CN113527455B - A grain development-related protein TaGSR1 and its application in wheat plant type breeding - Google Patents

Abstract

The invention discloses an application of a grain development related protein TaGSR1 or a coding gene thereof in wheat strain breeding; wherein the amino acid sequence of the grain development related protein TaGSR1 is shown as SEQ ID NO.1, and the nucleotide sequence of the encoding gene is shown as SEQ ID NO. 2; the application in wheat plant type breeding refers to the application of regulating the plant height of wheat or the application of regulating the included angle of wheat leaves. The invention confirms the relationship between the wheat TaGSR1 gene and the wheat plant height and leaf angle, and verifies that the high expression of TaGSR1 can reduce the plant height of wheat and the leaf angle. The invention realizes the rapid breeding of ideal wheat strain by using genetic engineering technology, provides a feasible method for improving the lodging resistance and reasonable close planting of wheat, and has important breeding application value and wide market application prospect.

Description

A grain development-related protein TaGSR1 and its application in wheat plant type breeding

Technical field

The invention relates to a grain development-related protein and its application, in particular to the application of a grain development-related protein TaGSR1 or its encoding gene in wheat plant type breeding; it belongs to the technical fields of plant genetic engineering and molecular breeding.

Background technique

Wheat is the world's second largest food crop, providing humans with 20% of carbohydrates and 23% of protein. Human food security issues are faced with the reduction of cultivated land area, global warming, continued population growth, and intensified environmental pollution. How to increase wheat production under limited resource conditions is of great significance to food security. The "green revolution" of wheat has reduced the plant height of wheat, increased its resistance to lodging, greatly increased wheat yield, and also proved the importance of the ideal plant type of crops to yield.

Brassinolide (BR), as the sixth largest phytohormone in plants, plays a very important role in cell elongation and division, leaf senescence, plant morphogenesis, and plant stress resistance. Due to the extensive role of BR in plant growth and development, its application in agricultural production has received increasing attention. BRI1 (Brassinosteroid-insensitive1) is a BR receptor located on the cell membrane. After binding to BR, it phosphorylates each other with the co-receptor BAK1 (BRI1-associated receptor), initiating downstream signal transduction, and then phosphorylates BSK/CDG1 kinase, which phosphorylates BSK/CDG1 further activates downstream BSU1 (BRI1-suppressor 1) through phosphorylation. BSU1, as a phosphatase, dephosphorylates BIN2 (Brassinosteroid-insensitive 2), thereby disabling BIN2, a negative regulator in the BR signaling pathway. activity, thereby regulating the expression of downstream genes.

The applicant's research found that TaGSR1 (Grain Size Regulator 1 of Triticum aestivum L.) is a transcription factor downstream of the BR signal and has the function of regulating wheat grain size. It also plays an important role in regulating wheat leaf angle, plant height and other plant types. This regulatory function provides very important information for studying the growth, development and yield breeding of BR in wheat. After searching, there are no reports on the coding sequence of the wheat TaGSR1 gene and the encoded grain development-related protein TaGSR1, as well as the application of the protein TaGSR1 in regulating wheat plant type breeding.

Contents of the invention

In view of the shortcomings of the existing technology, the problem to be solved by the present invention is to provide the application of a grain development-related protein TaGSR1 or its encoding gene in wheat plant type breeding.

The grain development-related protein of the present invention is characterized in that: the protein is named protein TaGSR1, derived from common wheat, and the protein is the protein described in 1) or 2) below:

1) A protein consisting of the amino acid sequence shown in SEQ ID NO.1 in the sequence listing;

2) The amino acid sequence shown in SEQ ID NO.1 in the sequence listing has undergone substitution and/or deletion and/or addition of one or several amino acid residues and is a protein derived from SEQ ID NO.1 that is related to grain development.

A further preferred embodiment is that the amino acid sequence of the protein TaGSR1 consists of 203 amino acids, and its amino acid sequence is shown in SEQ ID NO. 1.

The invention discloses a gene encoding the above-mentioned grain development-related protein TaGSR1, which is characterized in that: the nucleic acid sequence of the encoding gene is the nucleotide sequence of a) or b) or c) as follows:

a) The deoxyribonucleotide sequence shown in SEQ ID NO.2 in the sequence listing;

b) At least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96% identical to the deoxyribonucleotide sequence shown in SEQ ID NO.2 %, a deoxyribonucleotide sequence that has at least 97%, at least 98% or at least 99% homology and encodes the protein;

c) A ribonucleotide sequence that is complementary to the deoxyribonucleotide sequence defined in a) or b) and encodes said protein under stringent conditions.

A further preferred embodiment is that the coding gene consists of 612 deoxyribonucleotides, and its nucleotide sequence is shown in SEQ ID NO. 2.

The invention also discloses a binary vector capable of ectopically expressing the TaGSR1 gene in wheat, which is characterized in that: the binary vector is named pLGY02-TaGSR1, and its nucleotide sequence is as shown in SEQ ID NO.4 Shown; the binary vector contains the TaGSR1 gene expression cassette E1 which plays a major role in the genetic transformation process of wheat, wherein the nucleotide sequence of the expression cassette E1 is shown in SEQ ID NO.3, which from upstream to downstream is : The ubiquitin promoter Ubi from maize, the coding sequence of the TaGSR1 gene, the fluorescence-enhanced green fluorescent protein coding gene GFP from Aequorea, and the terminator T1.

Wherein: the nucleotide sequence of the maize ubiquitin promoter Ubi is shown in SEQ ID NO.5, the coding sequence of the TaGSR1 gene is shown in SEQ ID NO.2, the fluorescence-enhanced green color from Aequorea The nucleotide sequence of the fluorescent protein encoding gene GFP is shown in SEQ ID NO.6; the nucleotide sequence of the terminator T1 is shown in SEQ ID NO.7.

The present invention further discloses the application of grain development-related protein TaGSR1 or its encoding gene in wheat plant type breeding; wherein the amino acid sequence of the grain development-related protein TaGSR1 is shown in SEQ ID NO. 1, and the nucleotide of its encoding gene The sequence is shown in SEQ ID NO. 2; the application in wheat plant type breeding refers to the application of regulating wheat plant height or the application of regulating the angle of wheat leaves.

Wherein, the application method is: by constructing a binary expression vector pLGY02-TaGSR1 that ectopically expresses the TaGSR1 gene, transforming the binary expression vector into wheat to achieve high ectopic expression of the TaGSR1 gene; or converting the binary expression vector The vector is transformed into common wheat through Agrobacterium to obtain transgenic wheat with overexpression of the TaGSR1 gene, thereby reducing the wheat plant height and leaf angle; the nucleotide sequence of the TaGSR1 gene is as shown in SEQ ID NO.2 Show.

The present invention provides a grain development-related protein TaGSR1 or its encoding gene and its application in optimizing ideal wheat plant type breeding; wherein, the grain development-related protein TaGSR1 or its encoding gene is disclosed for the first time. The present invention further confirms that TaGSR1 is a transcription factor downstream of BR signal, has the function of regulating wheat grain size, and also plays an important role in regulating wheat leaf angle, plant height and other plant types. At the same time, experiments were used to clarify the relationship between the wheat TaGSR1 gene and wheat plant height and leaf angle, and verified that high expression of TaGSR1 can reduce the decrease in wheat plant height and reduce the leaf angle. The outstanding effect of the invention is that by constructing a binary expression vector pLGY02-TaGSR1 that can ectopically express the TaGSR1 gene, and genetically transforming it into common wheat, offspring with high expression of the TaGSR1 gene were obtained, thereby achieving overexpression of the TaGSR1 gene. Experiments confirmed that the obtained transgenic wheat with overexpression of TaGSR1 gene achieved reduced wheat plant height and erect leaves. This plant type is more suitable for dense planting. The present invention uses genetic engineering technology to achieve rapid breeding of ideal wheat plant types, provides a practical method for improving wheat lodging resistance and reasonable dense planting, and has important breeding application value and broad market application prospects.

Description of the drawings

Figure 1 shows the TaGSR1 gene amplification electrophoresis pattern of Shixin 828.

Among them: lane 1 is Transgen 2K plus marker, lane 2 is TaGSR1 target band.

Figure 2 Sequencing results of the TaGSR1 gene inserted into the entry cloning vector pDonor221.

Figure 3 The TaGSR1 gene was ligated into the binary expression vector pLGY02 vector through LR reaction.

Among them: 1 is Transgen 2K plus marker, 2-6 are positive clones in which TaGSR1 was successfully ligated into the pLGY02 vector.

Figure 4 PCR identification of transgenic progeny overexpressing TaGSR1.

Among them: 1 is Transgen 2K plus marker, 2 to 9 are the target band detection results of transgenic lines 1 ^# to 8 ^# .

Figure 5 Identification of protein expression levels of transgenic progeny overexpressing TaGSR1.

Figure 6 Comparison of grain size between the OE-TaGSR1 overexpression transgenic line and its gene editing knockout line tagsr1-ko.

Among them: A&B, compared with the wild-type JW1 wheat, the grain length and width of the OE-TaGSR1 overexpression line are significantly reduced; C&D, compared with the wild-type JW1 wheat, the grain length of the homozygous gene knockout line tagsr1-ko is significantly reduced. increased, but there was no significant change in grain width. Bar=1cm.

Figure 7 Comparison of plant height traits of the OE-TaGSR1 overexpression transgenic line and its gene editing knockout line tagsr1-ko.

Among them: the plant height of TaGSR1 overexpression transgenic lines was significantly reduced, while the plant height of tagsr1-ko knockout lines had no significant change. Bar=5cm.

Figure 8 Comparison of leaf angle traits between the OE-TaGSR1 overexpression transgenic line and its gene editing knockout line tagsr1-ko.

Among them: the leaf angle of the TaGSR1 overexpression transgenic line was significantly reduced, and the leaf angle of the tagsr1-ko knockout line was significantly increased. Bar=1cm.

Detailed ways

The content of the present invention will be described in detail below with reference to specific drawings and embodiments. The following examples are only preferred embodiments of the present invention. It should be noted that the following descriptions are only for explaining the present invention and are not intended to limit the present invention in any form. Any simple modifications, equivalent changes and modifications made fall within the scope of the technical solution of the present invention.

The experimental methods in the following examples are all conventional methods unless otherwise specified.

The test materials used in the following examples were purchased from conventional biochemical reagent companies unless otherwise specified.

The high-fidelity enzyme required for PCR amplification is KOD-FX NEO (Toyobo); the vector used in the BP reaction is pDonor221 (Invitrogen); 2×PCR master mix was purchased from Beijing Jumei Biotechnology Co., Ltd.; T4 ligase was purchased from NEB Company; BPClonase and LR Clonase used for homologous recombination, gel recovery kits required for digestion fragment recovery, plasmid extraction kits, and antibodies for western blotting were all purchased from Thermo Fisher Scientific. The inorganic salts required for culture medium preparation were purchased from Sinopharm Group, vitamins and antibiotics were purchased from Sigma. The plasmid pLGY02 was obtained from the Crop Research Institute of Shandong Academy of Agricultural Sciences. The Escherichia coli strain used in the present invention is E.coli Transgen5α, purchased from Beijing Quanshijin Company; the primers used are synthesized by Qingdao Qingke Zixi Biotechnology Co., Ltd., and the relevant primer sequences are shown in Table 1:

Table 1: Primers used in the present invention

The wheat variety JW1 used in the present invention is a new germplasm with good tissue culture ability independently selected by the Crop Research Institute of the Shandong Academy of Agricultural Sciences. The public can obtain it from the Crop Research Institute of the Shandong Academy of Agricultural Sciences; Shixin 828 is a new wheat germplasm from Shijiazhuang City. The Variety New Technology Research Institute was bred in 2001 through eight generations of pedigree breeding methods, and was approved by the Hebei Provincial Variety Approval Committee in 2005 as a wheat variety with excellent traits such as cold resistance, drought resistance, and lodging resistance. The public can purchase it from Hebei Jiayuan Feng Seed Industry Co., Ltd. obtained.

Example 1 Construction of expression vector

1. Cloning of TaGSR1 coding sequence

In order to obtain the coding sequence of the TaGSR1 protein, this experiment amplified the sequence of the TaGSR1 gene in the wheat variety Shixin 828. The primers used were TaGSR1-F and TaGSR1-R. The PCR reaction system was: KOD-FX NEO buffer: 25 μL, dNTP ( 2mM): 10μL, TaGSR1-F (10μM): 1.5μL, TaGSR1-F (10μM): 1.5μL, Shixin 828gDNA (50ng/μL): 1μL, KOD-FX NEO: 1μL, add ddH ₂ O to make up 50μL. The PCR reaction program was: pre-denaturation at 98°C for 2 min, denaturation at 98°C for 12 sec; annealing at 58°C for 20 sec, extension at 68°C for 25 sec, 35 cycles of reaction; renaturation at 68°C for 5 min.

The PCR results are shown in Figure 1. Lane 1 is Transgen 2K plus marker, and lane 2 is the TaGSR1 target band.

The PCR product was sent to Qingdao Qingke Zixi Biotechnology Co., Ltd. for sequencing. The TaGSR1 gene in Shixin 828 wheat has a similarity with the known sequence of wheat in the Ensembl Plants database (http://plants.ensembl.org/index.html). 93.33%. The coding sequence of this gene is 612 bases in length and does not contain introns. Its nucleotide sequence is shown in SEQ ID NO.2.

2. BP reaction between TaGSR1 and pDonor221

Add homologous linkers attB1 and attB2 to the TaGSR1 gene.

TaGSR1 amplified in wheat variety Shixin 828 was used as the template. The primers used were TaGSR1-P1 and TaGSR1-P2. The PCR reaction system was: KOD-FX NEO buffer: 25 μL, dNTP (2mM): 10 μL, TaGSR1-P1 ( 10μM): 1.5μL, TaGSR1-P2 (10μM): 1.5μL, TaGSR1 PCR gel recovery product DNA (10ng/μL): 1μL, KOD-FX NEO: 1μL, add ddH ₂ O to make up 50μL. The PCR reaction program was: pre-denaturation at 98°C for 2 min, denaturation at 98°C for 12 sec; annealing at 58°C for 20 sec, extension at 68°C for 25 sec, 35 cycles of reaction; renaturation at 68°C for 5 min.

The PCR product was recovered by gel running, and then reacted with pDonor221 plasmid for BP reaction. The reaction system was: gel recovery product (about 50ng/μL): 1μL, pDonor221 (50ng/μL): 1μL, BP clonase: 0.5μL, ddH ₂ O was added to 10μL. The reaction system was allowed to react at 25°C for 1 hour. The reaction product was transformed into Escherichia coli Transgen 5α, and cultured on LB (containing kanamycin) plates until clones grew. Single clones were selected for bacterial liquid PCR identification and sequencing. The primer used was M13F. and M13R.

The positive recombinant strain was sent to Qingdao Qingke Zixi Biotechnology Co., Ltd. for sequencing. The sequencing results are shown in Figure 2, indicating that the TaGSR1 gene was connected into the entry vector pDonor221, and the recombinant vector was named pDonor221-TaGSR1.

3.Conversion

The PCR product was recovered by gel running and subjected to LR reaction with pLGY02 plasmid. The reaction system was: pDonor221-TaGSR1 plasmid (10ng/μL): 1μL, pLGY02 (50ng/μL): 1μL, LR clonase: 0.5μL, and ddH ₂ O was added to 10μL. The reaction system was allowed to react for 1 hour at 25°C. The reaction product was transformed into Escherichia coli Transgen 5α, and cultured on LB (containing kanamycin) plates until clones grew. Single clones were selected for bacterial liquid PCR identification and sequencing. The primer used was TaGSR1. -F and CCFP-R.

The electrophoresis results are shown in Figure 3. The clones with the target bands are positive clones carrying the binary vector pLGY02-TaGSR1. The nucleotide sequence of the binary expression vector pLGY02-TaGSR1 is shown in SEQ ID NO.4.

Example 2 Obtaining and Identification of Transgenic Progeny

1. Obtaining TaGSR1 transgenic offspring

Transform the binary expression vector pLGY02-TaGSR1 constructed in Experimental Example 1 into Agrobacterium EHA105 competent cells. Specifically: add the recombinant binary expression vector pLGY02-TaGSR1 to the Agrobacterium EHA105 competent cells, incubate on ice for 5 minutes, and quickly freeze in liquid nitrogen for 5 minutes. , heat shock reaction at 37°C for 5 minutes, followed by ice bath for 5 minutes, add anti-antibiotic LB, put in a shaker at 28°C for recovery for 2 hours, and then use an applicator to apply to LB (containing rifampicin, streptomycin and kanamycin) On the plate, invert culture at 28°C until colonies grow. Pick a single clone and inoculate it into LB culture medium containing the corresponding resistance, shake the bacteria at 28°C, 160 rpm, and culture for 24 hours.

Take JW1 wheat seeds about 15 days after pollination and peel off the young embryos. Agrobacterium suspension: Take 1ml of bacterial solution in a 1.5ml centrifuge tube, add 1.4ul of acetosyringone (0.1M) and mix well. Add the prepared bacterial solution for infection for 5 minutes, then place it on the co-culture medium and cultivate it in the dark at 23°C for 3 days. After co-cultivation, the cells were placed on resting medium and cultured in the dark at 25°C for 5 days. Transfer the calli to screening medium 1, seal the culture dish with a sealing film, and cultivate it in the dark in a 25.5°C incubator for 2 weeks. Cut the callus and transfer it to screening medium 2, seal the Petri dish again with a sealing film, and continue dark culture in a 25.5°C incubator for 2 weeks. After 2 weeks of callus cutting and screening, the resistant calli showing green shoots were transferred to the regeneration medium. Seal the petri dish and place it in a 25°C incubator with light/dark (16h/8h) culture for 2 weeks. After 2 weeks of regeneration, the healthy growing seedlings were transferred to new resistant regeneration boxes. When the seedlings grow to a certain size, samples can be taken for testing.

The various culture media involved in the above-mentioned wheat genetic transformation and their preparation can be found in the following documents:

Kan Wang (ed.), Agrobacterium Protocals: Volume 1, Methods in Molecular Biology, vol.1223DOI10.007/978-1-4939-1695-5_15, Spring Science+Businessed MediaNew York 2015.

2. Identification of TaGSR1 transgenic progeny

The CTAB method was used to extract DNA from young leaves of T0 generation wheat plants, and PCR identification of transgenic wheat was carried out using gDNA as template and LGY-DF and LGY-DR as primers. The reaction system is: 2×PCR master mix: 10 μL, LGY-DF (10 μM): 0.5 μL, LGY-DR (10 μM): 0.5 μL, gDNA (50ng/μL): 1 μL, and ddH ₂ O is added to 20 μL. PCR reaction conditions were: pre-denaturation at 94°C for 2 min; denaturation at 94°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 30 s, a total of 33 cycles; and final extension at 72°C for 5 min. The electrophoresis results are shown in Figure 4, with a total of 6 T0 generation transgenic positive plants.

The positive transgenic lines OE-TaGSR1-1 ^# , OE-TaGSR1-5 ^# and OE-TaGSR1-6 ^# were selected for westernblotting protein expression level identification. The main experimental operations are: extract total leaf protein by SDS method, use 12% SDS-PAGE gel for electrophoresis separation, and use the antibody of GFP fused with TaGSR1 protein to perform western blotting to detect the expression of transgenic wheat. The results are shown in Figure 5 , showing that the positive transgenic lines OE-TaGSR1-1 ^# and OE-TaGSR1-6 ^# have higher expression levels.

Example 3 Phenotypic identification of offspring overexpressing TaGSR1 gene in wheat

The wild-type receptor variety JW1, the overexpressed transgenic wheat OE-TaGSR1 and TaGSR1 and the homozygous knockout line tagsr1-ko of their homologous copies were planted together in the artificial climate chamber of Qingdao Campus of Shandong University. The culture conditions were: 16 hours of light, Dark for 8 hours; day temperature 22℃, night temperature 16℃; humidity 40%-50%; _CO2 concentration 500ppm-700ppm.

The grain length, width and thousand-grain weight of JW, OE-TaGSR1 and tagsr1-ko were analyzed.

Compared with wild-type JW1, the grain length and width of the overexpression strain OE-TaGSR1 were significantly reduced, while the grain length of the mutant strain tagsr1-ko was significantly increased, but the grain width did not change significantly (as shown in Table 2, Figure 6) . Measuring the 1,000-grain weight of the seeds showed that the 1,000-grain weight of the OE-TaGSR1 overexpression transgenic line was significantly reduced, while the 1,000-grain weight of tagsr1-ko was significantly increased (see Table 2). The results show that the TaGSR1 gene has a negative regulatory effect on wheat grain development.

Table 2 Statistics of yield traits of homozygous three-copy knockout mutant tagsr1-ko

Note: Different lowercase letter superscripts indicate significant differences at the 95% confidence level.

The plant height and leaf angle of the transgenic line OE-TaGSR1 overexpressing the TaGSR1 gene and the gene knockout line tagsr1-ko were analyzed.

The analysis results of plant height are shown in Figure 7. Compared with JW1, the plant height of OE-TaGSR1 is significantly reduced, while the plant height of the gene knockout line tagsr1-ko has no significant difference compared with JW1, indicating that the TaGSR1 gene has Wheat plant height has a significant inhibitory effect. At the same time, compared with the wild-type plant JW1, the leaf angle of OE-TaGSR1 is significantly reduced and the leaves are upright, while the leaf angle of the gene knockout line tagsr1-ko is significantly larger than that of JW1, as shown in Figure 8. The reduction in leaf angle leads to upright leaves, which in turn makes the plant shape of OE-TaGSR1 transgenic plants more compact, while the plant shape of the gene knockout line tagsr1-ko is more loose compared to JW1, as shown in Figure 7.

In this experiment, by constructing a TaGSR1 overexpression vector and infecting wheat immature embryo callus with Agrobacterium, the Ubi::TaGSR1 element was transferred into wheat, verifying that although TaGSR1 has a negative effect on grain size, its overexpression Significantly reduces the wheat plant height and reduces the leaf angle, thus producing a short stem and compact wheat plant type, which is of great significance for lodging resistance and high-density planting in crop production, and provides a basis for wheat plant type breeding. An excellent candidate gene locus and a practical method are provided.

sequence list

<110> Shandong University

<120> A grain development-related protein TaGSR1 and its application in wheat plant type breeding

<141> 2021-08-06

<160> 7

<210> 1

<211> 203

<212> PRT

<213> Wheat (Triticum aestivum L.)

<221> Amino acid sequence of protein TaGSR1

<222>(1)…(203)

<400> 1

MET Asp Ala Lys Gly Ala Thr Glu Ser Ser Asn Pro Asn Arg Arg Pro Ala Ser Val Pro

5 10 15 20

Ser Ala Ala Val Ala Ser Ala Ser Ala Pro Thr Lys Arg MET Leu Ala Phe His Phe Leu

25 30 35 40

Arg Ala Leu Ser Arg Ile His Gly Ala Ala Gly Pro Ala Arg Arg Arg Thr Arg Thr Ile

45 50 55 60

Arg Arg Ala Ala Tyr Ser Ser MET Ala Arg Ala Thr Gly Pro Arg Arg Ala Trp Ser Arg

65 70 75 80

Ala Leu Leu Leu Gln Ala Gln Ala Arg Ala Arg Arg Ser Arg Ala Glu Thr Ser Arg Arg

85 90 95 100

Ala Ala Val Leu Val Arg Arg Arg Val Val Ala Gly Pro Ala Val Ala Ala Ala Pro Ala

105 110 115 120

Pro Ala Arg Ala Ala Ser Val Gly Gly Gln Thr Ser Ser Ala Ala Ala Ile Arg Ala Ala

125 130 135 140

Leu Val Pro Pro Pro Pro Pro Ala Arg Gln Ala Gly Glu Pro Ala Arg Ser Asp Ala Leu

145 150 155 160

Arg Arg Leu Val Pro Gly Gly Ala Gly MET Glu Tyr Cys Ser Leu Leu Glu Glu Thr Ala

165 170 175 180

Asp Tyr Val Arg Cys Leu Arg Ala Gln Val Gln Leu MET Gln Gly Leu Ala Asp Leu Phe

185 190 195 200

Ser Cys Gln

203

<210> 2

<211> 612

<212> DNA

<213> Wheat (Triticum aestivum L.)

<221> Nucleotide sequence encoding protein TaGSR1

<222>(1)…(612)

<400> 2

atggacgcca agggcgcgac ggagagctca aaccctaacc gccgtccagc cagcgtaccg 60

tcggccgccg tggcgtcggc gtcggcgccg acgaagcgca tgctggcgtt ccacttcctg 120

cgcgcgctgt cccggatcca cggcgcggcc ggcccggcga ggcgccgcac gcgcaccatc 180

cgccgcgcgg cctactcctc catggcgcgc gccaccggcc cgcgccgcgc ctggagccgc 240

gcgctgctgc tccaggccca ggcgcgcgcg cggagatcca gggcggagac gtcgaggcgg 300

gccgccgtgc tcgtacggcg gcgcgtcgtc gccggaccgg cggtagcagc agcaccagca 360

ccggcacgcg ccgcttctgt cggcggacag acgtcgtcgg cggctgctat tcgcgcggcg 420

ctggtccctc cgccccctcc ggcgcggcag gcgggggagc cggccaggag cgacgcgctg 480

cggcggctcg tccccggagg cgccgggatg gagtactgca gcctgctgga ggagaccgcc 540

gactacgtcc gctgcctccg cgcgcaggtg cagctcatgc agggcctcgc cgacctcttc 600

tcctgccaatga 612

<210> 3

<211> 3705

<212> DNA

<213> Artificial sequence

<221> Nucleotide sequence of expression cassette E1

<222>(1)…(3705)

<400> 3

ctgcagtgca gcgtgacccg gtcgtgcccc tctctagaga taatgagcat tgcatgtcta 60

agttataaaa aattaccaca tatttttttt gtcacacttg tttgaagtgc agtttatcta 120

tctttataca tatatttaaa ctttactcta cgaataatat aatctatagt actacaataa 180

tatcagtgtt ttagagaatc atataaatga acagttagac atggtctaaa ggacaattga 240

gtattttgac aacaggactc tacagtttta tctttttagt gtgcatgtgt tctccttttt 300

ttttgcaaat agcttcacct atataatact tcatccattt tattagtaca tccatttagg 360

gtttagggtt aatggttttt atagactaat ttttttagta catctatttt attctatttt 420

agcctctaaa ttaagaaaac taaaactcta ttttagtttt tttatttaat aatttagata 480

taaaatagaa taaaataaag tgactaaaaa ttaaacaaat accctttaag aaattaaaaa 540

aactaaggaa acatttttct tgtttcgagt agataatgcc agcctgttaa acgccgtcga 600

cgagtctaac ggacaccaac cagcgaacca gcagcgtcgc gtcgggccaa gcgaagcaga 660

cggcacggca tctctgtcgc tgcctctgga cccctctcga gagttccgct ccaccgttgg 720

acttgctccg ctgtcggcat ccagaaattg cgtggcggag cggcagacgt gagccggcac 780

ggcaggcggc ctcctcctcc tctcacggca ccggcagcta cgggggattc ctttcccacc 840

gctccttcgc tttcccttcc tcgcccgccg taataaatag acaccccctc cacaccctct 900

ttccccaacc tcgtgttgtt cggagcgcac acacacacaa ccagatctcc cccaaatcca 960

cccgtcggca cctccgcttc aaggtacgcc gctcgtcctc cccccccccc cctctctacc 1020

ttctctagat cggcgttccg gtccatggtt agggcccggt agttctactt ctgttcatgt 1080

ttgtgttaga tccgtgtttg tgttagatcc gtgctgctag cgttcgtaca cggatgcgac 1140

ctgtacgtca gacacgttct gattgctaac ttgccagtgt ttctctttgg ggaatcctgg 1200

gatggctcta gccgttccgc agacgggatc gatttcatga ttttttttgt ttcgttgcat 1260

agggtttggt ttgccctttt cctttatttc aatatatgcc gtgcacttgt ttgtcgggtc 1320

atcttttcat gctttttttt gtcttggttg tgatgatgtg gtctggttgg gcggtcgttc 1380

tagatcggag tagaattctg tttcaaacta cctggtggat ttattaattt tggatctgta 1440

tgtgtgtgcc atacatattc atagttacga attgaagatg atggatggaa atatcgatct 1500

aggataggta tacatgttga tgcgggtttt actgatgcat atacagagat gctttttgtt 1560

cgcttggttg tgatgatgtg gtgtggttgg gcggtcgttc attcgttcta gatcggagta 1620

gaatactgtt tcaaactacc tggtgtattt attaattttg gaactgtatg tgtgtgtcat 1680

acatcttcat agttacgagt ttaagatgga tggaaatatc gatctaggat aggtatacat 1740

gttgatgtgg gttttatactga tgcatataca tgatggcata tgcagcatct attcatatgc 1800

tctaaccttg agtacctatc tattataata aacaagtatg ttttataatt attttgatct 1860

tgatatactt ggatgatggc atatgcagca gctatatgtg gattttttta gccctgcctt 1920

catacgctat ttatttgctt ggtactgttt cttttgtcga tgctcaccct gttgtttggt 1980

gttacttctg caggtcgact ctagaggatc cacaagtttg tacaaaaaag caggctctat 2040

ggacgccaag ggcgcgacgg agagctcaaa ccctaaccgc cgtccagcca gcgtaccgtc 2100

ggccgccgtg gcgtcggcgt cggcgccgac gaagcgcatg ctggcgttcc acttcctgcg 2160

cgcgctgtcc cggatccacg gcgcggccgg cccggcgagg cgccgcacgc gcaccatccg 2220

ccgcgcggcc tactcctcca tggcgcgcgc caccggcccg cgccgcgcct ggagccgcgc 2280

gctgctgctc caggcccagg cgcgcgcgcg gagatccagg gcggagacgt cgaggcgggc 2340

cgccgtgctc gtacggcggc gcgtcgtcgc cggaccggcg gtagcagcag caccagcacc 2400

ggcacgcgcc gcttctgtcg gcggacagac gtcgtcggcg gctgctattc gcgcggcgct 2460

ggtccctccg ccccctccgg cgcggcaggc gggggagccg gccaggagcg acgcgctgcg 2520

gcggctcgtc cccggaggcg ccgggatgga gtactgcagc ctgctggagg agaccgccga 2580

ctacgtccgc tgcctccgcg cgcaggtgca gctcatgcag ggcctcgccg acctcttctc 2640

ctgccaaagt attggggata agggtgggcg cgccgaccca gctttcttgt acaaagtggt 2700

gcctatggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct 2760

ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac 2820

ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc 2880

caccctcgtg accaccttcg gctacggcct gcagtgcttc gcccgctacc ccgaccacat 2940

gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat 3000

cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac 3060

cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg 3120

gcacaagctg gagtacaact acaacagcca caacgtctat atcatggccg acaagcagaa 3180

gaacggcatc aaggtgaact tcaagatccg ccacaacatc gaggacggca gcgtgcagct 3240

cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa 3300

ccactacctg agctaccagt ccgccctgag caaagacccc aacgagaagc gcgatcacat 3360

ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa 3420

gtagactagt tctagagagc tcgaatttcc ccgatcgttc aaacatttgg caataaagtt 3480

tcttaagatt gaatcctgtt gccggtcttg cgatgattat catataattt ctgttgaatt 3540

acgttaagca tgtaataatt aacatgtaat gcatgacgtt atttatgaga tgggttttta 3600

tgattagagt cccgcaatta tacatttaat acgcgataga aaacaaaata tagcgcgcaa 3660

actaggataa attatcgcgc gcggtgtcat ctatgttat agatc 3705

<210> 4

<211> 12699

<212> DNA

<213> Artificial sequence

<221> Nucleotide sequence of recombinant vector pLGY02-TaGSR1

<222>(1)…(12699)

<400> 4

cgtcagacac gttctgattg ctaacttgcc agtgtttctc tttggggaat cctggggatgg 60

ctctagccgt tccgcagacg ggatcgattt catgattttt tttgtttcgt tgcatagggt 120

ttggtttgcc cttttccttt atttcaatat atgccgtgca cttgtttgtc gggtcatctt 180

ttcatgcttt tttttgtctt ggttgtgatg atgtggtctg gttgggcggt cgttctagat 240

cggagtagaa ttctgtttca aactacctgg tggatttatt aattttggat ctgtatgtgt 300

gtgccataca tattcatagt tacgaattga agatgatgga tggaaatatc gatctaggat 360

aggtatacat gttgatgcgg gttttactga tgcatataca gagatgcttt ttgttcgctt 420

ggttgtgatg atgtggtgtg gttgggcggt cgttcattcg ttctagatcg gagtagaata 480

ctgtttcaaa ctacctggtg tatttattaa ttttggaact gtatgtgtgt gtcatacatc 540

ttcatagtta cgagtttaag atggatggaa atatcgatct aggataggta tacatgttga 600

tgtgggtttt actgatgcat atacatgatg gcatatgcag catctattca tatgctctaa 660

ccttgagtac ctatctatta taataaacaa gtatgtttta taattatttt gatcttgata 720

tacttggatg atggcatatg cagcagctat atgtggattt ttttagccct gccttcatac 780

gctatttatt tgcttggtac tgtttctttt gtcgatgctc accctgttgt ttggtgttac 840

ttctgcaggt cgactctaga ggatccacaa gtttgtacaa aaaagcaggc tctatggacg 900

ccaagggcgc gacggagagc tcaaacccta accgccgtcc agccagcgta ccgtcggccg 960

ccgtggcgtc ggcgtcggcg ccgacgaagc gcatgctggc gttccacttc ctgcgcgcgc 1020

tgtcccggat ccacggcgcg gccggcccgg cgaggcgccg cacgcgcacc atccgccgcg 1080

cggcctactc ctccatggcg cgcgccaccg gcccgcgccg cgcctggagc cgcgcgctgc 1140

tgctccaggc ccaggcgcgc gcgcggagat ccagggcgga gacgtcgagg cgggccgccg 1200

tgctcgtacg gcggcgcgtc gtcgccggac cggcggtagc agcagcacca gcaccggcac 1260

gcgccgcttc tgtcggcgga cagacgtcgt cggcggctgc tattcgcgcg gcgctggtcc 1320

ctccgccccc tccggcgcgg caggcggggg agccggccag gagcgacgcg ctgcggcggc 1380

tcgtccccgg aggcgccggg atggagtact gcagcctgct ggaggagacc gccgactacg 1440

tccgctgcct ccgcgcgcag gtgcagctca tgcagggcct cgccgacctc ttctcctgcc 1500

aaagtattgg ggataagggt gggcgcgccg acccagcttt cttgtacaaa gtggtgccta 1560

tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg 1620

gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg 1680

gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc 1740

tcgtgaccac cttcggctac ggcctgcagt gcttcgcccg ctaccccgac cacatgaagc 1800

agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct 1860

tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg 1920

tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca 1980

agctggagta caactacaac agccacaacg tctatatcat ggccgacaag cagaagaacg 2040

gcatcaaggt gaacttcaag atccgccaca acatcgagga cggcagcgtg cagctcgccg 2100

accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact 2160

acctgagcta ccagtccgcc ctgagcaaag acccccaacga gaagcgcgat cacatggtcc 2220

tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tacaagtaga 2280

ctagttctag agagctcgaa tttccccgat cgttcaaaca tttggcaata aagtttctta 2340

agattgaatc ctgttgccgg tcttgcgatg attatcatat aatttctgtt gaattacgtt 2400

aagcatgtaa taattaacat gtaatgcatg acgttattta tgagatgggt ttttatgatt 2460

agagtcccgc aattatacat ttaatacgcg atagaaaaca aaatatagcg cgcaaactag 2520

gataaattat cgcgcgcggt gtcatctatg ttactagatc gggaattcgt aatcatgtca 2580

tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga 2640

agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg 2700

cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc 2760

caacgcgcgg ggagaggcgg tttgcgtatt ggctagagca gcttgccaaa catggtggag 2820

cacgacactc tcgtctactc caagaatatc aaagatacag tctcagaaga ccaaagggct 2880

attgagacttttcaacaaag ggtaatatcg ggaaacctcc tcggattcca ttgcccagct 2940

atctgtcact tcatcaaaag gacagtagaa aaggaaggtg gcacctacaa atgccatcat 3000

tgcgataaag gaaaggctat cgttcaagat gcctctgccg acagtggtcc caaagatgga 3060

cccccaccca cgaggagcat cgtggaaaaa gaagacgttc caaccacgtc ttcaaagcaa 3120

gtggattgat gtgataacat ggtggagcac gacactctcg tctactccaa gaatatcaaa 3180

gatacagtct cagaagacca aagggctatt gagacttttc aacaaagggt aatatcggga 3240

aacctcctcg gattccattg cccagctatc tgtcacttca tcaaaaggac agtagaaaag 3300

gaaggtggca cctacaaatg ccatcattgc gataaaggaa aggctatcgt tcaagatgcc 3360

tctgccgaca gtggtcccaa agatggaccc ccacccacga ggagcatcgt ggaaaaagaa 3420

gacgttccaa ccacgtcttc aaagcaagtg gattgatgtg atatctccac tgacgtaagg 3480

gatgacgcac aatcccacta tccttcgcaa gaccttcctc tatataagga agttcatttc 3540

atttggagag gacacgctga aatcaccagt ctctctctac aaatctatct cctcgagctt 3600

tcgcagatcc cggggggcaa tgagatatga aaaagcctga actcaccgcg acgtctgtcg 3660

agaagtttct gatcgaaaag ttcgacagcg tctccgacct gatgcagctc tcggagggcg 3720

aagaatctcg tgctttcagc ttcgatgtag gagggcgtgg atatgtcctg cgggtaaata 3780

gctgcgccga tggtttctac aaagatcgtt atgtttatcg gcactttgca tcggccgcgc 3840

tcccgattcc ggaagtgctt gacattgggg agtttagcga gagcctgacc tattgcatct 3900

cccgccgtgc acagggtgtc acgttgcaag acctgcctga aaccgaactg cccgctgttc 3960

tacaaccggt cgcggaggct atggatgcga tcgctgcggc cgatcttagc cagacgagcg 4020

ggttcggccc attcggaccg caaggaatcg gtcaatacac tacatggcgt gatttcatat 4080

gcgcgattgc tgatccccat gtgtatcact ggcaaactgt gatggacgac accgtcagtg 4140

cgtccgtcgc gcaggctctc gatgagctga tgctttgggc cgaggactgc cccgaagtcc 4200

ggcacctcgt gcacgcggat ttcggctcca acaatgtcct gacggacaat ggccgcataa 4260

cagcggtcat tgactggagc gaggcgatgt tcggggattc ccaatacgag gtcgccaaca 4320

tcttcttctg gaggccgtgg ttggcttgta tggagcagca gacgcgctac ttcgagcgga 4380

ggcatccgga gcttgcagga tcgccacgac tccgggcgta tatgctccgc attggtcttg 4440

accaactcta tcagagcttg gttgacggca atttcgatga tgcagcttgg gcgcagggtc 4500

gatgcgacgc aatcgtccga tccggagccg ggactgtcgg gcgtacacaa atcgcccgca 4560

gaagcgcggc cgtctggacc gatggctgtg tagaagtact cgccgatagt ggaaaccgac 4620

gccccagcac tcgtccgagg gcaaagaaat agagtagatg ccgaccggat ctgtcgatcg 4680

acaagctcga gtttctccat aataatgtgt gagtagttcc cagataaggg aattagggtt 4740

cctatagggt ttcgctcatg tgttgagcat ataagaaacc cttagtatgt atttgtattt 4800

gtaaaatact tctatcaata aaatttctaa ttcctaaaac caaaatccag tactaaaatc 4860

cagatccccc gaattaattc ggcgttaatt cagtacatta aaaacgtccg caatgtgtta 4920

ttaagttgtc taagcgtcaa tttgtttaca ccacaatata tcctgccacc agccagccaa 4980

cagctccccg accggcagct cggcacaaaa tcaccactcg atacaggcag cccatcagtc 5040

cgggacggcg tcagcggggag agccgttgta aggcggcaga ctttgctcat gttaccgatg 5100

ctattcggaa gaacggcaac taagctgccg ggtttgaaac acggatgatc tcgcggaggg 5160

tagcatgttg attgtaacga tgacagagcg ttgctgcctg tgatcaccgc ggtttcaaaa 5220

tcggctccgt cgatactatg ttatacgcca actttgaaaa caactttgaa aaagctgttt 5280

tctggtattt aaggttttag aatgcaagga acagtgaatt ggagttcgtc ttgttataat 5340

tagcttcttg gggtatcttt aaatactgta gaaaagagga aggaaataat aaatggctaa 5400

aatgagaata tcaccggaat tgaaaaaact gatcgaaaaa taccgctgcg taaaagatac 5460

ggaaggaatg tctcctgcta aggtatataa gctggtggga gaaaatgaaa acctatattt 5520

aaaaatgacg gacagccggt ataaagggac cacctatgat gtggaacggg aaaaggacat 5580

gatgctatgg ctggaaggaa agctgcctgt tccaaaggtc ctgcactttg aacggcatga 5640

tggctggagc aatctgctca tgagtgaggc cgatggcgtc ctttgctcgg aagagtatga 5700

agatgaacaa agccctgaaa agattatcga gctgtatgcg gagtgcatca ggctctttca 5760

ctccatcgac atatcggatt gtccctatac gaatagctta gacagccgct tagccgaatt 5820

ggattactta ctgaataacg atctggccga tgtggattgc gaaaactggg aagaagacac 5880

tccatttaaa gatccgcgcg agctgtatga ttttttaaag acggaaaagc ccgaagagga 5940

acttgtcttt tcccacggcg acctgggaga cagcaacatc tttgtgaaag atggcaaagt 6000

aagtggcttt attgatcttg ggagaagcgg cagggcggac aagtggtatg acattgcctt 6060

ctgcgtccgg tcgatcaggg aggatatcgg ggaagaacag tatgtcgagc tattttttga 6120

cttactgggg atcaagcctg attgggagaa aataaaatat tatattttac tggatgaatt 6180

gttttagtac ctagaatgca tgaccaaaat cccttaacgt gagttttcgt tccactgagc 6240

gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat 6300

ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga 6360

gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt 6420

ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata 6480

cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac 6540

cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg 6600

ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg 6660

tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag 6720

cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct 6780

ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc 6840

aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt 6900

ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg 6960

tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga 7020

gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat tttctcctta cgcatctgtg 7080

cggtatttca caccgcatat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt 7140

aagccagtat acactccgct atcgctacgt gactgggtca tggctgcgcc ccgacacccg 7200

ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa 7260

gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc 7320

gcgaggcagg gtgccttgat gtgggcgccg gcggtcgagt ggcgacggcg cggcttgtcc 7380

gcgccctggt agattgcctg gccgtaggcc agccattttt gagcggccag cggccgcgat 7440

aggccgacgc gaagcggcgg ggcgtaggga gcgcagcgac cgaagggtag gcgctttttg 7500

cagctcttcg gctgtgcgct ggccagacag ttatgcacag gccaggcggg ttttaagagt 7560

tttaatagtt ttccgtctgt cgaagcgtga ccgacgagct ggcgaggtga tccgctacga 7620

gcttccagac gggcacgtag aggtttccgc agggccggcc ggcatggcca gtgtgtggga 7680

ttacgacctg gtactgatgg cggtttccca tctaaccgaa tccatgaacc gataccggga 7740

agggaaggga gacaagcccg gccgcgtgtt ccgtccacac gttgcggacg tactcaagtt 7800

ctgccggcga gccgatggcg gaaagcagaa agacgacctg gtagaaacct gcattcggtt 7860

aaacacccacg cacgttgcca tgcagcgtac gaagaaggcc aagaacggcc gcctggtgac 7920

ggtatccgag ggtgaagcct tgattagccg ctacaagatc gtaaagagcg aaaccgggcg 7980

gccggagtac atcgagatcg agctagctga ttggatgtac cgcgagatca cagaaggcaa 8040

gaacccggac gtgctgacgg ttcaccccga ttactttttg atcgatcccg gcatcggccg 8100

ttttctctac cgcctggcac gccgcgccgc aggcaaggca gaagccagat ggttgttcaa 8160

gacgatctac gaacgcagtg gcagcgccgg agagttcaag aagttctgtt tcaccgtgcg 8220

caagctgatc gggtcaaatg acctgccgga gtacgatttg aaggaggagg cggggcaggc 8280

tggcccgatc ctagtcatgc gctaccgcaa cctgatcgag ggcgaagcat ccgccggttc 8340

ctaatgtacg gagcagatgc tagggcaaat tgccctagca ggggaaaaag gtcgaaaagg 8400

tctctttcct gtggatagca cgtacattgg gaacccaaag ccgtacattg ggaaccggaa 8460

cccgtacatt gggaacccaa agccgtacat tgggaaccgg tcacacatgt aagtgactga 8520

tataaaagag aaaaaaggcg atttttccgc ctaaaactct ttaaaactgg ccacgtccat 8580

gatgctgcga ctatcgcggg tgcccacgtc atagagcatc ggaacgaaaa aatctggttg 8640

ctcgtcgccc ttgggcggct tcctaatcga cggcgcaccg gctgccggcg gttgccggga 8700

ttctttgcgg attcgatcag cggccgcttg ccacgattca ccggggcgtg cttctgcctc 8760

gatgcgttgc cgctgggcgg cctgcgcggc cttcaacttc tccaccaggt catcacccag 8820

cgccgcgccg atttgtaccg ggccggatgg tttgcgaccg tcacgccgat tcctcgggct 8880

tgggggttcc agtgccattg cagggccggc agacaaccca gccgcttacg cctggccaac 8940

cgcccgttcc tccacacatg gggcattcca cggcgtcggt gcctggttgt tcttgatttt 9000

ccatgccgcc tcctttagcc gctaaaattc atctactcat ttattcattt gctcatttac 9060

tctggtagct gcgcgatgta ttcagatagc agctcggtaa tggtcttgcc ttggcgtacc 9120

gcgtacatct tcagcttggt gtgatcctcc gccggcaact gaaagttgac ccgcttcatg 9180

gctggcgtgt ctgccaggct ggccaacgtt gcagccttgc tgctgcgtgc gctcggacgg 9240

ccggcactta gcgtgtttgt gcttttgctc attttctctt tacctcatta actcaaatga 9300

gttttgattt aatttcagcg gccagcgcct ggacctcgcg ggcagcgtcg ccctcgggtt 9360

ctgattcaag aacggttgtg ccggcggcgg cagtgcctgg gtagctcacg cgctgcgtga 9420

tacgggactc aagaatgggc agctcgtacc cggccagcgc ctcggcaacc tcaccgccga 9480

tgcgcgtgcc tttgatcgcc cgcgacacga caaaggccgc ttgtagcctt ccatccgtga 9540

cctcaatgcg ctgcttaacc agctccacca ggtcggcggt ggcccatatg tcgtaagggc 9600

ttggctgcac cggaatcagc acgaagtcgg ctgccttgat cgcggacaca gccaagtccg 9660

ccgcctgggg cgctccgtcg atcactacga agtcgcgccg gccgatggcc ttcacgtcgc 9720

ggtcaatcgt cgggcggtcg atgccgacaa cggttagcgg ttgatcttcc cgcacggccg 9780

cccaatcgcg ggcactgccc tggggatcgg aatcgactaa cagaacatcg gccccggcga 9840

gttgcagggc gcgggctaga tgggttgcga tggtcgtctt gcctgacccg cctttctggt 9900

taagtacagc gataaccttc atgcgttccc cttgcgtatt tgtttattta ctcatcgcat 9960

catatacgca gcgaccgcat gacgcaagct gttttactca aatacacatc acctttttag 10020

acggcggcgc tcggtttctt cagcggccaa gctggccggc caggccgcca gcttggcatc 10080

agacaaaccg gccaggattt catgcagccg cacggttgag acgtgcgcgg gcggctcgaa 10140

cacgtacccg gccgcgatca tctccgcctc gatctcttcg gtaatgaaaa acggttcgtc 10200

ctggccgtcc tggtgcggtt tcatgcttgt tcctcttggc gttcattctc ggcggccgcc 10260

agggcgtcgg cctcggtcaa tgcgtcctca cggaaggcac cgcgccgcct ggcctcggtg 10320

ggcgtcactt cctcgctgcg ctcaagtgcg cggtacaggg tcgagcgatg cacgccaagc 10380

agtgcagccg cctctttcac ggtgcggcct tcctggtcga tcagctcgcg ggcgtgcgcg 10440

atctgtgccg gggtgagggt agggcggggg ccaaacttca cgcctcgggc cttggcggcc 10500

tcgcgcccgc tccgggtgcg gtcgatgatt agggaacgct cgaactcggc aatgccggcg 10560

aacacggtca acaccatgcg gccggccggc gtggtggtgt cggcccacgg ctctgccagg 10620

ctacgcaggc ccgcgccggc ctcctggatg cgctcggcaa tgtccagtag gtcgcgggtg 10680

ctgcgggcca ggcggtctag cctggtcact gtcacaacgt cgccagggcg taggtggtca 10740

agcatcctgg ccagctccgg gcggtcgcgc ctggtgccgg tgatcttctc ggaaaacagc 10800

ttggtgcagc cggccgcgtg cagttcggcc cgttggttgg tcaagtcctg gtcgtcggtg 10860

ctgacgcggg catagcccag caggccagcg gcggcgctct tgttcatggc gtaatgtctc 10920

cggttctagt cgcaagtatt ctactttatg cgactaaaac acgcgacaag aaaacgccag 10980

gaaaagggca gggcggcagc ctgtcgcgta acttaggact tgtgcgacat gtcgttttca 11040

gaagacggct gcactgaacg tcagaagccg actgcactat agcagcggag gggttggatc 11100

aaagtacttt gatcccgagg ggaaccctgt ggttggcatg cacatacaaa tggacgaacg 11160

gataaacctt ttcacgccct tttaaatatc cgttattcta ataaacgctc ttttctctta 11220

ggtttacccg ccaatatatc ctgtcaaaca ctgatagttt aattcccgat ctagtaacat 11280

agatgacacc gcgcgcgata atttatccta gtttgcgcgc tatattttgt tttctatcgc 11340

gtattaaatg tataattgcg ggactctaat cataaaaacc catctcataa ataacgtcat 11400

gcattacatg ttaattatta catgcttaac gtaattcaac agaaattata tgataatcat 11460

cgcaagaccg gcaacaggat tcaatcttaa gaaactttat tgccaaatgt ttgaacgatc 11520

ggggaaattc gagctggtca ccaagcttgc atgcctgcag tgcagcgtga cccggtcgtg 11580

cccctctcta gagataatga gcattgcatg tctaagttat aaaaaattac cacatatttt 11640

ttttgtcaca cttgtttgaa gtgcagttta tctatcttta tacatatatt taaactttac 11700

tctacgaata atataatcta tagtactaca ataatatcag tgttttagag aatcatataa 11760

atgaacagtt agacatggtc taaaggacaa ttgagtattt tgacaacagg actctacagt 11820

tttatctttt tagtgtgcat gtgttctcct ttttttttgc aaatagcttc acctatataa 11880

tacttcatcc attttattag tacatccatt tagggtttag ggttaatggt ttttatagac 11940

taattttttt agtacatcta ttttattcta ttttagcctc taaattaaga aaactaaaac 12000

tctattttag tttttttatt taataattta gatataaaat agaataaaat aaagtgacta 12060

aaaattaaac aaataccctt taagaaatta aaaaaactaa ggaaacattt ttcttgtttc 12120

gagtagataa tgccagcctg ttaaacgccg tcgacgagtc taacggacac caaccagcga 12180

accagcagcg tcgcgtcggg ccaagcgaag cagacggcac ggcatctctg tcgctgcctc 12240

tggacccctc tcgagagttc cgctccaccg ttggacttgc tccgctgtcg gcatccagaa 12300

attgcgtggc ggagcggcag acgtgagccg gcacggcagg cggcctcctc ctcctctcac 12360

ggcaccggca gctacggggg attcctttcc caccgctcct tcgctttccc ttcctcgccc 12420

gccgtaataa atagacaccc cctccacacc ctctttcccc aacctcgtgt tgttcggagc 12480

gcacacacac acaaccagat ctcccccaaa tccacccgtc ggcacctccg cttcaaggta 12540

cgccgctcgt cctccccccc cccccctctc taccttctct agatcggcgt tccggtccat 12600

ggttagggcc cggtagttct acttctgttc atgtttgtgt tagatccgtg tttgtgttag 12660

atccgtgctg ctagcgttcg tacacggatg cgacctgta 12699

<210> 5

<211> 1992

<212> DNA

<213> Corn (Zea mays L.)

<221> Nucleotide sequence of maize ubiquitin promoter Ubi

<222>(1)…(1992)

<400> 5

ctgcagtgca gcgtgacccg gtcgtgcccc tctctagaga taatgagcat tgcatgtcta 60

agttataaaa aattaccaca tatttttttt gtcacacttg tttgaagtgc agtttatcta 120

tctttataca tatatttaaa ctttactcta cgaataatat aatctatagt actacaataa 180

tatcagtgtt ttagagaatc atataaatga acagttagac atggtctaaa ggacaattga 240

gtattttgac aacaggactc tacagtttta tctttttagt gtgcatgtgt tctccttttt 300

ttttgcaaat agcttcacct atataatact tcatccattt tattagtaca tccatttagg 360

gtttagggtt aatggttttt atagactaat ttttttagta catctatttt attctatttt 420

agcctctaaa ttaagaaaac taaaactcta ttttagtttt tttatttaat aatttagata 480

taaaatagaa taaaataaag tgactaaaaa ttaaacaaat accctttaag aaattaaaaa 540

aactaaggaa acatttttct tgtttcgagt agataatgcc agcctgttaa acgccgtcga 600

cgagtctaac ggacaccaac cagcgaacca gcagcgtcgc gtcgggccaa gcgaagcaga 660

cggcacggca tctctgtcgc tgcctctgga cccctctcga gagttccgct ccaccgttgg 720

acttgctccg ctgtcggcat ccagaaattg cgtggcggag cggcagacgt gagccggcac 780

ggcaggcggc ctcctcctcc tctcacggca cggcagctac gggggattcc tttcccaccg 840

ctccttcgct ttcccttcct cgcccgccgt aataaataga caccccctcc acaccctctt 900

tccccaacct cgtgttgttc ggagcgcaca cacacacaac cagatctccc ccaaatccac 960

ccgtcggcac ctccgcttca aggtacgccg ctcgtcctcc cccccccccc ctctctacct 1020

tctctagatc ggcgttccgg tccatggtta gggcccggta gttctacttc tgttcatgtt 1080

tgtgttagat ccgtgtttgt gttagatccg tgctgctagc gttcgtacac ggatgcgacc 1140

tgtacgtcag acacgttctg attgctaact tgccagtgtt tctctttggg gaatcctggg 1200

atggctctag ccgttccgca gacggggatcg atttcatgat tttttttgtt tcgttgcata 1260

gggtttggtt tgcccttttc ctttatttca atatatgccg tgcacttgtt tgtcgggtca 1320

tcttttcatg cttttttttg tcttggttgt gatgatgtgg tctggttggg cggtcgttct 1380

agatcggagt agaattctgt ttcaaactac ctggtggatt tattaatttt ggatctgtat 1440

gtgtgtgcca tacatattca tagttacgaa ttgaagatga tggatggaaa tatcgatcta 1500

ggataggtat acatgttgat gcgggtttta ctgatgcata tacagagatg ctttttgttc 1560

gcttggttgt gatgatgtgg tgtggttggg cggtcgttca ttcgttctag atcggagtag 1620

aatactgttt caaactacct ggtgtattta ttaattttgg aactgtatgt gtgtgtcata 1680

catcttcata gttacgagtt taagatggat ggaaatatcg atctaggata ggtatacatg 1740

ttgatgtggg ttttactgat gcatatacat gatggcatat gcagcatcta ttcatatgct 1800

ctaaccttga gtacctatct attataataa acaagtatgt tttataatta ttttgatctt 1860

gatatacttg gatgatggca tatgcagcag ctatatgtgg atttttttag ccctgccttc 1920

atacgctatt tatttgcttg gtactgtttc ttttgtcgat gctcaccctg ttgtttggtg 1980

ttacttctgc ag 1992

<210> 6

<211> 720

<212> DNA

<213> Artificial sequence

<221> Nucleotide sequence of green fluorescent protein GFP

<222>(1)…(720)

<400> 6

atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60

ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 120

ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 180

ctcgtgacca ccttcggcta cggcctgcag tgcttcgccc gctaccccga ccacatgaag 240

cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300

ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360

gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420

aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac 480

ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 540

gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600

tacctgagct accagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660

ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtag 720

<210> 7

<211> 253

<212> DNA

<213> Artificial sequence

<221> Nucleotide sequence of terminator T1

<222>(1)…(253)

<400> 7

gatcgttcaa acatttggca ataaagtttc ttaagattga atcctgttgc cggtcttgcg 60

atgattatca tataatttct gttgaattac gttaagcatg taataattaa catgtaatgc 120

atgacgttat ttatgagatg ggtttttatg attagagtcc cgcaattata catttaatac 180

gcgatagaaa acaaaatata gcgcgcaaac taggataaat tatcgcgcgc ggtgtcatct 240

atgtactag atc 253

Claims (2)

1. Application of grain development related protein TaGSR1 or coding gene thereof in wheat strain type breeding; wherein the amino acid sequence of the grain development related protein TaGSR1 is shown as SEQ ID NO.1, and the nucleotide sequence of the encoding gene is shown as SEQ ID NO. 2; the application in wheat plant type breeding refers to the application of regulating the plant height of wheat or the application of regulating the included angle of wheat leaves.

2. The use according to claim 1, characterized in that the application method is: by constructing ectopic expressionTaGSR1Binary expression vector pLGY02-TaGSR 1of gene, and the binary expression vector is transformed into wheat to realizeTaGSR1Ectopic high expression of the gene; or transforming the binary expression vector into common wheat by agrobacterium to obtainTaGSR1Transgenic wheat with over-expressed genes realizes the reduction of the plant height of the wheat and the reduction of the included angle of leaves; wherein the nucleotide sequence of the binary expression vector pLGY02-TaGSR1 is shown as SEQ ID NO. 4; the binary carrier containsTaGSR1The expression cassette E1 of the gene plays a main role in the genetic transformation process of wheat, the nucleotide sequence of the expression cassette E1 is shown as SEQ ID NO.3, and the expression cassette E1 comprises the following components in sequence from upstream to downstream: ubiquitin promoter Ubi from maize,TaGSR1coding sequence of gene, fluorescence enhanced green fluorescent protein coding gene from multiple-tube jellyfishGFPA terminator T1; wherein the nucleotide sequence of the maize ubiquitin promoter Ubi is shown as SEQ ID NO.5, theTaGSR1The coding sequence of the gene is shown as SEQ ID NO.2, and the fluorescence enhanced green fluorescent protein coding gene from the jellyfishGFPThe nucleotide sequence of (2) is shown as SEQ ID NO. 6; the nucleotide sequence of the terminator T1 is shown in SEQ ID NO. 7.