Glycoside hydrolase gene and promoter for improving yield traits of cotton and application thereof

文档序号：81229 发布日期：2021-10-08 浏览：36次中文

阅读说明：本技术 一个改良棉花产量性状的糖苷水解酶基因和启动子及其应用 (Glycoside hydrolase gene and promoter for improving yield traits of cotton and application thereof ) 是由张天真韩泽刚何璐胡艳方磊于 2021-06-24 设计创作，主要内容包括：本发明公开了一个改良棉花产量性状的糖苷水解酶基因及其启动子及其应用。该基因GHLP1基因组序列为：SEQ ID NO.2或SEQ ID NO.12,启动子序列为：SEQ ID NO.3或SEQ ID NO.13。基因GHLP1的外显子区域包含一个非同义突变的单核苷酸多态性位点,位于编码区序列的1174bp位置处,该位点碱基是T或G,对应的氨基酸为Ser或Arg；同时该基因上游471bp的启动子区域中包含一个单核苷酸多态性变异位点,该位点碱基是A或C。该基因启动子区域的单核苷酸多态性变异与棉花衣分性状和籽指性状密切关联,并且启动子单倍型CC的棉花品种(系)的产量性状显著优于单倍型AA的棉花材料。该基因在高效鉴定高产陆地棉品种、提升棉花产量性状以及培育棉花高产新品种中具有重要的研究价值和应用前景。(The invention discloses a glycoside hydrolase gene for improving yield traits of cotton, a promoter thereof and application thereof. The genome sequence of the gene GHLP1 is as follows: SEQ ID No.2 or SEQ ID No.12, the promoter sequence is: SEQ ID NO.3 or SEQ ID NO. 13. The exon region of the gene GHLP1 contains a single nucleotide polymorphism site with non-synonymous mutation, which is positioned at the position of 1174bp of the coding region sequence, the base of the site is T or G, and the corresponding amino acid is Ser or Arg; meanwhile, the promoter region of 471bp upstream of the gene contains a single nucleotide polymorphism variation site, and the base of the site is A or C. The single nucleotide polymorphism variation of the gene promoter region is closely related to the coat division character and the seed finger character of cotton, and the yield character of the cotton variety (line) of the promoter haplotype CC is obviously superior to that of the cotton material of the haplotype AA. The gene has important research value and application prospect in efficiently identifying high-yield upland cotton varieties, improving the yield characters of cotton and cultivating high-yield new varieties of cotton.)

1. A glycoside hydrolase gene GHLP1 for improving cotton yield traits is characterized in that the cDNA sequence of the gene is shown as SEQ ID NO.1 or SEQ ID NO.11, and the genome sequence of the gene is shown as SEQ ID NO.2 or SEQ ID NO. 12.

2. Use of the glycoside hydrolase gene GHLP1 of claim 1 in the identification of high yielding upland cotton varieties and in improving cotton yield traits.

3. The use of the glycoside hydrolase gene GHLP1 as defined in claim 1 in breeding new varieties of cotton with high yield by genetic engineering means.

4. A promoter of a glycoside hydrolase gene GHLP1 for improving yield traits of cotton, which is characterized in that the sequence of the promoter is shown as SEQ ID NO.3 or SEQ ID NO. 13.

5. Use of the glycoside hydrolase gene GHLP1 promoter of claim 4 for identifying high yielding upland cotton varieties and improving cotton yield traits.

6. The use of the promoter of the glycoside hydrolase gene GHLP1 as set forth in claim 4 in breeding new varieties of high-yield cotton by genetic engineering means.

7. A single nucleotide polymorphism variation site based on the glycoside hydrolase gene GHLP1 of claim 4, wherein the single nucleotide polymorphism variation site is located at 471bp of the sequence shown in SEQ ID NO.3, and the base is A or C.

8. The use of the single nucleotide polymorphism variant site of claim 7 for identifying a high yielding upland cotton variety.

9. The use of claim 7, wherein the high-yield cotton variety is obtained by detecting the genotype of the SNP variant site in a cotton genome to be detected, wherein the SNP variant site is C.

10. The use according to claim 7, wherein the primers for detecting the SNP variant sites are specifically: the upstream primer is shown as SEQ ID NO.8, and the downstream primer is shown as SEQ ID NO. 9.

Technical Field

The invention belongs to the field of biotechnology application, and relates to a glycosidic hydrolase gene related to cotton yield traits and a promoter thereof.

Background

Cotton, as a major source of natural fiber, is an important commercial crop. The cotton production not only has important influence on the development of agriculture and even national economy in China, but also plays a very important role in the world cotton trade market. In addition, cotton fiber is an excellent natural fiber which is most widely used, is an important raw material of textile industry, and plays a significant role in national economic development. With the improvement of living standard of people, the demand of natural pure cotton fabrics is continuously increased, and the requirements on the quality of fibers are higher and higher. Therefore, it is extremely important to deeply excavate and utilize the genetic variation related to the quality of cotton.

Genome-wide association analysis (GWAS) is a new strategy which takes millions of Single Nucleotide Polymorphisms (SNPs) in a Genome as molecular genetic markers, analyzes the association on the Genome level and discovers genetic variation affecting complex traits through comparison. With the improvement of genome sequencing technology and the reduction of sequencing cost, combined with the high development of bioinformatics, GWAS becomes one of the most effective methods for mining and analyzing genes for human diseases, crop agronomic traits and resistance traits and their related genetic mechanisms. The agronomic trait related genes are mined and cloned by utilizing whole genome association analysis, candidate genes do not need to be assumed in advance, the detection capability is strong, the precision is high, and the method is a hotspot of molecular breeding research. Belo et al (2008) performed GWAS analysis of the SNP of 8,950 from 553 elite inbred lines and identified loci associated with oleic acid content, the first true genome-wide association analysis in maize. Huang et al (2011) re-sequenced 517 rice cultivars by using a second generation sequencing technology to obtain millions of SNPs, then carried out GWAS analysis on 14 agronomic traits of rice, and successfully identified 80 trait-associated sites. In addition, they also re-sequenced up to 950 rice populations, subjected to GWAS analysis for flowering phase and 10 yield-related traits, and identified many known functional genes (Huang et al.2012). Lin and the like (2014) carry out whole genome re-sequencing on 360 parts of tomato germplasm all over the world, and through population differentiation analysis, a key mutation site determining the color of the pink fruit pericarp, namely 603bp deletion of a SlMYB12 gene promoter region is found for the first time, so that the expression of the gene is inhibited, and flavonoid cannot be accumulated in the mature pink fruit tomato pericarp, thereby causing the difference between fresh-eating tomatoes and processed tomatoes. Zhou et al (2015) re-sequence 302 soybean wild, local and improved varieties, and find that 96 GWAS associated sites are associated with the previously reported QTL by combining GWAS analysis technology, and identify new associated sites related to oil content, plant height and trichogenous formation. Fang et al (2017) identified 25 selection signals during cotton improvement by genome-wide re-sequencing of 318 pieces of upland cotton material, and identified 119 association sites in total by GWAS analysis, of which 71 were yield-related association sites, 45 were fiber quality-related association sites, and 3 were associated with verticillium wilt resistance (Fang et al, 2017). Ma et al (2018) re-sequencing 419 core germplasm upland cotton material, found 7383 SNPs significantly associated with these traits, located within or near 4820 genes. And a key analysis was performed on some candidate genes that control flowering, affect fiber length, and fiber strength (Ma et al, 2018). Liu et al (2021) utilizes natural population composed of 290 upland cotton cultivation seeds to perform field identification for many years, and combines high-density SNP markers to perform whole genome association analysis on cotton wilt resistance, so as to identify and obtain a main effect disease-resistant site Fov7, and determines that the gene GhGLR4.8 is a novel plant atypical main effect disease-resistant gene (Liu et al, 2021). The results fully show that the whole genome association analysis has high positioning precision, even can reach the level of a single gene, and the obtained functional marker related to the target character is utilized to screen the target character, so that the breeding process and efficiency can be greatly accelerated.

Plant glycoside hydrolases (glycohydrolases) play an important role in the hydrolysis and synthesis of sugars and glycoconjugates in organisms, they can participate in a variety of signaling processes in plant growth and development and in response to environmental responses, and they play important biological functions in plant life activities (liuli, 2010). Among them, 1, 4-beta-endoxylanase (Endo-1,4-beta-xylanase) is the most critical enzyme for complete hydrolysis of xylan, and it can act on xylan-glycoside bonds in xylan backbone, randomly cut off glycoside bonds in backbone, hydrolyze xylan to generate oligosaccharides with different molecular weights (Zhang Yingchun, 2020). Xylanase plays an important role in seed germination, plant pollination, fruit ripening and softening, and stem development (Wangqingdong, 2020).

Disclosure of Invention

The present invention aims to provide a glycoside hydrolase gene line percent related (GHLP1) and a promoter thereof. The whole genome correlation analysis result shows that the gene and the promoter thereof are closely related to the coat character of cotton.

The invention also aims to provide application of the gene and a promoter thereof.

The purpose of the invention can be realized by the following technical scheme:

glycoside hydrolase (glycosylhydrolase) gene GHLP1, which belongs to the family of xylanases, 1, 4-beta-endoxylanases (Endo-1, 4-beta-xylanase). The cDNA sequence of the glycoside hydrolase gene GHLP1 in tetraploid uploid upland cotton TM-1 is as follows: SEQ ID NO.1, the genomic sequence is: SEQ ID NO.2, the promoter sequence is: SEQ ID No. 3; the glycoside hydrolase gene GHLP1 contains SNP locus, which is located at the position of 1174bp of the coding region sequence, the base of the SNP locus is mutated from T to G (cDNA sequence: SEQ ID NO.11, genome sequence: SEQ ID NO.12), and the corresponding amino acid is mutated from Ser to Arg; meanwhile, the promoter region of 471bp at the upstream of the gene comprises an SNP locus, and the base of the SNP locus is mutated from A to C (genome sequence: SEQ ID NO. 13). And the yield traits of cotton varieties with the genotype of CC, such as seeds and the like, are obviously higher than those of cotton varieties with the genotype of AA.

The glycoside hydrolase gene GHLP1 and the application of the promoter thereof in identifying high-yield upland cotton varieties are provided. And if the genome of the cotton to be detected contains the genotype C of the promoter of the glycoside hydrolase gene GHLP1, identifying the cotton as a high-yield upland cotton variety.

The glycoside hydrolase gene GHLP1 and the application of the promoter thereof in improving the yield traits of cotton.

The glycoside hydrolase gene GHLP1 and the application of the promoter thereof in breeding high-yield new varieties of cotton by means of genetic engineering. The method specifically comprises the following steps:

the SNP locus in the genome GHLP1 promoter haplotype AA of the low-yield upland cotton variety is subjected to site-directed mutagenesis to be transformed into a high-yield haplotype so as to culture a new high-yield cotton variety and improve the yield character of the cotton.

The invention also provides a primer pair for detecting the SNP locus of the GHLP1 promoter, wherein the upstream primer is as follows: SEQ ID NO.8, the downstream primer is: SEQ ID NO. 9.

The primer pair is applied to screening high-yield cotton varieties.

A method for screening high-yield cotton variety includes detecting said SNP site, selecting cotton whose base is C at 471bp position of upstream promoter sequence of said gene as high-yield cotton variety, and its amplification product sequence is shown in SEQ ID NO. 10.

The invention has the advantages that:

according to the invention, a glycoside hydrolase gene GHLP1 closely related to the coat character of cotton is excavated through cotton population weight sequencing and whole genome association analysis. The glycoside hydrolase gene GHLP1 and the promoter thereof are closely related to cotton coat traits in whole genome association analysis. The GHLP1 cDNA, the genome sequence and the two-thousand base pair promoter sequence provided by the invention are obtained by a PCR technology, and the technology has the advantages of small quantity of initial templates, simple and easy test steps and high sensitivity.

Analysis of the expression levels of GHLP1 in different tissues and developmental stages of cotton was performed by transcriptome sequencing. The gene is expressed dominantly in ovule seeds at-3 days, -1 day and 0 day before cotton blossoming and at 1 day, 3 days, 5 days, 10 days, 20 days and 25 days after cotton blossoming, and shows that the gene is related to the yield traits of cotton.

The SNP genotype of GHLP1 in relatively high-yield and low-product populations is verified by PCR technology, and the method is easy to operate, high in sensitivity and good in accuracy.

The breed population can be divided into two main groups according to different SNP genotypes of GHLP1, namely, the haplotype is CC and the haplotype is AA. The statistical analysis method finds that the two groups of clusters have significant differences in the clothes and seed indexes, and further proves the correlation between the gene and the cotton yield traits.

The relative expression level of different SNP genotypes of GHLP1 in ovule and fibrous tissues of Sibang 3 (haplotype is CC) and Xinluohao 42 (haplotype is AA) was different, and particularly in 20-day ovule tissue of Sibang 3, the relative expression level was significantly higher than that of Xinluohao 42 of low-coat.

Drawings

FIG. 1 shows the analysis result of cotton coat-dividing character GWAS association.

The abscissa represents the position (Mb) on the chromosome and the ordinate represents the significance of the SNP site association, expressed as-log₁₀(P value) is shown.

Figure 2 manhattan plot of cotton coat-dividing trait GWAS associations and linkage disequilibrium attenuation heatmap near association sites.

The top half of the graph is a Manhattan graph of cotton coat trait GWAS associations, and the bottom half is a linkage disequilibrium attenuation heat map near SNP sites.

FIG. 3 sequence information of GHLP1 and identification of different haplotypes.

Detecting the 1174bp position of the GHLP1 coding region sequence in the breed population, wherein the base of the SNP site is mutated from T to G, and the corresponding amino acid is mutated from Ser to Arg; meanwhile, a 471bp upstream promoter region of the gene comprises an SNP locus, and the base of the SNP locus is mutated from A to C.

FIG. 4 expression levels of GHLP1 in different tissues and developmental stages of cotton.

The abscissa represents the expression level of the gene (expressed in the number of sequencing fragments contained in each thousand transcript sequencing bases per million sequencing bases FPKM). The ordinate represents different tissues including roots, stems, leaves, ovules and fibres. Ovule tissue includes 3 and 1 days before flowering, the day of flowering, and 1 to 25 days after flowering; fibrous tissue includes 5 to 25 days after flowering.

FIG. 5. comparative analysis of yield traits between different haplotypes of GHLP 1.

The box plot represents the distribution of yield traits for the breed population. The abscissa is different planting environments, and the ordinate is corresponding yield character numerical values which are respectively the coat character and the seed finger character in the yield characters. The varieties containing both AA and CC haplotypes were 114 and 276, respectively. The horizontal line within the box represents the median of the trait distribution. Indicates a difference at the 0.01 level.

FIG. 6 expression analysis of GHLP1 in ovules and fibrous tissues of two cotton varieties.

The abscissa represents ovule and fibrous tissue at different times; the ordinate represents the relative expression level of GHLP1 gene. Black represents Simian 3, and its haplotype is CC; white represents New Luzao No. 42, and its haplotype is AA. Indicates a difference at the 0.05 level and indicates a difference at the 0.01 level.

Detailed Description

Example 1 excavation of cotton coat trait-associated glycoside hydrolase gene GHLP 1:

aiming at 486 modern upland cotton varieties or lines, detailed investigation on clothes traits is repeatedly carried out by planting in fields of each variety of Kuerle Xinjiang and Stone river Xinjiang in 2016 to 2017. Meanwhile, the 486 cotton varieties (lines) were subjected to whole genome re-sequencing to obtain 7.55Tb sequencing data, and the average sequencing depth was 10.51X. The sequences are aligned to the genome sequence of cotton upland cotton TM-1, the identification of whole genome SNP is carried out by utilizing bioinformatics software, and 4489601 high-quality SNPs (minimum gene frequency) are mined in total>0.05) for subsequent analysis. First, whole genome association analysis is performed, and then P is used<1×10^-6And screening SNP related signal sites. By analyzing the association sites, we found that an SNP signal association site (D01:54812810) on the D01 chromosome can be associated with the cotton coat trait (FIG. 1). In the hot spot region of linkage disequilibrium attenuation of the association signal (FIG. 2), a SNP site (D01:54689390, T/G) was found to be right in the exonic region of the gene and caused mutation of the amino acid sequence, and a SNP variation (D01:54691881, A/C) was found to be right in the 471bp upstream promoter region of the gene (FIG. 3). The gene is glycoside hydrolase gene, belongs to 1, 4-beta-endoxylanase in xylanase class and is named as GHLP 1.

Example 2 obtaining of glycoside hydrolase gene GHLP 1:

the cDNA sequence, genome sequence and promoter sequence of GHLP1 are obtained from upland cotton genome sequence, shown as SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO. 3. Designing gene full-length primers according to two ends of the cDNA, and carrying out PCR amplification, wherein the primer sequence is F1: SEQ ID NO.4 and R1: SEQ ID NO. 5. Designing gene full-length primers according to two ends of the promoter, and carrying out PCR amplification, wherein the primer sequence is F2: SEQ ID NO.6 and R2: SEQ ID NO. 7. The PCR reaction procedure was as follows: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30sec, annealing at 60 ℃ for 1min, extension at 72 ℃ for 1min, and 30 cycles; finally, extension is carried out for 10min at 72 ℃. Sequencing the PCR amplification product, and further comparing the sequence with the cDNA and the promoter sequence to determine the accuracy of the sequence.

Example 3 analysis of expression levels of GHLP1 in different tissues and developmental stages of cotton:

RNA samples of different tissues and different development periods of the cotton TM-1 variety are collected for transcriptome sequencing in the experiment. Sample material included roots, stems, leaves, ovules, and fibers. The ovule tissue includes 3 and 1 days before flowering, the day of flowering, and 1 to 25 days after flowering. Fibrous tissue includes 5 to 25 days after flowering. Transcriptome sequencing adopts an Illumina HiSeq 2500 platform, and the average sequencing depth of each sample reaches 6 Gb. The gene expression level is calculated by comparing the reads obtained by sequencing with the upland cotton genome, and the calculated expression level is expressed by the number of sequencing Fragments (FPKM) contained in each thousand transcript sequencing bases in each million sequencing bases. The experimental result is shown in FIG. 4, the gene is expressed dominantly in ovule seeds before-3 days and-1 day before and after flowering of TM-1 cotton, and in 1 day, 3 days, 5 days, 10 days and 20 days after flowering (FIG. 4), which shows that the gene is related to yield character constitutive factors.

Example 4 application of GHLP1 gene and its promoter in identifying high-yielding cotton varieties and improving yield traits:

the genotype of each breed population at this SNP site (D01:54691881) was analyzed based on the results of genome-wide re-sequencing and bioinformatics analysis. We confirmed that the promoter region 471bp upstream of the GHLP1 gene contains a nonsynonymous mutated SNP site, and the base of the SNP site is mutated from A to C. Based on the base information of the SNP site, the modern variety (line) of upland cotton is divided into two haplotypes, AA and CC (FIG. 5).

Based on the SNP variation in the promoter region 471bp upstream of the GHLP1 gene, we identified 114 haplotype AA materials and 276 haplotype CC materials from this natural population (FIG. 5 and Table 1). The Dai cotton 15 which makes outstanding contribution to the breeding work of upland cotton varieties in China, the Si cotton 2B is high-yield haplotype CC, the 108 phi, KK1543, C1470 and the like are low-yield haplotype AA, and further reflects the profound influence of American cotton varieties on the improvement of cotton varieties in China and the positive effect on the improvement of cotton varieties in cotton areas in Xinjiang after cotton areas in the Huanghe river basin and the Changjiang river basin are introduced to the cotton areas in Xinjiang (Fan et al., 2017; Han et al., 2020).

Using the t-test statistical detection method, we calculated the correlation of yield traits between the two sets of haplotypes (FIG. 5). The result shows that the coat score of the haplotype CC is superior to that of an AA haplotype cotton material in four environments of 2016 stone river, two-year two-point (2016 stone river, 2016 Korla, 2017 stone river, 2017 Korla) mean values, 2016 and 2017 mean values and the like, and the obvious difference is shown (P is less than 0.01); meanwhile, the seed of the cotton material of haplotype CC is lower than that of the AA haplotype material in 2016 Couler, 2017 Couler, and the mean value of 2016 and 2017 in two years, and the like, and the cotton material of haplotype CC also shows very significant difference (P < 0.01).

In addition, the GHLP1 promoter has different SNP genotypes in Sibanzo No.3 (haplotype: CC) and Xinluohao No. 42 (haplotype: AA), and the GHLP1 gene has a difference in relative expression level in ovule and fibrous tissue, and particularly in 20-day ovule tissue of Sibanzo No.3, the relative expression level is significantly higher than that of Xinluohao No. 42 of low-coat (fig. 6). The gene promoter is proved to be capable of improving the yield of cotton by improving the gene expression quantity of GHLP1 after being mutated.

The results are combined to show that the gene GHLP1 has important research value in improving the yield traits of cotton and breeding high-yield and new varieties of cotton. On one hand, the molecular marker can be designed according to the haplotype of the gene GHLP1, so that the yield traits of cotton can be effectively identified, and the molecular marker has good application value in the breeding research of high-yield cotton varieties. On the other hand, the SNP site in the GHLP1 promoter haplotype AA can be subjected to site-directed mutagenesis to be modified into a high-yield haplotype, so that a new high-yield cotton variety is cultured.

TABLE 1 identification of high-and Low-yield haplotypes in population variety material

Sequence listing

<110> Zhejiang university

<120> glucoside hydrolase gene and promoter for improving cotton yield traits and application thereof

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aaggtggatt tgttcttgat tcaccttcaa atttagcttt actgctattc caggtgaacc 1020

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gcaataacta taaaccaagt ctcaaaggat tttccatttg gttctgcaat agcacacacc 1440

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caaagaagaa ttcatacact gggatgtcag taatgagatg ctccactttg atttttacga 1860

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aacagttgac acttacattg aaaggataag agagcttgaa agcggtggca tgtacatgga 2040

tggaattggg ttggaaagtc attttacagt gccaaacctt cctctaatgc gagccgtgat 2100

agacaaattg gctaccctca gactccccat atggctaaca gaggtagata ttagcagttc 2160

ggttggtaaa gaattacagg gggtgtacct agaacaggta ctaagagaag ggttttcaca 2220

cccttcagtg aatgggataa tgctgtggac ggctctccat ccaaaagggt gttacgaaat 2280

gtgtttaact gatgggaatt ttaagaatct cccagcgggt gatgtggttg acaagctgct 2340

gaaagaatgg gaaagcgggg agatgaaggg ggtaacaggt gagcatggtt catacagctt 2400

ttatggattc ttaggcgaat acaaggtcaa tgttggatat ggagacaggg ctgcaaattt 2460

tactttctca ctccccccta gcggtgaaac taaacacttc agcattcatt tgtaa 2515

<210> 3

<211> 2000

<212> DNA

<213> Gossypium hirsutum

<400> 3

gttcctgttt tgaacaaact gctattgaat tgaacaaatg ctgaagctta cattatatac 60

catacagaaa aaaaatatga aatgatttcg gatatcagga gaaagagggg gagtgtacct 120

aattaaggtt tgtggtgggg atcacaaggg aacagtgaca ggcctatgag cacccgtgcc 180

ttgctcacat gttttactta agggtttcat ttgatgtgaa cttcaaataa acttgtcttt 240

gctgctaaat gtccatttat ggttatacag gcttagactc aacgttatat aagtgggaaa 300

tttatttatt ttctatccac ccttaatatt agtcacttta gctttaatgg agagttggct 360

tgttgctaag gactaagtga tgagtccaag ttaagtgttt ttttaatcta agatttggtt 420

gtgtgccaaa ctctggtttt tgtcagcaaa ctgaaacaat gttcgtttaa acaatattat 480

tagaacttcg gttgcttttg gcgaccaact tgggctgctt tgagaagtgt aaaagaagaa 540

atttatataa tggacgttcg aggattaggt gaccagtgta accaagcact gttgctaata 600

ctgagagatt ccgaaattag tgattggggg atagcttagt ttttgttttg atcatggaga 660

ttggaaaaaa aaaagcacaa gctgtacaag gattgaagat gggtaattaa tgggaagtgg 720

gttggaaata ggtgaagtct gctttagatt aaagtctagt cctccattgt cagaccagtt 780

tgattccagg ggtgcttcat tcatggctag aacttgcacg cgtgaagcgt gcatgatatt 840

gggaccatgc attcacatgc tttaaaagtc aaatcacatt gaatgcccaa tccctcggtt 900

cctccatgga tgtggaacct gttgggcatt gaaggaaaag gtgaagacat tttcattttc 960

actgcaattc tagtatcact gtagggaggg taggacaatt tgggaaggaa aataatttat 1020

caaagtaatt tttcttttaa cttgataatt ttgacttttt aactttagat aaggtaaata 1080

tttatcaaaa caatgtagtt ttgtcttttt tttatttgga ttttttaata acttgaatta 1140

accgcgagcg ttcagttgaa tcgagtgaga ttgtttgagt taaattagaa aaattaatat 1200

tgtcaaatta aaatcttgtt acaatataat tatttccatg ttagagtata taaatttaaa 1260

actatatata tttgcaaact tgtttaaagc ataataataa aatattttaa tacgataaac 1320

ttgaatcatt aatcattcca aaattattat tttataaaat ttttaaaatt tcaacttttt 1380

atatattttc tagatttctt taaaaaaatt aaattttaaa ataattttaa aatttttttg 1440

caatttttgt taaaagagac caatttgctc attttcaaat ttgataggga ccaaaatggt 1500

atttaaatta atctgttatt cgaattattc gagttatttg aattgtaaaa attcaactcg 1560

actcgaactc aaagtcagaa atttcttatt caagctgact caaataattc atttcgatta 1620

actcaaaatt taattttttt cgattttttt atcgaatcaa attttgttca cctctaatta 1680

gaaggatgtt gttcaaatca agatagtgtc aaaatgtggg cctctaaaac catatttttg 1740

agcccagagg ctttaatctg tcgatcaatt agataccgaa gacaaactcg actcatgtga 1800

cccaaaaaca caaacaagac tttcagagat tcctccctta ttttcttaga ccagaccggg 1860

ccaaatgaac cattttttat catgggcaaa cccaaatagc tgcaagattg taagttttat 1920

aattatttga accaagaaca ttaatatata tcttgataat cgtagtaaat gcatttaacc 1980

aaatgacatg cctaccaata 2000

<210> 4

<211> 20

<212> DNA