CN110894222B - Application of 1Dx2m1/2/3 protein and coding gene thereof in cultivation of wheat variety with improved processing quality - Google Patents
Application of 1Dx2m1/2/3 protein and coding gene thereof in cultivation of wheat variety with improved processing quality Download PDFInfo
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- CN110894222B CN110894222B CN201911273308.2A CN201911273308A CN110894222B CN 110894222 B CN110894222 B CN 110894222B CN 201911273308 A CN201911273308 A CN 201911273308A CN 110894222 B CN110894222 B CN 110894222B
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Abstract
The invention discloses an application of 1Dx2m1/2/3 protein and a coding gene thereof in cultivating and processing a wheat variety with improved quality. The invention adopts a large primer PCR mutation technology to carry out site-directed mutagenesis on the wheat 1Dx2 gene to obtain a three-site mutation gene 1Dx2m1/2/3, and the three-site mutation gene is introduced into a 1Dx2 subunit deletion mutant of a wheat fine variety Kenong 199 to obtain a stable transgenic line TL1Dx2m 1/2/3. Quality analysis experiments prove that the processing quality of the wheat flour can be remarkably improved by the mutated high molecular weight glutenin subunit 1Dx2m 1/2/3. The invention improves the processing quality of wheat by site-directed mutagenesis of HMW-GS gene 1Dx2 and a transgenic method, fills the blank of improving the processing quality of wheat by using the site-directed mutagenesis gene at home and abroad, and opens up a new way for improving the processing quality of wheat.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to an application of 1Dx2m1/2/3 protein and a coding gene thereof in cultivation of a wheat variety with improved processing quality.
Background
Common wheat (Triticum aestivum l., AABBDD, 2n ═ 6x ═ 42) belongs to the gramineous (Poaceae) wheat (Triticum) genus, and is the most important food crop for human beings. Because the wheat dough has unique elasticity and ductility, the wheat dough can be processed into various foods such as bread, biscuits, noodles, steamed bread, dumplings, fried bread sticks and the like through the processes of baking, cooking, frying and the like. The quality of the food is closely related to the quality of the wheat. Wheat quality refers to the degree to which a wheat variety adapts and meets a particular end use and can be divided into nutritional quality and processing quality. The nutritional quality refers to the degree of satisfaction of nutritional ingredients contained in wheat to human (livestock) nutritional needs, including the amount of nutritional ingredients, whether the nutritional ingredients are balanced, the content of protein, the composition of amino acids, and the like. The processing quality is divided into primary processing quality and secondary processing quality. The quality of primary processing is also called grinding quality, and mainly comprises flour yield, flour color, ash content and the like. The secondary processing quality, also called eating quality, refers to the degree of adaptation and satisfaction of wheat flour to the preparation of different foods, and is mainly related to the physicochemical properties of the dough. The edible quality parameters are many and mainly comprise gluten content, sedimentation value, forming time, stabilizing time, resistance to elongation, stretching area and specific indexes aiming at various baked, steamed and fried wheaten foods.
Currently, the quality improvement of wheat is mainly focused on the processing quality, and is also mainly focused on the baking quality of industrially processed bread, biscuits, cakes and the like, and the baking quality mainly depends on the gluten content and quality of flour. After the flour is mixed with water and subjected to a suitable kneading, a dough is formed, the dough is washed with a suitable concentration of weak saline water to remove bran, starch and proteins dissolved in water or dilute salt, and the remaining material having a viscoelastic network structure is called gluten (gluten), which consists of glutenin and gliadin. The properties of gluten determine the rheological properties of the dough and thus the food processing quality of the wheat variety. In many studies, gluten strength is often used to describe the food processing quality of wheat, and wheat can be classified into strong gluten wheat, medium gluten wheat and weak gluten wheat according to the strength of gluten. The method for measuring and identifying the quality of wheat mainly comprises physicochemical detection (including using various special instruments and analysis methods) and food processing detection (including baking, cooking and the like). The main indexes for measuring the processing quality of the wheat flour comprise gluten content, sedimentation value, water absorption, forming time, stabilization time, resistance to elongation, stretching area, swelling index, various kneading and mixing instrument parameters and the like.
SDS-sedimentation value (SDS-SV) is a comprehensive index reflecting wheat gluten content and quality, and is deeply valued by wheat quality breeding workers. The principle is that a certain amount of wheat flour absorbs water to expand under the action of weak acid medium to form floccule and slowly precipitate, and the sedimentation speed and volume reflect the content and quality of gluten. The sedimentation volume in the specified standard time is the sedimentation value, and is taken as the unit of ml. The larger the measured value, the higher the gluten content, the higher the gluten strength, and the better the baking quality of the flour. The Swelling Index (SIG) of glutenin is an important index for wheat quality evaluation, and is in obvious positive correlation with the SDS-sedimentation value. SIG indirectly reflects the content and quality of glutenin based on the swelling capacity of glutenin at different swelling times. SIG measurement has the characteristics of rapidness, simplicity, convenience, small sample consumption and the like, and is often used for quality detection of early-generation materials in wheat quality breeding.
The Farinograph (Farinograph) is one of the most widely used wheat quality detection instruments, and can be used for quality detection and quality control of wheat grains or flour in the processes of wheat breeding, storage, flour processing, food production and the like. The flour quality instrument is used for kneading by adding a proper amount of water into quantitative wheat flour, and a coordinate graph of the change of stirring resistance along with time, namely a flour quality curve, is drawn by computer software. And calculating the quality characteristic parameters of the flour according to the water adding amount and the flour quality curve so as to evaluate the strength of the gluten of the flour. The main parameters measured by the flour quality meter are Water Absorption (WA), dough formation time (DT), dough Stability Time (ST), degree of weakness (DS), and Flour Quality Number (FQN). WA is affected by factors such as protein content and starch granule breakage rate in wheat flour, and the higher the protein content and starch granule breakage rate, the higher the water absorption rate. DT and ST reflect the stability and degree of dough resistance. The longer DT and ST of the dough, indicating greater gluten strength, generally better bake processing characteristics. DS represents the rate of dough breakdown during mixing, reflecting the strength of the gluten. The larger the DS, the weaker the gluten, the more rheological the dough, the less processable and the less good the baking quality. FQN is an important index reflecting rheological characteristics of dough, and has a close relation with gluten strength, the FQN of strong gluten flour is higher, and the FQN of weak gluten flour is lower.
The mixer (mixogrph) is an important instrument for measuring the quality of wheat processing by measuring the change in rheological properties during the stirring of the dough. In the continuous kneading process of the dough, the kneading instrument measures and records the kneading resistance of the dough and draws a kneading curve map to provide the optimal kneading time, stirring endurance, rheological characteristics of other dough, baking estimated water absorption value and other indexes of the dough. The kneading and mixing instrument has the advantages of high detection speed, small flour consumption, high correlation with baking characteristics and the like, and has good guiding function on wheat quality improvement, wheat quality chemical research and development of special flour and flour products. Among the parameters obtained by the kneading apparatus, the Middle Peak Time (MPT) is a very important parameter. Generally, it is considered that the MPT has high correlation with the kneading time of actual and experimental baking and is an important prediction index of bread quality. The midline peak height (MPV) is related to the extensibility of the dough and the toasted volume of the bread, the Midline Peak Width (MPW) is related to the stretch resistance of the dough, and the midline peak area (MPI) is significantly related to the maximum stretch resistance of the dough, the stretch area, and the toasted volume. The fade resistance value (RBD) is related to the kneading resistance of the dough, with the greater the RBD, the weaker the strength of the dough and the poorer the kneading resistance.
Disclosure of Invention
The first object of the present invention is to provide a protein, which is obtained by mutating serine at position 118 to cysteine, glycine at position 402 to cysteine, and tyrosine at position 722 to cysteine of an amino acid sequence of 1Dx2 protein, and which is denoted as 1Dx2m1/2/3 protein.
The 1Dx2 protein is a protein shown in a) or b) as follows:
a) the amino acid sequence is a protein shown in a sequence 4;
b) and (b) a fusion protein obtained by connecting a tag to the N-terminal and/or the C-terminal of the protein shown in the sequence 4.
The 1Dx2m1/2/3 protein is a protein shown in the following c) or d):
c) the amino acid sequence is a protein shown in a sequence 14;
d) and (b) a fusion protein obtained by connecting a tag to the N-terminal and/or C-terminal of the protein shown in the sequence 14.
In the above protein, the tag is a polypeptide or protein that is expressed by fusion with a target protein using in vitro recombinant DNA technology, so as to facilitate expression, detection, tracking and/or purification of the target protein. The tag may be a Flag tag, a His tag, an MBP tag, an HA tag, a myc tag, a GST tag, and/or a SUMO tag, among others.
It is a second object of the present invention to provide a biomaterial that is related to the 1Dx2m1/2/3 protein.
The biomaterial is any one of the following A1) to A8):
A1) a nucleic acid molecule encoding a 1Dx2m1/2/3 protein;
A2) an expression cassette comprising the nucleic acid molecule of a 1);
A3) a recombinant vector comprising the nucleic acid molecule of a 1);
A4) a recombinant vector comprising the expression cassette of a 2);
A5) a recombinant microorganism comprising the nucleic acid molecule of a 1);
A6) a recombinant microorganism comprising the expression cassette of a 2);
A7) a recombinant microorganism comprising a3) said recombinant vector;
A8) a recombinant microorganism comprising the recombinant vector of a 4).
In the above biological material, the nucleic acid molecule of A1) is a gene represented by the following 1) or 2) or 3):
1) the coding sequence is a cDNA molecule or a DNA molecule shown in sequence 13;
2) a cDNA molecule or a genome DNA molecule which has 95 percent or more than 95 percent of identity with the nucleotide sequence limited by 1) and codes 1Dx2m1/2/3 protein;
3) a cDNA molecule or a genomic DNA molecule which hybridizes with the nucleotide sequence defined in 1) or 2) under stringent conditions and encodes a 1Dx2m1/2/3 protein.
Wherein the nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA, etc.
The nucleotide sequence of the present invention encoding 1Dx2m1/2/3 can be easily mutated by one of ordinary skill in the art using known methods, such as directed evolution and point mutation. Those nucleotides which have been artificially modified to have 75% or more identity to the nucleotide sequence encoding 1Dx2m1/2/3 are derived from and identical to the nucleotide sequence of the present invention as long as they encode 1Dx2m1/2/3 and have the same function.
The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "identity" includes nucleotide sequences that are 75% or greater, or 85% or greater, or 90% or greater, or 95% or greater, identical to the nucleotide sequence of a protein consisting of the amino acid sequence set forth in coding sequence 14 of the present invention. Identity can be assessed visually or by computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to assess the identity between related sequences.
The above-mentioned identity of 75% or more may be 80%, 85%, 90% or 95% or more.
In the above biological material, the stringent conditions are hybridization and membrane washing at 68 ℃ for 2 times, 5min each, in a solution of 2 XSSC, 0.1% SDS, and hybridization and membrane washing at 68 ℃ for 2 times, 15min each, in a solution of 0.5 XSSC, 0.1% SDS; alternatively, hybridization was carried out at 65 ℃ in a solution of 0.1 XSSPE (or 0.1 XSSC), 0.1% SDS, and the membrane was washed.
In the above biological material, the vector may be a plasmid, a cosmid, a phage, or a viral vector. The recombinant vector can be specifically a recombinant vector p1Bx14PRO-1Dx2m 1/2/3.
In the above biological material, the microorganism may be yeast, bacteria, algae or fungi, such as Agrobacterium.
The third purpose of the invention is to provide a new application of the 1Dx2m1/2/3 protein or biological material.
The invention provides an application of the 1Dx2m1/2/3 protein or biomaterial in any one of the following B1) -B10):
B1) regulating and controlling the processing quality of the wheat flour;
B2) regulating and controlling the baking quality of the wheat flour;
B3) regulating and controlling the gluten property and/or gluten strength and/or gluten quality of the wheat flour;
B4) regulating the dough rheological property and/or the extensibility and/or the stretch resistance and/or the kneading resistance of the wheat flour;
B5) regulating and controlling the SDS-sedimentation value of the wheat flour;
B6) regulating and controlling the glutenin swelling index of the wheat flour;
B7) regulating and controlling the dough forming time and/or dough stabilizing time and/or weakening degree in flour quality instrument detection parameters of the wheat flour;
B8) regulating and controlling peak time and/or peak area and/or anti-fading value in the parameters of the kneading and mixing instrument of the wheat flour;
B9) cultivating transgenic wheat with improved flour processing quality and/or baking quality and/or gluten quality;
B10) and (5) wheat breeding.
In the application, the regulation and control of the processing quality of the wheat flour is specifically to improve the processing quality of the wheat flour;
the method is characterized in that the baking quality of the wheat flour is regulated and controlled, and specifically the baking quality of the wheat flour is improved;
the gluten property and/or the gluten strength and/or the gluten quality of the wheat flour are regulated and controlled, and specifically the gluten property of the wheat flour is improved and/or the gluten strength of the wheat flour is improved and/or the gluten quality of the wheat flour is improved;
the dough rheological property and/or the extensibility and/or the stretch resistance and/or the kneading resistance of the wheat flour are/is regulated, and particularly the dough rheological property and/or the extensibility and/or the stretch resistance and/or the kneading resistance of the wheat flour are improved;
the SDS-sedimentation value of the wheat flour is specifically adjusted and controlled to be improved;
the glutenin swelling index of the wheat flour is regulated and controlled, and is specifically the glutenin swelling index of the wheat flour is improved;
the dough forming time and/or dough stabilizing time and/or weakening degree in the flour quality instrument detection parameters of the wheat flour are/is specifically increased, the dough forming time in the flour quality instrument detection parameters of the wheat flour is/is increased, the dough stabilizing time in the flour quality instrument detection parameters of the wheat flour is/is increased, and/or the weakening degree in the flour quality instrument detection parameters of the wheat flour is reduced;
the peak time and/or the peak area and/or the anti-fading value in the detection parameters of the kneading and mixing instrument for regulating and controlling the wheat flour are/is specifically to improve the peak time in the detection parameters of the kneading and mixing instrument for regulating and controlling the wheat flour and/or improve the peak area in the detection parameters of the kneading and mixing instrument for regulating and controlling the wheat flour and/or reduce the anti-fading value in the detection parameters of the kneading and mixing instrument for regulating and controlling the wheat flour;
the aim of the wheat breeding is to improve the processing quality and/or baking quality and/or gluten quality of wheat flour.
A fourth object of the present invention is to provide a method for breeding transgenic wheat having improved flour processing quality and/or baking quality and/or gluten quality.
The method for cultivating the transgenic wheat with improved flour processing quality and/or baking quality and/or gluten quality comprises the steps of improving the content and/or activity of 1Dx2m1/2/3 protein in receptor wheat to obtain transgenic wheat; the transgenic wheat has a higher flour processing quality and/or baking quality and/or gluten quality than the recipient wheat.
In the above method, the transgenic wheat has higher flour processing quality than the recipient wheat as embodied in any one of C1) -C8):
C1) the SDS-sedimentation value of the transgenic wheat flour is higher than that of receptor wheat;
C2) the swelling index of glutenin of the transgenic wheat flour is higher than that of receptor wheat;
C3) the dough forming time in the flour quality instrument detection parameters of the transgenic wheat flour is longer than that of receptor wheat;
C4) the dough stability time in the flour quality instrument detection parameters of the transgenic wheat flour is longer than that of receptor wheat;
C5) the attenuation degree of the detection parameters of the flour quality instrument of the transgenic wheat flour is lower than that of receptor wheat;
C6) the peak time in the detection parameters of the kneading and mixing instrument of the transgenic wheat flour is higher than that of receptor wheat;
C7) the peak value area in the detection parameters of the kneading and mixing instrument of the transgenic wheat flour is higher than that of receptor wheat;
C8) the anti-fading value of the parameters detected by the kneading and mixing instrument of the transgenic wheat flour is lower than that of receptor wheat.
Further, the method for improving the content and/or the activity of the 1Dx2m1/2/3 protein in the receptor wheat is to over-express the 1Dx2m1/2/3 protein in the receptor wheat.
Furthermore, the method for over-expressing the 1Dx2m1/2/3 protein in the recipient wheat is to introduce a coding gene of the 1Dx2m1/2/3 protein into the recipient wheat. The nucleotide sequence of the coding gene of the 1Dx2m1/2/3 protein is specifically a DNA molecule shown as a sequence 13.
In a specific embodiment of the invention, the coding gene of the 1Dx2m1/2/3 protein is introduced into recipient wheat through a recombinant vector p1Bx14PRO-1Dx2m 1/2/3. The recombinant vector p1Bx14PRO-1Dx2m1/2/3 is a vector obtained by replacing a sequence 19 in a sequence table with a vector pUBI, wherein the DNA fragment is positioned between the Pst I and Kpn I enzyme cutting sites of cas, and other sequence structures are kept unchanged.
In the above method, the transgenic plant is understood to include not only the first generation transgenic plant obtained by transforming a plant of interest with the 1Dx2m1/2/3 gene, but also the progeny thereof. For transgenic plants, the gene can be propagated in the species, and can also be transferred into other varieties of the same species, including particularly commercial varieties, using conventional breeding techniques. The transgenic plants include seeds, callus, whole plants and cells.
In the method, the receptor wheat is a 1Dx2 subunit deletion mutant (kn 2) of wheat elite Kenong 199-Mutant). The kn2-The mutant is a 1Dx2 deletion mutant obtained by deleting only 1Dx2 gene (sequence 3) in the genome sequence of the common wheat variety Kenong 199(KN 199).
The invention adopts a large primer PCR mutation technology to carry out site-directed mutagenesis on the wheat 1Dx2 gene to obtain a three-site mutant gene 1Dx2m1/2/3, the mutant gene 1Dx2m1/2/3 can be translated into a mutant high molecular weight glutenin subunit 1Dx2m1/2/3 in vivo, and a gene gun mediated method is adopted to introduce the three-site mutant gene 1Dx2m1/2/3 into a 1Dx2 subunit deletion mutant of a wheat fine variety Kenong 199 to obtain a stable transgenic line TL1Dx2m 1/2/3. Quality analysis experiments prove that the processing quality of the wheat flour can be remarkably improved by the mutated high molecular weight glutenin subunit 1Dx2m 1/2/3. The invention improves the processing quality of wheat by site-directed mutagenesis of HMW-GS gene 1Dx2 and a transgenic method, fills the blank of improving the processing quality of wheat by using the site-directed mutagenesis gene at home and abroad, and opens up a new way for improving the processing quality of wheat.
Drawings
FIG. 1 is an electrophoretogram of PCR amplification products of the full-length sequence of the coding region of 1Dx2 gene. Note: lane M is DNAmarker; the KN199 Lane is a product obtained by performing PCR amplification by using a specific primer combination Dx2F and Dx2R for amplifying the full-length sequence of the coding region of the 1Dx2 gene and taking the genome DNA of the common wheat variety Kenong 199 as a template, and the band marked by 1Dx2 is an amplified fragment of the full-length sequence of the coding region of the 1Dx2 gene.
FIG. 2 is a schematic diagram of the mutation site of 1Dx2m1/2/3 gene. Note: SP is a signal peptide sequence; NT is an N-terminal sequence; CRD is the middle repeat region; CT is C-terminal sequence; m1 is the site-directed mutation site of 1Dx2m1/2/3 gene at the 5 'end of CRD, namely 353 rd base C is mutated into base G, m2 is the site-directed mutation site of 1Dx2m1/2/3 gene at the 3' end of CRD, namely 2165 th base A is mutated into base G, m3 is the site-directed mutation site of 1Dx2m1/2/3 gene at the CRD region, namely 1204 th base G is mutated into base T.
FIG. 3 shows the acquisition of 1Dx2m1/2/3 gene. A: FRm3Preparing site-directed mutagenesis fragments; b is the amplification of the full-length sequence of the coding region of the 1Dx2m3 gene; c is site-directed mutagenesis large primer Dx2m2Amplification of the FR; d: amplification of the full-length sequence of the coding region of the 1Dx2m2/3 gene; e: FRm1Amplifying the site-directed mutagenesis primer; f: amplification of the full-length sequence of the coding region of the 1Dx2m1/2/3 gene. And the lane M is a DNA marker.
FIG. 4 shows the general wheat variety Kenong 199 and its 1Dx2 subunit deletion line kn2-SDS-PAGE profile of glutenin. Note: kn2-1Dx2 subunit deletion line for konong 199; KN199 is wild type Kenong 199; the right side labeled 1Dx2 etc. are the corresponding subunits.
FIG. 5 shows the construction process of 1Dx2m1/2 gene transformation vector p1Bx14PRO-1Dx2m 1/2/3.
FIG. 6 is T0PCR detection of 1Dx2m1/2/3 transgenic wheat plants. Note: m is DNAmarker; + represents a positive plant; -represents a negative plant; kn is a transgenic receptor, namely 1Dx2 subunit deletion mutant kn2 of Kenong 199-(ii) a KN is wild type Kenong 199; b is blank control. The 414bp band shown by an arrow is a target amplified fragment of the positive plant.
FIG. 7 is T3SDS-PAGE detection of the 1Dx2m1/2/3 transgenic line HMW-GS. Note: kn2-Is a transgenic receptor, namely a deletion mutant of 1Dx2 subunit of Kenong 199; KN199 is wild type Kenong 199; TL1Dx2m1/2/3 is a transgenic line of mutant gene 1Dx2m 1/2/3; TL1Dx2 is a 1Dx2 transgenic line.
FIG. 8 is a powdery mass map of the 1Dx2m1/2/3 transgenic line. Note: kn2-Is a transgenic receptor, namely a deletion mutant of 1Dx2 subunit of Kenong 199; KN199 is wild type Kenong 199; TL1Dx2 is a 1Dx2 transgenic line; TL1Dx2m1/2/3 is a transgenic line of mutant gene 1Dx2m 1/2/3.
Detailed Description
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The powder mass apparatus (Farinograph-E) in the examples below is a product of Brabender, OHG Duisburg, Germany.
The kneading apparatus (mixogrph) in the following examples is a product of National company (National mfg.co., Lincoln, USA).
The vector pAHC20 containing the selection marker gene Bar in the following examples is a product of Pubaezin Biotechnology (Beijing) Ltd.
The general wheat cultivar kong 199 in the following examples was bred by Lijunming researcher of institute of genetics and developmental biology, national institute of sciences, and the cultivar was examined and approved: national scrutiny wheat 2006017.
Example 1 obtaining of wheat mutant Gene 1Dx2m1/2/3
1. PCR amplification of wheat 1Dx2 gene coding region full-length sequence
PCR amplification was performed using genomic DNA of Nongman 199 of the family Triticum aestivum variety as a template, using specific primer combinations Dx2F and Dx2R, PrimeSTAR HS DNA polymerase with high fidelity, and 2 XPrimeSTAR GC Buffer (Takara, Dalian). The resulting PCR-amplified fragment 1Dx2 (FIG. 1) was the full-length sequence of the coding region of 1Dx2 gene. The nucleotide sequences of the primer combination Dx2F and Dx2R are respectively sequence 1 and sequence 2 in a sequence table.
The PCR procedure for amplification of the full length sequence of the coding region of 1Dx2 was: pre-denaturation at 98 ℃ for 3 min; denaturation at 98 deg.C for 30sec, renaturation at 72 deg.C and extension for 3min, and circulation for 38 times; finally, extension is carried out for 10min at 72 ℃.
2. Recovery and clone sequencing of 1Dx2 gene PCR amplification product
After the PCR amplification product was separated by 1% agarose gel electrophoresis, the gel fraction (FIG. 1) containing the target band 1Dx2 (about 2500bp) was cut under an ultraviolet lamp and placed in a 1.5ml centrifuge tube, and the amplified fragment 1Dx2 was recovered using an agarose gel recovery kit (Tiangen, Beijing). The recovered fragment was ligated to the Blunt-end cloning vector pEasy blast (all-trans gold, Beijing) and sequenced (Huada gene, Beijing). The sequencing result shows that the length of the cloned DNA fragment is 2523bp, and the nucleotide sequence is the sequence 3 in the sequence table, namely the full-length sequence of the coding region of the 1Dx2 gene. 1Dx2 is a gene without intron, and the coded 1Dx2 protein consists of 839 amino acid residues, and the amino acid sequence of the protein is the sequence 4 in the sequence table.
3. Creation of mutant Gene 1Dx2m3 Using PCR and restriction Endonzyme method
(1) Using the full-length sequence of the coding region of 1Dx2 gene as a template, PrimeSTAR HS DNA polymerase and 2 XPimeSTAR GC Buffer (Takara Shuzo, Dalian) were used with a large primer set Dx2F and Dx2Rm3PCR amplification was performed. Primer combination Dx2F and Dx2Rm3The nucleotide sequences of (A) are respectively sequence 2 and sequence 5 in the sequence table.
The large primer PCR reaction program adopts a two-step method, and the specific program is pre-denaturation at 98 ℃ for 3 min; denaturation at 98 ℃ for 30sec, annealing at 72 ℃ and extension for 1min for 30 cycles; 10min at 72 ℃.
(2) After the PCR product was identified by 1% agarose gel electrophoresis, a 1240bp large fragment containing the mutation site was recovered (FIG. 3A), which was named Dx2m3And (6) FR. Large primer fragment Dx2m3The nucleotide sequence of FR is sequence 6 in the sequence table.
(3) Dx2 ligated to pEasy blast vector (all-trans gold, Beijing) and verified for insertion direction was separately ligated with restriction enzymes BamHI and BlpIm3FR and 1Dx2 genes were subjected to double digestion. Recovering the enzyme cutting fragment Dx2 with the mutation sitem3FR, replacing the corresponding fragment on the 1Dx2 gene to obtain a vector pEasy blast-1 Dx2m3 containing the mutant gene, verifying by a restriction endonuclease enzyme cutting method (figure 3B) and sequencing to obtain the mutant gene 1Dx2m3, wherein the nucleotide sequence of the mutant gene is shown as a sequence 7 in the sequence table. The coding region sequence of the 1Dx2m3 gene is that the 1204 st guanine (G) of the coding region sequence of the wild type 1Dx2 gene shown in the sequence 3 in the sequence table is mutated into thymine (T), and the 1Dx2 gene is keptThe resulting nucleotide sequence was not changed.
4. Acquisition of two-site mutant Gene 1Dx2m2/3 of 1Dx2
The m2 site of 1Dx2m3 is subjected to site-directed mutagenesis by a large primer PCR method to obtain a two-site mutant gene 1Dx2m2/3, wherein the mutation site m2 refers to that the 2165 th adenine (A) in the coding region sequence of the 1Dx2 gene is mutated into guanine (G).
The specific operation steps are as follows:
(1) using the full-length sequence of the coding region of 1Dx2 gene as a template, PrimeSTAR HS DNA polymerase and 2 XPrimeSTAR GC Buffer (Takara Shuzo, Dalian) were used, and a large primer combination Dx2F was usedm2And Dx2R for PCR amplification. Large primer combination Dx2Fm2And the nucleotide sequences of Dx2R are respectively sequence 8 and sequence 2 in the sequence table.
The large primer PCR reaction program adopts a two-step method, and the specific program is pre-denaturation at 98 ℃ for 3 min; denaturation at 98 ℃ for 30sec, annealing at 72 ℃ and extension for 1min for 30 cycles; 10min at 72 ℃.
(2) After the PCR product was identified by 1% agarose gel electrophoresis, a large primer fragment of 369bp in size and containing a mutation site was recovered (FIG. 3C), which was designated as Dx2m2And (6) FR. Large primer fragment Dx2m2The nucleotide sequence of FR is the sequence 9 in the sequence table.
(3) Wild type gene 1Dx2 is used as template, and large primer segment Dx2 is adoptedm2And performing PCR amplification on the FR and a corresponding primer Dx2F on the other side, wherein the nucleotide sequence of the primer Dx2F is the sequence 1 in the sequence table. The amplified product is separated, recovered, cloned and sequenced to obtain the full-length sequence of the coding region of the two-site mutant gene 1Dx2m2/3 of 1Dx 2. The full-length sequence of the coding region of 1Dx2m2/3 is the sequence 10 in the sequence table. In fact, the coding region sequence of the 1Dx2m2/3 gene is a nucleotide sequence obtained by mutating guanine (G) to thymine (T) at position 1204 of the coding region sequence of the wild-type 1Dx2 gene shown in sequence 3 in the sequence table, and mutating adenine (a) to guanine (G) at position 2165, while keeping the other sequences of the 1Dx2 gene unchanged.
5. Obtaining of three-site mutant Gene 1Dx2m1/2/3 of 1Dx2
Taking 1Dx2m2/3 as a template, and carrying out site-directed mutagenesis on an m1 site by a large primer PCR method to obtain a three-site mutant gene 1Dx2m1/2/3 of 1Dx2, wherein the mutation site m1 refers to that 353 rd cytosine (C) in a coding region sequence of the 1Dx2 gene is mutated into guanine (G). The specific operation steps are as follows:
(1) using the full-length sequence of the coding region of 1Dx2 gene as a template, PrimeSTAR HS DNA polymerase and 2 XPimeSTAR GC Buffer (Takara Shuzo, Dalian) were used with a large primer set Dx2F and Dx2Rm1PCR amplification was performed. Large primer combinations Dx2F and Dx2Rm1The nucleotide sequences of (A) are respectively sequence 1 and sequence 11 in the sequence table.
The large primer PCR reaction program adopts a two-step method, and the specific program is pre-denaturation at 98 ℃ for 3 min; denaturation at 98 ℃ for 30sec, annealing at 72 ℃ and extension for 1min for 30 cycles; 10min at 72 ℃.
(2) After the PCR product was identified by 1% agarose gel electrophoresis, a large primer fragment of 364bp in size and containing a mutation site was recovered (FIG. 3E), which was designated as Dx2FRm1. Large primer fragment Dx2FRm1The nucleotide sequence of (A) is sequence 12 in the sequence table.
(3) 1Dx2m2 is used as a template, and a large primer segment Dx2FR is adoptedm1And carrying out PCR amplification with a corresponding primer Dx2R on the other side, wherein the nucleotide sequence of the primer Dx2R is the sequence 2 in the sequence table. The amplified product is separated, recovered, cloned and sequenced to obtain the full-length sequence of the coding region of the 1Dx2 three-site mutant gene 1Dx2m 1/2/3. The full-length sequence of the coding region of 1Dx2m1/2/3 is the sequence 13 in the sequence table. In fact, the coding region sequence of the 1Dx2m1/2/3 gene is a nucleotide sequence obtained by mutating the 353 rd cytosine (C) to guanine (G), the 1204 nd guanine (G) to thymine (T), and the 2165 th adenine (a) to guanine (G) of the coding region sequence of the wild-type 1Dx2 gene shown in sequence 3 in the sequence table, while keeping the other sequences of the 1Dx2 gene unchanged. The protein coded by the mutant gene 1Dx2m1/2/3 is 1Dx2m1/2/3 protein. The amino acid sequence of the 1Dx2m1/2/3 protein is the sequence 14 in the sequence table. In fact, the amino acid sequence of the 1Dx2m1/2/3 protein is defined as SEQ ID No. 4The amino acid sequence obtained by mutating the serine residue (Ser) at position 118 to the cysteine residue (Cys), the glycine residue (Gly) at position 402 to the cysteine residue (Cys), and the tyrosine residue (Tyr) at position 722 to the cysteine residue (Cys) of the 1Dx2 protein shown, while keeping the other sequences of the 1Dx2 protein unchanged.
Example 2, 1Dx2m1/2/3 obtaining of transgenic wheat
1. Creation of transgenic recipient Material
The transgenic receptor material used by the invention is a high molecular weight glutenin subunit 1Dx2 deletion mutant of common wheat variety Kenong 199(KN199), which is marked as KN2-。kn2-Mutants were obtained by EMS mutagenesis. The specific process is as follows: (1) 1000 filled wild type farmer 199 seeds were selected and treated with 0.4% EMS (Sigma, M0880) in the dark for 24 hours and washed with running water for 24 hours, and the seeds were marked M0；(2)M0The seeds are planted in the Beijing Changping experiment base of the institute of genetics and development biology of Chinese academy of sciences after normal germination and vernalization treatment (4 ℃) for 4 weeks, normal field management is carried out, and the harvested seeds are recorded as M1(ii) a From M by SDS-PAGE1Screening out 1Dx2 subunit deletion mutant from seeds, and marking as kn2-(FIG. 4); m1Generation kn2-The seeds are backcrossed and selfed for two successive generations to obtain enough M3Seeds are used as the receptor material for genetic transformation of mutant gene 1Dx2m 1/2/3. Sequencing verification shows that: kn2-The mutant is a 1Dx2 deletion mutant obtained by deleting only 1Dx2 gene (sequence 3) in the genome sequence of the common wheat variety Kenong 199(KN199), and the rest sequences are the same as the genome sequence of the common wheat variety Kenong 199(KN 199).
2. Construction of transformation vectors for mutant Gene 1Dx2m1/2/3 and wild-type Gene 1Dx2
The construction process of the 1Dx2m1/2/3 gene transformation vector is shown in FIG. 5. The specific operation steps are as follows:
(1) the genome DNA of common wheat variety Elytrigia tritici 54 is used as a template, and PCR amplification is carried out by adopting primer combination Bx14proF and Bx14proR to obtain a 1Bx14 promoter sequence. The nucleotide sequences of the primer combination Bx14proF and Bx14proR are respectively a sequence 15 and a sequence 16 in a sequence table. The obtained 1Bx14 promoter sequence is the sequence 17 in the sequence table.
(2) And (2) inserting the 1Bx14 promoter sequence (sequence 17 in a sequence table) obtained in the step (1) into a vector pUBI: (sequence 18 in the sequence table) between Pst I and BamH I enzyme digestion sites of cas to replace the Ubiquitin promoter sequence, and keeping other sequences of the vector pUBI: (sequence 18 in the sequence table) unchanged to obtain an intermediate vector p1Bx14 PRO.
(3) The coding region sequence (sequence 13 in the sequence table) of the three-site mutation gene 1Dx2m1/2/3 is inserted between the BamH I and Kpn I enzyme cutting sites of the intermediate vector p1Bx14PRO, namely, the coding region sequence is connected to the downstream of the promoter sequence of 1Bx14, and other sequences of the intermediate vector p1Bx14PRO are kept unchanged, so that the recombinant vector p1Bx14PRO-1Dx2m1/2/3 is obtained, namely, the recombinant vector of the 1Dx2m1/2/3 gene.
(4) The recombinant vector p1Bx14PRO-1Dx2m1/2/3 was sequenced. The sequencing result shows that the recombinant vector p1Bx14PRO-1Dx2m1/2/3 is a vector obtained by replacing a DNA molecule (a sequence of a 1Bx14 promoter sequence and a 1Dx2m1/2/3 coding region sequence) of a sequence 19 in a sequence table with a DNA fragment between the Pst I and Kpn I enzyme cutting sites of the vector pUBI:: cas and keeping other sequences of the vector pUBI:: cas unchanged.
The construction process and operation procedure of the wild-type 1Dx2 gene transformation vector are the same as those of the mutant gene 1Dx2m1/2/3 transformation vector, except that the coding region sequence of the wild-type 1Dx2 (sequence 3 in the sequence table) is used to replace the coding region sequence of the mutant gene 1Dx2m1/2/3 (sequence 13 in the sequence table). The transformation vector was designated as p1Bx14PRO-1Dx 1/2/3.
3. Genetic transformation of 1Dx2m1/2/3 Gene
Genetic transformation was performed by the particle gun method. The transformation acceptor material is 1Dx2 subunit deletion mutation system kn2 of common wheat variety Kenong 199-Young embryos about 14 days after flowering. Co-transforming the transformation vector p1Bx14PRO-1Dx2m1/2/3 with the vector pAHC20 containing the selection marker gene Bar to obtain T of the mutation-transferred gene 1Dx2m1/2/30And (5) plant generation. Genetic transformation in the genetic transformation platform of institute of genetics and developmental biology of Chinese academy of sciencesThe process is carried out. The genetic transformation of the wild-type 1Dx2 gene was performed in the same manner as the genetic transformation of the mutant gene 1Dx2m1/2/3 except that the transformation vector was p1Bx14PRO-1Dx 2.
4. PCR identification and propagation of 1Dx2m1/2/3 transgenic wheat
T of transgenic 1Dx2m1/2/3 with primer combination Dx2nosF and Dx2nosR0Performing PCR identification on the generation plants by using a receptor kn2-Is a negative control. If the PCR amplification product is a 414bp fragment (FIG. 6), the plant is a positive plant of the mutation-transferred gene 1Dx2m1/2/3, and if the 414bp amplification fragment does not exist, the plant is a negative plant. The nucleotide sequences of the primers Dx2nosF and Dx2nosR are respectively a sequence 20 and a sequence 21 in a sequence table. Identification of wild-type 1Dx2 Gene transformant T was detected by SDS-PAGE0In the method for generating the glutenin subunit, a plant expressing the 1Dx2 subunit is a positive plant of the 1Dx2 gene, otherwise, the plant is a negative plant.
Planting T0Generations of 1Dx2m1/2/3 transgenic positive plants and harvesting seeds in individual plants. The T with stable HMW-GS expression is obtained by propagating 3 to 4 generations continuously and performing PCR identification on the exogenous gene by generations (figure 6) and SDS-PAGE identification on HMW-GS (figure 7)3Generations (2016 years) or T4The generation (2017) 1Dx2m1/2/3 transgenic homozygous line is used for determining the processing quality traits. Meanwhile, the 1Dx2 transgenic line is propagated and identified by the same method as the 1Dx2m1/2/3 transgenic plant, and the transgenic receptor kn2 is propagated in parallel-And seeds of wild type Kenong 199 were used as a control for the quality analysis of the processing of the transgenic line with the mutant gene 1Dx2m 1/2/3.
Example 3, determination of processing quality traits of 1Dx2m1/2/3 transgenic lines and comparative analysis with recipient, wild type Kenong 199 and 1Dx2 transgenic lines
The invention provides a method for determining the T obtained in example 2 in 2016 and 2017, respectively3Generation 1Dx2m1/2/3 transgenic line (hybrid line) TL1Dx2m1/2/3 and 2T4The generation 1Dx2m1/2/3 transgenic lines TL1Dx2m1/2/3-1 and TL1Dx2m1/2/3-12 are subjected to processing quality character determination, and are compared with a transgenic line TL1Dx2 and a transgenic receptor kn2 of a wild type gene 1Dx2-Comparative analysis was performed with wild type Kenong 199. All assay samples were assigned 3 biological replicates. Data ofThe collection, treatment and comparative analyses were performed using Excel (Microsoft, USA) software, One-way ANOVA (One-way ANOVA) and multiple comparisons using SPSS 19.0(IBM, USA) software, and multiple comparisons were performed using the Duncon method in One-way ANOVA.
1. Determination of SDS-sedimentation values and comparative analysis
The SDS-sedimentation value (SDS-SV) of the 1Dx2m1/2/3 transgenic line was determined in 2016 and 2017, respectively, according to the methods described in "Zhang L., Chen Q., Su M., Yan B., Zhang X., Jiano Z., high-molecular-weight glutathione-specific properties induced by beam and the effects of Glu-1loci deletion on quality properties J., Sci., food Agents, 2016,96: 1289-1296". The specific operation steps are as follows: weighing 1g flour (14% water content) in 35ml glass test tube with plug scale, adding 16.8ml bromophenol blue aqueous solution (4mg/L), and mixing by reverse rotation for 10-20 sec; placing the test tube on CAU-B sedimentation value analyzer (China university of agriculture), and shaking for 5min for 40 times/min; adding 16.8ml of SDS-lactic acid solution (2% SDS, 0.19% lactic acid), reversing, mixing, placing on a sedimentation value analyzer, and shaking for 5min for 40 times/min; and (5) reversing and mixing for 10-20sec, standing for 5min, and observing and recording sedimentation value results. The experiment uses 1DX2 transgenic line TL1Dx2 and transgenic receptor kn2 respectively-Comparative analysis was performed against wild type Kenong 199.
The results of SDS-sedimentation of the transgenic lines 1Dx2m1/2/3 and their control material are shown in Table 1. As can be seen from table 1: in 2016, the SDS-sedimentation value of the 1Dx2m1/2/3 transgenic line TL1Dx2m1/2/3 is obviously larger than that of the 1Dx2 transgenic line TL1Dx2 and the transgenic receptor kn2-And wild type Kenong 199(KN 199). The SDS-sedimentation value of 21 Dx2m1/2/3 transgenic lines TL1Dx2m1/2/3-1 and TL1Dx2m1/2/3-12 determined in 2017 is also obviously larger than that of a transgenic receptor kn2-1Dx2 transgenic line TL1Dx2 and wild type konong 199. These results fully demonstrate that the mutant subunit 1Dx2m1/2/3 can significantly increase the SDS-sedimentation value of flour compared to the wild-type subunit 1Dx 2.
TABLE 1 determination and analysis of SDS-sedimentation value
Note: (1) TL1Dx2m1/2/3 measured in 2016 as T3Mixed samples of the generation 1Dx2m1/2/3 transgenic line, TL1Dx2 sample T3Mixed samples of passage 1Dx2 transgenic line. TL1Dx2m1/2/3-1 and TL1Dx2m1/2/3-12 determined in 2017 are two T4Generation 1Dx2m1/2/3 transgenic single line, TL1Dx2-2 and TL1Dx-12 are two T4Generation 1Dx2 transgenic single line. (2) The data in the table are the average of 3 biological replicates, with different letters representing significant differences at the 0.05 level after the assay.
2. Measurement and comparative analysis of glutenin swelling index
The glutenin Swelling Index (SIG) of the 1Dx2m1/2/3 transgenic lines was determined in 2016 and 2017, respectively, and likewise with the transgenic receptor kn2, according to the method described in "Wang, C., and Kovacs, M.I.P.spinning index of glutenin test.II.application in the prediction of dough properties and end-use quality. Central chem.J.2002,79:190--1Dx2 transgenic line TL1Dx2 and wild type Kenong 199 were controls. The specific operation steps are as follows: (1) 50ml centrifuge tubes were weighed one by one and recorded as M0. To each tube was added 1.0g of flour of the sample to be tested and weighed as M1. (2) 15ml of distilled water was added to the centrifuge tube, vortexed for 10sec, and allowed to stand at 24 ℃ for 20min, during which vortexed for 2 times for 10 sec. (3) 15ml of SDS-lactic acid solution (3% SDS, 0.19% lactic acid) was added to each centrifuge tube, vortexed for 10sec, and allowed to stand at 24 ℃ for hydration for 20min, during which vortexed for 2 times for 10 sec. (4) Centrifuging at 450 Xg for 5min, taking out the centrifuge tube, sucking most of supernatant liquid by a pipette under the condition of not touching the interface, centrifuging the precipitate at 450 Xg for 3min, immediately inverting the centrifuge tube on a piece of absorbent filter paper, sucking off residual water on the tube wall one by one, and weighing, wherein the mark is M2. (5) The weight of wet batter formed per weight of flour (M2-M0). times.100/(M1-M0). times.100-W, where W is the moisture content of the sample, was calculated as the SIG value.
The SIG measurement results are shown in Table 2. As can be seen from table 2: in 2016, the SIG value of the 1Dx2m1/2/3 transgenic line TL1Dx2m1/2/3 is significantly greater than that of the transgenic receptorBody kn2-1Dx2 transgenic line TL1Dx2 and wild type konong 199(KN 199). In 2017, the SIG values of 21 Dx2m1/2/3 transgenic lines TL1Dx2m1/2/3-1 and TL1Dx2m1/2/3-12 are also significantly higher than that of a transgenic receptor kn2-1Dx2 and wild type cronong 199. The expression of the mutant subunit 1Dx2m1/2/3 is proved to be capable of obviously improving the swelling index of glutenin compared with the wild type 1Dx2 subunit.
TABLE 2 measurement and analysis of swelling index of glutenin
3. Powder quality instrument parameter determination and comparative analysis
According to American society of cereal chemistry, AACCthSt.MN, USA American Association of Central Chemistry,2000 "and the program" AACC 54-21 "were used to perform powder quality analysis on the transgenic line expressing the mutant gene 1Dx2m1/2/3 and flour of its control material, respectively. The experiment was repeated 3 times using a 50g dough bowl. The specific operation steps are as follows: (1) 50g of flour (14% wet basis, 50g) of each sample to be tested was weighed according to the moisture content of the flour and the data given by the instrument software. After the instrument is opened and zero calibration is carried out, flour is put into a dough kneading bowl of 50g of the flour quality instrument, and a dough kneading bowl cover is covered. (2) Starting the dough kneading device, stirring, kneading and mixing for 1min, rapidly adding water, and covering with a dough kneading pot cover. After dough kneading until the peak is formed, it is observed whether the peak of the curve is between 500 + -20 FU. (3) If the peak value is between 500 +/-20 FU, continuing kneading dough, continuing softening for 12min after the curve starts to obviously decrease, finishing the powder quality map, and performing data acquisition and comparative analysis.
The results of the powder quality testing are shown in Table 3, from which it can be seen that: the dough forming time, dough stabilizing time and flour quality index of TL1Dx2m1/2/3 in 2016 and 2017 are all significantly higher than that of the control KN199 and transgenic receptor kn2-The attenuation degree is obviously lower than that of KN99 and a transgenic receptor KN2-The water absorption did not change significantly. The dough forming time of TL1Dx2m1/2/3 is more than 2 times of KN199, the dough stabilizing time is nearly 4 times of KN199, the flour quality index is more than 3 times of KN199, and the weakening degree is about 1/2 times of KN199, which shows that the mutant gene 1Dx2m1/2/3 can remarkably improve the rheological property of wheat dough and improve the gluten strength of wheat and the processing quality of flour.
TABLE 3 determination of powder quality Instrument parameters
4. Kneading and mixing instrument analysis
The dough peak time (MPT), peak height (MPV), peak width (MPW), peak area (MPI) and anti-fade value (RBD) of the 1Dx2m1/2/3 transgenic line were determined in 2016 and 2017, respectively, using a kneader according to the methods of documents "AACC 54-40A" and "Sun et al (2015) (Sun, J., Yang, W., Liu, D., ZHao, J., Luo, G., Li, X., Liu, Y., Guo, J., and Zhang, A. (2015)., Improvement on Mixograph test through water addition and parameter conversion.J.integer.Agr.14: 1715-1722.". The method comprises the following specific steps: a kneading bowl of 10g was used. The total kneading time of the standard kneading machine is 10min, and the weighing amount M of the flour is (10g × 86)/(100-W), wherein W is the water content of the sample. The data obtained were analyzed with MixSmart software (AEW Consulting, Lincoln, Nebraska, USA). The curve of the kneading and mixing instrument is automatically drawn by a computer, the parameters exceed 40, and the analysis usually adopts the middle line (Midline) parameters which are divided into 4 categories of belt height, bandwidth, area and slope. Each index of the kneading and mixing instrument spectrum reflects the rheological characteristics and quality of the wheat dough.
The results of measurement of the main parameters measured by the kneading machine are shown in Table 4. As can be seen from the table: in 2016 and 2017, the dough peak time and the dough peak area of TL1Dx2m1/2/3 are obviously higher than those of a control, the fading resistance value is obviously lower than that of the control, and the peak height and the peak width are reduced compared with those of the control, so that TL1Dx2m1/2/3 reduces the extensibility of the dough, improves the elasticity and the kneading resistance of the dough, and further shows that the 1Dx2m1/2/3 gene has the functions of improving the gluten strength and improving the baking quality of the flour.
TABLE 4 measurement of various indexes of kneading and mixing apparatus
The SDS-sedimentation value (SDS-SV) and the glutenin Swelling Index (SIG) are comprehensive indexes for evaluating the quality and quantity of wheat gluten, and are highly positively correlated with the gluten strength. The peak time (MPT) and the peak area (MPI) in the parameters of the kneading and mixing device, the forming time and the Stable Time (ST) in the parameters of the flour quality device, the flour quality index (FQN) are positively correlated with the gluten quality, and the anti-fading value (RBD) in the parameters of the kneading and mixing device and the weakening Degree (DS) in the parameters of the flour quality device are negatively correlated with the gluten quality.
The results of the above measurements are combined to show that the SDS, SIG, MPT, MPI and FQN of the transgenic line of the mutant gene 1Dx2m1/2/3 are obviously improved compared with the control, the dough forming time and the stability time are obviously prolonged compared with the control, and the RBD and DS are obviously reduced compared with the control, which shows that the 1Dx2m1/2/3 transgenic line has better gluten quality and dough rheological property, thereby proving that the mutant gene 1Dx2m1/2/3 can obviously improve the processing quality of wheat flour.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the technical principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
<110> institute of genetics and developmental biology of Chinese academy of sciences
<120>1Dx2m1/2/3 protein and application of coding gene thereof in breeding wheat variety with improved processing quality
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atattttggg gaatacctgc actactaaaa aggtattacc caagtgtaac ttctccgcag 360
caggtttcat actatccagg ccaagcttct ccgcaacggc caggacaagg tcagcagcca 420
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gggcaacaac caggacaagg gcaacaaggt cagcagctcg gacaaggaca acaagggtac 840
tacccaactt ctctgcaaca gtcgggacaa gggcaaccag ggtactaccc aacttctctg 900
cagcagctag gacaagggca atcagggtac tacccaactt ctccgcagca accaggacaa 960
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Met Ala Lys Arg Leu Val Leu Phe Val Ala Val Val Val Ala Leu Val
1 5 10 15
Ala Leu Thr Val Ala Glu Gly Glu Ala Ser Glu Gln Leu Gln Cys Glu
20 25 30
Arg Glu Leu Gln Glu Leu Gln Glu Arg Glu Leu Lys Ala Cys Gln Gln
35 40 45
Val Met Asp Gln Gln Leu Arg Asp Ile Ser Pro Glu Cys His Pro Val
50 55 60
Val Val Ser Pro Val Ala Gly Gln Tyr Glu Gln Gln Ile Val Val Pro
65 70 75 80
Pro Lys Gly Gly Ser Phe Tyr Pro Gly Glu Thr Thr Pro Pro Gln Gln
85 90 95
Leu Gln Gln Arg Ile Phe Trp Gly Ile Pro Ala Leu Leu Lys Arg Tyr
100 105 110
Tyr Pro Ser Val Thr Ser Pro Gln Gln Val Ser Tyr Tyr Pro Gly Gln
115 120 125
Ala Ser Pro Gln Arg Pro Gly Gln Gly Gln Gln Pro Gly Gln Gly Gln
130 135 140
Gln Ser Gly Gln Gly Gln Gln Gly Tyr Tyr Pro Thr Ser Pro Gln Gln
145 150 155 160
Pro Gly Gln Trp Gln Gln Pro Glu Gln Gly Gln Pro Gly Tyr Tyr Pro
165 170 175
Thr Ser Pro Gln Gln Pro Gly Gln Leu Gln Gln Pro Ala Gln Gly Gln
180 185 190
Gln Pro Gly Gln Gly Gln Gln Gly Arg Gln Pro Gly Gln Gly Gln Pro
195 200 205
Gly Tyr Tyr Pro Thr Ser Ser Gln Leu Gln Pro Gly Gln Leu Gln Gln
210 215 220
Pro Ala Gln Gly Gln Gln Gly Gln Gln Pro Gly Gln Gly Gln Gln Gly
225 230 235 240
Gln Gln Pro Gly Gln Gly Gln Gln Pro Gly Gln Gly Gln Gln Gly Gln
245 250 255
Gln Pro Gly Gln Gly Gln Gln Pro Gly Gln Gly Gln Gln Gly Gln Gln
260 265 270
Leu Gly Gln Gly Gln Gln Gly Tyr Tyr Pro Thr Ser Leu Gln Gln Ser
275 280 285
Gly Gln Gly Gln Pro Gly Tyr Tyr Pro Thr Ser Leu Gln Gln Leu Gly
290 295 300
Gln Gly Gln Ser Gly Tyr Tyr Pro Thr Ser Pro Gln Gln Pro Gly Gln
305 310 315 320
Gly Gln Gln Pro Gly Gln Leu Gln Gln Pro Ala Gln Gly Gln Gln Pro
325 330 335
Glu Gln Gly Gln Gln Gly Gln Gln Pro Gly Gln Gly Gln Gln Gly Gln
340 345 350
Gln Pro Gly Gln Gly Gln Gln Pro Gly Gln Gly Gln Pro Gly Tyr Tyr
355 360 365
Pro Thr Ser Pro Gln Gln Ser Gly Gln Gly Gln Pro Gly Tyr Tyr Pro
370 375 380
Thr Ser Ser Gln Gln Pro Thr Gln Ser Gln Gln Pro Gly Gln Gly Gln
385 390 395 400
Gln Gly Gln Gln Val Gly Gln Gly Gln Gln Ala Gln Gln Pro Gly Gln
405 410 415
Gly Gln Gln Pro Gly Gln Gly Gln Pro Gly Tyr Tyr Pro Thr Ser Pro
420 425 430
Leu Gln Ser Gly Gln Gly Gln Pro Gly Tyr Tyr Leu Thr Ser Pro Gln
435 440 445
Gln Ser Gly Gln Gly Gln Gln Pro Gly Gln Leu Gln Gln Ser Ala Gln
450 455 460
Gly Gln Lys Gly Gln Gln Pro Gly Gln Gly Gln Gln Pro Gly Gln Gly
465 470 475 480
Gln Gln Gly Gln Gln Pro Gly Gln Gly Gln Gln Gly Gln Gln Pro Gly
485 490 495
Gln Gly Gln Pro Gly Tyr Tyr Pro Thr Ser Pro Gln Gln Ser Gly Gln
500 505 510
Gly Gln Gln Pro Gly Gln Trp Gln Gln Pro Gly Gln Gly Gln Pro Gly
515 520 525
Tyr Tyr Pro Thr Ser Pro Leu Gln Pro Gly Gln Gly Gln Pro Gly Tyr
530 535 540
Asp Pro Thr Ser Pro Gln Gln Pro Gly Gln Gly Gln Gln Pro Gly Gln
545 550 555 560
Leu Gln Gln Pro Ala Gln Gly Gln Gln Gly Gln Gln Leu Ala Gln Gly
565 570 575
Gln Gln Gly Gln Gln Pro Ala Gln Val Gln Gln Gly Gln Gln Pro Ala
580 585 590
Gln Gly Gln Gln Gly Gln Gln Leu Gly Gln Gly Gln Gln Gly Gln Gln
595 600 605
Pro Gly Gln Gly Gln Gln Pro Ala Gln Gly Gln Gln Gly Gln Gln Pro
610 615 620
Gly Gln Gly Gln Gln Gly Gln Gln Pro Gly Gln Gly Gln Gln Pro Gly
625 630 635 640
Gln Gly Gln Pro Trp Tyr Tyr Pro Thr Ser Pro Gln Glu Ser Gly Gln
645 650 655
Gly Gln Gln Pro Gly Gln Trp Gln Gln Pro Gly Gln Trp Gln Gln Pro
660 665 670
Gly Gln Gly Gln Pro Gly Tyr Tyr Leu Thr Ser Pro Leu Gln Leu Gly
675 680 685
Gln Gly Gln Gln Gly Tyr Tyr Pro Thr Ser Leu Gln Gln Pro Gly Gln
690 695 700
Gly Gln Gln Pro Gly Gln Trp Gln Gln Ser Gly Gln Gly Gln His Gly
705 710 715 720
Tyr Tyr Pro Thr Ser Pro Gln Leu Ser Gly Gln Gly Gln Arg Pro Gly
725 730 735
Gln Trp Leu Gln Pro Gly Gln Gly Gln Gln Gly Tyr Tyr Pro Thr Ser
740 745 750
Pro Gln Gln Ser Gly Gln Gly Gln Gln Leu Gly Gln Trp Leu Gln Pro
755 760 765
Gly Gln Gly Gln Gln Gly Tyr Tyr Pro Thr Ser Leu Gln Gln Thr Gly
770 775 780
Gln Gly Gln Gln Ser Gly Gln Gly Gln Gln Gly Tyr Tyr Ser Ser Tyr
785 790 795 800
His Val Ser Val Glu His Gln Ala Ala Ser Leu Lys Val Ala Lys Ala
805 810 815
Gln Gln Leu Ala Ala Gln Leu Pro Ala Met Cys Arg Leu Glu Gly Gly
820 825 830
Asp Ala Leu Ser Ala Ser Gln
835
<210>5
<211>43
<212>DNA
<213>Artificial Sequence
<400>5
gctgctgagc ttgttgccct tgtcctacct gctgacattg ttg 43
<210>6
<211>1240
<212>DNA
<213>Artificial Sequence
<400>6
atggctaagc ggttagtcct ctttgtggcg gtagtcgtcg ccctcgtggc tctcaccgtc 60
gctgaaggtg aggcctctga gcaactacag tgtgagcgcg agctccagga gctccaggag 120
cgcgagctca aggcatgcca gcaggtcatg gaccagcagc tccgagacat tagccccgag 180
tgccaccccg tcgtcgtcag cccggtcgcg ggacaatacg agcagcaaat cgtggtgccg 240
cccaagggcg gatctttcta ccccggcgag accacgccac cgcagcaact ccaacaacgt 300
atattttggg gaatacctgc actactaaaa aggtattacc caagtgtaac ttctccgcag 360
caggtttcat actatccagg ccaagcttct ccgcaacggc caggacaagg tcagcagcca 420
ggacaagggc aacaatcagg acaaggacaa caagggtact acccaacttc tccgcaacag 480
ccaggacaat ggcaacaacc ggaacaaggg caaccagggt actacccaac ttctccgcag 540
cagccaggac aattgcaaca accagcacaa gggcagcaac caggacaagg acaacaaggt 600
cggcagccag gacaagggca accagggtac tacccaactt cttcgcagct gcagccagga 660
caattgcaac aaccagcaca agggcaacaa gggcagcaac caggacaagg gcaacaaggt 720
caacagccag gacaagggca acaaccagga caaggacaac aaggtcaaca gccaggacaa 780
gggcaacaac caggacaagg gcaacaaggt cagcagctcg gacaaggaca acaagggtac 840
tacccaactt ctctgcaaca gtcgggacaa gggcaaccag ggtactaccc aacttctctg 900
cagcagctag gacaagggca atcagggtac tacccaactt ctccgcagca accaggacaa 960
gggcagcagc caggacaatt gcaacaacca gcacaagggc agcaaccaga acaagggcaa 1020
caaggtcagc agccaggaca agggcaacaa ggccagcagc caggacaagg gcagcaaccg 1080
ggacaagggc aaccagggta ctacccaact tctccgcagc agtcaggaca agggcaacca 1140
gggtactacc caacttcttc gcagcagcca acacaatcgc agcaaccagg acaagggcaa 1200
caatgtcagc aggtaggaca agggcaacaa gctcagcagc 1240
<210>7
<211>2523
<212>DNA
<213>Artificial Sequence
<400>7
atggctaagc ggttagtcct ctttgtggcg gtagtcgtcg ccctcgtggc tctcaccgtc 60
gctgaaggtg aggcctctga gcaactacag tgtgagcgcg agctccagga gctccaggag 120
cgcgagctca aggcatgcca gcaggtcatg gaccagcagc tccgagacat tagccccgag 180
tgccaccccg tcgtcgtcag cccggtcgcg ggacaatacg agcagcaaat cgtggtgccg 240
cccaagggcg gatctttcta ccccggcgag accacgccac cgcagcaact ccaacaacgt 300
atattttggg gaatacctgc actactaaaa aggtattacc caagtgtaac ttctccgcag 360
caggtttcat actatccagg ccaagcttct ccgcaacggc caggacaagg tcagcagcca 420
ggacaagggc aacaatcagg acaaggacaa caagggtact acccaacttc tccgcaacag 480
ccaggacaat ggcaacaacc ggaacaaggg caaccagggt actacccaac ttctccgcag 540
cagccaggac aattgcaaca accagcacaa gggcagcaac caggacaagg acaacaaggt 600
cggcagccag gacaagggca accagggtac tacccaactt cttcgcagct gcagccagga 660
caattgcaac aaccagcaca agggcaacaa gggcagcaac caggacaagg gcaacaaggt 720
caacagccag gacaagggca acaaccagga caaggacaac aaggtcaaca gccaggacaa 780
gggcaacaac caggacaagg gcaacaaggt cagcagctcg gacaaggaca acaagggtac 840
tacccaactt ctctgcaaca gtcgggacaa gggcaaccag ggtactaccc aacttctctg 900
cagcagctag gacaagggca atcagggtac tacccaactt ctccgcagca accaggacaa 960
gggcagcagc caggacaatt gcaacaacca gcacaagggc agcaaccaga acaagggcaa 1020
caaggtcagc agccaggaca agggcaacaa ggccagcagc caggacaagg gcagcaaccg 1080
ggacaagggc aaccagggta ctacccaact tctccgcagc agtcaggaca agggcaacca 1140
gggtactacc caacttcttc gcagcagcca acacaatcgc agcaaccagg acaagggcaa 1200
caatgtcagc aggtaggaca agggcaacaa gctcagcagc caggacaagg gcagcaaccg 1260
ggacaagggc agccagggta ctacccaact tctccgctgc agtcaggaca agggcaacca 1320
gggtactacc taacttctcc gcagcagtca ggacaagggc agcagccagg acaattgcaa 1380
caatcagcac aagggcaaaa aggacagcaa ccaggacaag gtcaacagcc agggcaaggg 1440
caacaaggtc agcagccagg acaagggcaa caaggtcagc aaccggggca agggcagcca 1500
gggtactacc caacttctcc gcagcaatca ggacaagggc aacagccagg acaatggcaa 1560
caaccaggac aagggcaacc aggatactac ccaacttctc cgttgcagcc aggacaaggg 1620
caaccagggt acgacccaac ttctccgcaa cagccaggac aagggcagca accaggacaa 1680
ttgcaacaac cagcacaagg gcaacaaggg cagcaactag cacaagggca acaagggcag 1740
caaccagcac aagtgcaaca agggcagcag ccagcacaag ggcaacaagg tcagcagcta 1800
ggacaagggc aacaaggtca gcagccagga caagggcagc aaccagcaca agggcaacaa 1860
ggtcagcagc caggacaagg gcaacaaggt cagcagccag gacaagggca gcaaccggga 1920
caagggcagc catggtacta cccaacttct ccgcaggagt caggacaagg gcaacagcca 1980
ggacaatggc aacaaccagg acaatggcaa caaccaggac aagggcaacc agggtactac 2040
ctaacttctc cgttgcagct aggacaaggg caacaagggt actacccaac ttctctgcaa 2100
caaccaggac aagggcagca accaggacaa tggcaacaat cgggacaagg gcaacatggg 2160
tactacccaa cttctccgca gctgtcagga caagggcaac ggccaggaca atggctgcaa 2220
ccaggacaag ggcaacaagg gtactaccca acttctccgc aacagtcagg acaagggcaa 2280
caactaggac aatggctgca accaggacaa gggcaacaag ggtactaccc aacttctctg 2340
caacagacag gacaagggca gcaatcagga caagggcaac aaggctacta cagctcatac 2400
catgttagcg tggagcacca ggcggccagc ctaaaggtgg caaaggcgca gcagctcgcg 2460
gcacagctgc cggcaatgtg ccggctggag ggcggcgacg cattgtcggc cagccagtga 2520
tag 2523
<210>8
<211>28
<212>DNA
<213>Artificial Sequence
<400>8
catgggtact gcccaacttc tccgcagc 28
<210>9
<211>369
<212>DNA
<213>Artificial Sequence
<400>9
catgggtact gcccaacttc tccgcagctg tcaggacaag ggcaacggcc aggacaatgg 60
ctgcaaccag gacaagggca acaagggtac tacccaactt ctccgcaaca gtcaggacaa 120
gggcaacaac taggacaatg gctgcaacca ggacaagggc aacaagggta ctacccaact 180
tctctgcaac agacaggaca agggcagcaa tcaggacaag ggcaacaagg ctactacagc 240
tcataccatg ttagcgtgga gcaccaggcg gccagcctaa aggtggcaaa ggcgcagcag 300
ctcgcggcac agctgccggc aatgtgccgg ctggagggcg gcgacgcatt gtcggccagc 360
cagtgatag 369
<210>10
<211>2523
<212>DNA
<213>Artificial Sequence
<400>10
atggctaagc ggttagtcct ctttgtggcg gtagtcgtcg ccctcgtggc tctcaccgtc 60
gctgaaggtg aggcctctga gcaactacag tgtgagcgcg agctccagga gctccaggag 120
cgcgagctca aggcatgcca gcaggtcatg gaccagcagc tccgagacat tagccccgag 180
tgccaccccg tcgtcgtcag cccggtcgcg ggacaatacg agcagcaaat cgtggtgccg 240
cccaagggcg gatctttcta ccccggcgag accacgccac cgcagcaact ccaacaacgt 300
atattttggg gaatacctgc actactaaaa aggtattacc caagtgtaac ttctccgcag 360
caggtttcat actatccagg ccaagcttct ccgcaacggc caggacaagg tcagcagcca 420
ggacaagggc aacaatcagg acaaggacaa caagggtact acccaacttc tccgcaacag 480
ccaggacaat ggcaacaacc ggaacaaggg caaccagggt actacccaac ttctccgcag 540
cagccaggac aattgcaaca accagcacaa gggcagcaac caggacaagg acaacaaggt 600
cggcagccag gacaagggca accagggtac tacccaactt cttcgcagct gcagccagga 660
caattgcaac aaccagcaca agggcaacaa gggcagcaac caggacaagg gcaacaaggt 720
caacagccag gacaagggca acaaccagga caaggacaac aaggtcaaca gccaggacaa 780
gggcaacaac caggacaagg gcaacaaggt cagcagctcg gacaaggaca acaagggtac 840
tacccaactt ctctgcaaca gtcgggacaa gggcaaccag ggtactaccc aacttctctg 900
cagcagctag gacaagggca atcagggtac tacccaactt ctccgcagca accaggacaa 960
gggcagcagc caggacaatt gcaacaacca gcacaagggc agcaaccaga acaagggcaa 1020
caaggtcagc agccaggaca agggcaacaa ggccagcagc caggacaagg gcagcaaccg 1080
ggacaagggc aaccagggta ctacccaact tctccgcagc agtcaggaca agggcaacca 1140
gggtactacc caacttcttc gcagcagcca acacaatcgc agcaaccagg acaagggcaa 1200
caatgtcagc aggtaggaca agggcaacaa gctcagcagc caggacaagg gcagcaaccg 1260
ggacaagggc agccagggta ctacccaact tctccgctgc agtcaggaca agggcaacca 1320
gggtactacc taacttctcc gcagcagtca ggacaagggc agcagccagg acaattgcaa 1380
caatcagcac aagggcaaaa aggacagcaa ccaggacaag gtcaacagcc agggcaaggg 1440
caacaaggtc agcagccagg acaagggcaa caaggtcagc aaccggggca agggcagcca 1500
gggtactacc caacttctcc gcagcaatca ggacaagggc aacagccagg acaatggcaa 1560
caaccaggac aagggcaacc aggatactac ccaacttctc cgttgcagcc aggacaaggg 1620
caaccagggt acgacccaac ttctccgcaa cagccaggac aagggcagca accaggacaa 1680
ttgcaacaac cagcacaagg gcaacaaggg cagcaactag cacaagggca acaagggcag 1740
caaccagcac aagtgcaaca agggcagcag ccagcacaag ggcaacaagg tcagcagcta 1800
ggacaagggc aacaaggtca gcagccagga caagggcagc aaccagcaca agggcaacaa 1860
ggtcagcagc caggacaagg gcaacaaggt cagcagccag gacaagggca gcaaccggga 1920
caagggcagc catggtacta cccaacttct ccgcaggagt caggacaagg gcaacagcca 1980
ggacaatggc aacaaccagg acaatggcaa caaccaggac aagggcaacc agggtactac 2040
ctaacttctc cgttgcagct aggacaaggg caacaagggt actacccaac ttctctgcaa 2100
caaccaggac aagggcagca accaggacaa tggcaacaat cgggacaagg gcaacatggg 2160
tactgcccaa cttctccgca gctgtcagga caagggcaac ggccaggaca atggctgcaa 2220
ccaggacaag ggcaacaagg gtactaccca acttctccgc aacagtcagg acaagggcaa 2280
caactaggac aatggctgca accaggacaa gggcaacaag ggtactaccc aacttctctg 2340
caacagacag gacaagggca gcaatcagga caagggcaac aaggctacta cagctcatac 2400
catgttagcg tggagcacca ggcggccagc ctaaaggtgg caaaggcgca gcagctcgcg 2460
gcacagctgc cggcaatgtg ccggctggag ggcggcgacg cattgtcggc cagccagtga 2520
tag 2523
<210>11
<211>29
<212>DNA
<213>Artificial Sequence
<400>11
cctgctgcgg acaagttaca cttgggtaa 29
<210>12
<211>364
<212>DNA
<213>Artificial Sequence
<400>12
atggctaagc ggttagtcct ctttgtggcg gtagtcgtcg ccctcgtggc tctcaccgtc 60
gctgaaggtg aggcctctga gcaactacag tgtgagcgcg agctccagga gctccaggag 120
cgcgagctca aggcatgcca gcaggtcatg gaccagcagc tccgagacat tagccccgag 180
tgccaccccg tcgtcgtcag cccggtcgcg ggacaatacg agcagcaaat cgtggtgccg 240
cccaagggcg gatctttcta ccccggcgag accacgccac cgcagcaact ccaacaacgt 300
atattttggg gaatacctgc actactaaaa aggtattacc caagtgtaac ttgtccgcag 360
cagg 364
<210>13
<211>2523
<212>DNA
<213>Artificial Sequence
<400>13
atggctaagc ggttagtcct ctttgtggcg gtagtcgtcg ccctcgtggc tctcaccgtc 60
gctgaaggtg aggcctctga gcaactacag tgtgagcgcg agctccagga gctccaggag 120
cgcgagctca aggcatgcca gcaggtcatg gaccagcagc tccgagacat tagccccgag 180
tgccaccccg tcgtcgtcag cccggtcgcg ggacaatacg agcagcaaat cgtggtgccg 240
cccaagggcg gatctttcta ccccggcgag accacgccac cgcagcaact ccaacaacgt 300
atattttggg gaatacctgc actactaaaa aggtattacc caagtgtaac ttgtccgcag 360
caggtttcat actatccagg ccaagcttct ccgcaacggc caggacaagg tcagcagcca 420
ggacaagggc aacaatcagg acaaggacaa caagggtact acccaacttc tccgcaacag 480
ccaggacaat ggcaacaacc ggaacaaggg caaccagggt actacccaac ttctccgcag 540
cagccaggac aattgcaaca accagcacaa gggcagcaac caggacaagg acaacaaggt 600
cggcagccag gacaagggca accagggtac tacccaactt cttcgcagct gcagccagga 660
caattgcaac aaccagcaca agggcaacaa gggcagcaac caggacaagg gcaacaaggt 720
caacagccag gacaagggca acaaccagga caaggacaac aaggtcaaca gccaggacaa 780
gggcaacaac caggacaagg gcaacaaggt cagcagctcg gacaaggaca acaagggtac 840
tacccaactt ctctgcaaca gtcgggacaa gggcaaccag ggtactaccc aacttctctg 900
cagcagctag gacaagggca atcagggtac tacccaactt ctccgcagca accaggacaa 960
gggcagcagc caggacaatt gcaacaacca gcacaagggc agcaaccaga acaagggcaa 1020
caaggtcagc agccaggaca agggcaacaa ggccagcagc caggacaagg gcagcaaccg 1080
ggacaagggc aaccagggta ctacccaact tctccgcagc agtcaggaca agggcaacca 1140
gggtactacc caacttcttc gcagcagcca acacaatcgc agcaaccagg acaagggcaa 1200
caatgtcagc aggtaggaca agggcaacaa gctcagcagc caggacaagg gcagcaaccg 1260
ggacaagggc agccagggta ctacccaact tctccgctgc agtcaggaca agggcaacca 1320
gggtactacc taacttctcc gcagcagtca ggacaagggc agcagccagg acaattgcaa 1380
caatcagcac aagggcaaaa aggacagcaa ccaggacaag gtcaacagcc agggcaaggg 1440
caacaaggtc agcagccagg acaagggcaa caaggtcagc aaccggggca agggcagcca 1500
gggtactacc caacttctcc gcagcaatca ggacaagggc aacagccagg acaatggcaa 1560
caaccaggac aagggcaacc aggatactac ccaacttctc cgttgcagcc aggacaaggg 1620
caaccagggt acgacccaac ttctccgcaa cagccaggac aagggcagca accaggacaa 1680
ttgcaacaac cagcacaagg gcaacaaggg cagcaactag cacaagggca acaagggcag 1740
caaccagcac aagtgcaaca agggcagcag ccagcacaag ggcaacaagg tcagcagcta 1800
ggacaagggc aacaaggtca gcagccagga caagggcagc aaccagcaca agggcaacaa 1860
ggtcagcagc caggacaagg gcaacaaggt cagcagccag gacaagggca gcaaccggga 1920
caagggcagc catggtacta cccaacttct ccgcaggagt caggacaagg gcaacagcca 1980
ggacaatggc aacaaccagg acaatggcaa caaccaggac aagggcaacc agggtactac 2040
ctaacttctc cgttgcagct aggacaaggg caacaagggt actacccaac ttctctgcaa 2100
caaccaggac aagggcagca accaggacaa tggcaacaat cgggacaagg gcaacatggg 2160
tactgcccaa cttctccgca gctgtcagga caagggcaac ggccaggaca atggctgcaa 2220
ccaggacaag ggcaacaagg gtactaccca acttctccgc aacagtcagg acaagggcaa 2280
caactaggac aatggctgca accaggacaa gggcaacaag ggtactaccc aacttctctg 2340
caacagacag gacaagggca gcaatcagga caagggcaac aaggctacta cagctcatac 2400
catgttagcg tggagcacca ggcggccagc ctaaaggtgg caaaggcgca gcagctcgcg 2460
gcacagctgc cggcaatgtg ccggctggag ggcggcgacg cattgtcggc cagccagtga 2520
tag 2523
<210>14
<211>839
<212>PRT
<213>Artificial Sequence
<400>14
Met Ala Lys Arg Leu Val Leu Phe Val Ala Val Val Val Ala Leu Val
1 5 10 15
Ala Leu Thr Val Ala Glu Gly Glu Ala Ser Glu Gln Leu Gln Cys Glu
20 25 30
Arg Glu Leu Gln Glu Leu Gln Glu Arg Glu Leu Lys Ala Cys Gln Gln
35 40 45
Val Met Asp Gln Gln Leu Arg Asp Ile Ser Pro Glu Cys His Pro Val
50 55 60
Val Val Ser Pro Val Ala Gly Gln Tyr Glu Gln Gln Ile Val Val Pro
65 70 75 80
Pro Lys Gly Gly Ser Phe Tyr Pro Gly Glu Thr Thr Pro Pro Gln Gln
85 90 95
Leu Gln Gln Arg Ile Phe Trp Gly Ile Pro Ala Leu Leu Lys Arg Tyr
100 105 110
Tyr Pro Ser Val Thr Cys Pro Gln Gln Val Ser Tyr Tyr Pro Gly Gln
115 120 125
Ala Ser Pro Gln Arg Pro Gly Gln Gly Gln Gln Pro Gly Gln Gly Gln
130 135 140
Gln Ser Gly Gln Gly Gln Gln Gly Tyr Tyr Pro Thr Ser Pro Gln Gln
145 150 155 160
Pro Gly Gln Trp Gln Gln Pro Glu Gln Gly Gln Pro Gly Tyr Tyr Pro
165 170 175
Thr Ser Pro Gln Gln Pro Gly Gln Leu Gln Gln Pro Ala Gln Gly Gln
180 185 190
Gln Pro Gly Gln Gly Gln Gln Gly Arg Gln Pro Gly Gln Gly Gln Pro
195 200 205
Gly Tyr Tyr Pro Thr Ser Ser Gln Leu Gln Pro Gly Gln Leu Gln Gln
210 215 220
Pro Ala Gln Gly Gln Gln Gly Gln Gln Pro Gly Gln Gly Gln Gln Gly
225 230 235 240
Gln Gln Pro Gly Gln Gly Gln Gln Pro Gly Gln Gly Gln Gln Gly Gln
245 250 255
Gln Pro Gly Gln Gly Gln Gln Pro Gly Gln Gly Gln Gln Gly Gln Gln
260 265 270
Leu Gly Gln Gly Gln Gln Gly Tyr Tyr Pro Thr Ser Leu Gln Gln Ser
275 280 285
Gly Gln Gly Gln Pro Gly Tyr Tyr Pro Thr Ser Leu Gln Gln Leu Gly
290 295 300
Gln Gly Gln Ser Gly Tyr Tyr Pro Thr Ser Pro Gln Gln Pro Gly Gln
305 310 315 320
Gly Gln Gln Pro Gly Gln Leu Gln Gln Pro Ala Gln Gly Gln Gln Pro
325 330 335
Glu Gln Gly Gln Gln Gly Gln Gln Pro Gly Gln Gly Gln Gln Gly Gln
340 345 350
Gln Pro Gly Gln Gly Gln Gln Pro Gly Gln Gly Gln Pro Gly Tyr Tyr
355 360 365
Pro Thr Ser Pro Gln Gln Ser Gly Gln Gly Gln Pro Gly Tyr Tyr Pro
370 375 380
Thr Ser Ser Gln Gln Pro Thr Gln Ser Gln Gln Pro Gly Gln Gly Gln
385 390 395 400
Gln Cys Gln Gln Val Gly Gln Gly Gln Gln Ala Gln Gln Pro Gly Gln
405 410 415
Gly Gln Gln Pro Gly Gln Gly Gln Pro Gly Tyr Tyr Pro Thr Ser Pro
420 425 430
Leu Gln Ser Gly Gln Gly Gln Pro Gly Tyr Tyr Leu Thr Ser Pro Gln
435 440 445
Gln Ser Gly Gln Gly Gln Gln Pro Gly Gln Leu Gln Gln Ser Ala Gln
450 455 460
Gly Gln Lys Gly Gln Gln Pro Gly Gln Gly Gln Gln Pro Gly Gln Gly
465 470 475 480
Gln Gln Gly Gln Gln Pro Gly Gln Gly Gln Gln Gly Gln Gln Pro Gly
485 490 495
Gln Gly Gln Pro Gly Tyr Tyr Pro Thr Ser Pro Gln Gln Ser Gly Gln
500 505 510
Gly Gln Gln Pro Gly Gln Trp Gln Gln Pro Gly Gln Gly Gln Pro Gly
515 520 525
Tyr Tyr Pro Thr Ser Pro Leu Gln Pro Gly Gln Gly Gln Pro Gly Tyr
530 535 540
Asp Pro Thr Ser Pro Gln Gln Pro Gly Gln Gly Gln Gln Pro Gly Gln
545 550 555 560
Leu Gln Gln Pro Ala Gln Gly Gln Gln Gly Gln Gln Leu Ala Gln Gly
565 570 575
Gln Gln Gly Gln Gln Pro Ala Gln Val Gln Gln Gly Gln Gln Pro Ala
580 585 590
Gln Gly Gln Gln Gly Gln Gln Leu Gly Gln Gly Gln Gln Gly Gln Gln
595 600 605
Pro Gly Gln Gly Gln Gln Pro Ala Gln Gly Gln Gln Gly Gln Gln Pro
610 615 620
Gly Gln Gly Gln Gln Gly Gln Gln Pro Gly Gln Gly Gln Gln Pro Gly
625 630 635 640
Gln Gly Gln Pro Trp Tyr Tyr Pro Thr Ser Pro Gln Glu Ser Gly Gln
645 650 655
Gly Gln Gln Pro Gly Gln Trp Gln Gln Pro Gly Gln Trp Gln Gln Pro
660 665 670
Gly Gln Gly Gln Pro Gly Tyr Tyr Leu Thr Ser Pro Leu Gln Leu Gly
675 680 685
Gln Gly Gln Gln Gly Tyr Tyr Pro Thr Ser Leu Gln Gln Pro Gly Gln
690 695 700
Gly Gln Gln Pro Gly Gln Trp Gln Gln Ser Gly Gln Gly Gln His Gly
705 710 715 720
Tyr Cys Pro Thr Ser Pro Gln Leu Ser Gly Gln Gly Gln Arg Pro Gly
725 730 735
Gln Trp Leu Gln Pro Gly Gln Gly Gln Gln Gly Tyr Tyr Pro Thr Ser
740 745 750
Pro Gln Gln Ser Gly Gln Gly Gln Gln Leu Gly Gln Trp Leu Gln Pro
755 760 765
Gly Gln Gly Gln Gln Gly Tyr Tyr Pro Thr Ser Leu Gln Gln Thr Gly
770 775 780
Gln Gly Gln Gln Ser Gly Gln Gly Gln Gln Gly Tyr Tyr Ser Ser Tyr
785 790 795 800
His Val Ser Val Glu His Gln Ala Ala Ser Leu Lys Val Ala Lys Ala
805 810 815
Gln Gln Leu Ala Ala Gln Leu Pro Ala Met Cys Arg Leu Glu Gly Gly
820 825 830
Asp Ala Leu Ser Ala Ser Gln
835
<210>15
<211>33
<212>DNA
<213>Artificial Sequence
<400>15
aactgcagag ggaaagacaa tggacatgca aag 33
<210>16
<211>33
<212>DNA
<213>Artificial Sequence
<400>16
cgggatccct cggtgaactg tcagtgaatt gat 33
<210>17
<211>1147
<212>DNA
<213>Artificial Sequence
<400>17
agggaaagac aatggacatg caaagaggta ggggcaggga agaaacactt ggagatcata 60
gaagaacata agaggttaaa cataggagca gtggcggagc ttgggctgat tttatggagg 120
ggcaaatggg ctggaggggc aagaaatatg agtttgggct gattttaact gggcatatgg 180
gctgaatact aggggatata tgctagtttt ctcatgggct gggggggcaa tggcccaggt 240
tgctctccac taagctccgc cactgcatag gagggcataa tggacaatta aatctacatt 300
aattgaactc atttgggaag taaacaaaat ccatattctg gtgtaaatca aactatttga 360
tgcggattta ctaagatcct atgttaattt tagacatgac tggccaaagg tttcagttag 420
ttcatttgtc acggaaaggt gttttcataa gtccaaaact ctaccaactt ttttgcacgt 480
catagcatag atagatgttg tgagtcattg gatagatatt gtgagtcagc atggatttgt 540
gttgcctgga aatccaacta aatgacaagc aacaaaacct gaaatgggct ttaggagaga 600
tggtttatca atttacatgt tccatgcagg ctaccttcca ctactcgaca tggttagaag 660
ttttgagtgc cgcatatttg cggaagcaat ggcactactc gacatggtta gaagttttga 720
gtgccgcata tttgcggaag caatggctaa cagatacata ttctgccaaa ccccaagaag 780
gataatcact cctcttagat aaaaagaaca gaccaatgta caaacatcca cacttctgca 840
aacaatacac cagaactagg attaagccca ttacgtggct ttagcagacc gtccaaaaat 900
ctgttttgca agcaccaatt gctccttact tatccagctt cttttgtgtt ggcaaactgc 960
ccttttccaa ccgattttgt tcttctcacg ctttcttcat aggctaaact aacctcggcg 1020
tgcacacaac catgtcctga accttcacct cgtccctata aaagcccatc caaccttcac 1080
aatctcatca tcacccacaa caccgagcac cccaatctac agatcaattc actgacagtt 1140
caccgag 1147
<210>18
<211>4691
<212>DNA
<213>Artificial Sequence
<400>18
gaaccctgca gtgcagcgtg acccggtcgt gcccctctct agagataatg agcattgcat 60
gtctaagtta taaaaaatta ccacatattt tttttgtcac acttgtttga agtgcagttt 120
atctatcttt atacatatat ttaaacttta ctctacgaat aatataatct atagtactac 180
aataatatca gtgttttaga gaatcatata aatgaacagt tagacatggt ctaaaggaca 240
attgagtatt ttgacaacag gactctacag ttttatcttt ttagtgtgca tgtgttctcc 300
tttttttttg caaatagctt cacctatata atacttcatc cattttatta gtacatccat 360
ttagggttta gggttaatgg tttttataga ctaatttttt tagtacatct attttattct 420
attttagcct ctaaattaag aaaactaaaa ctctatttta gtttttttat ttaataattt 480
agatataaaa tagaataaaa taaagtgact aaaaattaaa caaataccct ttaagaaatt 540
aaaaaaacta aggaaacatt tttcttgttt cgagtagata atgccagcct gttaaacgcc 600
gtcgacgagt ctaacggaca ccaaccagcg aaccagcagc gtcgcgtcgg gccaagcgaa 660
gcagacggca cggcatctct gtcgctgcct ctggacccct ctcgagagtt ccgctccacc 720
gttggacttg ctccgctgtc ggcatccaga aattgcgtgg cggagcggca gacgtgagcc 780
ggcacggcag gcggcctcct cctcctctca cggcacggca gctacggggg attcctttcc 840
caccgctcct tcgctttccc ttcctcgccc gccgtaataa atagacaccc cctccacacc 900
ctctttcccc aacctcgtgt tgttcggagc gcacacacac acaaccagat ctcccccaaa 960
tccacccgtc ggcacctccg cttcaaggta cgccgctcgt cctccccccc cccccctctc 1020
taccttctct agatcggcgt tccggtccat ggttagggcc cggtagttct acttctgttc 1080
atgtttgtgt tagatccgtg tttgtgttag atccgtgctg ctagcgttcg tacacggatg 1140
cgacctgtac gtcagacacg ttctgattgc taacttgcca gtgtttctct ttggggaatc 1200
ctgggatggc tctagccgtt ccgcagacgg gatcgatttc atgatttttt ttgtttcgtt 1260
gcatagggtt tggtttgccc ttttccttta tttcaatata tgccgtgcac ttgtttgtcg 1320
ggtcatcttt tcatgctttt ttttgtcttg gttgtgatga tgtggtctgg ttgggcggtc 1380
gttctagatc ggagtagaat tctgtttcaa actacctggt ggatttatta attttggatc 1440
tgtatgtgtg tgccatacat attcatagtt acgaattgaa gatgatggat ggaaatatcg 1500
atctaggata ggtatacatg ttgatgcggg ttttactgat gcatatacag agatgctttt 1560
tgttcgcttg gttgtgatga tgtggtgtgg ttgggcggtc gttcattcgt tctagatcgg 1620
agtagaatac tgtttcaaac tacctggtgt atttattaat tttggaactg tatgtgtgtg 1680
tcatacatct tcatagttac gagtttaaga tggatggaaa tatcgatcta ggataggtat 1740
acatgttgat gtgggtttta ctgatgcata tacatgatgg catatgcagc atctattcat 1800
atgctctaac cttgagtacc tatctattat aataaacaag tatgttttat aattattttg 1860
atcttgatat acttggatga tggcatatgc agcagctata tgtggatttt tttagccctg 1920
ccttcatacg ctatttattt gcttggtact gtttcttttg tcgatgctca ccctgttgtt 1980
tggtgttact tctgcaggtc gactctagag gatccccggg taccgagctc gaatttcccc 2040
gatcgttcaa acatttggca ataaagtttc ttaagattga atcctgttgc cggtcttgcg 2100
atgattatca tataatttct gttgaattac gttaagcatg taataattaa catgtaatgc 2160
atgacgttat ttatgagatg ggtttttatg attagagtcc cgcaattata catttaatac 2220
gcgatagaaa acaaaatata gcgcgcaaac taggataaat tatcgcgcgc ggtgtcatct 2280
atgttactag atcgggaatt catcgatgat atcagatctg ccggtctccc tatagtgagt 2340
cgtattaatt tcgataagcc aggttaacct gcattaatga atcggccaac gcgcggggag 2400
aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt 2460
cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga 2520
atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg 2580
taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa 2640
aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt 2700
tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct 2760
gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct 2820
cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc 2880
cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt 2940
atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc 3000
tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat 3060
ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa 3120
acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa 3180
aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga 3240
aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct 3300
tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga 3360
cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc 3420
catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg 3480
ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat 3540
aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat 3600
ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg 3660
caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc 3720
attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa 3780
agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc 3840
actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt 3900
ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag 3960
ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt 4020
gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag 4080
atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac 4140
cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc 4200
gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca 4260
gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg 4320
ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc taagaaacca ttattatcat 4380
gacattaacc tataaaaata ggcgtatcac gaggcccttt cgtctcgcgc gtttcggtga 4440
tgacggtgaa aacctctgac acatgcagct cccggagacg gtcacagctt gtctgtaagc 4500
ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg 4560
ctggcttaac tatgcggcat cagagcagat tgtactgaga gtgcaccata tggacatatt 4620
gtcgttagaa cgcggctaca attaatacat aaccttatgt atcatacaca tacgatttag 4680
gtgacactat a 4691
<210>19
<211>3676
<212>DNA
<213>Artificial Sequence
<400>19
agggaaagac aatggacatg caaagaggta ggggcaggga agaaacactt ggagatcata 60
gaagaacata agaggttaaa cataggagca gtggcggagc ttgggctgat tttatggagg 120
ggcaaatggg ctggaggggc aagaaatatg agtttgggct gattttaact gggcatatgg 180
gctgaatact aggggatata tgctagtttt ctcatgggct gggggggcaa tggcccaggt 240
tgctctccac taagctccgc cactgcatag gagggcataa tggacaatta aatctacatt 300
aattgaactc atttgggaag taaacaaaat ccatattctg gtgtaaatca aactatttga 360
tgcggattta ctaagatcct atgttaattt tagacatgac tggccaaagg tttcagttag 420
ttcatttgtc acggaaaggt gttttcataa gtccaaaact ctaccaactt ttttgcacgt 480
catagcatag atagatgttg tgagtcattg gatagatatt gtgagtcagc atggatttgt 540
gttgcctgga aatccaacta aatgacaagc aacaaaacct gaaatgggct ttaggagaga 600
tggtttatca atttacatgt tccatgcagg ctaccttcca ctactcgaca tggttagaag 660
ttttgagtgc cgcatatttg cggaagcaat ggcactactc gacatggtta gaagttttga 720
gtgccgcata tttgcggaag caatggctaa cagatacata ttctgccaaa ccccaagaag 780
gataatcact cctcttagat aaaaagaaca gaccaatgta caaacatcca cacttctgca 840
aacaatacac cagaactagg attaagccca ttacgtggct ttagcagacc gtccaaaaat 900
ctgttttgca agcaccaatt gctccttact tatccagctt cttttgtgtt ggcaaactgc 960
ccttttccaa ccgattttgt tcttctcacg ctttcttcat aggctaaact aacctcggcg 1020
tgcacacaac catgtcctga accttcacct cgtccctata aaagcccatc caaccttcac 1080
aatctcatca tcacccacaa caccgagcac cccaatctac agatcaattc actgacagtt 1140
caccgaggga tccatggcta agcggttagt cctctttgtg gcggtagtcg tcgccctcgt 1200
ggctctcacc gtcgctgaag gtgaggcctc tgagcaacta cagtgtgagc gcgagctcca 1260
ggagctccag gagcgcgagc tcaaggcatg ccagcaggtc atggaccagc agctccgaga 1320
cattagcccc gagtgccacc ccgtcgtcgt cagcccggtc gcgggacaat acgagcagca 1380
aatcgtggtg ccgcccaagg gcggatcttt ctaccccggc gagaccacgc caccgcagca 1440
actccaacaa cgtatatttt ggggaatacc tgcactacta aaaaggtatt acccaagtgt 1500
aacttgtccg cagcaggttt catactatcc aggccaagct tctccgcaac ggccaggaca 1560
aggtcagcag ccaggacaag ggcaacaatc aggacaagga caacaagggt actacccaac 1620
ttctccgcaa cagccaggac aatggcaaca accggaacaa gggcaaccag ggtactaccc 1680
aacttctccg cagcagccag gacaattgca acaaccagca caagggcagc aaccaggaca 1740
aggacaacaa ggtcggcagc caggacaagg gcaaccaggg tactacccaa cttcttcgca 1800
gctgcagcca ggacaattgc aacaaccagc acaagggcaa caagggcagc aaccaggaca 1860
agggcaacaa ggtcaacagc caggacaagg gcaacaacca ggacaaggac aacaaggtca 1920
acagccagga caagggcaac aaccaggaca agggcaacaa ggtcagcagc tcggacaagg 1980
acaacaaggg tactacccaa cttctctgca acagtcggga caagggcaac cagggtacta 2040
cccaacttct ctgcagcagc taggacaagg gcaatcaggg tactacccaa cttctccgca 2100
gcaaccagga caagggcagc agccaggaca attgcaacaa ccagcacaag ggcagcaacc 2160
agaacaaggg caacaaggtc agcagccagg acaagggcaa caaggccagc agccaggaca 2220
agggcagcaa ccgggacaag ggcaaccagg gtactaccca acttctccgc agcagtcagg 2280
acaagggcaa ccagggtact acccaacttc ttcgcagcag ccaacacaat cgcagcaacc 2340
aggacaaggg caacaatgtc agcaggtagg acaagggcaa caagctcagc agccaggaca 2400
agggcagcaa ccgggacaag ggcagccagg gtactaccca acttctccgc tgcagtcagg 2460
acaagggcaa ccagggtact acctaacttc tccgcagcag tcaggacaag ggcagcagcc 2520
aggacaattg caacaatcag cacaagggca aaaaggacag caaccaggac aaggtcaaca 2580
gccagggcaa gggcaacaag gtcagcagcc aggacaaggg caacaaggtc agcaaccggg 2640
gcaagggcag ccagggtact acccaacttc tccgcagcaa tcaggacaag ggcaacagcc 2700
aggacaatgg caacaaccag gacaagggca accaggatac tacccaactt ctccgttgca 2760
gccaggacaa gggcaaccag ggtacgaccc aacttctccg caacagccag gacaagggca 2820
gcaaccagga caattgcaac aaccagcaca agggcaacaa gggcagcaac tagcacaagg 2880
gcaacaaggg cagcaaccag cacaagtgca acaagggcag cagccagcac aagggcaaca 2940
aggtcagcag ctaggacaag ggcaacaagg tcagcagcca ggacaagggc agcaaccagc 3000
acaagggcaa caaggtcagc agccaggaca agggcaacaa ggtcagcagc caggacaagg 3060
gcagcaaccg ggacaagggc agccatggta ctacccaact tctccgcagg agtcaggaca 3120
agggcaacag ccaggacaat ggcaacaacc aggacaatgg caacaaccag gacaagggca 3180
accagggtac tacctaactt ctccgttgca gctaggacaa gggcaacaag ggtactaccc 3240
aacttctctg caacaaccag gacaagggca gcaaccagga caatggcaac aatcgggaca 3300
agggcaacat gggtactgcc caacttctcc gcagctgtca ggacaagggc aacggccagg 3360
acaatggctg caaccaggac aagggcaaca agggtactac ccaacttctc cgcaacagtc 3420
aggacaaggg caacaactag gacaatggct gcaaccagga caagggcaac aagggtacta 3480
cccaacttct ctgcaacaga caggacaagg gcagcaatca ggacaagggc aacaaggcta 3540
ctacagctca taccatgtta gcgtggagca ccaggcggcc agcctaaagg tggcaaaggc 3600
gcagcagctc gcggcacagc tgccggcaat gtgccggctg gagggcggcg acgcattgtc 3660
ggccagccag tgatag 3676
<210>20
<211>20
<212>DNA
<213>Artificial Sequence
<400>20
<210>21
<211>20
<212>DNA
<213>Artificial Sequence
<400>21
Claims (9)
1.1Dx2m1/2/3 protein, which is a protein represented by the following a) or b):
a) the amino acid sequence is a protein shown in a sequence 14;
b) and (b) a fusion protein obtained by connecting a tag to the N-terminal and/or C-terminal of the protein shown in the sequence 14.
2. The biomaterial related to the 1Dx2m1/2/3 protein of claim 1, which is any one of the following a1) to a 8):
A1) a nucleic acid molecule encoding the 1Dx2m1/2/3 protein of claim 1;
A2) an expression cassette comprising the nucleic acid molecule of a 1);
A3) a recombinant vector comprising the nucleic acid molecule of a 1);
A4) a recombinant vector comprising the expression cassette of a 2);
A5) a recombinant microorganism comprising the nucleic acid molecule of a 1);
A6) a recombinant microorganism comprising the expression cassette of a 2);
A7) a recombinant microorganism comprising a3) said recombinant vector;
A8) a recombinant microorganism comprising the recombinant vector of a 4).
3. The related biological material according to claim 2, wherein: A1) the nucleic acid molecule is a gene shown in the following 1) or 2) or 3):
1) the coding sequence is a cDNA molecule or a DNA molecule shown in sequence 13;
2) a cDNA molecule or a genomic DNA molecule having 95% or more identity to the nucleotide sequence defined in 1) and encoding the 1Dx2m1/2/3 protein of claim 1;
3) a cDNA molecule or a genomic DNA molecule which hybridizes under stringent conditions to the nucleotide sequence defined in 1) or 2) and encodes the 1Dx2m1/2/3 protein of claim 1.
4. Use of the protein of claim 1 or the related biomaterial of claim 2 or 3) in any one of the following B1) -B10):
B1) regulating and controlling the processing quality of the wheat flour;
B2) regulating and controlling the baking quality of the wheat flour;
B3) regulating and controlling the gluten property and/or gluten strength and/or gluten quality of the wheat flour;
B4) regulating the dough rheological property and/or the extensibility and/or the stretch resistance and/or the kneading resistance of the wheat flour;
B5) regulating and controlling the SDS-sedimentation value of the wheat flour;
B6) regulating and controlling the glutenin swelling index of the wheat flour;
B7) regulating and controlling the dough forming time and/or dough stabilizing time and/or weakening degree in flour quality instrument detection parameters of the wheat flour;
B8) regulating and controlling peak time and/or peak area and/or anti-fading value in the parameters of the kneading and mixing instrument of the wheat flour;
B9) cultivating transgenic wheat with improved flour processing quality and/or baking quality and/or gluten quality;
B10) and (5) wheat breeding.
5. A method of producing a transgenic wheat having an improved flour processing quality and/or baking quality and/or gluten quality, comprising the step of increasing the content and/or activity of the 1Dx2m1/2/3 protein of claim 1 in a recipient wheat to obtain a transgenic wheat; the transgenic wheat has a higher flour processing quality and/or baking quality and/or gluten quality than the recipient wheat.
6. The method of claim 5, wherein: the transgenic wheat has higher flour processing quality and/or baking quality and/or gluten quality than the acceptor wheat as shown in any one of C1-C8):
C1) the SDS-sedimentation value of the transgenic wheat flour is higher than that of receptor wheat;
C2) the swelling index of glutenin of the transgenic wheat flour is higher than that of receptor wheat;
C3) the dough forming time in the flour quality instrument detection parameters of the transgenic wheat flour is longer than that of receptor wheat;
C4) the dough stability time in the flour quality instrument detection parameters of the transgenic wheat flour is longer than that of receptor wheat;
C5) the attenuation degree of the detection parameters of the flour quality instrument of the transgenic wheat flour is lower than that of receptor wheat;
C6) the peak time in the detection parameters of the kneading and mixing instrument of the transgenic wheat flour is higher than that of receptor wheat;
C7) the peak value area in the detection parameters of the kneading and mixing instrument of the transgenic wheat flour is higher than that of receptor wheat;
C8) the anti-fading value of the parameters detected by the kneading and mixing instrument of the transgenic wheat flour is lower than that of receptor wheat.
7. The method according to claim 5 or 6, characterized in that: the method for increasing the content and/or activity of the 1Dx2m1/2/3 protein in receptor wheat is to overexpress the 1Dx2m1/2/3 protein in receptor wheat.
8. The method of claim 7, wherein: the method for over-expressing the 1Dx2m1/2/3 protein of claim 1 in recipient wheat is to introduce the gene encoding the 1Dx2m1/2/3 protein of claim 1 into recipient wheat.
9. The method of claim 8, wherein: the nucleotide sequence of the coding gene of the 1Dx2m1/2/3 protein is a DNA molecule shown as a sequence 13.
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Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2264271A1 (en) * | 1996-08-22 | 1998-02-26 | Vimla Vasil | Transformed wheat having improved breadmaking characteristics |
| JP2005117990A (en) * | 2003-10-17 | 2005-05-12 | National Agriculture & Bio-Oriented Research Organization | Method for creating superstrong wheat line |
| CN101798575A (en) * | 2009-12-08 | 2010-08-11 | 中国科学院成都生物研究所 | Wheat high-quality high-molecular weight glutenin subunit gene and expressed protein thereof |
| CN102863521B (en) * | 2011-07-04 | 2014-07-16 | 中国科学院遗传与发育生物学研究所 | Identification of high molecular weight glutenin subunits excellent allelic variation G330E and application |
| CN102719474B (en) * | 2012-07-06 | 2014-06-11 | 首都师范大学 | Method for improving quality of wheat |
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2019
- 2019-12-12 CN CN201911273308.2A patent/CN110894222B/en active Active
Non-Patent Citations (2)
| Title |
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| high molecular weight glutenin subunit 1Dx [Triticum spelta];Dong,Z.等;《Genbank》;20131024;AGH32931 * |
| high-molecular-weight glutenin subunit [Secale cereale x Triticum aestivum];Yuan,Z.等;《Genbank》;20150804;AGS18765 * |
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