CN114621972A - RNAi plant expression vector and application thereof - Google Patents

Abstract

The invention provides an RNAi plant expression vector and application thereof, wherein the RNAi plant expression vector contains a hairpin structure expression cassette, and the hairpin structure expression cassette contains a hairpin structure formed by DNA fragments shown in SEQ ID No. 1-3. The RNAi plant expression vector is used for transforming rice, so that the expression of the gene HIS1 can be successfully inhibited, the rice is changed from being resistant to herbicide to being sensitive, sensitive plants can be eliminated in a herbicide application mode, and the rice which is dominant sensitive to the herbicide is cultivated. The rice has important application value in the aspects of cultivating new transgenic varieties, producing hybrid rice seeds, preventing transgene escape and the like.

Description

RNAi plant expression vector and application thereof

Technical Field

The invention belongs to the technical field of agricultural biology, and particularly relates to an RNAi plant expression vector and application thereof.

Background

The application of the transgenic technology in various crop fields is more and more extensive, the transgenic product needs to be strictly controlled and supervised, and the prevention of the transgene drift from polluting other varieties and even other species is very important. Therefore, there is a need to establish a mechanism that allows targeted elimination of hybrid seeds or transgenic plants. The cultivation of sterile lines or restorer lines which are sensitive to herbicides is one of the effective means for solving the problems.

beta-Triketone herbicides (b-Triketone herbicides, bTHs) are 4-hydroxyphenylpyruvate dioxygenase (HPPD) inhibitors, are widely applied to agriculture, and can effectively prevent and kill various broadleaf weeds and grassy weeds. BTH Bicyclosulcotrione (BBC) for Weed Control in rice fields, has significant efficacy against rice weeds that can fight other types of Herbicides, including sulfonylureas (Kraehmer, et al, "Herbicides as well Control Agents: State of The Art: I. Wed Control Research and Safener Technology: The Path to model agriculture," Plant Physiology (2014)). Therefore, beta-triketone herbicides are commonly used to eliminate most broadleaf weeds and grassy weeds in rice fields, and studies have shown that a candidate gene for resistance of rice to BBC is HIS1(HPPD inhibitor sensitive 1). HIS1 encodes a 351 amino acid Fe (II)/2-oxoglutarate-dependent oxygenase protein, and the IV exon of BBC-sensitive varieties is deleted by 28bp, resulting in a loss of function (Maeda, Hideo, et al. "A edge gene at controls hybrid-specific resistance to β -triene binding: reference 365.6451(2019):393 396.).

If the rice sensitive to the beta-triketone herbicide dominance can be cultivated, the method has important application values in the aspects of cultivating new transgenic varieties, producing hybrid rice seeds, preventing transgene escape and the like.

Disclosure of Invention

The invention aims to provide an RNAi plant expression vector capable of effectively influencing expression of an HPPD inhibitor sensitive type 1 gene (HIS1) and application thereof.

In order to achieve the above purpose, the invention provides an RNAi plant expression vector for inhibiting expression of HPPD inhibitor sensitive type 1 gene HIS1, which comprises a hairpin structure expression cassette, wherein the hairpin structure expression cassette comprises a hairpin structure formed by DNA fragments shown in SEQ ID Nos. 1-3.

Wherein the DNA fragment shown in SEQ ID No.1 is a forward DNA fragment with the length of 228bp based on an HIS1 coding frame, and the DNA fragment shown in SEQ ID No.3 is a DNA fragment which is reversely complementary with the DNA fragment shown in SEQ ID No.1 with the length of 228bp based on an HIS1 coding frame. SEQ ID No.2 is an intron sequence (Rice intron) from the Rice Zinc finger gene.

The DNA fragments shown in SEQ ID Nos. 1 to 3 are arranged in order from the upstream to the downstream. The hairpin expression cassette can be transcribed in a transformed plant cell to form a hairpin-type secondary structure, the DNA fragment shown in SEQ ID No.2 forms a hairpin 'loop', and the DNA fragments shown in SEQ ID Nos. 1 and 3 are complemented to form a hairpin 'stem'.

Wherein the hairpin expression cassette further comprises a plant constitutive promoter or a plant tissue specific promoter upstream of the hairpin and a terminator downstream of the hairpin.

Wherein the plant constitutive promoter is a Ubi promoter, a Rubisco small subunit promoter, a Cab promoter, a CAMV 35S promoter or an Actin promoter of rice or corn. Preferably, when the promoter is a Ubi promoter of rice or maize, a very strong effect of interfering with the expression of the HIS1 gene can be obtained.

The terminator includes: DNA sequences capable of terminating gene transcription in plants, such as NOS terminator and Ubi terminator. Preferably, the terminator is a NOS terminator.

The RNAi plant expression vector also comprises a selective marker expression cassette, wherein the selective marker expression cassette contains a promoter, a marker gene and a terminator, the promoter is a Ubi promoter, a CAMV 35S promoter or an Actin promoter of rice or corn, and preferably, when the promoter is the CAMV 35S promoter, a good effect of driving the selective marker gene to be excessively expressed in plants can be achieved. The terminator is a NOS terminator or a Ubi terminator, preferably a NOS terminator.

The marker gene is a gene of an enzyme capable of producing a color change (e.g., a GUS gene, a luciferase gene, etc.), a fluorescent marker gene (e.g., a fluorescent protein gene), an antibiotic marker gene (an antibiotic marker for selection such as a hygromycin, gentamicin, a kanamycin gene), a herbicide selection marker gene (e.g., a glyphosate-resistant gene, a bispyribac-resistant gene, etc.), or a chemical agent resistance marker gene (e.g., an herbicide-resistant gene, etc.). Particularly preferably, the marker gene is the hygromycin gene.

In a preferred embodiment of the invention, the RNAi plant expression vector is obtained by connecting a DNA fragment shown in SEQ ID No.1-3 between SacI and BamHI sites of a plant binary transformation vector pTCK303 by overlapping method to obtain the RNAi plant expression vector pTCK303-HIS1 i-1.

Particularly preferably, the RNAi plant expression vector has a nucleotide sequence shown as SEQ ID No. 4.

Wherein the plant is rice, preferably, the plant is a transgenic rice line.

It is noted that intermediate vectors for constructing RNAi plant expression vectors, RNAi expression vectors with selectable marker genes, engineering bacteria, cells containing HIS1i-1 stem-loop structures, callus, transgenic seedlings, seeds and the like belong to the protection scope of the invention.

Another aspect of the invention also provides a construction method of RNAi plant expression vector, which comprises connecting the DNA fragment shown in SEQ ID No.1-3 to the plant binary transformation vector by overlapping method.

In a preferred embodiment of the present invention, the DNA fragment shown in SEQ ID Nos. 1-3 is ligated between SacI and BamHI sites of a plant binary transformation vector pTCK303 by overlapping to obtain an RNAi plant expression vector pTCK303-HIS1 i-1.

Specifically, the method can be performed according to the following steps:

(1) 3 pairs of primers are designed, 5' ends of two adjacent primers on the upstream and downstream of the DNA fragment shown in SEQ ID No.1-3 are respectively amplified to have about 15 nucleotide sequences which are repeated with corresponding connection positions of the fragment or the vector, so that the DNA fragment is recombined and connected by utilizing Gibson Assembly.

HIS1i-1-F: ATCGGGGAAATTCGAGCTcATGGCTGACGAGTCATGGAGG

HIS1i-1-Rv1: GAGATTTTCACAGGAAGAAGCCCCAATTCTGC

HIS1i-1-F2: CTTCTTCCTGTGAAAATCTCGAAACAGCCGTGT

HIS1i-1-Rv2: CTTCTTCCTGGGTAAGTTACTACAAACCTTTTTGTATTTATGTTCC

HIS1i-1-F3: AGTAACTTACCCAGGAAGAAGCCCCAATTCTGC

HIS1i-1-Rv: CTGCAGGTCGACTCTAGAGgatccATGGCTGACGAGTCATGGAGG

(2) Construction and transformation of RNAi plant expression vectors

Using rice cDNA as a template, the primers were used to perform PCR amplification of HIS1i-1 gene fragments 1 and 3. The amplification system and procedure were as follows:

the procedure is as follows: pre-denaturation at 94 deg.C for 5-10min, denaturation at 94 deg.C for 30s, annealing at 60 deg.C for 30s, extension at 72 deg.C for 30s, 30-35 cycles, and re-extension at 72 deg.C for 5 min; and finishing at 16 ℃.

The procedure is as follows: pre-denaturation at 94 deg.C for 5-10min, denaturation at 94 deg.C for 30s, annealing at 60 deg.C for 30s, extension at 72 deg.C for 30s, 30-35 cycles, and re-extension at 72 deg.C for 5 min; and finishing at 16 ℃.

Using rice DNA as a template, the primers were used to perform PCR amplification of HIS1i-1 gene fragment 2. The amplification system and procedure were as follows:

the procedure is as follows: pre-denaturation at 94 deg.C for 5-10min, denaturation at 94 deg.C for 30s, annealing at 58 deg.C for 30s, extension at 72 deg.C for 30s, 30-35 cycles, and re-extension at 72 deg.C for 5 min; and finishing at 16 ℃.

PCR product fragments were obtained and recovered and named HIS1i-1-1(SEQ ID No. 1), HIS1i-1-2(SEQ ID No. 2) and HIS1i-1-3(SEQ ID No. 3), respectively. The plant binary transformation vector pTCK303 is digested by SacI + BamHI, and target fragments HIS1i-1-1, HIS1i-1-2 and HIS1i-1-3 are directly connected into the digested pTCK303 plasmid to obtain the vector pTCK303-HIS1 i-1.

The invention also provides application of the RNAi plant expression vector in preparation of herbicide dominant sensitive plants by interfering expression of plant HPPD inhibitor sensitive type 1 gene HIS 1.

The RNAi plant expression vector of the present invention can be used to transform plant cells or tissues by conventional biological methods such as Agrobacterium-mediated genetic transformation, particle gun method, pollen tube channel method, etc.

Preferably, said use comprises introducing pTCK303-HIS1i-1 into Agrobacterium EHA105 strain and transforming callus. The callus can be anther-induced callus, mature embryo-induced callus, young embryo-induced callus, and young ear-induced callus.

In one aspect of the invention, the invention also provides a method for obtaining a plant sensitive to the dominance of the beta-triketone herbicide, which comprises the steps of transforming the plant with the RNAi plant expression vector and interfering the expression of the plant HPPD inhibitor sensitive type 1 gene HIS 1.

In one embodiment of the present invention, the method for obtaining a dominant sensitive plant to a β -triketone herbicide comprises:

(1) constructing RNAi plant expression vector: connecting the DNA fragment shown in SEQ ID No.1-3 to SacI and BamHI sites of a plant binary transformation vector pTCK303 by overlapping method to obtain an RNAi plant expression vector pTCK303-HIS1 i-1;

(2) and (3) transformation: pTCK303-HIS1i-1 is introduced into Agrobacterium EHA105 strain, and transformed callus is induced to differentiate to obtain dominant sensitive herbicide plant.

Wherein the RNAi plant expression vector pTCK303-HIS1i-1 has a nucleotide sequence shown in SEQ ID No. 4.

The beta-triketone herbicide provided by the invention comprises, but is not limited to, mesotrione and benzobicyclon. In the embodiment of the invention, the lowest concentration of the screened benzobicyclon sensitive transgenic line is 1800mg/L, and the lowest concentration of the screened benzobicyclon sensitive transgenic line is 9 mg/L.

The plant is rice, preferably, the plant is a transgenic rice line.

The invention provides application of the RNAi plant expression vector in cultivation of new transgenic varieties, hybrid rice seed production or prevention of transgene escape.

The invention constructs a plant binary transgenic vector aiming at the rice HPPD inhibitor sensitive type 1 gene HIS1 by screening based on RNAi technology, the vector can efficiently inhibit the vector of the HPPD inhibitor sensitive type 1 gene HIS1, the sequence of the vector is completely a rice endogenous sequence, and the risk and worry of the public to the exogenous gene transgene can be effectively eliminated. The expression of HIS1 gene is successfully inhibited by transforming the carrier into rice, and the rice is converted from being resistant to beta-triketone herbicide to being sensitive, thereby cultivating the rice which is dominant sensitive to the beta-triketone herbicide. So that the transgenic plants can be eliminated by means of applying herbicides. The rice has very important application value in the aspects of cultivating new transgenic varieties, producing hybrid rice seeds, preventing transgene escape and the like.

Drawings

FIG. 1 is an electrophoretogram obtained by amplifying and recovering 3 target fragments of HIS1i-1-1, HIS1i-1-2 and HIS1i-1-3 in example 2. Wherein lanes 1-6 are HIS1i-1-1 target fragments; lanes 8-13 are HIS1i-1-3 fragments of interest; lanes 15-20 are HIS1i-1-2 target fragments.

FIG. 2 is an electrophoresis diagram of the cleavage and recovery of pTCK303 backbone vector. Wherein CK is pTCK303 backbone vector as control; lanes 1-5 are Sac I/BamH I double-cleaved pTCK303 backbone vector.

FIG. 3 is an electrophoretogram of pTCK303-HIS1i-1 vector digested with SacI and BamHI. CK in the figure is pTCK303-HIS1i-1 monoclonal recombinant plasmid; lanes 1-4 are the digested monoclonal recombinant plasmids, M is marker, the size of the excised fragment is 934bp, including the forward and reverse complementary sequences of the target fragment and the rice intron sequence.

FIG. 4 is the electrophoresis diagram of PCR detection of hygromycin resistance gene of transgenic line. Lane 1, H₂O; lane 2 negative control (middle flower 11 non-transgenic plants); lane 3, positive control (pTCK303-HIS1i-1 plasmid); lanes 4-23, transgenic lines (where lanes 4,13,14 and 22 are transgenic negative lines); m, Marker.

FIG. 5 is a sensitivity test of pTCK303-HIS1i-1 transgenic T0 trilobate seedling leaves to 2250mg/L solution of diclosone. WT is a ZH11 non-transgenic plant, and the withered leaves are sensitive strains. 0d is before spraying; 7d and 14d are recorded 7 days and 14 days after spraying.

FIG. 6 is a sensitivity test of pTCK303-HIS1i-1 transgenic T0 trilobate seedling leaves to 15mg/L mesotrione solution for 14 d. WT is a ZH11 non-transgenic plant, 59-2 and 106-1 are sensitive lines.

FIG. 7 shows the result of 6000mg/L of benzobicyclon 14d sprayed on the transgenic seedlings after RNAi vectors are constructed after the loop sequence is replaced by the sequence shown in SEQ ID No.11 in comparative example 1. The sensitive lines are indicated by arrows.

Detailed Description

The present invention will be described in detail with reference to the following embodiments.

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are commercially available.

EXAMPLE 1 Strain, plasmid acquisition and PCR primer Synthesis

(1) Strains and plasmids

The plant binary transformation vector pTCK303 as a backbone vector comprises a hygromycin resistance gene, a maize Ubi promoter and a NOS terminator. The E.coli (Escherichia coli) strain is DH5 alpha; the Agrobacterium tumefaciens (Agrobacterium tumefaciens) strain was EHA 105.

(2) PCR primer sequences

3 pairs of primers are designed, 5' ends of two adjacent primers on the upstream and downstream of the DNA fragment shown in SEQ ID No.1-3 are respectively amplified to have about 15 nucleotide sequences which are repeated with corresponding connection positions of the fragment or the vector, so that the DNA fragment is recombined and connected by utilizing Gibson Assembly.

HIS1i-1-F: ATCGGGGAAATTCGAGCTcATGGCTGACGAGTCATGGAGG

HIS1i-1-Rv1: GAGATTTTCACAGGAAGAAGCCCCAATTCTGC

HIS1i-1-F2: CTTCTTCCTGTGAAAATCTCGAAACAGCCGTGT

HIS1i-1-Rv2: CTTCTTCCTGGGTAAGTTACTACAAACCTTTTTGTATTTATGTTCC

HIS1i-1-F3: AGTAACTTACCCAGGAAGAAGCCCCAATTCTGC

HIS1i-1-Rv: CTGCAGGTCGACTCTAGAGgatccATGGCTGACGAGTCATGGAGG

Example 2 construction and transformation of RNAi plant expression vectors

(1) In order to construct an interference vector of the HPPD inhibitor-sensitive type 1 gene HIS1, the primers of example 1 were used to amplify HIS1i-1 gene fragments 1 and 3 by PCR using rice cDNA as a template. The amplification system and procedure were as follows:

the procedure is as follows: pre-denaturation at 94 deg.C for 5-10min, denaturation at 94 deg.C for 30s, annealing at 60 deg.C for 30s, extension at 72 deg.C for 30s, 30-35 cycles, and re-extension at 72 deg.C for 5 min; and finishing at 16 ℃.

The procedure is as follows: pre-denaturation at 94 deg.C for 5-10min, denaturation at 94 deg.C for 30s, annealing at 60 deg.C for 30s, extension at 72 deg.C for 30s, 30-35 cycles, and re-extension at 72 deg.C for 5 min; and finishing at 16 ℃.

(2) Using rice DNA as a template, the primers were used to perform PCR amplification of HIS1i-1 gene fragment 2. The amplification system and procedure were as follows:

the procedure is as follows: pre-denaturation at 94 deg.C for 5-10min, denaturation at 94 deg.C for 30s, annealing at 58 deg.C for 30s, extension at 72 deg.C for 30s, 30-35 cycles, and re-extension at 72 deg.C for 5 min; and finishing at 16 ℃.

After agarose gel electrophoresis, e.z.n.a was used.

The PCR product fragments were recovered from the Extraction kit (Omega, the same below) and the sizes were 228bp, 478bp and 228bp, respectively, and named HIS1i-1-1, HIS1i-1-2 and HIS1i-1-3, respectively, as shown in FIG. 1. The vector plasmid pTCK303 was digested simultaneously with Sac I + BamH I, subjected to agarose gel electrophoresis, and then subjected to E.Z.N.A..

The Extraction kit (Omega, the same below) recovered a band of about 14kb in size, giving a pTCK303 linear fragment (FIG. 2).

The SacI + BamHI double digestion reaction system is as follows:

(3)2x lighting Cloning Kit connection Kit

Three fragments of HIS1i-1-1, HIS1i-1-2 and HIS1i-1-3 are connected to the pTCK303 vector by the following connection system:

and (3) connecting procedures: 30min at 50 ℃.

The ligation product is transformed into escherichia coli DH5 alpha competent cells, the recombinant plasmid is subjected to double enzyme digestion by Sac I and BamH I, the size of a target fragment accords with the expectation (figure 3), and the recombinants are subjected to sequencing verification to be correct, namely, a HIS1i-1RNAi vector is constructed by taking a Rice Zinc finger gene intron (Rice intron) as a ring, and the vector is named as pTCK303-HIS1 i-1.

Example 3 Agrobacterium transformation of Rice and identification of transgenic plants

Introducing the pTCK303-HIS1i-1 recombinant plasmid into agrobacterium EHA105 strain, transforming 11 calli of japonica rice flowers, screening hygromycin resistance, differentiating, rooting to obtain 20 regenerated transgenic strains, identifying the hygromycin resistance gene transferred into the transgenic strains by PCR, and transplanting the positive strains into soil to survive 15 strains, wherein the results show that 16 PCR positive strains (shown in figure 4) are obtained.

Example 4 acquisition of sensitive and resistant transgenic lines of the Rice beta-triketone herbicide Dometrione

To examine the effect of the designed interference vector fragment in transgenic plants, 1800mg/L to 4500mg/L bicyclosulfoncotrione solution was prepared from 25% bicyclosulfoncotrione stock solution (Japan Stachys biosciences, Ltd., lot No. 2019050603), and pTCK303-HIS1i-1 transgenic T0-th trilobate seedling leaves obtained in example 3 were sprayed and continuously observed after spraying.

Spraying 12 HIS1i-1 transgenic strains at 2250mg/L of the mesotrione, wherein 5 strains are white and withered after 14d of spraying, and belong to highly sensitive strains; 3 strains all have withered and yellow leaf tips and belong to a medium-sensitive strain (figure 5); there were 4 lines that did not respond and belonged to resistant lines.

Further spraying 1800mg/L of low-concentration benzobicyclon to 8 HIS1i-1 transgenic lines, wherein 14d after spraying, 3 lines have white leaves and wither leaves and belong to highly sensitive lines; 1 strain appears white to withered and yellow in leaves and belongs to a medium-sensitive strain; there are 4 strains with distinct withered and yellow leaf tips, and the strains are low-sensitive or insensitive.

Example 5 acquisition of sensitive and resistant transgenic lines of Paddy mesotrione (mesotrione)

In order to detect the effect of the designed interference vector fragment in the transgenic plant, mesotrione mother liquor (Jiangsu Fengshan group Co., Ltd., batch No. 20190119004) is used for preparing 15 mg/L-750 mg/L mesotrione solution, pTCK303-HIS1i-1 transgenic T0 trilobate seedlings obtained in example 3 are sprayed, and continuous observation is carried out after spraying.

15mg/L of mesotrione is sprayed with 3 lines of HIS1i-1 transgenic leaves, 7 days after spraying, the tips of partial lines of HIS1i-1 transgenic T0 seedlings are curled, and the leaves are slightly whitish; 14d after spraying, 2 strains appear that leaves are white to wither, and belong to highly sensitive strains (figure 6); the leaf reaction is not obvious before and after 1 strain is sprayed, and the strain belongs to a resistant strain.

It should be emphasized that although the example uses 15mg/L mesotrione spray to screen mesotrione sensitive transgenic lines, the screening sensitive lines are not limited to 15mg/L, and further experiments show that when 9mg/L mesotrione is sprayed to HIS1i-1 transgenic 3 lines of leaves, 14 days after spraying, 2 lines have white and curly leaves to wither, and belong to highly sensitive lines. 9mg/L is the minimum mesotrione concentration employed in the present invention. The results show that the transgenic material which is dominantly sensitive to the herbicide is successfully obtained.

Comparative example 1

In order to test the influence of different stem loops on the interference efficiency, after the intron sequence of SEQ ID No.2 is shortened to 300bp (see SEQ ID No.11), a vector is constructed and the medium flower 11 is transformed to prepare a transgenic line.

The transgenic line is sprayed with the dicyclosulfuron-ethyl with the concentration of 2250mg/L to 4500mg/L respectively, and the result shows that the transgenic line shows higher resistance. When the concentration of the benzobicylon is more than 6000mg/L, the benzobicylon has sensitive phenotype, and the proportion of sensitive strains is low, which shows that the OsHIS1i adopting SEQ ID No.11 to design stem loops has poor interference efficiency on HIS1 genes. The results of the transgenic line T0 generation 3-5 leaf stage seedlings tested on 6000mg/L mesotrione are shown in FIG. 7.

Comparative example 2

Further shortening the SEQ ID No.2 intron sequence to 240bp (see SEQ ID No.12), constructing a vector and transforming the medium flower 11 to prepare a transgenic line.

The transgenic lines are respectively sprayed with high-concentration benzobicylon, and the results show that the transgenic lines also show higher resistance, and the results are similar to those in comparative example 1, which shows that the OsHIS1i with stem loops designed according to the method has poor interference efficiency on HIS1 genes.

In addition to the 2 comparative examples, in the research process of the present invention, the entire intron sequence was selected for the intron sequence of OsHIS1 gene, and the sequences with different truncated sizes in the intron sequence of OsHIS1 gene were expected to form hairpin structures with the DNA fragments shown in SEQ ID Nos. 1 and 3, but it was found that the DNA fragments shown by these intron sequences could not form hairpin "loops" or the stem-loop structures formed were very inefficient, so that they could not make the DNA fragments shown in SEQ ID Nos. 1 and 3 complementary to form hairpin "stems" to form double-stranded short RNA in vivo successfully after replacing the sequence shown in SEQ ID No.2 of the present invention. The invention fully proves that the intron sequence of SEQ ID No.2 and the DNA segments shown in SEQ ID No.1 and SEQ ID No.3 form a hairpin structure, and the constructed plant expression vector can well inhibit the HIS1 gene, has high interference efficiency, and enables transgenic plants to show obvious sensitivity to beta-triketone herbicides.

Although the invention has been described in detail with respect to the general description and the specific embodiments thereof, it will be apparent to those skilled in the art that modifications and improvements can be made based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Sequence listing

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tcggtgaaaa accgcagcag ggaggcaaac aagctagcca ccaccaccac caccacgtgt 2040

gaattacagg tgaccagctc gaatttcccc gatcgttcaa acatttggca ataaagtttc 2100

ttaagattga atcctgttgc cggtcttgcg atgattatca tataatttct gttgaattac 2160

gttaagcatg taataattaa catgtaatgc atgacgttat ttatgagatg ggtttttatg 2220

attagagtcc cgcaattata catttaatac gcgatagaaa acaaaatata gcgcgcaaac 2280

taggataaat tatcgcgcgc ggtgtcatct atgttactag atcgggaatt aaactatcag 2340

tgtttgacag gatatattgg cgggtaaacc taagagaaaa gagcgtttat tagaataacg 2400

gatatttaaa agggcgtgaa aaggtttatc cgttcgtcca tttgtatgtg catgccaacc 2460

acagggttcc cctcgggatc aaagtacttt gatccaaccc ctccgctgct atagtgcagt 2520

cggcttctga cgttcagtgc agccgtcttc tgaaaacgac atgtcgcaca agtcctaagt 2580

tacgcgacag gctgccgccc tgcccttttc ctggcgtttt cttgtcgcgt gttttagtcg 2640

cataaagtag aatacttgcg actagaaccg gagacattac gccatgaaca agagcgccgc 2700

cgctggcctg ctgggctatg cccgcgtcag caccgacgac caggacttga ccaaccaacg 2760

ggccgaactg cacgcggccg gctgcaccaa gctgttttcc gagaagatca ccggcaccag 2820

gcgcgaccgc ccggagctgg ccaggatgct tgaccaccta cgccctggcg acgttgtgac 2880

agtgaccagg ctagaccgcc tggcccgcag cacccgcgac ctactggaca ttgccgagcg 2940

catccaggag gccggcgcgg gcctgcgtag cctggcagag ccgtgggccg acaccaccac 3000

gccggccggc cgcatggtgt tgaccgtgtt cgccggcatt gccgagttcg agcgttccct 3060

aatcatcgac cgcacccgga gcgggcgcga ggccgccaag gcccgaggcg tgaagtttgg 3120

cccccgccct accctcaccc cggcacagat cgcgcacgcc cgcgagctga tcgaccagga 3180

aggccgcacc gtgaaagagg cggctgcact gcttggcgtg catcgctcga ccctgtaccg 3240

cgcacttgag cgcagcgagg aagtgacgcc caccgaggcc aggcggcgcg gtgccttccg 3300

tgaggacgca ttgaccgagg ccgacgccct ggcggccgcc gagaatgaac gccaagagga 3360

acaagcatga aaccgcacca ggacggccag gacgaaccgt ttttcattac cgaagagatc 3420

gaggcggaga tgatcgcggc cgggtacgtg ttcgagccgc ccgcgcacgt ctcaaccgtg 3480

cggctgcatg aaatcctggc cggtttgtct gatgccaagc tggcggcctg gccggccagc 3540

ttggccgctg aagaaaccga gcgccgccgt ctaaaaaggt gatgtgtatt tgagtaaaac 3600

agcttgcgtc atgcggtcgc tgcgtatatg atgcgatgag taaataaaca aatacgcaag 3660

gggaacgcat gaaggttatc gctgtactta accagaaagg cgggtcaggc aagacgacca 3720

tcgcaaccca tctagcccgc gccctgcaac tcgccggggc cgatgttctg ttagtcgatt 3780

ccgatcccca gggcagtgcc cgcgattggg cggccgtgcg ggaagatcaa ccgctaaccg 3840

ttgtcggcat cgaccgcccg acgattgacc gcgacgtgaa ggccatcggc cggcgcgact 3900

tcgtagtgat cgacggagcg ccccaggcgg cggacttggc tgtgtccgcg atcaaggcag 3960

ccgacttcgt gctgattccg gtgcagccaa gcccttacga catatgggcc accgccgacc 4020

tggtggagct ggttaagcag cgcattgagg tcacggatgg aaggctacaa gcggcctttg 4080

tcgtgtcgcg ggcgatcaaa ggcacgcgca tcggcggtga ggttgccgag gcgctggccg 4140

ggtacgagct gcccattctt gagtcccgta tcacgcagcg cgtgagctac ccaggcactg 4200

ccgccgccgg cacaaccgtt cttgaatcag aacccgaggg cgacgctgcc cgcgaggtcc 4260

aggcgctggc cgctgaaatt aaatcaaaac tcatttgagt taatgaggta aagagaaaat 4320

gagcaaaagc acaaacacgc taagtgccgg ccgtccgagc gcacgcagca gcaaggctgc 4380

aacgttggcc agcctggcag acacgccagc catgaagcgg gtcaactttc agttgccggc 4440

ggaggatcac accaagctga agatgtacgc ggtacgccaa ggcaagacca ttaccgagct 4500

gctatctgaa tacatcgcgc agctaccaga gtaaatgagc aaatgaataa atgagtagat 4560

gaattttagc ggctaaagga ggcggcatgg aaaatcaaga acaaccaggc accgacgccg 4620

tggaatgccc catgtgtgga ggaacgggcg gttggccagg cgtaagcggc tgggttgtct 4680

gccggccctg caatggcact ggaaccccca agcccgagga atcggcgtga cggtcgcaaa 4740

ccatccggcc cggtacaaat cggcgcggcg ctgggtgatg acctggtgga gaagttgaag 4800

gccgcgcagg ccgcccagcg gcaacgcatc gaggcagaag cacgccccgg tgaatcgtgg 4860

caagcggccg ctgatcgaat ccgcaaagaa tcccggcaac cgccggcagc cggtgcgccg 4920

tcgattagga agccgcccaa gggcgacgag caaccagatt ttttcgttcc gatgctctat 4980

gacgtgggca cccgcgatag tcgcagcatc atggacgtgg ccgttttccg tctgtcgaag 5040

cgtgaccgac gagctggcga ggtgatccgc tacgagcttc cagacgggca cgtagaggtt 5100

tccgcagggc cggccggcat ggccagtgtg tgggattacg acctggtact gatggcggtt 5160

tcccatctaa ccgaatccat gaaccgatac cgggaaggga agggagacaa gcccggccgc 5220

gtgttccgtc cacacgttgc ggacgtactc aagttctgcc ggcgagccga tggcggaaag 5280

cagaaagacg acctggtaga aacctgcatt cggttaaaca ccacgcacgt tgccatgcag 5340

cgtacgaaga aggccaagaa cggccgcctg gtgacggtat ccgagggtga agccttgatt 5400

agccgctaca agatcgtaaa gagcgaaacc gggcggccgg agtacatcga gatcgagcta 5460

gctgattgga tgtaccgcga gatcacagaa ggcaagaacc cggacgtgct gacggttcac 5520

cccgattact ttttgatcga tcccggcatc ggccgttttc tctaccgcct ggcacgccgc 5580

gccgcaggca aggcagaagc cagatggttg ttcaagacga tctacgaacg cagtggcagc 5640

gccggagagt tcaagaagtt ctgtttcacc gtgcgcaagc tgatcgggtc aaatgacctg 5700

ccggagtacg atttgaagga ggaggcgggg caggctggcc cgatcctagt catgcgctac 5760

cgcaacctga tcgagggcga agcatccgcc ggttcctaat gtacggagca gatgctaggg 5820

caaattgccc tagcagggga aaaaggtcga aaaggtctct ttcctgtgga tagcacgtac 5880

attgggaacc caaagccgta cattgggaac cggaacccgt acattgggaa cccaaagccg 5940

tacattggga accggtcaca catgtaagtg actgatataa aagagaaaaa aggcgatttt 6000

tccgcctaaa actctttaaa acttattaaa actcttaaaa cccgcctggc ctgtgcataa 6060

ctgtctggcc agcgcacagc cgaagagctg caaaaagcgc ctacccttcg gtcgctgcgc 6120

tccctacgcc ccgccgcttc gcgtcggcct atcgcggccg ctggccgctc aaaaatggct 6180

ggcctacggc caggcaatct accagggcgc ggacaagccg cgccgtcgcc actcgaccgc 6240

cggcgcccac atcaaggcac cctgcctcgc gcgtttcggt gatgacggtg aaaacctctg 6300

acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca 6360

agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggcgcagcca tgacccagtc 6420

acgtagcgat agcggagtgt atactggctt aactatgcgg catcagagca gattgtactg 6480

agagtgcacc atatgcggtg tgaaataccg cacagatgcg taaggagaaa ataccgcatc 6540

aggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga 6600

gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca 6660

ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg 6720

ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt 6780

cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc 6840

ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct 6900

tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc 6960

gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta 7020

tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca 7080

gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag 7140

tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag 7200

ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt 7260

agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa 7320

gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg 7380

attttggtca tgcattctag gtactaaaac aattcatcca gtaaaatata atattttatt 7440

ttctcccaat caggcttgat ccccagtaag tcaaaaaata gctcgacata ctgttcttcc 7500

ccgatatcct ccctgatcga ccggacgcag aaggcaatgt cataccactt gtccgccctg 7560

ccgcttctcc caagatcaat aaagccactt actttgccat ctttcacaaa gatgttgctg 7620

tctcccaggt cgccgtggga aaagacaagt tcctcttcgg gcttttccgt ctttaaaaaa 7680

tcatacagct cgcgcggatc tttaaatgga gtgtcttctt cccagttttc gcaatccaca 7740

tcggccagat cgttattcag taagtaatcc aattcggcta agcggctgtc taagctattc 7800

gtatagggac aatccgatat gtcgatggag tgaaagagcc tgatgcactc cgcatacagc 7860

tcgataatct tttcagggct ttgttcatct tcatactctt ccgagcaaag gacgccatcg 7920

gcctcactca tgagcagatt gctccagcca tcatgccgtt caaagtgcag gacctttgga 7980

acaggcagct ttccttccag ccatagcatc atgtcctttt cccgttccac atcataggtg 8040

gtccctttat accggctgtc cgtcattttt aaatataggt tttcattttc tcccaccagc 8100

ttatatacct tagcaggaga cattccttcc gtatctttta cgcagcggta tttttcgatc 8160

agttttttca attccggtga tattctcatt ttagccattt attatttcct tcctcttttc 8220

tacagtattt aaagataccc caagaagcta attataacaa gacgaactcc aattcactgt 8280

tccttgcatt ctaaaacctt aaataccaga aaacagcttt ttcaaagttg ttttcaaagt 8340

tggcgtataa catagtatcg acggagccga ttttgaaacc gcggtgatca caggcagcaa 8400

cgctctgtca tcgttacaat caacatgcta ccctccgcga gatcatccgt gtttcaaacc 8460

cggcagctta gttgccgttc ttccgaatag catcggtaac atgagcaaag tctgccgcct 8520

tacaacggct ctcccgctga cgccgtcccg gactgatggg ctgcctgtat cgagtggtga 8580

ttttgtgccg agctgccggt cggggagctg ttggctggct ggtggcagga tatattgtgg 8640

tgtaaacaaa ttgacgctta gacaacttaa taacacattg cggacgtttt taatgtactg 8700

aattaacgcc gaattaattc gggggatctg gattttagta ctggattttg gttttaggaa 8760

ttagaaattt tattgataga agtattttac aaatacaaat acatactaag ggtttcttat 8820

atgctcaaca catgagcgaa accctatagg aaccctaatt cccttatctg ggaactactc 8880

acacattatt atggagaaac tcgagcttgt cgatcgacag atccggtcgg catctactct 8940

atttctttgc cctcggacga gtgctggggc gtcggtttcc actatcggcg agtacttcta 9000

cacagccatc ggtccagacg gccgcgcttc tgcgggcgat ttgtgtacgc ccgacagtcc 9060

cggctccgga tcggacgatt gcgtcgcatc gaccctgcgc ccaagctgca tcatcgaaat 9120

tgccgtcaac caagctctga tagagttggt caagaccaat gcggagcata tacgcccgga 9180

gtcgtggcga tcctgcaagc tccggatgcc tccgctcgaa gtagcgcgtc tgctgctcca 9240

tacaagccaa ccacggcctc cagaagaaga tgttggcgac ctcgtattgg gaatccccga 9300

acatcgcctc gctccagtca atgaccgctg ttatgcggcc attgtccgtc aggacattgt 9360

tggagccgaa atccgcgtgc acgaggtgcc ggacttcggg gcagtcctcg gcccaaagca 9420

tcagctcatc gagagcctgc gcgacggacg cactgacggt gtcgtccatc acagtttgcc 9480

agtgatacac atggggatca gcaatcgcgc atatgaaatc acgccatgta gtgtattgac 9540

cgattccttg cggtccgaat gggccgaacc cgctcgtctg gctaagatcg gccgcagcga 9600

tcgcatccat agcctccgcg accggttgta gaacagcggg cagttcggtt tcaggcaggt 9660

cttgcaacgt gacaccctgt gcacggcggg agatgcaata ggtcaggctc tcgctaaact 9720

ccccaatgtc aagcacttcc ggaatcggga gcgcggccga tgcaaagtgc cgataaacat 9780

aacgatcttt gtagaaacca tcggcgcagc tatttacccg caggacatat ccacgccctc 9840

ctacatcgaa gctgaaagca cgagattctt cgccctccga gagctgcatc aggtcggaga 9900

cgctgtcgaa cttttcgatc agaaacttct cgacagacgt cgcggtgagt tcaggctttt 9960

tcatatctca ttgccccccc ggatctgcga aagctcgaga gagatagatt tgtagagaga 10020

gactggtgat ttcagcgtgt cctctccaaa tgaaatgaac ttccttatat agaggaaggt 10080

cttgcgaagg atagtgggat tgtgcgtcat cccttacgtc agtggagata tcacatcaat 10140

ccacttgctt tgaagacgtg gttggaacgt cttctttttc cacgatgctc ctcgtgggtg 10200

ggggtccatc tttgggacca ctgtcggcag aggcatcttg aacgatagcc tttcctttat 10260

cgcaatgatg gcatttgtag gtgccacctt ccttttctac tgtccttttg atgaagtgac 10320

agatagctgg gcaatggaat ccgaggaggt ttcccgatat taccctttgt tgaaaagtct 10380

caatagccct ttggtcttct gagactgtat ctttgatatt cttggagtag acgagagtgt 10440

cgtgctccac catgttatca catcaatcca cttgctttga agacgtggtt ggaacgtctt 10500

ctttttccac gatgctcctc gtgggtgggg gtccatcttt gggaccactg tcggcagagg 10560

catcttgaac gatagccttt cctttatcgc aatgatggca tttgtaggtg ccaccttcct 10620

tttctactgt ccttttgatg aagtgacaga tagctgggca atggaatccg aggaggtttc 10680

ccgatattac cctttgttga aaagtctcaa tagccctttg gtcttctgag actgtatctt 10740

tgatattctt ggagtagacg agagtgtcgt gctccaccat gttggcaagc tgctctagcc 10800

aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag 10860

gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca 10920

ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag 10980

cggataacaa tttcacacag gaaacagcta tgaccatgat tacgaattcc ccgatctagt 11040

aacatagatg acaccgcgcg cgataattta tcctagtttg cgcgctatat tttgttttct 11100

atcgcgtatt aaatgtataa ttgcgggact ctaatcataa aaacccatct cataaataac 11160

gtcatgcatt acatgttaat tattacatgc ttaacgtaat tcaacagaaa ttatatgata 11220

atcatcgcaa gaccggcaac aggattcaat cttaagaaac tttattgcca aatgtttgaa 11280

cgatcgggga aattcgagct catggctgac gagtcatgga gggcgccggc gatagtgcaa 11340

gagctggcgg cagccggcgt cgaggagccg ccgagccgat acctgctacg ggagaaagac 11400

cgttctgacg tcaagctggt cgccgccgag ctgccggagc ccctccccgt cgttgatctc 11460

agccggctag atggtgccga ggaggccacc aagctcaggg tggctctgca gaattggggc 11520

ttcttcctgt gaaaatctcg aaacagccgt gtcatagtca atcattaggt gttataggaa 11580

caatcaaagg ttttttcaag tgttaatctt catactaata tatacagtgg gtactcttta 11640

tctactgccg tggaactgtc atatttgatt atgaaatttt agctctagaa aatatttgat 11700

catcaatgtc aagactttat gaccttgcaa aatacatttc ctaattgaga acagggtaaa 11760

attatgaact atgcctctga accttcatac acaggcagca cattttttgt tgtaaaattc 11820

atcttaatat cagcggaaag actggaccag agaaagaaaa agttaagaca ggcatatact 11880

cttgatcctc taaaagagat gaggcggtac aatgatcaac catgaacatt aaagtgatac 11940

gtggaacatg agaacacaaa taattgtcac tggaacataa atacaaaaag gtttgtagta 12000

acttacccag gaagaagccc caattctgca gagccaccct gagcttggtg gcctcctcgg 12060

caccatctag ccggctgaga tcaacgacgg ggaggggctc cggcagctcg gcggcgacca 12120

gcttgacgtc agaacggtct ttctcccgta gcaggtatcg gctcggcggc tcctcgacgc 12180

cggctgccgc cagctcttgc actatcgccg gcgccctcca tgactcgtca gccatggatc 12240

ctctagagtc gacctgcaga agtaacacca aacaacaggg tgagcatcga caaaagaaac 12300

agtaccaagc aaataaatag cgtatgaagg cagggctaaa aaaatccaca tatagctgct 12360

gcatatgcca tcatccaagt atatcaagat caaaataatt ataaaacata cttgtttatt 12420

ataatagata ggtactcaag gttagagcat atgaatagat gctgcatatg ccatcatgta 12480

tatgcatcag taaaacccac atcaacatgt atacctatcc tagatcgata tttccatcca 12540

tcttaaactc gtaactatga agatgtatga cacacacata cagttccaaa attaataaat 12600

acaccaggta gtttgaaaca gtattctact ccgatctaga acgaatgaac gaccgcccaa 12660

ccacaccaca tcatcacaac caagcgaaca aaaagcatct ctgtatatgc atcagtaaaa 12720

cccgcatcaa catgtatacc tatcctagat cgatatttcc atccatcatc ttcaattcgt 12780

aactatgaat atgtatggca cacacataca gatccaaaat taataaatcc accaggtagt 12840

ttgaaacaga attctactcc gatctagaac gaccgcccaa ccagaccaca tcatcacaac 12900

caagacaaaa aaaagcatga aaagatgacc cgacaaacaa gtgcacggca tatattgaaa 12960

taaaggaaaa gggcaaacca aaccctatgc aacgaaacaa aaaaaatcat gaaatcgatc 13020

ccgtctgcgg aacggctaga gccatcccag gattccccaa agagaaacac tggcaagtta 13080

gcaatcagaa cgtgtctgac gtacaggtcg catccgtgta cgaacgctag cagcacggat 13140

ctaacacaaa cacggatcta acacaaacat gaacagaagt agaactaccg ggccctaacc 13200

atggaccgga acgccgatct agagaaggta gagagggggg gggggggagg acgagcggcg 13260

taccttgaag cggaggtgcc gacgggtgga tttgggggag atctggttgt gtgtgtgtgc 13320

gctccgaaca acacgaggtt ggggaaagag ggtgtggagg gggtgtctat ttattacggc 13380

gggcgaggaa gggaaagcga aggagcggtg ggaaaggaat cccccgtagc tgccggtgcc 13440

gtgagaggag gaggaggccg cctgccgtgc cggctcacgt ctgccgctcc gccacgcaat 13500

ttctggatgc cgacagcgga gcaagtccaa cggtggagcg gaactctcga gaggggtcca 13560

gaggcagcga cagagatgcc gtgccgtctg cttcgcttgg cccgacgcga cgctgctggt 13620

tcgctggttg gtgtccgtta gactcgtcga cggcgtttaa caggctggca ttatctactc 13680

gaaacaagaa aaatgtttcc ttagtttttt taatttctta aagggtattt gtttaatttt 13740

tagtcacttt attttattct attttatatc taaattatta aataaaaaaa ctaaaataga 13800

gttttagttt tcttaattta gaggctaaaa tagaataaaa tagatgtact aaaaaaatta 13860

gtctataaaa accattaacc ctaaacccta aatggatgta ctaataaaat ggatgaagta 13920

ttatataggt gaagctattt gcaaaaaaaa aggagaacac atgcacacta aaaagataaa 13980

actgtagagt cctgttgtca aaatactcaa ttgtccttta gaccatgtct aactgttcat 14040

ttatatgatt ctctaaaaca ctgatattat tgtagtacta tagattatat tattcgtaga 14100

gtaaagttta aatatatgta taaagataga taaactgcac ttcaaacaag tgtgacaaaa 14160

aaaatatgtg gtaatttttt ataacttaga catgcaatgc tcattatctc tagagagggg 14220

cacgaccggg tcacgctgca caagcttggc actggccgtc gttttacaac gtcgtgactg 14280

ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg 14340

gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg 14400

cgaatgctag agcagcttga gcttggatca gattgtcgtt tcccgccttc agtttagctt 14460

catggagtca aagattcaaa tagaggacct aacagaactc gccgtaaaga ctggcgaaca 14520

gttcatacag agtctcttac gactcaatga caagaagaaa atcttcgtca acatggtgga 14580

gcacgacaca cttgtctact ccaaaaatat caaagataca gtctcagaag accaaagggc 14640

aattgagact tttcaacaaa gggtaatatc cggaaacctc ctcggattcc attgcccagc 14700

tatctgtcac tttattgtga agatagtgga aaaggaaggt ggctcctaca aatgccatca 14760

ttgcgataaa ggaaaggcca tcgttgaaga tgcctctgcc gacagtggtc ccaaagatgg 14820

acccccaccc acgaggagca tcgtggaaaa agaagacgtt ccaaccacgt cttcaaagca 14880

agtggattga tgtgatatct ccactgacgt aagggatgac gcacaatccc actatccttc 14940

gcaagaccct tcctctatat aaggaagttc atttcatttg gagagaacac gggggactct 15000

tgac 15004

<210> 5

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

atcggggaaa ttcgagctca tggctgacga gtcatggagg 40

<210> 6

<211> 32

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

gagattttca caggaagaag ccccaattct gc 32

<210> 7

<211> 33

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

cttcttcctg tgaaaatctc gaaacagccg tgt 33

<210> 8

<211> 46

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

cttcttcctg ggtaagttac tacaaacctt tttgtattta tgttcc 46

<210> 9

<211> 33

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

agtaacttac ccaggaagaa gccccaattc tgc 33

<210> 10

<211> 45

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

ctgcaggtcg actctagagg atccatggct gacgagtcat ggagg 45

<210> 11

<211> 300

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

tgaaaatctc gaaacagccg tgtcatagtc aatcattagg tgttatagga acaatcaaag 60

gttttttcaa gtgttaatct tcatactaat atatacagtg ggtactcttt atctactgcc 120

gtggaactgt catatttgat tatgaaattt tagctctaga aaatatttga tcatcaatgt 180

caagacttta tgaccttgca aaatacattt cctaattgag aacagggtaa aattatgaac 240

tatgcctctg aaccttcata cacaggcagc acattttttg ttgtaaaatt catcttaata 300

<210> 12

<211> 240

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

tgaaaatctc gaaacagccg tgtcatagtc aatcattagg tgttatagga acaatcaaag 60

gttttttcaa gtgttaatct tcatactaat atatacagtg ggtactcttt atctactgcc 120

gtggaactgt catatttgat tatgaaattt tagctctaga aaatatttga tcatcaatgt 180

caagacttta tgaccttgca aaatacattt cctaattgag aacagggtaa aattatgaac 240

Claims (10)

1. An RNAi plant expression vector, which is characterized by comprising a hairpin structure expression cassette, wherein the hairpin structure expression cassette comprises a hairpin structure formed by DNA fragments shown in SEQ ID No. 1-3.

2. The RNAi plant expression vector of claim 1, wherein the promoter of the hairpin expression cassette is one of a rice or maize Ubi promoter, Rubisco small subunit promoter, Cab promoter, CAMV 35S promoter or Actin promoter; preferably, the promoter is a Ubi promoter of rice or maize.

3. The RNAi plant expression vector of claim 1, further comprising a selectable marker expression cassette comprising a promoter, a marker gene, and a terminator, wherein the promoter is a Ubi promoter, CAMV 35S promoter, or an Actin promoter of rice or maize; the marker gene is a gene of enzyme capable of generating color change, a fluorescence marker gene, an antibiotic marker gene, a herbicide screening marker gene or an anti-chemical agent marker gene, and the terminator is a NOS terminator or a Ubi terminator.

4. The RNAi plant expression vector of any one of claims 1-3, having the nucleotide sequence shown in SEQ ID No. 4.

5. A biomaterial comprising the RNAi plant expression vector of any one of claims 1-4, wherein the biomaterial is an engineered bacterium, a cell, a callus, a plant, a seed.

6. The method for constructing an RNAi plant expression vector according to any one of claims 1 to 4, comprising linking the DNA fragment represented by SEQ ID Nos. 1 to 3 to a binary plant transformation vector by overlapping.

7. A method of interfering with the expression of plant HPPD inhibitor-sensitive gene HIS1, characterized in that a plant is transformed with the RNAi plant expression vector of any one of claims 1-4.

8. The method of claim 7, comprising:

(1) constructing RNAi plant expression vector: connecting the DNA fragment shown in SEQ ID No.1-3 to SacI and BamHI sites of a plant binary transformation vector pTCK303 by overlapping method to obtain an RNAi plant expression vector pTCK303-HIS1 i-1;

(2) and (3) transformation: introducing pTCK303-HIS1i-1 into Agrobacterium EHA105 strain, transforming callus, and inducing and differentiating the transformed callus to obtain dominant sensitive beta-triketone herbicide plant.

9. Use of the RNAi plant expression vector of any one of claims 1-4 to interfere with expression of plant HIS1 gene, to produce a beta-triketone herbicide dominant sensitive plant.

10. Use of the RNAi plant expression vector of any one of claims 1-4 for breeding new transgenic varieties, hybrid rice seed production, or preventing transgene escape.

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