims (5)

1. a kind of method of the normal wild rice stem of artificially breeding, it is characterised in that the following steps are included:

1) by wild rice smut (Ustilago esculenta) haploid strains UET1 △ Itd1 and wild rice smut (Ustilago esculenta) haploid strains UET2 △ Itd1 respectively 25-30 DEG C in YEPS fluid nutrient medium under the conditions of shake bacterium to OD₆₀₀For 2.0, then bacterium is shaken to OD under the conditions of 25-30 DEG C into YEPS fluid nutrient medium with the dilution proportion of 1:100₆₀₀For 0.8-1.0, from The heart collects thallus, is diluted to final concentration of OD with pure water₆₀₀For 2.0 bacterium solution, by wild rice smut haploid strains UET1 △ Itd1 It is mixed for connecing bacterium in equal volume with wild rice smut haploid strains bacterium solution, the wild rice smut (Ustilago esculenta) Haploid strains UET1 △ Itd1 deposit number be CGMCC No.16723, the wild rice smut (Ustilago esculenta) Haploid strains UET2 △ Itd1 deposit number is CGMCC No.16724；

2) the wild hay tubulose root progress nursery of selection, greenhouse cultivation 20-30 days, and transplant to small basin, Nutrition Soil slightly covers root, And continue to cultivate 7-10 days in the greenhouse of the same terms, select the wild hay seedling of 3-5 leaf phase as object of inoculation；

3) seedling base portion is injected to the mixed bacteria liquid syringe that step 1) obtains, until bacterium solution is overflowed above leaf sheath；

4) by treated, inoculation seedling is trimmed, and placing excessive transpiration leads to here plant withers；

5) seedling after inoculation is continued to be placed on 22-25 DEG C of greenhouse transition culture, completes inoculation after two weeks, outdoor dew can be transplanted Its cultivation；

6) time of the outdoor outdoor cultivation of transplanting should control in the 3-4 month and 9-10 month, prevent outdoor temperature too high or too low It is unfavorable for the growth of bacterium, fertilising plants standard with reference to normal wild rice stem.

2. a kind of method of the normal wild rice stem of artificially breeding as described in claim 1, it is characterised in that in the step 1) YEPS fluid nutrient medium contains following components in percentage by weight: peptone 2%, sucrose 2%, yeast powder 1% and water surplus.

3. a kind of method of the normal wild rice stem of artificially breeding as described in claim 1, it is characterised in that the step 2) medium temperature For the control of room breeding condition temperature at 22-25 DEG C, light application time is 8-12 hours.

4. a kind of method of the normal wild rice stem of artificially breeding as described in claim 1, it is characterised in that the step 2) is medium and small Basin size is 10 × 10cm.

5. a kind of method of the normal wild rice stem of artificially breeding as described in claim 1, it is characterised in that note in the step 3) Penetrate the above position of seedling base portion seedling apical point.