CN101709300B - Method for quickly constructing artificial mi RNA gene interference vector of paddy - Google Patents

Abstract

The invention relates to a method for quickly constructing an artificial mi RNA gene interference vector of paddy, in particular to a DNA sequence and a synthesizing method thereof for quickly modifying a paddy silencing vector, and the high efficiency paddy artificial mi RNA gene interference vector established by adopting the DNA sequence. The method comprises the following steps: firstly, placing invariant regions of two ends of a related silencing fragment in a transgenic target vector in advance; and then, connecting a core silencing fragment (wi-site) into a modified vector to directly complete the vector construction. When a head fragment and a tail fragment are selected, the DNA sequence is analyzed to ensure that an AfeI restriction enzyme cutting site can be acquired after the head fragment and the tail fragment are connected; after colibacillus is transformed, the vector can be prepared massively, can be linearized by using AfeI incision enzyme, and can be directly connected with the core silencing fragment synthesized by adopting the method to complete construction of the paddy silencing vector. The invention expands the invariant DNA regions on the target vector so asto simplify the method for constructing the single silencing vector, accelerate the constructing process, and improve the efficiency of vector construction.

Description

Method for quickly constructing artificial mi RNA gene interference vector of paddy

Technical field

The present invention relates to method for quickly constructing artificial mi RNA gene interference vector of paddy, relate in particular to the quick silent carrier transformation of paddy rice with the compound method of dna sequence dna and this dna sequence dna and adopt this dna sequence dna to be used to set up artificial mi RNA gene interference vector of paddy efficiently.

Technical background

It is core with Overlapping PCR that existing method makes up the paddy gene silent carrier, through the method structure paddy gene silent carrier (Warthmann et al., 2008) of PCR replacement rice Os-amiR528.Existing method makes up to single silent carrier and needs to adopt four PCR primers that have reticent correlated series; Cooperate with two consensus primers, the pNW55 carrier is increased, the 3 segment DNA fragments that amplification is obtained reclaim; Merge PCR again; Be connected into the T carrier, be connected into conversion carrier after enzyme is cut again, finally obtain the silent carrier that transgenic is used.Method specifically is divided into four steps:

The first step, through interfere synthetic 4 primers that length is 40bp of design to each different gene, synthetic two common combination of primers are used for and the reticent relevant dna fragmentation of gene specific primer combination amplification.

Second step, congratulate through reticent primer and two consensus primers of 4 gene specifics, amplification acquisition three segment length are 256bp respectively; 259bp, the dna fragmentation of 87bp is through the electrophoretic separation dna fragmentation; Cutting contains the gel of target sizes DNA, carries out dna fragmentation and reclaims.

In the 3rd step, three sections PCR products that recovery is obtained mix, and again with two public PCR that merge, obtaining length is the dna fragmentation of 554bp, and through the electrophoretic separation dna fragmentation, cutting contains the gel of target sizes DNA, carry out dna fragmentation and reclaim.

The 4th step was connected to T carrier or other intermediate carriers with the 554bp fragment after reclaiming, through connecting, transform the intestinal bacteria bacterium colony that obtains to carry respective carrier; Behind the amplification thalline, extraction has reticent segmental intermediate carrier, adopts restriction enzyme to carry out enzyme again and cuts; Obtain the terminal dna fragmentation of toughness, be connected with destination carrier again, through connecting, transform the intestinal bacteria bacterium colony that obtains to carry conversion carrier; Order-checking is used for Agrobacterium-mediated Transformation after detecting correctly.

Existing method carrier construction needs four big steps, and the content of each procedure is quite loaded down with trivial details again, needs the experimenter to have certain molecular biology operation basis.The method that this patent relates to is two steps of needs only, and each procedure is also very simple, and the experimenter with simple molecules Basic of Biology also can be quick, accomplishes the vector construction task accurately.

Patent content

In order to solve the technological deficiency of above-mentioned existence; First purpose of the present invention provides the quick silent carrier transformation of paddy rice with dna sequence dna and compound method thereof; Second purpose of the present invention provides and is used for carrier that efficiently makes up the paddy gene silent carrier and preparation method thereof, and the 3rd purpose of the present invention provides paddy gene silent carrier and preparation method thereof.The present invention has simplified the method that single silent carrier makes up, and has quickened building process, has improved the efficient of vector construction.

In order to realize first above-mentioned purpose, the technical scheme below the present invention has adopted:

Dna sequence dna is used in the quick silent carrier transformation of paddy rice, and this dna sequence dna comprises the dna sequence dna shown in SEQ ID NO:1, and SEQ ID NO:1 is as follows said:

Cagcagcagccacagcaaaatttggtttgggataggtaggtgttatgttaggtctg gttttttggctgtagcagcagcgctgctaggctgttctgtggaagtttgcagagtt tatattatgggtttaatcgtccatggcatcagcatcagcagc; Wherein, agcgct partly is the AfeI restriction enzyme site.

As preferably, dna sequence dna is used in the quick silent carrier transformation of described paddy rice, and this dna sequence dna is shown in SEQ IDNO:2, and SEQ ID NO:2 is as follows said:

TcGAGCTCcagcagcagccacagcaaaatttggtttgggataggtaggtgttatgt taggtctggttttttggctgtagcagcagcgctgctaggctgttctgtggaagttt gcagagtttatattatgggtttaatcgtccatggcatcagcatcagcagcGGTACC ga; Wherein, tcGAGCTC and GGTACCga partly are cloning site and protection base, and agcgct partly is the AfeI restriction enzyme site.

The quick silent carrier transformation of above-mentioned paddy rice is synthetic with the DNA joining method; This method adopts rice Os-amiR528DNA gene silencing sequence as the basis; Sequence is shown in SEQ ID NO:3; This sequence of paddy rice is divided into first fragment, cauda section and the reticent fragment (as shown in Figure 2) of core, through pcr amplification and merge first fragment and the cauda section, is used for the transformation of rapid gene silent carrier then.

SEQ ID NO:3 is as follows said:

cagcagcagccacagcaaaatttggtttgggataggtaggtgttatgttaggtctggttttttggctgtagcagcagcagtggaaggggcatgcagaggagcaggagattcagtttgaagctggacttcacttttgcctctctctcctgtgcttgcctcttccattcctgctgctaggctgttctgtggaagtttgcagagtttatattatgggtttaatcgtccatggcatcagcatcagcagc

First fragment such as SEQ ID NO:5, SEQ ID NO:5 is as follows said:

cagcagcagccacagcaaaatttggtttgggataggtaggtgttatgttaggtctggttttttggctgtagcagcagc；

The cauda section, SEQ ID NO:6, SEQ ID NO:6 is as follows said:

gctgctaggctgttctgtggaagtttgcagagtttatattatgggtttaatcgtccatggcatcagcatcagcagc；

Through sequential analysis and design, make wherein first fragment by the ending of AGC site, and interfere the cauda section initial by the GCT position.So first fragment, the splicing of cauda section obtain and just in time obtain to such an extent that dna sequence dna is used in a silent carrier transformation that comprises the AfeI restriction enzyme site; First segmental front end and the segmental tail end of tail have comprised restriction enzyme site and corresponding protection base simultaneously, cut and connect the destination carrier (shown in Fig. 3 a) of transformation in order to enzyme.

As preferably, the quick silent carrier transformation of above-mentioned paddy rice comprises the steps: with the compound method of dna sequence dna

1. primer is synthetic: synthetic 7 primers, and sequence is following:

2. adopt the substep salvage to transform fragment

Adopt synthetic this fragment of method of stepwise synthesis, fragment before amiR-F1, amiR-AfeI, amiR-R1 and amiR-R2 are synthetic, amiR-F4, amiR-AfeI, amiR-R2 and amiR-R4-G-R-KpnI synthesize the cauda section;

Preferred as again, synthetic leading portion and back segment PCR system concentration and reaction conditions are:

3. transform segmental splicing

Dilute 100 times respectively, adopt G-F-SacI and amiR-R4-G-R-KpnI primer again with merging two sections PCR products, the synthetic segmental DNA chain of complete transformation that has;

Preferred as again, corresponding PCR system concentration and reaction conditions are:

After the completion, be used to transform the reticent fragment sequence of paddy rice rapid gene shown in SEQ ID NO:1.

In order to realize second above-mentioned purpose, the technical scheme below the present invention has adopted:

Be used for efficiently making up the carrier of paddy gene silent carrier, this carrier contain just like SEQ ID NO:1 or shown in SEQ ID NO:2 the gene fragment of dna sequence dna.

The above-mentioned preparation method who is used for efficiently making up the carrier of paddy gene silent carrier, this method comprises the steps:

1. carrier is transformed being connected of fragment and destination carrier

Adopt the gene fragment of dna sequence dna shown in SEQ ID NO:1 or the SEQ ID N0:2; Adopt SacI and KpnI endonuclease digestion again; If other MCS of the employing of destination carrier then should be revised restriction enzyme site when design of primers; And carry out enzyme with corresponding restriction endonuclease herein and cut, be connected into unmodified destination carrier then;

Preferred linked system is following:

Linked system after mixing is spent the night 16 ℃ of water-baths;

2. transform the destination carrier transformed into escherichia coli of accomplishing

Get and connect product 5ul, the CaCl of adding 100ul ₂The DH5 α thermal shock competence of handling, 42 ℃ of thermal shocks were handled 90 seconds, added the LB nonreactive substratum of 800ul, recovered 1 hour with 250rpm at 37 ℃; Under aseptic condition, get 100ul and be coated on the LB culture plate that contains the 50ug/ml kantlex, cultivated 16 hours for 37 ℃, select transformant, insert and send order-checking to confirm after LB cultivates again; The correct bacterium of checking order adopts 25% glycerine frozen, in order to life-time service.

In order to realize the 3rd above-mentioned purpose, the technical scheme below the present invention has adopted:

Paddy rice silent carrier, this carrier contain the gene fragment of dna sequence dna shown in SEQ ID NO:1 or the SEQ ID NO:2.

The fast construction method of above-mentioned paddy rice silent carrier, this method comprises the steps:

1. plasmid is prepared and linearizing

The carrier that is used for efficiently making up the paddy gene silent carrier of above-mentioned acquisition is shaken the bacterium amplification, adopt the plasmid extraction test kit to carry out the DNA extracting, use the AfeI digested plasmid then;

It is following that enzyme is cut system:

Cut at 37 ℃ of following enzymes and to spend the night, the DNA after enzyme is cut adopts 1% agarose gel electrophoresis, with behind the ethidium bromide staining at uv lamp incision glue, adopt gel to reclaim test kit and reclaim enzyme and cut product; Carrier after the linearizing is the universal support fragment, can be used for the structure of all quick silent carriers, can be subsequent use-20 ℃ of long-time down preservations.

2. bridge-clip is synthetic

Two of the segmental primers of synthetic bridging, sequence is following:

Primer is diluted to 100uM, gets 5ul respectively, handled 5 minutes for 95 ℃, naturally cooling is annealed to room temperature state; Dilute 100 times subsequent use; This bridge-clip is general fragment, can be used for the structure of all quick silent carriers, can be subsequent use-20 ℃ of long-time down preservations.

3. the reticent segmental amplification of core

Be two of the reticent associated clip primers of each gene difference synthetic gene:

At first, exist Http:// wmd2.weigelworld.org/Design artificial mi RNA sequence (Warthmann etal., 2008).Artificial mi RNA fragment with design obtains increases AG and CAGGAGATTCAGTTTG at two and forms gene wi-Primerl; The artificial mi RNA fragment is carried out G-C with 12 and 16, and the A-T transversion increases AGGAA and AGAGAGGCAAAAGTG again at two, form wi-Primer2.Like following table, the black table worker miRNA fragment of leting others have a look at, grey color part is represented the sequence of adding, underscore representes to carry out the site of transversion.

Get the 1ul bridge-clip, adopt gene specific primer to carry out pcr amplification, preferred amplification condition is following:

4. the reticent fragment of core and carrier is connected

Need not to reclaim, the fragment PCR products that obtains of directly will increasing directly is connected with linearizing carrier, and preferably linked system is following:

Linked system after mixing is spent the night 16 ℃ of water-baths.

5. transformed into escherichia coli bacterial strain

This patent recommends to use conventional DH5 α bacterial strain to carry out the transformation of junction fragment.Get and connect product 5ul, the CaCl of adding 100ul ₂The DH5 α thermal shock competence bacteria of handling, 42 ℃ of thermal shocks were handled 90 seconds, added the LB nonreactive substratum of 800ul, recovered 1 hour with 250rpm at 37 ℃; Under aseptic condition, get 100ul and be coated on the LB culture plate that contains the 50ug/ml kantlex, cultivated 16 hours for 37 ℃, select transformant, insert LB and cultivate again.

6. the detection of gene silencing carrier

Owing to adopted flat terminal the connection, must select connection transformant in the right direction, hit efficient for improving order-checking, get rid of carrier simultaneously from situation about connecting.Before transformant checks order, at first need carry out bacterium liquid PCR.For this reason, need synthetic two detections to use primer:

With the flat board after connecting, selected clone shakes bacterium, after the incubated overnight, gets 1ul and carries out bacterium liquid PCR detection.Liquid PCR detection architecture is following:

The positive colony that PCR confirms can tentatively be judged as transformed clone, for confirming this result, send 2 positive colonies to check order.Press the operation of this patent method, positive colony order-checking back accuracy is more than 85%.

This patent is transformed the genetically modified destination carrier of paddy rice; Simplified experiment flow greatly; Only need carry out once conventional PCR reaction, a connection just can be accomplished the structure of gene silencing carrier, and time of vector construction was shortened to 4 days from 9 days; And, can carry out the interference vector structure work of a plurality of genes simultaneously because experimental implementation is very simple.This patent method makes up to the silent carrier of single specific gene, only need design synthetic two reticent relevant primers; And only adopt conventional PCR method, reduced greatly to cause the DNA cloning probability of errors because of Overlapping PCR; After PCR accomplishes, owing to there is not the interference of plasmid background, need not to carry out electrophoresis and reclaim dna fragmentation, reduced the consumption of reagent in electrophoresis and the reclaimer operation process, further saved experimental cost and experimental period.The important feature of this patent method is in addition, and the silent carrier that adopts present method to make up is in full accord with the silent carrier that adopts existing method to make up, and the downstream transgenic research does not receive fully that this method is improved to be influenced.

Through comparing, beneficial effect of the present invention is described below with existing method:

Existing method and this patent method flow comparison sheet

Can find out that from above-mentioned form the said method of this patent not only can effectively shorten the time that the paddy gene silent carrier makes up, saving related reagent consumptive material that can also be a large amount of.Compare with existing method, this patent makes up the method for single silent carrier, and the reagent consumptive material kind that not only needs is few, and the amount of reagent that is consumed also greatly reduces.

Except that table 1 is listed, need synthetic 4 primers to each silent carrier structures in the existing method, adopt this patent method, each silent carrier only makes up need two primers, cause the vector construction failed probability thereby also reduced because of primer synthetic mistake.

We can also find out from table 1, use this patent to shorten the vector construction time greatly, and existing method needs 9 days ability to accomplish vector construction approximately, and this patent method only needed just can accomplish in 4 days.And in operating process; Existing method need consume a large amount of energy of experimenter; In the overall process, approximately need take more than 20 hour experimental period, and present method only takies 9 hours experimental periods; And wherein be mainly PCR and tie-time, the experimenter can carry out other work fully simultaneously.

In sum, this patent method tool high efficiency can greatly shorten experimental period, reduces the improvement cost of single silent carrier.Simultaneously, because present method is simple and easy to do, easy to operate, make single experimenter can carry out a plurality of silent carrier transformations simultaneously.

Description of drawings

Fig. 1 is destination carrier transformation and core silent carrier fragment synoptic diagram.

Fig. 2 transforms segmental synthesis model figure for carrier.

Fig. 3 is a paddy gene silent carrier fast construction method synoptic diagram.

Embodiment

Embodiment 1 transforms the pCAMBIA1300 carrier and is used for quick silent carrier structure

1, in generation, transformed the preparation of conversion carrier

For improving this patent suitability, lay special stress on is explained the method that destination carrier is transformed together, to offer user's reference of the present invention, can be used for transforming other destination carrier when needed.

For head and the tail batch section is put into carrier; And can carry out linearization for enzyme restriction; We at first adopt transformed pCAMBIA1300 carrier is initial carrier; Be connected with Ubiqitin promotor and rbcs terminator in being somebody's turn to do, can be used as startup and stop the reticent segmental element of paddy rice, this carrier abbreviates the 1300UR carrier as.

At first, adopt SacI and KpnI restriction endonuclease that it is carried out enzyme and cuts, cut at 37 ℃ of following enzymes and spend the night, the DNA after enzyme is cut adopts 1% agarose gel electrophoresis, with behind the ethidium bromide staining at uv lamp incision glue, adopt gel to reclaim test kit recovery enzyme and cut product.Product be stored in-20 ℃ subsequent use.

2, transform the synthetic of fragment relevant primer

Press this patent method and transform conversion carrier, we synthesize 7 primers, are used to transform the 1300UR carrier, and primer sequence is following:

3, adopt the synthetic transformation fragment that contains the AfeI restriction enzyme site of substep stepwise process

By the explanation of patent, the method for stepwise synthesis is synthesized this fragment, and amiR-F1, amiR-AfeI, amiR-R1 and amiR-R2 synthesize first fragment, and amiR-F4, amiR-AfeI, amiR-R2 and amiR-R4-G-R-KpnI are combined into the cauda section, and be as shown in Figure 2.

Synthetic leading portion and back segment PCR system concentration and reaction conditions are:

4, merge two sections PCR products and obtain complete transformation fragment

Two sections PCR products that obtain are diluted 100 times respectively, adopt G-F-SacI and amiR-R4-G-R-KpnI primer again with merging two sections products, the synthetic complete transformation fragment that has restriction enzyme site.PCR system concentration and reaction conditions are:

5, make being connected of fragment and 1300UR carrier

Adopt gel to reclaim test kit and reclaim dna fragmentation,, be connected into unmodified destination carrier adopting SacI and KpnI endonuclease digestion.

Linked system is following:

Linked system after mixing is spent the night 16 ℃ of water-baths.

6, transformed into escherichia coli

Get and connect product 5ul, the CaCl of adding 100ul ₂The DH5 α thermal shock competence of handling, 42 ℃ of thermal shocks were handled 90 seconds, added the LB nonreactive substratum of 800ul, recovered 1 hour with 250rpm at 37 ℃.Under aseptic condition, get 100ul and be coated on the LB culture plate that contains the 50ug/ml kantlex, cultivated 16 hours for 37 ℃, select transformant, insert and send order-checking to confirm after LB cultivates again.The correct bacterium of checking order adopts 25% glycerine frozen, in order to life-time service.Transform back carrier sequence and see SEQ ID NO:4.

Embodiment 2 usefulness this patent methods make up 5 paddy gene silent carriers fast

1, the preparation work of the quick structure of silent carrier

(1) plasmid is prepared and linearizing

To shake bacterium amplification according to the carrier that embodiment 1 obtains, and adopt the plasmid extraction test kit to carry out the extracting of 1300URA DNA, and cut with the AfeI enzyme, it is following that enzyme is cut system:

Cut at 37 ℃ of following enzymes and to spend the night, the DNA after enzyme is cut adopts 1% agarose gel electrophoresis, with behind the ethidium bromide staining at uv lamp incision glue, adopt gel to reclaim test kit and reclaim enzyme and cut product.Carrier after the linearizing is the universal support fragment, can be used for the structure of all quick silent carriers, can be subsequent use-20 ℃ of long-time down preservations.

(2) bridge-clip is synthetic

Press two of the segmental primers of this patent method synthetic bridging, sequence is following:

Primer is diluted to 100uM, gets 5ul respectively, handled 5 minutes for 95 ℃, naturally cooling is annealed to room temperature state.Dilute 100 times subsequent use.This bridge-clip is general fragment, can be used for the structure of all quick silent carriers, can be subsequent use-20 ℃ of long-time down preservations.

2, the reticent segmental amplification of core

(1) the reticent segmental amplification of core

To two of the reticent associated clip primers of each gene synthetic gene,

The amplified fragments of 5 genes is respectively:

To each gene, get the 1ul bridge-clip respectively, adopt gene specific primer to carry out pcr amplification, amplification condition is following:

(2) the reticent fragment of core and carrier is connected

Need not to reclaim, the fragment PCR products that obtains of directly will increasing directly is connected with linearizing carrier, and linked system is following:

Linked system after mixing is spent the night 16 ℃ of water-baths.

(3) transformed into escherichia coli bacterial strain

This patent recommends to use conventional DH5 α bacterial strain to carry out the transformation of junction fragment.Get and connect product 5ul, the CaCl of adding 100ul ₂The DH5 α thermal shock competence bacteria of handling, 42 ℃ of thermal shocks were handled 90 seconds, added the LB nonreactive substratum of 800ul, recovered 1 hour with 250rpm at 37 ℃.Under aseptic condition, get 100ul and be coated on the LB culture plate that contains the 50ug/ml kantlex, cultivated 16 hours for 37 ℃, select transformant, insert LB and cultivate again.

3, the detection of gene silencing carrier

(1) detects the synthetic of primer

Owing to adopted flat terminal the connection, must select connection transformant in the right direction, hit efficient for improving order-checking, get rid of carrier from situation about connecting.Before transformant checks order, at first need carry out bacterium liquid PCR.For this reason, need synthetic two detections to use primer:

(2) bacterium liquid PCR detects

With the flat board after connecting, selected clone shakes bacterium, after the incubated overnight, gets 1ul and carries out bacterium liquid PCR detection.Bacterium liquid PCR detection architecture is following:

The positive colony that PCR confirms can tentatively be judged as transformed clone, for confirming this result, send 2 positive colony order-checkings.

(3) order-checking detects

Five genes that contain the LRR structural domain; After bacterium liquid PCR detected and accomplishes, the silent carrier of each gene sent two PCR positive colonies to check order respectively; Detect demonstration through sequencing result; 5 carriers all obtain correct clone, and wherein 4 carriers send two clones of survey correct, and the another one carrier send the carrier of survey to have a clone to have point mutation.Positive rate is 90% so these 5 gene silencing carriers of structure finally check order.

Sequence table

< 110>Zhejiang Academy of Agricultural Science

< 120>method for quickly constructing artificial mi RNA gene interference vector of paddy

<160>20

<210>1

<211>154

<212>DNA

< 213>synthetic

<222>(1)…(154)

<400>1

1 cagcagcagc?cacagcaaaa?tttggtttgg?gataggtagg?tgttatgtta?ggtctggttt

61 tttggctgta?gcagcagcgc?tgctaggctg?ttctgtggaa?gtttgcagag?tttatattat

121?gggtttaatc?gtccatggca?tcagcatcag?cagc

<210>2

<211>170

<212>DNA

< 213>synthetic

<222>(1)…(170)

<400>2

1 tcGAGCTCca?gcagcagcca?cagcaaaatt?tggtttggga?taggtaggtg?ttatgttagg

61 tctggttttt?tggctgtagc?agcagcgctg?ctaggctgtt?ctgtggaagt?ttgcagagtt

121?tatattatgg?gtttaatcgt?ccatggcatc?agcatcagca?gcGGTACCga

<210>3

<211>245

<212>DNA

< 213>synthetic

<222>(1)…(245)

<400>3

1 cagcagcagc?cacagcaaaa?tttggtttgg?gataggtagg?tgttatgtta?ggtctggttt

61 tttggctgta?gcagcagcag?tggaaggggc?atgcagagga?gcaggagatt?cagtttgaag

121?ctggacttca?cttttgcctc?tctctcctgt?gcttgcctct?tccattcctg?ctgctaggct

181?gttctgtgga?agtttgcaga?gtttatatta?tgggtttaat?cgtccatggc?atcagcatca

241?gcagc

<210>4

<211>11761

<212>DNA

< 213>synthetic

<222>(1)…(11761)

<400>4

1 aattgggctg?cagtgcagcg?tgacccggtc?gtgcccctct?ctagagataa?tgagcattgc

61 atgtctaagt?tataaaaaat?taccacatat?tttttttgtc?acacttgttt?gaagtgcagt

121 ttatctatct?ttatacatat?atttaaactt?tactctacga?ataatataat?ctatagtact

181 acaataatat?cagtgtttta?gagaatcata?taaatgaaca?gttagacatg?gtctaaagga

241 caattgagta?ttttgacaac?aggactctac?agttttatct?ttttagtgtg?catgtgttct

301 cctttttttt?tgcaaatagc?ttcacctata?taatacttca?tccattttat?tagtacatcc

361 atttagggtt?tagggttaat?ggtttttata?gactaatttt?tttagtacat?ctattttatt

421 ctattttagc?ctctaaatta?agaaaactaa?aactctattt?tagttttttt?atttaataat

481 ttagatataa?aatagaataa?aataaagtga?ctaaaaatta?aacaaatacc?ctttaagaaa

541 ttaaaaaaac?taaggaaaca?tttttcttgt?ttcgagtaga?taatgccagc?ctgttaaacg

601 ccgtcgacga?gtctaacgga?caccaaccag?cgaaccagca?gcgtcgcgtc?gggccaagcg

661 aagcagacgg?cacggcatct?ctgtcgctgc?ctctggaccc?ctctcgagag?ttccgctcca

721 ccgttggact?tgctccgctg?tcggcatcca?gaaattgcgt?ggcggagcgg?cagacgtgag

781 ccggcacggc?aggcggcctc?ctcctcctct?cacggcacgg?cagctacggg?ggattccttt

841 cccaccgctc?cttcgctttc?ccttcctcgc?ccgccgtaat?aaatagacac?cccctccaca

901 ccctctttcc?ccaacctcgt?gttgttcgga?gcgcacacac?acacaaccag?atctccccca

961 aatccacccg?tcggcacctc?cgcttcaagg?tacgccgctc?gtcctccccc?cccccccctc

1021?tctaccttct?ctagatcggc?gttccggtcc?atggttaggg?cccggtagtt?ctacttctgt

1081?tcatgtttgt?gttagatccg?tgtttgtgtt?agatccgtgc?tgctagcgtt?cgtacacgga

1141?tgcgacctgt?acgtcagaca?cgttctgatt?gctaacttgc?cagtgtttct?ctttggggaa

1201?tcctgggatg?gctctagccg?ttccgcagac?gggatcgatt?tcatgatttt?ttttgtttcg

1261?ttgcataggg?tttggtttgc?ccttttcctt?tatttcaata?tatgccgtgc?acttgtttgt

1321?cgggtcatct?tttcatgctt?ttttttgtct?tggttgtgat?gatgtggtct?ggttgggcgg

1381?tcgttctaga?tcggagtaga?attctgtttc?aaactacctg?gtggatttat?taattttgga

1441?tctgtatgtg?tgtgccatac?atattcatag?ttacgaattg?aagatgatgg?atggaaatat

1501?cgatctagga?taggtataca?tgttgatgcg?ggttttactg?atgcatatac?agagatgctt

1561?tttgttcgct?tggttgtgat?gatgtggtgt?ggttgggcgg?tcgttcattc?gttctagatc

1621?ggagtagaat?actgtttcaa?actacctggt?gtatttatta?attttggaac?tgtatgtgtg

1681?tgtcatacat?cttcatagtt?acgagtttaa?gatggatgga?aatatcgatc?taggataggt

1741?atacatgttg?atgtgggttt?tactgatgca?tatacatgat?ggcatatgca?gcatctattc

1801?atatgctcta?accttgagta?cctatctatt?ataataaaca?agtatgtttt?ataattattt

1861?tgatcttgat?atacttggat?gatggcatat?gcagcagcta?tatgtggatt?tttttagccc

1921?tgccttcata?cgctatttat?ttgcttggta?ctgtttcttt?tgtcgatgct?caccctgttg

1981?tttggtgtta?cttctgcagg?aattcgatat?caagctattc?gagctccagc?agcagccaca

2041?gcaaaatttg?gtttgggata?ggtaggtgtt?atgttaggtc?tggttttttg?gctgtagcag

2101?cagcgctgct?aggctgttct?gtggaagttt?gcagagttta?tattatgggt?ttaatcgtcc

2161?atggcatcag?catcagcagc?ggtacccggg?gatcctctag?agtcgacctg?cagagctttc

2221?gttcgtatca?tcggtttcga?caacgttcgt?caagttcaat?gcatcagttt?cattgcgcac

2281?acaccagaat?cctactgagt?ttgagtatta?tggcattggg?aaaactgttt?ttcttgtacc

2341?atttgttgtg?cttgtaattt?actgtgtttt?ttattcggtt?ttcgctatcg?aactgtgaaa

2401?tggaaatgga?tggagaagag?ttaatgaatg?atatggtcct?tttgttcatt?ctcaaattaa

2461?tattatttgt?tttttctctt?atttgttgtg?tgttgaattt?gaaattataa?gagatatgca

2521?aacattttgt?tttgagtaaa?aatgtgtcaa?atcgtggcct?ctaatgaccg?aagttaatat

2581?gaggagtaaa?acacttgtag?ttgtaccatt?atgcttattc?actaggcaac?aaatatattt

2641?tcagacctag?aaaagctgca?aatgttactg?aatacaagta?tgtcctcttg?tgttttagac

2701?atttatgaac?tttcctttat?gtaattttcc?agaatccttg?tcagattcta?atcattgctt

2761?tataattata?gttatactca?tggatttgta?gttgagtatg?aaaatatttt?ttaatgcatt

2821?ttatgacttg?ccaattgatt?gacaacatgc?atcaagcttg?gcactggccg?tcgttttaca

2881?acgtcgtgac?tgggaaaacc?ctggcgttac?ccaacttaat?cgccttgcag?cacatccccc

2941?tttcgccagc?tggcgtaata?gcgaagaggc?ccgcaccgat?cgcccttccc?aacagttgcg

3001?cagcctgaat?ggcgaatgct?agagcagctt?gagcttggat?cagattgtcg?tttcccgcct

3061?tcagtttaaa?ctatcagtgt?ttgacaggat?atattggcgg?gtaaacctaa?gagaaaagag

3121?cgtttattag?aataatcgga?tatttaaaag?ggcgtgaaaa?ggtttatccg?ttcgtccatt

3181?tgtatgtgca?tgccaaccac?agggttcccc?tcgggatcaa?agtactttga?tccaacccct

3241?ccgctgctat?agtgcagtcg?gcttctgacg?ttcagtgcag?ccgtcttctg?aaaacgacat

3301?gtcgcacaag?tcctaagtta?cgcgacaggc?tgccgccctg?cccttttcct?ggcgttttct

3361?tgtcgcgtgt?tttagtcgca?taaagtagaa?tacttgcgac?tagaaccgga?gacattacgc

3421?catgaacaag?agcgccgccg?ctggcctgct?gggctatgcc?cgcgtcagca?ccgacgacca

3481?ggacttgacc?aaccaacggg?ccgaactgca?cgcggccggc?tgcaccaagc?tgttttccga

3541?gaagatcacc?ggcaccaggc?gcgaccgccc?ggagctggcc?aggatgcttg?accacctacg

3601?ccctggcgac?gttgtgacag?tgaccaggct?agaccgcctg?gcccgcagca?cccgcgacct

3661?actggacatt?gccgagcgca?tccaggaggc?cggcgcgggc?ctgcgtagcc?tggcagagcc

3721?gtgggccgac?accaccacgc?cggccggccg?catggtgttg?accgtgttcg?ccggcattgc

3781?cgagttcgag?cgttccctaa?tcatcgaccg?cacccggagc?gggcgcgagg?ccgccaaggc

3841?ccgaggcgtg?aagtttggcc?cccgccctac?cctcaccccg?gcacagatcg?cgcacgcccg

3901?cgagctgatc?gaccaggaag?gccgcaccgt?gaaagaggcg?gctgcactgc?ttggcgtgca

3961?tcgctcgacc?ctgtaccgcg?cacttgagcg?cagcgaggaa?gtgacgccca?ccgaggccag

4021?gcggcgcggt?gccttccgtg?aggacgcatt?gaccgaggcc?gacgccctgg?cggccgccga

4081?gaatgaacgc?caagaggaac?aagcatgaaa?ccgcaccagg?acggccagga?cgaaccgttt

4141?ttcattaccg?aagagatcga?ggcggagatg?atcgcggccg?ggtacgtgtt?cgagccgccc

4201?gcgcacgtct?caaccgtgcg?gctgcatgaa?atcctggccg?gtttgtctga?tgccaagctg

4261?gcggcctggc?cggccagctt?ggccgctgaa?gaaaccgagc?gccgccgtct?aaaaaggtga

4321?tgtgtatttg?agtaaaacag?cttgcgtcat?gcggtcgctg?cgtatatgat?gcgatgagta

4381?aataaacaaa?tacgcaaggg?gaacgcatga?aggttatcgc?tgtacttaac?cagaaaggcg

4441?ggtcaggcaa?gacgaccatc?gcaacccatc?tagcccgcgc?cctgcaactc?gccggggccg

4501?atgttctgtt?agtcgattcc?gatccccagg?gcagtgcccg?cgattgggcg?gccgtgcggg

4561?aagatcaacc?gctaaccgtt?gtcggcatcg?accgcccgac?gattgaccgc?gacgtgaagg

4621?ccatcggccg?gcgcgacttc?gtagtgatcg?acggagcgcc?ccaggcggcg?gacttggctg

4681?tgtccgcgat?caaggcagcc?gacttcgtgc?tgattccggt?gcagccaagc?ccttacgaca

4741?tatgggccac?cgccgacctg?gtggagctgg?ttaagcagcg?cattgaggtc?acggatggaa

4801?ggctacaagc?ggcctttgtc?gtgtcgcggg?cgatcaaagg?cacgcgcatc?ggcggtgagg

4861?ttgccgaggc?gctggccggg?tacgagctgc?ccattcttga?gtcccgtatc?acgcagcgcg

4921?tgagctaccc?aggcactgcc?gccgccggca?caaccgttct?tgaatcagaa?cccgagggcg

4981?acgctgcccg?cgaggtccag?gcgctggccg?ctgaaattaa?atcaaaactc?atttgagtta

5041?atgaggtaaa?gagaaaatga?gcaaaagcac?aaacacgcta?agtgccggcc?gtccgagcgc

5101?acgcagcagc?aaggctgcaa?cgttggccag?cctggcagac?acgccagcca?tgaagcgggt

5161?caactttcag?ttgccggcgg?aggatcacac?caagctgaag?atgtacgcgg?tacgccaagg

5221?caagaccatt?accgagctgc?tatctgaata?catcgcgcag?ctaccagagt?aaatgagcaa

5281?atgaataaat?gagtagatga?attttagcgg?ctaaaggagg?cggcatggaa?aatcaagaac

5341?aaccaggcac?cgacgccgtg?gaatgcccca?tgtgtggagg?aacgggcggt?tggccaggcg

5401?taagcggctg?ggttgtctgc?cggccctgca?atggcactgg?aacccccaag?cccgaggaat

5461?cggcgtgacg?gtcgcaaacc?atccggcccg?gtacaaatcg?gcgcggcgct?gggtgatgac

5521?ctggtggaga?agttgaaggc?cgcgcaggcc?gcccagcggc?aacgcatcga?ggcagaagca

5581?cgccccggtg?aatcgtggca?agcggccgct?gatcgaatcc?gcaaagaatc?ccggcaaccg

5641?ccggcagccg?gtgcgccgtc?gattaggaag?ccgcccaagg?gcgacgagca?accagatttt

5701?ttcgttccga?tgctctatga?cgtgggcacc?cgcgatagtc?gcagcatcat?ggacgtggcc

5761?gttttccgtc?tgtcgaagcg?tgaccgacga?gctggcgagg?tgatccgcta?cgagcttcca

5821?gacgggcacg?tagaggtttc?cgcagggccg?gccggcatgg?ccagtgtgtg?ggattacgac

5881?ctggtactga?tggcggtttc?ccatctaacc?gaatccatga?accgataccg?ggaagggaag

5941?ggagacaagc?ccggccgcgt?gttccgtcca?cacgttgcgg?acgtactcaa?gttctgccgg

6001?cgagccgatg?gcggaaagca?gaaagacgac?ctggtagaaa?cctgcattcg?gttaaacacc

6061?acgcacgttg?ccatgcagcg?tacgaagaag?gccaagaacg?gccgcctggt?gacggtatcc

6121?gagggtgaag?ccttgattag?ccgctacaag?atcgtaaaga?gcgaaaccgg?gcggccggag

6181?tacatcgaga?tcgagctagc?tgattggatg?taccgcgaga?tcacagaagg?caagaacccg

6241?gacgtgctga?cggttcaccc?cgattacttt?ttgatcgatc?ccggcatcgg?ccgttttctc

6301?taccgcctgg?cacgccgcgc?cgcaggcaag?gcagaagcca?gatggttgtt?caagacgatc

6361?tacgaacgca?gtggcagcgc?cggagagttc?aagaagttct?gtttcaccgt?gcgcaagctg

6421?atcgggtcaa?atgacctgcc?ggagtacgat?ttgaaggagg?aggcggggca?ggctggcccg

6481?atcctagtca?tgcgctaccg?caacctgatc?gagggcgaag?catccgccgg?ttcctaatgt

6541?acggagcaga?tgctagggca?aattgcccta?gcaggggaaa?aaggtcgaaa?aggtctcttt

6601?cctgtggata?gcacgtacat?tgggaaccca?aagccgtaca?ttgggaaccg?gaacccgtac

6661?attgggaacc?caaagccgta?cattgggaac?cggtcacaca?tgtaagtgac?tgatataaaa

6721?gagaaaaaag?gcgatttttc?cgcctaaaac?tctttaaaac?ttattaaaac?tcttaaaacc

6781?cgcctggcct?gtgcataact?gtctggccag?cgcacagccg?aagagctgca?aaaagcgcct

6841?acccttcggt?cgctgcgctc?cctacgcccc?gccgcttcgc?gtcggcctat?cgcggccgct

6901?ggccgctcaa?aaatggctgg?cctacggcca?ggcaatctac?cagggcgcgg?acaagccgcg

6961?ccgtcgccac?tcgaccgccg?gcgcccacat?caaggcaccc?tgcctcgcgc?gtttcggtga

7021?tgacggtgaa?aacctctgac?acatgcagct?cccggagacg?gtcacagctt?gtctgtaagc

7081?ggatgccggg?agcagacaag?cccgtcaggg?cgcgtcagcg?ggtgttggcg?ggtgtcgggg

7141?cgcagccatg?acccagtcac?gtagcgatag?cggagtgtat?actggcttaa?ctatgcggca

7201?tcagagcaga?ttgtactgag?agtgcaccat?atgcggtgtg?aaataccgca?cagatgcgta

7261?aggagaaaat?accgcatcag?gcgctcttcc?gcttcctcgc?tcactgactc?gctgcgctcg

7321?gtcgttcggc?tgcggcgagc?ggtatcagct?cactcaaagg?cggtaatacg?gttatccaca

7381?gaatcagggg?ataacgcagg?aaagaacatg?tgagcaaaag?gccagcaaaa?ggccaggaac

7441?cgtaaaaagg?ccgcgttgct?ggcgtttttc?cataggctcc?gcccccctga?cgagcatcac

7501?aaaaatcgac?gctcaagtca?gaggtggcga?aacccgacag?gactataaag?ataccaggcg

7561?tttccccctg?gaagctccct?cgtgcgctct?cctgttccga?ccctgccgct?taccggatac

7621?ctgtccgcct?ttctcccttc?gggaagcgtg?gcgctttctc?atagctcacg?ctgtaggtat

7681?ctcagttcgg?tgtaggtcgt?tcgctccaag?ctgggctgtg?tgcacgaacc?ccccgttcag

7741?cccgaccgct?gcgccttatc?cggtaactat?cgtcttgagt?ccaacccggt?aagacacgac

7801?ttatcgccac?tggcagcagc?cactggtaac?aggattagca?gagcgaggta?tgtaggcggt

7861?gctacagagt?tcttgaagtg?gtggcctaac?tacggctaca?ctagaaggac?agtatttggt

7921?atctgcgctc?tgctgaagcc?agttaccttc?ggaaaaagag?ttggtagctc?ttgatccggc

7981?aaacaaacca?ccgctggtag?cggtggtttt?tttgtttgca?agcagcagat?tacgcgcaga

8041?aaaaaaggat?ctcaagaaga?tcctttgatc?ttttctacgg?ggtctgacgc?tcagtggaac

8101?gaaaactcac?gttaagggat?tttggtcatg?cattctaggt?actaaaacaa?ttcatccagt

8161?aaaatataat?attttatttt?ctcccaatca?ggcttgatcc?ccagtaagtc?aaaaaatagc

8221?tcgacatact?gttcttcccc?gatatcctcc?ctgatcgacc?ggacgcagaa?ggcaatgtca

8281 taccacttgt?ccgccctgcc?gcttctccca?agatcaataa?agccacttac?tttgccatct

8341 ttcacaaaga?tgttgctgtc?tcccaggtcg?ccgtgggaaa?agacaagttc?ctcttcgggc

8401 ttttccgtct?ttaaaaaatc?atacagctcg?cgcggatctt?taaatggagt?gtcttcttcc

8461 cagttttcgc?aatccacatc?ggccagatcg?ttattcagta?agtaatccaa?ttcggctaag

8521 cggctgtcta?agctattcgt?atagggacaa?tccgatatgt?cgatggagtg?aaagagcctg

8581 atgcactccg?catacagctc?gataatcttt?tcagggcttt?gttcatcttc?atactcttcc

8641 gagcaaagga?cgccatcggc?ctcactcatg?agcagattgc?tccagccatc?atgccgttca

8701 aagtgcagga?cctttggaac?aggcagcttt?ccttccagcc?atagcatcat?gtccttttcc

8761 cgttccacat?cataggtggt?ccctttatac?cggctgtccg?tcatttttaa?atataggttt

8821 tcattttctc?ccaccagctt?atatacctta?gcaggagaca?ttccttccgt?atcttttacg

8881 cagcggtatt?tttcgatcag?ttttttcaat?tccggtgata?ttctcatttt?agccatttat

8941 tatttccttc?ctcttttcta?cagtatttaa?agatacccca?agaagctaat?tataacaaga

9001 cgaactccaa?ttcactgttc?cttgcattct?aaaaccttaa?ataccagaaa?acagcttttt

9061 caaagttgtt?ttcaaagttg?gcgtataaca?tagtatcgac?ggagccgatt?ttgaaaccgc

9121 ggtgatcaca?ggcagcaacg?ctctgtcatc?gttacaatca?acatgctacc?ctccgcgaga

9181 tcatccgtgt?ttcaaacccg?gcagcttagt?tgccgttctt?ccgaatagca?tcggtaacat

9241 gagcaaagtc?tgccgcctta?caacggctct?cccgctgacg?ccgtcccgga?ctgatgggct

9301 gcctgtatcg?agtggtgatt?ttgtgccgag?ctgccggtcg?gggagctgtt?ggctggctgg

9361 tggcaggata?tattgtggtg?taaacaaatt?gacgcttaga?caacttaata?acacattgcg

9421 gacgttttta?atgtactgaa?ttaacgccga?attaattcgg?gggatctgga?ttttagtact

9481 ggattttggt?tttaggaatt?agaaatttta?ttgatagaag?tattttacaa?atacaaatac

9541 atactaaggg?tttcttatat?gctcaacaca?tgagcgaaac?cctataggaa?ccctaattcc

9601 cttatctggg?aactactcac?acattattat?ggagaaactc?gagcttgtcg?atcgacagat

9661 cccggtcggc?atctactcta?tttctttgcc?ctcggacgag?tgctggggcg?tcggtttcca

9721 ctatcggcga?gtacttctac?acagccatcg?gtccagacgg?ccgcgcttct?gcgggcgatt

9781 tgtgtacgcc?cgacagtccc?ggctccggat?cggacgattg?cgtcgcatcg?accctgcgcc

9841 caagctgcat?catcgaaatt?gccgtcaacc?aagctctgat?agagttggtc?aagaccaatg

9901 cggagcatat?acgcccggag?tcgtggcgat?cctgcaagct?ccggatgcct?ccgctcgaag

9961 tagcgcgtct?gctgctccat?acaagccaac?cacggcctcc?agaagaagat?gttggcgacc

10021?tcgtattggg?aatccccgaa?catcgcctcg?ctccagtcaa?tgaccgctgt?tatgcggcca

10081?ttgtccgtca?ggacattgtt?ggagccgaaa?tccgcgtgca?cgaggtgccg?gacttcgggg

10141?cagtcctcgg?cccaaagcat?cagctcatcg?agagcctgcg?cgacggacgc?actgacggtg

10201?tcgtccatca?cagtttgcca?gtgatacaca?tggggatcag?caatcgcgca?tatgaaatca

10261?cgccatgtag?tgtattgacc?gattccttgc?ggtccgaatg?ggccgaaccc?gctcgtctgg

10321?ctaagatcgg?ccgcagcgat?cgcatccata?gcctccgcga?ccggttgtag?aacagcgggc

10381?agttcggttt?caggcaggtc?ttgcaacgtg?acaccctgtg?aacggcggga?gatgcaatag

10441?gtcaggctct?cgctaaactc?cccaatgtca?agcacttccg?gaatcgggag?cgcggccgat

10501?gcaaagtgcc?gataaacata?acgatctttg?tagaaaccat?cggcgcagct?atttacccgc

10561?aggacatatc?cacgccctcc?tacatcgaag?ctgaaagcac?gagattcttc?gccctccgag

10621?agctgcatca?ggtcggagac?gctgtcgaac?ttttcgatca?gaaacttctc?gacagacgtc

10681?gcggtgagtt?caggcttttt?catatctcat?tgcccccccg?gatctgcgaa?agctcgagag

10741?agatagattt?gtagagagag?actggtgatt?tcagcgtgtc?ctctccaaat?gaaatgaact

10801?tccttatata?gaggaagggt?cttgcgaagg?atagtgggat?tgtgcgtcat?cccttacgtc

10861?agtggagata?tcacatcaat?ccacttgctt?tgaagacgtg?gttggaacgt?cttctttttc

10921?cacgatgctc?ctcgtgggtg?ggggtccatc?tttgggacca?ctgtcggcag?aggcatcttg

10981?aacgatagcc?tttcctttat?cgcaatgatg?gcatttgtag?gtgccacctt?ccttttctac

11041?tgtccttttg?atgaagtgac?agatagctgg?gcaatggaat?ccgaggaggt?ttcccgatat

11101?taccctttgt?tgaaaagtct?caatagccct?ttggtcttct?gagactgtat?ctttgatatt

11161?cttggagtag?acgagagtgt?cgtgctccac?catgttcaca?tcaatccact?tgctttgaag

11221?acgtggttgg?aacgtcttct?ttttccacga?tgctcctcgt?gggtgggggt?ccatctttgg

11281?gaccactgtc?ggcagaggca?tcttgaacga?tagcctttcc?tttatcgcaa?tgatggcatt

11341?tgtaggtgcc?accttccttt?tctactgtcc?ttttgatgaa?gtgacagata?gctgggcaat

11401?ggaatccgag?gaggtttccc?gatattaccc?tttgttgaaa?agtctcaata?gccctttggt

11461?cttctgagac?tgtatctttg?atattcttgg?agtagacgag?agtgtcgtgc?tccaccatgt

11521?tggcaagctg?ctctagccaa?tacgcaaacc?gcctctcccc?gcgcgttggc?cgattcatta

11581?atgcagctgg?cacgacaggt?ttcccgactg?gaaagcgggc?agtgagcgca?acgcaattaa

11641?tgtgagttag?ctcactcatt?aggcacccca?ggctttacac?tttatgcttc?cggctcgtat

11701?gttgtgtgga?attgtgagcg?gataacaatt?tcacacagga?aacagctatg?acatgattac

11761?g

<210>5

<211>78

<212>DNA

< 213>synthetic

<222>(1)…(78)

<400>5

1 cagcagcagc?cacagcaaaa?tttggtttgg?gataggtagg?tgttatgtta?ggtctggttt

61?tttggctgta?gcagcagc

<210>6

<211>76

<212>DNA

< 213>synthetic

<222>(1)…(76)

<400>6

1 gctgctaggc?tgttctgtgg?aagtttgcag?agtttatatt?atgggtttaa?tcgtccatgg

61?catcagcatc?agcagc

<210>7

<211>58

<212>DNA

< 213>primer

<222>(1)…(58)

<400>7

CCAGCAGCAG?CCACAGCAAA?ATTTGGTTTG?GGATAGGTAG?GTGTTATGTT?AGGTCTGG 58

<210>8

<211>38

<212>DNA

< 213>primer

<222>(1)…(38)

<400>8

CTGCTGCTGC?TACAGCCAAA?AAACCAGACC?TAACATAA 38

<210>9

<211>42

<212>DNA

< 213>primer

<222>(1)…(42)

<400>9

AGAGAGGCAA?AAGTGAAGTC?CAGCTTCAAA?CTGAATCTCC?TG?42

<210>10

<211>39

<212>DNA

< 213>primer

<222>(1)…(39)

<400>10

AACTCTGCAA?ACTTCCACAG?AACAGCCTAG?CAGCAGGAA 39

<210>11

<211>58

<212>DNA

< 213>primer

<222>(1)…(58)

<400>11

GAAGTTTGCA?GAGTTTATAT?TATGGGTTTA?ATCGTCCATG?GCATCAGCAT?CAGCAGCG 58

<210>12

<211>28

<212>DNA

< 213>primer

<222>(1)…(28)

<400>12

TCGGATCCGC?TGCTGATGCT GATGCCAT 28

<210>13

<211>27

<212>DNA

< 213>primer

<222>(1)…(27)

<400>13

TCGAGCTCCA?GCAGCAGCCA?CAGCAAA 27

<210>14

<211>28

<212>DNA

< 213>primer

<222>(1)…(28)

<400>14

TCGGTACCGC?TGCTGATGCT?GATGCCAT 28

<210>15

<211>20

<212>DNA

< 213>primer

<222>(1)…(20)

<400>15

ATTTTTTTAG?CCCTGCCTTC 20

<210>16

<211>21

<212>DNA

< 213>primer

<222>(1)…(21)

<400>16

AGAGAGGCAA?AAGTGAAGTC?C 21

<210>17

<211>39

<212>DNA

< 213>primer

<222>(1)…(39)

<400>17

AGTCTAATAA?TACCTGTCAC?GATCAGGAGA?TTCAGTTTG 39

<210>18

<211>41

<212>DNA

< 213>primer

<222>(1)…(41)

<400>18

AGGAATCTAA?TAATACGTGT?GACGATAGAG?AGGCAAAAGT?G?41

<210>19

<211>20

<212>DNA

< 213>primer

<222>(1)…(20)

<400>19

ATTTTTTTAG?CCCTGCCTTC 20

<210>20

<211>21

<212>DNA

< 213>primer

<222>(1)…(21)

<400>20

AGAGAGGCAA?AAGTGAAGTC?C 21

Claims (2)

1. the quick silent carrier transformation of paddy rice is with the compound method of dna sequence dna; It is characterized in that; This method adopts rice Os-amiR528DNA gene interference sequence, and described rice Os-amiR528DNA gene interference sequence is divided into the reticent fragment of first fragment, cauda section and core with rice Os-amiR528DNA gene interference sequence shown in SEQ ID NO:3; First fragments sequence is shown in SEQ ID NO:5, and the tail fragments sequence is shown in SEQ ID NO:6; Restriction enzyme site and corresponding protection base have been designed at segmental front end of head and the segmental tail end of tail; First fragment, the splicing of cauda section are obtained the silent carrier transformation shown in SEQ ID NO:2 that comprises the AfeI restriction enzyme site use dna sequence dna, wherein first fragment is by the ending of AGC site, and the cauda section is initial by the GCT position, and wherein SEQ ID NO:2 is as follows said:

tcGAGCTCcagcagcagccacagcaaaatttggtttgggataggtaggtgttatgttaggtctggttttttggctgtagcagcAGCGCTg

ctaggctgttctgtggaagtttgcagagtttatattatgggtttaatcgtccatggcatcagcatcagcagcGGTACCga；

Wherein, tcGAGCTC and GGTACCga partly are cloning site and protection base, and AGCGCT partly is the AfeI restriction enzyme site.

2. be used for efficiently making up the carrier of paddy gene silent carrier, it is characterized in that: this carrier contains the gene fragment just like the described dna sequence dna of SEQ ID NO:2, and wherein SEQ ID NO:2 is as follows said:

tcGAGCTCcagcagcagccacagcaaaatttggtttgggataggtaggtgttatgttaggtctggttttttggctgtagcagcAGCGCTg

ctaggctgttctgtggaagtttgcagagtttatattatgggtttaatcgtccatggcatcagcatcagcagcGGTACCga。

Images