CN111394322A - 银杏黄酮化合物生物合成基因中多个亚型的研究 - Google Patents

银杏黄酮化合物生物合成基因中多个亚型的研究 Download PDF

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CN111394322A
CN111394322A CN201811651100.5A CN201811651100A CN111394322A CN 111394322 A CN111394322 A CN 111394322A CN 201811651100 A CN201811651100 A CN 201811651100A CN 111394322 A CN111394322 A CN 111394322A
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于荣敏
陈珊
訾佳辰
朱建华
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Abstract

本发明通过建立和分析银杏的转录组，发现了5个可能的苯丙氨酸解氨酶(PAL)、2个可能的肉桂酸4‑羟化酶(C4H)、3个可能的香豆酰CoA连接酶(4CL)以及1个可能的查耳酮合成酶‑CHS。通过在酵母中异源表达并结合底物饲喂，表征了GbPAL2、GbPAL3、GbC4H1、GbC4H2、Gb4CL1和GbCHS的功能。Gb4CL1和Gb4CL3分别与GbCHS存在互作；GbC4H1和GbC4H2与其下游的Gb4CL1和Gb4CL3以及上游的GbPAL2和GbPAL3都存在互作，表明这些酶可能参与了黄酮的生物合成。进化树分析结果显示：GbPAL2和GbPAL3同属一个clade而远离其它PAL亚型；Gb4CL1和Gb4CL3同属一个分支而远离Gb4CL2。这结果进一步说明GbPAL2和GbPAL3同属黄酮生物合成路径，并且Gb4CL1和Gb4CL3也同属黄酮生物合成路径。以上结果为准确揭示银杏中黄酮的生物合成途径奠定了重要基础，为有效调控黄酮的含量提供重要依据。

Description

银杏黄酮化合物生物合成基因中多个亚型的研究
技术领域
本发明属植物基因资源和医药技术领域，涉及在银杏(Ginkgo biloba)中位于黄酮化合物有生物合成上游途径的多个基因亚型，揭示黄酮生物合成相关的多个亚型，为有效调控黄酮类化合物含量提供科学依据。
背景技术
银杏叶提取物具有抗氧化、抗衰老、清除自由基并提高免疫力的作用；它对冠心病、心绞痛、脑动脉硬化具有明显的改善作用，且广泛用于阿尔茨海默病和高血压的治疗。标准化的银杏叶提取物的主要成分为24％的黄酮类化合物和6％的萜内酯成分。黄酮类化合物是银杏提取物中含量最高，药效活性最强的成分。本发明基于发明人已经建立的银杏细胞悬浮体系，对银杏黄酮类化合物生物合成途径进行探究，挖掘参与黄酮类化合物生物合成基因。本发明通过对不同亚型的基因及其编码的蛋白进行功能表征、同源分析以及亚细胞定位等分析，从而确定在不同亚型中真正参与黄酮类生物合成途径相关的基因，并阐明此类基因是否真正与黄酮类化合物的生物合成途径相关。
发明内容
一、技术内容
本发明利用银杏幼苗，对银杏转录组进行高通量测序，以及黄酮类化合物生物合成的数据库，多序列比对，进行基因挖掘。成功找到黄酮类生物合成相关的候选基因，其中包括基因GbPAL、GbC4H、Gb4CL和GbCHS。使用BLAST软件将转录组测序序列与Swiss-Prot、Gene Ontology、Cluster of Orthologous Groups、eukaryotic Orthologous Groups和Kyoto Encyclopedia of Genes and Genomes数据库比对，获得转录组序列的功能注释。通过以上方式，寻找到候选基因GbPAL2、GbPAL3、GbPAL4、GbPAL5、GbC4H2、Gb4CL1、Gb4CL2及GbCHS。然后，结合Medicinal Plant Genomics Research数据库和NCBI中已报道的基因，利用BLAST比对，获得候选基因GbPAL1、GbC4H1和Gb4CL3。基因序列见附录1。每个基因分别带有SacI和EcoRI酶切位点的两对引物进行扩增GbPAL、GbC4H和Gb4CL基因，分别带有NheI和BamHI酶切位点的两对引物进行扩增GbCHS，扩增产物通过电泳纯化并连接平末端克隆载体pEASY-blunt(全式金)进行DNA测序。
本发明通过投入底物L-苯丙氨酸，对GbPAL进行功能验证，并检测到产物反式-肉桂酸。比对产物与标准品的紫外吸收光谱图及LC-MS的进一步验证，确认GbPAL2和GbPAL3具有苯丙氨酸解氨酶的生化功能(见图1，2)。反式肉桂酸的分子量为148，ESI阳离子扫描的质谱中可以检测到m/z 187为[M+K]，m/z 149为[M+H]，m/z 131为[M-COOH]的特征峰。通过投入底物反式肉桂酸，利用HPLC-DAD检测到产物，与标准品进行比对确证为对香豆酸。利用LC-MS检测Gb4CL1与GbCHS共表达产物，在ESI阳离子模式和阴离子模式扫描的离子流图中均检测到柚皮素查耳酮和柚皮素(图3、4)。其分子量均为272，在阳离子扫描模式对应的质谱中，找到其特征峰：m/z 273为[M+H]峰；m/z 295为[M+Na]峰；m/z 567为[2M+Na]峰以及特征碎片峰m/z 153。
本发明的亚细胞实验结果(图5、6)揭示了GbPAL，GbC4H，Gb4CL及GbCHS四个酶在细胞中的位置，其合成场所在内质网。黄酮类化合物生物合成的酶被定位在内质网的胞质一侧，它们以紧密结合在内质网上的细胞P450酶如C4H、F3’H和F3’5’H为中心，形成了多酶复合体。亚细胞实验结果进一步证明了前人的推测，即细胞色素P450酶(如C4H)起到了定位于内质网的作用，而其它酶通过相互作用与细胞色素P450酶形成多酶复合体，以此发挥合成黄酮类化合物的功能。
本发明以GbCHS为起点，通过GbCHS与Gb4CL的组合，获得与GbCHS蛋白间相互作用(图7)，且最有可能参与黄酮类生物合成途径的Gb4CL1、Gb4CL3。然后，将GbC4H分别与Gb4CL的组合，GbC4H1、GbC4H2均与Gb4CL1、Gb4CL3蛋白间有相互作用。GbPAL2与GbC4H2蛋白间存在相互作用。GbPAL3与GbC4H1、GbC4H2蛋白结合后均有YFP荧光的生成。据此推断GbPAL2、GbPAL3、GbC4H1、GbC4H2、Gb4CL1和Gb4CL3是最有可能参与黄酮类化合物生物合成的候选基因。
本发明利用进化树分析尚发现，五个不同亚型均分散在裸子植物和蕨类植物之间，但位于不同的分支中，差异较大(图8、9)，推测五个不同亚型的PAL参与不同的次生代谢产物生物合成途径。其中GbPAL2、GbPAL3与被子植物中的PAL亲缘关系最近，GbPAL1与GbPAL5之间的相似性更高。五个亚型的PAL都与相应的裸子植物相近，且与蕨类植物有较近的亲缘性。从进化树分析还可看出，只有GbPAL2和GbPAL3真正参与黄酮生物合成的途径。4CL分为两类大的进化分支：ClassI和ClassII。拟南芥、白杨和大豆中的ClassII普遍与黄酮的生物合成相关，而ClassI中的4CL更可能与木质素和其他苯丙素化合物的生物合成相关。Li等在研究拟南芥中不同亚型4CL的作用时，发现只有拟南芥4CL3(A.thaliana 4CL3)的突变体中，黄酮的含量减少了。这说明4CL3是真正参与黄酮的生物合成途径。4CL1和4CL2的突变体中，黄酮的含量没有减少的直接变化。从而，4CL1与4CL2更多的是参与木质素等其他化合物的生物合成。Chen等过表达火炬松Pinus taeda中的4CL3，黄酮的衍生化合物-单宁含量上升，而木质素没有影响，从而推测火炬松中的4CL3也是直接参与黄酮类生物合成途径的酶。根据我们构建的进化树分析候选的Gb4CL1、Gb4CL2和Gb4CL3的功能，Gb4CL2为单独的分支，相关性较低。Gb4CL1与Gb4CL3均与火炬松4CL3，拟南芥4CL3有很高的相似性。这说明Gb4CL1与Gb4CL3非常有可能直接参与黄酮的生物合成，属于ClassII。
二、附图说明
图1.表达GbPAL2的酵母与L-苯丙氨酸(L-phenylalanine)共发酵产物HPLC和MS图谱
图2.表达GbPAL3的酵母与L-苯丙氨酸共发酵产物HPLC和MS图谱
图3.表达GbC4H1和GbC4H2的酵母与反式肉桂酸(trans-cinnamic acid)共发酵产物HPLC图谱
图4.Gb4CL1与GbCHS在酵母中的共表达产物柚皮素查耳酮(1，naringeninchalcone)及柚皮素，2，naringenin)
a.选择性离子流图(阳离子模式,m/z 273)；b.对应a图中化合物1的质谱；
c.对应a图中产化合物2的质谱；
图5.亚细胞定位结果图a.亚细胞定位的载体构建示意图
b.绿色通道为GFP荧光，红色通道为叶绿体的自发荧光，Merged是GFP荧光与叶绿体自发荧光的组合；含空载体的拟南芥原生质体作为对照组，为35S::GFP；35S::GbPAL-GFP为已转入pBI221-GbPAL-GFP质粒的原生质体；Bars＝5μm；
图6.苯丙素途径及其黄酮分支途径中的GbPAL，GbC4H,Gb4CL及GbCHS酶在内质网形成大分子复合体模型
图7.GbPAL,GbC4H,Gb4CL,GbCHS蛋白间相互作用
图8.GbPAL的系统进化树分析图
图9.Gb4CL的系统进化树分析图
三、具体实施方式
下面结合具体实例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如Sambrook等人，分子克隆：实验室手册(New York:ColdSpring Harbor LaboratoryPress,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计。
实施例1
RNA提取与基因克隆
将总RNA提取所需要的实验器具进行灭菌和去RNA酶处理。取银杏叶片，用锡箔纸包住，浸泡在液氮中速冻，取出后放入研钵中，加入液氮，用研棒快速研成细粉，在研磨过程中始终补充液氮以保持低温。待其全部研磨成粉末后，按照北京全式金多糖多酚植物RNA的提取试剂盒的步骤进行总RNA的提取。利用南京诺维赞反转录试剂盒合成cDNA用于基因克隆。
通过利用本实验室的银杏幼苗，对银杏转录组进行高通量测序，以及黄酮类化合物生物合成的数据库多序列比对，进行基因挖掘。找到黄酮类生物合成相关的候选基因，其中包括基因GbPAL、GbC4H、Gb4CL和GbCHS。使用BLAST软件将转录组测序序列与Swiss-Prot、Gene Ontology、Cluster of Orthologous Groups、eukaryotic Orthologous Groups和Kyoto Encyclopedia of Genes and Genomes数据库比对，获得转录组序列的功能注释。通过以上方式，寻找到候选基因GbPAL2、GbPAL3、GbPAL4、GbPAL5、GbC4H2、Gb4CL1、Gb4CL2及GbCHS。然后，结合Medicinal Plant Genomics Research数据库和NCBI中已报道的基因，利用BLAST比对，获得候选基因GbPAL1、GbC4H1和Gb4CL3。基因序列见附录1。每个基因分别带有SacI和EcoRI酶切位点的两对引物进行扩增GbPAL、GbC4H和Gb4CL基因，分别带有NheI和BamHI酶切位点的两对引物进行扩增GbCHS，扩增产物通过电泳纯化并连接平末端克隆载体pEASY-blunt(全式金)进行DNA测序。引物序列见附录2。
实施例2
酿酒酵母表达载体的构建
为了验证基因的功能，我们选择了pESC系列酵母表达载体。测序成功后，同时用SacI和EcoRI酶对质粒和pESC-URA载体进行酶切，从而连接、构建成真核表达载体pESC-URA-GbPAL1、pESC-URA-GbPAL2、pESC-URA-GbPAL3、pESC-URA-GbPAL4、pESC-URA-GbPAL5、pESC-URA-GbC4H1和pESC-URA-GbC4H2。由于4CL的表达产物有CoA结构，如用以上独立基因表达验证功能，则在提取产物时，CoA结构会丢失。故，我们按照以下示意图，将4CL和CHS进行共表达，构建了pESC-URA-Gb4CL1-GbCHS、pESC-URA-Gb4CL2-GbCHS和pESC-URA-Gb4CL3-GbCHS。
基因的功能验证：
本实验所有酿酒酵母菌株的质粒转化均基于电击转化法：在EP管中加入40μL酿酒酵母电击感受态和300ng的质粒DNA，轻轻混匀并转移到预冷的2mm规格电击杯中，快速放入BIO-RAD MicroPulser电击槽中，选择Sc2模式进行电击转化；电击结束后立即用1M山梨醇将电击杯中的感受态细胞重悬并转移到EP管中，30℃孵育1h后涂布在相应营养缺陷的SD(Synthetic Dextrose Minimal Dropout Medium)培养中，30℃倒置培养48-72h。为验证PAL和C4H的功能，同时转化pESC-URA-GbPAL和pESC-URA-GbC4H，以及对照组空白质粒pESC-URA。验证4CL和CHS的功能则需同时转化pESC-URA-Gb4CL-GbCHS和对照组质粒pESC-URA-Gb4CL。转化后的菌株挑取单克隆接种到5mL SD培养基中，30℃220RPM条件下震荡培养过夜，次日以1:50的比例接种到50mL的SG(Synthetic Galactose Minimal Dropout Medium)培养基中，在30℃220RPM条件下的恒温振荡培养箱中培养24h后，投入1mg/mL的底物，发酵培养4天。
实施例3
发酵产物的检测方法
发酵培养4天后，以1:1的体积比加入乙酸乙酯终止反应。超声破碎细胞30min，使细胞内代谢物释放且化合物与乙酸乙酯充分混匀，静置。次日，用乙酸乙酯萃取三次，旋转蒸发仪蒸干，获得发酵产物。然后用1mL色谱甲醇将产物溶解。利用全波长HPLC检测，柱温30℃，流动相A为醋酸-水：1:1000，流动相B为醋酸-甲醇：1:1000；流速为1mL/min。检测波长分别为247nm和309nm。通过与标准品的比对，确证目标产物；然后，利用LC-MS进行确证。
实施例4
亚细胞定位
将基因构建在pBI221-GFP载体上，获得重组质粒。质粒的构建与pBI221-GbPAL1-GFP相同。通过大肠杆菌进行富集，提取质粒，使其浓度达到1μg/μL；再制备拟南芥叶片的原生质体，利用PEG方法进行转染。将重组质粒与内质网的标识物CNX(calnexin)一同转入拟南芥的原生质体，培养16h后，用剪过尖头的200μL枪头取一滴原生质体液至载玻片，盖上盖玻片，用滤纸吸干周边多余的水分；利用激光共聚焦显微镜观察候选基因的定位情况。
实施例5
蛋白间相互作用-双分子荧光互补技术
将候选基因分别构建在pUC-SPYCE和pUC-SPYNE载体上，通过大肠杆菌富集，获得重组质粒，使其浓度达到至少1μg/μL。利用PEG转染的方法，转入拟南芥的原生质体中，制备、转染及观察参照亚细胞定位的方法。
Figure BDA0001933018680000071
Figure BDA0001933018680000081
Figure BDA0001933018680000091
Figure BDA0001933018680000101
Figure BDA0001933018680000111
Figure BDA0001933018680000121
Figure BDA0001933018680000131
Figure BDA0001933018680000141
Figure BDA0001933018680000151
Figure BDA0001933018680000161
Figure BDA0001933018680000171
Figure BDA0001933018680000181
序列表
<110> 于荣敏
<120> 银杏黄酮化合物生物合成基因中多个亚型的研究
<130> 2018.12.27
<141> 2018-12-31
<160> 11
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2319
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 1
atgacggttg acgtaatggc agatgttgca gaaaccgaag ctggatcgat gctcagtggc 60
gtaacagcag tttgcataac aaatgagaat atggaaggtt tggatccttt gaattggtcc 120
agatctgccg attctctgaa aagaagccat gtgaatgaag tgaggtccat gatcaaagaa 180
tacgaggaga taaagcatgt gtctctcaga ggaacgaact tgagcatagc ccaagtagca 240
gcagtggctc ggaggcctga ggttaaggtg gagctggatg cagctactgc taaaaataga 300
gttgatgcta gctcagactg ggtcatcaat aatatcacca aggggacgga tacatatgga 360
gttactaccg gatttggtgc aacttcgcac cggaggacta atcaagcagt tgagttgcag 420
aaagagctta tccgttttct gaacgctgga gtgtttggga aaggggattc caattgcctt 480
ccgtcctctg caaccagagc tgcaatgctg gtgcgtacca atactctgat gcagggctat 540
tctgggattc gatgggaaat cctcgaggcc atgcagaaac tcatagattc tggcattact 600
cccaagttgc ctctccgagg tactatctcg gcttctggcg atctggttcc tctgtcatac 660
attgccggtc tgctcactgc ccggcctaac tctgtggcta ttgggcaaga tgggaaagaa 720
atgcctgcgg atgaggctct gagaatcgca gggatttctc agcgttttga gctttatccc 780
aaggaagggc tggctttggt aaatggtact gcagtagggg ctgcggttgc atgtactgtg 840
tgctacgatg ccaatgtttt ggctgttttt gcagagattg cgtctgctat gttctgcgag 900
gtgatgcatg gaaaaccaga gtttacagat ccactgactc acaagcttaa acaccatccc 960
ggtcagatgg aggctgctgc agtcatggaa tggattctgg acggaagttc gtatgttaaa 1020
tctgcagcta agctgaatga aacagatcca ttgaagaagc cgaagcagga tcgttatgcc 1080
ctgagaacat ccccacagtg gttgggtcct caggtggaag tgattcgcat ggccacgcat 1140
gcaatccaaa gagaaatcaa ttctgtcaat gataatcccc tcattgatgt tgtaagggac 1200
aaagctgtgc acggtgggaa cttccaaggg acgcccatag gcgtctccat ggacaatgta 1260
cgcttggctt tggctgccat agctaaactc atgttcgccc agttctcaga gcttgtgaat 1320
gactactaca acaatggctt accttctaat ctctctggcg gagccaatcc cagcctggac 1380
tatggcctca agggcgcaga gatcgccatg gcttcctaca cttctgaaat gcagtttttg 1440
gcaaatcctg tgacgaacca tgttcagagc gcagagcaac ataaccaaga cgtgaattcc 1500
ttgggtttgg tctctgcacg gaaaactgca gaggccattg agattctgaa gctcatggtt 1560
tcgacctact tgattgcact gtgccaggcg gtggatcttc ggcatctgga agagaacttc 1620
catggcggcg ttaagcaaac agtttgccaa gcggccagga agacgcttag cttaaccgcc 1680
gacggactgt tactgccatc tagattctgc gaaaaggagc ttctgcaggt tgtggatcat 1740
cagcccattt tcagctacat tgacgatccc gccggtcctt gttctccact gatgctgcag 1800
ctgcgacaag tgctggtcga acaggcgttg aattccgtat catcggcgtc tccaatggag 1860
gaaaccggat cgaaccctga gcattctctc ttcaacagaa tacctgtgtt tgaagaagaa 1920
ctgagaaata aactagccgt tgagattccc cttctgcgag aaaaatttga acgaggagat 1980
ttcgcagttg ctaacaaaat acgggattgt aggacatatc cagtgtatga atttgttcga 2040
ggagagcttg caacaagcct cttgtctgga ccccaaggaa gaacaccggg cgaggatatt 2100
gatagggttt atgtggcaat tacagaagga aagctcagtg ggcgcctgat ggaatgcttg 2160
gagggatgga atgaatcgcc tgggccattc gtggggttga agaagaaggg ctatggagtt 2220
gttaagacaa acaatccatg cgggtggagc tggttccaac agattggagg ccctcaggtt 2280
aatggaggca agggctattg gcttctgtct attgcttag 2319
<210> 1
<211> 2178
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 1
atgagtcaat ttgcattttc agaggatttc agatcaaccc acgtagccaa agccaatgga 60
gaagctctga attcctccaa attctgcgtg gaaaacaata tctctgatcc tctcaattgg 120
gtctctgcag cagaagcaat gaaaggatcc cacctggacc aagtgaagaa aatggtgcag 180
gaatacaata atccgcaagt aaagcttcaa ggatccaatc tcacagtagc ccaagtggcc 240
gctgcagcca gaagaatgga gtcggtgaga atcgaactgg agaccaatgc cagagaacga 300
gtggagcaaa gtagccgttg ggtgatggac agcatgaaca acggcactga cagctatggc 360
gtgacgacgg gattcggagc aacctcacac agaagaacac gccaaggaga agctttgcag 420
aaagagctta taaggtttct caacgctgga attttcggct gcggggattc gaacacgttg 480
cctcaggcca ccaccagagc cgccatgctc gtccgcgtca atactctgct tcaaggctac 540
tcgggcatac ggtgggaaat ccttgacgcc attagccgtc tgttgaacgc cggaattaca 600
ccgcagttgc ctctgcgcgg cacaatcacg gcttccggcg acctcgtccc gctctcatac 660
attgctggcc ttctgacagg ccgtcagaat tcacgagcgg ttacagttga cggacttgaa 720
ttgagtgcgc ttgacgccct gaaaaccgcc ggcgtcgttg acggaccctt cgaattgcag 780
cccaaggaag gcctggcgct cgtcaacggg accgccgtcg gatccggttt agcttcaatt 840
gtgctttttg acgccaatat tctagctctg ctcgccgagg ttctgtcagc gattttctgt 900
gaagtgatgc aaggtaagcc tgagtttaca gatcacttga cgcacaaatt gaagcatcat 960
cctggccaga tcgaggctgc ggccatcatg gaattcgtcc ttgagggaag ctcttacatg 1020
aaagccgctg caaaattgca cgaaatggat ccgctgcaga aaccgaagca ggatcgatat 1080
gcgctcagaa catcgcccca gtggcttgga cctcaagtcg aggtgattcg aaccgcgacc 1140
ctttcgattc agagagaaat caattccgtc aacgataatc cgttaattga cgtctccaga 1200
aacaaggcac tacatggagg aaatttccag gggacgccaa ttggggtgtc aatggataac 1260
accagattgg ccttggcggc aatcggaaaa ttgatgtttg cccaattctc agagctggtc 1320
aatgattttt acaacaatgg attgccgtca aatctcagcg gcggacctaa cccgagtttg 1380
gactatgggt ttaagggagc cgagatcgcc atggcttctt atacttcaga actccaattt 1440
ctggccaacc ctgttacgaa tcacgtccag agcgcagaac agcataacca ggacgttaat 1500
tccttgggtt taatttctgc ccggaagacg gccgaatccg tcgaaattct caagctcatg 1560
gcgtcaactt atctcgtcgc gctctgtcag gcagttgatc tccgtcatct ggaagaaaac 1620
atgcaggcca ccgtcaaaca tgccgtcagc caagtctgca agaaaacact ggcaattgga 1680
gcccaaggag aattgcttcc gtcaaggttt tgcgaaaagg atttgctcaa agtggtggag 1740
cgtgagccta tttttgcgta tattgacgac ccgtgcagcg cgtcgtatcc tctgacacag 1800
aaactcagac aagttcttgt agagcatgct ctgggcaatg gcgataatga gaaggacgag 1860
aacacttcca tcttccagag aattaatgcg ttcgaaaaag agctgaaagc ccatcttggc 1920
gcagaagttg accagaccag agatgcattc gacaaaggca ttgcttctgt tcctaatagg 1980
atcaagaact gtcgttcgta tccgctgtat gagttcgtga gattcgaact cgggacctct 2040
attctatcag gctcgaagac actctccccc ggccaagaat ttgacaaggt ttttgtcgct 2100
atcaatgaag gtaagttgat tgacccactt ctcaagtgtc tccaaggatg gaacggggct 2160
cctcttccta tatgttaa 2178
<210> 1
<211> 2175
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 1
atggttgcag gagcagaaag gatgcagtcg aatccgcaga atggatcaca gtatgttaag 60
agtggcggaa ttggagatct gtgtcagagt ttcgactcca ccactgatcc tctcaattgg 120
gctcgagctg ccaaggctct gcaaggaagc cacttcgaag aagtgaagca aatggtggat 180
tcgtatttca aatctgggga gatctcaatt gagggaaaaa ctctgactgt tgcagatgtt 240
actgctgttg ccaggaggcc ccaagtccaa gtaaaattgg atgctgctgc tgccaaatct 300
cgtgttgagg agagctctaa ctgggttctg caccagatga ctaagggaac ggatacctat 360
ggagtgacca ctgggtttgg ggctacttct cataggcgaa ccagccaggg agtcgaatta 420
cagaaggaat tgattcgttt cctgaatgct ggagtatttg gtagctgtga aggaaatgtg 480
cttccagagg ctactacacg ggctgcaatg ctcgtaagaa caaatactct cctgcaaggc 540
tactctggca taagatggtc tttactcgaa acaatcgaga agcttctgaa tgcaggaatt 600
acgcccaagt tgcccttgag ggggacgata acggcttctg gagatttggt tcctctgtct 660
tatattgccg ggctgttgac aggcaggcct aattcaaagg tcagaaccag agatggaact 720
gaaatgagcg gcttggaagc gctcaagcaa gtggggttgg aaaaaccctt tgaattgcag 780
cctaaggaag gattggccat tgtgaatggt acctctgttg gtgcagcctt ggcttcgatt 840
gtatgttttg atgcaaacgt gctggctgtg ctgtctgaag tgatgtctgc catgttctgt 900
gaagttatga acggaaaacc agaattcaca gatcctttga cgcacagact aaagcatcat 960
cccggccaaa tggaagccgc ggcaattatg gaatacgttt tggacggaag ttcgtatatg 1020
aaacaggctg ctaagctgca ggagctgaat cctctgcaaa agcccaagca ggatcgctat 1080
gcattgcgca cttctcctca gtggcttggt cctcaggttg aggttatcag agctgcaacg 1140
cacatgatcg agagggaaat caattctgtc aatgataatc cagtaattga tgtatccaga 1200
gacaaagctc tgcacggagg aaatttccaa ggtaccccta ttggtgtatc gatggataat 1260
ctccgtctgt ccattgcagc gattgggaag cttatgttcg cccaattctc agagctggtg 1320
aatgactact ataacggagg cttgccctca aatctaagcg gagggcctaa tcctagtctg 1380
gactatggtt taaagggcgc agagatagcc atggcctctt atacttccga gctcgagtac 1440
cttgccaacc cagtcactaa ccatgtacag agtgcggagc agcataacca ggatgtgaat 1500
tctttgggtt tagtttctgc cagaaaatcg gcagaagccc tagagattct gaagctcatg 1560
ctgtctactt atctgattgc attgtgtcaa gctgtcgatc ttcgtcattt ggaggagaat 1620
atgcaagcca cagtgaagca ggttgtggcg caggtggcga agaagactct gagtactggt 1680
aaaaacggag agcttttacc aggacggttc tgtgaaaagg atttgcttca ggcggtggat 1740
agccagcatg ttttcaccta cattgacgat ccctgcagtg ccacgtaccc gttgatgcag 1800
aaactcagac aagttctggt tgaacatgct ttcaagaaca ccgagagcga gaaggatccc 1860
aacacttcta ttttcaacaa gatcaatttg tttgaacaag aactcaaatc acaacttgaa 1920
tcagaagtgg atctgacaag ggctaattac gataaaggaa atagtgcagt ggctaacagg 1980
atccacgact gtcgttctta tcctctctat gaattcgtga ggactcagct tggcactaag 2040
ctcttgtgtg gcacacgagc tacgtctccg ggtgagtata ttgaaaaggt attcgacgca 2100
atttgcgagg acaagattat tgagcccatc attaaatgct tggatggctg gaaaggaact 2160
cctggtccgt tctga 2175
<210> 1
<211> 2055
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 1
atggagtgca atggggtcgc aacatctctc agctctcatg tgaaggacat tgcccttctc 60
atccaagttt tcaacgacac ggaatgcatc aatgtggacg gctctcaact gacagtagca 120
catgctgcgg ctctggcagt tcggccacaa gtaaaggtgg ttctggagga caaatgccga 180
ggccgggttg agcggtgctc gtcgtgggta cagcagaagg ccaaagatgg agcggatatc 240
tatggcgtca ctactggatt tggagcatgt tccagcaaaa gaaccaatca gctcagcctc 300
cttcaggaat cccttattcg atgcctgcta gccggagcct tcatgcgccc gtcttcggcc 360
tcgacctcct ccaccggtga gctggccacc agcacaaccc ggtgtgcaat gctgctgagg 420
atcaatagct tcatgcgtgg ctgctccgga attcgatggg aggttctgga ggctttgaag 480
gagcttctca atcaccatat tactcccaaa tgccctctgc gcggctctgt aagtgcttcc 540
gcagaccttg tccccttggc ctacatcgca ggccttctca ttggaagacc cactgtgaaa 600
gccagaacgg ccgatcatca agagctcact gccccacagg ctctcgctaa ggtagggctg 660
aagcccttta agcttcaggc caaggaaggt ctggcccttg tcaacggtac ttcatttgcc 720
accgccctgg cttccactgt catatacgat gccaatgttg ttttactctt tgtggaaatc 780
ctgacaggaa tgttctgtga agtggttttc ggcagagaag agtttgccca tccattgatt 840
cataggatga agccacaccc tggccagatt gagtccgcag cgctcttgga atggcttctc 900
aatgatagtc cgtttatgga gctttctcgc gagtattaca gcactgataa gctcaagaag 960
ccgaggcagg acagatacgc tctcaggtcg tctccccagt ggctgggccc tcttgtgcag 1020
atcatacgcg aagcaactgc aacaatacaa gtagaaatca attctgccaa cgataatccc 1080
atcatcgatc atctccatga caaggctctc catggcgcaa atttccaggg cagtgcaatc 1140
ggcttctaca tggaccacgt ccggattgct ttggctggac taggaaagct tatgtttgcc 1200
caattcactg agcttatgat cgaattctac agcaatgggc tccctggcaa cctctccctt 1260
ggtcctgacc tcggcgtaga ttatggttac aaaggagtcg atattgccat ggcttcttac 1320
tgctctgaac ttcagtatct cgcaaaccca gtcaccacgc acgtacagag cgctgaacaa 1380
cataaccaag acatcaactc tctggcactc atatctgccc ggaaaacaga agaggctctg 1440
gaaatcatga aattaatgct tgcctcattt ttaacagctc tatgtcaggc tgtggatctg 1500
cgtcaattgg aagaaatact tgtaaaagtt gtagtggatg tcatttctaa agtttcagac 1560
gagtgcggcc tgccggagtc catgaagaag gaacttgtga acgctgcaaa gggtattcct 1620
gtgtacagtt atctggaaaa cccatgcgac ccttcccttc ccctgctctc aaagctcaga 1680
caaacttgtc tggagtgcat tctcagatca ccgggaagca acgaggtatc gcacggtcct 1740
ttggtagaca agatcagaga gtttgaagcg cagctcatgt cagagcttga agctgaaatg 1800
actgcaacca ggctttctta cgagcgcaca caccctgcaa taacaacggt ggaaacggct 1860
agattcagct ctagaatccg tggctccaaa ttgtttcctc tgtatgcatt tgtccgccaa 1920
gagctgaata caaaactcat gactgcaagg actgatcaca cgccagaaga agacgttcag 1980
aaggtgtttg atgctatcat cgatggcacg atcacagtgc ctctccttca ttgtctcaaa 2040
ggattcctcg actag 2055
<210> 1
<211> 2358
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 1
atggcgggaa tagcaaatga gcaatctgtg cagagggcgg gtgaagacta cagcaagtct 60
ggtggaagta aattgagtcg accgagtgag atataccgtc cgcccactct gccgttgccg 120
cagggcggca gaaatcccat ctgcgtgagc ggtgaagggt atgtgcaggt ccctccacac 180
tggcggaagg cagcggaggc gatgcaatgc agccattttg aggaggtgag aaggatgatt 240
gagcaattca acaactcgca gaaagtggtg ttgcaaggca ctaatctcac ggtcgcccag 300
gtcgctgccc tcactcgcag acccgaggtg aaggtggagt tggacgaggg tgccgccaag 360
tcgagagtgg acgaaagctc caactgggtt ctgcacaaca ttgccaaggg caccgataca 420
tacggtgtca ccaccggctt cggagcaacc tctcacaggc gaaccgacca agccgtggat 480
ctccagaagg agttgatccg cttcttgaac gctggagtgc tgggcaagaa ggactctcaa 540
tgcctgccct ccgagtacgc aaaggcagcc atggcggttc ggaccaacac actgatgcag 600
ggctactccg gcatccgatg ggacatcctc cgctctctcc agaagctcat ggattgcaat 660
atcactccca agatgcctct cagaggcacc atcactgcct cgggagattt ggttccattg 720
tcctatattg caggcttgct cactgccagg cccaattctc gtgcggtcac cccagacggc 780
aaggagctga cggcgattga agctctcaag gctgcgggca tatcggagcc gtttcagctc 840
cagccaaagg agggtttggc ccttgtcaat ggaaccgcgg tgggatccgc agttgcctct 900
tccgtgtgct tcgatgccaa tgtcatggcg ctcctctcgg aggttctctc ggcacttttc 960
tgcgaggtga tgcagggcaa gcccgagttc acagaccctc tgacgcacga actcaaacat 1020
caccccgggc agatcgaagc cgccgccgtg atggaattcc tgctggacgg aagcgactac 1080
atgaaggagg ccaagaggct tcacgaaacg gatcctctgt ccaagcccaa gcaggatcgc 1140
tacgctctga gaacctcccc gcagtggttg gggcctcaga tagaggtgat tcgcatggct 1200
acccattcca tcgagaggga gatcaattcc gtcaacgaca acccgctcat cgacgtggcc 1260
agagacatgg ctctccacgg aggtaacttt caaggcactc ccatcggagt ttccatggac 1320
aacatgagga tcgctctcgc agccatcggc aaacttatgt tcgcccaatt ctccgaactg 1380
gtgtgcgatt actacaacaa tggcctgcca tccaatctca gcggcggccc aaatcccagt 1440
ctcgattacg gtttcaaagg tgccgagatc gcaatggctg catacacatc ggagcttcag 1500
tatcttgcca atccggtgac gactcatgtc cagagcgcag agcagcacaa ccaggacgtc 1560
aactctctag gtctcatttc ggctagaaag actgcagagg ccattgagat cctgaaactc 1620
atgtctgcaa catatctgat cgctctatgc caggcagtgg acttgagaca tgtggaagag 1680
aacatgagag cagttgtcaa acacgtggtg ctgcagactg ctagaaagac tctctatact 1740
gcagaggatg gatccttgct ctctacccgc ttctgcgaga aagagttgct gcaaatcatt 1800
gatcatcagc ccgttttctc ctatctggac gacccttcga atccttccta cgctctgatg 1860
ctgcagctga gagaggtgtt agtggagcag gctctcaagt gccctgtgga ggaggacact 1920
caacaaggca ctccctcctt gttccgaacc atacccgtct tccaagatga gctcaagcgt 1980
cgcttggagg aggagatccc caaggccaga cagagatacg acagcggaga ctaccctctc 2040
cccaacagaa ttcagaaatg ccgaacctac ccactttaca gattcgtcag atctgaggtt 2100
caaaccgagc tgctgactgg caccaagccc aggagcccag gggaagacat cgaaaaggtt 2160
ttcgacgcca tctgccaagg ccagttagca gagcccctat tgaaatgctt ggagatgtgg 2220
cgcggctcct ctggtccctt cacccctcgc acgcaagcag cttctcctgc ggcattcaat 2280
ccttcctact ggggatggtt tgacagtctc aaatcccctt ctgccaccag cggcagagga 2340
ttatggcaac aactctaa 2358
<210> 1
<211> 1518
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 1
atgttagaga gcatgaattt ggagaagggg ctgatagctc tgtttgttgt agtagtaagt 60
gccatatttg tatccaagct caaatctaag aagctcaagc tgccccctgg cccattcgca 120
ttgcccatat ttggcaactg gctccaagtt ggggatgatt taaatcacag aaatctcacg 180
gatttggcca agaaatatgg agaaatattt ctgctaaaaa tgggtcagag aaacctggtt 240
gttgtctcgt ctcccgagta tgccaaggag gtgctgcata cccaaggtgt ggagttcggt 300
tcccgcacaa gaaatgtggt ttttgatatt ttcaccggga agggacaaga tatggttttt 360
acggtttatg gagagcactg gaggaagatg cgcagaatta tgactgtacc tttcttcacc 420
aacaaagtcg ttcagcaata ccgatttgca tgggaagacg agataagccg ggcagttgag 480
gacgttaaaa accgccctga ggcttccacg accggtattg tgattagacg gcgtttgcag 540
ctgatgatgt ataatattat gtacagaatg atgtttgata ggcggtttga gagcgaggag 600
gatccattgt tcctcaagct taaggccttg aatggtgaaa gaagtcgatt ggcacagagc 660
tttgagtata actatggcga tttcatcccc attctcaggc cattccttag aggctatctc 720
aagatctgca aggaggtcaa ggaaatgagg ctttctttgt tcaaggatta tttcattaat 780
gagcgcaaga agttggctag caccaagggc tctagcagct taggcgagaa gtgtgccatt 840
gatcacatat tggatgcact ggacaaagga gaaatcaatg aggacaatgt cctgtacatt 900
gttgagaaca tcaacgtagc agcaatcgaa accactctct ggtccatgga atggggcctt 960
gctgagattg ttaaccatcc agacattcaa cagaaaattc gcaaagagct tgacactgtt 1020
cttggcccag gggtggagat aaccgagcca gacaccacca gattacctta cctgcaagct 1080
gtagtcaagg aaactctgcg tttgcacatg gcaatcccat tgctggttcc tcacatgaat 1140
ctcaaccagg ctaagctggg tggttatgac ataccagctg agagtaaaat ccttgtgaat 1200
gcatggtggc tggccaacaa ccctgaatgg tggaacaaac ccgaagagtt tattcctgaa 1260
agatttttgg aggacgagca gaagattgaa gccaacggaa atgatttcag gttcttgcca 1320
tttggtgttg gaaggagaag ttgcccagga attattctgg ctctgcccat tttggcactg 1380
tcccttggaa ggctggttca aaactttgac ctctcacccc cacctgggca ctccaaggtt 1440
gatgtctctg agaagggagg acaattcagc ttgcacattc tgaaccactc tgttgttgta 1500
gccaaaccca gagtttaa 1518
<210> 1
<211> 1518
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 1
atggaaatcc agacatcact aagctcattg acagagcaag catggcttcc agctctactg 60
gctatcacgg tcgccgccat agttctcatg aatctccgtg gcaagaagct gaagcttccc 120
cctggccctg tggcggtgcc ggttttcggc aactggctgc aggtgggcga tgatctcaac 180
caccgaaacc tgtccgaaat ggccaagaaa tatggcgaca ttttccttct gagaatgggc 240
caaaggaacc tcgtcatcgt ctcttctccc gacctggcca aagaagttct gcacacacaa 300
ggtgtcgagt ttggatcgcg cacgcgcaac gttgtcttcg atatcttcac caacaaaggg 360
caggacatgg tctttaccgt ttatggcgat cactggagac gaatgaggag gataatgacg 420
gtgccttttt tcacaaacaa agtcgttcag caatcgcggt ttgcttggga ggacgagatc 480
gaacacgtta tcaaggattt gaaggccgac cccagggccg tgtctgagag cggcatcgtg 540
atccggcgcc gtctgcagat gatgatgtac aatattatgt atcgaatgat gttcggcagg 600
agattcgaac gcgaggacga tcccctgttc gagaagctta agtttttgaa cggcgaaaga 660
agtcgtctgg cgcagagttt tgagtacaat tacggggatt ttattcccgt tctcaggccc 720
ttcttgaaga agtatttgga tacctgccag cgcgtgaagg accagcggat tggactcttt 780
aaggagttct ttgtcgatga acggaggaaa ctcgtcggaa aaattagtga tggcgagaaa 840
gttgctgtgg attacctttt cgaagcccta gaaaaagggg agattaacga ggaacagctg 900
ttgtatatca tcgagaatat caatgttgca gccattgaga caaccctgtg gtccatagaa 960
tggggcatcg cagagctggt gaacaaccct cgcatacaaa gaaaggttcg tgaagagctg 1020
gattcgatcc tgggacatac gcccatcaca gaaccagacg tagggaagct accttacctg 1080
gaagctgtag tgaaggaaac cctgcgccgc cgcatggcaa ttccttttct cgtcccacac 1140
atgaatctcc accaggccaa gttaggaggg tacgacattc ctgcagagag caagatcttg 1200
gtaaacgctt ggtggattgc caacaacccc aagctctggg accgccctga ggaattccga 1260
ccagaaaggt ttctgaacga aaagattgaa gccaacggaa acgacttcaa gttcctgccc 1320
ttcggctctg gccgaagaag ctgcccgggc atcataatcg ccattccatt gttgcacctt 1380
gtttttggca ggctgttgca gaatttcgaa atttctcttc catctggtgt caataaaatc 1440
gacatgacag agaagggagg gcaattcagt ttgaggattg ccaatcacta caatgtagtg 1500
gtcaaaccca gaacctga 1518
<210> 1
<211> 1773
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 1
atgattgagg tacctgccat ggcccctctt gaagataatt cgtctgtgac agtctgtact 60
gcatcgaccc aactagtgca catgaataat attgctgacc atacttctca tggcattggc 120
aagggaatac aaaaggagca gcagcagcag aagcaggaag agttgtatct gtatagatcc 180
aagcttccag acatagagat ctctaaccat cttccattgc atacatattg cttcgagaaa 240
ctgcacgagt tcagagaccg gccatgtctg atacaaggat caaccgggaa aatctacagc 300
tatggcgaag tagaattgat ttctcggaga gtggcttctg ggttatctaa attgggcatc 360
ggtaagggcg atgtagtgat gctgctgctg ccaaattgcc cggaattcgc cttcgtgttc 420
ttgggggcct cgttcagagg cgccatagct acgactgcca atcctttcta cactccgaac 480
gacattgcca agcaagtgaa tgcatctggt gcgaagctga tcgttaccca gagttcgtat 540
gtggagaaac tgcgggattt gatggcgaat gatgcactgt gtttgcaggt tgtgacgata 600
gacggccctc cggaggattg tctgcatatt tctctgctaa cggaggcgga tgagaatgag 660
tgcccaagtg tggatatcag tccggacgat gcagtggcgt tgccatactc ctctggaaca 720
accgggttgc ccaagggcgt catgctgact cacaaggggc ttgtatccag cgtggcgcag 780
caggtggatg gggaaaatcc taatctgtac ctgcgttccg aggacgttgt gctatgcgtt 840
ttgccgcttt ttcatattta ttcgcttaat tctgtgcttt tgtgctcgtt gagggccggg 900
tctacgatac tgttgatgca gaagtttgag attgggagtt tgttggattt gattcagagg 960
tttaaggtca ctgtggctcc tgttgtgcct ccgattgtgc ttgccattgc taagaatgca 1020
atggtggagg attatgactt gtcgtccata aggattgttt tgtctggcgc tgcaccgctt 1080
gggaaggagt tggaggaggc gcttcgaact cgggttccca atgccttgtt tgggcaggga 1140
tacggaatga ctgaggccgg cccagtgctt gcaatgtgcc tggcattcgc caaggaaccc 1200
tttccggtga agcctggctc atgcggaact gttgttcgca atgcccaagt gaagatcatc 1260
gatccagaga ccggagtgtc tcttccccat aacaaaccag gagaaatctg cattcgcgga 1320
ccccaaatta tgaaaggtta tcttaacgat gccgaggcta cggccagaac catagacgaa 1380
gatggttggc tgcatactgg cgatgttggt tatatagacg atgacgagga agtcttcatt 1440
gttgatcgag tcaaggagat tatcaaatac aagggatttc aggtgcctcc tgctgagttg 1500
gaagccattc ttatcagcca tccgtccatt gcagatgctg cagttgtacc tcaagtgaat 1560
gaagttgcgg gagaggtgcc ggtggccttc gttgtgagat caaatgggta tgaacccacg 1620
gagcaagaaa tcaaagattt cgtagctaaa caggttgtgt tctacaagaa gctgcacaag 1680
gtctacttca tccatgctat tcccaagtct ccctctggca agatactgcg aaaggatttg 1740
agagccaaac tcgctgcgcc accagtgcat taa 1773
<210> 1
<211> 1671
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 1
atggcagcag cagtatcatc aatgggtctc gagatcgatc tccgcagcgg atactgcaag 60
gcaaattcaa catactacag caaacgaaaa tctctgagtt ttccccaaga tgaatatctg 120
gatctcccat cctttgtttc ttcatctacc cacaacggcg aaattgcact gattgattcc 180
gccactggat acaaaatcac attctcagag ctctggttgt ctgtgaaatc agttgccgca 240
ggcctggccg gccttggggt taaaaagggc aatgtggttc ttcttctgtc cccaaattcc 300
atccattttc cagtgatttg catggcaatc atgtcccttg gagccgtggt taccaccaca 360
aatccgctaa atacagcgtc agaaatagcc aaacaggcca aggactccaa tgccatgctc 420
gcttttgtta cgccatctct ggcagagaaa gtagcaggcg ctaatttgcc attaatactg 480
attgggcaag aaaaagagat aaaaatgcga aaatccaatt cccaaaacat cgtttcttct 540
ctggagaagc tcatgggttc gaatcctgcc aagatgccgg ctgtgaaaat ccgtcaagac 600
gatactgcaa cgctgcttta ctcgtctgga actactggaa tgagcaaggg ggtcgtttct 660
actcatagaa atctgatcag catggtttgc gtcttgagaa acagatttag tttatctgat 720
catactcctg ctaatagttc ttatctctgc actgtgccaa tgtttcacat atatgggctt 780
gtggcttttg catgtggatt gctggcaaca ggggctacca ttgttgtaat gtctaagttt 840
gatctagtgg acatgcttgg ggctatacag aaatacagag taacatattt gcctctggtg 900
ccaccaattt tgttggctct cacaaagaca gatgttacga ataaatatga tctaagctct 960
ctgaaatctg ttctgtgtgg tggagctcca ttaggcaagg agtctatgga agaattcata 1020
tctaaatttc ccaatgtcac aatcatgcag ggttatggat tgacagaaac tacagcagtt 1080
ggagcgtcca cagacaccca agaagaaagt aggcactatg gaacagctgg aatgctgtct 1140
ccaaataccg atgcaaaagt agttgatcct gattctggaa ttcctctccc tccaaatcaa 1200
agaggagaat tgtggcttcg tggacccaca gttatgaaag gttattttag caatcctgaa 1260
gctactactt cagctctgga ctcagatggg tggcttcgta ctggtgatct ttgttacatt 1320
gatgatgaag gctacatctt tatagtagat agaataaaag agttgataaa gtacaaaggt 1380
tatcaggtcg ctcctgctga attagaagca ttactactct ctcatcctga ggttgcagag 1440
gttgcagtaa tcccgtttcc aaacaaagaa gcaggccaag ttccaatggc atacattgta 1500
aggaaaccag gcaccacact atctgaagcc agtgttatta actttgttgc tcagcaggtg 1560
gcaccctaca agaagattcg tcgggtggct tttgtgaacg agattcctaa aacagcagca 1620
ggaaagatct taagaaagga tttgattaaa ttggccacct ccaaactttg a 1671
<210> 1
<211> 1674
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 1
atggctctta ttgcgccttc taccttcgat cataaggtca tcagcgcacc ggctaaaatg 60
gaaaatggtg ccgatgagta tttgtttaga tccaagcttc ctgatattca gatcccttac 120
catcttccgc ttcatacata ttgcttcgag agattgtcag agtttgcaga gaagccatgc 180
ctgattgatg gagcgactgg taagatttac aactatggag aagtcgaatt gatttctcgt 240
aaagtggctg ccggtttggc caaattgggg ctgaagcagg gcgaagtggt tatgcttttg 300
ctgcaaaatt gcgttgaatt tgcatttgtg ttcctcggaa gctctatcag aggcgccatt 360
accacgactg ctaatccttt ttatactccg ggagagattg ccaagcaagc caatgcttct 420
ggagctcgta ttattgtcac tcaggcggcc tatgccgaca aactgactga tcttaagagt 480
gaaaatttga tagtaataac tatcgacagt ccgccagagg gatgtcaaca tatttctgtt 540
ctaacagaag cgcatgagag cgaatgcccg tctgttaaca tcgatccgga cgatgtcgtc 600
gccctgccat actcctctgg aaccacgggg cttccaaagg gcgtcatgct aactcacaaa 660
ggatgtgttt ccagcgttgc ccaacaggtg gatggcgaaa atcctaatct ttacatgcat 720
tcagaagacg tgatcctgtg cgtattgcca ttgtttcata tttattctct caatgccgcg 780
cttctctgcg ctctgagagc aggcgctgcg attctgataa tgcagaaatt caatactgtg 840
gctctgcttg agctcattca gagattcaag gtcacgatcg ccccctttgt gccgcccatt 900
gttctagaaa tggcgaagaa tccgattgtt ctaaattacg atgtgtcatc cattagggtc 960
atcatgtctg gcggtgctcc tctgggaaaa gaactggagg atgctctcag agcacggctt 1020
cccaaggcca aatttgggca gggttatggt atgacagaag ccgggccggt gctagcaatg 1080
aacctggcgt tcgccaagga accgtatcca gtgaaatctg gagcttgtgg aacagtcgtt 1140
ccgaatgccc agatgaagat catcgataca gagactggac aatgtctccc gcgcaacaaa 1200
cccggagaaa tctgtattcg tggaccccaa attatgaaag ggtatttaaa cgatccagag 1260
gcgacggcca gaacgattga tgaagaagga tggctgcata caggcgatgt tggattcatc 1320
gacaatgatg aagaaatctt cattgtcgat cgagttaaag agcttatcaa atataaggga 1380
tttcaggttg cccctgctga gctggaagcc atactcgtca accatccatt catagctgac 1440
gcagcagttg tccctcagaa gaacgaggct gcaggagaaa ttccagtagc attcgtggtg 1500
aaatcgaatg gggctgagat tagtgagcaa gagatcaaag aattcgtggc aaaacaggtg 1560
gtgttctaca agaagatcca gaaggtcttc ttcctggacg ccattcccaa gtctccatct 1620
ggcaaaatat tgcgcaagga tttgagagct agactgaatg caggcatgca ttag 1674
<210> 1
<211> 1191
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 1
atgcctgcag gagcgatgga ggatttggag gcattcagaa aggcacagag atcggatggt 60
ccagccacta tactggcaat cggcactgct actcctccca atgcggtcga gcagagcaat 120
tatcccgatt actacttccg aattaccaac agcgagcaca agacggagct caaggagaaa 180
ttcaagcgca tgtgcgacaa gtcagcgata aagaagagat acatgtactt gacggaggag 240
atactgaagg agaagccgga ggtgtgcgca tacatggcgc cttcgcttga cgcacggcag 300
gacatggtgg tggtggaggt gccgcggctg ggaaaagaag cggcggccaa ggcgataaag 360
gagtgggggc agccaaagtc gaagataact cacctcatat tctgcacaac aagcggggtg 420
gacatgccgg gggccgacta ccagctgacg aagctgctgg ggcctcgacc gggcgtgaag 480
agagttatga tgtaccagca aggctgcttc gcaggaggca ccgttctgag agtggcaaag 540
gatctcgccg agaacaacag aggagcgcgc gttctggtgg tttgcagtga aataacggcg 600
gtgacgttcc gggggccgag cgagactcac ttggacagtc tggtggggca ggcgctgttt 660
ggagacgggg cagcggcggt gatagtggga gccgatccca tcgcagaggt ggagaagccg 720
tccttccaac tcctctggac ctcccagacc attcttcccg acagcgacgg tgccatcgac 780
gggcacctgc gagaggtcgg cctaaccttc cacctcctca aggacgtccc cggcctcatc 840
tccaacaaca tcgagaagag cctcgtggag gccttccatc agttcggcat ctccgactgg 900
aatgagctct tctggatcgc tcaccctggc ggcccagcca ttctcgacca ggtagagtcc 960
aagctccacc tcaagtccct caagatgagg gccaccaggc acgttctgag cgaatatggc 1020
aacatgtcca gcgcctgcgt tctcttcatt ctcgacgaaa tgcgcaaatc ctctatcaaa 1080
aacggttgtt caacgaccgg cgaaggactg gactggggtg ttctctttgg cttcggccct 1140
ggcctcaccg tcgagaccgt agtcctcaga agcgttccct gcaacaagta a 1191

Claims (5)

1.银杏中11个与黄酮生物合成相关的候选基因序列，具体为：
一种编码苯丙氨酸解氨酶GbPAL1的核苷序列，其核苷序列为SEQ ID NO.1；
一种编码苯丙氨酸解氨酶GbPAL2的核苷序列，其核苷序列为SEQ ID NO.2；
一种编码苯丙氨酸解氨酶GbPAL3的核苷序列，其核苷序列为SEQ ID NO.3；
一种编码苯丙氨酸解氨酶GbPAL4的核苷序列，其核苷序列为SEQ ID NO.4；
一种编码苯丙氨酸解氨酶GbPAL5的核苷序列，其核苷序列为SEQ ID NO.5；
一种编码肉桂酸4-羟化酶GbC4H1的核苷序列，其核苷序列为SEQ ID NO.6；
一种编码肉桂酸4-羟化酶GbC4H2的核苷序列，其核苷序列为SEQ ID NO.7；
一种编码香豆酰CoA连接酶Gb4CL1的核苷序列，其核苷序列为SEQ ID NO.8；
一种编码香豆酰CoA连接酶Gb4CL2的核苷序列，其核苷序列为SEQ ID NO.9；
一种编码香豆酰CoA连接酶Gb4CL3的核苷序列，其核苷序列为SEQ ID NO.10；
一种编码查耳酮合成酶GbCHS的核苷序列，其核苷序列为SEQ ID NO.11。
2.根据权利要求1所述的候选基因的功能验证，其特征在于，利用银杏幼苗，提取RNA并反转录作为模板，每个基因分别带有SacI和EcoRI酶切位点的两对引物进行扩增GbPAL,GbC4H,Gb4CL基因，分别带有NheI和BamHI酶切位点的两对引物进行扩增GbCHS，并构建在酿酒酵母的载体pESC-URA载体上，获得真核表达载体pESC-URA-GbPAL1,pESC-URA-GbPAL2,pESC-URA-GbPAL3,pESC-URA-GbPAL4,pESC-URA-GbPAL5,pESC-URA-GbC4H1,pESC-URA-GbC4H2，pESC-URA-Gb4CL1-GbCHS,pESC-URA-Gb4CL2-GbCHS,pESC-URA-Gb4CL3-GbCHS的质粒。采用电击转化法、底物喂养的方式进行功能验证。
3.根据权利要求1所述的候选基因与黄酮生物合成途径的相关性，其特征在于，将候选基因构建在pBI221-GFP载体上，获得重组质粒。利用亚细胞定位的方法，PEG转染拟南芥叶片，采用激光共聚焦观察的结果进行判断。
4.根据权利要求1所述的候选基因与黄酮生物合成途径的相关性，其特征在于，将候选基因分别构建在pUC-SPYCE、pUC-SPYNE的载体上，利用双分子荧光互补技术的方法，PEG转染拟南芥叶片，采用激光共聚焦观察的结果进行判断。
5.根据权利要求1所述的候选基因与黄酮生物合成途径的相关性，其特征在于，利用进化树分析进一步验证。
CN201811651100.5A 2018-12-31 2018-12-31 银杏黄酮化合物生物合成基因中多个亚型的研究 Pending CN111394322A (zh)

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CN117363779A (zh) * 2023-10-31 2024-01-09 南京林业大学 一种银杏GbPAL10基因的DNA甲基化分子标记及应用

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