具体实施方式

以下通过实施例说明本发明的具体步骤，但本发明的范围不受此实施例限制。

DNS试剂配制：酒石酸钾钠18.2g，溶于50ml蒸馏水中，加热，于热溶液中依次加入3，5-二硝基水杨酸0.63g，NaOH2.1g，苯酚0.5g，搅拌至溶，冷却后用蒸馏水定容至100ml，贮于棕色瓶中，室温保存。

实施例1 HPLC分析条件

色谱柱：YMC-Pack ODS-A(150×4.6mm,5μm,12nm)；流动相A：超纯水(含0.1％甲酸)；流动相B：乙腈(含0.1％甲酸)；进样体积：2μL；系统流速：1.0mL/min；柱温：30℃；检测波长：232nm；洗脱梯度：10％B～50％B线性洗脱6min，50％B～70％B线性洗脱16min。

实施例2 DNS显色法

配制不同浓度的果糖溶液(见表1)，取每个浓度的果糖溶液各0.3mL，依次加入0.3mL DNS试剂，充分混匀后，依次加入0.3mL DNS试剂，充分混匀后，12 000rpm离心30s；沸水浴5min，12000rpm条件下将离心30s，冷却至室温后测定OD₅₄₀值，根据果糖浓度及其对应的OD₅₄₀值绘制果糖标准曲线。标准曲线方程为y＝0.4497x-0.0345(r²＝0.999)，其中y为OD_540nm，x为果糖浓度。

表1果糖溶液的配制

双酶偶联反应结束后，将反应溶液80度水浴5min，以终止反应，离心后取上清液0.3mL，加入0.3mL DNS试剂，充分混匀后，12000rpm离心30s；沸水浴5min，12000rpm条件下将离心30s，冷却至室温后测定OD_540nm值。测定时，需将浓度过高的测定液稀释至OD_540nm至1.0以内。

实施例3λRed基因重组法敲除malZ基因

在KEGG代谢通路数据库(KEGG Pathway Database)中，通过分类检索，选择碳水化合物代谢(1.1 Carbohydrate metabolism)下的淀粉和蔗糖代谢(00500 Starch andsucrose metabolism)模块，选择待分析菌株E.coli BL21(DE3)，获得其淀粉和蔗糖代谢通路图(https://www.kegg.jp/kegg-bin/show_pathway？ebl00500)，据此分析蔗糖分解代谢途径，确定蔗糖水解的关键酶α-葡萄糖苷酶MalZ(SEQ ID NO:1)。

选取麦芽糊精葡萄糖苷酶基因malZ上下游50bp作为同源臂，然后分别将其添加至pKD3质粒通用扩增引物P1和P2的5’端，最终获得用于同源替代片段扩增的引物malZ-homo-F(SEQ ID NO:11)和malZ-homo-R(SEQ ID NO:12)。

malZ-homo-F：

5’-TGCATTAGGCTATGGCAAGGTGATCAGATTTTCATCACAGGGGAATTATGGTGTAGGCTGGAGCTGCTTC-3’

malZ-homo-R：

5’-GTTTTATCCGCGGATGATGGCGCAGGCGTCACGCAAGGCGTTATAAAACGATGGGAATTAGCCATGGTCC-3’

以pKD3质粒为模板，扩增带有同源臂的替代片段(该片段含有FRT位点和氯霉素抗性基因)，PCR反应结束后，利用1％琼脂糖凝胶电泳分离PCR扩增片段，回收同源替代片段。

将pKD46质粒转化至E.coli BL21感受态，同源替代片段加入至E.coli BL21电转化感受态中，进行同源重组，然后42℃培养使阳性重组菌中的pKD46质粒丢失，然后将pCP20质粒转化至重组菌感受态，该质粒通过作用于FRT位点，消除菌体的氯霉素抗性，42℃培养使该质粒丢失。最终得到E.coli malZ-KO菌株。

实施例4 E.coli malZ-KO的背景检测

超声波破碎E.coli malZ-KO和E.coli BL21，离心后获得其上清液。按照反应体系总体积为1mL，各组分终浓度分别为：蔗糖溶液0.2mol/L，粗酶液0.5mL，缓冲液0.05mol/L磷酸盐缓冲液(pH 7.5)进行DNS显色反应。结果表明，E.coli BL21产生的果糖浓度为2.326mmol/L，而E.coli malZ-KO产生的果糖浓度为0.074mmol/L，其果糖浓度降低了31.5倍，完全消除了E.coli BL21的背景干扰。

实施例5 OleD-ASP和蔗糖合酶SUS1的可溶性表达及检测方法验证

将重组质粒pET22b-sus1，pET22b-OleD-ASP转化至E.coli malZ-KO，在0.5mMIPTG和20℃诱导表达12h。从SDS-PAGE可看出，OleD-ASP(SEQ ID NO:2)和蔗糖合酶SUS1(SEQ ID NO:5)均可正常可溶性表达(图1)。

在双酶可溶性表达的基础上，构建5mL反应体系，以验证基于颜色反应的UDP-葡萄糖活性测定方法的可行性。5mL反应体系含4-甲基伞形酮1mg/ml,UDP-葡萄糖3.5mg/ml，蔗糖0.2M，OleD-ASP粗酶液1.25ml，蔗糖合酶SUS1粗酶液1.25ml，2.5ml Tris-HCl缓冲液(pH8.0，含5mM MgCl₂)。在30℃、220r/min条件下，反应12h。结果如图2所示，与E.coli malZ-KO的空菌对照相比，双酶反应组显示出明显的橙色，OD_540nm为1.37。该结果表明，基于DNS法检测果糖的UDP-葡萄糖活性测定方法是可行的。

实施例6共表达菌株的构建

采用共表达载体pETDuet-1，借助Nco I/Hind III和Nde I/Xho I等酶切位点，构建蔗糖合酶(Arabidopsis thaliana蔗糖合酶简称AtSUS1)和OleD-ASP的共表达质粒pETDuet-1-1ATSUS1-oleD-Asp(见图5)，导入E.coli malZ-KO获得共表达菌株E.coli K&1和E.coli K&2(表2)。上述工程菌株IPTG的诱导表达条件为0.4mM IPTG，20℃、14小时，SDS-PAGE检测表达量(图3)，然后构建5mL反应体系：4-甲基伞形酮1mg/ml,UDP-葡萄糖3.5mg/ml，蔗糖0.2M，粗酶液2.5ml，2.5ml Tris-HCl缓冲液(pH8.0，含5mM MgCl2)。在30℃、220r/min条件下，反应12h后进行DNS显色反应。结果如图4所示，E.coli K&1和E.coli K&2均能正常催化蔗糖产生果糖，并且E.coli K&2的活力(OD_540nm＝1.85)略高于E.coli K&1(OD_540nm＝1.73)，故选择E.coli K&2进行后续研究。

表2共表达菌株

实施例7双酶偶联反应条件的优化

按照实施例6构建双酶反应体系，分别考察反应温度(25℃～40℃)，反应pH(6.5～9.0)，反应时间(2h～14h)对OD_540nm和4-甲基伞形酮转化率的影响。1)最佳反应温度：30℃，OD_540nm值最大，为1.83。4-甲基伞形酮的转化率为66.5％；2)最佳反应pH：pH 8.0，OD_540nm值最大，为1.83。4-甲基伞形酮的转化率为67.5％；2)最佳反应时间：10h，OD_540nm值最大，为2.36。4-甲基伞形酮的转化率为82.8％。因此，最终确定该双酶反应最佳条件为30℃，pH8.0，反应10小时，见图6。

实施例8颜色反应检测限的考察

采用DNS显色法检测空白试验组(不含果糖)的OD₅₄₀值，重复测定10次。根据实施例2的果糖标准曲线，计算检测限，检测限的计算公式如下：

LOD＝3s/b

(LOD：检测限；s：测定值的标准偏差；b：线性回归方程的斜率)

通过计算得出颜色反应的检测限为0.026mmol/L。

实施例9颜色反应稳定性的考察

将不同浓度的果糖溶液(0.5mmol/L、0.75mmol/L、1.0mmol/L、1.25mmol/L、1.5mmol/L)经处理后，在常温条件下放置于室内，分别于0min、10min、20min、30min、45min、60min测定OD_540nm值。结果显示，在常温条件下，不同浓度的果糖溶液与DNS试剂作用产生的显色物质3-氨基-5-硝基水杨酸于室内放置60min后，其OD_540nm值未发生变化。由此表明，颜色反应的稳定性较好。

实施例10基于颜色反应糖基转移酶筛选方法的应用

以酶法催化诺西肽的糖基化修饰为模型反应，以验证基于颜色反应的活力测定方法的实用性。选择发明人前期发明专利(申请号：202010921312.1)的UDP-葡萄糖依赖糖基转移酶GT-2和GT-5为催化剂，分别与蔗糖合酶AtSUS1构建相应共表达质粒，转化E.colimalZ-KO后分别获得共表达菌株E.coli K&3和E.coli K&4。IPTG的诱导表达条件为0.4mMIPTG，20℃、14小时。

按照实施例6构建5mL反应体系：诺西肽1mg/ml,UDP-葡萄糖3.5mg/ml，蔗糖0.2M，粗酶液2.5ml，2.5ml Tris-HCl缓冲液(pH8.0，含5mM MgCl₂)。在30℃、220r/min条件下，反应10h后进行DNS显色反应。结果显示，E.coli K&4反应组的OD_540nm为1.17，显著高于E.coliK&3反应组的0.52。以上结果的趋势与发明人前期发明专利(申请号：202010921312.1)的酶活力大小趋势一致。综上所述，基于颜色反应的UDP-葡萄糖依赖糖基转移酶活性筛选方法可用于酶活力测定、突变体活性筛选等，是一种有效的酶活初筛方法。

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Gly Arg Val Pro Met Val Phe Asn Val Val Ile Leu Ser Pro His Gly

275 280 285

Tyr Phe Ala Gln Asp Asn Val Leu Gly Tyr Pro Asp Thr Gly Gly Gln

290 295 300

Val Val Tyr Ile Leu Asp Gln Val Arg Ala Leu Glu Ile Glu Met Leu

305 310 315 320

Gln Arg Ile Lys Gln Gln Gly Leu Asn Ile Lys Pro Arg Ile Leu Ile

325 330 335

Leu Thr Arg Leu Leu Pro Asp Ala Val Gly Thr Thr Cys Gly Glu Arg

340 345 350

Leu Glu Arg Val Tyr Asp Ser Glu Tyr Cys Asp Ile Leu Arg Val Pro

355 360 365

Phe Arg Thr Glu Lys Gly Ile Val Arg Lys Trp Ile Ser Arg Phe Glu

370 375 380

Val Trp Pro Tyr Leu Glu Thr Tyr Thr Glu Asp Ala Ala Val Glu Leu

385 390 395 400

Ser Lys Glu Leu Asn Gly Lys Pro Asp Leu Ile Ile Gly Asn Tyr Ser

405 410 415

Asp Gly Asn Leu Val Ala Ser Leu Leu Ala His Lys Leu Gly Val Thr

420 425 430

Gln Cys Thr Ile Ala His Ala Leu Glu Lys Thr Lys Tyr Pro Asp Ser

435 440 445

Asp Ile Tyr Trp Lys Lys Leu Asp Asp Lys Tyr His Phe Ser Cys Gln

450 455 460

Phe Thr Ala Asp Ile Phe Ala Met Asn His Thr Asp Phe Ile Ile Thr

465 470 475 480

Ser Thr Phe Gln Glu Ile Ala Gly Ser Lys Glu Thr Val Gly Gln Tyr

485 490 495

Glu Ser His Thr Ala Phe Thr Leu Pro Gly Leu Tyr Arg Val Val His

500 505 510

Gly Ile Asp Val Phe Asp Pro Lys Phe Asn Ile Val Ser Pro Gly Ala

515 520 525

Asp Met Ser Ile Tyr Phe Pro Tyr Thr Glu Glu Lys Arg Arg Leu Thr

530 535 540

Lys Phe His Ser Glu Ile Glu Glu Leu Leu Tyr Ser Asp Val Glu Asn

545 550 555 560

Lys Glu His Leu Cys Val Leu Lys Asp Lys Lys Lys Pro Ile Leu Phe

565 570 575

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

580 585 590

Trp Tyr Gly Lys Asn Thr Arg Leu Arg Glu Leu Ala Asn Leu Val Val

595 600 605

Val Gly Gly Asp Arg Arg Lys Glu Ser Lys Asp Asn Glu Glu Lys Ala

610 615 620

Glu Met Lys Lys Met Tyr Asp Leu Ile Glu Glu Tyr Lys Leu Asn Gly

625 630 635 640

Gln Phe Arg Trp Ile Ser Ser Gln Met Asp Arg Val Arg Asn Gly Glu

645 650 655

Leu Tyr Arg Tyr Ile Cys Asp Thr Lys Gly Ala Phe Val Gln Pro Ala

660 665 670

Leu Tyr Glu Ala Phe Gly Leu Thr Val Val Glu Ala Met Thr Cys Gly

675 680 685

Leu Pro Thr Phe Ala Thr Cys Lys Gly Gly Pro Ala Glu Ile Ile Val

690 695 700

His Gly Lys Ser Gly Phe His Ile Asp Pro Tyr His Gly Asp Gln Ala

705 710 715 720

Ala Asp Thr Leu Ala Asp Phe Phe Thr Lys Cys Lys Glu Asp Pro Ser

725 730 735

His Trp Asp Glu Ile Ser Lys Gly Gly Leu Gln Arg Ile Glu Glu Lys

740 745 750

Tyr Thr Trp Gln Ile Tyr Ser Gln Arg Leu Leu Thr Leu Thr Gly Val

755 760 765

Tyr Gly Phe Trp Lys His Val Ser Asn Leu Asp Arg Leu Glu Ala Arg

770 775 780

Arg Tyr Leu Glu Met Phe Tyr Ala Leu Lys Tyr Arg Pro Leu Ala Gln

785 790 795 800

Ala Val Pro Leu Ala Gln Asp Asp

805

<210> 6

<211> 1815

<212> DNA

<213> MalZ的核苷酸序列(2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 6

atgttaaatg catggcacct gccggtgccc ccatttgtta aacaaagcaa agatcaactg 60

ctcataacac tgtggctgac gggcgaagac ccaccgcagc gcattatgct gcgtacagaa 120

cacgataacg aagaaatgtc agtaccaatg cataagcagc gcagtcagcc gcagccaggc 180

gtcaccgcat ggcgtgcggc gattgatctc tccagcggac aaccccggcg gcgttacagt 240

ttcaaactgc tgtggcacga tcgccagcgt tggtttacac cgcagggctt cagccgaatg 300

ccgccggcac gactggagca gtttgccgtc gatgtaccgg atatcggccc acaatgggct 360

gcggatcaga ttttttatca gatcttccct gatcgttttg cgcgtagtct tcctcgtgaa 420

gctgaacagg atcatgtcta ttaccatcat gcagccggac aagagatcat cttgcgtgac 480

tgggatgaac cggtcacggc gcaggcgggc ggatcaacgt tctatggcgg cgatctggac 540

gggataagcg aaaaactgcc gtatctgaaa aagcttggcg tgacagcgct gtatctcaat 600

ccggtgttta aagctcccag cgtacataaa tacgataccg aggattatcg ccatgtcgat 660

ccgcagtttg gcggtgatgg ggcgttgctg cgtttgcgac acaatacgca gcagctggga 720

atgcggctgg tgctggacgg cgtgtttaac cacagtggcg attcccatgc ctggtttgac 780

aggcacaatc gtggcacggg gggagcttgt cacaaccccg aatcgccctg gcgcgactgg 840

tactcgttta gtgatgatgg cacggcgctc gactggcttg gctatgccag cttgccgaag 900

ctggattatc agtcggaaag tctggtgaat gaaatttatc gcggggaaga cagtattgtc 960

cgccattggc tgaaagcgcc gtggagtatg gacggctggc ggctggatgt ggtgcatatg 1020

ctgggggagg cgggtggggc gcgcaataat atgcagcacg ttgctgggat caccgaagcg 1080

gcgaaagaaa cccagccgga agcgtatatt gtcggcgaac attttggcga tgcacggcaa 1140

tggttacagg ccgatgtgga agatgccgcc atgaactatc gtggcttcac attcccgttg 1200

tggggatttc ttgccaatac cgatatctct tacgatccgc agcaaattga tgcccaaacc 1260

tgtatggcct ggatggataa ttaccgcgca gggctttctc atcaacaaca attacgtatg 1320

tttaatcagc tcgacagcca cgatactgcg cgatttaaaa cgctgctcgg tcgggatatt 1380

gcgcgcctgc cgctggcggt ggtctggctg ttcacctggc ctggtgtacc gtgcatttat 1440

tacggtgatg aagtaggact ggatggcaaa aacgatccgt tttgccgtaa accgttcccc 1500

tggcaggtgg aaaagcagga tacggcgtta ttcgcgctgt accagcgaat gattgcgctg 1560

cgtaagaaaa gtcaggcgct acgtcatggc ggttgtcagg tgctgtatgc ggaagataac 1620

gtggtggtat ttgtccgcgt gctgaatcag caacgtgtac tggtggcaat caaccgtggc 1680

gaggcctgtg aagtggtgct acccgcgtca ccgtttctca atgccgtgca atggcaatgc 1740

aaagaagggc atgggcaact gactgacggg attctggctt tgcctgccat ttcggctacg 1800

gtatggatga actaa 1815

<210> 7

<211> 1248

<212> DNA

<213> OleD-ASP的核苷酸序列(2 Ambystoma laterale x Ambystomajeffersonianum)

<400> 7

atgaccaccc agaccactcc cgcccacatc gccatgttct ccatcgccgc ccacggccat 60

gtgaacccca gcctggaggt gatccgtgaa ctcgtcgccc gcggccaccg ggtcacgtac 120

gccattccgc ccgtcttcgc cgacaaggtg gccgccaccg gcgcccggcc cgtcctctac 180

cactccaccc tgcccggcac cgacgccgac ccggaggcat ggggaagcac cctgctggac 240

aacgtcgaac cgttcctgaa cgacgcgatc caggcgctcc cgcagctcgc cgatgcctac 300

gccgacgaca tccccgatct cgtcctgcac gacatcacct cctacccggc ccgcgtcctg 360

gcccgccgct ggggcgtccc ggcggtctcc ctctttccga acctcgtcgc ctggaagggt 420

tacgaggagg aggtcgccga gccgatgtgg cgcgaacccc ggcagaccga gcgcggacgg 480

gcctactacg cccggttcga ggcatggctg aaggagaacg ggatcaccga gcacccggac 540

acgttcgcca gtcatccgcc gcgctccctg gtgctcatcc cgaaggcgct ccagccgcac 600

gccgaccggg tggacgaaga cgtgtacacc ttcgtcggcg cctgccaggg agaccgcgcc 660

gaggaaggcg gctggcagcg gcccgccggc gcggagaagg tcgtcctggt gtcgctcggc 720

tcggttttca ccaagcagcc cgccttctac cgggagtgcg tgcgcgcctt cgggaacctg 780

cccggctggc acctcgtcct ccagatcggc cggaaggtga cccccgccga actgggggag 840

ctgccggaca acgtggaggt gcacgactgg gtgccgcagc tcgcgatcct gcgccaggcc 900

gatctgttcg tcacccacgc gggcgccggc ggcagccagg aggggctggc caccgcgacg 960

cccatgatcg ccgtaccgca ggccgtcgac cagttcggca acgccgacat gctccaaggg 1020

ctcggcgtcg cccggaagct ggcgaccgag gaggccaccg ccgacctgct ccgcgagacc 1080

gccctcgctc tggtggacga cccggaggtc gcgcgccggc tccggcggat ccaggcggag 1140

atggcccagg agggcggcac ccggcgggcg gccgacctca tcgaggccga actgcccgcg 1200

cgccacgagc ggcaggagcc ggtgggcgac cgacccaacg gtgggtga 1248

<210> 8

<211> 1248

<212> DNA

<213> GT-2核苷酸序列(2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 8

atgaccaccc agaccacgcc ggcccatatc gcgatgttca gcatcgccgc ccatggccat 60

gtgaatccga gtctggaagt gatccgtgaa ctggttgccc gtggccatcg cgtgacctat 120

gcgatcccgc cggtgttcgc cgataaagtt gccgccaccg gtgcccgtcc ggttctgtac 180

cacagtacgc tgccgggtcc agatgccgac ccagaagcgt ggggcagtac gctgctggat 240

aacgttgaac cgttcctcaa cgacgcgatc caagcgctgc cacagctggc ggatgcgtat 300

gcggatgaca tcccagatct ggttctccac gatatcacca gctatccagc gcgtgttctg 360

gcgcgtcgct ggggtgttcc agccgttagt ctgagtccga acctcgttgc gtggaaaggc 420

tatgaggaag aagtggcgga accgatgtgg cgcgaaccgc gtcagacgga acgtggtcgc 480

gcctattatg cgcgctttga ggcgtggctg aaagagaatg gcatcacgga acacccggat 540

acctttgcca gccatccacc acgcagtctg gttctgatcc caaaagcgct gcaaccgcat 600

gccgatcgcg tggatgagga cgtgtacacc tttgtgggtg cgtgccaagg tgaccgtgcg 660

gaagaaggtg gttggcaacg tccggcgggt gccgaaaagg ttgttctggt tagtctgggc 720

agcgccttca ccaaacagcc agccttttac cgcgaatgcg tgcgcgcctt cggtaatctg 780

ccgggctggc atctggttct gcaaatcggc cgcaaagtga ccccagcgga actgggtgaa 840

ctgccagata acgtggaggt gcatgactgg gttccgcagc tggatattct gaccaaagcg 900

agcgcgttca tcacgcatgc cggtatgggc agcaccatgg aagcgctgag caatgccgtt 960

ccgatgatcg cggttccgca agccgtggat caattcggca atgcggatat gctgcaaggt 1020

ctgggtgttg cgcgcaaact ggcgacggaa gaggccacgg ccgatctgct gcgtgaaacc 1080

gcgctggcgc tggtggatga tccggaagtt gcccgccgtc tgcgtcgtat tcaagccgag 1140

atggcgcaag aaggtggtac ccgtcgcgcc gccgatctga ttgaagccga actgccagcg 1200

cgccatgaac gccaagaacc agttggtgac cgcccgaatg gcggttaa 1248

<210> 9

<211> 1227

<212> DNA

<213> GT-5的核苷酸序列(2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 9

atgaccagtg agcatcgtag tgccagcgtt accccggcac atatcgccat gtttagcatc 60

gccgcacacg gtcacgtgaa tccgagcctg gaagttattc gcgaactggt ggcacgtggc 120

caccgtgtta cctacgccat tccgcctgtt ttcgccgata aagttgccgc aaccggtgca 180

cgtccggtgc tgtaccatag caccctgccg aaaccgagta atccggaaga aagctggccg 240

gaagatcagg aaagcgccat gggcctgttt ctgaatgacg ccattcaggc actgccgcag 300

ttagccgatg cctacgccga tgatatccct gatctggtgc tgcacgatat caccagttat 360

ccggcacgtg ttctggcacg tcgctggggt gtgcctgccg tgagcctgag cccgaatctg 420

gtggcctgga aaggctacga agaagaagtt gccgagccga tgtggcgtga accgcgtcag 480

acagaacgtg gtcgcgccta ctatgcccgc ttcgaagcct ggctgaaaga gaacggcatc 540

accgaacatc cggatacctt cgcaagccat ccgccgcgca gtctggttct gatcccgaaa 600

gccctgcagc cgcatgccga tcgtgtggat gaggacgttt acaccttcgt tggcgcctgt 660

cagggtgatc gtgccgaaga aggtggctgg cagcgccctg caggtgcaga gaaagtggtg 720

ctggtgagcc tgggcagtgc ctttaccaag cagccggcat tctatcgcga atgtgtgcgt 780

gcctttggca acctgccggg ctggcacctg gttctgcaga tcggccgtaa agtgaccccg 840

gccgaactgg gtgaactgcc gcctaatgtg gaagtgcatc agtgggttcc gcagctggat 900

atcctgacca aagccagtgc cttcatcacc catgcaggta tgggcagcac aatggaagcc 960

ctgagtaatg ccgttccgat gatcgccgtt ccgcaggccg tggaccagtt tggcaacgca 1020

gatatgctgc agggtctggg cgtggcacgt aaactggcca ccgaagaagc aaccgcagat 1080

ctgctgcgtg agaccgccct ggccctggtt gacgatccgg aagttgcccg tcgcctgcgt 1140

cgtattcagg ccgaaatggc acaggaaggt ggcacccgtc gtgcagccga tctgattgag 1200

gccgaactgc cggcccgtca tggctaa 1227

<210> 10

<211> 2445

<212> DNA

<213> 蔗糖合酶AtSUS1的核苷酸序列(2 Ambystoma laterale x Ambystomajeffersonianum)

<400> 10

atggccaatg cagagcgcat gatcacacgt gtgcacagtc aacgtgaacg tctgaacgag 60

accctggtta gcgagcgcaa cgaggttctg gcactgttaa gccgcgtgga agcaaagggc 120

aaaggcatcc tgcagcaaaa ccagatcatt gccgagtttg aagccctgcc ggaacagacc 180

cgtaagaagc tggagggtgg cccgttcttt gatctgctga aaagcaccca ggaagcaatt 240

gtgttacctc cgtgggtggc actggcagtt cgtccgcgtc cgggcgtttg ggaatacctg 300

cgtgtgaatc tgcatgcact ggtggttgag gagctgcagc ctgccgaatt cctgcatttc 360

aaggaagaac tggtggatgg cgttaaaaac ggtaatttta cattagagct ggactttgaa 420

ccgtttaatg ccagcattcc gcgcccgacc ctgcataaat atatcggtaa cggcgtggat 480

tttctgaatc gccatctgag cgccaagctg tttcatgaca aggagagctt actgcctctg 540

ctgaaatttc tgcgtctgca tagtcaccag ggcaagaacc tgatgctgag cgaaaagatc 600

caaaatctga acaccctgca gcacaccctg cgtaaagccg aggaatatct ggccgaactg 660

aagagcgaaa ccctgtatga ggagtttgag gccaagttcg aggagatcgg cctggagcgt 720

ggctggggtg acaacgcaga acgtgtgctg gacatgattc gtctgctgct ggacctgctg 780

gaggcaccgg atccgtgcac actggagaca ttcctgggcc gcgtgccgat ggttttcaat 840

gtggtgattc tgagcccgca cggctacttt gcacaggaca acgttctggg ttatccggat 900

acaggtggcc aagtggtgta cattctggat caggtgcgtg ccttagagat cgagatgctg 960

cagcgcatta aacagcaggg cctgaatatc aaaccgcgca tcctgatcct gacccgtctg 1020

ttacctgatg ccgtgggcac aacctgcggt gaacgcctgg aacgcgtgta tgatagcgaa 1080

tactgtgaca ttctgcgtgt gccgttccgt acagagaaag gcatcgtgcg taaatggatt 1140

agccgcttcg aggtttggcc ttacctggaa acctacaccg aggatgcagc agtggagtta 1200

agcaaagagc tgaacggcaa gccggacctg attattggca actacagcga cggcaacctg 1260

gtggccagcc tgttagccca caaattaggt gtgacccagt gtaccatcgc ccacgcactg 1320

gaaaagacaa aatacccgga cagcgatatc tactggaaaa agttagatga taaatatcac 1380

ttcagctgcc agtttaccgc cgacatcttt gccatgaacc acaccgattt tattatcaca 1440

agcacattcc aggaaatcgc aggcagtaaa gagaccgttg gccagtacga gagccataca 1500

gcctttacac tgcctggcct gtatcgtgtt gtgcacggca tcgatgtgtt tgatcctaaa 1560

tttaacattg ttagcccggg tgcagacatg agtatctact tcccgtacac cgaggagaag 1620

cgccgtctga ccaagtttca cagtgaaatc gaggaactgc tgtacagtga cgtggagaac 1680

aaggagcatc tgtgcgtgtt aaaggataag aaaaaaccga tcttatttac aatggcacgc 1740

ctggatcgcg tgaagaatct gagtggcctg gttgagtggt atggcaaaaa tacccgcctg 1800

cgcgaactgg ccaatctggt tgtggttggt ggcgaccgtc gtaaagaaag caaggacaac 1860

gaggagaagg ccgagatgaa gaaaatgtac gatctgatcg aagagtataa gctgaatggc 1920

cagtttcgct ggatcagcag tcagatggac cgtgtgcgca atggcgaact gtatcgctac 1980

atttgtgaca caaagggcgc attcgttcag ccggcactgt atgaggcctt cggcctgaca 2040

gtggtggaag ccatgacctg cggcctgccg acctttgcaa cctgcaaagg cggcccggca 2100

gaaatcatcg ttcatggcaa gagcggcttc catatcgatc cgtatcatgg tgaccaggcc 2160

gccgatacac tggcagactt ttttaccaaa tgtaaagagg atccgagcca ctgggatgag 2220

attagcaagg gtggtctgca gcgcatcgaa gaaaaataca cctggcagat ctacagccaa 2280

cgtctgctga ccctgaccgg tgtgtatggt ttctggaaac acgtgagtaa tctggaccgt 2340

ctggaagccc gccgttacct ggaaatgttc tatgcactga aatatcgccc gctggcacaa 2400

gccgttcctc tggcacaaga cgatcatcac catcaccatc attaa 2445

<210> 11

<211> 70

<212> DNA

<213> malZ-homo-F(2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 11

tgcattaggc tatggcaagg tgatcagatt ttcatcacag gggaattatg gtgtaggctg 60

gagctgcttc 70

<210> 12

<211> 70

<212> DNA

<213> malZ-homo-R(2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 12

gttttatccg cggatgatgg cgcaggcgtc acgcaaggcg ttataaaacg atgggaatta 60

gccatggtcc 70