CN110564728A - Application of osa-miR529b and precursor gene thereof in improving bacterial blight resistance of rice - Google Patents
Application of osa-miR529b and precursor gene thereof in improving bacterial blight resistance of rice Download PDFInfo
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Abstract
The invention discloses application of osa-miR529b obtained from rice and a precursor gene thereof in improving rice bacterial leaf blight resistance. The invention claims a miRNA (osa-miR529b) as shown in sequence 1 of a sequence table. The invention also protects the precursor gene of the miRNA. The invention also provides a method for preparing a transgenic plant, which comprises the following steps: and (3) over-expressing the miRNA in a plant to obtain a transgenic plant with improved disease resistance. The invention also provides a method for preparing a transgenic plant, which comprises the following steps: the precursor gene is introduced into a plant to obtain a transgenic plant with improved disease resistance. The invention can be used for improving the resistance of rice to bacterial blight, has important significance for cultivating new varieties of bacterial blight resistant rice, and is suitable for popularization and application.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to application of osa-miR529b obtained from rice and a precursor gene thereof in improving bacterial blight resistance of rice.
Background
Rice is an important grain crop in the world, and bacterial blight of rice caused by xanthomonas oryzae rice pathogenic varieties is a main bacterial disease in rice production and is particularly serious in Asian regions. In the southern rice area of China, the rice bacterial leaf blight disease has wide disease area, high morbidity and quick infection, and the production safety of grains in China is severely restricted. The cultivation and planting of disease-resistant varieties are currently economical and effective measures for preventing and treating the bacterial blight of rice. To date, 44 rice genes for resisting bacterial blight have been officially reported, and only genes such as Xa3, Xa4, Xa21 and Xa23 are widely applied in breeding. As the xanthomonas oryzae rice pathopoiesia varieties have high variation speed and complex diversity, production practices show that after the varieties carrying single disease-resistant genes are popularized and planted in a large area, dominant toxic microspecies are increased or pathogenic bacteria are mutated into new toxic microspecies, and the resistance of the varieties is easy to lose.
microRNA (miRNA) is a non-coding single-stranded RNA molecule which is coded by endogenous genes and has the length of about 20-24nt, and plays a key role in the processes of plant growth and development, cell metabolism, signal transduction, biological stress, abiotic stress and the like.
Compared with the disease-resistant gene, the miRNA can timely and accurately regulate the expression of the disease-resistant related gene, start disease-resistant reaction, enhance the disease resistance of the plant and avoid adverse effects caused by the continuous expression of the disease-resistant gene in the plant body. Therefore, the miRNA related to rice disease resistance is identified, and the application value in rice disease resistance breeding is important.
Disclosure of Invention
The invention aims to provide application of osa-miR529b obtained from rice and a precursor gene thereof in improving bacterial blight resistance of rice.
The invention claims a miRNA (osa-miR529b) as shown in sequence 1 of a sequence table.
The invention also protects the precursor gene of the miRNA. Specifically, the invention protects the precursor gene of the miRNA in rice. The precursor gene can be a DNA molecule shown in a sequence 2 of a sequence table.
The invention also protects the application of the miRNA in regulating and controlling the disease resistance of plants. The modulation is positive modulation. The positive regulation means that: miRNA level is increased, and disease resistance is improved. The plant may be a dicot or a monocot. The plant may be a graminaceous plant. The plant may be a plant of the genus oryza. The plant may be rice, for example, Nipponbare. The disease resistance may be resistance to bacterial blight. The disease resistance may be a disease resistance to bacterial blight of rice.
The invention also protects the application of the precursor gene in preparing transgenic plants with improved disease resistance. The plant may be a dicot or a monocot. The plant may be a graminaceous plant. The plant may be a plant of the genus oryza. The plant may be rice, for example, Nipponbare. The disease resistance may be resistance to bacterial blight. The disease resistance may be a disease resistance to bacterial blight of rice.
The invention also provides a method for preparing a transgenic plant, which comprises the following steps: and (3) over-expressing the miRNA in a plant to obtain a transgenic plant with improved disease resistance. The plant may be a dicot or a monocot. The plant may be a graminaceous plant. The plant may be a plant of the genus oryza. The plant may be rice, for example, Nipponbare. The disease resistance may be resistance to bacterial blight. The disease resistance may be a disease resistance to bacterial blight of rice.
The invention also provides a method for preparing a transgenic plant, which comprises the following steps: the precursor gene is introduced into a plant to obtain a transgenic plant with improved disease resistance. The plant may be a dicot or a monocot. The plant may be a graminaceous plant. The plant may be a plant of the genus oryza. The plant may be rice, for example, Nipponbare. The disease resistance may be resistance to bacterial blight. The disease resistance may be a disease resistance to bacterial blight of rice.
The invention also provides a method for preparing a transgenic plant, which comprises the following steps: the precursor gene is introduced into a plant and expressed to obtain a transgenic plant with improved disease resistance. The plant may be a dicot or a monocot. The plant may be a graminaceous plant. The plant may be a plant of the genus oryza. The plant may be rice, for example, Nipponbare. The disease resistance may be resistance to bacterial blight. The disease resistance may be a disease resistance to bacterial blight of rice.
The invention also protects the application of any one of the methods in plant breeding. The plant may be a dicot or a monocot. The plant may be a graminaceous plant. The plant may be a plant of the genus oryza. The plant may be rice, for example, Nipponbare. The breeding aims to obtain plants with improved disease resistance. The disease resistance may be resistance to bacterial blight. The disease resistance may be a disease resistance to bacterial blight of rice.
Any one of the above bacterial blight diseases of rice may be specifically caused by a rice pathogenic variety of Xanthomonas oryzae.
Any one of the bacterial leaf blight of rice can be specifically caused by rice bacterial leaf blight PXO 99. The rice bacterial leaf blight PXO99 is a Philippine virulent strain.
The invention discloses a miRNA, namely osa-miR529b, and provides a mature sequence and a precursor gene sequence thereof. osa-miR529b has a function of positively regulating disease resistance of rice. The overexpression of the osa-miR529b precursor gene can obviously improve the disease resistance of rice to bacterial blight. The invention can be used for improving the resistance of rice to bacterial blight, has important significance for cultivating new varieties of bacterial blight resistant rice, and is suitable for popularization and application.
Drawings
FIG. 1 shows the relative expression amounts of osa-miR529b precursor genes in different materials after inoculation of Rhizoctonia solani PXO99 in example 1.
FIG. 2 is a partial result of the PCR identification in example 2; m: DL2000 DNA marker; 1: a positive control; 2: negative control; 3: OE-8 plants; 4: OE-11 plants; 5: OE-22 plants.
FIG. 3 shows the results of measuring the relative expression level of the osa-miR529b precursor gene in example 2.
FIG. 4 shows the results of measuring the length of lesion in example 2.
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.
The rice variety Nip, named Nipponbare for short, belongs to the common cultivated japonica rice variety. The rice bacterial leaf blight PXO99 is a Philippine virulent strain. Nipponbare showed moderate level of susceptibility to rice bacterial blight PXO 99.
pCUbi1390 vector: biovector NTCC plasmid vector bacterial cell Gene Collection.
Agrobacterium tumefaciens EHA 105: biovector NTCC type culture Collection.
miRcute enhanced miRNA cDNA first strand synthesis kit: tiangen Biochemical technology (Beijing) Ltd, Cat # KR 211. Website address: https:// www.docin.com/p-2044311570. html. The miRcute enhanced miRNA fluorescent quantitative detection kit comprises: tiangen Biochemical technology (Beijing) Ltd, Cat number FP 411; website address: http:// www.docin.com/p-2042264874. html.
Example 1 obtaining of osa-miR529b
Putting rice seeds (a disease-resistant introduction line FF329 and recurrent parents HHZ) in a seed soaking bag, accelerating germination for 2 days at 37 ℃, then transferring the rice seeds into a seedling raising tray, transplanting the rice seeds into a field after 2 weeks, and planting 10 rows of rice seeds in each row of 8 rice seeds. And (3) when the tillering stage is full, performing rice leaf blight strain PXO99 infection on the plants, inoculating 5-6 pieces of the plants by a leaf cutting method, respectively sampling 0h, 12h, 24h and 36h after inoculation, and taking 2 times of repetition at each time point to obtain 16 samples in total. The rice inoculated portion and about 1cm of its downward extension should be taken at the time of collecting the sample, immediately after sampling, frozen in liquid nitrogen, and stored at-80 ℃.
The 16 samples were extracted using a total plant RNA extraction kit (DP432) from TIANGEN, Inc., and the extracted RNAs were subjected to quality control and small molecule RNA library construction. The concrete library building process comprises the following steps: adopting a qualified sample, taking 1.5 mu g of RNA as a sample starting amount, utilizing T4 RNA Ligase1 and T4 RNA Ligase2 to connect a linker at the 3 'end and the 5' end of small RNA respectively, carrying out reverse transcription to synthesize cDNA, carrying out PCR amplification, adopting a gel separation technology to screen a target fragment, cutting gel and recycling the obtained fragment to obtain a small RNA library. Sequencing is carried out by using an Illumina Hiseq 2000 platform, and the data volume of each sample is about 10M.
And (3) filtering and screening the original data obtained by sequencing to obtain the unannotated Reads containing miRNA, and using miRDeep2 software to carry out sequence alignment on the unannotated Reads and the rice reference genome (MSU _ v7.0), wherein the Reads on the alignment is the Mapped Reads. Then comparing the Mapped Reads with a miRBase database to obtain known miRNA; predicting the secondary structure of the new miRNA by using the known miRNA in the miRBase database and Randfold software, and further predicting the new miRNA. Differential expression screening of miRNA among influenza materials is carried out by using DESeq2 in R toolkit, the screening standard is FoldChange >2 or Fold Change <1/2 and FDR <0.05, and some miRNA which are possibly involved in bacterial blight resistance are obtained. Wherein one of the identified miRNAs is osa-miR529 b.
Mature sequence of osa-miR529b (sequence 1 in sequence Listing): AGAAGAGAGAGAGUACAGCUU are provided.
In Nipponbare of rice, the precursor gene sequence of osa-miR529b (sequence 2 in the sequence table): ATCGTGAAGGCTAAATGGAGAAGAGAGAGAGTACAGCTTTGGGCGGTTTAGTTGTTAGATGGTGGAAGTACTTGCGACGCCTACTACCCTTTTCACCAAGGCTGTACGCTCCCTCTTCTTCTCTTAGCTTTTTATGAT are provided.
Based on the results of the differential expression screening (see table 1), it was found that: the osa-miR529b of the disease-resistant material FF329 and the disease-susceptible material HHZ hardly expresses or has extremely low expression level at two time points of 0h and 12h after PXO99 inoculation; at two time points of 24h and 36h after PXO99 inoculation, the expression quantity of osa-miR529b of the disease-resistant material FF329 is about 2-3 times of that of the disease-sensitive material HHZ, and it is preliminarily presumed that osa-miR529b can positively control the bacterial leaf blight resistance of rice.
TABLE 1 expression levels of osa-miR529b between different materials at different time points after inoculation of PXO99
| #ID | H-0 | F-0 | H-12 | F-12 | H-24 | F-24 | H-36 | F-36 |
| osa-miR529b | 0.00 | 0.00 | 0.00 | 0.48 | 33.00 | 63.55 | 38.15 | 86.19 |
note: h-0 means 0H after the susceptible material HHZ is inoculated with PXO 99; f-0 means 0h after the disease-resistant material FF329 is inoculated with PXO99, and the rest samples are analogized in turn.
In order to verify the reliability of the sequencing data, the expression level of osa-miR529b in the 16 samples is detected by a fluorescent quantitative PCR method.
(1) And synthesizing the cDNA of the mature miRNA by adopting a MIRcute enhanced miRNA cDNA first strand synthesis kit. Reverse transcription system: total RNA 2. mu.g, 2 × miRNA RT Reaction Buffer 10. mu.l, miRNA RT Enzyme Mix 2. mu.l, RNase-Free ddH2O was supplemented to 20. mu.l. Reverse transcription program: 60min at 42 ℃ and 3min at 95 ℃. After the reaction is finishedDiluting by 10 times, and storing at-20 deg.C for use.
(2) Designing a forward specific primer of the fluorescence quantitative PCR of osa-miR529b, taking a downstream primer as a Kit general primer, taking U6 as an internal reference gene, and carrying out qPCR detection by using a SYBR Green method of miRcute Plus miRNA qPCR Kit. Reaction system: 2 × miRcute Plus miRNA PreMix (SYBR)&ROX) 10. mu.l, osa-miR529b-qF or U6-qF (10. mu.M) 0.4. mu.l, Reverse Primer (10. mu.M) 0.4. mu.l, miRNA first strand cDNA 2. mu.l, plus dd ddH2O was supplemented to 20. mu.l. Reaction procedure: 5min at 95 ℃; 94 ℃ for 20s, 60 ℃ for 34s,40 cycles; and (4) analyzing a dissolution curve.
The primers used for fluorescent quantitative PCR were as follows:
U6-qF：CGATAAAATTGGAACGATACAGA；
osa-miR529b-qF：GCCGAGAGAAGAGAGAGAGTACAGCTT。
(3) By using 2-ΔΔCtAnd calculating the expression level of osa-miR529b, and comparing and analyzing the expression levels of the disease-resistant material FF329 and the disease-susceptible material HHZ. The results are shown in figure 1, in the disease-resistant material FF329, the expression amount of osa-miR529b is far higher than that of the disease-sensitive material HHZ at 0h, 12h, 24h and 36h after PXO99 inoculation, and is about 2-10 times that of HHZ.
Compared to the sequencing data in table 1, the fold difference exhibited by fluorescent quantitative PCR was slightly less than the sequencing data, but the expression trends were consistent. In general, after PXO99 is inoculated, the expression level of osa-miR529b in the disease-resistant material FF329 is higher than that in the disease-susceptible material HHZ, and the bacterial leaf blight resistance of rice can be positively regulated.
Example 2 obtaining and identification of transgenic plants
Construction of osa-miR529b precursor gene overexpression vector
Prepared from pCUbi1390 vector "TGCAGGTCGACThe recombinant plasmid pUbi is obtained by replacing double-stranded DNA molecules shown in a sequence 2 in a sequence table, and premiR529b is obtained. The pCUbi1390 vector has been sequenced as shown in sequence 3 of the sequence Listing. The recombinant plasmid pUbi shows that premiR529b has been subjected to sequencing verification and is shown as a sequence 4 in a sequence table.
The double-stranded DNA molecule shown in the sequence 2 of the sequence table is obtained by PCR amplification from Nipponbare genomic DNA.
Second, obtaining transgenic rice plant
The recombinant plasmid pUbi is introduced into premiR529b to obtain the recombinant agrobacterium tumefaciens EHA 105. Carrying out genetic transformation on the embryogenic callus of Nipponbare by adopting an agrobacterium infection method, carrying out resistance screening by adopting 50mg/L hygromycin, and then carrying out regeneration culture to obtain a regeneration plant (T0 generation plant).
Transgenic plants were selected from regenerated plants by PCR identification. The specific method for PCR identification comprises the following steps: extracting genome DNA of plant leaves, carrying out PCR amplification by using a primer pair consisting of pCUbi1390-VF and miR529b-JR, and then carrying out 1% agarose gel electrophoresis, wherein if a specific amplification product (183bp) exists, the plant is a transgenic plant (also called an overexpression plant). The recombinant plasmid pUbi:: premiR529b was used as a positive control. Genomic DNA from Nipponbare was used as a negative control. The electrophoretogram of the plant parts is shown in FIG. 2.
pCUbi1390-VF：5’-TCGATGCTCACCCTGTTGTT-3’；
miR529b-JR：5’-ATCATAAAAAGCTAAGAGAAGAAGA-3’。
T0 transgenic plants were selfed and seeds were harvested to give T1 seeds. And (4) breeding the T1 generation seeds into plants, namely T1 generation plants. T1 plant was selfed and seeds were harvested to give T2 seeds. And (4) breeding the T2 generation seeds into plants, namely T2 generation plants. The plants were identified by PCR (as above).
For a certain T0 generation transgenic plant, if the T2 generation plants obtained by selfing are all transgenic plants, the T0 generation plant is homozygous transgenic plant, and the selfed T0 generation plant is a homozygous transgenic line.
Randomly taking 3 homozygous transgenic strains (OE-8 strain, OE-11 strain and OE-22 strain), and performing identification in the fourth step and the fifth step.
Third, obtaining empty carrier rice plant
Replacing the recombinant plasmid pUbi with the pCUbi1390 vector, and operating according to the step two to obtain a transgenic empty vector plant, wherein the recombinant plasmid pUbi is premiR529 b.
expression quantity analysis of osa-miR529b precursor gene overexpression strain
The test plants were: t2-generation plants of the OE-8 strain, T2-generation plants of the OE-11 strain, T2-generation plants of the OE-22 strain, Nipponbare plants (wild type control).
Taking leaves of a test plant, providing total RNA, carrying out reverse transcription by adopting a miRcute enhanced miRNA cDNA first strand synthesis kit to obtain cDNA, and carrying out fluorescence quantitative PCR by adopting a miRcute enhanced miRNA fluorescence quantitative detection kit to detect the expression quantity of osa-miR529b precursor gene. Fluorescent quantitative PCR used the U6 gene as an internal reference gene. And adding an upstream primer aiming at the target gene during fluorescent quantitative PCR.
The upstream primers used to detect the U6 gene were as follows:
U6-qF：CGATAAAATTGGAACGATACAGA。
The upstream primers used for detecting the osa-miR529b gene were as follows:
osa-miR529b-qF：GCCGAGAGAAGAGAGAGAGTACAGCTT。
The results of relative expression of the osa-miR529b precursor gene are shown in FIG. 3. Compared with Nipponbare, the relative expression quantity of osa-miR529b precursor genes in the three transgenic strains (OE-8 strain, OE-11 strain and OE-22 strain) is obviously up-regulated and is 23-33 times of that of Nipponbare.
Fifth, identification of resistance to bacterial blight of transgenic line
The test plants were: t2-generation plants of the OE-8 strain, T2-generation plants of the OE-11 strain, T2-generation plants of the OE-22 strain, Nipponbare plants (wild type control), and T2-generation plants of the empty vector transfer strain.
1. The test plants were cultivated in a greenhouse for about 25 days and then transplanted into a net room and planted individually.
2. When the plant grows to the full tillering stage, inoculating the rice bacterial leaf blight strain PXO99 by a leaf cutting method (namely, the top end of the leaf is cut by 1cm after the rice bacterial leaf blight strain PXO99 bacterial liquid is dipped by scissors, and the bacterial concentration of the rice bacterial leaf blight strain PXO99 bacterial liquid is 1 multiplied by 109cfu/mL), 5-6 flag leaves were inoculated per plant.
3. After 3 weeks of completion of step 2, lesion length was measured for each inoculated leaf.
The lesion length of 20 plants was measured per line and averaged, and the results are shown in FIG. 4.
The average lesion length of Nipponbare is 10.2cm, and the average lesion lengths of OE-8 strain, OE-11 strain and OE-22 strain are respectively 6.6cm, 5.7cm and 7.3cm, which are all obviously smaller than the lesion length of wild type control. The length of the lesion of the empty vector plant is not obviously different from that of Nipponbare. Results show that the overexpression of the osa-miR529b precursor gene can obviously improve the disease resistance of rice to bacterial blight.
the results show that the osa-miR529b precursor gene positively regulates the resistance of the rice to bacterial blight.
SEQUENCE LISTING
<110> institute of crop science of Chinese academy of agricultural sciences
<120> osa-miR529b and application of precursor gene thereof in improving bacterial blight resistance of rice
<130> GNCYX192055
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 21
<212> RNA
<213> Oryza sativa
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agaagagaga gaguacagcu u 21
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<213> Oryza sativa
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atcgtgaagg ctaaatggag aagagagaga gtacagcttt gggcggttta gttgttagat 60
ggtggaagta cttgcgacgc ctactaccct tttcaccaag gctgtacgct ccctcttctt 120
ctcttagctt tttatgat 138
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<213> Artificial Sequence
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ctagccacca ccaccaccac cacgtgtgaa ttacaggtga ccagctcgaa tttccccgat 60
cgttcaaaca tttggcaata aagtttctta agattgaatc ctgttgccgg tcttgcgatg 120
attatcatat aatttctgtt gaattacgtt aagcatgtaa taattaacat gtaatgcatg 180
acgttattta tgagatgggt ttttatgatt agagtcccgc aattatacat ttaatacgcg 240
atagaaaaca aaatatagcg cgcaaactag gataaattat cgcgcgcggt gtcatctatg 300
ttactagatc gggaattaaa ctatcagtgt ttgacaggat atattggcgg gtaaacctaa 360
gagaaaagag cgtttattag aataacggat atttaaaagg gcgtgaaaag gtttatccgt 420
tcgtccattt gtatgtgcat gccaaccaca gggttcccct cgggatcaaa gtactttgat 480
ccaacccctc cgctgctata gtgcagtcgg cttctgacgt tcagtgcagc cgtcttctga 540
aaacgacatg tcgcacaagt cctaagttac gcgacaggct gccgccctgc ccttttcctg 600
gcgttttctt gtcgcgtgtt ttagtcgcat aaagtagaat acttgcgact agaaccggag 660
acattacgcc atgaacaaga gcgccgccgc tggcctgctg ggctatgccc gcgtcagcac 720
cgacgaccag gacttgacca accaacgggc cgaactgcac gcggccggct gcaccaagct 780
gttttccgag aagatcaccg gcaccaggcg cgaccgcccg gagctggcca ggatgcttga 840
ccacctacgc cctggcgacg ttgtgacagt gaccaggcta gaccgcctgg cccgcagcac 900
ccgcgaccta ctggacattg ccgagcgcat ccaggaggcc ggcgcgggcc tgcgtagcct 960
ggcagagccg tgggccgaca ccaccacgcc ggccggccgc atggtgttga ccgtgttcgc 1020
cggcattgcc gagttcgagc gttccctaat catcgaccgc acccggagcg ggcgcgaggc 1080
cgccaaggcc cgaggcgtga agtttggccc ccgccctacc ctcaccccgg cacagatcgc 1140
gcacgcccgc gagctgatcg accaggaagg ccgcaccgtg aaagaggcgg ctgcactgct 1200
tggcgtgcat cgctcgaccc tgtaccgcgc acttgagcgc agcgaggaag tgacgcccac 1260
cgaggccagg cggcgcggtg ccttccgtga ggacgcattg accgaggccg acgccctggc 1320
ggccgccgag aatgaacgcc aagaggaaca agcatgaaac cgcaccagga cggccaggac 1380
gaaccgtttt tcattaccga agagatcgag gcggagatga tcgcggccgg gtacgtgttc 1440
gagccgcccg cgcacgtctc aaccgtgcgg ctgcatgaaa tcctggccgg tttgtctgat 1500
gccaagctgg cggcctggcc ggccagcttg gccgctgaag aaaccgagcg ccgccgtcta 1560
aaaaggtgat gtgtatttga gtaaaacagc ttgcgtcatg cggtcgctgc gtatatgatg 1620
cgatgagtaa ataaacaaat acgcaagggg aacgcatgaa ggttatcgct gtacttaacc 1680
agaaaggcgg gtcaggcaag acgaccatcg caacccatct agcccgcgcc ctgcaactcg 1740
ccggggccga tgttctgtta gtcgattccg atccccaggg cagtgcccgc gattgggcgg 1800
ccgtgcggga agatcaaccg ctaaccgttg tcggcatcga ccgcccgacg attgaccgcg 1860
acgtgaaggc catcggccgg cgcgacttcg tagtgatcga cggagcgccc caggcggcgg 1920
acttggctgt gtccgcgatc aaggcagccg acttcgtgct gattccggtg cagccaagcc 1980
cttacgacat atgggccacc gccgacctgg tggagctggt taagcagcgc attgaggtca 2040
cggatggaag gctacaagcg gcctttgtcg tgtcgcgggc gatcaaaggc acgcgcatcg 2100
gcggtgaggt tgccgaggcg ctggccgggt acgagctgcc cattcttgag tcccgtatca 2160
cgcagcgcgt gagctaccca ggcactgccg ccgccggcac aaccgttctt gaatcagaac 2220
ccgagggcga cgctgcccgc gaggtccagg cgctggccgc tgaaattaaa tcaaaactca 2280
tttgagttaa tgaggtaaag agaaaatgag caaaagcaca aacacgctaa gtgccggccg 2340
tccgagcgca cgcagcagca aggctgcaac gttggccagc ctggcagaca cgccagccat 2400
gaagcgggtc aactttcagt tgccggcgga ggatcacacc aagctgaaga tgtacgcggt 2460
acgccaaggc aagaccatta ccgagctgct atctgaatac atcgcgcagc taccagagta 2520
aatgagcaaa tgaataaatg agtagatgaa ttttagcggc taaaggaggc ggcatggaaa 2580
atcaagaaca accaggcacc gacgccgtgg aatgccccat gtgtggagga acgggcggtt 2640
ggccaggcgt aagcggctgg gttgtctgcc ggccctgcaa tggcactgga acccccaagc 2700
ccgaggaatc ggcgtgacgg tcgcaaacca tccggcccgg tacaaatcgg cgcggcgctg 2760
ggtgatgacc tggtggagaa gttgaaggcc gcgcaggccg cccagcggca acgcatcgag 2820
gcagaagcac gccccggtga atcgtggcaa gcggccgctg atcgaatccg caaagaatcc 2880
cggcaaccgc cggcagccgg tgcgccgtcg attaggaagc cgcccaaggg cgacgagcaa 2940
ccagattttt tcgttccgat gctctatgac gtgggcaccc gcgatagtcg cagcatcatg 3000
gacgtggccg ttttccgtct gtcgaagcgt gaccgacgag ctggcgaggt gatccgctac 3060
gagcttccag acgggcacgt agaggtttcc gcagggccgg ccggcatggc cagtgtgtgg 3120
gattacgacc tggtactgat ggcggtttcc catctaaccg aatccatgaa ccgataccgg 3180
gaagggaagg gagacaagcc cggccgcgtg ttccgtccac acgttgcgga cgtactcaag 3240
ttctgccggc gagccgatgg cggaaagcag aaagacgacc tggtagaaac ctgcattcgg 3300
ttaaacacca cgcacgttgc catgcagcgt acgaagaagg ccaagaacgg ccgcctggtg 3360
acggtatccg agggtgaagc cttgattagc cgctacaaga tcgtaaagag cgaaaccggg 3420
cggccggagt acatcgagat cgagctagct gattggatgt accgcgagat cacagaaggc 3480
aagaacccgg acgtgctgac ggttcacccc gattactttt tgatcgatcc cggcatcggc 3540
cgttttctct accgcctggc acgccgcgcc gcaggcaagg cagaagccag atggttgttc 3600
aagacgatct acgaacgcag tggcagcgcc ggagagttca agaagttctg tttcaccgtg 3660
cgcaagctga tcgggtcaaa tgacctgccg gagtacgatt tgaaggagga ggcggggcag 3720
gctggcccga tcctagtcat gcgctaccgc aacctgatcg agggcgaagc atccgccggt 3780
tcctaatgta cggagcagat gctagggcaa attgccctag caggggaaaa aggtcgaaaa 3840
ggtctctttc ctgtggatag cacgtacatt gggaacccaa agccgtacat tgggaaccgg 3900
aacccgtaca ttgggaaccc aaagccgtac attgggaacc ggtcacacat gtaagtgact 3960
gatataaaag agaaaaaagg cgatttttcc gcctaaaact ctttaaaact tattaaaact 4020
cttaaaaccc gcctggcctg tgcataactg tctggccagc gcacagccga agagctgcaa 4080
aaagcgccta cccttcggtc gctgcgctcc ctacgccccg ccgcttcgcg tcggcctatc 4140
gcggccgctg gccgctcaaa aatggctggc ctacggccag gcaatctacc agggcgcgga 4200
caagccgcgc cgtcgccact cgaccgccgg cgcccacatc aaggcaccct gcctcgcgcg 4260
tttcggtgat gacggtgaaa acctctgaca catgcagctc ccggagacgg tcacagcttg 4320
tctgtaagcg gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg 4380
gtgtcggggc gcagccatga cccagtcacg tagcgatagc ggagtgtata ctggcttaac 4440
tatgcggcat cagagcagat tgtactgaga gtgcaccata tgcggtgtga aataccgcac 4500
agatgcgtaa ggagaaaata ccgcatcagg cgctcttccg cttcctcgct cactgactcg 4560
ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg 4620
ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag 4680
gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac 4740
gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga 4800
taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt 4860
accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc 4920
tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc 4980
cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta 5040
agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat 5100
gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca 5160
gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct 5220
tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt 5280
acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct 5340
cagtggaacg aaaactcacg ttaagggatt ttggtcatgc attctaggta ctaaaacaat 5400
tcatccagta aaatataata ttttattttc tcccaatcag gcttgatccc cagtaagtca 5460
aaaaatagct cgacatactg ttcttccccg atatcctccc tgatcgaccg gacgcagaag 5520
gcaatgtcat accacttgtc cgccctgccg cttctcccaa gatcaataaa gccacttact 5580
ttgccatctt tcacaaagat gttgctgtct cccaggtcgc cgtgggaaaa gacaagttcc 5640
tcttcgggct tttccgtctt taaaaaatca tacagctcgc gcggatcttt aaatggagtg 5700
tcttcttccc agttttcgca atccacatcg gccagatcgt tattcagtaa gtaatccaat 5760
tcggctaagc ggctgtctaa gctattcgta tagggacaat ccgatatgtc gatggagtga 5820
aagagcctga tgcactccgc atacagctcg ataatctttt cagggctttg ttcatcttca 5880
tactcttccg agcaaaggac gccatcggcc tcactcatga gcagattgct ccagccatca 5940
tgccgttcaa agtgcaggac ctttggaaca ggcagctttc cttccagcca tagcatcatg 6000
tccttttccc gttccacatc ataggtggtc cctttatacc ggctgtccgt catttttaaa 6060
tataggtttt cattttctcc caccagctta tataccttag caggagacat tccttccgta 6120
tcttttacgc agcggtattt ttcgatcagt tttttcaatt ccggtgatat tctcatttta 6180
gccatttatt atttccttcc tcttttctac agtatttaaa gataccccaa gaagctaatt 6240
ataacaagac gaactccaat tcactgttcc ttgcattcta aaaccttaaa taccagaaaa 6300
cagctttttc aaagttgttt tcaaagttgg cgtataacat agtatcgacg gagccgattt 6360
tgaaaccgcg gtgatcacag gcagcaacgc tctgtcatcg ttacaatcaa catgctaccc 6420
tccgcgagat catccgtgtt tcaaacccgg cagcttagtt gccgttcttc cgaatagcat 6480
cggtaacatg agcaaagtct gccgccttac aacggctctc ccgctgacgc cgtcccggac 6540
tgatgggctg cctgtatcga gtggtgattt tgtgccgagc tgccggtcgg ggagctgttg 6600
gctggctggt ggcaggatat attgtggtgt aaacaaattg acgcttagac aacttaataa 6660
cacattgcgg acgtttttaa tgtactgaat taacgccgaa ttaattcggg ggatctggat 6720
tttagtactg gattttggtt ttaggaatta gaaattttat tgatagaagt attttacaaa 6780
tacaaataca tactaagggt ttcttatatg ctcaacacat gagcgaaacc ctataggaac 6840
cctaattccc ttatctggga actactcaca cattattatg gagaaactcg agcttgtcga 6900
tcgacagatc cggtcggcat ctactctatt tctttgccct cggacgagtg ctggggcgtc 6960
ggtttccact atcggcgagt acttctacac agccatcggt ccagacggcc gcgcttctgc 7020
gggcgatttg tgtacgcccg acagtcccgg ctccggatcg gacgattgcg tcgcatcgac 7080
cctgcgccca agctgcatca tcgaaattgc cgtcaaccaa gctctgatag agttggtcaa 7140
gaccaatgcg gagcatatac gcccggagtc gtggcgatcc tgcaagctcc ggatgcctcc 7200
gctcgaagta gcgcgtctgc tgctccatac aagccaacca cggcctccag aagaagatgt 7260
tggcgacctc gtattgggaa tccccgaaca tcgcctcgct ccagtcaatg accgctgtta 7320
tgcggccatt gtccgtcagg acattgttgg agccgaaatc cgcgtgcacg aggtgccgga 7380
cttcggggca gtcctcggcc caaagcatca gctcatcgag agcctgcgcg acggacgcac 7440
tgacggtgtc gtccatcaca gtttgccagt gatacacatg gggatcagca atcgcgcata 7500
tgaaatcacg ccatgtagtg tattgaccga ttccttgcgg tccgaatggg ccgaacccgc 7560
tcgtctggct aagatcggcc gcagcgatcg catccatagc ctccgcgacc ggttgtagaa 7620
cagcgggcag ttcggtttca ggcaggtctt gcaacgtgac accctgtgca cggcgggaga 7680
tgcaataggt caggctctcg ctaaactccc caatgtcaag cacttccgga atcgggagcg 7740
cggccgatgc aaagtgccga taaacataac gatctttgta gaaaccatcg gcgcagctat 7800
ttacccgcag gacatatcca cgccctccta catcgaagct gaaagcacga gattcttcgc 7860
cctccgagag ctgcatcagg tcggagacgc tgtcgaactt ttcgatcaga aacttctcga 7920
cagacgtcgc ggtgagttca ggctttttca tatctcattg ccccccggga tctgcgaaag 7980
ctcgagagag atagatttgt agagagagac tggtgatttc agcgtgtcct ctccaaatga 8040
aatgaacttc cttatataga ggaaggtctt gcgaaggata gtgggattgt gcgtcatccc 8100
ttacgtcagt ggagatatca catcaatcca cttgctttga agacgtggtt ggaacgtctt 8160
ctttttccac gatgctcctc gtgggtgggg gtccatcttt gggaccactg tcggcagagg 8220
catcttgaac gatagccttt cctttatcgc aatgatggca tttgtaggtg ccaccttcct 8280
tttctactgt ccttttgatg aagtgacaga tagctgggca atggaatccg aggaggtttc 8340
ccgatattac cctttgttga aaagtctcaa tagccctttg gtcttctgag actgtatctt 8400
tgatattctt ggagtagacg agagtgtcgt gctccaccat gttatcacat caatccactt 8460
gctttgaaga cgtggttgga acgtcttctt tttccacgat gctcctcgtg ggtgggggtc 8520
catctttggg accactgtcg gcagaggcat cttgaacgat agcctttcct ttatcgcaat 8580
gatggcattt gtaggtgcca ccttcctttt ctactgtcct tttgatgaag tgacagatag 8640
ctgggcaatg gaatccgagg aggtttcccg atattaccct ttgttgaaaa gtctcaatag 8700
ccctttggtc ttctgagact gtatctttga tattcttgga gtagacgaga gtgtcgtgct 8760
ccaccatgtt gggcccggcg cgccaagctt ctagtgcagt gcagcgtgac ccggtcgtgc 8820
ccctctctag agataatgag cattgcatgt ctaagttata aaaaattacc acatattttt 8880
tttgtcacac ttgtttgaag tgcagtttat ctatctttat acatatattt aaactttact 8940
ctacgaataa tataatctat agtactacaa taatatcagt gttttagaga atcatataaa 9000
tgaacagtta gacatggtct aaaggacaat tgagtatttt gacaacagga ctctacagtt 9060
ttatcttttt agtgtgcatg tgttctcctt tttttttgca aatagcttca cctatataat 9120
acttcatcca ttttattagt acatccattt agggtttagg gttaatggtt tttatagact 9180
aattttttta gtacatctat tttattctat tttagcctct aaattaagaa aactaaaact 9240
ctattttagt ttttttattt aataatttag atataaaata gaataaaata aagtgactaa 9300
aaattaaaca aatacccttt aagaaattaa aaaaactaag gaaacatttt tcttgtttcg 9360
agtagataat gccagcctgt taaacgccgt cgacgagtct aacggacacc aaccagcgaa 9420
ccagcagcgt cgcgtcgggc caagcgaagc agacggcacg gcatctctgt cgctgcctct 9480
ggacccctct cgagagttcc gctccaccgt tggacttgct ccgctgtcgg catccagaaa 9540
ttgcgtggcg gagcggcaga cgtgagccgg cacggcaggc ggcctcctcc tcctctcacg 9600
gcacggcagc tacgggggat tcctttccca ccgctccttc gctttccctt cctcgcccgc 9660
cgtaataaat agacaccccc tccacaccct ctttccccaa cctcgtgttg ttcggagcgc 9720
acacacacac aaccagatct cccccaaatc cacccgtcgg cacctccgct tcaaggtacg 9780
ccgctcgtcc tccccccccc cccctctcta ccttctctag atcggcgttc cggtccatgg 9840
ttagggcccg gtagttctac ttctgttcat gtttgtgtta gatccgtgtt tgtgttagat 9900
ccgtgctgct agcgttcgta cacggatgcg acctgtacgt cagacacgtt ctgattgcta 9960
acttgccagt gtttctcttt ggggaatcct gggatggctc tagccgttcc gcagacggga 10020
tcgatttcat gatttttttt gtttcgttgc atagggtttg gtttgccctt ttcctttatt 10080
tcaatatatg ccgtgcactt gtttgtcggg tcatcttttc atgctttttt ttgtcttggt 10140
tgtgatgatg tggtctggtt gggcggtcgt tctagatcgg agtagaattc tgtttcaaac 10200
tacctggtgg atttattaat tttggatctg tatgtgtgtg ccatacatat tcatagttac 10260
gaattgaaga tgatggatgg aaatatcgat ctaggatagg tatacatgtt gatgcgggtt 10320
ttactgatgc atatacagag atgctttttg ttcgcttggt tgtgatgatg tggtgtggtt 10380
gggcggtcgt tcattcgttc tagatcggag tagaatactg tttcaaacta cctggtgtat 10440
ttattaattt tggaactgta tgtgtgtgtc atacatcttc atagttacga gtttaagatg 10500
gatggaaata tcgatctagg ataggtatac atgttgatgt gggttttact gatgcatata 10560
catgatggca tatgcagcat ctattcatat gctctaacct tgagtaccta tctattataa 10620
taaacaagta tgttttataa ttattttgat cttgatatac ttggatgatg gcatatgcag 10680
cagctatatg tggatttttt tagccctgcc ttcatacgct atttatttgc ttggtactgt 10740
ttcttttgtc gatgctcacc ctgttgtttg gtgttacttc tgcactaggt acctgcaggt 10800
cgacggatcc ccgggaattc taagaggagt ccaccatggt agatctgact agtgttaacg 10860
<210> 4
<211> 10987
<212> DNA
<213> Artificial Sequence
<400> 4
ctagccacca ccaccaccac cacgtgtgaa ttacaggtga ccagctcgaa tttccccgat 60
cgttcaaaca tttggcaata aagtttctta agattgaatc ctgttgccgg tcttgcgatg 120
attatcatat aatttctgtt gaattacgtt aagcatgtaa taattaacat gtaatgcatg 180
acgttattta tgagatgggt ttttatgatt agagtcccgc aattatacat ttaatacgcg 240
atagaaaaca aaatatagcg cgcaaactag gataaattat cgcgcgcggt gtcatctatg 300
ttactagatc gggaattaaa ctatcagtgt ttgacaggat atattggcgg gtaaacctaa 360
gagaaaagag cgtttattag aataacggat atttaaaagg gcgtgaaaag gtttatccgt 420
tcgtccattt gtatgtgcat gccaaccaca gggttcccct cgggatcaaa gtactttgat 480
ccaacccctc cgctgctata gtgcagtcgg cttctgacgt tcagtgcagc cgtcttctga 540
aaacgacatg tcgcacaagt cctaagttac gcgacaggct gccgccctgc ccttttcctg 600
gcgttttctt gtcgcgtgtt ttagtcgcat aaagtagaat acttgcgact agaaccggag 660
acattacgcc atgaacaaga gcgccgccgc tggcctgctg ggctatgccc gcgtcagcac 720
cgacgaccag gacttgacca accaacgggc cgaactgcac gcggccggct gcaccaagct 780
gttttccgag aagatcaccg gcaccaggcg cgaccgcccg gagctggcca ggatgcttga 840
ccacctacgc cctggcgacg ttgtgacagt gaccaggcta gaccgcctgg cccgcagcac 900
ccgcgaccta ctggacattg ccgagcgcat ccaggaggcc ggcgcgggcc tgcgtagcct 960
ggcagagccg tgggccgaca ccaccacgcc ggccggccgc atggtgttga ccgtgttcgc 1020
cggcattgcc gagttcgagc gttccctaat catcgaccgc acccggagcg ggcgcgaggc 1080
cgccaaggcc cgaggcgtga agtttggccc ccgccctacc ctcaccccgg cacagatcgc 1140
gcacgcccgc gagctgatcg accaggaagg ccgcaccgtg aaagaggcgg ctgcactgct 1200
tggcgtgcat cgctcgaccc tgtaccgcgc acttgagcgc agcgaggaag tgacgcccac 1260
cgaggccagg cggcgcggtg ccttccgtga ggacgcattg accgaggccg acgccctggc 1320
ggccgccgag aatgaacgcc aagaggaaca agcatgaaac cgcaccagga cggccaggac 1380
gaaccgtttt tcattaccga agagatcgag gcggagatga tcgcggccgg gtacgtgttc 1440
gagccgcccg cgcacgtctc aaccgtgcgg ctgcatgaaa tcctggccgg tttgtctgat 1500
gccaagctgg cggcctggcc ggccagcttg gccgctgaag aaaccgagcg ccgccgtcta 1560
aaaaggtgat gtgtatttga gtaaaacagc ttgcgtcatg cggtcgctgc gtatatgatg 1620
cgatgagtaa ataaacaaat acgcaagggg aacgcatgaa ggttatcgct gtacttaacc 1680
agaaaggcgg gtcaggcaag acgaccatcg caacccatct agcccgcgcc ctgcaactcg 1740
ccggggccga tgttctgtta gtcgattccg atccccaggg cagtgcccgc gattgggcgg 1800
ccgtgcggga agatcaaccg ctaaccgttg tcggcatcga ccgcccgacg attgaccgcg 1860
acgtgaaggc catcggccgg cgcgacttcg tagtgatcga cggagcgccc caggcggcgg 1920
acttggctgt gtccgcgatc aaggcagccg acttcgtgct gattccggtg cagccaagcc 1980
cttacgacat atgggccacc gccgacctgg tggagctggt taagcagcgc attgaggtca 2040
cggatggaag gctacaagcg gcctttgtcg tgtcgcgggc gatcaaaggc acgcgcatcg 2100
gcggtgaggt tgccgaggcg ctggccgggt acgagctgcc cattcttgag tcccgtatca 2160
cgcagcgcgt gagctaccca ggcactgccg ccgccggcac aaccgttctt gaatcagaac 2220
ccgagggcga cgctgcccgc gaggtccagg cgctggccgc tgaaattaaa tcaaaactca 2280
tttgagttaa tgaggtaaag agaaaatgag caaaagcaca aacacgctaa gtgccggccg 2340
tccgagcgca cgcagcagca aggctgcaac gttggccagc ctggcagaca cgccagccat 2400
gaagcgggtc aactttcagt tgccggcgga ggatcacacc aagctgaaga tgtacgcggt 2460
acgccaaggc aagaccatta ccgagctgct atctgaatac atcgcgcagc taccagagta 2520
aatgagcaaa tgaataaatg agtagatgaa ttttagcggc taaaggaggc ggcatggaaa 2580
atcaagaaca accaggcacc gacgccgtgg aatgccccat gtgtggagga acgggcggtt 2640
ggccaggcgt aagcggctgg gttgtctgcc ggccctgcaa tggcactgga acccccaagc 2700
ccgaggaatc ggcgtgacgg tcgcaaacca tccggcccgg tacaaatcgg cgcggcgctg 2760
ggtgatgacc tggtggagaa gttgaaggcc gcgcaggccg cccagcggca acgcatcgag 2820
gcagaagcac gccccggtga atcgtggcaa gcggccgctg atcgaatccg caaagaatcc 2880
cggcaaccgc cggcagccgg tgcgccgtcg attaggaagc cgcccaaggg cgacgagcaa 2940
ccagattttt tcgttccgat gctctatgac gtgggcaccc gcgatagtcg cagcatcatg 3000
gacgtggccg ttttccgtct gtcgaagcgt gaccgacgag ctggcgaggt gatccgctac 3060
gagcttccag acgggcacgt agaggtttcc gcagggccgg ccggcatggc cagtgtgtgg 3120
gattacgacc tggtactgat ggcggtttcc catctaaccg aatccatgaa ccgataccgg 3180
gaagggaagg gagacaagcc cggccgcgtg ttccgtccac acgttgcgga cgtactcaag 3240
ttctgccggc gagccgatgg cggaaagcag aaagacgacc tggtagaaac ctgcattcgg 3300
ttaaacacca cgcacgttgc catgcagcgt acgaagaagg ccaagaacgg ccgcctggtg 3360
acggtatccg agggtgaagc cttgattagc cgctacaaga tcgtaaagag cgaaaccggg 3420
cggccggagt acatcgagat cgagctagct gattggatgt accgcgagat cacagaaggc 3480
aagaacccgg acgtgctgac ggttcacccc gattactttt tgatcgatcc cggcatcggc 3540
cgttttctct accgcctggc acgccgcgcc gcaggcaagg cagaagccag atggttgttc 3600
aagacgatct acgaacgcag tggcagcgcc ggagagttca agaagttctg tttcaccgtg 3660
cgcaagctga tcgggtcaaa tgacctgccg gagtacgatt tgaaggagga ggcggggcag 3720
gctggcccga tcctagtcat gcgctaccgc aacctgatcg agggcgaagc atccgccggt 3780
tcctaatgta cggagcagat gctagggcaa attgccctag caggggaaaa aggtcgaaaa 3840
ggtctctttc ctgtggatag cacgtacatt gggaacccaa agccgtacat tgggaaccgg 3900
aacccgtaca ttgggaaccc aaagccgtac attgggaacc ggtcacacat gtaagtgact 3960
gatataaaag agaaaaaagg cgatttttcc gcctaaaact ctttaaaact tattaaaact 4020
cttaaaaccc gcctggcctg tgcataactg tctggccagc gcacagccga agagctgcaa 4080
aaagcgccta cccttcggtc gctgcgctcc ctacgccccg ccgcttcgcg tcggcctatc 4140
gcggccgctg gccgctcaaa aatggctggc ctacggccag gcaatctacc agggcgcgga 4200
caagccgcgc cgtcgccact cgaccgccgg cgcccacatc aaggcaccct gcctcgcgcg 4260
tttcggtgat gacggtgaaa acctctgaca catgcagctc ccggagacgg tcacagcttg 4320
tctgtaagcg gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg 4380
gtgtcggggc gcagccatga cccagtcacg tagcgatagc ggagtgtata ctggcttaac 4440
tatgcggcat cagagcagat tgtactgaga gtgcaccata tgcggtgtga aataccgcac 4500
agatgcgtaa ggagaaaata ccgcatcagg cgctcttccg cttcctcgct cactgactcg 4560
ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg 4620
ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag 4680
gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac 4740
gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga 4800
taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt 4860
accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc 4920
tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc 4980
cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta 5040
agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat 5100
gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca 5160
gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct 5220
tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt 5280
acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct 5340
cagtggaacg aaaactcacg ttaagggatt ttggtcatgc attctaggta ctaaaacaat 5400
tcatccagta aaatataata ttttattttc tcccaatcag gcttgatccc cagtaagtca 5460
aaaaatagct cgacatactg ttcttccccg atatcctccc tgatcgaccg gacgcagaag 5520
gcaatgtcat accacttgtc cgccctgccg cttctcccaa gatcaataaa gccacttact 5580
ttgccatctt tcacaaagat gttgctgtct cccaggtcgc cgtgggaaaa gacaagttcc 5640
tcttcgggct tttccgtctt taaaaaatca tacagctcgc gcggatcttt aaatggagtg 5700
tcttcttccc agttttcgca atccacatcg gccagatcgt tattcagtaa gtaatccaat 5760
tcggctaagc ggctgtctaa gctattcgta tagggacaat ccgatatgtc gatggagtga 5820
aagagcctga tgcactccgc atacagctcg ataatctttt cagggctttg ttcatcttca 5880
tactcttccg agcaaaggac gccatcggcc tcactcatga gcagattgct ccagccatca 5940
tgccgttcaa agtgcaggac ctttggaaca ggcagctttc cttccagcca tagcatcatg 6000
tccttttccc gttccacatc ataggtggtc cctttatacc ggctgtccgt catttttaaa 6060
tataggtttt cattttctcc caccagctta tataccttag caggagacat tccttccgta 6120
tcttttacgc agcggtattt ttcgatcagt tttttcaatt ccggtgatat tctcatttta 6180
gccatttatt atttccttcc tcttttctac agtatttaaa gataccccaa gaagctaatt 6240
ataacaagac gaactccaat tcactgttcc ttgcattcta aaaccttaaa taccagaaaa 6300
cagctttttc aaagttgttt tcaaagttgg cgtataacat agtatcgacg gagccgattt 6360
tgaaaccgcg gtgatcacag gcagcaacgc tctgtcatcg ttacaatcaa catgctaccc 6420
tccgcgagat catccgtgtt tcaaacccgg cagcttagtt gccgttcttc cgaatagcat 6480
cggtaacatg agcaaagtct gccgccttac aacggctctc ccgctgacgc cgtcccggac 6540
tgatgggctg cctgtatcga gtggtgattt tgtgccgagc tgccggtcgg ggagctgttg 6600
gctggctggt ggcaggatat attgtggtgt aaacaaattg acgcttagac aacttaataa 6660
cacattgcgg acgtttttaa tgtactgaat taacgccgaa ttaattcggg ggatctggat 6720
tttagtactg gattttggtt ttaggaatta gaaattttat tgatagaagt attttacaaa 6780
tacaaataca tactaagggt ttcttatatg ctcaacacat gagcgaaacc ctataggaac 6840
cctaattccc ttatctggga actactcaca cattattatg gagaaactcg agcttgtcga 6900
tcgacagatc cggtcggcat ctactctatt tctttgccct cggacgagtg ctggggcgtc 6960
ggtttccact atcggcgagt acttctacac agccatcggt ccagacggcc gcgcttctgc 7020
gggcgatttg tgtacgcccg acagtcccgg ctccggatcg gacgattgcg tcgcatcgac 7080
cctgcgccca agctgcatca tcgaaattgc cgtcaaccaa gctctgatag agttggtcaa 7140
gaccaatgcg gagcatatac gcccggagtc gtggcgatcc tgcaagctcc ggatgcctcc 7200
gctcgaagta gcgcgtctgc tgctccatac aagccaacca cggcctccag aagaagatgt 7260
tggcgacctc gtattgggaa tccccgaaca tcgcctcgct ccagtcaatg accgctgtta 7320
tgcggccatt gtccgtcagg acattgttgg agccgaaatc cgcgtgcacg aggtgccgga 7380
cttcggggca gtcctcggcc caaagcatca gctcatcgag agcctgcgcg acggacgcac 7440
tgacggtgtc gtccatcaca gtttgccagt gatacacatg gggatcagca atcgcgcata 7500
tgaaatcacg ccatgtagtg tattgaccga ttccttgcgg tccgaatggg ccgaacccgc 7560
tcgtctggct aagatcggcc gcagcgatcg catccatagc ctccgcgacc ggttgtagaa 7620
cagcgggcag ttcggtttca ggcaggtctt gcaacgtgac accctgtgca cggcgggaga 7680
tgcaataggt caggctctcg ctaaactccc caatgtcaag cacttccgga atcgggagcg 7740
cggccgatgc aaagtgccga taaacataac gatctttgta gaaaccatcg gcgcagctat 7800
ttacccgcag gacatatcca cgccctccta catcgaagct gaaagcacga gattcttcgc 7860
cctccgagag ctgcatcagg tcggagacgc tgtcgaactt ttcgatcaga aacttctcga 7920
cagacgtcgc ggtgagttca ggctttttca tatctcattg ccccccggga tctgcgaaag 7980
ctcgagagag atagatttgt agagagagac tggtgatttc agcgtgtcct ctccaaatga 8040
aatgaacttc cttatataga ggaaggtctt gcgaaggata gtgggattgt gcgtcatccc 8100
ttacgtcagt ggagatatca catcaatcca cttgctttga agacgtggtt ggaacgtctt 8160
ctttttccac gatgctcctc gtgggtgggg gtccatcttt gggaccactg tcggcagagg 8220
catcttgaac gatagccttt cctttatcgc aatgatggca tttgtaggtg ccaccttcct 8280
tttctactgt ccttttgatg aagtgacaga tagctgggca atggaatccg aggaggtttc 8340
ccgatattac cctttgttga aaagtctcaa tagccctttg gtcttctgag actgtatctt 8400
tgatattctt ggagtagacg agagtgtcgt gctccaccat gttatcacat caatccactt 8460
gctttgaaga cgtggttgga acgtcttctt tttccacgat gctcctcgtg ggtgggggtc 8520
catctttggg accactgtcg gcagaggcat cttgaacgat agcctttcct ttatcgcaat 8580
gatggcattt gtaggtgcca ccttcctttt ctactgtcct tttgatgaag tgacagatag 8640
ctgggcaatg gaatccgagg aggtttcccg atattaccct ttgttgaaaa gtctcaatag 8700
ccctttggtc ttctgagact gtatctttga tattcttgga gtagacgaga gtgtcgtgct 8760
ccaccatgtt gggcccggcg cgccaagctt ctagtgcagt gcagcgtgac ccggtcgtgc 8820
ccctctctag agataatgag cattgcatgt ctaagttata aaaaattacc acatattttt 8880
tttgtcacac ttgtttgaag tgcagtttat ctatctttat acatatattt aaactttact 8940
ctacgaataa tataatctat agtactacaa taatatcagt gttttagaga atcatataaa 9000
tgaacagtta gacatggtct aaaggacaat tgagtatttt gacaacagga ctctacagtt 9060
ttatcttttt agtgtgcatg tgttctcctt tttttttgca aatagcttca cctatataat 9120
acttcatcca ttttattagt acatccattt agggtttagg gttaatggtt tttatagact 9180
aattttttta gtacatctat tttattctat tttagcctct aaattaagaa aactaaaact 9240
ctattttagt ttttttattt aataatttag atataaaata gaataaaata aagtgactaa 9300
aaattaaaca aatacccttt aagaaattaa aaaaactaag gaaacatttt tcttgtttcg 9360
agtagataat gccagcctgt taaacgccgt cgacgagtct aacggacacc aaccagcgaa 9420
ccagcagcgt cgcgtcgggc caagcgaagc agacggcacg gcatctctgt cgctgcctct 9480
ggacccctct cgagagttcc gctccaccgt tggacttgct ccgctgtcgg catccagaaa 9540
ttgcgtggcg gagcggcaga cgtgagccgg cacggcaggc ggcctcctcc tcctctcacg 9600
gcacggcagc tacgggggat tcctttccca ccgctccttc gctttccctt cctcgcccgc 9660
cgtaataaat agacaccccc tccacaccct ctttccccaa cctcgtgttg ttcggagcgc 9720
acacacacac aaccagatct cccccaaatc cacccgtcgg cacctccgct tcaaggtacg 9780
ccgctcgtcc tccccccccc cccctctcta ccttctctag atcggcgttc cggtccatgg 9840
ttagggcccg gtagttctac ttctgttcat gtttgtgtta gatccgtgtt tgtgttagat 9900
ccgtgctgct agcgttcgta cacggatgcg acctgtacgt cagacacgtt ctgattgcta 9960
acttgccagt gtttctcttt ggggaatcct gggatggctc tagccgttcc gcagacggga 10020
tcgatttcat gatttttttt gtttcgttgc atagggtttg gtttgccctt ttcctttatt 10080
tcaatatatg ccgtgcactt gtttgtcggg tcatcttttc atgctttttt ttgtcttggt 10140
tgtgatgatg tggtctggtt gggcggtcgt tctagatcgg agtagaattc tgtttcaaac 10200
tacctggtgg atttattaat tttggatctg tatgtgtgtg ccatacatat tcatagttac 10260
gaattgaaga tgatggatgg aaatatcgat ctaggatagg tatacatgtt gatgcgggtt 10320
ttactgatgc atatacagag atgctttttg ttcgcttggt tgtgatgatg tggtgtggtt 10380
gggcggtcgt tcattcgttc tagatcggag tagaatactg tttcaaacta cctggtgtat 10440
ttattaattt tggaactgta tgtgtgtgtc atacatcttc atagttacga gtttaagatg 10500
gatggaaata tcgatctagg ataggtatac atgttgatgt gggttttact gatgcatata 10560
catgatggca tatgcagcat ctattcatat gctctaacct tgagtaccta tctattataa 10620
taaacaagta tgttttataa ttattttgat cttgatatac ttggatgatg gcatatgcag 10680
cagctatatg tggatttttt tagccctgcc ttcatacgct atttatttgc ttggtactgt 10740
ttcttttgtc gatgctcacc ctgttgtttg gtgttacttc tgcactaggt accatcgtga 10800
aggctaaatg gagaagagag agagtacagc tttgggcggt ttagttgtta gatggtggaa 10860
gtacttgcga cgcctactac ccttttcacc aaggctgtac gctccctctt cttctcttag 10920
ctttttatga tggatccccg ggaattctaa gaggagtcca ccatggtaga tctgactagt 10980
gttaacg 10987
Claims (10)
1. An miRNA is shown as a sequence 1 in a sequence table.
2. A precursor gene of the miRNA described in claim 1.
3. The precursor gene of miRNA of claim 1, which is represented by sequence 2 in the sequence table of the sequence table.
4. Use of the miRNA of claim 1 for modulating disease resistance in a plant.
5. Use of the miRNA of claim 1 for positively modulating disease resistance in a plant.
6. Use of the precursor gene of claim 2 or 3 for the preparation of transgenic plants with improved disease resistance.
7. A method of making a transgenic plant comprising the steps of: overexpressing the miRNA of claim 1 in a plant to produce a transgenic plant with increased disease resistance.
8. A method of making a transgenic plant comprising the steps of: a transgenic plant having improved disease resistance, which is obtained by introducing the precursor gene according to claim 2 or 3 into a plant.
9. A method of making a transgenic plant comprising the steps of: a transgenic plant having improved disease resistance, which is obtained by introducing and expressing the precursor gene according to claim 2 or 3 into a plant.
10. Use of the method of any one of claims 7 to 9 in plant breeding.
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| CN103589721A (en) * | 2012-08-15 | 2014-02-19 | 北京命码生科科技有限公司 | Extraction, preparation and application of plant micro ribonucleic acid |
| CN110157707A (en) * | 2019-05-27 | 2019-08-23 | 中国农业科学院作物科学研究所 | The application of one rice miRNA and its precursor-gene in rice bacterial blight resistance |
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| CN103589721A (en) * | 2012-08-15 | 2014-02-19 | 北京命码生科科技有限公司 | Extraction, preparation and application of plant micro ribonucleic acid |
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| CN110157707A (en) * | 2019-05-27 | 2019-08-23 | 中国农业科学院作物科学研究所 | The application of one rice miRNA and its precursor-gene in rice bacterial blight resistance |
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