CN114574500A - Clone and application of rice sword-leaf sheath and panicle whitening trait gene OsWSSP - Google Patents

Clone and application of rice sword-leaf sheath and panicle whitening trait gene OsWSSP Download PDF

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CN114574500A
CN114574500A CN202210280493.3A CN202210280493A CN114574500A CN 114574500 A CN114574500 A CN 114574500A CN 202210280493 A CN202210280493 A CN 202210280493A CN 114574500 A CN114574500 A CN 114574500A
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向勇
赵波
徐建龙
张会
陈凯
刘瑞芳
朱双兵
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Abstract

The application relates to cloning and application of a rice scabbard and panicle albino trait gene OsWSSP. The application discovers for the first time that the expression quantity of the gene coding protein is reduced due to the inverted mutation of the OsWSSP promoter, so that the albino phenotype appears on the rice scabbard and the ear. Meanwhile, the application also develops and verifies a molecular marker tightly linked with the target gene OsWSSP so as to be used as a screening marker of different rice lines and hybrids derived from the rice lines.

Description

Clone and application of rice sword-leaf sheath and panicle whitening trait gene OsWSSP
Technical Field
The application relates to cloning and application of rice scabbard and panicle albino character gene OsWSSP, and belongs to the technical field of molecular biology.
Background
Rice is one of the important food crops in China. The rice photosynthetic efficiency is related to the rice yield, and the photosynthetic efficiency is closely related to the chloroplast development and the photosynthetic pigment content. Therefore, the deep analysis of the molecular mechanism of chloroplast development in the green tissues of rice has important significance for improving the photosynthetic efficiency of rice. Related researches in rice mainly focus on the development and control of chloroplast of leaves, and the development of chloroplast of panicle parts and sword-leaved sheaths and photosynthetic efficiency of chloroplast are deficient in research on the influence of rice yield.
At present, related research in rice mainly focuses on the chloroplast development regulation of leaves, people utilize leaf color mutation as a screening marker of sterile lines and pure varieties of two-line hybrid rice, but light green and light yellow leaves are difficult to distinguish, so that the efficiency of using the leaf color mutation as a screening tool is very low. Therefore, if the related genes influencing the whitening of the rice panicle and the scab of the sword-shaped leaf can be found, the molecular mechanism research of the development of rice chloroplast is facilitated, and the application research of the cob and the scab whitening regulation gene in rice crossbreeding is facilitated to be expanded.
In addition, in variety identification, although the field planting identification has the advantages of wide application range, reliable identification result and the like, the method has long time, low efficiency, large workload and lagged result. Generally speaking, the target characters to be identified are mostly quantitative characters, which are greatly influenced by the environment, and people with certain field inspection experience are needed to ensure the detection accuracy, while the DNA fingerprint spectrum technology has the advantages of high efficiency, accuracy, simple experimental operation, no influence by the environment and the like, and is widely applied to the authenticity identification and purity detection research of plant varieties.
Mature and widely used DNA fingerprinting techniques including RAPD, ISSR, InDel, etc. are known, each of which has advantages. InDel refers to the phenomenon that insertion or deletion of nucleotide fragments with different sizes occurs in sequences of the same site of genome between related species or different individuals of the same species, and gaps (gap) are generated by homologous sequence alignment. The InDel molecular marker has the characteristics of rich quantity, high polymorphism, high precision, short detection time, mature technology and the like, and has wide application prospect in the aspects of plant variety identification and purity detection. However, InDel production is mainly related to genomic features and DNA replication errors, and the mechanism of generation of most insertion/deletion mutations is still unknown at present. Since the ratio of insertions and deletions increases due to sequencing errors and the true InDel polymorphism rate also increases in these sequencing error regions, the challenge is presented to obtain effective molecular marker primers for tens of thousands of insertions and deletions in the enormous genome sequencing data, which locations are operational problems, which locations are sequencing errors, which locations are true InDel variations, and so on.
Disclosure of Invention
The application discovers a gene OsWSSP for controlling the panicle part of rice to show an obvious albino phenotype in the booting stage, the flowering stage and the seed filling stage for the first time, and a protein coded by CDS of the gene, and further determines a molecular mechanism of the gene or the protein for regulating the phenotype.
The inventor finds that the promoter of the gene has inversion mutation, and the mutation causes the expression level of target protein to be reduced, thereby causing the scab of the tassel during the booting stage of the rice and the albino phenotype of the tassel during the flowering stage and the filling stage of seeds.
Meanwhile, the optimal identifying primer is screened out to improve the efficiency of molecular assisted breeding. Specifically, the method comprises the following steps:
the application provides an OsWSSP gene, the nucleic acid sequence of which is shown in SEQ ID No.1 or SEQ ID No. 2.
Wherein, SEQ ID.NO.1 is the complete sequence of the genomic DNA of the OsWSSP gene; SEQ ID No.2 is the genomic DNA complete sequence of the mutated OsWSSP gene.
The application also provides a promoter, and the nucleic acid sequence of the promoter is shown in SEQ ID No.5 or SEQ ID No. 6.
Wherein, SEQ ID.NO.5 is a promoter sequence in the genomic DNA of the OsWSSP gene; SEQ ID No.6 is the promoter sequence in the genomic DNA of the mutated OsWSSP gene.
The difference of the promoters causes the corresponding protein expression quantity of the OsWSSP gene to be different.
The application also provides the application of the gene or the protein with the sequence shown in SEQ ID No.4 or the coding nucleic acid thereof or the promoter in identifying rice varieties or rice breeding.
The application also provides application of the gene or the protein with the sequence shown in SEQ ID No.4 or the coding nucleic acid thereof or the promoter in regulating and controlling chloroplast development and/or chlorophyll synthesis of rice scabbard and spike.
In one embodiment, the sequence of the nucleic acid encoding the above protein is as shown in SEQ id No. 3.
Wherein, SEQ ID No.3 is CDS sequence in OsWSSP gene genome DNA, and its coded protein sequence is shown in SEQ ID No. 4.
In some embodiments, the protein is expressed in rice in which albino is present in the scabbard and ear of the rice, in a lower amount than in normal rice.
The present application further provides a primer set, comprising at least the following 2 primer pairs:
1, the sequence of an upstream primer is shown as SEQ ID.NO.7, and the sequence of a downstream primer is shown as SEQ ID.NO. 8;
in the 2 nd pair of primers, the sequence of the upstream primer is shown in SEQ ID.NO.9, and the sequence of the downstream primer is shown in SEQ ID.NO. 10.
The application also provides a primer group, which at least comprises the following 4 primer pairs:
1, the sequence of an upstream primer is shown as SEQ ID.NO.11, and the sequence of a downstream primer is shown as SEQ ID.NO. 12;
in the 2 nd pair of primers, the sequence of the upstream primer is shown in SEQ ID.NO.13, and the sequence of the downstream primer is shown in SEQ ID.NO. 14;
the 3 rd pair of primers has an upstream primer sequence shown in SEQ ID No.15 and a downstream primer sequence shown in SEQ ID No. 16;
in the 4 th pair of primers, the sequence of the upstream primer is shown in SEQ ID.NO.17, and the sequence of the downstream primer is shown in SEQ ID.NO. 18.
The present application provides a kit comprising the above-described gene, and/or the above-described promoter, and/or the above-described primer set.
The application provides a method for identifying rice varieties, which comprises the step of identifying the genes in a rice genome, and/or the promoters, and/or the proteins with the sequences shown in SEQ ID No.4 or the coding nucleic acids thereof.
The application provides a method for identifying rice varieties, which comprises the following steps: (1) extracting the genome DNA of the rice sample; (2) carrying out PCR amplification on the genome DNA by using the primer group; (3) analyzing the size of the amplified product.
The application also provides the application of the gene, the protein or the coding nucleic acid thereof, the promoter, the primer group, the kit or the method in identifying rice varieties or rice breeding.
In some embodiments, the rice comprises wakame, nippon, a light-or temperature-sensitive sterile line, and/or hybrids derived therefrom.
In some embodiments, the rice has a sheath and ear that is normally non-albino, or has a albino phenotype.
In the present application, a primer set or a primer pair may refer to the same concept.
Drawings
FIG. 1 comparison of the phenotypes of conventional oryza sativa HHZ (wild type) and the near isogenic line NIL (mutant).
FIG. 2 Fine localization of OsWSSP gene of interest.
FIG. 3 is a PCR verification of the OsWSSP inversion mutation breakpoint.
FIG. 4 overexpression of the WSSP gene restores the cob albino phenotype of the mutant oswssp.
FIG. 5 polymorphism detection of closely linked markers in the target region.
FIG. 6 OsWSSP flanking markers of target genes assist in identifying heterozygous individuals of BC3F 1.
Detailed Description
The present application is further illustrated by the following examples, but any examples or combination thereof should not be construed as limiting the scope or implementation of the present invention. The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1 identification of Rice cob and Sword sheath albino phenotype
A mutant showing a significantly albino phenotype in panicle stage, flowering stage and seed filling stage is selected from natural population materials of rice and named oswssp (white Sword leaf shear and Panicum). To determine the genetic basis of this trait, we constructed F by crossing oswssp with the albino normal variety Huanghuazhan (HHZ)2Isolating the population.
Further, the target gene OsWSSP was initially located within a physical interval of about 1.61Mb in the short arm of chromosome 2 by BSA-seq analysis. Near Isogenic Lines (NILs) with HHZ background were selected using parental deep resequencing (>30X), developed in this interval and using InDels markers to assist backcrossing for 5 generations.
Compared with the conventional cultivar Huanghuazhan (HHZ), the near isogenic line NIL is mainly characterized by comprising the following components in percentage by weight: the scabbard at the booting stage and the ear at the flowering and seed filling stages showed a significantly albino phenotype (fig. 1), while the chlorophyll content of these two sites was reduced (fig. 1).
Example 2 localization and albinism mechanism of cob and scabbard albinism Gene OsWSSP
After the target gene was localized to a physical region of 1.61Mb using BSA-seq, the region was further encrypted with a molecular marker, and F was used2The target gene OsWSSP is finely positioned, and finally the target gene is determined in a 146kb physical interval (figure 2).
Using IGV (Integrated Genomics viewer) software, visually comparing Paired-end reads sequences of candidate genes with parents covering the interval, and searching potential sense mutation sites in the candidate genes so as to search potential candidate genes. As a result, it was found that: one candidate gene promoter region has inverted variation.
To confirm this conclusion, the inventors designed 2 pairs of identifying primers for this inversion site (table 1):
the 1 st pair of primers (identification 1), the sequence of the upstream primer is shown as SEQ ID.NO.7 (UP _ F), and the sequence of the downstream primer is shown as SEQ ID.NO.8 (UP _ R);
in the 2 nd primer pair (identification 2), the sequence of the upstream primer is shown as SEQ ID.NO.9 (Down _ F), and the sequence of the downstream primer is shown as SEQ ID.NO.10 (Down _ R).
The authenticity of the inversions was finally confirmed by PCR amplification (FIG. 3).
Furthermore, a binary overexpression vector proUBI of a CDS sequence of a wild-type Nipponbare WSSP gene driven by a maize Ubiquitin promoter is constructed, WSSP is genetically transformed by taking a cob albino mutant ospsp as a receptor, and the cob albino phenotype of positive transgenic materials (com _ #1, com _ #2 and com _ #3) is restored to the wild-type phenotype (as shown in figure 4). As can be seen, these results all substantiate that the mutation of the promoter of the candidate gene of interest is a direct cause of oswssp encapsium and panicle albino phenotype.
TABLE 1 primer sequences
Figure BDA0003557647500000081
Figure BDA0003557647500000091
Figure BDA0003557647500000101
Figure BDA0003557647500000111
Figure BDA0003557647500000121
Example 3 development and application of marker closely linked with target gene OsWSSP
Using the data of the parental sequencing, InDel molecular marker primers linked to the target gene were designed in all regions of the target region, and after a large number of preliminary screens, 19 pairs of primer sets were selected, and polymorphisms between wild type HHZ and mutant oswssp could be resolved by 2.5% agarose horizontal gel using these primers (FIG. 5). It can be seen that in the 19 primer pairs (table 1) of the present application, there are still some bands amplified by the primers with defects such as deletion, unclear or indistinguishable (e.g. 930269, 938043, 941198, etc.), and therefore, the inventors further screened and finally confirmed through experiments that 4 pairs of InDel labeled primers with the best specificity and the strongest stability (fig. 6, table 1):
the 1 st primer (screening 4), the sequence of the upstream primer is shown as SEQ ID.NO.11 (827382_ F), and the sequence of the downstream primer is shown as SEQ ID.NO.12 (827382_ R);
the 2 nd primer (screening 6), the sequence of the upstream primer is shown as SEQ ID.NO.13 (894514_ F), and the sequence of the downstream primer is shown as SEQ ID.NO.14 (894514_ R);
the 3 rd pair of primers (screening 12), wherein the sequence of the upstream primer is shown as SEQ ID No.15 (973145_ F), and the sequence of the downstream primer is shown as SEQ ID No.16 (973145_ R);
in the 4 th primer pair (screening 13), the sequence of the upstream primer is shown as SEQ ID.NO.17 (998331_ F), and the sequence of the downstream primer is shown as SEQ ID.NO.18 (998331_ R).
These labeled primers can aid in the selection of BC3F1For example, the 4 th, 6 th, 8 th, 17 th, 19 th, 21 th in FIG. 6, and used for the creation of a rice near isogenic line.
In summary, the applicant has endeavored to describe the idea and proof of the effect of the invention. The scope of the invention is defined by the appended claims, and it will be apparent to those skilled in the art from this description and general knowledge in the art that the scope of the invention is defined by the claims. Those skilled in the art can make any modification or change to the technical solution of the present invention without departing from the spirit and scope of the present invention, and such modifications and changes are also included in the scope of the present invention.
SEQUENCE LISTING
<110> Shenzhen agricultural genome institute of Chinese agricultural science institute
<120> clone and application of rice Jianyebao and panicle albino character gene OsWSSP
<130> 20220310
<160> 18
<170> PatentIn version 3.3
<210> 1
<211> 4677
<212> DNA
<213> Artificial sequence
<400> 1
tttggcgaga agtgggtcca gtctgtagga ttatatcttg ggccgtgcca ggacagggca 60
gcaatcgact gcagcaacag taaaaccgct tgtcaaacaa ccccaataga agttatagcc 120
gcttattaca aaattctttg ataaactaaa tgcaaatgat aaaaaaaagt atgatgaaaa 180
gatagacgcc ggaaaaaaac atattcggca actaataatt ttactgctaa atcttcatct 240
ataggttgat aaaaatttat aaaaccgttg tctttaaatg caaaatgtag gagcttatta 300
tcttcatcat attttagttg gacccaaaat cgaaaacatt aactagtatt gcctgcatat 360
agaatttagt aagagctcta taaaaaaaat gtaccattgc tactaagccg aaatactcaa 420
cgtaatttct agtaaggtta ggctcggtag aaccatatgt gtaatgatat aatatctaga 480
gtatatatat tgcacatgtc gcgacgttga tgactggcta tgtgagctta atctcaatta 540
atttccaaga aaaaaaatac ctagagtata tatattgcac atgttgcgac gttgataacg 600
ggctatgcga gccttatctt aattaatgtc caagaaaaaa ccgacctagt aattatcgtc 660
ggtgacaaga agtttgcttg tgtcaaaact attgggccag gcatgggtta cccgttggta 720
aaaattgatt cccaattttt gcccactgcg ctttcggttt cggacggacg tgtccacatg 780
aataccccgg acacatgggt aaaattgcca tccctacata catacataca tacatatata 840
tatatacata catatacata tatatacata tgtatatata catatatata tatatatata 900
tatatatata tatatatata tatatatata gacacacaca tcaacaacaa caaaaaccct 960
aacctaatac atatccctcc atccgcttca aggcggcgac ggctggcagc tggctctgag 1020
ggcaacgcgg ccgagcatgg cgccgaggcg gactcaggca gtgagcggcg agcagactcc 1080
acgtcgtcac caacttcctt cctcctggtc gccgagtccg gcccagaggc cgccgtcgag 1140
ctccgcttcg cttcctcgcg tcctggtcca aggccaaggc cctcctcctc caccatcgcc 1200
cggcagctgg tgacacggcg gcgcacggca ggcaagccca gtgagtagca agcacgcatc 1260
gtgatgtgac attctagctt agcttccgtc gtcttcttct tcggctgtgt ttagatttaa 1320
agtttggatc caaacttcag tcttttttat cacatcaact tatcatacac acataatttt 1380
tcatttttca gtcacatcat ctccaatttc aaccaaaatt caaactttgc gctaaactaa 1440
acacagcctt cgtcaggtca tcgccgatca gttaatactc ctactaggct actactccca 1500
gctgaattag ctgcacaatt aaatatcgga gaagacgacg agggtgactt gttgggccta 1560
agtgggccgt gtgccgaaca aatggcccaa gtctaggaca atattttttg ggccgtgata 1620
cgacattttg ccacgagggc ccaatatgga tattttttgc attttgctcc ctgatttgaa 1680
ctgtatttct gcattttttg acggcatctc atccaggagc ccaggtcggc gccgccgcac 1740
acgcgtggcg gcctcgccgg cgactcggct gctcagagcg gcgcgaagaa aggccgcgct 1800
ggagtctgca gccgctgcgt gtggcagagt agagaccagc cagggagagc gcagctgtca 1860
gcaacctata ccagccagtg agcttgctcg cgccgttcgc cggggcgggg gaagagaacc 1920
atctggcgac aaagcacagg tgaaatccag tctctgccaa gcaatctcta attcccccca 1980
tggttcccta attgtgtgtg tatttcccat cattagtgct gttctaattc atcatttgtg 2040
ctgtttgttc tcaagcgtat tgctgagcga atagaatgcc caaagtgctt ctaatttgta 2100
tgcataggaa ttcttgacct gtcaagttac acactgcatg agttgatatg ggaacgtgcg 2160
gatagaaaaa ttcgtatttt ttggcgaaat ttcatgaaac ttgattgcat gttttgttct 2220
ctgcgtttca ttttggaatt tggggttcaa ctgcagttga aacgagtcat ctcaaattcg 2280
ttctgaacaa ttcccttcac gctgttcaaa cgaaagcaaa gagcctctct tataaacatg 2340
tttgcaatcc ggtggggatg atggcaattt caaagcttcc gaaacctgca aggcaaaagt 2400
aaaaaatttc acattggaaa gctggcttga tttgaaagcc ctataatttt ttttagtata 2460
aaatctctgt ttcgaagtca aacaattctg atcctgagta ggaatcactg atttgctaag 2520
ctgaaatctg aatttgtttt tgcactgaaa ttttatttgg aaattgctca aaaagaaagt 2580
ccagtacatg tttagaaaat tcctgatttc aggaaacttt taagccgaaa acctgagttg 2640
gaagttgttc ttgggaattt accaattagt gtttttttgt aattataaat acaaatagta 2700
aggacaacaa aatcctcccg ctactttgtg acctaactgt tggtttgcat attagaaaca 2760
cccctcgtgc taaatccagt agtttatgtt gtctgttagt ttttgaggca aacttctata 2820
tttgtgcagg tggctgtgat gggaacataa gagaggctct tatgaacttc taattcatac 2880
tagtagtata tttatgctta ttatacatgc tgacttgcaa ccttttgctt tatcatgcta 2940
ctatctggaa tcaatattgt tctaagtaac aacattttgc ctaatcttaa aacaggaagc 3000
atgtcactgg ttaaccggcc gctccctgca ctgtatggaa tttgcacggc ctcggcgaag 3060
accagagctt ggtggctctg cagagatggc aatctcccat caacaagtcg aatttcttgc 3120
acagagccat cgaatggggg ttctgtcatg gagttggagg taatggaccg caatgagcag 3180
acataccatg agaattcttc tgcttctgag gatgaggacg atgatgagga agaggctgta 3240
gagtggagca aagatgagct tgatgccatc tcggcactct ttgaccggcc gatgcgccag 3300
aagccaccaa agccgccaaa cccggtgagg cagcgacctc tcccgctgcc actgcctcac 3360
aagacacgac tgcccaatgc tcctgcacca aagcagcata tcaggctggc agcaagggct 3420
gcactttctt cacgctcttc attcagtgac caggtgtgca agaacccgga ggtcctcatt 3480
gggattgccc gggagattgc agcgctgcca ccggagtctg acgtgtccat tgtgctcgat 3540
cggtgggttc ggttcctccg gaaaggatcc ttgtcaatga ccattcggga gcttgggcac 3600
atggggctcc cagagagggc attgcagaca ctttgctggg ctcagaggca gactgtcgtg 3660
ccactcttcc ccgacgaccg gattctcgct tcgacaattg aggtcttggc acgcttcgac 3720
cagctgaaga tggaggatgc gctagagcag tgcgtccctt cagcgagccg tgctgttctt 3780
gaagccatgg tcagtgggtt catcagggca ggtaaagtag gccttgcccg aaaactcctc 3840
gagttcgcca cgatcaacaa gaggacactg agcccgagcg tccacgtgaa gctgatgctg 3900
gaggccgtcc gtacgcctga gggctacggg ctcgccgcgg cgctgctgga cgagctcggc 3960
gagaggccgg agctgcacgt tcggcagcag gactgcacgg ccgtgatgaa ggtgtgcgtg 4020
aagctccggc ggtacgccgc cgtggagagc ctgttcggct ggttcaggga caccggcggg 4080
aggcccacgg tggtgatgta cacggcggtg atccacagcc ggtgccgcga cgggcggcac 4140
cgggaggcgc tgtccctggc gtgggagatg gagcggcacg ccggcggcct cctcgacctg 4200
ccggcgtacc gggtgctggt gaagctgtgc gtggcgttgc gcgaccacga gaggggcgtc 4260
cggtacttgg cgaggatgaa ggacgccggg ttcgtcccga ccggcgacat gtacggcggc 4320
ctgatcggag gctacgcggc ggaggggagg atgggcaggt gccggaggct gatcagggag 4380
gcggagttgg ccggcgtgaa gctggagagg aggctgcttt cgcgcttgtc tgagatgggg 4440
gttgagcatt ctcagctctg aaagaaagaa actgatcttg ctgttggcat ctgactagca 4500
tggacattgc acagaaattg aactgtaaaa ttaggctctg cgtaattgcc tttaacctgt 4560
agtaacagaa cattacatta tgaatatatt tgttttgttg ttggcttgtt atcctctatt 4620
acactgtcca aaaaaaaatc tagctacgaa gtttgtagtt agaattgagt ttttttt 4677
<210> 2
<211> 4677
<212> DNA
<213> Artificial sequence
<400> 2
tgccatcgat agcttagctc atcccctttc tcttgatcgg tcggccaaag tttgacatgc 60
ttgcagttgc agctaacgtc tctgcatttt ctttcttgtt tctcagatta atttcaggct 120
gcttaattat tctgtaagaa cgaggtgctc tttgtgctag ctagctatgg ctcagctaca 180
ccctcgttta tcataaacat acacgtttct caaactgtaa gctataaaca gtgtgtttcg 240
tacaaaatct ttttatacta tataaaagaa tttaatggtg atggtgaatc taccatcatt 300
tagagttggt gagatcttct aatgctacat aaatacatct ttgtgtgtgg tttggttttc 360
tattggatgt ttgattgcaa ttatgtttga caaatacaat ttctaaatca agaaattcac 420
ataaatcttt taaaatccgt ccgttgcaga gcacgttact ttgctagtct ttaaaatttt 480
aaataaattt agttttcaag tttataatag gtaatactga gtttatcatg tactattcac 540
ctgttccggt tttcatgcca ctaactagct cagtcaaact cctttatcga acacagccca 600
gccagaatgc acccttgtct ttttacctag tgctataaga ggaaccaatt caagatgatg 660
atgattgatt taccttgtag cattactacg cagttcaaat tactaacctt tcagattagg 720
tacggtctga atatgtgatg gaagatcgaa gcagctgtat ctcataaaat aaattacaga 780
ttccgtctgg aaactgcaag acgaatttat taagcctaat taatctatta ttagcaaata 840
tttattgtag caccaaattg tcaaatcatg gcgcaattag gcttaaaaga tttgtctcgt 900
aatttcaagg cgaaatgttt aattgttttt tttctacatt tagtactcta tgcatgtgtc 960
caaacatcct atgtaacagc gtgaaaaatt ttgtttggga actaaacaga ccctgattgt 1020
gatgcaaccc ataagtaatt gcaaaactag ctagctatct acagcatcat cagcatatat 1080
cttggtggtc taaacttctc aggaaacaaa agatgagaaa ttgttctgtt cttagtccac 1140
gctgatgata acgataggtg tgctgtggtg ggcacaagct tttttctttc tttttttttt 1200
tgaggggaaa gttgcttaag atgaagtgac ataaacaaga ttaattcgag gaaaatgtgc 1260
gcatctcaga tgtgtactcc atttatacaa aacccagcaa attaagaagg tacaattatc 1320
tgacagacta gcgaacaaac tgaaaattat agtcgtagta ctacaaattt gcagtttgtg 1380
gctgagggga atctatccca tctggtatat gctgtcactt tgtgctagta ctgtactatg 1440
tactagcctg tagcctctac ttttgtaatt gaatgagaat atttgaatgt tttacaccac 1500
ataatggaac agctcaaatc tcataagatc ttggagagat attcttgatt actctgatta 1560
atgggtcatg tgttgatgca tatatatata tatatatata tatatatata tatatatata 1620
tatatatata tatatatatg ttaatgaatc aaaacgctgc attttgctcc ctgatttgaa 1680
ctgtatttct gcattttttg acggcatctc atccaggagc ccaggtcggc gccgccgcac 1740
acgcgtggcg gcctcgccgg cgactcggct gctcagagcg gcgcgaagaa aggccgcgct 1800
ggagtctgca gccgctgcgt gtggcagagt agagaccagc cagggagagc gcagctgtca 1860
gcaacctata ccagccagtg agcttgctcg cgccgttcgc cggggcgggg gaagagaacc 1920
atctggcgac aaagcacagg tgaaatccag tctctgccaa gcaatctcta attcccccca 1980
tggttcccta attgtgtgtg tatttcccat cattagtgct gttctaattc atcatttgtg 2040
ctgtttgttc tcaagcgtat tgctgagcga atagaatgcc caaagtgctt ctaatttgta 2100
tgcataggaa ttcttgacct gtcaagttac acactgcatg agttgatatg ggaacgtgcg 2160
gatagaaaaa ttcgtatttt ttggcgaaat ttcatgaaac ttgattgcat gttttgttct 2220
ctgcgtttca ttttggaatt tggggttcaa ctgcagttga aacgagtcat ctcaaattcg 2280
ttctgaacaa ttcccttcac gctgttcaaa cgaaagcaaa gagcctctct tataaacatg 2340
tttgcaatcc ggtggggatg atggcaattt caaagcttcc gaaacctgca aggcaaaagt 2400
aaaaaatttc acattggaaa gctggcttga tttgaaagcc ctataatttt ttttagtata 2460
aaatctctgt ttcgaagtca aacaattctg atcctgagta ggaatcactg atttgctaag 2520
ctgaaatctg aatttgtttt tgcactgaaa ttttatttgg aaattgctca aaaagaaagt 2580
ccagtacatg tttagaaaat tcctgatttc aggaaacttt taagccgaaa acctgagttg 2640
gaagttgttc ttgggaattt accaattagt gtttttttgt aattataaat acaaatagta 2700
aggacaacaa aatcctcccg ctactttgtg acctaactgt tggtttgcat attagaaaca 2760
cccctcgtgc taaatccagt agtttatgtt gtctgttagt ttttgaggca aacttctata 2820
tttgtgcagg tggctgtgat gggaacataa gagaggctct tatgaacttc taattcatac 2880
tagtagtata tttatgctta ttatacatgc tgacttgcaa ccttttgctt tatcatgcta 2940
ctatctggaa tcaatattgt tctaagtaac aacattttgc ctaatcttaa aacaggaagc 3000
atgtcactgg ttaaccggcc gctccctgca ctgtatggaa tttgcacggc ctcggcgaag 3060
accagagctt ggtggctctg cagagatggc aatctcccat caacaagtcg aatttcttgc 3120
acagagccat cgaatggggg ttctgtcatg gagttggagg taatggaccg caatgagcag 3180
acataccatg agaattcttc tgcttctgag gatgaggacg atgatgagga agaggctgta 3240
gagtggagca aagatgagct tgatgccatc tcggcactct ttgaccggcc gatgcgccag 3300
aagccaccaa agccgccaaa cccggtgagg cagcgacctc tcccgctgcc actgcctcac 3360
aagacacgac tgcccaatgc tcctgcacca aagcagcata tcaggctggc agcaagggct 3420
gcactttctt cacgctcttc attcagtgac caggtgtgca agaacccgga ggtcctcatt 3480
gggattgccc gggagattgc agcgctgcca ccggagtctg acgtgtccat tgtgctcgat 3540
cggtgggttc ggttcctccg gaaaggatcc ttgtcaatga ccattcggga gcttgggcac 3600
atggggctcc cagagagggc attgcagaca ctttgctggg ctcagaggca gactgtcgtg 3660
ccactcttcc ccgacgaccg gattctcgct tcgacaattg aggtcttggc acgcttcgac 3720
cagctgaaga tggaggatgc gctagagcag tgcgtccctt cagcgagccg tgctgttctt 3780
gaagccatgg tcagtgggtt catcagggca ggtaaagtag gccttgcccg aaaactcctc 3840
gagttcgcca cgatcaacaa gaggacactg agcccgagcg tccacgtgaa gctgatgctg 3900
gaggccgtcc gtacgcctga gggctacggg ctcgccgcgg cgctgctgga cgagctcggc 3960
gagaggccgg agctgcacgt tcggcagcag gactgcacgg ccgtgatgaa ggtgtgcgtg 4020
aagctccggc ggtacgccgc cgtggagagc ctgttcggct ggttcaggga caccggcggg 4080
aggcccacgg tggtgatgta cacggcggtg atccacagcc ggtgccgcga cgggcggcac 4140
cgggaggcgc tgtccctggc gtgggagatg gagcggcacg ccggcggcct cctcgacctg 4200
ccggcgtacc gggtgctggt gaagctgtgc gtggcgttgc gcgaccacga gaggggcgtc 4260
cggtacttgg cgaggatgaa ggacgccggg ttcgtcccga ccggcgacat gtacggcggc 4320
ctgatcggag gctacgcggc ggaggggagg atgggcaggt gccggaggct gatcagggag 4380
gcggagttgg ccggcgtgaa gctggagagg aggctgcttt cgcgcttgtc tgagatgggg 4440
gttgagcatt ctcagctctg aaagaaagaa actgatcttg ctgttggcat ctgactagca 4500
tggacattgc acagaaattg aactgtaaaa ttaggctctg cgtaattgcc tttaacctgt 4560
agtaacagaa cattacatta tgaatatatt tgttttgttg ttggcttgtt atcctctatt 4620
acactgtcca aaaaaaaatc tagctacgaa gtttgtagtt agaattgagt ttttttt 4677
<210> 3
<211> 1461
<212> DNA
<213> Artificial sequence
<400> 3
atgtcactgg ttaaccggcc gctccctgca ctgtatggaa tttgcacggc ctcggcgaag 60
accagagctt ggtggctctg cagagatggc aatctcccat caacaagtcg aatttcttgc 120
acagagccat cgaatggggg ttctgtcatg gagttggagg taatggaccg caatgagcag 180
acataccatg agaattcttc tgcttctgag gatgaggacg atgatgagga agaggctgta 240
gagtggagca aagatgagct tgatgccatc tcggcactct ttgaccggcc gatgcgccag 300
aagccaccaa agccgccaaa cccggtgagg cagcgacctc tcccgctgcc actgcctcac 360
aagacacgac tgcccaatgc tcctgcacca aagcagcata tcaggctggc agcaagggct 420
gcactttctt cacgctcttc attcagtgac caggtgtgca agaacccgga ggtcctcatt 480
gggattgccc gggagattgc agcgctgcca ccggagtctg acgtgtccat tgtgctcgat 540
cggtgggttc ggttcctccg gaaaggatcc ttgtcaatga ccattcggga gcttgggcac 600
atggggctcc cagagagggc attgcagaca ctttgctggg ctcagaggca gactgtcgtg 660
ccactcttcc ccgacgaccg gattctcgct tcgacaattg aggtcttggc acgcttcgac 720
cagctgaaga tggaggatgc gctagagcag tgcgtccctt cagcgagccg tgctgttctt 780
gaagccatgg tcagtgggtt catcagggca ggtaaagtag gccttgcccg aaaactcctc 840
gagttcgcca cgatcaacaa gaggacactg agcccgagcg tccacgtgaa gctgatgctg 900
gaggccgtcc gtacgcctga gggctacggg ctcgccgcgg cgctgctgga cgagctcggc 960
gagaggccgg agctgcacgt tcggcagcag gactgcacgg ccgtgatgaa ggtgtgcgtg 1020
aagctccggc ggtacgccgc cgtggagagc ctgttcggct ggttcaggga caccggcggg 1080
aggcccacgg tggtgatgta cacggcggtg atccacagcc ggtgccgcga cgggcggcac 1140
cgggaggcgc tgtccctggc gtgggagatg gagcggcacg ccggcggcct cctcgacctg 1200
ccggcgtacc gggtgctggt gaagctgtgc gtggcgttgc gcgaccacga gaggggcgtc 1260
cggtacttgg cgaggatgaa ggacgccggg ttcgtcccga ccggcgacat gtacggcggc 1320
ctgatcggag gctacgcggc ggaggggagg atgggcaggt gccggaggct gatcagggag 1380
gcggagttgg ccggcgtgaa gctggagagg aggctgcttt cgcgcttgtc tgagatgggg 1440
gttgagcatt ctcagctctg a 1461
<210> 4
<211> 486
<212> PRT
<213> Artificial sequence
<400> 4
Met Ser Leu Val Asn Arg Pro Leu Pro Ala Leu Tyr Gly Ile Cys Thr
1 5 10 15
Ala Ser Ala Lys Thr Arg Ala Trp Trp Leu Cys Arg Asp Gly Asn Leu
20 25 30
Pro Ser Thr Ser Arg Ile Ser Cys Thr Glu Pro Ser Asn Gly Gly Ser
35 40 45
Val Met Glu Leu Glu Val Met Asp Arg Asn Glu Gln Thr Tyr His Glu
50 55 60
Asn Ser Ser Ala Ser Glu Asp Glu Asp Asp Asp Glu Glu Glu Ala Val
65 70 75 80
Glu Trp Ser Lys Asp Glu Leu Asp Ala Ile Ser Ala Leu Phe Asp Arg
85 90 95
Pro Met Arg Gln Lys Pro Pro Lys Pro Pro Asn Pro Val Arg Gln Arg
100 105 110
Pro Leu Pro Leu Pro Leu Pro His Lys Thr Arg Leu Pro Asn Ala Pro
115 120 125
Ala Pro Lys Gln His Ile Arg Leu Ala Ala Arg Ala Ala Leu Ser Ser
130 135 140
Arg Ser Ser Phe Ser Asp Gln Val Cys Lys Asn Pro Glu Val Leu Ile
145 150 155 160
Gly Ile Ala Arg Glu Ile Ala Ala Leu Pro Pro Glu Ser Asp Val Ser
165 170 175
Ile Val Leu Asp Arg Trp Val Arg Phe Leu Arg Lys Gly Ser Leu Ser
180 185 190
Met Thr Ile Arg Glu Leu Gly His Met Gly Leu Pro Glu Arg Ala Leu
195 200 205
Gln Thr Leu Cys Trp Ala Gln Arg Gln Thr Val Val Pro Leu Phe Pro
210 215 220
Asp Asp Arg Ile Leu Ala Ser Thr Ile Glu Val Leu Ala Arg Phe Asp
225 230 235 240
Gln Leu Lys Met Glu Asp Ala Leu Glu Gln Cys Val Pro Ser Ala Ser
245 250 255
Arg Ala Val Leu Glu Ala Met Val Ser Gly Phe Ile Arg Ala Gly Lys
260 265 270
Val Gly Leu Ala Arg Lys Leu Leu Glu Phe Ala Thr Ile Asn Lys Arg
275 280 285
Thr Leu Ser Pro Ser Val His Val Lys Leu Met Leu Glu Ala Val Arg
290 295 300
Thr Pro Glu Gly Tyr Gly Leu Ala Ala Ala Leu Leu Asp Glu Leu Gly
305 310 315 320
Glu Arg Pro Glu Leu His Val Arg Gln Gln Asp Cys Thr Ala Val Met
325 330 335
Lys Val Cys Val Lys Leu Arg Arg Tyr Ala Ala Val Glu Ser Leu Phe
340 345 350
Gly Trp Phe Arg Asp Thr Gly Gly Arg Pro Thr Val Val Met Tyr Thr
355 360 365
Ala Val Ile His Ser Arg Cys Arg Asp Gly Arg His Arg Glu Ala Leu
370 375 380
Ser Leu Ala Trp Glu Met Glu Arg His Ala Gly Gly Leu Leu Asp Leu
385 390 395 400
Pro Ala Tyr Arg Val Leu Val Lys Leu Cys Val Ala Leu Arg Asp His
405 410 415
Glu Arg Gly Val Arg Tyr Leu Ala Arg Met Lys Asp Ala Gly Phe Val
420 425 430
Pro Thr Gly Asp Met Tyr Gly Gly Leu Ile Gly Gly Tyr Ala Ala Glu
435 440 445
Gly Arg Met Gly Arg Cys Arg Arg Leu Ile Arg Glu Ala Glu Leu Ala
450 455 460
Gly Val Lys Leu Glu Arg Arg Leu Leu Ser Arg Leu Ser Glu Met Gly
465 470 475 480
Val Glu His Ser Gln Leu
485
<210> 5
<211> 3000
<212> DNA
<213> Artificial sequence
<400> 5
tttggcgaga agtgggtcca gtctgtagga ttatatcttg ggccgtgcca ggacagggca 60
gcaatcgact gcagcaacag taaaaccgct tgtcaaacaa ccccaataga agttatagcc 120
gcttattaca aaattctttg ataaactaaa tgcaaatgat aaaaaaaagt atgatgaaaa 180
gatagacgcc ggaaaaaaac atattcggca actaataatt ttactgctaa atcttcatct 240
ataggttgat aaaaatttat aaaaccgttg tctttaaatg caaaatgtag gagcttatta 300
tcttcatcat attttagttg gacccaaaat cgaaaacatt aactagtatt gcctgcatat 360
agaatttagt aagagctcta taaaaaaaat gtaccattgc tactaagccg aaatactcaa 420
cgtaatttct agtaaggtta ggctcggtag aaccatatgt gtaatgatat aatatctaga 480
gtatatatat tgcacatgtc gcgacgttga tgactggcta tgtgagctta atctcaatta 540
atttccaaga aaaaaaatac ctagagtata tatattgcac atgttgcgac gttgataacg 600
ggctatgcga gccttatctt aattaatgtc caagaaaaaa ccgacctagt aattatcgtc 660
ggtgacaaga agtttgcttg tgtcaaaact attgggccag gcatgggtta cccgttggta 720
aaaattgatt cccaattttt gcccactgcg ctttcggttt cggacggacg tgtccacatg 780
aataccccgg acacatgggt aaaattgcca tccctacata catacataca tacatatata 840
tatatacata catatacata tatatacata tgtatatata catatatata tatatatata 900
tatatatata tatatatata tatatatata gacacacaca tcaacaacaa caaaaaccct 960
aacctaatac atatccctcc atccgcttca aggcggcgac ggctggcagc tggctctgag 1020
ggcaacgcgg ccgagcatgg cgccgaggcg gactcaggca gtgagcggcg agcagactcc 1080
acgtcgtcac caacttcctt cctcctggtc gccgagtccg gcccagaggc cgccgtcgag 1140
ctccgcttcg cttcctcgcg tcctggtcca aggccaaggc cctcctcctc caccatcgcc 1200
cggcagctgg tgacacggcg gcgcacggca ggcaagccca gtgagtagca agcacgcatc 1260
gtgatgtgac attctagctt agcttccgtc gtcttcttct tcggctgtgt ttagatttaa 1320
agtttggatc caaacttcag tcttttttat cacatcaact tatcatacac acataatttt 1380
tcatttttca gtcacatcat ctccaatttc aaccaaaatt caaactttgc gctaaactaa 1440
acacagcctt cgtcaggtca tcgccgatca gttaatactc ctactaggct actactccca 1500
gctgaattag ctgcacaatt aaatatcgga gaagacgacg agggtgactt gttgggccta 1560
agtgggccgt gtgccgaaca aatggcccaa gtctaggaca atattttttg ggccgtgata 1620
cgacattttg ccacgagggc ccaatatgga tattttttgc attttgctcc ctgatttgaa 1680
ctgtatttct gcattttttg acggcatctc atccaggagc ccaggtcggc gccgccgcac 1740
acgcgtggcg gcctcgccgg cgactcggct gctcagagcg gcgcgaagaa aggccgcgct 1800
ggagtctgca gccgctgcgt gtggcagagt agagaccagc cagggagagc gcagctgtca 1860
gcaacctata ccagccagtg agcttgctcg cgccgttcgc cggggcgggg gaagagaacc 1920
atctggcgac aaagcacagg tgaaatccag tctctgccaa gcaatctcta attcccccca 1980
tggttcccta attgtgtgtg tatttcccat cattagtgct gttctaattc atcatttgtg 2040
ctgtttgttc tcaagcgtat tgctgagcga atagaatgcc caaagtgctt ctaatttgta 2100
tgcataggaa ttcttgacct gtcaagttac acactgcatg agttgatatg ggaacgtgcg 2160
gatagaaaaa ttcgtatttt ttggcgaaat ttcatgaaac ttgattgcat gttttgttct 2220
ctgcgtttca ttttggaatt tggggttcaa ctgcagttga aacgagtcat ctcaaattcg 2280
ttctgaacaa ttcccttcac gctgttcaaa cgaaagcaaa gagcctctct tataaacatg 2340
tttgcaatcc ggtggggatg atggcaattt caaagcttcc gaaacctgca aggcaaaagt 2400
aaaaaatttc acattggaaa gctggcttga tttgaaagcc ctataatttt ttttagtata 2460
aaatctctgt ttcgaagtca aacaattctg atcctgagta ggaatcactg atttgctaag 2520
ctgaaatctg aatttgtttt tgcactgaaa ttttatttgg aaattgctca aaaagaaagt 2580
ccagtacatg tttagaaaat tcctgatttc aggaaacttt taagccgaaa acctgagttg 2640
gaagttgttc ttgggaattt accaattagt gtttttttgt aattataaat acaaatagta 2700
aggacaacaa aatcctcccg ctactttgtg acctaactgt tggtttgcat attagaaaca 2760
cccctcgtgc taaatccagt agtttatgtt gtctgttagt ttttgaggca aacttctata 2820
tttgtgcagg tggctgtgat gggaacataa gagaggctct tatgaacttc taattcatac 2880
tagtagtata tttatgctta ttatacatgc tgacttgcaa ccttttgctt tatcatgcta 2940
ctatctggaa tcaatattgt tctaagtaac aacattttgc ctaatcttaa aacaggaagc 3000
<210> 6
<211> 3000
<212> DNA
<213> Artificial sequence
<400> 6
tgccatcgat agcttagctc atcccctttc tcttgatcgg tcggccaaag tttgacatgc 60
ttgcagttgc agctaacgtc tctgcatttt ctttcttgtt tctcagatta atttcaggct 120
gcttaattat tctgtaagaa cgaggtgctc tttgtgctag ctagctatgg ctcagctaca 180
ccctcgttta tcataaacat acacgtttct caaactgtaa gctataaaca gtgtgtttcg 240
tacaaaatct ttttatacta tataaaagaa tttaatggtg atggtgaatc taccatcatt 300
tagagttggt gagatcttct aatgctacat aaatacatct ttgtgtgtgg tttggttttc 360
tattggatgt ttgattgcaa ttatgtttga caaatacaat ttctaaatca agaaattcac 420
ataaatcttt taaaatccgt ccgttgcaga gcacgttact ttgctagtct ttaaaatttt 480
aaataaattt agttttcaag tttataatag gtaatactga gtttatcatg tactattcac 540
ctgttccggt tttcatgcca ctaactagct cagtcaaact cctttatcga acacagccca 600
gccagaatgc acccttgtct ttttacctag tgctataaga ggaaccaatt caagatgatg 660
atgattgatt taccttgtag cattactacg cagttcaaat tactaacctt tcagattagg 720
tacggtctga atatgtgatg gaagatcgaa gcagctgtat ctcataaaat aaattacaga 780
ttccgtctgg aaactgcaag acgaatttat taagcctaat taatctatta ttagcaaata 840
tttattgtag caccaaattg tcaaatcatg gcgcaattag gcttaaaaga tttgtctcgt 900
aatttcaagg cgaaatgttt aattgttttt tttctacatt tagtactcta tgcatgtgtc 960
caaacatcct atgtaacagc gtgaaaaatt ttgtttggga actaaacaga ccctgattgt 1020
gatgcaaccc ataagtaatt gcaaaactag ctagctatct acagcatcat cagcatatat 1080
cttggtggtc taaacttctc aggaaacaaa agatgagaaa ttgttctgtt cttagtccac 1140
gctgatgata acgataggtg tgctgtggtg ggcacaagct tttttctttc tttttttttt 1200
tgaggggaaa gttgcttaag atgaagtgac ataaacaaga ttaattcgag gaaaatgtgc 1260
gcatctcaga tgtgtactcc atttatacaa aacccagcaa attaagaagg tacaattatc 1320
tgacagacta gcgaacaaac tgaaaattat agtcgtagta ctacaaattt gcagtttgtg 1380
gctgagggga atctatccca tctggtatat gctgtcactt tgtgctagta ctgtactatg 1440
tactagcctg tagcctctac ttttgtaatt gaatgagaat atttgaatgt tttacaccac 1500
ataatggaac agctcaaatc tcataagatc ttggagagat attcttgatt actctgatta 1560
atgggtcatg tgttgatgca tatatatata tatatatata tatatatata tatatatata 1620
tatatatata tatatatatg ttaatgaatc aaaacgctgc attttgctcc ctgatttgaa 1680
ctgtatttct gcattttttg acggcatctc atccaggagc ccaggtcggc gccgccgcac 1740
acgcgtggcg gcctcgccgg cgactcggct gctcagagcg gcgcgaagaa aggccgcgct 1800
ggagtctgca gccgctgcgt gtggcagagt agagaccagc cagggagagc gcagctgtca 1860
gcaacctata ccagccagtg agcttgctcg cgccgttcgc cggggcgggg gaagagaacc 1920
atctggcgac aaagcacagg tgaaatccag tctctgccaa gcaatctcta attcccccca 1980
tggttcccta attgtgtgtg tatttcccat cattagtgct gttctaattc atcatttgtg 2040
ctgtttgttc tcaagcgtat tgctgagcga atagaatgcc caaagtgctt ctaatttgta 2100
tgcataggaa ttcttgacct gtcaagttac acactgcatg agttgatatg ggaacgtgcg 2160
gatagaaaaa ttcgtatttt ttggcgaaat ttcatgaaac ttgattgcat gttttgttct 2220
ctgcgtttca ttttggaatt tggggttcaa ctgcagttga aacgagtcat ctcaaattcg 2280
ttctgaacaa ttcccttcac gctgttcaaa cgaaagcaaa gagcctctct tataaacatg 2340
tttgcaatcc ggtggggatg atggcaattt caaagcttcc gaaacctgca aggcaaaagt 2400
aaaaaatttc acattggaaa gctggcttga tttgaaagcc ctataatttt ttttagtata 2460
aaatctctgt ttcgaagtca aacaattctg atcctgagta ggaatcactg atttgctaag 2520
ctgaaatctg aatttgtttt tgcactgaaa ttttatttgg aaattgctca aaaagaaagt 2580
ccagtacatg tttagaaaat tcctgatttc aggaaacttt taagccgaaa acctgagttg 2640
gaagttgttc ttgggaattt accaattagt gtttttttgt aattataaat acaaatagta 2700
aggacaacaa aatcctcccg ctactttgtg acctaactgt tggtttgcat attagaaaca 2760
cccctcgtgc taaatccagt agtttatgtt gtctgttagt ttttgaggca aacttctata 2820
tttgtgcagg tggctgtgat gggaacataa gagaggctct tatgaacttc taattcatac 2880
tagtagtata tttatgctta ttatacatgc tgacttgcaa ccttttgctt tatcatgcta 2940
ctatctggaa tcaatattgt tctaagtaac aacattttgc ctaatcttaa aacaggaagc 3000
<210> 7
<211> 20
<212> DNA
<213> Artificial sequence
<400> 7
tgtggctgag gggaatctat 20
<210> 8
<211> 20
<212> DNA
<213> Artificial sequence
<400> 8
ggacggttca ccaactagca 20
<210> 9
<211> 20
<212> DNA
<213> Artificial sequence
<400> 9
gcaagctcac tggctggtat 20
<210> 10
<211> 27
<212> DNA
<213> Artificial sequence
<400> 10
ttatcacatc aacttatcat acacaca 27
<210> 11
<211> 20
<212> DNA
<213> Artificial sequence
<400> 11
ccagccatgc atgtttgaag 20
<210> 12
<211> 23
<212> DNA
<213> Artificial sequence
<400> 12
ttggtcgatt atcagaaact tcc 23
<210> 13
<211> 22
<212> DNA
<213> Artificial sequence
<400> 13
tggaatcatg tttggtcttc ac 22
<210> 14
<211> 20
<212> DNA
<213> Artificial sequence
<400> 14
tgattgaaac cgttgcacag 20
<210> 15
<211> 20
<212> DNA
<213> Artificial sequence
<400> 15
cagcgtatac ccatgtctgc 20
<210> 16
<211> 20
<212> DNA
<213> Artificial sequence
<400> 16
ggacagaggc atcatcaacc 20
<210> 17
<211> 20
<212> DNA
<213> Artificial sequence
<400> 17
gtcgggcagc actgagtaat 20
<210> 18
<211> 20
<212> DNA
<213> Artificial sequence
<400> 18
caactgattg cgccactcta 20

Claims (10)

1. A nucleic acid sequence of the OsWSSP gene is shown in SEQ ID No.1 or SEQ ID No. 2.
2. A promoter has a nucleic acid sequence shown as SEQ ID No.5 or SEQ ID No. 6.
3. The gene or the protein with the sequence shown in SEQ ID No.4 or the coding nucleic acid thereof according to claim 1 or the promoter according to claim 2 is used for regulating and controlling chloroplast development and/or chlorophyll synthesis of rice scabbard and panicle.
4. A primer set, comprising at least the following 2 primer pairs:
1, the sequence of an upstream primer is shown as SEQ ID.NO.7, and the sequence of a downstream primer is shown as SEQ ID.NO. 8;
in the 2 nd pair of primers, the sequence of the upstream primer is shown in SEQ ID.NO.9, and the sequence of the downstream primer is shown in SEQ ID.NO. 10.
5. A primer set comprising at least 4 primer pairs selected from the group consisting of:
1, the sequence of an upstream primer is shown as SEQ ID.NO.11, and the sequence of a downstream primer is shown as SEQ ID.NO. 12;
in the 2 nd pair of primers, the sequence of the upstream primer is shown in SEQ ID.NO.13, and the sequence of the downstream primer is shown in SEQ ID.NO. 14;
the 3 rd pair of primers has an upstream primer sequence shown in SEQ ID No.15 and a downstream primer sequence shown in SEQ ID No. 16;
in the 4 th pair of primers, the sequence of the upstream primer is shown in SEQ ID No.17, and the sequence of the downstream primer is shown in SEQ ID No. 18.
6. A kit, characterized in that: comprising the gene of claim 1, and/or the promoter of claim 2, and/or the primer set of claim 4 or 5.
7. A method for identifying a rice variety, comprising the steps of identifying the gene of claim 1, and/or the promoter of claim 2, and/or the protein of SEQ id No.4 or the nucleic acid encoding the protein in the genome of rice; or:
the method comprises the following steps: (1) extracting the genome DNA of the rice sample; (2) performing PCR amplification on the genomic DNA by using the primer set of claim 4 or 5; (3) analyzing the size of the amplified product.
8. Use of the gene of claim 1, or a protein having the sequence shown in SEQ ID No.4 or a nucleic acid encoding the same, or the promoter of claim 2, or the primer set of claim 4 or 5, or the kit of claim 6, or the method of claim 7 for identifying rice varieties or in rice breeding.
9. The use according to claim 3, or the method according to claim 7, or the use according to claim 8, wherein the rice comprises wakame, nipponlily, a light-or temperature-sensitive sterile line, and/or hybrids derived therefrom.
10. The use, method or use according to claim 9 wherein the rice has a sheath and panicle albino phenotype.
CN202210280493.3A 2022-03-22 2022-03-22 Clone and application of rice sword-leaf sheath and panicle whitening trait gene OsWSSP Active CN114574500B (en)

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