CN112575028A - RNAi plant expression vector for inhibiting expression of HIS1 gene and application thereof - Google Patents

Abstract

The invention provides an RNAi plant expression vector for inhibiting HIS1 gene expression and application thereof, a method for interfering plant HIS1 expression and a method for obtaining a plant dominantly sensitive to a beta-triketone herbicide. 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 for inhibiting expression of HIS1 gene and application thereof

Technical Field

The invention belongs to the technical field of agricultural biology, and particularly relates to a plant expression vector for inhibiting expression of HPPD inhibitor sensitive type 1(HIS1) gene, and a construction method 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-tribeno herbicides, bTHs) are 4-hydroxyphenylpyruvate dioxygenase (HPPD) inhibitors, are widely applied to agriculture, and can effectively prevent and kill various broadleaf weeds and gramineous 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 branched-specific resistance to β -tribetone binding." science365.6451(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 a plant expression vector capable of effectively influencing expression of an HPPD inhibitor sensitive type 1 gene (HIS1) and a construction method 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 325bp based on the HIS1 coding frame, and the DNA fragment shown in SEQ ID No.3 is a DNA fragment with the length of 325bp based on the HIS1 coding frame and is reversely complementary with the DNA fragment shown in SEQ ID No. 1. 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-3.

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-3 stem-loop structures, callus, transgenic seedlings, seeds and the like all 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-3.

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-3-F:ATCGGGGAAATTCGAGCTcGGATGTTCTGAACAAGTATGCATCAG

HIS1i-3-Rv1:GAGATTTTCACTCCATGGTGTCACCTAAGTTGAT

HIS1i-3-F2:ACACCATGGAGTGAAAATCTCGAAACAGCCGTGT

HIS1i-3-Rv2:ACACCATGGAGGGTAAGTTACTACAAACCTTTTTGTATTTATGTTCC

HIS1i-3-F3:AGTAACTTACCCTCCATGGTGTCACCTAAGTTGAT

HIS1i-3-Rv:CTGCAGGTCGACTCTAGAGgatccGGATGTTCTGAACAAGTATGCATCAG

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

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-3 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 herbicide dominant-sensitive plant, which comprises the step of transforming a 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 herbicide dominant sensitive plant 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-3;

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

Wherein the RNAi plant expression vector pTCK303-HIS1i-3 has a nucleotide sequence shown in SEQ ID No. 4.

Wherein the herbicide may be a beta-triketone herbicide.

The beta-triketone herbicide provided by the invention comprises, but is not limited to, mesotrione and benzobicyclon. In the embodiment of the invention, the verification shows that the minimum concentration of the mesotrione sprayed and screened mesotrione sensitive transgenic strain is 2250mg/L, and the minimum concentration of the mesotrione sprayed and screened mesotrione sensitive transgenic strain is 15 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 cultivating new transgenic varieties, hybrid rice seed production or preventing transgene escape.

The invention constructs a plant binary transgenic vector aiming at a rice HPPD inhibitor sensitive type 1 gene (HIS1) by screening based on an RNAi technology, the vector can efficiently inhibit the HPPD inhibitor sensitive type 1 gene HIS1 vector, the sequence of the vector is a rice endogenous sequence completely, 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 HIS1i-3-1, HIS1i-3-2 and HIS1 i-3-33 target fragments in example 2 of the present application. Wherein lanes 1-6 are HIS1i-3-1 target fragments; lanes 7-12 are HIS1i-3-2 target fragments; lanes 13-18 are HIS1i-3-3 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-3 vector digested with SacI and BamHI. CK in the figure is pTCK303-HIS1i-3 monoclonal recombinant plasmid; lanes 1-5 are the digested monoclonal recombinant plasmids, M is marker, the size of the excised fragment is 1128bp, 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. Lanes 1, H in the figure₂O; lane 2 negative control (middle flower 11 non-transgenic plants); lane 3, positive control (pTCK303-HIS1i-3 plasmid); lanes 4-24, transgenic lines (where lanes 4,10,12,19 and 23 are transgenic negative lines); m, Marker.

FIG. 5 is a sensitivity test of pTCK303-HIS1i-3 transgenic T0 trilobate seedlings to 2250mg/L bicyclosulfulone solution for 14 d. Control WT is shown in outline as a ZH11 non-transgenic plant; the whole plant is withered, and the dying plant is a highly sensitive plant; the leaves are whitish, and the leaf tips are withered and yellow, so that the plants are moderate sensitive plants; no response is a resistant strain.

FIG. 6 is a sensitivity test of pTCK303-HIS1i-3 transgenic T0 trilobate seedling leaves to 15mg/L mesotrione solution for 14 d. WT is a ZH11 non-transgenic plant, 77-2 and 101-1 are sensitive lines.

FIG. 7 shows the result of 6000mg/L of benzobicyclon 14d sprayed on transgenic seedlings after RNAi vectors are constructed after different loop sequences are used to replace SEQ ID No.2 sequences 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-3-F:ATCGGGGAAATTCGAGCTcGGATGTTCTGAACAAGTATGCATCAG(SEQ ID No.5)

HIS1i-3-Rv1:GAGATTTTCACTCCATGGTGTCACCTAAGTTGAT(SEQ ID No.6)

HIS1i-3-F2:ACACCATGGAGTGAAAATCTCGAAACAGCCGTGT(SEQ ID No.7)

HIS1i-3-Rv2:ACACCATGGAGGGTAAGTTACTACAAACCTTTTTGTATTTATGTTCC(SEQ ID No.8)

HIS1i-3-F3:AGTAACTTACCCTCCATGGTGTCACCTAAGTTGAT(SEQ ID No.9)

HIS1i-3-Rv:CTGCAGGTCGACTCTAGAGgatccGGATGTTCTGAACAAGTATGCATCAG(SEQ ID No.10)

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 perform PCR amplification of HIS1i-3 gene fragments 1 and 3 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-3 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 325bp, 478bp and 325bp, respectively, and named HIS1i-3-1, HIS1i-3-2 and HIS1i-3-3, 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-3-1, HIS1i-3-2 and HIS1i-3-3 are connected to the pTCK303 vector by the following connection system:

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

The ligation product is transformed into an escherichia coli DH5 alpha competent cell, 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 a recombinant is sent to sequencing to verify the correctness, namely, a HIS1i-3RNAi 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-3.

Example 3 Agrobacterium transformation of Rice and identification of transgenic plants

Introducing the pTCK303-HIS1i-3 recombinant plasmid into an Agrobacterium EHA105 strain, transforming 11 calli of japonica rice flowers, screening hygromycin resistance, differentiating and rooting to obtain 21 regenerated transgenic strains, identifying the hygromycin resistance gene transferred into the transgenic strains through PCR (polymerase chain reaction), and transplanting the positive strains into soil to survive 15 strains as shown by the result, wherein 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, a 2250mg/L-4500mg/L solution of bicyclosulfoncotrione was prepared from a 25% mother liquor of bicyclosulfoncotrione (Shishy bioscience, Japan, lot No. 2019050603), and pTCK303-HIS1i-3 transgenic T0 trilobate seedlings obtained in example 3 were sprayed and continuously observed after spraying.

Spraying HIS1i-3 transgenic 11 strains at 2250mg/L of mesotrione, wherein 14d after spraying, the 3 strains have white and withered leaves, and belong to highly sensitive strains; 7 strains all have white leaves to withered yellow tips of the leaves, and belong to middle sensitive strains; 1 line appeared withered and yellow leaf tips, belonging to a low-sensitive line (FIG. 5). The results show that the invention successfully obtains the transgenic material with dominant sensitivity to the herbicide.

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

To test the effect of the designed interference vector fragments in the transgenic plants, mesotrione mother liquor (Jiangsu Fengshan group Co., Ltd., batch No. 20190119004) was used to prepare a 15mg/L-750mg/L mesotrione solution, and the pTCK303-HIS1i-3 transgenic T0-generation trifoliate-stage seedling leaves obtained in example 3 were sprayed and continuously observed after spraying.

Spraying HIS1i-3 transgenic 4 strain leaves on mesotrione, and after spraying 15mg/L mesotrione for 7d, curling the leaf tips of partial strains of HIS1i-3 transgenic T0 seedlings, wherein 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 spraying of 2 strains, and the strain belongs to a resistant strain. The results show that the invention successfully obtains the transgenic material with dominant sensitivity to the herbicide.

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 2250mg/L-4500mg/L bicyclosulfuron-methyl respectively, and the result shows that the transgenic line shows higher resistance. When the concentration of the benzobicylon is more than 6000mg/L, sensitive phenotype exists, and the proportion of sensitive strains is low, which indicates that the interference efficiency of OsHIS1i of the stem-loop designed according to the method on HIS1 genes is poor. The test results of the OsHIS1i transgenic line T0 generation 3-5 leaf stage seedlings designed by the method on 6000mg/L dicyclo sulcotrione are shown in figure 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 hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

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tgcactcaat gtacaccgac atgtggagtg aagagtatca gtgtgcatgg ctggatatgt 1800

atcaccgcgt ctttgatcgc gtcagcgccg tcgtcggtga acaggtatgg aatttcgccg 1860

attttgcgac ctcgcaaggc atattgcgcg ttggcggtaa caagaaaggg atcttcactc 1920

gcgaccgcaa accgaagtcg gcggcttttc tgctgcaaaa acgctggact ggcatgaact 1980

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 cggatgttct gaacaagtat gcatcaggaa ccaaaagaat 11340

tagagacgat atcattcagg ctatggccaa gcttcttgag cttgatgagg attacttctt 11400

ggaccgactc aacgaagctc ctgcatttgc aagattcaac tactaccctc cctgtccaag 11460

gcctgacctt gtgttcggca tcaggcctca ctccgacggc accctcttga cgattcttct 11520

cgtcgacaaa gatgtcagtg gcctgcaagt tcagagggat ggcaagtggt ccaacgttga 11580

ggcaactcct cacacattgc tgatcaactt aggtgacacc atggagtgaa aatctcgaaa 11640

cagccgtgtc atagtcaatc attaggtgtt ataggaacaa tcaaaggttt tttcaagtgt 11700

taatcttcat actaatatat acagtgggta ctctttatct actgccgtgg aactgtcata 11760

tttgattatg aaattttagc tctagaaaat atttgatcat caatgtcaag actttatgac 11820

cttgcaaaat acatttccta attgagaaca gggtaaaatt atgaactatg cctctgaacc 11880

ttcatacaca ggcagcacat tttttgttgt aaaattcatc ttaatatcag cggaaagact 11940

ggaccagaga aagaaaaagt taagacaggc atatactctt gatcctctaa aagagatgag 12000

gcggtacaat gatcaaccat gaacattaaa gtgatacgtg gaacatgaga acacaaataa 12060

ttgtcactgg aacataaata caaaaaggtt tgtagtaact taccctccat ggtgtcacct 12120

aagttgatca gcaatgtgtg aggagttgcc tcaacgttgg accacttgcc atccctctga 12180

acttgcaggc cactgacatc tttgtcgacg agaagaatcg tcaagagggt gccgtcggag 12240

tgaggcctga tgccgaacac aaggtcaggc cttggacagg gagggtagta gttgaatctt 12300

gcaaatgcag gagcttcgtt gagtcggtcc aagaagtaat cctcatcaag ctcaagaagc 12360

ttggccatag cctgaatgat atcgtctcta attcttttgg ttcctgatgc atacttgttc 12420

agaacatccg gatcctctag agtcgacctg cagaagtaac accaaacaac agggtgagca 12480

tcgacaaaag aaacagtacc aagcaaataa atagcgtatg aaggcagggc taaaaaaatc 12540

cacatatagc tgctgcatat gccatcatcc aagtatatca agatcaaaat aattataaaa 12600

catacttgtt tattataata gataggtact caaggttaga gcatatgaat agatgctgca 12660

tatgccatca tgtatatgca tcagtaaaac ccacatcaac atgtatacct atcctagatc 12720

gatatttcca tccatcttaa actcgtaact atgaagatgt atgacacaca catacagttc 12780

caaaattaat aaatacacca ggtagtttga aacagtattc tactccgatc tagaacgaat 12840

gaacgaccgc ccaaccacac cacatcatca caaccaagcg aacaaaaagc atctctgtat 12900

atgcatcagt aaaacccgca tcaacatgta tacctatcct agatcgatat ttccatccat 12960

catcttcaat tcgtaactat gaatatgtat ggcacacaca tacagatcca aaattaataa 13020

atccaccagg tagtttgaaa cagaattcta ctccgatcta gaacgaccgc ccaaccagac 13080

cacatcatca caaccaagac aaaaaaaagc atgaaaagat gacccgacaa acaagtgcac 13140

ggcatatatt gaaataaagg aaaagggcaa accaaaccct atgcaacgaa acaaaaaaaa 13200

tcatgaaatc gatcccgtct gcggaacggc tagagccatc ccaggattcc ccaaagagaa 13260

acactggcaa gttagcaatc agaacgtgtc tgacgtacag gtcgcatccg tgtacgaacg 13320

ctagcagcac ggatctaaca caaacacgga tctaacacaa acatgaacag aagtagaact 13380

accgggccct aaccatggac cggaacgccg atctagagaa ggtagagagg gggggggggg 13440

gaggacgagc ggcgtacctt gaagcggagg tgccgacggg tggatttggg ggagatctgg 13500

ttgtgtgtgt gtgcgctccg aacaacacga ggttggggaa agagggtgtg gagggggtgt 13560

ctatttatta cggcgggcga ggaagggaaa gcgaaggagc ggtgggaaag gaatcccccg 13620

tagctgccgg tgccgtgaga ggaggaggag gccgcctgcc gtgccggctc acgtctgccg 13680

ctccgccacg caatttctgg atgccgacag cggagcaagt ccaacggtgg agcggaactc 13740

tcgagagggg tccagaggca gcgacagaga tgccgtgccg tctgcttcgc ttggcccgac 13800

gcgacgctgc tggttcgctg gttggtgtcc gttagactcg tcgacggcgt ttaacaggct 13860

ggcattatct actcgaaaca agaaaaatgt ttccttagtt tttttaattt cttaaagggt 13920

atttgtttaa tttttagtca ctttatttta ttctatttta tatctaaatt attaaataaa 13980

aaaactaaaa tagagtttta gttttcttaa tttagaggct aaaatagaat aaaatagatg 14040

tactaaaaaa attagtctat aaaaaccatt aaccctaaac cctaaatgga tgtactaata 14100

aaatggatga agtattatat aggtgaagct atttgcaaaa aaaaaggaga acacatgcac 14160

actaaaaaga taaaactgta gagtcctgtt gtcaaaatac tcaattgtcc tttagaccat 14220

gtctaactgt tcatttatat gattctctaa aacactgata ttattgtagt actatagatt 14280

atattattcg tagagtaaag tttaaatata tgtataaaga tagataaact gcacttcaaa 14340

caagtgtgac aaaaaaaata tgtggtaatt ttttataact tagacatgca atgctcatta 14400

tctctagaga ggggcacgac cgggtcacgc tgcacaagct tggcactggc cgtcgtttta 14460

caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc 14520

cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg 14580

cgcagcctga atggcgaatg ctagagcagc ttgagcttgg atcagattgt cgtttcccgc 14640

cttcagttta gcttcatgga gtcaaagatt caaatagagg acctaacaga actcgccgta 14700

aagactggcg aacagttcat acagagtctc ttacgactca atgacaagaa gaaaatcttc 14760

gtcaacatgg tggagcacga cacacttgtc tactccaaaa atatcaaaga tacagtctca 14820

gaagaccaaa gggcaattga gacttttcaa caaagggtaa tatccggaaa cctcctcgga 14880

ttccattgcc cagctatctg tcactttatt gtgaagatag tggaaaagga aggtggctcc 14940

tacaaatgcc atcattgcga taaaggaaag gccatcgttg aagatgcctc tgccgacagt 15000

ggtcccaaag atggaccccc acccacgagg agcatcgtgg aaaaagaaga cgttccaacc 15060

acgtcttcaa agcaagtgga ttgatgtgat atctccactg acgtaaggga tgacgcacaa 15120

tcccactatc cttcgcaaga cccttcctct atataaggaa gttcatttca tttggagaga 15180

acacggggga ctcttgac 15198

<210> 5

<211> 45

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

atcggggaaa ttcgagctcg gatgttctga acaagtatgc atcag 45

<210> 6

<211> 34

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

gagattttca ctccatggtg tcacctaagt tgat 34

<210> 7

<211> 34

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

acaccatgga gtgaaaatct cgaaacagcc gtgt 34

<210> 8

<211> 47

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

acaccatgga gggtaagtta ctacaaacct ttttgtattt atgttcc 47

<210> 9

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

agtaacttac cctccatggt gtcacctaag ttgat 35

<210> 10

<211> 50

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

ctgcaggtcg actctagagg atccggatgt tctgaacaag tatgcatcag 50

<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 for inhibiting expression of HPPD inhibitor sensitive type 1 gene, 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-3;

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

9. Use of the RNAi plant expression vector of any one of claims 1-4 for interfering with expression of plant HIS1 gene, in the preparation of plants dominantly sensitive to triketones.

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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