阅读说明：本技术 一种调控番茄耐旱性的转录因子SpbHLH89及其应用 (Transcription factor SpbHLH89 for regulating and controlling drought tolerance of tomato and application thereof ) 是由 王娟 郭斌 王柏柯 李宁 胡佳蕙 杨涛 余庆辉 周涛 于 2021-04-09 设计创作，主要内容包括：本发明属于植物基因工程技术领域,具体公开一种调控番茄耐旱性的转录因子SpbHLH89其核苷酸序列如SEQ ID NO：1所示,并且通过转基因技术对SpbHLH89进行功能验证、创制耐旱番茄材料的应用方法,研究发现与野生型番茄植株相比,超表达SpbHLH89的转基因番茄株系的耐旱能力增加,且积累了大量有机渗调物质,提高了抗氧化酶活性,有效缓解了胁迫下活性氧的积累,表明SpbHLH89是调节番茄耐旱的正响应因子,进一步认识植物耐旱的调控机制,对于番茄的耐旱改良具有重要意义。(The invention belongs to the technical field of plant genetic engineering, and particularly discloses a transcription factor SpbHLH89 for regulating and controlling drought tolerance of tomatoes, and a nucleotide sequence of the transcription factor SpbHLH89 is shown as SEQ ID NO:1, and through the application method of functional verification and creation of drought-tolerant tomato materials to SpbHLH89 by transgenic technology, researches show that compared with wild tomato plants, the drought tolerance of transgenic tomato lines over-expressing SpbHLH89 is increased, a large amount of organic osmoregulation substances are accumulated, the antioxidant enzyme activity is improved, the accumulation of active oxygen under stress is effectively relieved, and the SpbHLH89 is a positive response factor for regulating the drought tolerance of tomatoes, further recognizes the regulation and control mechanism of plant drought tolerance, and has important significance for improving the drought tolerance of tomatoes.)

1. A transcription factor SpbHLH89 for regulating tomato drought tolerance has a nucleotide sequence shown in SEQ ID NO:1 is shown.

2. The transcription factor SpbHLH89 for regulating tomato drought tolerance as claimed in claim 1, wherein the cDNA sequence is shown in SEQ ID NO:2, respectively.

3. The protein of transcription factor SpbHLH89 for regulating tomato drought tolerance as claimed in claim 1, wherein the amino acid sequence is shown in SEQ ID NO:3, respectively.

4. A construction method of a transcription factor SpbHLH89 overexpression vector for regulating and controlling drought tolerance of tomatoes is characterized by comprising the following steps: the construction method comprises the following steps:

the method comprises the following steps: using Pennelli tomato leaf cDNA as template, using KOD enzyme to make PCR amplification to obtain target fragment, detecting PCR product by means of 1.5% agarose gel electrophoresis, using recovery kit to recover target fragmentKpnI andXhoi, double enzyme digestion pSUP1300 is unloaded, a linearized skeleton vector is recovered and is subjected to homologous recombination with a target fragment to construct an over-expression vector;

step two: transforming T5-Zero competent cells by a heat shock method, and carrying out colony PCR on the monoclonal to identify positive clones;

step three: the pSUP1300 vector selective marker gene is kanamycin, PCR positive clone plasmids are extracted for sequencing verification, and the recombinant expression vector of the SpbHLH89 gene is pSUP1300-SpbHLH 89;

step four: introducing the expression vector pSUP-SpbHLH89 into GV3101 in the agrobacterium to obtain the overexpression vector agrobacterium containing the target segment; infecting the leaves with agrobacterium;

step five: inducing the callus, and screening kanamycin resistance to obtain a transgenic positive plant;

step six: after a positive transgenic strain T0 generation is obtained, extracting genome DNA, and detecting an over-expression plant; designing primer pairs on two sides of the insertion site, carrying out PCR amplification on the target fragment, carrying out sequencing after purifying PCR amplification products, screening over-expression strains according to sequencing results, and carrying out drought phenotype observation and detection by using T2 generation plants.

5. The method for constructing the overexpression vector of the transcription factor SpbHLH89 for regulating the drought tolerance of tomato according to claim 4, wherein the construction method comprises the following steps: and in the second step, a primer sequence used for identifying the positive clone by colony PCR is carried out, wherein the forward primer is shown as SEQ ID NO: 4, 5'-GCTCTAGAATGGATCCTCATTCTACTATAATGAGTGC-3', reverse primer is shown in SEQ ID NO: and 5, as follows: 5'-GGGGTACCTGTTGTTCTTTCAAATCCACCA-3' are provided.

6. The method for constructing the overexpression vector of the transcription factor SpbHLH89 for regulating the drought tolerance of tomato according to claim 4, wherein the construction method comprises the following steps: designing primer pairs on two sides of the locus as described in the sixth step, wherein the primer pairs are respectively: SpbHLH 89-F: 5'-GCTCTAGAATGGATCCTCAT TCTACTATAA TGAGTGC-3', SEQ ID NO: 6, Flag-R: 5'-TTCTTGTACAGCTCGTCCATGCC-3' SEQ ID NO: shown at 7.

7. The application of a transcription factor SpbHLH89 for regulating and controlling the drought resistance of tomatoes is characterized in that the SpbHLH89 gene can increase the drought resistance of tomatoes by influencing osmotic regulatory substances, an ROS system and oxidase.

Technical Field

The invention belongs to the technical field of plant genetic engineering, and particularly discloses a transcription factor SpbHLH89 for regulating and controlling drought tolerance of tomatoes, and an application method for carrying out functional verification on SpbHLH89 and creating a drought-tolerant tomato material through a transgenic technology.

Background

Tomato (A)Solanum lycopersicum) Is an important vegetable and economic crop and is widely popular worldwide. China is the first major producing country for fresh eating tomatoes and the third major producing country for processing tomatoes, and plays a significant role in the world tomato market. In recent years, global population growth and frequent extreme weather and water resource scarcity have exacerbated the impact of drought stress on tomato production. Therefore, the analysis of the molecular mechanism of drought resistance of the tomatoes and the cultivation of drought-resistant tomato varieties become problems to be solved urgently in the quality-improving and efficiency-improving process.

The growth and development of plants are inevitably affected by many abiotic stresses. To adapt to the environment, the plants themselves need to respond to stress through various physiological and biochemical regulation. The bHLH transcription factor is a transcription factor containing basic helix-loop-helix (bHLH) structural domain, and the bHLH family is the second largest plant transcription factor, but the research on the plant stress response is relatively lagged. The bHLH class of transcription factors have a highly conserved basic/helix-loop-helix domain, which consists of two parts, a basic amino acid domain and a helix-loop-helix region, containing about 60 amino acids. The basic amino acid region is positioned at the N end of the bHLH structural domain, contains about 15 amino acids, and has the main function of recognizing and specifically binding a DNA motif of a target gene promoter; the helix-loop-helix region is located C-terminal to the bHLH domain, contains about 40-50 amino acids, at least 5 basic amino acids, and has a highly conserved HER motif (His 5-Glu9-Arg 13) in more than 50% of plant bHLH. Studies have shown that the bHLH protein binds predominantly to the core DNA sequence motifs of E-box (5 '-CANNTG-3'), with G-box (5 '-CACGTG-3') being the most common form, which determines several conserved amino acids in the basic amino acid region of the core recognition site of different boxes. bHLH proteins are classified based on evolutionary relationships, taking into account a variety of characteristics such as DNA binding specificity, conservation of amino acids at certain positions, and the presence or absence of conserved domains in addition to the bHLH domain. Recent results of phylogenetic analysis have shown that over 500 plant bHLH can be divided into 26 subgroups, while phylogenetic analysis of some atypical bHLH proteins extends their classification number to 32 subgroups. According to current studies on the function of the bHLH protein, plant bHLH transcription factors regulate the expression of a large number of genes, involving multiple overlapping and specific regulatory pathways. For example, bHLH transcription factors regulate carpel, epidermal cell and airway development, seed germination, flowering time and root hair formation. In addition, bHLH is also involved in the regulation of metabolic processes, such as flavonoid synthesis. Many bHLH play a role in light signal regulation and are involved in hormone signaling pathways, such as ABA, jasmonic acid, and the like. In addition, the bHLH transcription factor is also involved in the response of plants to abiotic stresses such as low temperature and iron deficiency.

At present, a plurality of beneficial genes are gradually lost along with continuous intraspecific hybridization after a plurality of existing tomato germplasms are selected under high pressure for a long time, so that the genetic basis of the modern cultivated tomatoes becomes very narrow and drought-resistant germplasms are lacking. This will make the loss of tomatoes more severe in extreme climatic conditions. Therefore, the discovery and utilization of beneficial genes of wild germplasm resources have important significance for clarifying gene functions, innovating tomato breeding materials, analyzing the drought-resistant molecular mechanism of tomatoes and improving the drought resistance of tomatoes. Panaili tomato (A)Solanum pennellii) Is a tomato closely-related wild species, found in the west slope of Peru Andes mountain, the epidermis of which covers dense glandular hair and secretes viscous glands.S. pennelliiHas drought tolerance characteristic different from other tomato varieties, can survive in extreme drought environment, and is one of the breeding for improving the drought tolerance of tomatoA particularly valuable genetic resource. In addition to this, the present invention is,S. pennelliithe publication of the genome sequencing result provides an ideal experimental model for analyzing the molecular mechanism of drought tolerance of the tomato, so that researchers can quickly identify and identify candidate genes related to the drought tolerance.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a transcription factor SpbHLH89 for regulating and controlling drought tolerance of tomato and an application method thereof.

The technical scheme of the invention is as follows: a transcription factor SpbHLH89 for regulating tomato drought tolerance has a nucleotide sequence shown in SEQ ID NO:1 is shown.

Secondly, a transcription factor SpbHLH89 for regulating tomato drought tolerance, wherein the cDNA sequence is shown as SEQ ID NO:2, respectively.

And thirdly, the amino acid sequence of the protein of the transcription factor SpbHLH89 for regulating the drought tolerance of the tomato is shown as SEQ ID NO:3, respectively.

Fourthly, a construction method of a transcription factor SpbHLH89 overexpression vector for regulating and controlling drought tolerance of tomatoes, wherein the construction method comprises the following steps:

the method comprises the following steps: using Pennelli tomato leaf cDNA as template, using KOD enzyme to make PCR amplification to obtain target fragment, detecting PCR product by means of 1.5% agarose gel electrophoresis, using recovery kit to recover target fragmentKpnI andXhoi, double enzyme digestion pSUP1300 is unloaded, a linearized skeleton vector is recovered and is subjected to homologous recombination with a target fragment to construct an over-expression vector;

step two: transforming T5-Zero competent cells by a heat shock method, and carrying out colony PCR on the monoclonal to identify positive clones;

step three: the pSUP1300 vector selective marker gene is kanamycin, PCR positive clone plasmids are extracted for sequencing verification, and the recombinant expression vector of the SpbHLH89 gene is pSUP1300-SpbHLH 89;

step four: introducing the expression vector pSUP-SpbHLH89 into GV3101 in the agrobacterium to obtain the overexpression vector agrobacterium containing the target segment; infecting the leaves with agrobacterium;

step five: inducing the callus, and screening kanamycin resistance to obtain a transgenic positive plant;

step six: after a positive transgenic strain T0 generation is obtained, extracting genome DNA, and respectively detecting over-expression plants; designing primer pairs on two sides of the insertion site, carrying out PCR amplification on the target fragment, carrying out sequencing after purifying PCR amplification products, screening over-expression strains according to sequencing results, and carrying out drought phenotype observation and detection by using T2 generation plants.

And B, performing colony PCR in the step B to identify a primer sequence used by the positive clone, wherein the forward primer is shown as SEQ ID NO: 4, 5'-GCTCTAGAATGGATCCTCATTCTACTATAATGAGTGC-3', reverse primer is shown in SEQ ID NO: and 5, as follows: 5'-GGGGTACCTGTTGTTCTTTCAAATCCACCA-3' are provided.

Wherein, in the sixth step, primer pairs are designed at two sides of the insertion site, and the primer pairs respectively are as follows: SpbHLH 89-F: 5'-GCTCTAGAATGGATCCTCAT TCTACTATAATGAGTGC-3', SEQ ID NO: 6, Flag-R: 5'-TTCTTGTACAGCTCGTCCATGCC-3' SEQ ID NO: shown at 7.

And fifthly, the application of a transcription factor SpbHLH89 for regulating the drought tolerance of the tomato is realized, and the SpbHLH89 gene can increase the drought tolerance of the tomato by influencing osmotic regulatory substances, an ROS system and oxidase.

Has the advantages that: the SpbHLH (Sopen 04g 001150) gene is cloned from solanum pennelli by using transcriptome sequencing analysis. The result of the alignment according to Sol genomics and NCBI was named SpbHLH 89. The nucleotide sequence of the gene is shown in a list SEQ ID NO:1, it comprises 3324 base pairs; the cDNA sequence is shown in the list SEQ ID NO:2, it comprises 684 base pairs; the amino acid sequence of the amino acid coding region is shown as SEQ ID NO:3, it comprises 227 amino acids. According to the SpbHLH89 gene coding region sequence, a transgenic experiment is designed, and finally, the fact that the gene can be used for breeding for improving the drought tolerance of tomatoes is determined.

The invention uses transcriptome sequencing to sequence Panaili tomato (tomato) under normal growth and drought stressSolanum pennellii) And cultivated tomato variety 'M82' ((R))S. lycopersicum) Is analyzed atS. pennelliiIdentified a gene-gene 30159 that is responding to drought, whose expression level is significantly higher than 'M82' in solanum pennellii, functionally annotated as bHLH type transcription factors, which was subsequently found to map to chromosome 4 in https:// solgenomics. Further research finds that compared with wild tomato plants, transgenic tomato lines over-expressing SpbHLH89 have increased drought tolerance, accumulate a large amount of organic osmoregulation substances, improve the activity of antioxidant enzyme, and effectively relieve the accumulation of active oxygen under stress, which indicates that SpbHLH89 is a positive response factor for regulating the drought tolerance of tomatoes, further recognizes the regulation mechanism of plant drought tolerance, and has important significance for improving the drought tolerance of tomatoes.

Drawings

FIG. 1 is a flow chart of SpbHLH89 gene for regulating tomato drought tolerance and an application method thereof, which are provided by the embodiment of the invention; FIG. 2 is a PCR amplification map, a restriction enzyme identification map and a colony PCR map of SpbHLH89 gene in the present invention;

FIG. 3 is a flow chart of a transgene experiment provided by an embodiment of the present invention, wherein A is: carrying out co-culture on agrobacteria infected explants; b is: screening and inducing callus, cotyledon (left) and hypocotyl (right); c is: inducing to bud; d is: inducing to root; e is: hardening seedlings; f is: regenerating plants;

FIG. 4 is a schematic diagram of detection of SpbHLH89 gene in the overexpression strain provided by the embodiment of the invention;

FIG. 5 is a phenotypic chart of transgenic SpbHLH89 strain in drought tolerance of tomato, wherein A is: before drought stress treatment of wild type and over-expression strain tomato seedlings; b is: naturally drought for 14 days; c is: after 24 hours of rehydration;

fig. 6 is a significance analysis of differences in osmoregulation substances of each transgenic line under drought stress conditions provided by the embodiment of the present invention, wherein a refers to proline content (μ g/g fresh weight): b is betaine content (mg/g fresh weight): c is as follows: soluble sugar content (mg/g fresh weight);

FIG. 7 shows the analysis of the oxidase system and ROS accumulation difference significance of each transgenic line under drought stress conditions provided by the embodiments of the present inventionWherein A is: o is₂ ^-Content (mg/g fresh weight); b is H₂O₂Content (μmol/g fresh weight): c is as follows: MDA content (nmol/g fresh weight); d is as follows: SOD activity (U/g fresh weight); e is as follows: POD activity (U/g fresh weight); f is as follows: CAT activity (U/g fresh weight).

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the reagents used are all conventional commercial products.

Example 1 a transcription factor SpbHLH89 for regulating drought tolerance in tomato, having a nucleotide sequence shown in SEQ ID NO:1 is shown.

Example 2 transcription factor SpbHLH89 for regulating tomato drought tolerance has the cDNA sequence shown in SEQ ID NO:2, respectively.

Example 3, a protein of transcription factor SpbHLH89 for regulating tomato drought tolerance, which has an amino acid sequence shown in SEQ ID NO:3, respectively.

Example 4, a method for constructing a transcription factor SpbHLH89 overexpression vector for regulating drought tolerance of tomato, the method comprising the following steps:

the method comprises the following steps: using Pennelli tomato leaf cDNA as template, using KOD enzyme to make PCR amplification to obtain target fragment, detecting PCR product by means of 1.5% agarose gel electrophoresis, using recovery kit to recover target fragmentKpnI andXhoi, double enzyme digestion pSUP1300 is unloaded, a linearized skeleton vector is recovered and is subjected to homologous recombination with a target fragment to construct an over-expression vector;

step two: transforming T5-Zero competent cells by a heat shock method, and carrying out colony PCR on the monoclonal to identify positive clones;

step three: the pSUP1300 vector selective marker gene is kanamycin, PCR positive clone plasmids are extracted for sequencing verification, and the recombinant expression vector of the SpbHLH89 gene is pSUP1300-SpbHLH 89;

step four: introducing the expression vector pSUP-SpbHLH89 into GV3101 in the agrobacterium to obtain the overexpression vector agrobacterium containing the target segment; infecting the leaves with agrobacterium;

step five: inducing the callus, and screening kanamycin resistance to obtain a transgenic positive plant;

step six: after a positive transgenic strain T0 generation is obtained, extracting genome DNA, and respectively detecting over-expression plants; designing primer pairs on two sides of the insertion site, carrying out PCR amplification on the target fragment, carrying out sequencing after purifying a PCR amplification product, screening an overexpression strain and a homozygous mutant according to a sequencing result, and carrying out drought phenotype observation and detection by using T2 generation plants.

And the primer sequence used for colony PCR identification of positive clones in the second step is shown as SEQ ID NO: 4, 5'-GCTCTAGAATGGATCCTCATTCTACTATAATGAGTGC-3', reverse primer is shown in SEQ ID NO: and 5, as follows: 5'-GGGGTACCTGTTGTTCTTTCAAATCCACCA-3' are provided.

Wherein, in the sixth step, primer pairs are designed at two sides of the insertion site, and the primer pairs respectively comprise: SpbHLH 89-F: 5'-GCTCTAGAATGGATCCTCAT TCTACTATAA TGAGTGC-3', SEQ ID NO: 6, Flag-R: 5'-TTCTTGTACAGCTCGTCCATGCC-3' SEQ ID NO: shown at 7.

Example 5 application of transcription factor SpbHLH89 for regulating drought tolerance of tomato, SpbHLH89 gene can increase drought tolerance of tomato by influencing osmotic regulatory substances, ROS system and oxidase.

The application of the principles of the present invention will be described in detail with reference to the accompanying drawings.

As shown in fig. 1, the application method of the SpbHLH89 gene for regulating tomato drought tolerance provided by the embodiment of the present invention comprises:

selecting under normal growth and drought stressS. pennelliiAnd transcriptome sequencing the leaves of cultivated tomato variety 'M82', and analyzing the differentially expressed genes inS. pennelliiIdentified a gene-gene 30159 that is responding to drought, whose expression increased significantly under drought stress, functionally annotated as a bHLH-type transcription factor, and subsequently found to map to chromosome 4 in https:// solgenomics.

Transgenic experiments were designed to determine gene function based on the SpbHLH89 gene sequence.

The nucleotide sequence of the SpbHLH89 gene provided by the invention is shown in a sequence table SEQ ID NO. 1, and the SpbHLH89 gene comprises 3324 base pairs; the cDNA sequence is shown as sequence table SEQ ID NO. 2, which comprises 684 base pairs; the amino acid sequence of the coding region is shown as the sequence table SEQ ID NO. 3, and the coding region comprises 227 amino acids.

Designing a primer according to the cDNA full-length sequence of SEQ ID NO. 2 toS. pennelliiThe leaf cDNA is used as template, and RT-PCR technology is used to obtain full-length PCR product, as shown in figure 2.

And (3) PCR reaction system: cDNA template 1. mu.L, KOD PCR Master Mix (2X) 25. mu.L, primer SpbHLH 89-F2. mu.L, primer SpbHLH 89-F2. mu.L, ddH₂O20 mu L; PCR reaction procedure: pre-denaturation at 95 ℃ for 1 min; denaturation at 95 ℃ for 30 seconds, annealing at 60 ℃ for 30 seconds, extension at 68 ℃ for 45 seconds, and 30 cycles; extension at 68 ℃ for 5 minutes. And recovering and purifying PCR products, and sequencing.

The recombinant plasmid pSUP1300-SpbHLH89 is transformed into agrobacterium GV3101, and the recombinant agrobacterium-mediated transformation is utilized to transform a tomato cultivar 'M82', as shown in figure 3.

Shaking and culturing the identified pSUP1300-SpbHLH89 recombinant agrobacterium single colony in a liquid YEB culture medium for 14 h for activation until bacterial liquid OD₆₀₀= 0.5. The cells were collected by centrifugation at 3000 rpm for 5 min and suspended in modified MS liquid medium. The infected explants were returned to the co-cultivation medium and cultured in the dark at 28 ℃ for 2 d.

Explant differentiation is carried out, resistance screening is carried out through kanamycin, and 3 transgenic positive individuals are obtained through differentiation and regeneration of resistant callus, wherein the transgenic positive individuals are OE1, OE2 and OE 3.

Detection of SpbHLH89 gene in transgenic tomato: according to the gene of interest,

when the primers SpbHLH89-F and Flag-R are used as detection primers, the sequences are respectively as follows: SpbHLH 89-F: 5'-GCTCTAGAAT GGATCCTCAT TCTACTATA ATGAGTGC-3', respectively;

Flag-R：5’-TTCTTGTACAGCTCGTCCAT GCC-3’。

the genomic DNA of the obtained 3 transgenic positive plants and wild plants is extracted respectively. The above DNA was used as a template for PCR reaction, and the PCR product was detected by 1.5% agarose gel electrophoresis as shown in FIG. 3.

Three lines of T2 transgenic tomatoes (OE 1, OE2 and OE 3) and wild type tomato seeds are sown in a matrix with the same volume and mass, the matrix is illuminated for 16 h/dark for 8 h, the matrix is cultured for 3-4 weeks at 25 ℃, transgenic plants with consistent growth vigor and wild type plants are selected after normal watering and culturing for 30 days, natural drought treatment is started, and plant phenotypes are observed and recorded every day.

After the plants are withered due to severe water shortage (about 14 days of drought), the plant phenotypes are observed after watering is resumed for 24 hours, and transgenic tomatoes which overexpress SpbHLH89 are found to enhance the drought tolerance of the plants, as shown in figure 5.

Under drought stress, the relevance of the SpbHLH89 gene to osmotic regulatory substances, ROS accumulation and an oxidase system is verified.

And (3) simulating drought stress by using mannitol, irrigating tomato seedlings, and detecting the response of plants to the mannitol. The method comprises the following specific steps: the transgenic tomato T2 generation strains (OE 1, OE2 and OE 3) of the pannelli tomato SpbHLH89 and the seeds of the wild type material M82' are sowed in a seedling raising pot, and when the plants grow to 6 weeks, plants with consistent growth vigor are selected for drought stress treatment. And (3) pouring 300 mmol/L mannitol solution (prepared by 1/2 Hoagland nutrient solution) from the surface layer of the substrate to the seedling raising pot completely (to avoid splashing on leaves), ensuring that all the original water in the seedling raising pot is replaced and drained, placing the seedling raising pot in a tray containing the corresponding mannitol solution, and sampling for later use after processing for 24 hours. Plants without any treatment were used as controls.

Respectively detecting osmoregulation substances (proline, betaine, soluble sugar) according to kit method, as shown in figure 6, ROS accumulation (MDA, H)₂O₂、O^2-) As shown in figure 7, and the content and activity of oxidase systems (SOD, CAT and POD), the transgenic line which over-expresses SpbHLH89 can obviously improve the osmotic regulation capacity, increase the activity of oxidase and enhance the ability of eliminating ROS, and the SpbHLH89 can improve the drought tolerance of tomato plants, as shown in figure 7.

Sequence listing

<110> research institute of horticultural crops of Sinkiang academy of agricultural sciences

<120> transcription factor SpbHLH89 for regulating and controlling drought tolerance of tomato and application thereof

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<170> SIPOSequenceListing 1.0

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<211> 3324

<212> DNA

<213> SpbHLH89 Gene ()

<400> 1

cctttatcaa attttaaagg gacgacgaat caatcttatc agaaattgaa tggcgggagc 60

tgccgtactg cgaatatcat cactatcttc cttgagatta tactcaataa cttcatcttc 120

tccaatttct cactcgttag ctctcccttt acgaaaaccc agattcacaa attgccatgc 180

caaggtcgat ctcattttgc tttatttgga cctatttaat tttgttgatt taatctcttg 240

cttgtgtttt agtttgatgg atccattgga gaagcagctg aaaggatgta ccgaatgaat 300

tttgaagatg atgaatcatt ggaggatgat gaagagagcg atgaggaaga ggagacggag 360

agcagtattg acttactagt tagatttgtg cagagtatgt tcacaaaggt ttctaagcgt 420

gctaggaaag ctactcgttc tatactgccg gacgtcattt ctccacagct tgtaaatctt 480

caaactcact cttttatctc ttcaataatt tgttcagcat ataaattgat ccaataggtt 540

tcaactttgt gtgaattcta agtgactttg ttcatgaaat cagtgtcctt tcctaggcac 600

aaccataatg ttataggcta tagtgcagct tattctaccc gaattagaaa ctcctatcaa 660

ttcccagggg agacaaaaag cacactaggt gattctagcc ttggtggaca gagttacctg 720

gtacttgttg ctggtgcatc ctgtggaagt agtcatgatg tgtgaaagct gactcggaca 780

ccacagtcct caaaaagaag aactatattt cttggactgg aggtgactaa gtaattgact 840

gtgttaggat gctgaaaaac atggcattct tctggttctt ttgtagatgt atggtggcca 900

ttgtttcgga tggaatgata agctttcttt tatttatgtg tcgagggagg caagggcctt 960

tcttcacctt ttacataaat gttattaggg gtcaactctc ctacagagag agcggccttg 1020

aacgcttttg gtcacgttaa actactaccc agtttactct catctcaagc cttcatacac 1080

aatctggtgc catggtcgtg ggatctaagt aaagtagcag aatactctca tgcttcctag 1140

tgccttaagc atcggctcgg tgaaaatctc atcatggtag agcttgtatt cactggggtt 1200

tttgaatcac atcaggacac tcattcgtgc ttgaagcttt gactataact agttagtata 1260

ctcatggcaa ggaactctta aattgtgtat gtgcatctgt aaatggctaa ggctattggt 1320

tgcctcttcc ttgatactga gcattgctcg agtaaggagt aaggacatat tcagttctta 1380

agtttgtgca cttggcttgt ggttgtgtca acacaaccaa cttatgcatc accataagag 1440

ggtgattctt aagtccttga cttgctttct agtgcatttg tcaataatcc ccactcttag 1500

catgtggggt tacagaacat ttcccaggcc tgatgtattt cacacgatat aatattgctc 1560

ctcaactaca gttctaagcc aattgatcct cctcccaaga ttgaacccta gccaagctca 1620

aaaattaaca catgagattt taaatagtgg tgcctcgagt gtttaaaagc atggaggtga 1680

ttttaactct caaaatttag cctgcaaaac atggccatca catgatgtca catccaacta 1740

ctaacaccca attttagtac taagattgtg tcgtccatat tcaacgcgaa tatgtctagt 1800

tcccaactag gatgggtgca ttctttagcc acccgtttaa aagggaaaat tatgtttaac 1860

caaattgcaa tgacttgtca gattgcatgg tagtaattgt cacatctaca aatgtgtaag 1920

aattatgctt cttagttttc agagttaagt tgatttgtgt tttcatgtca acataagtcc 1980

atccaccttc ataaagtttt aatcagttca caccatctat aatacaatgt ttcttctttt 2040

actaacatat ttgggctact agcgttttcg gtgttttgtt tagttttgat cttctagttg 2100

aattatatac ataaattatg ttcaaaatcc tctcttccat ctgttctcat tgttggagga 2160

atgcaggtaa cttttgcagt tgatggcgtt ctaattttgg ctttactctc catcttgaag 2220

gcatttcttg aggtattact ttgccatgtc attttttgaa tgttttacct gaattaagca 2280

cccccccaaa cacacacact ctgtaattac ttgttgcttg ttttatgaat ttactcattg 2340

accaaatctg gctttctgct gcttttgtgg atgcaagctg cctttaatcc actaattaaa 2400

gtaaacagat atgcaattcg aagaatgcaa tataaaagag atattataat ttatcaaaca 2460

taagacttgc aaaatcaaaa tcttcagcca cttaagaaga tcacagtatt acacatcaca 2520

atttctgaaa gttcttgtac aaaacaattt tatgtcttct attctgattt agagcatggt 2580

tagggtaaat agagcaactt ctcctcttgt gtgggtgaaa tattgatctt caattgaact 2640

ctgctagtga tggttctgtc cttctaggtg gtctgctcac ttggtggtgc cgtctttgta 2700

gcaattctgt tattacgtgt gctctggtcc gctgtttcct actttcagtc taatggttca 2760

gatttcaaca gtgctggaag ttcatatggc agaacacgac ccgctgcatg attgagccta 2820

gtcacatacc tttcatctgg tgaatcatta attccttaaa aaacaaaaga tgttattagc 2880

attagaatgt tctataagta atagtgactt aatttggcaa tgtgaatttt tacatgtgtg 2940

aagatctcag cagatcttat agggagtaga gtgtgtttat aagtttcttt ttgacttcat 3000

tagccttctt cccgtgcttg ggaatcttct ataatgaata gttaagagtc ttaactctta 3060

acacaccact tataatttga gctatatata atactcatgt tgtcgcagca cactaacaac 3120

tgtttaacgc ccaaggctca tattttctag ttgtgatagt tttggcagag caaagcagca 3180

catatgaatg cttttcagag cgcaggcaca tattacagct tcttgatata ccttactaac 3240

catgtcaaat acgcattctc accagtatac tattcattta gcatttttgt ttcagttgat 3300

gtgaaagaaa tgtgctgtta aatt 3324

<210> 2

<211> 684

<212> DNA

<213> cDNA sequence of SpbHLH89 Gene ()

<400> 2

atggatcctc attctactat aatgagtgcg tttcaaactg cgactaattt ggcggagatc 60

tggccctatc accatcttat cgaccacaca acaaatcacg ccgccagaaa gcgacgcgac 120

gatgatgaat ctgctattgg agtttcaact agtggaaatg ccttgactga atctgatagt 180

aagcggctga aggccacaag atcaaacgag aatggggaat attcaggagg gaattcagga 240

aaatcttcag aacaacctgc aaagccacca gctgaaccac ctaaggacta catccatgtg 300

cgagcgagga gaggtcaagc taccgatagt catagcctag cagaaagagc cagaagagaa 360

aagattagtg acaggatgaa aatcctacaa gacttggtcc ctggttgtaa caaggttatt 420

ggaaaagctc ttgtccttga tgagataatc aattatgtcc aatcattaca acgtcaagtt 480

gagttcctat caatgaagct tgaagcagtt aatacaagag taaccccaac catcgaagga 540

attcctacta aagactttgg gcagcaaaca ttcgagacaa acgctatggc atttggttca 600

caaggtacaa gggaatatgc tggggggaca tcaccagact ggttgcacat gcagataggt 660

ggtggatttg aaagaacaac ataa 684

<210> 3

<211> 227

<212> PRT

<213> protein encoded by SpbHLH89 Gene ()

<400> 3

Met Asp Pro His Ser Thr Ile Met Ser Ala Phe Gln Thr Ala Thr Asn

1 5 10 15

Leu Ala Glu Ile Trp Pro Tyr His His Leu Ile Asp His Thr Thr Asn

20 25 30

His Ala Ala Arg Lys Arg Arg Asp Asp Asp Glu Ser Ala Ile Gly Val

35 40 45

Ser Thr Ser Gly Asn Ala Leu Thr Glu Ser Asp Ser Lys Arg Leu Lys

50 55 60

Ala Thr Arg Ser Asn Glu Asn Gly Glu Tyr Ser Gly Gly Asn Ser Gly

65 70 75 80

Lys Ser Ser Glu Gln Pro Ala Lys Pro Pro Ala Glu Pro Pro Lys Asp

85 90 95

Tyr Ile His Val Arg Ala Arg Arg Gly Gln Ala Thr Asp Ser His Ser

100 105 110

Leu Ala Glu Arg Ala Arg Arg Glu Lys Ile Ser Asp Arg Met Lys Ile

115 120 125

Leu Gln Asp Leu Val Pro Gly Cys Asn Lys Val Ile Gly Lys Ala Leu

130 135 140

Val Leu Asp Glu Ile Ile Asn Tyr Val Gln Ser Leu Gln Arg Gln Val

145 150 155 160

Glu Phe Leu Ser Met Lys Leu Glu Ala Val Asn Thr Arg Val Thr Pro

165 170 175

Thr Ile Glu Gly Ile Pro Thr Lys Asp Phe Gly Gln Gln Thr Phe Glu

180 185 190

Thr Asn Ala Met Ala Phe Gly Ser Gln Gly Thr Arg Glu Tyr Ala Gly

195 200 205

Gly Thr Ser Pro Asp Trp Leu His Met Gln Ile Gly Gly Gly Phe Glu

210 215 220

Arg Thr Thr

225

<210> 4

<211> 37

<212> DNA

<213> Artificial sequence ()

<400> 4

gctctagaat ggatcctcat tctactataa tgagtgc 37

<210> 5

<211> 23

<212> DNA

<213> Artificial sequence ()

<400> 5

ggggtacctg ttgttctttc aaatccacca 30

<210> 6

<211> 37

<212> DNA

<213> Artificial sequence ()

<400> 6

gctctagaat ggatcctcat tctactataa tgagtgc 37

<210> 7

<211> 23

<212> DNA

<213> Artificial sequence ()

<400> 7

ttcttgtaca gctcgtccat gcc 23