CN110157746B - Method for synthesizing auxin by microorganisms - Google Patents
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Abstract
The invention discloses a method for synthesizing auxin by microorganisms, which is characterized in that genes TrpR, dkgB, yeaE, yqhC, yqhD, dkgA, yahK and yjgB are knocked out in the microorganisms, and aroG is highly expressed fbr 、pheA fbr KDC, ALDH and ARO8 genes, and the constructed target engineering microorganism can realize the synthesis of phenylacetic acid by fermenting glucose. Further replacement of pheA fbr The gene is tyrA fbr The gene enables the modified target microorganism to realize the synthesis of p-hydroxyphenylacetic acid by fermenting glucose. Or replacing pheA fbr The gene is TrpE fbr serA fbr And trpABCD gene, so that the modified target microorganism can realize the synthesis of indole acetic acid by fermenting glucose. The microorganism transformed by the invention can realize the microbial transformation from glucose to phenylacetic acid, p-hydroxyphenylacetic acid and indoleacetic acid, and provides a feasible way for large-scale biosynthesis of the auxin.
Description
Technical Field
The invention belongs to the field of microbial metabolic engineering, and particularly relates to a method for directly synthesizing auxin from monosaccharide by modifying a microbial metabolic pathway.
Background
The auxin is one of the most important pheromones in plants, is an endogenous auxin commonly existing in plants, is vital to the growth and development of the plants, has the functions of promoting cell division, vascular bundle differentiation, stem elongation, apical dominance, seed germination, lateral root and adventitious root formation, root nodule formation, ovary wall growth, seed and fruit growth, fruit setting and the like, and has wide application prospect and economic position in agricultural production. These auxins include indoleacetic acid, phenylacetic acid, p-hydroxyphenylacetic acid, and the like.
At present, the commercial auxin is synthesized by a chemical method. The chemical synthesis of auxin involves many reaction steps resulting in high synthesis cost. Through a biotechnology means, the synthesis of various auxins by fermenting monosaccharides by microorganisms is realized, and the cost can be effectively reduced.
Disclosure of Invention
The invention aims to provide a method for synthesizing auxin in a microorganism, which constructs a metabolic pathway for converting monosaccharide into the auxin in the microorganism.
In order to realize the purpose, the invention provides a method for synthesizing auxin phenylacetic acid, p-hydroxyphenylacetaldehyde and indoleacetic acid in escherichia coli and pichia pastoris, which mainly comprises the following steps:
(1) Knock-out gene construction strain GDY
The strain was prepared by using Escherichia coli MG1655 as a starting strain, and removing TrpR gene and seven aldehyde reductase genes (dkgB, yeaE, yqhC, yqhD, dkgA, yahK and yjgB) in the strain in this order, and the 8 genes-removed strain was designated as GDY.
(2) Construction of Phenylacetic acid-producing engineering bacteria GDY1
KDC and ARO8 genes from saccharomyces cerevisiae and ALDH gene from escherichia coli are recombined onto a vector pET28a (+) to obtain a vector pDY001. aroG from E.coli fbr Gene and pheA fbr The gene was recombined into the vector pBBR1MCS1 to obtain vector pDY002. And transferring the vectors pDY001 and pDY002 into Escherichia coli GDY to realize high-amount expression of the 5 genes in Escherichia coli, thereby obtaining an Escherichia coli engineering strain GDY1. Expression of feedback-released aroG fbr Gene and pheA fbr The gene is used for enhancing the synthesis of the intracellular phenylalanine of the escherichia coli. The expression ARO8 is used for catalyzing phenylalanine to convert phenylpyruvic acid, the expression KDC is used for catalyzing the decarboxylation of the phenylpyruvic acid into phenylacetaldehyde, and the expression ALDH is used for catalyzing the oxidation of the phenylacetaldehyde to form phenylacetic acid. The GDY1 engineering bacteria constructed above can effectively convert monosaccharide into phenylacetic acid.
(3) Construction of P-hydroxyphenylacetic acid-producing engineering bacteria GDY2
aroG from E.coli fbr Gene and tyrA fbr The gene was recombined into the vector pBBR1MCS1 to give the vector pDY003. pDY003 and the previously constructed vector pDY001 were simultaneously transferred into Escherichia coli GDY to realize aroG fbr 、tyrA fbr And KDC, ARO8 and ALDH5 genes are expressed in Escherichia coli in high quantity to obtain an Escherichia coli engineering strain GDY2. Expressing aroG released from feedback inhibition fbr Gene and tyrA fbr The gene is used for enhancing the synthesis of the intracellular tyrosine of the Escherichia coli. ARO8 is expressed to catalyze tyrosine to convert p-hydroxyphenylpyruvic acid, KDC is expressed to catalyze the decarboxylation of p-hydroxyphenylpyruvic acid to p-hydroxyphenylacetaldehyde, and ALDH is expressed to catalyze the oxidation of p-hydroxyphenylacetaldehyde to form p-hydroxyphenylacetic acid. The GDY2 engineering bacteria constructed above can effectively convert monosaccharide into p-hydroxyphenylacetic acid.
(4) Construction of Indoleacetic acid-producing engineering bacterium GDY3
aroG from E.coli fbr 、TrpE fbr And serA fbr The gene is recombined on a vector pBBR1MCS1 to obtain a vector pDY004. the trpABCD gene (including the four genes trpA, trpB, trpC, and trpD) was recombined onto vector pDY001 to obtain vector pDY005. The vectors pDY004 and pDY005 were transferred into Escherichia coli GDY at the same time to realize aroG fbr 、TrpE fbr 、serA fbr And high-quantity expression of trpABCD, KDC, ARO8 and ALDH7 genes in escherichia coli to obtain an escherichia coli engineering strain GDY3. Expressing aroG released from feedback inhibition fbr 、TrpE fbr And serA fbr The gene and the trpABCD gene are used for enhancing the synthesis of tryptophan in the escherichia coli cells. Expression of ARO8 for catalyzing conversion of tryptophan to indolylpyruvic acid, expressionKDC is used to catalyze the decarboxylation of indole pyruvate to indole acetaldehyde, and ALDH is expressed to subsequently catalyze the oxidation of indole acetaldehyde to form indole acetic acid. The GDY3 engineering bacteria constructed by the method can effectively convert monosaccharide into indoleacetic acid.
(5) Construction of auxin-producing engineering bacteria GDY4
The ALDH gene derived from E.coli was recombined into vector pGAPZa to obtain vector pDY006. KDC and ARO8 genes from saccharomyces cerevisiae are respectively recombined into a vector pGAPZa to obtain vectors pDY14 and pDY15. pDY14 and pDY15 were digested with BamHI and BglII, respectively, to obtain a KDC gene expression cassette (containing GAP promoter, KDC gene and AOX1 TT) and an ARO8 gene expression cassette (containing GAP promoter, ARO8 gene and AOX1 TT). And respectively recombining the KDC gene expression frame and the ARO8 gene expression frame to a pAO815 vector to obtain vectors pDY007 and pDY008. After linearization, vectors pDY006, pDY007 and pDY008 are respectively introduced into Pichia pastoris GS115 to realize constitutive expression of ARO8, ALDH and KDC genes, and the engineering bacterium is named as GDY4. ARO8 is expressed for catalyzing the conversion of phenylalanine, tyrosine and tryptophan to phenylpyruvic acid, p-hydroxyacetone and indolylpyruvic acid. KDC is expressed to catalyze the conversion of phenylpyruvic acid, p-hydroxyphenylpyruvic acid and indolylpyruvic acid into phenylacetaldehyde, p-hydroxyphenylacetaldehyde and indolylacetylaldehyde respectively. The ALDH is expressed to catalyze the conversion of phenylacetaldehyde, p-hydroxyphenylacetaldehyde and indoleacetaldehyde to phenylacetic acid, p-hydroxyphenylacetic acid and indoleacetic acid, respectively.
The invention has the advantages that the exogenous gene is introduced into the microorganism to construct a metabolic pathway for synthesizing the auxin, the aim of synthesizing the auxin by taking monosaccharide as a raw material is finally realized, excessive chemical synthesis reaction is not needed, the release of toxic substances in the chemical synthesis process is reduced, and the synthesis cost is also reduced. Meanwhile, as the growth speed of the microorganism is high, the genetic operation technology is mature, and the fermentation process cannot cause pollution damage to the environment. The pichia pastoris strain can be used as an agricultural microorganism, and can directly utilize nutrient substances in environmental soil to synthesize auxin, thereby promoting the growth of plants.
Drawings
FIG. 1 shows the GC-MS detection result of the fermentation product of the engineering bacteria GDY1 of Escherichia coli. Peak 1 represents the internal standard benzoic acid and peak 2 represents the product phenylacetic acid.
FIG 2 shows the GC-MS detection result of the fermentation product of the engineering bacteria GDY2 of Escherichia coli. Peak 1 represents internal standard benzoic acid, peak 2 represents by-product phenylacetic acid, and peak 3 represents product p-hydroxyphenylacetic acid.
FIG. 3 shows the GC-MS detection result of the fermentation product of the engineering bacteria GDY3 of Escherichia coli. Peak 1 represents internal standard benzoic acid, peak 2 represents byproduct phenylacetic acid, peak 3 represents byproduct p-hydroxyphenylacetic acid, and peak 4 represents product indoleacetic acid.
FIG. 4 shows the GC-MS detection result of the fermentation product of Pichia pastoris GDY4. Peak 1 represents internal standard benzoic acid, peak 2 represents product indoleacetic acid, peak 3 represents p-hydroxyphenylacetic acid, and peak 4 represents indoleacetic acid.
Detailed Description
The following examples are intended to further illustrate the invention but should not be construed as limiting it.
Example 1 construction of Phenylacetic acid-producing Escherichia coli
1. Construction of Gene knockout Strain GDY
PCR amplification of the targeting fragment using pKD4 plasmid as template, the fragment containing (kan) r Sequence and homology arms complementary to both ends of the target gene to be knocked out); the amplified targeting fragment was electroporated into E.coli harboring pKD46 plasmid. And (3) recombining the target gene to be knocked out and the targeting fragment by using a Red homologous recombination system carried on the pKD46 plasmid, wherein the target gene is replaced by the resistance gene. Then, a temperature-sensitive plasmid pCP20 is introduced into the strain, and the FLP flippase on the plasmid is utilized to remove the resistance gene on the chromosome of the target strain. The genes of TrpR, dkgB, yeaE, yqhC, yqhD, dkgA, yahK and yjgB in the Escherichia coli MG1655 strain were sequentially knocked out by the above method to obtain Escherichia coli DGY. The TrpR gene has an amino acid sequence of SEQ ID NO: 25; the TrpR gene has an amino acid sequence of SEQ ID NO: 26; the dkgB gene has the sequence shown in SEQ ID NO:27, or a pharmaceutically acceptable salt thereof; the ALDH gene has an amino acid sequence shown in SEQ ID NO: 28; the yeaE gene has an amino acid sequence shown in SEQ ID NO:29 under the general formula ofColumn encoding; yeaE has the sequence shown in SEQ ID NO: 30; the yqhC gene has the sequence of SEQ ID NO: 31; the yqhC gene has the sequence shown in SEQ ID NO: 32; the yqhD gene has the sequence of SEQ ID NO: 33; the yqhD gene has the sequence of SEQ ID NO: 34; the dkgA gene has the nucleotide sequence shown in SEQ ID NO: 35; the dkgA gene has the nucleotide sequence shown in SEQ ID NO: 36; the yahK gene has the sequence shown in SEQ ID NO: 37; the yahK gene has the sequence shown in SEQ ID NO: 38; the yjgB gene has the sequence shown in SEQ ID NO: 39; the yjgB gene has the sequence shown in SEQ ID NO: 40; the primers for gene knockout were: atggccaacaatcacctattcagcagcaggcagcgatggcagaacaggtgtgtaggctggagctgctgcttc
TrpR-R:tcaatcgcttttcagcaacacctcttccagccactggcgcacatatgaatatcctcctta
DkgB-F:atggctatccctgcatttggtttaggtactttccgtctgagtgtaggctggagctgcttc
DkgB-R:ttaatcccattcaggagccagaccttccgggctaaccaggcggcatatgaatatcctcctta
yeaE-F:atgcaacaaaaaatgattcaatttagtggcgatgtctcactggtgtaggctggagctgcttc
yeaE-R:tcacaccatatccagcgcagtttttccttttggtgccggatatcatatgaatatcctcctta
yqhC-F:atgctacaaaattgcgcacaatcaaattgccgcattattcctaagtgtaggctggagctgcttc
yqhC-R:ttaattcccctgcatcgcccgcattcttgccgcatcttcccccatatgaatatcctcctta
yqhD-F:atgaacaactttaatctgcacaccccaacccgcattctgttgtgtaggctggagctgcttc
yqhD-R:ttagcgggcggcttcgtatatacggcggctgacatccaacgcatatgaatatcctcctta
dkgA-F：atggctaatccaaccgttattaagctacaggatggcaatgtcgtgtaggctggagctgcttc
dkgA-R：ttagccgccgaactggtcaggatcgggaccgagacgcttgccatatgaatatcctcctta
yahK-F:atgaagatcaaagctgttggtgcatattccgctaaacaaccgtgtaggctggagctgcttc
yahK-R:tcagtctgttagtgtgcgattatcgataacaaaacgatattcatatgaatatcctcctta
yjgB-F:atgtcgatgataaaaagctatgccgcaaaagaagcgggcggcgtgtaggctggagctgcttc
yjgB-R:tcaaaaatcggctttcaacaccacgcggtaacgcgccttaccgtcatatgaatatcctcctta
2. Construction of vector pDY001
KDC gene from Saccharomyces cerevisiae YPH499 was PCR amplified using primers KDC-XbaI (ATCTCTAGATTTAAGAAGGAGATATATATATAATGGCACCTGTTTACAATTGAA AAGTTC) and KDC-NheI-BamHI (TCTGGATCCGCTAGCCTATTTTTTTTATTTTTTAAG TGCCGCTG), and the amplified fragment was subsequently inserted into pET28a (+) as XbaI and BamHI to give vector pDY01. The ALDH gene from E.coli was PCR amplified using primers ALDH-XbaI (ATCTCTAGATTTAAGAAGGAGATATATATAATGAATTTCATCATCTGGCTTACTG) and ALDH-NheI-BamHI (TCAGGATCCGCTCAGCGGCCTCCAGGCTTTATCC) and inserted into pET28a (+) using XbaI and BamHI to give vector pDY02. KDC gene from Saccharomyces cerevisiae YPH499 was PCR amplified using primers ARO8-XbaI (AACTCTAGATTTAAGAAGGAGATATATATATATATATATATGATGATGACTTTACCTGAATCTAAAGACTTT TC) and ARO8-NheI-BamHI (ACAGGATCGCCTAGCCTATTTGGAAATACCAATTCTTTCGTATA A A), and the amplified fragment was subsequently inserted into pET28a (+) with XbaI and BamHI to give vector pDY03. pDY02 was double-digested with XbaI and XhoI to give an ALDH expression cassette, which was inserted into vector pDY01 double-digested with NheI and XhoI to give vector pDY04. pDY03 was double-digested with XbaI and XhoI to obtain an ARO8 expression cassette, which was inserted into vector pDY04 double-digested with NheI and XhoI to obtain vector pDY001. The KDC gene has the sequence shown in SEQ ID NO: 19; the KDC has the sequence shown in SEQ ID NO: 20; the ALDH gene has an amino acid sequence shown in SEQ ID NO: 21; the ALDH gene has an amino acid sequence shown in SEQ ID NO: 22; the ARO8 gene has an amino acid sequence shown in SEQ ID NO: 23; the ARO8 gene has an amino acid sequence shown in SEQ ID NO: 24;
3. construction of vector pDY002
aroG for releasing feedback inhibition fbr And pheA fbr Gene entrusted Jinwei ZhishengSynthesized by Biotech Ltd, using the synthesized gene as a template and using a primer aroG fbr XbaI (GTATCTAGAAAGAGGAGGATATATAATGAATTTAT CAGAACGACGATTTACGC) and aroG fbr PCR amplification of aroG with SpeI-BamHI (TATGGATCCATAGTTACCCGCGCGACG CGCTTTTA) fbr The amplified fragment was then inserted into pET28a (+) with XbaI and BamHI to give vector pDY05. Using the synthesized gene as a template and using a primer pheA fbrr XbaI (GTATCTAGAAAGAGGAGATATAATGACATCGGAAACCCGTTACTG) and pheA fbr -SpeI-SalI (ATATGTCGACATCTAGTTCACAACGTGGTTTTCGCCGGA) PCR amplification from pheA fbr The gene, and the amplified fragment were inserted into pET28a (+) with XbaI and SalI to give vector pDY06. Obtaining pheA by double digestion of pDY06 with XbaI and XhoI fbr And inserting the expression cassette into a vector pDY05 which is subjected to double digestion by SpeI and XhoI to obtain a vector pDY07. Double digestion of pDY07 with XbaI and XhoI gave aroG fbr And pheA fb The expression cassette was inserted into the vector pBBR1MCS1 digested with XbaI and XhoI to obtain vector pDY002. The aroG fbr The gene has the sequence shown in SEQ ID NO: 1; the aroG fbr The gene has the sequence similar to SEQ ID NO: 2; the pheA fbr The gene has the sequence shown in SEQ ID NO: 3; the pheA fbr The gene has the sequence shown in SEQ ID NO: 4; 4. and simultaneously introducing the recombinant vectors pDY001 and pDY002 into the Escherichia coli GDY to obtain the GDY1 engineering bacteria. The engineering bacterium can realize aroG fbr 、pheA fbr High expression of KDC, ALDH and ARO8 genes can effectively convert monosaccharide into phenylacetic acid.
Example 2 construction of P-hydroxyphenylacetic acid-producing Escherichia coli engineering bacteria
1. Construction of vector pDY003
tyrA with feedback suppression removed fbr The gene is synthesized by Jinzhi Biotechnology GmbH, and the synthesized gene is used as a template to utilize a primer tyrA fbrr XbaI (GTATCTAGAAAGAGGAGATAATGGTTGCTGAATT GACCGCAT) and tyrA fbr PCR amplification of SpeI-SalI (ATATGTCGACATCTAGTTTACTGGCGATTGTCATTCGCC) to tyrA fbr Gene, followed by insertion of the amplified fragment into pET28a (+) with XbaI and SalIThe vector pDY08 was obtained. Double digestion of pDY08 with XbaI and XhoI to give tyrA fbr And inserting the expression cassette into a vector pDY05 which is subjected to double digestion by SpeI and XhoI to obtain a vector pDY09. Double digestion of pDY09 with XbaI and XhoI to obtain aroG fbr And tyrA fbr The expression cassette was inserted into the vector pBBR1MCS1 digested simultaneously with XbaI and XhoI to obtain vector pDY003. Said tyrA fbr The gene has the sequence shown in SEQ ID NO: 5; the tyrA fbr The gene has the sequence shown in SEQ ID NO: 6;
2. and simultaneously introducing the recombinant vectors pDY001 and pDY003 into the Escherichia coli GDY to obtain the GDY2 engineering bacteria. The engineering bacterium can realize aroG fbr 、tyrA fbr High expression of KDC, ALDH and ARO8 genes can effectively convert monosaccharide into p-hydroxyphenylacetic acid.
Example 3 construction of Indoleacetic acid-producing Escherichia coli
1. Construction of vector pDY004
TrpE for removing feedback inhibition fbr And serA fbr The gene is entrusted to Jinzhi Biotechnology, inc. Using the synthesized gene as a template and using a primer TrpE fbr XbaI (GTATCTAGAAAGAGGAGGATATATAATGCAAACAAAACCGACTCTC) and TrpE fbr PCR amplification of TrpE with SpeI-BamHI (TATGGATCCATAGTTCAGAAAGTCTCCTGTGCATGGATTG) fbr The amplified fragment was then inserted into pET28a (+) with XbaI and BamHI to obtain a vector pDY10. Using the synthesized gene as a template and the primer serA fbr XbaI (GTATCTAGAAAGAGGAGATATAATGACATCG GAAAACCCGTTACTG) and serA fbr PCR amplification of SpeI-SalI (ATATGTCGACCTAGTTCACAACGTGGTTTT CGCCGGA) to SerA fbr The amplified fragment was then inserted into pET28a (+) with XbaI and SalI to give vector pDY11. Double digestion of pDY10 with XbaI and XhoI to give TrpE fbr And inserting the expression cassette into a vector pDY01 which is subjected to double digestion by SpeI and XhoI to obtain a vector pDY12. Double digestion of pDY10 with XbaI and XhoI to obtain TrpE fbr And inserting the expression cassette into a vector pDY12 which is subjected to double enzyme digestion by SpeI and XhoI to obtain a vector pDY13. Obtaining serA by double digestion of pDY13 with XbaI and XhoI fbr And TrpE fbr Expression cassette, insert it intoVector pDY004 was obtained from vector pBBR1MCS1 digested with XbaI and XhoI. The TrpE fbr The gene has the sequence shown in SEQ ID NO: 7; the TrpE fbr The gene has the sequence shown in SEQ ID NO: 8; serA fbr The serA fbr The gene has the sequence shown in SEQ ID NO: 9; the serA fbr The gene has the sequence shown in SEQ ID NO: 10;
2. construction of vector pDY005
TrpABCD gene (containing trpA, trpB, trpC, and trpD four genes) was PCR-amplified using TrpABCD-XbaI (CCATCTAGATTTAAGAGAGGATATAATGGCTGGACATTCTGCTGC TCGATA) and TrpABCD-XhoI (GATTCTCGAGTTAACTGCGCGTCGCCGCT) primers using the genome of Escherichia coli MG1655 as a template, and the amplified fragment was inserted into pDY001 with XbaI and XhoI to obtain vector pDY005. The trpA has the sequence shown in SEQ ID NO: 11; the trpA gene has the sequence shown in SEQ ID NO: 12; the trpB has the nucleotide sequence shown in SEQ ID NO:13 is coded by the nucleotide sequence shown in the specification; the trpB gene has the sequence shown in SEQ ID NO: 14; the trpC has the nucleotide sequence shown in SEQ ID NO: 15; the trpC gene has the sequence shown in SEQ ID NO: 16; the trpD has the amino acid sequence of SEQ ID NO:17 encodes the polypeptide; the trpD gene has the nucleotide sequence shown in SEQ ID NO: 18;
3. and simultaneously introducing the recombinant vectors pDY004 and pDY005 into Escherichia coli GDY to obtain GDY3 engineering bacteria. The engineering bacterium can realize aroG fbr 、TrpE fbr 、SerA fbr High expression of TrpABCD, KDC, ALDH and ARO8 genes can effectively convert monosaccharide into indoleacetic acid.
Example 4 construction of Pichia pastoris engineering bacteria GDY4 for producing auxin
The ALDHr gene is amplified by PCR using primers ALDH-BstBI (AGCATTCGAAAAT GAATTTTCATCTGGCTTACTGGC) and ALDH-XhoI (GAACCTCGAGTCAGGCCTCCAGGCTTATCCAGAT) by taking an Escherichia coli MG1655 genome as a template, and then the amplified fragment is inserted into pGAPZa by BstBI and XhoI to obtain a vector pDY006. KDC and ARO8 genes were PCR amplified using Saccharomyces cerevisiae YPH499 genome as template and primers KDC-BstBI (AGCATTCGAAAATGGCACCTGTTAAATTGAAAGTTC)/KDC-XhoI (CTTCCTCTGAGCTATTTTTTATTTCTTTTAAGTGCCGCTG) and ARO8-BstBI (AGCATTCGAAAATGACTT TACCTGAATCAAAGACTTTTC)/ARO 8-XhoI (CTTCCTCGAGCTATTTGGAAATACCAATCTTTCTCTCTTC GTATAA), and then the amplified fragments were inserted into pGAPZa with BstBI and XhoI, respectively, to obtain vectors pDY14 and pDY15. pDY14 and pDY15 were digested with BamHI and BglII, respectively, to obtain a KDC gene expression cassette (containing GAP promoter, KDC gene and AOX1 TT) and an ARO8 gene expression cassette (containing GAP promoter, ARO8 gene and AOX1 TT). And recombining the KDC gene expression frame to a pAO815 vector which is cut by BamHI enzyme to obtain a vector pDY007. The ARO8 gene expression cassette is recombined to a pPIC3.5K vector which is digested by BamHI, and a vector pDY008 is obtained. After the vectors pDY006, pDY007 and pDY008 are respectively linearized, the linearized vectors are sequentially introduced into Pichia pastoris GS115 to realize the constitutive expression of ARO8, ALDH and KDC genes, and the engineering bacterium is named as GDY4. ARO8 is expressed for catalyzing the conversion of phenylalanine, tyrosine and tryptophan to phenylpyruvic acid, p-hydroxyacetone and indolylpyruvic acid. KDC is expressed for catalyzing the conversion of phenylpyruvic acid, p-hydroxyphenylpyruvic acid and indolylpyruvic acid to phenylacetaldehyde, p-hydroxyphenylacetaldehyde and indolylacetylacetaldehyde, respectively. The ALDH is expressed to catalyze the conversion of phenylacetaldehyde, p-hydroxyphenylacetaldehyde and indoleacetaldehyde into phenylacetic acid, p-hydroxyphenylacetic acid and indoleacetic acid respectively. The KDC gene has the sequence shown in SEQ ID NO: 19; the KDC has the sequence shown in SEQ ID NO: 20; the ALDH gene has an amino acid sequence shown in SEQ ID NO: 21; the ALDH gene has an amino acid sequence shown in SEQ ID NO: 22; the ARO8 gene has an amino acid sequence shown in SEQ ID NO:23 is shown in the specification; the ARO8 gene has an amino acid sequence shown in SEQ ID NO: 24;
example 5 fermentation experiment of Escherichia coli engineering bacteria for producing auxin
1. Respectively inoculating engineering bacteria GDY1, GDY2 and GDY3 of Escherichia coli cultured overnight in 37 degree LB into 50ml M9 fermentation medium (containing 2% glucose), performing 30 degree shake flask fermentation to obtain thallus OD 600 About 0.8, 0.1mM IPTG was added to induce expression of the desired gene.
2. After 24 hours of fermentation culture, the thalli are collected, cells are broken by ultrasonic waves, and products are extracted by ethyl acetate.
3. The solvent ethyl acetate was spin dried using a rotary evaporator and the product was dissolved with a quantity of ethyl acetate.
4. And detecting the sample by GC-MS (gas chromatography-mass spectrometer).
5. The experimental results are as follows: the results of GC-MS detection of the Escherichia coli engineering bacteria for synthesizing auxin by using glucose are shown in FIG. 1, FIG. 2 and FIG. 3. FIG. 1 shows the GC-MS detection result of the fermentation product of the engineering bacteria GDY1 of Escherichia coli, which shows that the strain GDY1 can effectively utilize glucose to synthesize auxin phenylacetic acid, and the yield of the shaking table is 1.67g/L. FIG. 2 is a GC-MS detection result of a fermentation product of Escherichia coli engineering bacteria GDY2, which shows that the strain GDY2 can effectively utilize glucose to synthesize auxin p-hydroxyphenylacetic acid, and the yield of a shaker is 1.07g/L. FIG. 3 is a GC-MS detection result of a fermentation product of Escherichia coli engineering bacteria GDY3, which shows that the strain GDY3 can effectively utilize glucose to synthesize auxin-indolacetic acid, and the yield of the shaker is 1.16g/L.
Example 6 fermentation experiment for producing auxin by Pichia pastoris engineering bacteria
6. Pichia pastoris GDY4 cultured overnight in 30-degree YPD was inoculated into 50ml of YPD fermentation medium (containing 2% glucose) and subjected to 30-degree shake flask fermentation.
7. After fermentation culture for 28 hours, the thalli are collected, cells are broken by ultrasonic waves, and products are extracted by ethyl acetate.
8. The solvent ethyl acetate was spin dried using a rotary evaporator and the product was dissolved with a quantity of ethyl acetate.
9. GC-MS (gas chromatography-mass spectrometer) detects the sample.
10. The experimental results are as follows: the GC-MS detection result of the Pichia pastoris engineering bacteria for synthesizing the plant auxin by using glucose is shown in figure 4. The result shows that the strain GDY4 can effectively utilize glucose to synthesize auxin phenylacetic acid, p-hydroxyphenylacetic acid and indoleacetic acid, and the yield is 108mg/L,36mg/L and 33mg/L respectively.
The foregoing is directed to the preferred embodiment of the present invention and it is appreciated that those skilled in the art may now make appropriate changes without departing from the principles of the invention, and that such changes are deemed to be within the scope of the invention.
Sequence listing
<110> university of Jiangxian teachers
<120> method for synthesizing auxin by microorganisms
<160> 55
<170> SIPOSequenceListing 1.0
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<211> 1053
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 1
atgaattatc agaacgacga tttacgcatc aaagaaatca aagagttact tcctcctgtc 60
gcattgctgg aaaaattccc cgctactgaa aatgccgcga atacggttgc ccatgcccga 120
aaagcgatcc ataagatcct gaaaggtaat gatgatcgcc tgttggttgt gattggccca 180
tgctcaattc atgatcctgt cgcggcaaaa gagtatgcca ctcgcttgct ggcgctgcgt 240
gaagagctga aagatgagct ggaaatcgta atgcgcgtct attttgaaaa gccgcgtacc 300
acggtgggct ggaaagggct gattaacgat ccgcatatgg ataatagctt ccagatcaac 360
gacggtctgc gtatagcccg taaattgctg cttgatatta acgacagcgg tctgccagcg 420
gcaggtgagt ttctcaatat gatcacccca caatatctcg ctgacctgat gagctggggc 480
gcaattggcg cacgtaccac cgaatcgcag gtgcaccgcg aactggcatc agggctttct 540
tgtccggtcg gcttcaaaaa tggcaccgac ggtacgatta aagtggctat cgatgccatt 600
aatgccgccg gtgcgccgca ctgcttcctg tccgtaacga aatgggggca ttcggcgatt 660
gtgaatacca gcggtaacgg cgattgccat atcattctgc gcggcggtaa agagcctaac 720
tacagcgcga agcacgttgc tgaagtgaaa gaagggctga acaaagcagg cctgccagca 780
caggtgatga tcgatttcag ccatgctaac tcgtccaaac aattcaaaaa gcagatggat 840
gtttgtgctg acgtttgcca gcagattgcc ggtggcgaaa aggccattat tggcgtgatg 900
gtggaaagcc atctggtgga aggcaatcag agcctcgaga gcggggagcc gctggcctac 960
ggtaagagca tcaccgatgc ctgcatcggc tgggaagata ccgatgctct gttacgtcaa 1020
ctggcgaatg cagtaaaagc gcgtcgcggg taa 1053
<210> 3
<211> 350
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 3
Met Asn Tyr Gln Asn Asp Asp Leu Arg Ile Lys Glu Ile Lys Glu Leu
1 5 10 15
Leu Pro Pro Val Ala Leu Leu Glu Lys Phe Pro Ala Thr Glu Asn Ala
20 25 30
Ala Asn Thr Val Ala His Ala Arg Lys Ala Ile His Lys Ile Leu Lys
35 40 45
Gly Asn Asp Asp Arg Leu Leu Val Val Ile Gly Pro Cys Ser Ile His
50 55 60
Asp Pro Val Ala Ala Lys Glu Tyr Ala Thr Arg Leu Leu Ala Leu Arg
65 70 75 80
Glu Glu Leu Lys Asp Glu Leu Glu Ile Val Met Arg Val Tyr Phe Glu
85 90 95
Lys Pro Arg Thr Thr Val Gly Trp Lys Gly Leu Ile Asn Asp Pro His
100 105 110
Met Asp Asn Ser Phe Gln Ile Asn Asp Gly Leu Arg Ile Ala Arg Lys
115 120 125
Leu Leu Leu Asp Ile Asn Asp Ser Gly Leu Pro Ala Ala Gly Glu Phe
130 135 140
Leu Asn Met Ile Thr Pro Gln Tyr Leu Ala Asp Leu Met Ser Trp Gly
145 150 155 160
Ala Ile Gly Ala Arg Thr Thr Glu Ser Gln Val His Arg Glu Leu Ala
165 170 175
Ser Gly Leu Ser Cys Pro Val Gly Phe Lys Asn Gly Thr Asp Gly Thr
180 185 190
Ile Lys Val Ala Ile Asp Ala Ile Asn Ala Ala Gly Ala Pro His Cys
195 200 205
Phe Leu Ser Val Thr Lys Trp Gly His Ser Ala Ile Val Asn Thr Ser
210 215 220
Gly Asn Gly Asp Cys His Ile Ile Leu Arg Gly Gly Lys Glu Pro Asn
225 230 235 240
Tyr Ser Ala Lys His Val Ala Glu Val Lys Glu Gly Leu Asn Lys Ala
245 250 255
Gly Leu Pro Ala Gln Val Met Ile Asp Phe Ser His Ala Asn Ser Ser
260 265 270
Lys Gln Phe Lys Lys Gln Met Asp Val Cys Ala Asp Val Cys Gln Gln
275 280 285
Ile Ala Gly Gly Glu Lys Ala Ile Ile Gly Val Met Val Glu Ser His
290 295 300
Leu Val Glu Gly Asn Gln Ser Leu Glu Ser Gly Glu Pro Leu Ala Tyr
305 310 315 320
Gly Lys Ser Ile Thr Asp Ala Cys Ile Gly Trp Glu Asp Thr Asp Ala
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<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
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atgacatcgg aaaacccgtt actggcgctg cgagagaaaa tcagcgcgct ggatgaaaaa 60
ttattagcgt tactggcaga acggcgcgaa ctggccgtcg aggtgggaaa agccaaactg 120
ctctcgcatc gcccggtacg tgatattgat cgtgaacgcg atttgctgga aagattaatt 180
acgctcggta aagcgcacca tctggacgcc cattacatta ctcgcctgtt ccagctcatc 240
attgaagatt ccgtattaac tcagcaggct ttgctccaac aacatctcaa taaaattaat 300
ccgcactcag cacgcatcgc ttttctcggc cccaaaggtt cttattccca tcttgcggcg 360
cgccagtatg ctgcccgtca ctttgagcaa ttcattgaaa gtggctgcgc caaatttgcc 420
gatattttta atcaggtgga aaccggccag gccgactatg ccgtcgtacc gattgaaaat 480
accagctccg gtgccataaa cgacgtttac gatctgctgc aacataccag cttgtcgatt 540
gttggcgaga tgacgttaac tatcgaccat tgtttgttgg tctccggcac tactgattta 600
tccaccatca atacggtcta cagccatccg cagccattcc agcaatgcag caaattcctt 660
aatcgttatc cgcactggaa gattgaatat accgaaagta cgtctgcggc aatggaaaag 720
gttgcacagg caaaatcacc gcatgttgct gcgttgggaa gcgaagctgg cggcactttg 780
tacggtttgc aggtactgga gcgtattgaa gcaaatcagc gacaaaactt cacccgattt 840
gtggtgttgg cgcgtaaagc cattaacgtg tctgatcagg ttccggcgaa aaccacgttg 900
tga 903
<210> 4
<211> 300
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 4
Met Thr Ser Glu Asn Pro Leu Leu Ala Leu Arg Glu Lys Ile Ser Ala
1 5 10 15
Leu Asp Glu Lys Leu Leu Ala Leu Leu Ala Glu Arg Arg Glu Leu Ala
20 25 30
Val Glu Val Gly Lys Ala Lys Leu Leu Ser His Arg Pro Val Arg Asp
35 40 45
Ile Asp Arg Glu Arg Asp Leu Leu Glu Arg Leu Ile Thr Leu Gly Lys
50 55 60
Ala His His Leu Asp Ala His Tyr Ile Thr Arg Leu Phe Gln Leu Ile
65 70 75 80
Ile Glu Asp Ser Val Leu Thr Gln Gln Ala Leu Leu Gln Gln His Leu
85 90 95
Asn Lys Ile Asn Pro His Ser Ala Arg Ile Ala Phe Leu Gly Pro Lys
100 105 110
Gly Ser Tyr Ser His Leu Ala Ala Arg Gln Tyr Ala Ala Arg His Phe
115 120 125
Glu Gln Phe Ile Glu Ser Gly Cys Ala Lys Phe Ala Asp Ile Phe Asn
130 135 140
Gln Val Glu Thr Gly Gln Ala Asp Tyr Ala Val Val Pro Ile Glu Asn
145 150 155 160
Thr Ser Ser Gly Ala Ile Asn Asp Val Tyr Asp Leu Leu Gln His Thr
165 170 175
Ser Leu Ser Ile Val Gly Glu Met Thr Leu Thr Ile Asp His Cys Leu
180 185 190
Leu Val Ser Gly Thr Thr Asp Leu Ser Thr Ile Asn Thr Val Tyr Ser
195 200 205
His Pro Gln Pro Phe Gln Gln Cys Ser Lys Phe Leu Asn Arg Tyr Pro
210 215 220
His Trp Lys Ile Glu Tyr Thr Glu Ser Thr Ser Ala Ala Met Glu Lys
225 230 235 240
Val Ala Gln Ala Lys Ser Pro His Val Ala Ala Leu Gly Ser Glu Ala
245 250 255
Gly Gly Thr Leu Tyr Gly Leu Gln Val Leu Glu Arg Ile Glu Ala Asn
260 265 270
Gln Arg Gln Asn Phe Thr Arg Phe Val Val Leu Ala Arg Lys Ala Ile
275 280 285
Asn Val Ser Asp Gln Val Pro Ala Lys Thr Thr Leu
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<210> 5
<211> 1122
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 5
atggttgctg aattgaccgc attacgcgat caaattgatg aagtcgataa agcgctgctg 60
aatttattag cgaagcgtct ggaactggtt gctgaagtgg gcgaggtgaa aagccgcttt 120
ggactgccta tttatgttcc ggagcgcgag gcatctattt tggcctcgcg gcgcgcagag 180
gcggaagctc tgggtgtacc gccagatctg attgaggatg ttttgcgtcg ggtgatgcgt 240
gaatcttact ccagtgaaaa cgacaaagga tttaaaacac tttgtccgtc actgcgtccg 300
gtggttatcg tcggcggtgg cggtcagatg ggacgcctgt tcgagaagat gctgacacta 360
tcgggttatc aggtgcggat tctggagcaa catgactggg atcgagcggc tgatattgtt 420
gccgatgccg gaatggtgat tgttagtgtg ccaatccacg ttactgagca agttatcggc 480
aaattaccgc ctttaccgaa agattgtatt ctggttgatc tggcatcagt gaaaaatgga 540
ccattacagg ccatgctggc ggcgcacgat ggcccggtac tggggttaca cccgatgttc 600
ggcccggaca gcggtagcct ggcaaagcaa gttgtggtct ggtgtgatgg acgtaagccg 660
gaagcatacc aatggtttct ggagcaaatt caggtctggg gcgctcggct gcatcgtatt 720
agcgctgtcg agcacgatca gaatatggcg tttattcagg ctctgcgcca ctttgctact 780
tttgcttatg ggctgcatct ggcagaagaa aatgttcagc ttgagcaact tctggcgctc 840
tcttcgccga tttaccgcct tgagctggcg atggtcgggc gactgtttgc tcaggatccg 900
cagctttatg ccgacattat tatgtcgtca gagcgtaatc tggcgttaat caaacgttac 960
tataagcgtt tcggcgaggc gattgagttg ctggagcagg gcgataagca ggcgtttatt 1020
gacagtttcc gcaaggtgga gcactggttc ggcgattacg ttcagcgttt tcagagtgaa 1080
agccgcgtgt tattgcgtca ggcgaatgac aatcgccagt aa 1122
<210> 6
<211> 373
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 6
Met Val Ala Glu Leu Thr Ala Leu Arg Asp Gln Ile Asp Glu Val Asp
1 5 10 15
Lys Ala Leu Leu Asn Leu Leu Ala Lys Arg Leu Glu Leu Val Ala Glu
20 25 30
Val Gly Glu Val Lys Ser Arg Phe Gly Leu Pro Ile Tyr Val Pro Glu
35 40 45
Arg Glu Ala Ser Ile Leu Ala Ser Arg Arg Ala Glu Ala Glu Ala Leu
50 55 60
Gly Val Pro Pro Asp Leu Ile Glu Asp Val Leu Arg Arg Val Met Arg
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Glu Ser Tyr Ser Ser Glu Asn Asp Lys Gly Phe Lys Thr Leu Cys Pro
85 90 95
Ser Leu Arg Pro Val Val Ile Val Gly Gly Gly Gly Gln Met Gly Arg
100 105 110
Leu Phe Glu Lys Met Leu Thr Leu Ser Gly Tyr Gln Val Arg Ile Leu
115 120 125
Glu Gln His Asp Trp Asp Arg Ala Ala Asp Ile Val Ala Asp Ala Gly
130 135 140
Met Val Ile Val Ser Val Pro Ile His Val Thr Glu Gln Val Ile Gly
145 150 155 160
Lys Leu Pro Pro Leu Pro Lys Asp Cys Ile Leu Val Asp Leu Ala Ser
165 170 175
Val Lys Asn Gly Pro Leu Gln Ala Met Leu Ala Ala His Asp Gly Pro
180 185 190
Val Leu Gly Leu His Pro Met Phe Gly Pro Asp Ser Gly Ser Leu Ala
195 200 205
Lys Gln Val Val Val Trp Cys Asp Gly Arg Lys Pro Glu Ala Tyr Gln
210 215 220
Trp Phe Leu Glu Gln Ile Gln Val Trp Gly Ala Arg Leu His Arg Ile
225 230 235 240
Ser Ala Val Glu His Asp Gln Asn Met Ala Phe Ile Gln Ala Leu Arg
245 250 255
His Phe Ala Thr Phe Ala Tyr Gly Leu His Leu Ala Glu Glu Asn Val
260 265 270
Gln Leu Glu Gln Leu Leu Ala Leu Ser Ser Pro Ile Tyr Arg Leu Glu
275 280 285
Leu Ala Met Val Gly Arg Leu Phe Ala Gln Asp Pro Gln Leu Tyr Ala
290 295 300
Asp Ile Ile Met Ser Ser Glu Arg Asn Leu Ala Leu Ile Lys Arg Tyr
305 310 315 320
Tyr Lys Arg Phe Gly Glu Ala Ile Glu Leu Leu Glu Gln Gly Asp Lys
325 330 335
Gln Ala Phe Ile Asp Ser Phe Arg Lys Val Glu His Trp Phe Gly Asp
340 345 350
Tyr Val Gln Arg Phe Gln Ser Glu Ser Arg Val Leu Leu Arg Gln Ala
355 360 365
Asn Asp Asn Arg Gln
370
<210> 7
<211> 1563
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 7
atgcaaacac aaaaaccgac tctcgaacag ctaacctgcg aaggcgctta tcgcgacaat 60
cccaccgcgc tttttcacca gttgtgtggg gatcgtccgg caacgctgct gctggaattt 120
gcagatatcg acagcaaaga tgatttaaaa agcctgctgc tggtagacag tgcgctgcgc 180
attacagctt taggtgacac tgtcacaatc caggcacttt ccggcaacgg cgaagccctg 240
ctggcactac tggataacgc cctgcctgcg ggtgtggaaa gtgaacaatc accaaactgc 300
cgtgtgctgc gcttcccccc tgtcagtcca ctgctggatg aagacgcccg cttatgctcc 360
ctttcggttt ttgacgcttt ccgtttattg cagaatctgt tgaatgtacc gaaggaagaa 420
cgagaagcca tgttcttcgg cggcctgttc tcttatgacc ttgtggcggg atttgaagat 480
ttaccgcaac tgtcagcgga aaataactgc cctgatttct gtttttatct cgctgaaacg 540
ctgatggtga ttgaccatca gaaaaaaagc acccgtattc aggccagcct gtttgctccg 600
aatgaagaag aaaaacaacg tctcactgct cgcctgaacg aactacgtca gcaactgacc 660
gaagccgcgc cgccgctgcc agtggtttcc gtgccgcata tgcgttgtga atgtaatcag 720
agcgatgaag agttcggtgg cgtagtgcgt ttgttgcaaa aagcgattcg cgctggagaa 780
attttccagg tggtgccatc tcgccgtttc tctctgccct gcccgtcacc gctggcggcc 840
tattacgtgc tgaaaaagag taatcccagc ccgtacatgt tttttatgca ggataatgat 900
ttcaccctat ttggcgcgtc gccggaaagc tcgctcaagt atgatgccac cagccgccag 960
attgagatct acccgattgc cggaacacgc ccacgcggtc gtcgcgccga tggttcactg 1020
gacagagatc tcgacagccg tattgaactg gaaatgcgta ccgatcataa agagctgtct 1080
gaacatctga tgctggttga tctcgcccgt aatgatctgg cacgcatttg cacccccggc 1140
agccgctacg tcgccgatct caccaaagtt gaccgttatt cctatgtgat gcacctcgtc 1200
tctcgcgtag tcggcgaact gcgtcacgat cttgacgccc tgcacgctta tcgcgcctgt 1260
atgaatatgg ggacgttaag cggtgcgccg aaagtacgcg ctatgcagtt aattgccgag 1320
gcggaaggtc gtcgccgcgg cagctacggc ggcgcggtag gttatttcac cgcgcatggc 1380
gatctcgaca cctgcattgt gatccgctcg gcgctggtgg aaaacggtat cgccaccgtg 1440
caagcgggtg ctggtgtagt ccttgattct gttccgcagt cggaagccga cgaaacccgt 1500
aacaaagccc gcgctgtact gcgcgctatt gccaccgcgc atcatgcaca ggagactttc 1560
tga 1563
<210> 8
<211> 520
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 8
Met Gln Thr Gln Lys Pro Thr Leu Glu Gln Leu Thr Cys Glu Gly Ala
1 5 10 15
Tyr Arg Asp Asn Pro Thr Ala Leu Phe His Gln Leu Cys Gly Asp Arg
20 25 30
Pro Ala Thr Leu Leu Leu Glu Phe Ala Asp Ile Asp Ser Lys Asp Asp
35 40 45
Leu Lys Ser Leu Leu Leu Val Asp Ser Ala Leu Arg Ile Thr Ala Leu
50 55 60
Gly Asp Thr Val Thr Ile Gln Ala Leu Ser Gly Asn Gly Glu Ala Leu
65 70 75 80
Leu Ala Leu Leu Asp Asn Ala Leu Pro Ala Gly Val Glu Ser Glu Gln
85 90 95
Ser Pro Asn Cys Arg Val Leu Arg Phe Pro Pro Val Ser Pro Leu Leu
100 105 110
Asp Glu Asp Ala Arg Leu Cys Ser Leu Ser Val Phe Asp Ala Phe Arg
115 120 125
Leu Leu Gln Asn Leu Leu Asn Val Pro Lys Glu Glu Arg Glu Ala Met
130 135 140
Phe Phe Gly Gly Leu Phe Ser Tyr Asp Leu Val Ala Gly Phe Glu Asp
145 150 155 160
Leu Pro Gln Leu Ser Ala Glu Asn Asn Cys Pro Asp Phe Cys Phe Tyr
165 170 175
Leu Ala Glu Thr Leu Met Val Ile Asp His Gln Lys Lys Ser Thr Arg
180 185 190
Ile Gln Ala Ser Leu Phe Ala Pro Asn Glu Glu Glu Lys Gln Arg Leu
195 200 205
Thr Ala Arg Leu Asn Glu Leu Arg Gln Gln Leu Thr Glu Ala Ala Pro
210 215 220
Pro Leu Pro Val Val Ser Val Pro His Met Arg Cys Glu Cys Asn Gln
225 230 235 240
Ser Asp Glu Glu Phe Gly Gly Val Val Arg Leu Leu Gln Lys Ala Ile
245 250 255
Arg Ala Gly Glu Ile Phe Gln Val Val Pro Ser Arg Arg Phe Ser Leu
260 265 270
Pro Cys Pro Ser Pro Leu Ala Ala Tyr Tyr Val Leu Lys Lys Ser Asn
275 280 285
Pro Ser Pro Tyr Met Phe Phe Met Gln Asp Asn Asp Phe Thr Leu Phe
290 295 300
Gly Ala Ser Pro Glu Ser Ser Leu Lys Tyr Asp Ala Thr Ser Arg Gln
305 310 315 320
Ile Glu Ile Tyr Pro Ile Ala Gly Thr Arg Pro Arg Gly Arg Arg Ala
325 330 335
Asp Gly Ser Leu Asp Arg Asp Leu Asp Ser Arg Ile Glu Leu Glu Met
340 345 350
Arg Thr Asp His Lys Glu Leu Ser Glu His Leu Met Leu Val Asp Leu
355 360 365
Ala Arg Asn Asp Leu Ala Arg Ile Cys Thr Pro Gly Ser Arg Tyr Val
370 375 380
Ala Asp Leu Thr Lys Val Asp Arg Tyr Ser Tyr Val Met His Leu Val
385 390 395 400
Ser Arg Val Val Gly Glu Leu Arg His Asp Leu Asp Ala Leu His Ala
405 410 415
Tyr Arg Ala Cys Met Asn Met Gly Thr Leu Ser Gly Ala Pro Lys Val
420 425 430
Arg Ala Met Gln Leu Ile Ala Glu Ala Glu Gly Arg Arg Arg Gly Ser
435 440 445
Tyr Gly Gly Ala Val Gly Tyr Phe Thr Ala His Gly Asp Leu Asp Thr
450 455 460
Cys Ile Val Ile Arg Ser Ala Leu Val Glu Asn Gly Ile Ala Thr Val
465 470 475 480
Gln Ala Gly Ala Gly Val Val Leu Asp Ser Val Pro Gln Ser Glu Ala
485 490 495
Asp Glu Thr Arg Asn Lys Ala Arg Ala Val Leu Arg Ala Ile Ala Thr
500 505 510
Ala His His Ala Gln Glu Thr Phe
515 520
<210> 9
<211> 1233
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 9
atggcaaagg tatcgctgga gaaagacaag attaagtttc tgctggtaga aggcgtgcac 60
caaaaggcgc tggaaagcct tcgtgcagct ggttacacca acatcgaatt tcacaaaggc 120
gcgctggatg atgaacaatt aaaagaatcc atccgcgatg cccacttcat cggcctgcga 180
tcccgtaccc atctgactga agacgtgatc aacgccgcag aaaaactggt cgctattggc 240
tgtttctgta tcggaacaaa ccaggttgat ctggatgcgg cggcaaagcg cgggatcccg 300
gtatttaacg caccgttctc aaatacgcgc tctgttgcgg agctggtgat tggcgaactg 360
ctgctgctat tgcgcggcgt gccggaagcc aatgctaaag cgcaccgtgg cgtgtggaac 420
aaactggcgg cgggttcttt tgaagcgcgc ggcaaaaagc tgggtatcat cggctacggt 480
catattggta cgcaattggg cattctggct gaatcgctgg gaatgtatgt ttacttttat 540
gatattgaaa ataaactgcc gctgggcaac gccactcagg tacagcatct ttctgacctg 600
ctgaatatga gcgatgtggt gagtctgcat gtaccagaga atccgtccac caaaaatatg 660
atgggcgcga aagaaatttc actaatgaag cccggctcgc tgctgattaa tgcttcgcgc 720
ggtactgtgg tggatattcc ggcgctgtgt gatgcgctgg cgagcaaaca tctggcgggg 780
gcggcaatcg acgtattccc gacggaaccg gcgaccaata gcgatccatt tacctctccg 840
ctgtgtgaat tcgacaacgt ccttctgacg ccacacattg gcggttcgac tcaggaagcg 900
caggagaata tcggcctgga agttgcgggt aaattgatca agtattctga caatggctca 960
acgctctctg cggtgaactt cccggaagtc tcgctgccac tgcacggtgg gcgtcgtctg 1020
atgcacatcg cagaaaaccg tccgggcgtg ctaactgcgc tgaacaaaat cttcgccgag 1080
cagggcgtcg caatcgccgc gcaatatctg caaacttccg cccagatggg ttatgtggtt 1140
attgatattg aagccgacga agacgttgcc gaaaaagcgc tgcaggcaat gaaagctatt 1200
ccgggtacca ttcgcgcccg tctgctgtac taa 1233
<210> 10
<211> 410
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 10
Met Ala Lys Val Ser Leu Glu Lys Asp Lys Ile Lys Phe Leu Leu Val
1 5 10 15
Glu Gly Val His Gln Lys Ala Leu Glu Ser Leu Arg Ala Ala Gly Tyr
20 25 30
Thr Asn Ile Glu Phe His Lys Gly Ala Leu Asp Asp Glu Gln Leu Lys
35 40 45
Glu Ser Ile Arg Asp Ala His Phe Ile Gly Leu Arg Ser Arg Thr His
50 55 60
Leu Thr Glu Asp Val Ile Asn Ala Ala Glu Lys Leu Val Ala Ile Gly
65 70 75 80
Cys Phe Cys Ile Gly Thr Asn Gln Val Asp Leu Asp Ala Ala Ala Lys
85 90 95
Arg Gly Ile Pro Val Phe Asn Ala Pro Phe Ser Asn Thr Arg Ser Val
100 105 110
Ala Glu Leu Val Ile Gly Glu Leu Leu Leu Leu Leu Arg Gly Val Pro
115 120 125
Glu Ala Asn Ala Lys Ala His Arg Gly Val Trp Asn Lys Leu Ala Ala
130 135 140
Gly Ser Phe Glu Ala Arg Gly Lys Lys Leu Gly Ile Ile Gly Tyr Gly
145 150 155 160
His Ile Gly Thr Gln Leu Gly Ile Leu Ala Glu Ser Leu Gly Met Tyr
165 170 175
Val Tyr Phe Tyr Asp Ile Glu Asn Lys Leu Pro Leu Gly Asn Ala Thr
180 185 190
Gln Val Gln His Leu Ser Asp Leu Leu Asn Met Ser Asp Val Val Ser
195 200 205
Leu His Val Pro Glu Asn Pro Ser Thr Lys Asn Met Met Gly Ala Lys
210 215 220
Glu Ile Ser Leu Met Lys Pro Gly Ser Leu Leu Ile Asn Ala Ser Arg
225 230 235 240
Gly Thr Val Val Asp Ile Pro Ala Leu Cys Asp Ala Leu Ala Ser Lys
245 250 255
His Leu Ala Gly Ala Ala Ile Asp Val Phe Pro Thr Glu Pro Ala Thr
260 265 270
Asn Ser Asp Pro Phe Thr Ser Pro Leu Cys Glu Phe Asp Asn Val Leu
275 280 285
Leu Thr Pro His Ile Gly Gly Ser Thr Gln Glu Ala Gln Glu Asn Ile
290 295 300
Gly Leu Glu Val Ala Gly Lys Leu Ile Lys Tyr Ser Asp Asn Gly Ser
305 310 315 320
Thr Leu Ser Ala Val Asn Phe Pro Glu Val Ser Leu Pro Leu His Gly
325 330 335
Gly Arg Arg Leu Met His Ile Ala Glu Asn Arg Pro Gly Val Leu Thr
340 345 350
Ala Leu Asn Lys Ile Phe Ala Glu Gln Gly Val Ala Ile Ala Ala Gln
355 360 365
Tyr Leu Gln Thr Ser Ala Gln Met Gly Tyr Val Val Ile Asp Ile Glu
370 375 380
Ala Asp Glu Asp Val Ala Glu Lys Ala Leu Gln Ala Met Lys Ala Ile
385 390 395 400
Pro Gly Thr Ile Arg Ala Arg Leu Leu Tyr
405 410
<210> 11
<211> 807
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 11
atggaacgct acgaatctct gtttgcccag ttgaaggagc gcaaagaagg cgcattcgtt 60
cctttcgtca cgctcggtga tccgggcatt gagcagtcat tgaaaattat cgatacgcta 120
attgaagccg gtgctgacgc gctggagtta ggtatcccct tctccgaccc actggcggat 180
ggcccgacga ttcaaaacgc cactctgcgc gcctttgcgg caggtgtgac tccggcacaa 240
tgttttgaaa tgctggcact gattcgccag aaacacccga ccattcccat tggcctgttg 300
atgtatgcca atctggtgtt taacaaaggc attgatgagt tttatgccca gtgcgaaaaa 360
gtcggcgtcg attcggtgct ggttgccgat gtgccagttg aagagtccgc gcccttccgc 420
caggccgcgt tgcgtcataa tgtcgcacct atcttcatct gcccgccaaa tgccgatgac 480
gacctgctgc gccagatagc ctcttacggt cgtggttaca cctatttgct gtcacgagca 540
ggcgtgaccg gcgcagaaaa ccgcgccgcg ttacccctca atcatctggt tgcgaagctg 600
aaagagtaca acgctgcacc tccattgcag ggatttggta tttccgcccc ggatcaggta 660
aaagcagcga ttgatgcagg agctgcgggc gcgatttctg gttcggccat tgttaaaatc 720
atcgagcaac atattaatga gccagagaaa atgctggcgg cactgaaagt ttttgtacaa 780
ccgatgaaag cggcgacgcg cagttaa 807
<210> 12
<211> 268
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 12
Met Glu Arg Tyr Glu Ser Leu Phe Ala Gln Leu Lys Glu Arg Lys Glu
1 5 10 15
Gly Ala Phe Val Pro Phe Val Thr Leu Gly Asp Pro Gly Ile Glu Gln
20 25 30
Ser Leu Lys Ile Ile Asp Thr Leu Ile Glu Ala Gly Ala Asp Ala Leu
35 40 45
Glu Leu Gly Ile Pro Phe Ser Asp Pro Leu Ala Asp Gly Pro Thr Ile
50 55 60
Gln Asn Ala Thr Leu Arg Ala Phe Ala Ala Gly Val Thr Pro Ala Gln
65 70 75 80
Cys Phe Glu Met Leu Ala Leu Ile Arg Gln Lys His Pro Thr Ile Pro
85 90 95
Ile Gly Leu Leu Met Tyr Ala Asn Leu Val Phe Asn Lys Gly Ile Asp
100 105 110
Glu Phe Tyr Ala Gln Cys Glu Lys Val Gly Val Asp Ser Val Leu Val
115 120 125
Ala Asp Val Pro Val Glu Glu Ser Ala Pro Phe Arg Gln Ala Ala Leu
130 135 140
Arg His Asn Val Ala Pro Ile Phe Ile Cys Pro Pro Asn Ala Asp Asp
145 150 155 160
Asp Leu Leu Arg Gln Ile Ala Ser Tyr Gly Arg Gly Tyr Thr Tyr Leu
165 170 175
Leu Ser Arg Ala Gly Val Thr Gly Ala Glu Asn Arg Ala Ala Leu Pro
180 185 190
Leu Asn His Leu Val Ala Lys Leu Lys Glu Tyr Asn Ala Ala Pro Pro
195 200 205
Leu Gln Gly Phe Gly Ile Ser Ala Pro Asp Gln Val Lys Ala Ala Ile
210 215 220
Asp Ala Gly Ala Ala Gly Ala Ile Ser Gly Ser Ala Ile Val Lys Ile
225 230 235 240
Ile Glu Gln His Ile Asn Glu Pro Glu Lys Met Leu Ala Ala Leu Lys
245 250 255
Val Phe Val Gln Pro Met Lys Ala Ala Thr Arg Ser
260 265
<210> 13
<211> 1194
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 13
atgacaacat tacttaaccc ctattttggt gagtttggcg gcatgtacgt gccacaaatc 60
ctgatgcctg ctctgcgcca gctggaagaa gcttttgtca gtgcgcaaaa agatcctgaa 120
tttcaggctc agttcaacga cctgctgaaa aactatgccg ggcgtccaac cgcgctgacc 180
aaatgccaga acattacagc cgggacgaac accacgctgt atctcaagcg tgaagatttg 240
ctgcacggcg gcgcgcataa aactaaccag gtgctggggc aggcgttgct ggcgaagcgg 300
atgggtaaaa ccgaaatcat cgccgaaacc ggtgccggtc agcatggcgt ggcgtcggcc 360
cttgccagcg ccctgctcgg cctgaaatgc cgtatttata tgggtgccaa agacgttgaa 420
cgccagtcgc ctaacgtttt tcgtatgcgc ttaatgggtg cggaagtgat cccggtgcat 480
agcggttccg cgacgctgaa agatgcctgt aacgaggcgc tgcgcgactg gtccggtagt 540
tacgaaaccg cgcactatat gctgggcacc gcagctggcc cgcatcctta tccgaccatt 600
gtgcgtgagt ttcagcggat gattggcgaa gaaaccaaag cgcagattct ggaaagagaa 660
ggtcgcctgc cggatgccgt tatcgcctgt gttggcggcg gttcgaatgc catcggcatg 720
tttgctgatt tcatcaatga aaccaacgtc ggcctgattg gtgtggagcc aggtggtcac 780
ggtatcgaaa ctggcgagca cggcgcaccg ctaaaacatg gtcgcgtggg tatctatttc 840
ggtatgaaag cgccgatgat gcaaaccgaa gacgggcaga ttgaagaatc ttactccatc 900
tccgccggac tggatttccc gtctgtcggc ccacaacacg cgtatcttaa cagcactgga 960
cgcgctgatt acgtgtctat taccgatgat gaagcccttg aagccttcaa aacgctgtgc 1020
ctgcacgaag ggatcatccc ggcgctggaa tcctcccacg ccctggccca tgcgttgaaa 1080
atgatgcgcg aaaacccgga taaagagcag ctactggtgg ttaacctttc cggtcgcggc 1140
gataaagaca tcttcaccgt tcacgatatt ttgaaagcac gaggggaaat ctga 1194
<210> 14
<211> 397
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 14
Met Thr Thr Leu Leu Asn Pro Tyr Phe Gly Glu Phe Gly Gly Met Tyr
1 5 10 15
Val Pro Gln Ile Leu Met Pro Ala Leu Arg Gln Leu Glu Glu Ala Phe
20 25 30
Val Ser Ala Gln Lys Asp Pro Glu Phe Gln Ala Gln Phe Asn Asp Leu
35 40 45
Leu Lys Asn Tyr Ala Gly Arg Pro Thr Ala Leu Thr Lys Cys Gln Asn
50 55 60
Ile Thr Ala Gly Thr Asn Thr Thr Leu Tyr Leu Lys Arg Glu Asp Leu
65 70 75 80
Leu His Gly Gly Ala His Lys Thr Asn Gln Val Leu Gly Gln Ala Leu
85 90 95
Leu Ala Lys Arg Met Gly Lys Thr Glu Ile Ile Ala Glu Thr Gly Ala
100 105 110
Gly Gln His Gly Val Ala Ser Ala Leu Ala Ser Ala Leu Leu Gly Leu
115 120 125
Lys Cys Arg Ile Tyr Met Gly Ala Lys Asp Val Glu Arg Gln Ser Pro
130 135 140
Asn Val Phe Arg Met Arg Leu Met Gly Ala Glu Val Ile Pro Val His
145 150 155 160
Ser Gly Ser Ala Thr Leu Lys Asp Ala Cys Asn Glu Ala Leu Arg Asp
165 170 175
Trp Ser Gly Ser Tyr Glu Thr Ala His Tyr Met Leu Gly Thr Ala Ala
180 185 190
Gly Pro His Pro Tyr Pro Thr Ile Val Arg Glu Phe Gln Arg Met Ile
195 200 205
Gly Glu Glu Thr Lys Ala Gln Ile Leu Glu Arg Glu Gly Arg Leu Pro
210 215 220
Asp Ala Val Ile Ala Cys Val Gly Gly Gly Ser Asn Ala Ile Gly Met
225 230 235 240
Phe Ala Asp Phe Ile Asn Glu Thr Asn Val Gly Leu Ile Gly Val Glu
245 250 255
Pro Gly Gly His Gly Ile Glu Thr Gly Glu His Gly Ala Pro Leu Lys
260 265 270
His Gly Arg Val Gly Ile Tyr Phe Gly Met Lys Ala Pro Met Met Gln
275 280 285
Thr Glu Asp Gly Gln Ile Glu Glu Ser Tyr Ser Ile Ser Ala Gly Leu
290 295 300
Asp Phe Pro Ser Val Gly Pro Gln His Ala Tyr Leu Asn Ser Thr Gly
305 310 315 320
Arg Ala Asp Tyr Val Ser Ile Thr Asp Asp Glu Ala Leu Glu Ala Phe
325 330 335
Lys Thr Leu Cys Leu His Glu Gly Ile Ile Pro Ala Leu Glu Ser Ser
340 345 350
His Ala Leu Ala His Ala Leu Lys Met Met Arg Glu Asn Pro Asp Lys
355 360 365
Glu Gln Leu Leu Val Val Asn Leu Ser Gly Arg Gly Asp Lys Asp Ile
370 375 380
Phe Thr Val His Asp Ile Leu Lys Ala Arg Gly Glu Ile
385 390 395
<210> 15
<211> 1362
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 15
atgatgcaaa ccgttttagc gaaaatcgtc gcagacaagg cgatttgggt agaagcccgc 60
aaacagcagc aaccgctggc cagttttcag aatgaggttc agccgagcac gcgacatttt 120
tatgatgcgc tacagggtgc gcgcacggcg tttattctgg agtgcaagaa agcgtcgccg 180
tcaaaaggcg tgatccgtga tgatttcgat ccagcacgca ttgccgccat ttataaacat 240
tacgcttcgg caatttcggt gctgactgat gagaaatatt ttcaggggag ctttaatttc 300
ctccccatcg tcagccaaat cgccccgcag ccgattttat gtaaagactt cattatcgac 360
ccttaccaga tctatctggc gcgctattac caggccgatg cctgcttatt aatgctttca 420
gtactggatg acgaccaata tcgccagctt gccgccgtcg ctcacagtct ggagatgggg 480
gtgctgaccg aagtcagtaa tgaagaggaa caggagcgcg ccattgcatt gggagcaaag 540
gtcgttggca tcaacaaccg cgatctgcgt gatttgtcga ttgatctcaa ccgtacccgc 600
gagcttgcgc cgaaactggg gcacaacgtg acggtaatca gcgaatccgg catcaatact 660
tacgctcagg tgcgcgagtt aagccacttc gctaacggtt ttctgattgg ttcggcgttg 720
atggcccatg acgatttgca cgccgccgtg cgccgggtgt tgctgggtga gaataaagta 780
tgtggcctga cgcgtgggca agatgctaaa gcagcttatg acgcgggcgc gatttacggt 840
gggttgattt ttgttgcgac atcaccgcgt tgcgtcaacg ttgaacaggc gcaggaagtg 900
atggctgcgg caccgttgca gtatgttggc gtgttccgca atcacgatat tgccgatgtg 960
gtggacaaag ctaaggtgtt atcgctggcg gcagtgcaac tgcatggtaa tgaagaacag 1020
ctgtatatcg atacgctgcg tgaagctctg ccagcacatg ttgccatctg gaaagcatta 1080
agcgtcggtg aaaccctgcc cgcccgcgag tttcagcacg ttgataaata tgttttagac 1140
aacggccagg gtggaagcgg gcaacgtttt gactggtcac tattaaatgg tcaatcgctt 1200
ggcaacgttc tgctggcggg gggcttaggc gcagataact gcgtggaagc ggcacaaacc 1260
ggctgcgccg gacttgattt taattctgct gtagagtcgc aaccgggcat caaagacgca 1320
cgtcttttgg cctcggtttt ccagacgctg cgcgcatatt aa 1362
<210> 16
<211> 453
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 16
Met Met Gln Thr Val Leu Ala Lys Ile Val Ala Asp Lys Ala Ile Trp
1 5 10 15
Val Glu Ala Arg Lys Gln Gln Gln Pro Leu Ala Ser Phe Gln Asn Glu
20 25 30
Val Gln Pro Ser Thr Arg His Phe Tyr Asp Ala Leu Gln Gly Ala Arg
35 40 45
Thr Ala Phe Ile Leu Glu Cys Lys Lys Ala Ser Pro Ser Lys Gly Val
50 55 60
Ile Arg Asp Asp Phe Asp Pro Ala Arg Ile Ala Ala Ile Tyr Lys His
65 70 75 80
Tyr Ala Ser Ala Ile Ser Val Leu Thr Asp Glu Lys Tyr Phe Gln Gly
85 90 95
Ser Phe Asn Phe Leu Pro Ile Val Ser Gln Ile Ala Pro Gln Pro Ile
100 105 110
Leu Cys Lys Asp Phe Ile Ile Asp Pro Tyr Gln Ile Tyr Leu Ala Arg
115 120 125
Tyr Tyr Gln Ala Asp Ala Cys Leu Leu Met Leu Ser Val Leu Asp Asp
130 135 140
Asp Gln Tyr Arg Gln Leu Ala Ala Val Ala His Ser Leu Glu Met Gly
145 150 155 160
Val Leu Thr Glu Val Ser Asn Glu Glu Glu Gln Glu Arg Ala Ile Ala
165 170 175
Leu Gly Ala Lys Val Val Gly Ile Asn Asn Arg Asp Leu Arg Asp Leu
180 185 190
Ser Ile Asp Leu Asn Arg Thr Arg Glu Leu Ala Pro Lys Leu Gly His
195 200 205
Asn Val Thr Val Ile Ser Glu Ser Gly Ile Asn Thr Tyr Ala Gln Val
210 215 220
Arg Glu Leu Ser His Phe Ala Asn Gly Phe Leu Ile Gly Ser Ala Leu
225 230 235 240
Met Ala His Asp Asp Leu His Ala Ala Val Arg Arg Val Leu Leu Gly
245 250 255
Glu Asn Lys Val Cys Gly Leu Thr Arg Gly Gln Asp Ala Lys Ala Ala
260 265 270
Tyr Asp Ala Gly Ala Ile Tyr Gly Gly Leu Ile Phe Val Ala Thr Ser
275 280 285
Pro Arg Cys Val Asn Val Glu Gln Ala Gln Glu Val Met Ala Ala Ala
290 295 300
Pro Leu Gln Tyr Val Gly Val Phe Arg Asn His Asp Ile Ala Asp Val
305 310 315 320
Val Asp Lys Ala Lys Val Leu Ser Leu Ala Ala Val Gln Leu His Gly
325 330 335
Asn Glu Glu Gln Leu Tyr Ile Asp Thr Leu Arg Glu Ala Leu Pro Ala
340 345 350
His Val Ala Ile Trp Lys Ala Leu Ser Val Gly Glu Thr Leu Pro Ala
355 360 365
Arg Glu Phe Gln His Val Asp Lys Tyr Val Leu Asp Asn Gly Gln Gly
370 375 380
Gly Ser Gly Gln Arg Phe Asp Trp Ser Leu Leu Asn Gly Gln Ser Leu
385 390 395 400
Gly Asn Val Leu Leu Ala Gly Gly Leu Gly Ala Asp Asn Cys Val Glu
405 410 415
Ala Ala Gln Thr Gly Cys Ala Gly Leu Asp Phe Asn Ser Ala Val Glu
420 425 430
Ser Gln Pro Gly Ile Lys Asp Ala Arg Leu Leu Ala Ser Val Phe Gln
435 440 445
Thr Leu Arg Ala Tyr
450
<210> 17
<211> 1596
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 17
atggctgaca ttctgctgct cgataatatc gactctttta cgtacaacct ggcagatcag 60
ttgcgcagca atgggcataa cgtggtgatt taccgcaacc atattccggc gcaaacctta 120
attgaacgcc tggcgaccat gagcaatccg gtgctgatgc tttctcctgg ccccggtgtg 180
ccgagcgaag ccggttgtat gccggaactc ctcacccgct tgcgtggcaa gctgcccatt 240
attggcattt gcctcggaca tcaggcgatt gtcgaagctt acgggggcta tgtcggtcag 300
gcgggcgaaa ttctccacgg taaagcctcc agcattgaac atgacggtca ggcgatgttt 360
gccggattaa caaacccgct gccggtggcg cgttatcact cgctggttgg cagtaacatt 420
ccggccggtt taaccatcaa cgcccatttt aatggcatgg tgatggcagt acgtcacgat 480
gcggatcgcg tttgtggatt ccagttccat ccggaatcca ttctcaccac ccagggcgct 540
cgcctgctgg aacaaacgct ggcctgggcg cagcagaaac tagagccagc caacacgctg 600
caaccgattc tggaaaaact gtatcaggcg cagacgctta gccaacaaga aagccaccag 660
ctgttttcag cggtggtgcg tggcgagctg aagccggaac aactggcggc ggcgctggtg 720
agcatgaaaa ttcgcggtga gcacccgaac gagatcgccg gggcagcaac cgcgctactg 780
gaaaacgcag cgccgttccc gcgcccggat tatctgtttg ctgatatcgt cggtactggc 840
ggtgacggca gcaacagtat caatatttct accgccagtg cgtttgtcgc cgcggcctgt 900
gggctgaaag tggcgaaaca cggcaaccgt agcgtctcca gtaaatctgg ttcgtccgat 960
ctgctggcgg cgttcggtat taatcttgat atgaacgccg ataaatcgcg ccaggcgctg 1020
gatgagttag gtgtatgttt cctctttgcg ccgaagtatc acaccggatt ccgccacgcg 1080
atgccggttc gccagcaact gaaaacccgc accctgttca atgtgctggg gccattgatt 1140
aacccggcgc atccgccgct ggcgttaatt ggtgtttata gtccggaact ggtgctgccg 1200
attgccgaaa ccttgcgcgt gctggggtat caacgcgcgg cggtggtgca cagcggcggg 1260
atggatgaag tttcattaca cgcgccgaca atcgttgccg aactgcatga cggcgaaatt 1320
aaaagctatc agctcaccgc agaagacttt ggcctgacac cctaccacca ggagcaactg 1380
gcaggcggaa caccggaaga aaaccgtgac attttaacac gtttgttaca aggtaaaggc 1440
gacgccgccc atgaagcagc cgtcgctgcg aacgtcgcca tgttaatgcg cctgcatggc 1500
catgaagatc tgcaagccaa tgcgcaaacc gttcttgagg tactgcgcag tggttccgct 1560
tacgacagag tcaccgcact ggcggcacga gggtaa 1596
<210> 18
<211> 531
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 18
Met Ala Asp Ile Leu Leu Leu Asp Asn Ile Asp Ser Phe Thr Tyr Asn
1 5 10 15
Leu Ala Asp Gln Leu Arg Ser Asn Gly His Asn Val Val Ile Tyr Arg
20 25 30
Asn His Ile Pro Ala Gln Thr Leu Ile Glu Arg Leu Ala Thr Met Ser
35 40 45
Asn Pro Val Leu Met Leu Ser Pro Gly Pro Gly Val Pro Ser Glu Ala
50 55 60
Gly Cys Met Pro Glu Leu Leu Thr Arg Leu Arg Gly Lys Leu Pro Ile
65 70 75 80
Ile Gly Ile Cys Leu Gly His Gln Ala Ile Val Glu Ala Tyr Gly Gly
85 90 95
Tyr Val Gly Gln Ala Gly Glu Ile Leu His Gly Lys Ala Ser Ser Ile
100 105 110
Glu His Asp Gly Gln Ala Met Phe Ala Gly Leu Thr Asn Pro Leu Pro
115 120 125
Val Ala Arg Tyr His Ser Leu Val Gly Ser Asn Ile Pro Ala Gly Leu
130 135 140
Thr Ile Asn Ala His Phe Asn Gly Met Val Met Ala Val Arg His Asp
145 150 155 160
Ala Asp Arg Val Cys Gly Phe Gln Phe His Pro Glu Ser Ile Leu Thr
165 170 175
Thr Gln Gly Ala Arg Leu Leu Glu Gln Thr Leu Ala Trp Ala Gln Gln
180 185 190
Lys Leu Glu Pro Ala Asn Thr Leu Gln Pro Ile Leu Glu Lys Leu Tyr
195 200 205
Gln Ala Gln Thr Leu Ser Gln Gln Glu Ser His Gln Leu Phe Ser Ala
210 215 220
Val Val Arg Gly Glu Leu Lys Pro Glu Gln Leu Ala Ala Ala Leu Val
225 230 235 240
Ser Met Lys Ile Arg Gly Glu His Pro Asn Glu Ile Ala Gly Ala Ala
245 250 255
Thr Ala Leu Leu Glu Asn Ala Ala Pro Phe Pro Arg Pro Asp Tyr Leu
260 265 270
Phe Ala Asp Ile Val Gly Thr Gly Gly Asp Gly Ser Asn Ser Ile Asn
275 280 285
Ile Ser Thr Ala Ser Ala Phe Val Ala Ala Ala Cys Gly Leu Lys Val
290 295 300
Ala Lys His Gly Asn Arg Ser Val Ser Ser Lys Ser Gly Ser Ser Asp
305 310 315 320
Leu Leu Ala Ala Phe Gly Ile Asn Leu Asp Met Asn Ala Asp Lys Ser
325 330 335
Arg Gln Ala Leu Asp Glu Leu Gly Val Cys Phe Leu Phe Ala Pro Lys
340 345 350
Tyr His Thr Gly Phe Arg His Ala Met Pro Val Arg Gln Gln Leu Lys
355 360 365
Thr Arg Thr Leu Phe Asn Val Leu Gly Pro Leu Ile Asn Pro Ala His
370 375 380
Pro Pro Leu Ala Leu Ile Gly Val Tyr Ser Pro Glu Leu Val Leu Pro
385 390 395 400
Ile Ala Glu Thr Leu Arg Val Leu Gly Tyr Gln Arg Ala Ala Val Val
405 410 415
His Ser Gly Gly Met Asp Glu Val Ser Leu His Ala Pro Thr Ile Val
420 425 430
Ala Glu Leu His Asp Gly Glu Ile Lys Ser Tyr Gln Leu Thr Ala Glu
435 440 445
Asp Phe Gly Leu Thr Pro Tyr His Gln Glu Gln Leu Ala Gly Gly Thr
450 455 460
Pro Glu Glu Asn Arg Asp Ile Leu Thr Arg Leu Leu Gln Gly Lys Gly
465 470 475 480
Asp Ala Ala His Glu Ala Ala Val Ala Ala Asn Val Ala Met Leu Met
485 490 495
Arg Leu His Gly His Glu Asp Leu Gln Ala Asn Ala Gln Thr Val Leu
500 505 510
Glu Val Leu Arg Ser Gly Ser Ala Tyr Asp Arg Val Thr Ala Leu Ala
515 520 525
Ala Arg Gly
530
<210> 19
<211> 1908
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 19
atggcacctg ttacaattga aaagttcgta aatcaagaag aacgacacct tgtttccaac 60
cgatcagcaa caattccgtt tggtgaatac atatttaaaa gattgttgtc catcgatacg 120
aaatcagttt tcggtgttcc tggtgacttc aacttatctc tattagaata tctctattca 180
cctagtgttg aatcagctgg cctaagatgg gtcggcacgt gtaatgaact gaacgccgct 240
tatgcggccg acggatattc ccgttactct aataagattg gctgtttaat aaccacgtat 300
ggcgttggtg aattaagcgc cttgaacggt atagccggtt cgttcgctga aaatgtcaaa 360
gttttgcaca ttgttggtgt ggccaagtcc atagattcgc gttcaagtaa ctttagtgat 420
cggaacctac atcatttggt cccacagcta catgattcaa attttaaagg gccaaatcat 480
aaagtatatc atgatatggt aaaagataga gtcgcttgct cggtagccta cttggaggat 540
attgaaactg catgtgacca agtcgataat gttatccgcg atatttacaa gtattctaaa 600
cctggttata tttttgttcc tgcagatttt gcggatatgt ctgttacatg tgataatttg 660
gttaatgttc cacgtatatc tcaacaagat tgtatagtat acccttctga aaaccaattg 720
tctgacataa tcaacaagat tactagttgg atatattcca gtaaaacacc tgcgatcctt 780
ggagacgtac tgactgatag gtatggtgtg agtaactttt tgaacaagct tatctgcaaa 840
actgggattt ggaatttttc cactgttatg ggaaaatctg taattgatga gtcaaaccca 900
acttatatgg gtcaatataa tggtaaagaa ggtttaaaac aagtctatga acattttgaa 960
ctgtgcgact tggtcttgca ttttggagtc gacatcaatg aaattaataa tgggcattat 1020
acttttactt ataaaccaaa tgctaaaatc attcaatttc atccgaatta tattcgcctt 1080
gtggacacta ggcagggcaa tgagcaaatg ttcaaaggaa tcaattttgc ccctatttta 1140
aaagaactat acaagcgcat tgacgtttct aaactttctt tgcaatatga ttcaaatgta 1200
actcaatata cgaacgaaac aatgcggtta gaagatccta ccaatggaca atcaagcatt 1260
attacacaag ttcacttaca aaagacgatg cctaaatttt tgaaccctgg tgatgttgtc 1320
gtttgtgaaa caggctcttt tcaattctct gttcgtgatt tcgcgtttcc ttcgcaatta 1380
aaatatatat cgcaaggatt tttcctttcc attggcatgg cccttcctgc cgccctaggt 1440
gttggaattg ccatgcaaga ccactcaaac gctcacatca atggtggcaa cgtaaaagag 1500
gactataagc caagattaat tttgtttgaa ggtgacggtg cagcacagat gacaatccaa 1560
gaactgagca ccattctgaa gtgcaatatt ccactagaag ttatcatttg gaacaataac 1620
ggctacacta ttgaaagagc catcatgggc cctaccaggt cgtataacga cgttatgtct 1680
tggaaatgga ccaaactatt tgaagcattc ggagacttcg acggaaagta tactaatagc 1740
actctcattc aatgtccctc taaattagca ctgaaattgg aggagcttaa gaattcaaac 1800
aaaagaagcg ggatagaact tttagaagtc aaattaggcg aattggattt ccccgaacag 1860
ctaaagtgca tggttgaagc agcggcactt aaaagaaata aaaaatag 1908
<210> 20
<211> 635
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 20
Met Ala Pro Val Thr Ile Glu Lys Phe Val Asn Gln Glu Glu Arg His
1 5 10 15
Leu Val Ser Asn Arg Ser Ala Thr Ile Pro Phe Gly Glu Tyr Ile Phe
20 25 30
Lys Arg Leu Leu Ser Ile Asp Thr Lys Ser Val Phe Gly Val Pro Gly
35 40 45
Asp Phe Asn Leu Ser Leu Leu Glu Tyr Leu Tyr Ser Pro Ser Val Glu
50 55 60
Ser Ala Gly Leu Arg Trp Val Gly Thr Cys Asn Glu Leu Asn Ala Ala
65 70 75 80
Tyr Ala Ala Asp Gly Tyr Ser Arg Tyr Ser Asn Lys Ile Gly Cys Leu
85 90 95
Ile Thr Thr Tyr Gly Val Gly Glu Leu Ser Ala Leu Asn Gly Ile Ala
100 105 110
Gly Ser Phe Ala Glu Asn Val Lys Val Leu His Ile Val Gly Val Ala
115 120 125
Lys Ser Ile Asp Ser Arg Ser Ser Asn Phe Ser Asp Arg Asn Leu His
130 135 140
His Leu Val Pro Gln Leu His Asp Ser Asn Phe Lys Gly Pro Asn His
145 150 155 160
Lys Val Tyr His Asp Met Val Lys Asp Arg Val Ala Cys Ser Val Ala
165 170 175
Tyr Leu Glu Asp Ile Glu Thr Ala Cys Asp Gln Val Asp Asn Val Ile
180 185 190
Arg Asp Ile Tyr Lys Tyr Ser Lys Pro Gly Tyr Ile Phe Val Pro Ala
195 200 205
Asp Phe Ala Asp Met Ser Val Thr Cys Asp Asn Leu Val Asn Val Pro
210 215 220
Arg Ile Ser Gln Gln Asp Cys Ile Val Tyr Pro Ser Glu Asn Gln Leu
225 230 235 240
Ser Asp Ile Ile Asn Lys Ile Thr Ser Trp Ile Tyr Ser Ser Lys Thr
245 250 255
Pro Ala Ile Leu Gly Asp Val Leu Thr Asp Arg Tyr Gly Val Ser Asn
260 265 270
Phe Leu Asn Lys Leu Ile Cys Lys Thr Gly Ile Trp Asn Phe Ser Thr
275 280 285
Val Met Gly Lys Ser Val Ile Asp Glu Ser Asn Pro Thr Tyr Met Gly
290 295 300
Gln Tyr Asn Gly Lys Glu Gly Leu Lys Gln Val Tyr Glu His Phe Glu
305 310 315 320
Leu Cys Asp Leu Val Leu His Phe Gly Val Asp Ile Asn Glu Ile Asn
325 330 335
Asn Gly His Tyr Thr Phe Thr Tyr Lys Pro Asn Ala Lys Ile Ile Gln
340 345 350
Phe His Pro Asn Tyr Ile Arg Leu Val Asp Thr Arg Gln Gly Asn Glu
355 360 365
Gln Met Phe Lys Gly Ile Asn Phe Ala Pro Ile Leu Lys Glu Leu Tyr
370 375 380
Lys Arg Ile Asp Val Ser Lys Leu Ser Leu Gln Tyr Asp Ser Asn Val
385 390 395 400
Thr Gln Tyr Thr Asn Glu Thr Met Arg Leu Glu Asp Pro Thr Asn Gly
405 410 415
Gln Ser Ser Ile Ile Thr Gln Val His Leu Gln Lys Thr Met Pro Lys
420 425 430
Phe Leu Asn Pro Gly Asp Val Val Val Cys Glu Thr Gly Ser Phe Gln
435 440 445
Phe Ser Val Arg Asp Phe Ala Phe Pro Ser Gln Leu Lys Tyr Ile Ser
450 455 460
Gln Gly Phe Phe Leu Ser Ile Gly Met Ala Leu Pro Ala Ala Leu Gly
465 470 475 480
Val Gly Ile Ala Met Gln Asp His Ser Asn Ala His Ile Asn Gly Gly
485 490 495
Asn Val Lys Glu Asp Tyr Lys Pro Arg Leu Ile Leu Phe Glu Gly Asp
500 505 510
Gly Ala Ala Gln Met Thr Ile Gln Glu Leu Ser Thr Ile Leu Lys Cys
515 520 525
Asn Ile Pro Leu Glu Val Ile Ile Trp Asn Asn Asn Gly Tyr Thr Ile
530 535 540
Glu Arg Ala Ile Met Gly Pro Thr Arg Ser Tyr Asn Asp Val Met Ser
545 550 555 560
Trp Lys Trp Thr Lys Leu Phe Glu Ala Phe Gly Asp Phe Asp Gly Lys
565 570 575
Tyr Thr Asn Ser Thr Leu Ile Gln Cys Pro Ser Lys Leu Ala Leu Lys
580 585 590
Leu Glu Glu Leu Lys Asn Ser Asn Lys Arg Ser Gly Ile Glu Leu Leu
595 600 605
Glu Val Lys Leu Gly Glu Leu Asp Phe Pro Glu Gln Leu Lys Cys Met
610 615 620
Val Glu Ala Ala Ala Leu Lys Arg Asn Lys Lys
625 630 635
<210> 21
<211> 1488
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 21
atgaattttc atcatctggc ttactggcag gataaagcgt taagtctcgc cattgaaaac 60
cgcttattta ttaacggtga atatactgct gcggcggaaa atgaaacctt tgaaaccgtt 120
gatccggtca cccaggcacc gctggcgaaa attgcccgcg gcaagagcgt cgatatcgac 180
cgtgcgatga gcgcagcacg cggcgtattt gaacgcggcg actggtcact ctcttctccg 240
gctaaacgta aagcggtact gaataaactc gccgatttaa tggaagccca cgccgaagag 300
ctggcactgc tggaaactct cgacaccggc aaaccgattc gtcacagtct gcgtgatgat 360
attcccggcg cggcgcgcgc cattcgctgg tacgccgaag cgatcgacaa agtgtatggc 420
gaagtggcga ccaccagtag ccatgagctg gcgatgatcg tgcgtgaacc ggtcggcgtg 480
attgccgcca tcgtgccgtg gaacttcccg ctgttgctga cttgctggaa actcggcccg 540
gcgctggcgg cgggaaacag cgtgattcta aaaccgtctg aaaaatcacc gctcagtgcg 600
attcgtctcg cggggctggc gaaagaagca ggcttgccgg atggtgtgtt gaacgtggtg 660
acgggttttg gtcatgaagc cgggcaggcg ctgtcgcgtc ataacgatat cgacgccatt 720
gcctttaccg gttcaacccg taccgggaaa cagctgctga aagatgcggg cgacagcaac 780
atgaaacgcg tctggctgga agcgggcggc aaaagcgcca acatcgtttt cgctgactgc 840
ccggatttgc aacaggcggc aagcgccacc gcagcaggca ttttctacaa ccagggacag 900
gtgtgcatcg ccggaacgcg cctgttgctg gaagagagca tcgccgatga attcttagcc 960
ctgttaaaac agcaggcgca aaactggcag ccgggccatc cacttgatcc cgcaaccacc 1020
atgggcacct taatcgactg cgcccacgcc gactcggtcc atagctttat tcgggaaggc 1080
gaaagcaaag ggcaactgtt gttggatggc cgtaacgccg ggctggctgc cgccatcggc 1140
ccgaccatct ttgtggatgt ggacccgaat gcgtccttaa gtcgcgaaga gattttcggt 1200
ccggtgctgg tggtcacgcg tttcacatca gaagaacagg cgctacagct tgccaacgac 1260
agccagtacg gccttggcgc ggcggtatgg acgcgcgacc tctcccgcgc gcaccgcatg 1320
agccgacgcc tgaaagccgg ttccgtcttc gtcaataact acaacgacgg cgatatgacc 1380
gtgccgtttg gcggctataa gcagagcggc aacggtcgcg acaaatccct gcatgccctt 1440
gaaaaattca ctgaactgaa aaccatctgg ataagcctgg aggcctga 1488
<210> 22
<211> 495
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 22
Met Asn Phe His His Leu Ala Tyr Trp Gln Asp Lys Ala Leu Ser Leu
1 5 10 15
Ala Ile Glu Asn Arg Leu Phe Ile Asn Gly Glu Tyr Thr Ala Ala Ala
20 25 30
Glu Asn Glu Thr Phe Glu Thr Val Asp Pro Val Thr Gln Ala Pro Leu
35 40 45
Ala Lys Ile Ala Arg Gly Lys Ser Val Asp Ile Asp Arg Ala Met Ser
50 55 60
Ala Ala Arg Gly Val Phe Glu Arg Gly Asp Trp Ser Leu Ser Ser Pro
65 70 75 80
Ala Lys Arg Lys Ala Val Leu Asn Lys Leu Ala Asp Leu Met Glu Ala
85 90 95
His Ala Glu Glu Leu Ala Leu Leu Glu Thr Leu Asp Thr Gly Lys Pro
100 105 110
Ile Arg His Ser Leu Arg Asp Asp Ile Pro Gly Ala Ala Arg Ala Ile
115 120 125
Arg Trp Tyr Ala Glu Ala Ile Asp Lys Val Tyr Gly Glu Val Ala Thr
130 135 140
Thr Ser Ser His Glu Leu Ala Met Ile Val Arg Glu Pro Val Gly Val
145 150 155 160
Ile Ala Ala Ile Val Pro Trp Asn Phe Pro Leu Leu Leu Thr Cys Trp
165 170 175
Lys Leu Gly Pro Ala Leu Ala Ala Gly Asn Ser Val Ile Leu Lys Pro
180 185 190
Ser Glu Lys Ser Pro Leu Ser Ala Ile Arg Leu Ala Gly Leu Ala Lys
195 200 205
Glu Ala Gly Leu Pro Asp Gly Val Leu Asn Val Val Thr Gly Phe Gly
210 215 220
His Glu Ala Gly Gln Ala Leu Ser Arg His Asn Asp Ile Asp Ala Ile
225 230 235 240
Ala Phe Thr Gly Ser Thr Arg Thr Gly Lys Gln Leu Leu Lys Asp Ala
245 250 255
Gly Asp Ser Asn Met Lys Arg Val Trp Leu Glu Ala Gly Gly Lys Ser
260 265 270
Ala Asn Ile Val Phe Ala Asp Cys Pro Asp Leu Gln Gln Ala Ala Ser
275 280 285
Ala Thr Ala Ala Gly Ile Phe Tyr Asn Gln Gly Gln Val Cys Ile Ala
290 295 300
Gly Thr Arg Leu Leu Leu Glu Glu Ser Ile Ala Asp Glu Phe Leu Ala
305 310 315 320
Leu Leu Lys Gln Gln Ala Gln Asn Trp Gln Pro Gly His Pro Leu Asp
325 330 335
Pro Ala Thr Thr Met Gly Thr Leu Ile Asp Cys Ala His Ala Asp Ser
340 345 350
Val His Ser Phe Ile Arg Glu Gly Glu Ser Lys Gly Gln Leu Leu Leu
355 360 365
Asp Gly Arg Asn Ala Gly Leu Ala Ala Ala Ile Gly Pro Thr Ile Phe
370 375 380
Val Asp Val Asp Pro Asn Ala Ser Leu Ser Arg Glu Glu Ile Phe Gly
385 390 395 400
Pro Val Leu Val Val Thr Arg Phe Thr Ser Glu Glu Gln Ala Leu Gln
405 410 415
Leu Ala Asn Asp Ser Gln Tyr Gly Leu Gly Ala Ala Val Trp Thr Arg
420 425 430
Asp Leu Ser Arg Ala His Arg Met Ser Arg Arg Leu Lys Ala Gly Ser
435 440 445
Val Phe Val Asn Asn Tyr Asn Asp Gly Asp Met Thr Val Pro Phe Gly
450 455 460
Gly Tyr Lys Gln Ser Gly Asn Gly Arg Asp Lys Ser Leu His Ala Leu
465 470 475 480
Glu Lys Phe Thr Glu Leu Lys Thr Ile Trp Ile Ser Leu Glu Ala
485 490 495
<210> 23
<211> 1503
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 23
atgactttac ctgaatcaaa agacttttct tacttgtttt cggatgaaac caatgctcgt 60
aaaccatccc cattgaaaac ctgcatccat cttttccaag atcctaacat tatctttttg 120
ggtggtggcc tgccattaaa agattatttc ccatgggata atctatctgt agattcaccc 180
aagcctcctt ttccccaggg tattggagct ccaattgacg agcagaattg cataaaatac 240
accgtcaaca aagattacgc tgataaaagt gccaatcctt ccaacgatat tcctttgtca 300
agagctttgc aatacgggtt cagtgctggt caacctgaac tattaaactt cattagagat 360
cataccaaga ttatccacga tttgaagtat aaggactggg acgttttagc cactgcaggt 420
aacacaaatg cctgggaatc tactttaaga gtcttttgta accgaggtga tgtcatctta 480
gttgaggcac attctttttc ctcttcattg gcttctgcag aggctcaagg tgtcattacc 540
ttccccgtgc caattgacgc tgatggtatc attcctgaaa aattagctaa agtcatggaa 600
aactggacac ctggtgctcc taaaccaaag ttgttataca ctattccaac gggccaaaat 660
ccaactggta cttccattgc agaccataga aaggaggcaa tttacaagat cgctcaaaag 720
tacgacttcc taattgtgga agatgaacct tattatttct tacaaatgaa tccctacatc 780
aaagacttga aggaaagaga gaaggcacaa agttctccaa agcaggacca tgacgaattt 840
ttgaagtcct tggcaaacac tttcctttcc ttggatacag aaggccgtgt tattagaatg 900
gattcctttt caaaagtttt ggccccaggg acaagattgg gttggattac tggttcatcc 960
aaaatcttga agccttactt gagtttgcat gaaatgacga ttcaagcccc agcaggtttt 1020
acacaagttt tggtcaacgc tacgctatcc aggtggggtc aaaagggtta cttggactgg 1080
ttgcttggcc tgcgtcatga atacactttg aaacgtgact gtgccatcga tgccctttac 1140
aagtatctac cacaatctga tgctttcgtg atcaatcctc caattgcagg tatgtttttc 1200
accgtgaaca ttgacgcatc tgtccaccct gagtttaaaa caaaatacaa ctcagaccct 1260
taccagctag aacagagtct ttaccacaaa gtggttgaac gtggtgtttt agtggttccc 1320
ggttcttggt tcaagagtga gggtgagacg gaacctcctc aacccgctga atctaaagaa 1380
gtcagtaatc caaacataat tttcttcaga ggtacctatg cagctgtctc tcctgagaaa 1440
ctgactgaag gtctgaagag attaggtgat actttatacg aagaatttgg tatttccaaa 1500
tag 1503
<210> 24
<211> 500
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 24
Met Thr Leu Pro Glu Ser Lys Asp Phe Ser Tyr Leu Phe Ser Asp Glu
1 5 10 15
Thr Asn Ala Arg Lys Pro Ser Pro Leu Lys Thr Cys Ile His Leu Phe
20 25 30
Gln Asp Pro Asn Ile Ile Phe Leu Gly Gly Gly Leu Pro Leu Lys Asp
35 40 45
Tyr Phe Pro Trp Asp Asn Leu Ser Val Asp Ser Pro Lys Pro Pro Phe
50 55 60
Pro Gln Gly Ile Gly Ala Pro Ile Asp Glu Gln Asn Cys Ile Lys Tyr
65 70 75 80
Thr Val Asn Lys Asp Tyr Ala Asp Lys Ser Ala Asn Pro Ser Asn Asp
85 90 95
Ile Pro Leu Ser Arg Ala Leu Gln Tyr Gly Phe Ser Ala Gly Gln Pro
100 105 110
Glu Leu Leu Asn Phe Ile Arg Asp His Thr Lys Ile Ile His Asp Leu
115 120 125
Lys Tyr Lys Asp Trp Asp Val Leu Ala Thr Ala Gly Asn Thr Asn Ala
130 135 140
Trp Glu Ser Thr Leu Arg Val Phe Cys Asn Arg Gly Asp Val Ile Leu
145 150 155 160
Val Glu Ala His Ser Phe Ser Ser Ser Leu Ala Ser Ala Glu Ala Gln
165 170 175
Gly Val Ile Thr Phe Pro Val Pro Ile Asp Ala Asp Gly Ile Ile Pro
180 185 190
Glu Lys Leu Ala Lys Val Met Glu Asn Trp Thr Pro Gly Ala Pro Lys
195 200 205
Pro Lys Leu Leu Tyr Thr Ile Pro Thr Gly Gln Asn Pro Thr Gly Thr
210 215 220
Ser Ile Ala Asp His Arg Lys Glu Ala Ile Tyr Lys Ile Ala Gln Lys
225 230 235 240
Tyr Asp Phe Leu Ile Val Glu Asp Glu Pro Tyr Tyr Phe Leu Gln Met
245 250 255
Asn Pro Tyr Ile Lys Asp Leu Lys Glu Arg Glu Lys Ala Gln Ser Ser
260 265 270
Pro Lys Gln Asp His Asp Glu Phe Leu Lys Ser Leu Ala Asn Thr Phe
275 280 285
Leu Ser Leu Asp Thr Glu Gly Arg Val Ile Arg Met Asp Ser Phe Ser
290 295 300
Lys Val Leu Ala Pro Gly Thr Arg Leu Gly Trp Ile Thr Gly Ser Ser
305 310 315 320
Lys Ile Leu Lys Pro Tyr Leu Ser Leu His Glu Met Thr Ile Gln Ala
325 330 335
Pro Ala Gly Phe Thr Gln Val Leu Val Asn Ala Thr Leu Ser Arg Trp
340 345 350
Gly Gln Lys Gly Tyr Leu Asp Trp Leu Leu Gly Leu Arg His Glu Tyr
355 360 365
Thr Leu Lys Arg Asp Cys Ala Ile Asp Ala Leu Tyr Lys Tyr Leu Pro
370 375 380
Gln Ser Asp Ala Phe Val Ile Asn Pro Pro Ile Ala Gly Met Phe Phe
385 390 395 400
Thr Val Asn Ile Asp Ala Ser Val His Pro Glu Phe Lys Thr Lys Tyr
405 410 415
Asn Ser Asp Pro Tyr Gln Leu Glu Gln Ser Leu Tyr His Lys Val Val
420 425 430
Glu Arg Gly Val Leu Val Val Pro Gly Ser Trp Phe Lys Ser Glu Gly
435 440 445
Glu Thr Glu Pro Pro Gln Pro Ala Glu Ser Lys Glu Val Ser Asn Pro
450 455 460
Asn Ile Ile Phe Phe Arg Gly Thr Tyr Ala Ala Val Ser Pro Glu Lys
465 470 475 480
Leu Thr Glu Gly Leu Lys Arg Leu Gly Asp Thr Leu Tyr Glu Glu Phe
485 490 495
Gly Ile Ser Lys
500
<210> 25
<211> 327
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 25
atggcccaac aatcacccta ttcagcagcg atggcagaac agcgtcacca ggagtggtta 60
cgttttgtcg acctgcttaa gaatgcctac caaaacgatc tccatttacc gttgttaaac 120
ctgatgctga cgccagatga gcgcgaagcg ttggggactc gcgtgcgtat tgtcgaagag 180
ctgttgcgcg gcgaaatgag ccagcgtgag ttaaaaaatg aactcggcgc aggcatcgcg 240
acgattacgc gtggatctaa cagcctgaaa gccgcgcccg tcgagctgcg ccagtggctg 300
gaagaggtgt tgctgaaaag cgattga 327
<210> 26
<211> 108
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 26
Met Ala Gln Gln Ser Pro Tyr Ser Ala Ala Met Ala Glu Gln Arg His
1 5 10 15
Gln Glu Trp Leu Arg Phe Val Asp Leu Leu Lys Asn Ala Tyr Gln Asn
20 25 30
Asp Leu His Leu Pro Leu Leu Asn Leu Met Leu Thr Pro Asp Glu Arg
35 40 45
Glu Ala Leu Gly Thr Arg Val Arg Ile Val Glu Glu Leu Leu Arg Gly
50 55 60
Glu Met Ser Gln Arg Glu Leu Lys Asn Glu Leu Gly Ala Gly Ile Ala
65 70 75 80
Thr Ile Thr Arg Gly Ser Asn Ser Leu Lys Ala Ala Pro Val Glu Leu
85 90 95
Arg Gln Trp Leu Glu Glu Val Leu Leu Lys Ser Asp
100 105
<210> 27
<211> 804
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 27
atggctatcc ctgcatttgg tttaggtact ttccgtctga aagacgacgt tgttatttca 60
tctgtgataa cggcgcttga acttggttat cgcgcaattg ataccgcaca aatctatgat 120
aacgaagccg cagtaggtca ggcgattgca gaaagtggcg tgccacgtca tgaactctac 180
atcaccacta aaatctggat tgaaaatctc agcaaagaca aattgatccc aagtctgaaa 240
gagagcctgc aaaaattgcg taccgattat gttgatctga cgctaatcca ctggccgtca 300
ccaaacgatg aagtctctgt tgaagagttt atgcaggcgc tgctggaagc caaaaaacaa 360
gggctgacgc gtgagatcgg tatttccaac ttcacgatcc cgttgatgga aaaagcgatt 420
gctgctgttg gtgctgaaaa catcgctact aaccagattg aactctctcc ttatctgcaa 480
aaccgtaaag tggttgcctg ggctaaacag cacggcatcc atattacttc ctatatgacg 540
ctggcgtatg gtaaggccct gaaagatgag gttattgctc gtatcgcagc taaacacaat 600
gcgactccgg cacaagtgat tctggcgtgg gctatggggg aaggttactc agtaattcct 660
tcttctacta aacgtaaaaa cctggaaagt aatcttaagg cacaaaattt acagcttgat 720
gccgaagata aaaaagcgat cgccgcactg gattgcaacg accgcctggt tagcccggaa 780
ggtctggctc ctgaatggga ttaa 804
<210> 28
<211> 267
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 28
Met Ala Ile Pro Ala Phe Gly Leu Gly Thr Phe Arg Leu Lys Asp Asp
1 5 10 15
Val Val Ile Ser Ser Val Ile Thr Ala Leu Glu Leu Gly Tyr Arg Ala
20 25 30
Ile Asp Thr Ala Gln Ile Tyr Asp Asn Glu Ala Ala Val Gly Gln Ala
35 40 45
Ile Ala Glu Ser Gly Val Pro Arg His Glu Leu Tyr Ile Thr Thr Lys
50 55 60
Ile Trp Ile Glu Asn Leu Ser Lys Asp Lys Leu Ile Pro Ser Leu Lys
65 70 75 80
Glu Ser Leu Gln Lys Leu Arg Thr Asp Tyr Val Asp Leu Thr Leu Ile
85 90 95
His Trp Pro Ser Pro Asn Asp Glu Val Ser Val Glu Glu Phe Met Gln
100 105 110
Ala Leu Leu Glu Ala Lys Lys Gln Gly Leu Thr Arg Glu Ile Gly Ile
115 120 125
Ser Asn Phe Thr Ile Pro Leu Met Glu Lys Ala Ile Ala Ala Val Gly
130 135 140
Ala Glu Asn Ile Ala Thr Asn Gln Ile Glu Leu Ser Pro Tyr Leu Gln
145 150 155 160
Asn Arg Lys Val Val Ala Trp Ala Lys Gln His Gly Ile His Ile Thr
165 170 175
Ser Tyr Met Thr Leu Ala Tyr Gly Lys Ala Leu Lys Asp Glu Val Ile
180 185 190
Ala Arg Ile Ala Ala Lys His Asn Ala Thr Pro Ala Gln Val Ile Leu
195 200 205
Ala Trp Ala Met Gly Glu Gly Tyr Ser Val Ile Pro Ser Ser Thr Lys
210 215 220
Arg Lys Asn Leu Glu Ser Asn Leu Lys Ala Gln Asn Leu Gln Leu Asp
225 230 235 240
Ala Glu Asp Lys Lys Ala Ile Ala Ala Leu Asp Cys Asn Asp Arg Leu
245 250 255
Val Ser Pro Glu Gly Leu Ala Pro Glu Trp Asp
260 265
<210> 29
<211> 855
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 29
atgcaacaaa aaatgattca atttagtggc gatgtctcac tgccagccgt agggcaggga 60
acatggtata tgggcgaaga tgccagtcag cgcaaaacag aagttgctgc actacgcgcg 120
ggcattgaac tcggtttaac cctcattgat accgccgaaa tgtatgccga tggcggtgcc 180
gaaaaggtgg ttggggaagc attaaccggt ctgcgagaga aggtctttct cgtctctaaa 240
gtctatccgt ggaatgctgg cgggcaaaaa gcgataaatg catgcgaagc cagtttacgc 300
cgtctcaata ctgattatct cgatctttac ttattacact ggtctggcag tttcgctttt 360
gaagagactg tcgcagcgat ggaaaaattg atcgcccagg gaaaaatccg ccgctggggc 420
gtttctaacc ttgattatgc tgatatgcag gaactctggc agctgccggg gggaaatcag 480
tgtgccacta atcaggtgct ttaccatctc ggttcacgag gaattgagta cgatctactc 540
ccctggtgcc agcaacagca gatgccggtg atggcttaca gtccgttagc ccaggccggg 600
cggttgcgca atggactgtt aaaaaacgcg gtagtcaacg aaattgcaca tgctcacaat 660
atcagcgcgg cacaagtatt gttggcgtgg gtgatcagtc atcagggtgt gatggcgatt 720
ccaaaagcgg ccacgattgc ccatgtccaa caaaatgcgg ctgtgcttga ggtcgaactt 780
tcttcagcgg aattagctat gctggataag gcatatccgg caccaaaagg aaaaactgcg 840
ctggatatgg tgtga 855
<210> 30
<211> 284
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 30
Met Gln Gln Lys Met Ile Gln Phe Ser Gly Asp Val Ser Leu Pro Ala
1 5 10 15
Val Gly Gln Gly Thr Trp Tyr Met Gly Glu Asp Ala Ser Gln Arg Lys
20 25 30
Thr Glu Val Ala Ala Leu Arg Ala Gly Ile Glu Leu Gly Leu Thr Leu
35 40 45
Ile Asp Thr Ala Glu Met Tyr Ala Asp Gly Gly Ala Glu Lys Val Val
50 55 60
Gly Glu Ala Leu Thr Gly Leu Arg Glu Lys Val Phe Leu Val Ser Lys
65 70 75 80
Val Tyr Pro Trp Asn Ala Gly Gly Gln Lys Ala Ile Asn Ala Cys Glu
85 90 95
Ala Ser Leu Arg Arg Leu Asn Thr Asp Tyr Leu Asp Leu Tyr Leu Leu
100 105 110
His Trp Ser Gly Ser Phe Ala Phe Glu Glu Thr Val Ala Ala Met Glu
115 120 125
Lys Leu Ile Ala Gln Gly Lys Ile Arg Arg Trp Gly Val Ser Asn Leu
130 135 140
Asp Tyr Ala Asp Met Gln Glu Leu Trp Gln Leu Pro Gly Gly Asn Gln
145 150 155 160
Cys Ala Thr Asn Gln Val Leu Tyr His Leu Gly Ser Arg Gly Ile Glu
165 170 175
Tyr Asp Leu Leu Pro Trp Cys Gln Gln Gln Gln Met Pro Val Met Ala
180 185 190
Tyr Ser Pro Leu Ala Gln Ala Gly Arg Leu Arg Asn Gly Leu Leu Lys
195 200 205
Asn Ala Val Val Asn Glu Ile Ala His Ala His Asn Ile Ser Ala Ala
210 215 220
Gln Val Leu Leu Ala Trp Val Ile Ser His Gln Gly Val Met Ala Ile
225 230 235 240
Pro Lys Ala Ala Thr Ile Ala His Val Gln Gln Asn Ala Ala Val Leu
245 250 255
Glu Val Glu Leu Ser Ser Ala Glu Leu Ala Met Leu Asp Lys Ala Tyr
260 265 270
Pro Ala Pro Lys Gly Lys Thr Ala Leu Asp Met Val
275 280
<210> 31
<211> 957
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 31
atgctacaaa attgcgcaca atcaaattgc cgcattattc ctaagaaatt acgcgatatg 60
aaacgtgaag agatttgccg cttgctggcg gataaagtta ataaactgaa aaataaagaa 120
aatagtttgt caggactgtt gcccgatgtg cgtttgttgt atggcgagac gcctttcgca 180
cgtacaccgg tgatgtacga gcctggcatc ataattctct tttccgggca taaaatcggt 240
tatatcaatg aacgcgtgtt tcgttatgat gccaatgaat acctgctgct gacggtgccg 300
ttgccgtttg agtgcgaaac ctatgccacg tcagaggtgc cgctggcagg gttgcgtctc 360
aatgtcgata ttttgcagtt acaggaactg ttgatggaca ttggcgaaga tgagcatttc 420
cagccgtcga tggcagccag cgggattaac tccgccacgt tatcagaaga gattttatgc 480
gcggcggagc ggttactcga cgtgatggag cgaccactgg atgcgcgtat tctcggcaaa 540
cagatcatcc gcgaaattct gtactacgtg ctgaccggac cttgcggcgg cgcgttactg 600
gcgctggtca gtcgccagac tcacttcagt ctgattagcc gcgtgctgaa acggattgag 660
aataaataca ccgaaaacct gagcgtcgag caactggcgg cagaagccaa catgagcgta 720
tcggcgttcc accataattt taagtctgtc accagtacct cgccgttgca gtatttgaag 780
aattaccgtc tgcataaggc gcggatgatg atcatccatg acggcatgaa ggccagcgca 840
gcagcgatgc gcgtcggcta tgaaagcgca tcgcaattta gccgtgagtt taaacgttac 900
ttcggtgtga cgccggggga agatgcggca agaatgcggg cgatgcaggg gaattaa 957
<210> 32
<211> 318
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 32
Met Leu Gln Asn Cys Ala Gln Ser Asn Cys Arg Ile Ile Pro Lys Lys
1 5 10 15
Leu Arg Asp Met Lys Arg Glu Glu Ile Cys Arg Leu Leu Ala Asp Lys
20 25 30
Val Asn Lys Leu Lys Asn Lys Glu Asn Ser Leu Ser Gly Leu Leu Pro
35 40 45
Asp Val Arg Leu Leu Tyr Gly Glu Thr Pro Phe Ala Arg Thr Pro Val
50 55 60
Met Tyr Glu Pro Gly Ile Ile Ile Leu Phe Ser Gly His Lys Ile Gly
65 70 75 80
Tyr Ile Asn Glu Arg Val Phe Arg Tyr Asp Ala Asn Glu Tyr Leu Leu
85 90 95
Leu Thr Val Pro Leu Pro Phe Glu Cys Glu Thr Tyr Ala Thr Ser Glu
100 105 110
Val Pro Leu Ala Gly Leu Arg Leu Asn Val Asp Ile Leu Gln Leu Gln
115 120 125
Glu Leu Leu Met Asp Ile Gly Glu Asp Glu His Phe Gln Pro Ser Met
130 135 140
Ala Ala Ser Gly Ile Asn Ser Ala Thr Leu Ser Glu Glu Ile Leu Cys
145 150 155 160
Ala Ala Glu Arg Leu Leu Asp Val Met Glu Arg Pro Leu Asp Ala Arg
165 170 175
Ile Leu Gly Lys Gln Ile Ile Arg Glu Ile Leu Tyr Tyr Val Leu Thr
180 185 190
Gly Pro Cys Gly Gly Ala Leu Leu Ala Leu Val Ser Arg Gln Thr His
195 200 205
Phe Ser Leu Ile Ser Arg Val Leu Lys Arg Ile Glu Asn Lys Tyr Thr
210 215 220
Glu Asn Leu Ser Val Glu Gln Leu Ala Ala Glu Ala Asn Met Ser Val
225 230 235 240
Ser Ala Phe His His Asn Phe Lys Ser Val Thr Ser Thr Ser Pro Leu
245 250 255
Gln Tyr Leu Lys Asn Tyr Arg Leu His Lys Ala Arg Met Met Ile Ile
260 265 270
His Asp Gly Met Lys Ala Ser Ala Ala Ala Met Arg Val Gly Tyr Glu
275 280 285
Ser Ala Ser Gln Phe Ser Arg Glu Phe Lys Arg Tyr Phe Gly Val Thr
290 295 300
Pro Gly Glu Asp Ala Ala Arg Met Arg Ala Met Gln Gly Asn
305 310 315
<210> 33
<211> 1164
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 33
atgaacaact ttaatctgca caccccaacc cgcattctgt ttggtaaagg cgcaatcgct 60
ggtttacgcg aacaaattcc tcacgatgct cgcgtattga ttacctacgg cggcggcagc 120
gtgaaaaaaa ccggcgttct cgatcaagtt ctggatgccc tgaaaggcat ggacgtgctg 180
gaatttggcg gtattgagcc aaacccggct tatgaaacgc tgatgaacgc cgtgaaactg 240
gttcgcgaac agaaagtgac tttcctgctg gcggttggcg gcggttctgt actggacggc 300
accaaattta tcgccgcagc ggctaactat ccggaaaata tcgatccgtg gcacattctg 360
caaacgggcg gtaaagagat taaaagcgcc atcccgatgg gctgtgtgct gacgctgcca 420
gcaaccggtt cagaatccaa cgcaggcgcg gtgatctccc gtaaaaccac aggcgacaag 480
caggcgttcc attctgccca tgttcagccg gtatttgccg tgctcgatcc ggtttatacc 540
tacaccctgc cgccgcgtca ggtggctaac ggcgtagtgg acgcctttgt acacaccgtg 600
gaacagtatg ttaccaaacc ggttgatgcc aaaattcagg accgtttcgc agaaggcatt 660
ttgctgacgc taatcgaaga tggtccgaaa gccctgaaag agccagaaaa ctacgatgtg 720
cgcgccaacg tcatgtgggc ggcgactcag gcgctgaacg gtttgattgg cgctggcgta 780
ccgcaggact gggcaacgca tatgctgggc cacgaactga ctgcgatgca cggtctggat 840
cacgcgcaaa cactggctat cgtcctgcct gcactgtgga atgaaaaacg cgataccaag 900
cgcgctaagc tgctgcaata tgctgaacgc gtctggaaca tcactgaagg ttccgatgat 960
gagcgtattg acgccgcgat tgccgcaacc cgcaatttct ttgagcaatt aggcgtgccg 1020
acccacctct ccgactacgg tctggacggc agctccatcc cggctttgct gaaaaaactg 1080
gaagagcacg gcatgaccca actgggcgaa aatcatgaca ttacgttgga tgtcagccgc 1140
cgtatatacg aagccgcccg ctaa 1164
<210> 34
<211> 387
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 34
Met Asn Asn Phe Asn Leu His Thr Pro Thr Arg Ile Leu Phe Gly Lys
1 5 10 15
Gly Ala Ile Ala Gly Leu Arg Glu Gln Ile Pro His Asp Ala Arg Val
20 25 30
Leu Ile Thr Tyr Gly Gly Gly Ser Val Lys Lys Thr Gly Val Leu Asp
35 40 45
Gln Val Leu Asp Ala Leu Lys Gly Met Asp Val Leu Glu Phe Gly Gly
50 55 60
Ile Glu Pro Asn Pro Ala Tyr Glu Thr Leu Met Asn Ala Val Lys Leu
65 70 75 80
Val Arg Glu Gln Lys Val Thr Phe Leu Leu Ala Val Gly Gly Gly Ser
85 90 95
Val Leu Asp Gly Thr Lys Phe Ile Ala Ala Ala Ala Asn Tyr Pro Glu
100 105 110
Asn Ile Asp Pro Trp His Ile Leu Gln Thr Gly Gly Lys Glu Ile Lys
115 120 125
Ser Ala Ile Pro Met Gly Cys Val Leu Thr Leu Pro Ala Thr Gly Ser
130 135 140
Glu Ser Asn Ala Gly Ala Val Ile Ser Arg Lys Thr Thr Gly Asp Lys
145 150 155 160
Gln Ala Phe His Ser Ala His Val Gln Pro Val Phe Ala Val Leu Asp
165 170 175
Pro Val Tyr Thr Tyr Thr Leu Pro Pro Arg Gln Val Ala Asn Gly Val
180 185 190
Val Asp Ala Phe Val His Thr Val Glu Gln Tyr Val Thr Lys Pro Val
195 200 205
Asp Ala Lys Ile Gln Asp Arg Phe Ala Glu Gly Ile Leu Leu Thr Leu
210 215 220
Ile Glu Asp Gly Pro Lys Ala Leu Lys Glu Pro Glu Asn Tyr Asp Val
225 230 235 240
Arg Ala Asn Val Met Trp Ala Ala Thr Gln Ala Leu Asn Gly Leu Ile
245 250 255
Gly Ala Gly Val Pro Gln Asp Trp Ala Thr His Met Leu Gly His Glu
260 265 270
Leu Thr Ala Met His Gly Leu Asp His Ala Gln Thr Leu Ala Ile Val
275 280 285
Leu Pro Ala Leu Trp Asn Glu Lys Arg Asp Thr Lys Arg Ala Lys Leu
290 295 300
Leu Gln Tyr Ala Glu Arg Val Trp Asn Ile Thr Glu Gly Ser Asp Asp
305 310 315 320
Glu Arg Ile Asp Ala Ala Ile Ala Ala Thr Arg Asn Phe Phe Glu Gln
325 330 335
Leu Gly Val Pro Thr His Leu Ser Asp Tyr Gly Leu Asp Gly Ser Ser
340 345 350
Ile Pro Ala Leu Leu Lys Lys Leu Glu Glu His Gly Met Thr Gln Leu
355 360 365
Gly Glu Asn His Asp Ile Thr Leu Asp Val Ser Arg Arg Ile Tyr Glu
370 375 380
Ala Ala Arg
385
<210> 35
<211> 828
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 35
atggctaatc caaccgttat taagctacag gatggcaatg tcatgcccca gctgggactg 60
ggcgtctggc aagcaagtaa tgaggaagta atcaccgcca ttcaaaaagc gttagaagtg 120
ggttatcgct cgattgatac cgccgcggcc tacaagaacg aagaaggtgt cggcaaagcc 180
ctgaaaaatg cctcagtcaa cagagaagaa ctgttcatca ccactaagct gtggaacgac 240
gaccacaagc gcccccgcga agccctgctc gacagcctga aaaaactcca gcttgattat 300
atcgacctct acttaatgca ctggcccgtt cccgctatcg accattatgt cgaagcatgg 360
aaaggcatga tcgaattgca aaaagaggga ttaatcaaaa gcatcggcgt gtgcaacttc 420
cagatccatc acctgcaacg cctgattgat gaaactggcg tgacgcctgt gataaaccag 480
atcgaacttc atccgctgat gcaacaacgc cagctacacg cctggaacgc gacacacaaa 540
atccagaccg aatcctggag cccattagcg caaggaggga aaggcgtttt cgatcagaaa 600
gtcattcgcg atctggcaga taaatacggc aaaaccccgg cgcagattgt tatccgctgg 660
catctggata gcggcctggt ggtgatcccg aaatcggtca caccttcacg tattgccgaa 720
aactttgatg tctgggattt ccgtctcgac aaagacgaac tcggcgaaat tgcaaaactc 780
gatcagggca agcgtctcgg tcccgatcct gaccagttcg gcggctaa 828
<210> 36
<211> 275
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 36
Met Ala Asn Pro Thr Val Ile Lys Leu Gln Asp Gly Asn Val Met Pro
1 5 10 15
Gln Leu Gly Leu Gly Val Trp Gln Ala Ser Asn Glu Glu Val Ile Thr
20 25 30
Ala Ile Gln Lys Ala Leu Glu Val Gly Tyr Arg Ser Ile Asp Thr Ala
35 40 45
Ala Ala Tyr Lys Asn Glu Glu Gly Val Gly Lys Ala Leu Lys Asn Ala
50 55 60
Ser Val Asn Arg Glu Glu Leu Phe Ile Thr Thr Lys Leu Trp Asn Asp
65 70 75 80
Asp His Lys Arg Pro Arg Glu Ala Leu Leu Asp Ser Leu Lys Lys Leu
85 90 95
Gln Leu Asp Tyr Ile Asp Leu Tyr Leu Met His Trp Pro Val Pro Ala
100 105 110
Ile Asp His Tyr Val Glu Ala Trp Lys Gly Met Ile Glu Leu Gln Lys
115 120 125
Glu Gly Leu Ile Lys Ser Ile Gly Val Cys Asn Phe Gln Ile His His
130 135 140
Leu Gln Arg Leu Ile Asp Glu Thr Gly Val Thr Pro Val Ile Asn Gln
145 150 155 160
Ile Glu Leu His Pro Leu Met Gln Gln Arg Gln Leu His Ala Trp Asn
165 170 175
Ala Thr His Lys Ile Gln Thr Glu Ser Trp Ser Pro Leu Ala Gln Gly
180 185 190
Gly Lys Gly Val Phe Asp Gln Lys Val Ile Arg Asp Leu Ala Asp Lys
195 200 205
Tyr Gly Lys Thr Pro Ala Gln Ile Val Ile Arg Trp His Leu Asp Ser
210 215 220
Gly Leu Val Val Ile Pro Lys Ser Val Thr Pro Ser Arg Ile Ala Glu
225 230 235 240
Asn Phe Asp Val Trp Asp Phe Arg Leu Asp Lys Asp Glu Leu Gly Glu
245 250 255
Ile Ala Lys Leu Asp Gln Gly Lys Arg Leu Gly Pro Asp Pro Asp Gln
260 265 270
Phe Gly Gly
275
<210> 37
<211> 1050
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 37
atgaagatca aagctgttgg tgcatattcc gctaaacaac cacttgaacc gatggatatc 60
acccggcgtg aaccgggacc gaatgatgtc aaaatcgaaa tcgcttactg tggcgtttgc 120
cattccgatc tccaccaggt ccgttccgag tgggcgggga cggtttaccc ctgcgtgccg 180
ggtcatgaaa ttgtggggcg tgtggtagcc gttggtgatc aggtagaaaa atatgcgccg 240
ggcgatctgg tcggtgtcgg ctgcattgtc gacagttgta aacattgcga agagtgtgaa 300
gacgggttgg aaaactactg tgatcacatg accggcacct ataactcgcc gacgccggac 360
gaaccgggcc atactctggg cggctactca caacagatcg tcgttcatga gcgatatgtt 420
ctgcgtattc gtcacccgca agagcagctg gcggcggtgg ctcctttgtt gtgtgcaggg 480
atcaccacgt attcgccgct acgtcactgg caggccgggc cgggtaaaaa agtgggcgtg 540
gtcggcatcg gcggtctggg acatatgggg attaagctgg cccacgcgat gggggcacat 600
gtggtggcat ttaccacttc tgaggcaaaa cgcgaagcgg caaaagccct gggggccgat 660
gaagttgtta actcacgcaa tgccgatgag atggcggctc atctgaagag tttcgatttc 720
attttgaata cagtagctgc gccacataat ctcgacgatt ttaccacctt gctgaagcgt 780
gatggcacca tgacgctggt tggtgcgcct gcgacaccgc ataaatcgcc ggaagttttc 840
aacctgatca tgaaacgccg tgcgatagcc ggttctatga ttggcggcat tccagaaact 900
caggagatgc tcgatttttg cgccgaacat ggcatcgtgg ctgatataga gatgattcgg 960
gccgatcaaa ttaatgaagc ctatgagcga atgctgcgcg gtgatgtgaa atatcgtttt 1020
gttatcgata atcgcacact aacagactga 1050
<210> 38
<211> 349
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 38
Met Lys Ile Lys Ala Val Gly Ala Tyr Ser Ala Lys Gln Pro Leu Glu
1 5 10 15
Pro Met Asp Ile Thr Arg Arg Glu Pro Gly Pro Asn Asp Val Lys Ile
20 25 30
Glu Ile Ala Tyr Cys Gly Val Cys His Ser Asp Leu His Gln Val Arg
35 40 45
Ser Glu Trp Ala Gly Thr Val Tyr Pro Cys Val Pro Gly His Glu Ile
50 55 60
Val Gly Arg Val Val Ala Val Gly Asp Gln Val Glu Lys Tyr Ala Pro
65 70 75 80
Gly Asp Leu Val Gly Val Gly Cys Ile Val Asp Ser Cys Lys His Cys
85 90 95
Glu Glu Cys Glu Asp Gly Leu Glu Asn Tyr Cys Asp His Met Thr Gly
100 105 110
Thr Tyr Asn Ser Pro Thr Pro Asp Glu Pro Gly His Thr Leu Gly Gly
115 120 125
Tyr Ser Gln Gln Ile Val Val His Glu Arg Tyr Val Leu Arg Ile Arg
130 135 140
His Pro Gln Glu Gln Leu Ala Ala Val Ala Pro Leu Leu Cys Ala Gly
145 150 155 160
Ile Thr Thr Tyr Ser Pro Leu Arg His Trp Gln Ala Gly Pro Gly Lys
165 170 175
Lys Val Gly Val Val Gly Ile Gly Gly Leu Gly His Met Gly Ile Lys
180 185 190
Leu Ala His Ala Met Gly Ala His Val Val Ala Phe Thr Thr Ser Glu
195 200 205
Ala Lys Arg Glu Ala Ala Lys Ala Leu Gly Ala Asp Glu Val Val Asn
210 215 220
Ser Arg Asn Ala Asp Glu Met Ala Ala His Leu Lys Ser Phe Asp Phe
225 230 235 240
Ile Leu Asn Thr Val Ala Ala Pro His Asn Leu Asp Asp Phe Thr Thr
245 250 255
Leu Leu Lys Arg Asp Gly Thr Met Thr Leu Val Gly Ala Pro Ala Thr
260 265 270
Pro His Lys Ser Pro Glu Val Phe Asn Leu Ile Met Lys Arg Arg Ala
275 280 285
Ile Ala Gly Ser Met Ile Gly Gly Ile Pro Glu Thr Gln Glu Met Leu
290 295 300
Asp Phe Cys Ala Glu His Gly Ile Val Ala Asp Ile Glu Met Ile Arg
305 310 315 320
Ala Asp Gln Ile Asn Glu Ala Tyr Glu Arg Met Leu Arg Gly Asp Val
325 330 335
Lys Tyr Arg Phe Val Ile Asp Asn Arg Thr Leu Thr Asp
340 345
<210> 39
<211> 1020
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 39
atgtcgatga taaaaagcta tgccgcaaaa gaagcgggcg gcgaactgga agtttatgag 60
tacgatcccg gtgagctgag gccacaagat gttgaagtgc aggtggatta ctgcgggatc 120
tgccattccg atctgtcgat gatcgataac gaatggggat tttcacaata tccgctggtt 180
gccgggcatg aggtgattgg gcgcgtggtg gcactcggga gcgccgcgca ggataaaggt 240
ttgcaggtcg gtcagcgtgt cgggattggc tggacggcgc gtagctgtgg tcactgcgac 300
gcctgtatta gcggtaatca gatcaactgc gagcaaggtg cggtgccgac gattatgaat 360
cgcggtggct ttgccgagaa gttgcgtgcg gactggcaat gggtgattcc actgccagaa 420
aatattgata tcgagtccgc cgggccgctg ttgtgcggcg gtatcacggt ctttaaacca 480
ctgttgatgc accatatcac tgctaccagc cgcgttgggg taattggtat tggcgggctg 540
gggcatatcg ctataaaact tctgcacgca atgggatgcg aggtgacagc ctttagttct 600
aatccggcga aagagcagga agtgctggcg atgggtgccg ataaagtggt gaatagccgc 660
gatccgcagg cactgaaagc actggcgggg cagtttgatc tcattatcaa caccgtcaac 720
gtcagcctcg actggcagcc ctattttgag gcgctgacct atggcggtaa tttccatacg 780
gtcggtgcgg ttctcacgcc gctgtctgtt ccggccttta cgttaattgc gggcgatcgc 840
agcgtctctg gttctgctac cggcacgcct tatgagctgc gtaagctgat gcgttttgcc 900
gcccgcagca aggttgcgcc gaccaccgaa ctgttcccga tgtcgaaaat taacgacgcc 960
atccagcatg tgcgcgacgg taaggcgcgt taccgcgtgg tgttgaaagc cgatttttga 1020
<210> 40
<211> 339
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 40
Met Ser Met Ile Lys Ser Tyr Ala Ala Lys Glu Ala Gly Gly Glu Leu
1 5 10 15
Glu Val Tyr Glu Tyr Asp Pro Gly Glu Leu Arg Pro Gln Asp Val Glu
20 25 30
Val Gln Val Asp Tyr Cys Gly Ile Cys His Ser Asp Leu Ser Met Ile
35 40 45
Asp Asn Glu Trp Gly Phe Ser Gln Tyr Pro Leu Val Ala Gly His Glu
50 55 60
Val Ile Gly Arg Val Val Ala Leu Gly Ser Ala Ala Gln Asp Lys Gly
65 70 75 80
Leu Gln Val Gly Gln Arg Val Gly Ile Gly Trp Thr Ala Arg Ser Cys
85 90 95
Gly His Cys Asp Ala Cys Ile Ser Gly Asn Gln Ile Asn Cys Glu Gln
100 105 110
Gly Ala Val Pro Thr Ile Met Asn Arg Gly Gly Phe Ala Glu Lys Leu
115 120 125
Arg Ala Asp Trp Gln Trp Val Ile Pro Leu Pro Glu Asn Ile Asp Ile
130 135 140
Glu Ser Ala Gly Pro Leu Leu Cys Gly Gly Ile Thr Val Phe Lys Pro
145 150 155 160
Leu Leu Met His His Ile Thr Ala Thr Ser Arg Val Gly Val Ile Gly
165 170 175
Ile Gly Gly Leu Gly His Ile Ala Ile Lys Leu Leu His Ala Met Gly
180 185 190
Cys Glu Val Thr Ala Phe Ser Ser Asn Pro Ala Lys Glu Gln Glu Val
195 200 205
Leu Ala Met Gly Ala Asp Lys Val Val Asn Ser Arg Asp Pro Gln Ala
210 215 220
Leu Lys Ala Leu Ala Gly Gln Phe Asp Leu Ile Ile Asn Thr Val Asn
225 230 235 240
Val Ser Leu Asp Trp Gln Pro Tyr Phe Glu Ala Leu Thr Tyr Gly Gly
245 250 255
Asn Phe His Thr Val Gly Ala Val Leu Thr Pro Leu Ser Val Pro Ala
260 265 270
Phe Thr Leu Ile Ala Gly Asp Arg Ser Val Ser Gly Ser Ala Thr Gly
275 280 285
Thr Pro Tyr Glu Leu Arg Lys Leu Met Arg Phe Ala Ala Arg Ser Lys
290 295 300
Val Ala Pro Thr Thr Glu Leu Phe Pro Met Ser Lys Ile Asn Asp Ala
305 310 315 320
Ile Gln His Val Arg Asp Gly Lys Ala Arg Tyr Arg Val Val Leu Lys
325 330 335
Ala Asp Phe
<210> 41
<211> 62
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 41
atggcccaac aatcacccta ttcagcagcg atggcagaac aggtgtaggc tggagctgct 60
tc 62
<210> 42
<211> 60
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 42
tcaatcgctt ttcagcaaca cctcttccag ccactggcgc acatatgaat atcctcctta 60
<210> 43
<211> 60
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 43
atggctatcc ctgcatttgg tttaggtact ttccgtctga gtgtaggctg gagctgcttc 60
<210> 44
<211> 62
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 44
ttaatcccat tcaggagcca gaccttccgg gctaaccagg cggcatatga atatcctcct 60
ta 62
<210> 45
<211> 62
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 45
atgcaacaaa aaatgattca atttagtggc gatgtctcac tggtgtaggc tggagctgct 60
tc 62
<210> 46
<211> 64
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 46
atgctacaaa attgcgcaca atcaaattgc cgcattattc ctaagtgtag gctggagctg 60
cttc 64
<210> 47
<211> 61
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 47
ttaattcccc tgcatcgccc gcattcttgc cgcatcttcc cccatatgaa tatcctcctt 60
a 61
<210> 48
<211> 61
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 48
atgaacaact ttaatctgca caccccaacc cgcattctgt tgtgtaggct ggagctgctt 60
c 61
<210> 49
<211> 60
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 49
ttagcgggcg gcttcgtata tacggcggct gacatccaac gcatatgaat atcctcctta 60
<210> 50
<211> 62
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 50
atggctaatc caaccgttat taagctacag gatggcaatg tcgtgtaggc tggagctgct 60
tc 62
<210> 51
<211> 60
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 51
ttagccgccg aactggtcag gatcgggacc gagacgcttg ccatatgaat atcctcctta 60
<210> 52
<211> 61
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 52
atgaagatca aagctgttgg tgcatattcc gctaaacaac cgtgtaggct ggagctgctt 60
c 61
<210> 53
<211> 60
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 53
tcagtctgtt agtgtgcgat tatcgataac aaaacgatat tcatatgaat atcctcctta 60
<210> 54
<211> 62
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 54
atgtcgatga taaaaagcta tgccgcaaaa gaagcgggcg gcgtgtaggc tggagctgct 60
tc 62
<210> 55
<211> 63
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 55
tcaaaaatcg gctttcaaca ccacgcggta acgcgcctta ccgtcatatg aatatcctcc 60
tta 63
Claims (1)
1. A method for synthesizing phenylacetic acid, p-hydroxyphenylacetic acid and indoleacetic acid by microbial fermentation of glucose is characterized by comprising the following steps:
knocking out a TrpR gene, a dkgB gene, a yeaE gene, a yqhC gene, a yqhD gene, a dkgA gene, a yahK gene and a yjgB gene in a microorganism, wherein the TrpR gene is represented by SEQ ID NO: 25; the dkgB gene is SEQ ID NO:27, or a pharmaceutically acceptable salt thereof; the yeaE gene is SEQ ID NO: 29; the yqhC gene is SEQ ID NO: 31; the yqhD gene is SEQ ID NO: 33; the dkgA gene is SEQ ID NO: 35; the yahK gene is SEQ ID NO: 37; the yjgB gene is SEQ ID NO: 39; the microorganism is escherichia coli or pichia pastoris;
KDC gene, ALDH gene, ARO8 gene and aroG fbr Gene and pheA fbr Gene recombination is carried out on a carrier, and the carrier is transferred into a gene knockout strain, so that the target microorganism which can ferment glucose to synthesize phenylacetic acid is obtained;
or replacing pheA fbr The gene is tyrA fbr The modified target microorganism can realize the synthesis of p-hydroxyphenylacetic acid by fermenting glucose;
or replacing pheA fbr The gene is TrpE fbr Gene, serA fbr The gene and the TrpABCD gene, and the modified target microorganism can realize the synthesis of indoleacetic acid by fermenting glucose;
the KDC gene is SEQ ID NO: 19; the ALDH gene is SEQ ID NO: 21; the ARO8 gene is the amino acid sequence shown in SEQ ID NO: 23; the aroG fbr The gene is SEQ ID NO: 1; the pheA fbr The gene is SEQ ID NO: 3; the tyrA fbr The gene is SEQ ID NO: 5; the TrpE fbr The gene is SEQ ID NO: 7; the serA fbr The gene is SEQ ID NO: 9; the TrpABCD gene comprises four genes of trpA, trpB, trpC and trpD, wherein the trpA is SEQ ID NO: 11; the trpB is SEQ ID NO:13 is coded by the nucleotide sequence shown in the specification; the trpC is SEQ ID NO:15, and trpD is the nucleotide sequence code shown in SEQ ID NO:17 is coded by the nucleotide sequence shown in the specification.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810115170.2A CN110157746B (en) | 2018-02-06 | 2018-02-06 | Method for synthesizing auxin by microorganisms |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810115170.2A CN110157746B (en) | 2018-02-06 | 2018-02-06 | Method for synthesizing auxin by microorganisms |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110157746A CN110157746A (en) | 2019-08-23 |
| CN110157746B true CN110157746B (en) | 2022-12-13 |
Family
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Family Applications (1)
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