具体实施方式

下面结合实施例对本发明作进一步的详细描述。

本领域技术人员将会理解，下列实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用材料或设备未注明生产厂商者，均为可以通过购买获得的常规产品。

本发明中我们从光黑壳属真菌(Preussia isomera,GenBank编号为MK300824.1，菌株现保存于中南民族大学药学院杨小龙教授课题组)中克隆到与红粉苔酸合成相关的聚酮合酶PreuA，并基于酿酒酵母异源表达技术，构建了一株可高效生产聚酮分子红粉苔酸(化合物分子结构见图1)的酵母突变株。本实验的大致步骤为：首先基于同源重组原理，构建聚酮合酶PreuA的异源表达载体YEpADH2p-URA-PreuA(质粒图谱见图2)；然后采用PEG4000/LiAc转化法，将构建的异源表达载体转化酿酒酵母，获得阳性酵母转化株，分析其代谢产物并进行结构鉴定，发现构建的酵母突变株可高产红粉苔酸(～0.5g/L)；最后，以制备的红粉苔酸为对象，研究了其对七种农作物病原真菌(苹果轮纹病菌、棉花枯萎病菌、玉米小斑病菌、马铃薯黄萎病菌、油菜菌核菌、水稻纹枯病菌、草莓黑斑病菌)的抑制活性(实验结果见表3)。具体实验过程如下。

1.聚酮合酶PreuA异源表达载体YEpADH2p-URA-PreuA的构建

1.1聚酮合酶基因preuA的克隆：聚酮合酶preuA的mRNA序列全长6594-bp，如SEQID NO.2，将其均分为3段进行克隆，克隆引物见表1。

PCR反应体系配置(100μL)：ddH₂O 55.5μL；5×Phusion HF buffer 20μL；dNTPs(2.5mmol/L)8μL；正向引物(10pmol/μL)5μL；反向引物(10pmol/μL)5μL；DMSO 3μL；PhusionDNA Ploymerase(2U/μL)1μL；cDNA 2.5μL。PCR程序是：98℃3min；98℃30s，58℃30s，72℃2min，共34cycles；72℃10min。

注：第一段用引物PreuA-E-1F、PreuA-E-1R；第二段引物用PreuA-E-2F、PreuA-E-2R；第三段引物用PreuA-E-3F、PreuA-E-3R。

DNA片段回收采用Thermo Scientific GeneJET凝胶回收试剂盒(K0692)，具体步骤参见其说明书。

1.2异源表达载体片段的制备：片段4(6.2-kb)通过限制性内切酶Nde I/Pme I酶切质粒YEpADH2p-FLAG-URA获得。质粒YEpADH2p-FLAG-URA为亚利桑那大学农业与生命科学学院Istvan Molnar教授馈赠。

酶切体系配置(180μL)：ddH₂O 132μL，10×FastDigest Green Buffer 18μL，质粒YEpADH2p-FLAG-URA 18μL，限制性内切酶①(Nde I)6μL，限制性内切酶②(Pme I)6μL。37℃酶切2h后，采用Thermo Scientific GeneJET凝胶回收试剂盒(K0692)，具体步骤参见其说明书。

1.3异源表达载体的构建与转化：采用SE无缝克隆和组装试剂盒(庄盟生物，ZC231)将1.1制备的preuA基因片段1-3与1.2制备的载体片段4进行重组构建异源表达载体YEpADH2p-URA-PreuA。

重组反应体系配置(10μL)：5×SE Cloning Buffer 2μL；片段1 2μL；片段2 2μL；片段3 2μL；片段4 1μL；SE Recombinase 1μL。37℃反应0.5h，冰上2min，然后转化T1PhageResistant感受态细胞(庄盟生物，ZC102)具体步骤参见其说明书。

1.4异源表达质粒的提取及验证：大肠杆菌质粒的提取采用Axygen^○R AxyPrepPlasmid Miniprep Kit(AP-MN-P-250)，具体步骤参见其说明书。对构建的异源表达载体进行Eco RI/Eco RV酶切验证(7877/2530/1382/1067-bp)并测序，确证载体序列的正确性(载体酶切验证见图3)。

酶切验证体系配置(10μL)：ddH₂O 5μL，10×FastDigest Green Buffer 1μL，异源表达质粒3μL，限制性内切酶①(Eco RI)0.5μL，限制性内切酶②(Eco RV)0.5μL。37℃酶切反应0.5h后跑胶。

2.聚酮合酶PreuA酿酒酵母异源表达突变株的构建及其代谢产物分析鉴定

2.1聚酮合酶PreuA酿酒酵母异源表达突变株的构建：采用LiAc/PEG4000介导的转化法将载体YEpADH2p-URA-PreuA转化酿酒酵母。

将制备的100μL酵母感受态细胞3600r/min，离心5min，弃上清，然后依次向管中加入78μL ddH₂O，36μL LiAc(1mol/L)，240μL PEG4000溶液(50％(w/v))，3μL异源表达载体(YEpADH2p-URA-PreuA)，将混合液混合均匀；30℃放置30min，然后转移至42℃放置30min；将转化液3600r/min，离心5min，弃上清，用200μL无菌水重悬菌体，然后均匀涂布于SC-Ura固体平板上，30℃培养箱中培养2-4d。待酵母转化菌株长出后，将其进行发酵及代谢产物分析。

SC-Ura固体平板的配置：YNB 6.7g/L，葡糖糖20g/L，0.77g/L-ura Do Supplement(美国Clontech公司)，15g琼脂，121℃灭菌15min。

2.2酵母突变株的小量发酵：在转化的平板上随机挑取两个克隆，在SC-Ura固体平板上划线，平板置于30℃培养箱过夜培养。取适量菌体接种于25mL SC-Ura液体培养基中(用125mL三角瓶)，30℃，220r/min，培养1d；第2d，加入25mL 1％YPD培养基，继续培养3d后进行次级代谢产物分析。

SC-Ura液体培养基的配置：YNB 6.7g/L，葡糖糖20g/L，0.77g/L-ura DoSupplement(美国Clontech公司)，121℃灭菌15min。

1％YPD培养基的配置：酵母提取物10g/L，蛋白胨20g/L，葡萄糖10g/L，121℃灭菌15min。

2.3酵母突变株代谢产物分析(色谱图见图4)：用等体积的乙酸乙酯萃取酿酒酵母发酵液，取乙酸乙酯相装入圆底烧瓶中旋蒸。待溶剂悬干后，加入800μL甲醇溶解代谢产物，进行LC-MS分析。色谱条件是：Reverse-phase C₁₈column(Kromasil 100-5-C18，4.6×250mm，5μm)；流动相是水(A)/甲醇(B，加0.1％冰醋酸)；流速0.8mL/min；进样体积5μL；DAD检测器，检测波长为300nm；柱温25℃；采用梯度洗脱，程序是起始流动相比例5％B保持5min,30min内B从5％线性升至100％，保持100％B 10min，然后1min内B从100％线性降至5％，并保持5％B 4min。质谱条件是：Q Exactive检测器；电喷雾离子(ESI)源；正、负离子切换采集；一级质谱全扫描(150-1000m/z)；喷雾电压3.8kv；离子传输管温度325℃；鞘气流速40arb；辅助气流速20arb；辅助气加热温度350℃。

2.4酵母突变株目标代谢产物的分离纯化：将可产生目标代谢产物的酵母突变株大规模发酵3L，发酵及萃取方法分别同2.2，2.3，制得粗浸膏2.1g。将粗浸膏依次采用150mL正己烷、氯仿、二氯甲烷、乙酸乙酯、甲醇对粗浸膏进行洗涤，回收乙酸乙酯、甲醇洗涤液，浓缩后合并，即得目标单体化合物约1.5g。

2.5酵母突变株目标代谢产物的结构解析：目标代谢产物为白色针晶，最大紫外吸收波长为320nm(图5)，其结构经高分辨质谱(HRMS)及一维核磁共振波谱(¹H-NMR、¹³C-NMR)鉴定，HRMS测得其分子离子峰m/z为317.06784[M-H]-以及319.08127[M+H]+(图6)，分子式为C₁₆H₁₄O₇，其结构解析所需的核磁数据见图7-图8以及表2，最终解析其结构为红粉苔酸(分子结构见图1)。

3.红粉苔酸抗农作物病原真菌活性实验

3.1将红粉苔酸以及酮康唑(阳性对照)配制成1mg/mL的母液。

3.2将目标农作物病原真菌(苹果轮纹病菌、棉花枯萎病菌、玉米小斑病菌、马铃薯黄萎病菌、油菜菌核菌、水稻纹枯病菌、草莓黑斑病菌)在PDB(马铃薯葡糖糖肉汤)培养基中活化2-3d，活化好后取1mL菌液加入到100mL PDB培养基中得到稀释菌液。

PDB培养基配制：取23g PDB粉末(北京奥博星生物技术有限责任公司)，加入1000mL蒸馏水，121℃高压灭菌15min。

3.3以酮康唑为阳性对照，取2uL目标化合物加入到198uL的目标菌液里面，采用二倍稀释法测试红粉苔酸对以上七种农作物病原真菌的拮抗活性。(活性结果见表3)

4.结论

实验结果表明，我们首次从光黑壳属真菌(Preussia isomera)中克隆到与红粉苔酸合成相关的聚酮合酶PreuA，并基于酿酒酵母异源表达技术，构建了一株可高效生产红粉苔酸的酵母突变株(～0.5g/L)；并且制备的红粉苔酸对苹果轮纹病菌、玉米小斑病菌、马铃薯黄萎病菌、油菜菌核菌四种农作物病原真菌均有不同程度的抑制作用，其中对苹果轮纹病菌有很强的拮抗作用(MIC，25μg/mL)。

表1：聚酮合酶基因preuA的克隆相关引物信息

表2：化合物红粉苔酸的¹H与¹³C核磁数据(500 MHz,CD3OD,δin ppm,J in Hz)

表3：红粉苔酸对七种农作物病原真菌的抑制活性(以酮康唑为阳性对照)

聚酮分子红粉苔酸具有抗菌、抗肿瘤、抗氧化和抗糖尿病等生物活性，故可作为食品与医药行业天然抗菌剂、抗氧化剂等的潜在生物资源。但目前，市售红粉苔酸(CAS:480-56-8)均通过化学合成方式生产，售价约2000元/mg，产量低，售价高，故导致其在食品、医药等领域应用受限。在本发明中，我们首次从光黑壳属真菌(Preussia isomera)中克隆到与红粉苔酸合成相关的聚酮合酶PreuA，并基于酿酒酵母异源表达技术，构建了一株可高效生产红粉苔酸的酵母突变株(～0.5g/L)；并以制备的红粉苔酸为对象，研究了其对多株农作物病原真菌的拮抗作用。研究发现，红粉苔酸对苹果轮纹病菌、玉米小斑病菌、马铃薯黄萎病菌、油菜菌核菌四种农作物病原真菌均有不同程度的抑制作用，其中对苹果轮纹病菌有很强的拮抗作用(MIC，25μg/mL)。本发明极大地丰富了红粉苔酸的生产来源，对于拓展其衍生化途径，研发新型生物农药有重要的科学价值和应用前景。

以上显示和描述了本发明的基本原理、主要特征和本发明的优点。本行业的技术人员应该了解，本发明不受上述实施例的限制，上述实施例和说明书中描述的只是说明本发明的原理，在不脱离本发明精神和范围的前提下，本发明还会有各种变化和改进，这些变化和改进都落入要求保护的本发明范围内。本发明要求保护范围由所附的权利要求书及其等效物界定。

序列表

<110> 中南民族大学

<120> 聚酮合酶PreuA及其在制备红粉苔酸中的应用

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Ile Ser Ser Ile Ile Leu Arg Glu Asn Glu Ser Leu Ala Thr Ile Lys

1460 1465 1470

Leu Pro Pro Asn Gln Pro Gly Leu His Glu Ser Thr Ala Trp Lys Arg

1475 1480 1485

Cys Asp Ala Val Phe Leu Asp Gly Phe Ile Ser Ser Ser Gly Leu Leu

1490 1495 1500

Leu Asn Ser Ser Ser Val Val Gln Ser Gly His Val Leu Ile Ala Val

1505 1510 1515 1520

Gly Val Glu Arg Ala Ile Leu Thr Ala Ala Phe Gln Ala Ser Leu Ala

1525 1530 1535

Ser Ser Trp Gln Ala Tyr Ala Thr Phe Thr Met Val Gly Glu Thr His

1540 1545 1550

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

1555 1560 1565

Met Met Thr Gly Val Arg Phe Asn Lys Met Glu Ile Ser Lys Leu Ala

1570 1575 1580

Lys Ser Leu Ser Ser Val Asn Ala Ser Ser Pro Thr Gly Gly Arg Thr

1585 1590 1595 1600

Gln Pro Pro Ala Ala Pro Lys Thr Gln Ala Gln Pro Met Ala Ser Arg

1605 1610 1615

Pro Ser Pro Thr Pro Leu Gln Val Ser Phe Ala Thr Ala Glu Pro Ala

1620 1625 1630

Ala Pro Glu Pro Val Gln Gln Ser Thr Ala Ala Leu Ala Arg Asn Asp

1635 1640 1645

Ile Gly Pro Val Leu Lys Ser Leu Ile Ser Asn Tyr Thr Gly Leu Ile

1650 1655 1660

Glu Glu Asp Val Ser Glu Asp Ser Pro Leu Val Asp Leu Gly Leu Asp

1665 1670 1675 1680

Ser Leu Ser Ser Val Glu Phe Ala Ser Glu Ile Gly Thr Lys Phe Gly

1685 1690 1695

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

1700 1705 1710

Cys Gln Arg Leu Ser Gly Thr Ser Asn Val Val Ser Gln Lys Met Ser

1715 1720 1725

Glu Thr Pro Ala Ala Ala Pro Val Lys Glu Leu Ile Glu Thr Val Pro

1730 1735 1740

Ser Pro Ile Val Thr Phe Ser Ser Pro Val Ser Asn Ser Ile Thr Ser

1745 1750 1755 1760

Val Leu Lys Ser Leu Leu Gly Ser Tyr Thr Gly Leu Gln Glu Glu Asp

1765 1770 1775

Met Pro Asp Asp Val Pro Leu Ile Asp Leu Gly Leu Asp Ser Leu Ser

1780 1785 1790

Ser Val Glu Phe Ala Ser Glu Leu Asn Asp Lys Met Gly Ala Asp Ile

1795 1800 1805

Asp Ser Ala Val Val Ala Asp Met Thr Leu Ser Ala Leu Glu Gln Gln

1810 1815 1820

Leu Gly Ala Ser Ala Thr Pro Pro Ser Thr Thr Gly Ser Ser Thr Pro

1825 1830 1835 1840

Gly Asp Ile Ser Thr Ala Ala Thr Thr Pro Tyr Ala Thr Gly Ala Ser

1845 1850 1855

Thr Pro Asp Tyr Leu Val His Gly Asn Lys Pro Ser Ile Ser Asn Gly

1860 1865 1870

Val Val Ala Ala Lys Asp Ser Tyr Gln Val Lys Thr Val Glu Tyr Lys

1875 1880 1885

Arg Val Ser Gly Val Pro Ile His Ala Asp Ile Tyr Val Pro Leu Val

1890 1895 1900

Gln Arg Val Ser Pro Met Pro Leu Ala Leu Met Ile His Gly Gly Gly

1905 1910 1915 1920

His Met Thr Leu Ser Arg Lys Ala Val Arg Pro Thr Gln Ala Lys Tyr

1925 1930 1935

Leu Leu Ser His Gly Phe Leu Pro Ile Ser Ile Asp Tyr Arg Leu Cys

1940 1945 1950

Pro Glu Val Asn Leu Ile Asp Gly Pro Ile Ala Asp Val Arg Asp Ala

1955 1960 1965

Tyr Val Trp Ala Cys Gln Asn Leu Gly Thr His Leu Ala Glu His Ser

1970 1975 1980

Ile Ser Val Asp Gly Gly Arg Val Val Val Val Gly Trp Ser Thr Gly

1985 1990 1995 2000

Gly His Leu Ala Met Ser Leu Gly Trp Ser Leu Glu Glu Ala Gly Val

2005 2010 2015

Pro Pro Pro Lys Ala Val Leu Ser Phe Tyr Ala Pro Val Asp Phe Glu

2020 2025 2030

Ser Gly Glu Leu Asp Asn Gln Lys Asn Pro Ala Leu Pro Lys Pro Arg

2035 2040 2045

Met Thr Leu Asp Gln Ile Thr Lys Ala Leu Pro Arg Thr Pro Val Thr

2050 2055 2060

Gln Tyr Gly Ala Ser Ser Thr Asp Glu Thr Asn Leu Gly Trp Leu His

2065 2070 2075 2080

Pro Gly Asp Pro Arg Ser Glu Leu Leu Leu His Val Phe His Ser Asp

2085 2090 2095

Ile Gly Leu Pro Leu Ile Leu His Gly Leu Pro Ile Ser Gly Ser Gly

2100 2105 2110

Arg Pro Ser Pro Ser Leu Val Ala Ser Ile Ser Pro Leu Ala Arg Leu

2115 2120 2125

Arg Asn Gly Ser Tyr Thr Ile Pro Thr Phe Ile Ile His Gly Thr Lys

2130 2135 2140

Asp Val Ile Ala Pro Tyr Ala Ala Ala Glu Arg Phe Val Lys Ile Met

2145 2150 2155 2160

Ser Glu Lys Gly Val Lys Ser Gly Phe Leu Ser Leu Ser Gly Thr Gly

2165 2170 2175

His Val Phe Asp Val Thr Met Lys Pro Asp Ser Lys Gly Trp Glu Asp

2180 2185 2190

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

2195 2200 2205

<210> 1

<211> 6618

<212> DNA/RNA

<213> 人工序列()

<400> 1

atgtctaatt ctacacgtga ctatcccata tccgcggcct tcttttgtcc acagagtagg 60

gcacccccag cggaatacct ccacgccctt tattcttttc tcagccaaaa cacccttgga 120

aaggctttcc ttcgccacat tgcgtccctt gacgaagtct ggcccatctt ctccgaagcg 180

agggacgata ttctcagact gcctgatgcg cgccaaaata tcaatgtgct cgtcgattgg 240

gcaaagggtg gctcttccac tcccattgct gaagcccgat ctggagtgat cgctcttcca 300

tcagtcttca ttgtccagct tggacaatac tttcgttacc tcgaggcaaa tcggctatcc 360

cacggcgact ttatcggcca gctcaaggat attggtggtg ttcatggata ctgtggaggc 420

gctgctgcag cactctccgt tgcgtgtgca gccgatgaga cccagctcat tgaccatgct 480

gcagtgttgc tacgtttatt cgttggtatc ggctgttgca tcgaggcagt ggatgattgg 540

accacaactg agtccactgt cattgcctgc cgtctcaaat acgaaggaca gggtgatgaa 600

ctctgtagcc gatttccagg tacatacgtc tctgccatta cagagcctaa gtcaataagc 660

attactggca atgcccgcac actgtcagag ctttttgatt atgcggtggg ccttggactt 720

cccacccata agatggaaat caccggtaaa gcacacaacc cagaaaacgc tgaactggcc 780

aaagatttta tcaacttata tcgtcggact ccggctctgc aactgccccc taccttcaag 840

ttgcaagcaa cagtgcgctc aaatcgtact gcggagaagt tgaccaacga aggcattatt 900

gaggacatga tcacgatgat tatagcgtcc caatgtgact ggaacacgct tctgacaaga 960

gtcgccgagg acatgaaggt ctctggtcga ccatttcaca agatggtgtc ctttgggatg 1020

aacgattgcg ttcctgtaac acctttcaat cgacagcggc ttaagaccac caaatttgag 1080

gctcatgtcc tcatcgagcc cctgaagcct tcgcgtattt ccgctgcgca gtatcctacc 1140

ttctcagatg atgcaatcgc cataacaggc gcttctttgc gcctaccagg tgcaaataat 1200

cttgacgaat tatgggacct gatctccaaa ggtaccgact gtcacaggga aataccaaaa 1260

gacagattcg atccgcacaa catttatcgg acctctcaga gtggcttcag caaagcccag 1320

aagtattttg gcaactttct tgaggacatc aaagggttcg atagggcgta tttcagcatg 1380

ggtgtacggg aagctgccaa catcgaccca caacagcgat tactcctgga gcttgcagtt 1440

gaagcccttg aggcaagtgg ctatctcgcc aaccatgtac gagaagctgg tgacccggtc 1500

ggctgctttg ttggagccag ctttatagaa tacctggaaa atactggtgc ccaccctcca 1560

acagcttaca ccgctcccgg aaccatcaga gcctttttat gtggcagact cagctattac 1620

tttggatgga cagccccagc ggaagttatc gatactgcct gctcggcttc catggtcgcg 1680

atcaaccgcg cagtcaaatc tatccaagca ggagaatgtg agatggcgct tgctggaggc 1740

gtaaacctga tcactggaat gaacaactat ctcgatttgg ccaaagccgg atttctgagc 1800

ccaacaggcc aatgcaagcc attcgaccaa tctggagatg gctattgtcg ctctgatgga 1860

gcaggatttg ttgttctgaa gaagttgtcg caggctctgg taaatggcga tccgatcatg 1920

ggtgttattc ccagtatcga aaccaatcaa ggtggtctat ccgggtcact cactgttcca 1980

tcatccactg cactacaagc actttacaaa cgcgtccttt cgaaatctgg tctggaacct 2040

gcacagatta cctatgttga agcccatggt acaggaaccc aagcaggtga cccgattgag 2100

gtggagagcg ttcgtgcagt tctcggagac cccacgcgag cccattctct ttccctaggc 2160

tctgtgaaag gaaacattgg ccattgcgaa actggcgctg gcgtcgctgg tctgctgaaa 2220

gtacttgcaa tgatcaaaca tggaggtatc ccgcctctgg caagccacaa agcgctcaat 2280

cctaaaatac cagccttgga gacacatcac atggaaattg caaagcagct taagccttgg 2340

gatgtcccac tgagggcagc atttgtcaac agttatggcg ctgctggctc gaatgctgcc 2400

gtgatctgtg tcgagccacc accagtcgtc accgacggat catctttgat tggcactgag 2460

cctcaaaagg taacgctccc agtcatcgtc agtggcgcta caagaaagag cttggtcctg 2520

aatgcacgag cattggcaag ctacctctcg caagacggat cacacctcag catacatgat 2580

gttgcgttta ccgtcaacca acggagaaaa cggaatagat tttgcgccga ggtctctggc 2640

accgatttgc catctttggt tcagtcactc cgcgctgtag actctcccag tttcgagagc 2700

cctggaaagt ccaagcctgt ggtgctcgtc ttcagcggac aaaacaccaa tgcagtagcc 2760

ttggaccgca cgatatacga tacctaccca gtatttaaag cctatattga cgcctgtgac 2820

tccgaaattg tgaaacttgg cttcccaagt atcatggagg ccattttcca gaaggagccc 2880

ataagcactg ctgttgcttt gcaaggcagt atcttcgcaa tgcagtatgc gtgtgcccgt 2940

tcttggatcg acgcaggcct caaacctcga gcaatcatcg gccacagttt cggcgaactt 3000

actgctttgg ctgtctctgg agctctgtca cttgcagaca gcttgaagct ggtcacatgc 3060

cgcggtcacc tcatcgacac taagtggggt gaggaaaggg gcggtatgct tgtcattcat 3120

gcggatgtgg ccacggtcga acgcttccaa tcccggttca aagcgcagca tgacggagct 3180

gaactggaga ttgcttgcta taactctcca actaccacag tggttgctgg gccagtggca 3240

tacatggatg cagctgagca gatgctggcc acagatccag atttccaggg ccttcgtaag 3300

ctgcgcattg taacgagtaa cgccttccat tcttcgttgt cggatccgat cctagccgat 3360

ttggactcca tggcagatac cttgacctgg aatgagccca gtatcccact ggaagcttgc 3420

acgagtgaag gcctggcgtc gatcaaagag tggagtgcct ccagacatac cagaggctca 3480

gtgtacttca ccaaagcggt ggagcgtatc gaaggacgac tgggtgcatg catctgggtt 3540

gaagccggcc ttgactccgc catcatagca atggctcgaa aagcttcttc aaagccagac 3600

tcgcaggtct ttcaatctgt cagcacgaag gctggagcta cttctttcat tgatggaatc 3660

gtaaacaatc tttggcgtca aggcgtgcct ctttctcact tgaacgcgct atcggcgact 3720

gtcaaaccca accctgtctg gcttccacca taccagttcg aaagagaaca gcattggacc 3780

gagcacatcg atagggcgac cgaagcaagt caagcaagca ccacaagcga cactattcag 3840

tcgactccaa cgcaaaccgt ccaaagtcca ccaaagctga tttccagact cgcatctctg 3900

cagtatcaga tcaacacgca gtgcgagcgc ttccaaaaga tcaccgaagg ccatgcggtg 3960

ttgtatgaac ccttatgccc tgcatccttg tacatggaat gtgttgtcat ggctctccaa 4020

gaactagcag gcgaccttgg ttcccgcact cttgacttcg aaaatctgga cttccatgcg 4080

gggttgggcc tacagactga ccgccgtgtg cttctcgact tggaagaagc gcgccctcac 4140

tcatggactt tcaaggtgca atccacgaaa gctgggtcat ctcggtcatt gttacattgc 4200

tcgggccggg tgatcctaac tgagagttcg gtacctacca cgttccagcg tctggtcgat 4260

ggccccagat cacgtctcga ccaggacaag gatgctgaga agcttatgtc gtctcgcgcc 4320

tacggtctgt tttccaacat catgacctat tcggaattcc tcaagccgat ttcgtcgatc 4380

atcctgcgcg aaaacgaatc cttggctacc atcaaacttc caccgaacca gcctggtctg 4440

catgaaagca cggcttggaa aagatgcgac gcagtgttcc tggatggctt catctcctct 4500

tcgggacttc tgctcaatag cagcagcgtg gttcagtcag gccatgtttt gatcgctgtt 4560

ggagtcgagc gggccatcct cacagctgct ttccaagcat cccttgcttc ctcatggcag 4620

gcgtacgcaa catttaccat ggttggcgaa actcacgctc tctgcgatgt tttcgcttgc 4680

actcctgacg gtgaagtggt agccatgatg acaggcgtga ggttcaacaa gatggagatc 4740

tcaaagttag cgaaatcgct ctcgtcggtc aacgcctcgt caccgacagg tggaagaact 4800

cagccaccag ccgcgccaaa aacccaagct cagccgatgg cttctagacc ctcacctacc 4860

ccactgcagg tttcctttgc aacggcagaa cctgccgcac cagagcctgt tcaacagtca 4920

acagctgccc tagcgcgtaa tgacataggt ccagtcctta agtctctcat ctccaactac 4980

accggcctaa tcgaagagga tgtctcggaa gatagtcctc tcgtcgactt aggtcttgac 5040

tcgctctctt ccgttgagtt cgcgtctgaa atcggaacca agttcggagt cactctggat 5100

gcggatacag tgggagactt gacgttacac tcgctttgcc agaggctcag tggcacctcg 5160

aacgttgtat cccagaagat gtccgagaca cctgcagcgg ctccggtgaa ggagctgatt 5220

gaaactgtac catcacccat cgtgactttc agcagtcctg tgtcaaacag catcacatcg 5280

gtcctaaagt ctcttctcgg gagctacacc ggcttacaag aagaagacat gcccgacgat 5340

gtacctctta ttgatcttgg actggattcc ttgtcatccg tcgagtttgc gtcggaactg 5400

aacgacaaaa tgggagcaga tatcgactcc gctgttgttg cagacatgac cttatctgcg 5460

ctcgagcaac agctgggagc atctgcgaca ccacctagca ccaccggctc atccactcct 5520

ggggatattt caactgctgc cacgacccct tatgctacgg gagcctctac gccagactac 5580

ttggtgcatg gaaataaacc gtctatttca aatggagttg tcgctgcaaa agactcctat 5640

caagtcaaga ccgtcgaata caaacgggta agtggtgttc ccatccacgc agacatctac 5700

gtccctcttg ttcaacgcgt ctctcctatg cccttagcgc tcatgatcca cggcggtggc 5760

cacatgacct tgtctcggaa ggccgtccgt cccacacaag ccaaatatct tctgtcgcac 5820

ggcttcctcc ccattagtat tgactaccgt ctgtgtccgg aggtcaatct catcgatgga 5880

cccatcgccg acgtacgtga tgcttatgtg tgggcttgtc aaaatctggg gacgcatctt 5940

gcggaacaca gtatctctgt cgatggtggc cgcgtggtgg tggtggggtg gtcgactgga 6000

ggtcatttgg caatgtcact gggatggtcg cttgaagagg ctggcgtgcc gccaccaaag 6060

gctgtgttga gcttctatgc cccagtggat tttgagtctg gtgagctcga taatcaaaag 6120

aaccccgctc tcccgaaacc gcgcatgact ttggaccaaa ttacaaaggc acttcctagg 6180

accccagtga cgcagtatgg tgcctcttcc acggacgaaa caaatctcgg atggctgcac 6240

ccaggcgatc cgcgatccga actccttctc cacgtgttcc actccgatat tggcttaccg 6300

ctcatactac acggtcttcc catttcgggt tctggcaggc catcgccttc ccttgttgct 6360

tccataagtc cgctggcgcg cttgcgcaac ggctcataca ctatcccgac cttcatcatt 6420

catggcacga aggacgtcat cgctccatat gcagcagcgg agcgatttgt caagattatg 6480

tccgagaaag gggtaaagag tggatttcta tcgttgtcag ggacgggtca tgttttcgat 6540

gttacgatga agcctgatag taaggggtgg gaggacaagg ttaagccggg gcttgacttt 6600

ttgattcaga acgcttga 6618

<210> 3

<211> 60

<212> DNA

<213> 人工序列()

<400> 3

gactacaaag acgatgacga caagcttcat atgtctaatt ctacacgtga ctatcccata 60

<210> 4

<211> 52

<212> DNA

<213> 人工序列()

<400> 4

catgtttgat cattgcaagt actttcagca gaccagcgac gccagcgcca gt 52

<210> 5

<211> 43

<212> DNA

<213> 人工序列()

<400> 5

gctgaaagta cttgcaatga tcaaacatgg aggtatcccg cct 43

<210> 6

<211> 25

<212> DNA

<213> 人工序列()

<400> 6

tcggcttgag gaattccgaa taggt 25

<210> 7

<211> 56

<212> DNA

<213> 人工序列()

<400> 7

tcatgaccta ttcggaattc ctcaagccga tttcgtcgat catcctgcgc gaaaac 56

<210> 8

<211> 59

<212> DNA

<213> 人工序列()

<400> 8

agtgatggtg atggtgatgt ccgtttaaac tcaagcgttc tgaatcaaaa agtcaagcc 59