香雪兰二氢黄酮醇-4还原酶基因的cDNA
阅读：144发布：2020-05-13
专利汇可以提供香雪兰二氢黄酮醇-4还原酶基因的cDNA专利检索，专利查询，专利分析的服务。并且本 发明 首次从红花香 雪 兰花瓣中克隆得到三个二氢 黄 酮 醇 -4还原酶（DFR）基因的新cDNA，并确定了它的核苷酸序列和由此得出 氨 基酸的序列。在香雪兰花朵着色过程中，三者基因表达和花色苷合成呈现明显的相关性，另外，将3个FhDFR 的真核表达载体 转染 到拟南芥tt3突变体中，其 幼苗 和种皮都恢复了表型。，下面是香雪兰二氢黄酮醇-4还原酶基因的cDNA专利的具体信息内容。
1.香雪兰二氢黄酮醇-4还原酶基因的cDNA，其特征如下：
其中1：FhDFR2基因的全长核苷酸序列（SEQ ID NO.1）
GGGGCCTAGGAAAGAACGATGGGGACGGTTGTCGTGACCGGAGCTAGCGGATACGTCGGCTCATGGCTCCTCATGAAGCTTCTGCAGAATGGCTACGCCGTCAGGGCCACCGTCAGAGACCCCACCAACCTGAGAAAGACGAAGCCTCTGTTAGACCTTTCAGGAGCAGACAGGCTGCTCACAATTTGGAAAGCTGACCTCGGAGAGGAAGGAAGTTTCGACGAGGTGGTACAAGGATGCACTGCTGTTTTCCATGTCGCAACTCCGATGGATTTCGAGTCACAAGACCCAGAAAATGAAGTGATCAAGCCGGCGATAAATGGTATGCTGAGCATCATGAGGTCCTGTAAAAGGGCTGGCAGCGTCGAGCGAGTAATTTTCACATCATCTGCGGGGACCGTGAACGTCGAAGAACATCAACGAGCAGAGTACGATGAGAATTGTTGGAGTGACGTCGAGTTCTGCAGACGTGTGAAGATGACTGGCTGGATGTATTTTGTGTCGAAGACACTCGCGGAGAAAGCCGCTTGGGATTTCGCACTAGAGAATGGAATTCATCTTATAACGATCATTCCAACATTAGTGGTCGGTCCTTTCATCACCACAACTATGCCACCAAGTATGATCACTGCATTATCATTGATCACAGGAAATGAAGGTCACTATTCGATAATAAAGCAAGCGCAACTTGTTCACCTAGATGACTTGTGTGCTGCTCACATTTACCTCCTGAACCATCCTGAAGCAAAAGGGAGGTACATATGCTCTTCTCATGATGCTACCATCTATGATCTGGCAAGGATGCTCAGAGAGAGGCATCCTTGGTGTTCCATTCCTCAAAAGTTCAAAGGGATTAATGAGGGTATTGAAACTGTGCACTTCTCTTCAAAGAAGCTGATGGACCTTGGATTCAGGTTTAAGTACTCGATGGAGGAGATGTTCGATGAGGCCATCCAGACATGCATCGAGAAGAAGCTCATAACACTACCTCAGAGTGGTGATCGCAAAGTCGAGAATGGCTGATTGGATGCGGTCGGAGAGGAAGTAGCTATGGTTAAAGTGTAGTTCACGGTCTTCATATGGAGACTAGCTGGAAGGTGAAATAAGGGC；
其中2：由FhDFR2基因核苷酸序列推测得到的蛋白质序列（SEQ ID NO. 2）MGTVVVTGASGYVGSWLLMKLLQNGYAVRATVRDPTNLRKTKPLLDLSGADRLLTIWKADLGEEGSFDEVVQGCTAVFHVATPMDFESQDPENEVIKPAINGMLSIMRSCKRAGSVERVIFTSSAGTVNVEEHQRAEYDENCWSDVEFCRRVKMTGWMYFVSKTLAEKAAWDFALENGIHLITIIPTLVVGPFITTTMPPSMITALSLITGNEGHYSIIKQAQLVHLDDLCAAHIYLLNHPEAKGRYICSSHDATIYDLARMLRERHPWCSIPQKFKGINEGIETVHFSSKKLMDLGFRFKYSMEEMFDEAIQTCIEKKLITLPQSGDRKVENG-；
其中3：FhDFR3基因的全长核苷酸序列（SEQ ID NO.3）
ATGGGGAAACGAGAGTTGAGCTTAATTAGCCTTTACTAATTACTTGATTAGGATACTATCATCAATGGCCATTACAGCCGACACAGAGAGTAAGGGGCCAGTCGTCGTCACCGGAGCTAGCGGTTACGTCGGTTCATGGCTGGTTATGCTGCTTCTCAAGGATGGTTATGCTGTCAGGGCAACAGTTAGAGATCCAACTGACAAGAGGAAGACGAAGCCGCTGCTTGACCTTCCGGGGGCTGACACGCTGCTTACGCTGTGGAAAGCAGACCTCAGCGAGGATGGAAGTTTCGATGAGGCAATTGCAGGATGCACTGCAGTTTTCCATGTTGCAACCCCAATGGATTTTGAGTCCAAAGACCCTGAAAATGAAGTGATCAAGCCTACGGTAGATGGCGTGTTAAGTATAATGAGGTCTTGTAAGAATTCCGGTACTGTCAAGCGCGTGATCTTCACGTCATCTGCCGGGACCGTCGACGTCCAAGAACATCAGCTGGCAGAGTACGACGAGAACTCGTGGAGTGACATCGAGTTTTGCAGACGTGTGAAGATGACTGGCTGGATGTATTTTGTATCGAAGACTCTAGCCGAGCAAGCCGCATCGGGTTTCGCTAGGGAGAATGACATTGATCTAATAAGCATCATTCCTACCTTAGTTGTGGGTCCTTTCATCACCACAAGCATGCCACCAAGCATGATCACTGCATTGTCATTGATCACAGGAAATGAAGACCATTATCATATAATTAAGCAAGCCCAACTTGTTCACTTGGATGACTTGTGTGCTGCTCACATTTACCTCCTAAACCATCCTGAAGCCAAAGGAAGGTACATATGCTCTTCCCATGAAGTTACCATCTTCGAGTTGGCGAGGATGATCAAAGAGAGACATCCCTGGTGTTCCGTCCCGGAGAAGTTCGAAGGCATTGATGAGAATATTAAACCTGTGCACTTCTCTTCGAAGAAGCTGACGGACCTGGGATTCAAGTTTCAACACACGATGGAGGAAATGTTCGACGAAGCCATCGAGTCGTGCATTCAGAAGAAGCTTATAGAACTCCCTCAAACTGAAAAATCAGTACCTGAAAATGTGATTCAGGGAGCAAAGGAACAAGCCACAGTTGCTGCTGAAAACTGAGATTAGTGCCCTGGAGTG；
其中4：由FhDFR3基因核苷酸序列推测得到的蛋白质序列（SEQ ID NO. 4）MAITADTESKGPVVVTGASGYVGSWLVMLLLKDGYAVRATVRDPTDKRKTKPLLDLPGADTLLTLWKADLSEDGSFDEAIAGCTAVFHVATPMDFESKDPENEVIKPTVDGVLSIMRSCKNSGTVKRVIFTSSAGTVDVQEHQLAEYDENSWSDIEFCRRVKMTGWMYFVSKTLAEQAASGFARENDIDLISIIPTLVVGPFITTSMPPSMITALSLITGNEDHYHIIKQAQLVHLDDLCAAHIYLLNHPEAKGRYICSSHEVTIFELARMIKERHPWCSVPEKFEGIDENIKPVHFSSKKLTDLGFKFQHTMEEMFDEAIESCIQKKLIELPQTEKSVPENVIQGAKEQATVAAEN
其中5：FhDFR6基因的全长核苷酸序列（SEQ ID NO.5）；GGGGCCTAGGAAAGAACGATGGGGACGGTTGTCGTGACCGGAGCTAGCGGATACGTCGGCTCATGGCTCCTCATGAAGCTTCTGCAGAATGGCTACGCCGTCAGGGCCACCGTCAGAGACCCCACCAACCTGAGAAAGACGAAGCCTCTGTTAGACCTTTCAGGAGCAGACAGGCTGCTCACAATTTGGAAAGCTGACCTCGGAGAGGAAGGAAGTTTCGACGAGGTGGTACAAGGATGCACTGCTGTTTTCCATGTCGCAACTCCGATGGATTTCGAGTCACAAGACCCAGAAAATGAAGTGATCAAGCCGACGATAAATGGTATGCTAAGCATCATGAGGTCCTGTAAAAGGGCTGGCAGCGTCGAGCGAGTAATTTTCACATCATCTGCGGGGACCGTGAACGTCGAAGAACATCAACGAGCAGAGTACGATGAGAATTGTTGGAGTGACGTCGAGTTCTGCAGACGTGTGAAGATGACTGGCTGGATGTATTTTGTGTCGAAGACACTCGCGGAGAAAGCCGCTTGGGATTTCGCACGTGAGAATGGAATTCATCTTATAACGATCATTCCAACATTAGTTGTCGGTCCTTTCATCACCACAACTATGCCACCAAGTATGATCACTGCATTATCATTGATCACAGGAAATGAAGCTCACTATTCGATAATAAAGCAAGCGCAACTTGTTCACCTAGATGACTTGTGTGCTGCTCACATTTACCTCCTGAACCATCCTGAAGCAAAAGGGAGGTACATATGCTCTTCTCATGATGCTACCATCTATGATCTGGCAAGGATGCTCAGAGAGAGGCATCCTTGGTGTTCCATTCCTCAAAAGTTCAAAGGGATTAATGAGGGTATTGAAATTGTGCACTTCTCTTCAAAGAAGCTGATGGACCTTGGATTCCGGTTTAAGTACTCGATGGAGGAGATGTTCGATGAGGCCATCCAGACATGCATCGAGAAGAAGCTCATAACACTACCTCAGAGTGGTGATCGCAAAGTCGAGAATGGCCGATTGGATGCGGTCGGAGAGGAAGTAGCTATGGTTAAAGTGTAGTTCATGGTCTT；
其中6：由FhDFR6基因核苷酸序列推测得到的蛋白质序列（SEQ ID NO. 6）MGTVVVTGASGYVGSWLLMKLLQNGYAVRATVRDPTNLRKTKPLLDLSGADRLLTIWKADLGEEGSFDEVVQGCTAVFHVATPMDFESQDPENEVIKPTINGMLSIMRSCKRAGSVERVIFTSSAGTVNVEEHQRAEYDENCWSDVEFCRRVKMTGWMYFVSKTLAEKAAWDFARENGIHLITIIPTLVVGPFITTTMPPSMITALSLITGNEAHYSIIKQAQLVHLDDLCAAHIYLLNHPEAKGRYICSSHDATIYDLARMLRERHPWCSIPQKFKGINEGIEIVHFSSKKLMDLGFRFKYSMEEMFDEAIQTCIEKKLITLPQSGDRKVENGRLDAVGEEVAMVKV。
香雪兰二氢黄酮醇-4还原酶基因的cDNA
技术领域
背景技术
[0002] 自1985年人们首次从金鱼草和玉米中分离获得DFR基因以来，许多物种的DFR基因相继被克隆出来，比如矮牵牛、拟南芥、水稻、紫薯、苹果、葡萄、花生、玫瑰、银杏以及百合等。近年来，获得的DFR基因数量急剧增加，但大都来自双子叶植物（592条），单子叶植物只有52条，其中禾本科17条，而汇聚了大量观赏植物的鸢尾科只有1条，且其功能还没有得到验证。
[0003] DFR属于NADPH依赖性短链还原酶（SDR）家族，该基因在植物基因组中一般为单拷贝，在少数物种中会以一个小的基因家族存在，如矮牵牛、杨树和银杏等。不同植物中，DFR基因的非编码区长度差异较大，开放阅读框(ORF)长度差异较小。
[0004] DFR对于底物的特异性选择和表达水平的差异，是植物呈现出不同花色的重要原因之一。DFR为酸性蛋白，属于稳定类蛋白质。相关研究显示， “VTGASGFVGSWLVM -RLLEHGY”和“TVNVEEKQKPVYDETCWSDVDFCRRV”是DFR蛋白都含有的两个特征性的保守结合域，前者能与NADP（H）特异性结合，后者能与底物特异性结合，这两个结合域在不同来源的植物中高度保守。有研究表明，底物结合域的第134位与145位氨基酸残基可直接影响DFR的底物特异性，且大多数物种在第134位含有Asp（D）或者Asn（N），但在其他以DHK为底物的双子叶植物（除矮牵牛）中，其高度保守的145位为Glu（E）。
[0005] 根据DFR的134位氨基酸的种类可将DFR分为三类，即 Asn型DFR、Asp型DFR和非Asn/Asp型DFR。研究发现，一种植物中可同时存在几种不同类型的DFR，但不同类型的DFR在不同物种或同一物种中对三种底物所表现出的亲和程度不同。如百脉根(Lotus aponcus)的DFR2与DFR3均属于Asn型DFR ， DFR4和DFR5属于Asp型DFR，而 DFRl和银杏(Ginkgo biloba)的DFR1都属于非Asn/Asp型，百脉根的DFR1在第134位氨基酸残基为丝氨酸(Ser)，而银杏的DFR1则为赖氨酸(Lys)。苜蓿(Medicago truncatula)的DFR1属于Asn型，能够有效还原DHK；DFR2属于Asp型，对DHK的活性十分有限。
[0006] 由此可见，二氢黄酮醇-4还原酶(Dihydroflavonol 4-Reductase，DFR)是作为催化二氢黄酮醇形成花色素反应的第一个酶，在不同花色形成中起决定性作用。DFR催化底物的反应需要NADPH的参与。不同物种中所含有的DFR类型不同，同种类型的DFR在不同物种中对底物DHK、DHM和DHQ的亲和程度也存在差异，进而合成了不同的花色素，也就产生了丰富多彩的花色。研究表明，不同物种的DFR基因在其不同发育期与不同组织的时空表达特性有所不同，其表达模式是多种内外因素共同影响的结果。
[0007]
发明内容
[0008] 本发明的主要目的是提供编码二氢黄酮醇-4还原酶（DFR）的新cDNA和由此推断的氨基酸序列。
[0009] 本发明首次从红花香雪兰花瓣中克隆得到三个二氢黄酮醇-4还原酶（DFR）基因的新cDNA，并确定了它的核苷酸序列和由此得出氨基酸的序列。在香雪兰花朵着色过程中，三者基因表达和花色苷合成呈现明显的相关性，另外，将3个FhDFR的真核表达载体转染到拟南芥tt3突变体中，其幼苗和种皮都恢复了表型。
[0010] 本发明的技术方案：为了从香雪兰中克隆得到FhDFR基因，首先利用拟南芥DFR基因为探针，从建立的香雪兰花瓣转录组测序得到的EST数据库中检索出高度同源的FhDFR基因片段，随后利用RACE技术成功扩增得到了FhDFR2、FhDFR3和 FhDFR6全长cDNA序列。对这3条序列的cDNA和DNA进行生物信息学分析，结果表明：全长cDNA序列长度分别为1119 bp、1156 bp、1239 bp，开放阅读框分别长1005 bp、1074 bp、1047 bp，推测其分别编码334、357、348个氨基酸。
预测其编码的蛋白均为亲水性蛋白，分子量（Mr）为37.4 kD、39.6 kD、39.0 kD，等电点（PI）为5.95、5.10、5.96。蛋白序列分析表明，推测的FhDFR蛋白与其他植物DFR同源性都在80%以上，其中与荷兰鸢尾（Iris x hollandica）的DFR蛋白序列同源性最高，达90%； FhDFR2和FhDFR6蛋白为Asn型DFR，FhDFR3蛋白为Asp型DFR。
预测其编码的蛋白均为亲水性蛋白，分子量（Mr）为37.4 kD、39.6 kD、39.0 kD，等电点（PI）为5.95、5.10、5.96。蛋白序列分析表明，推测的FhDFR蛋白与其他植物DFR同源性都在80%以上，其中与荷兰鸢尾（Iris x hollandica）的DFR蛋白序列同源性最高，达90%； FhDFR2和FhDFR6蛋白为Asn型DFR，FhDFR3蛋白为Asp型DFR。
[0011] 分别构建携带FhDFR2、FhDFR3和 FhDFR6的真核表达载体，转入到农杆菌GV3101细胞中，利用蘸花法侵染拟南芥tt5突变体。结果表明：3条香雪兰 FhDFR基因的导入都能够使突变体的表型得到不同程度的恢复。
[0012] 利用半定量RT-PCR技术分析了三个FhDFR基因在开花过程中的表达模式，同时利用高效液相质谱串联技术分析了香雪兰开花过程中花色苷积累的规律，二者的相关性分析表明：FhDFR2、FhDFR3和 FhDFR6基因的表达与花色苷的积累显著相关。
[0013] 3条香雪兰FhDFRs基因的cDNA的序列和氨基酸序列信息见序列表：其中1：FhDFR2基因的全长核苷酸序列（SEQ ID NO.1）
GGGGCCTAGGAAAGAACGATGGGGACGGTTGTCGTGACCGGAGCTAGCGGATACGTCGGCTCATGGCTCCTCATGAAGCTTCTGCAGAATGGCTACGCCGTCAGGGCCACCGTCAGAGACCCCACCAACCTGAGAAAGACGAAGCCTCTGTTAGACCTTTCAGGAGCAGACAGGCTGCTCACAATTTGGAAAGCTGACCTCGGAGAGGAAGGAAGTTTCGACGAGGTGGTACAAGGATGCACTGCTGTTTTCCATGTCGCAACTCCGATGGATTTCGAGTCACAAGACCCAGAAAATGAAGTGATCAAGCCGGCGATAAATGGTATGCTGAGCATCATGAGGTCCTGTAAAAGGGCTGGCAGCGTCGAGCGAGTAATTTTCACATCATCTGCGGGGACCGTGAACGTCGAAGAACATCAACGAGCAGAGTACGATGAGAATTGTTGGAGTGACGTCGAGTTCTGCAGACGTGTGAAGATGACTGGCTGGATGTATTTTGTGTCGAAGACACTCGCGGAGAAAGCCGCTTGGGATTTCGCACTAGAGAATGGAATTCATCTTATAACGATCATTCCAACATTAGTGGTCGGTCCTTTCATCACCACAACTATGCCACCAAGTATGATCACTGCATTATCATTGATCACAGGAAATGAAGGTCACTATTCGATAATAAAGCAAGCGCAACTTGTTCACCTAGATGACTTGTGTGCTGCTCACATTTACCTCCTGAACCATCCTGAAGCAAAAGGGAGGTACATATGCTCTTCTCATGATGCTACCATCTATGATCTGGCAAGGATGCTCAGAGAGAGGCATCCTTGGTGTTCCATTCCTCAAAAGTTCAAAGGGATTAATGAGGGTATTGAAACTGTGCACTTCTCTTCAAAGAAGCTGATGGACCTTGGATTCAGGTTTAAGTACTCGATGGAGGAGATGTTCGATGAGGCCATCCAGACATGCATCGAGAAGAAGCTCATAACACTACCTCAGAGTGGTGATCGCAAAGTCGAGAATGGCTGATTGGATGCGGTCGGAGAGGAAGTAGCTATGGTTAAAGTGTAGTTCACGGTCTTCATATGGAGACTAGCTGGAAGGTGAAATAAGGGC
其中2：由FhDFR2基因核苷酸序列推测得到的蛋白质序列（SEQ ID NO. 2）
MGTVVVTGASGYVGSWLLMKLLQNGYAVRATVRDPTNLRKTKPLLDLSGADRLLTIWKADLGEEGSFDEVVQGCTAVFHVATPMDFESQDPENEVIKPAINGMLSIMRSCKRAGSVERVIFTSSAGTVNVEEHQRAEYDENCWSDVEFCRRVKMTGWMYFVSKTLAEKAAWDFALENGIHLITIIPTLVVGPFITTTMPPSMITALSLITGNEGHYSIIKQAQLVHLDDLCAAHIYLLNHPEAKGRYICSSHDATIYDLARMLRERHPWCSIPQKFKGINEGIETVHFSSKKLMDLGFRFKYSMEEMFDEAIQTCIEKKLITLPQSGDRKVENG-
其中3：FhDFR3基因的全长核苷酸序列（SEQ ID NO.3）
ATGGGGAAACGAGAGTTGAGCTTAATTAGCCTTTACTAATTACTTGATTAGGATACTATCATCAATGGCCATTACAGCCGACACAGAGAGTAAGGGGCCAGTCGTCGTCACCGGAGCTAGCGGTTACGTCGGTTCATGGCTGGTTATGCTGCTTCTCAAGGATGGTTATGCTGTCAGGGCAACAGTTAGAGATCCAACTGACAAGAGGAAGACGAAGCCGCTGCTTGACCTTCCGGGGGCTGACACGCTGCTTACGCTGTGGAAAGCAGACCTCAGCGAGGATGGAAGTTTCGATGAGGCAATTGCAGGATGCACTGCAGTTTTCCATGTTGCAACCCCAATGGATTTTGAGTCCAAAGACCCTGAAAATGAAGTGATCAAGCCTACGGTAGATGGCGTGTTAAGTATAATGAGGTCTTGTAAGAATTCCGGTACTGTCAAGCGCGTGATCTTCACGTCATCTGCCGGGACCGTCGACGTCCAAGAACATCAGCTGGCAGAGTACGACGAGAACTCGTGGAGTGACATCGAGTTTTGCAGACGTGTGAAGATGACTGGCTGGATGTATTTTGTATCGAAGACTCTAGCCGAGCAAGCCGCATCGGGTTTCGCTAGGGAGAATGACATTGATCTAATAAGCATCATTCCTACCTTAGTTGTGGGTCCTTTCATCACCACAAGCATGCCACCAAGCATGATCACTGCATTGTCATTGATCACAGGAAATGAAGACCATTATCATATAATTAAGCAAGCCCAACTTGTTCACTTGGATGACTTGTGTGCTGCTCACATTTACCTCCTAAACCATCCTGAAGCCAAAGGAAGGTACATATGCTCTTCCCATGAAGTTACCATCTTCGAGTTGGCGAGGATGATCAAAGAGAGACATCCCTGGTGTTCCGTCCCGGAGAAGTTCGAAGGCATTGATGAGAATATTAAACCTGTGCACTTCTCTTCGAAGAAGCTGACGGACCTGGGATTCAAGTTTCAACACACGATGGAGGAAATGTTCGACGAAGCCATCGAGTCGTGCATTCAGAAGAAGCTTATAGAACTCCCTCAAACTGAAAAATCAGTACCTGAAAATGTGATTCAGGGAGCAAAGGAACAAGCCACAGTTGCTGCTGAAAACTGAGATTAGTGCCCTGGAGTG
其中4：由FhDFR3基因核苷酸序列推测得到的蛋白质序列（SEQ ID NO. 4）
MAITADTESKGPVVVTGASGYVGSWLVMLLLKDGYAVRATVRDPTDKRKTKPLLDLPGADTLLTLWKADLSEDGSFDEAIAGCTAVFHVATPMDFESKDPENEVIKPTVDGVLSIMRSCKNSGTVKRVIFTSSAGTVDVQEHQLAEYDENSWSDIEFCRRVKMTGWMYFVSKTLAEQAASGFARENDIDLISIIPTLVVGPFITTSMPPSMITALSLITGNEDHYHIIKQAQLVHLDDLCAAHIYLLNHPEAKGRYICSSHEVTIFELARMIKERHPWCSVPEKFEGIDENIKPVHFSSKKLTDLGFKFQHTMEEMFDEAIESCIQKKLIELPQTEKSVPENVIQGAKEQATVAAEN
其中5：FhDFR6基因的全长核苷酸序列（SEQ ID NO.5）GGGGCCTAGGAAAGAACGATGGGGACGGTTGTCGTGACCGGAGCTAGCGGATACGTCGGCTCATGGCTCCTCATGAAGCTTCTGCAGAATGGCTACGCCGTCAGGGCCACCGTCAGAGACCCCACCAACCTGAGAAAGACGAAGCCTCTGTTAGACCTTTCAGGAGCAGACAGGCTGCTCACAATTTGGAAAGCTGACCTCGGAGAGGAAGGAAGTTTCGACGAGGTGGTACAAGGATGCACTGCTGTTTTCCATGTCGCAACTCCGATGGATTTCGAGTCACAAGACCCAGAAAATGAAGTGATCAAGCCGACGATAAATGGTATGCTAAGCATCATGAGGTCCTGTAAAAGGGCTGGCAGCGTCGAGCGAGTAATTTTCACATCATCTGCGGGGACCGTGAACGTCGAAGAACATCAACGAGCAGAGTACGATGAGAATTGTTGGAGTGACGTCGAGTTCTGCAGACGTGTGAAGATGACTGGCTGGATGTATTTTGTGTCGAAGACACTCGCGGAGAAAGCCGCTTGGGATTTCGCACGTGAGAATGGAATTCATCTTATAACGATCATTCCAACATTAGTTGTCGGTCCTTTCATCACCACAACTATGCCACCAAGTATGATCACTGCATTATCATTGATCACAGGAAATGAAGCTCACTATTCGATAATAAAGCAAGCGCAACTTGTTCACCTAGATGACTTGTGTGCTGCTCACATTTACCTCCTGAACCATCCTGAAGCAAAAGGGAGGTACATATGCTCTTCTCATGATGCTACCATCTATGATCTGGCAAGGATGCTCAGAGAGAGGCATCCTTGGTGTTCCATTCCTCAAAAGTTCAAAGGGATTAATGAGGGTATTGAAATTGTGCACTTCTCTTCAAAGAAGCTGATGGACCTTGGATTCCGGTTTAAGTACTCGATGGAGGAGATGTTCGATGAGGCCATCCAGACATGCATCGAGAAGAAGCTCATAACACTACCTCAGAGTGGTGATCGCAAAGTCGAGAATGGCCGATTGGATGCGGTCGGAGAGGAAGTAGCTATGGTTAAAGTGTAGTTCATGGTCTT
其中6：由FhDFR6基因核苷酸序列推测得到的蛋白质序列（SEQ ID NO. 6）
MGTVVVTGASGYVGSWLLMKLLQNGYAVRATVRDPTNLRKTKPLLDLSGADRLLTIWKADLGEEGSFDEVVQGCTAVFHVATPMDFESQDPENEVIKPTINGMLSIMRSCKRAGSVERVIFTSSAGTVNVEEHQRAEYDENCWSDVEFCRRVKMTGWMYFVSKTLAEKAAWDFARENGIHLITIIPTLVVGPFITTTMPPSMITALSLITGNEAHYSIIKQAQLVHLDDLCAAHIYLLNHPEAKGRYICSSHDATIYDLARMLRERHPWCSIPQKFKGINEGIEIVHFSSKKLMDLGFRFKYSMEEMFDEAIQTCIEKKLITLPQSGDRKVENGRLDAVGEEVAMVKV
附图说明
GGGGCCTAGGAAAGAACGATGGGGACGGTTGTCGTGACCGGAGCTAGCGGATACGTCGGCTCATGGCTCCTCATGAAGCTTCTGCAGAATGGCTACGCCGTCAGGGCCACCGTCAGAGACCCCACCAACCTGAGAAAGACGAAGCCTCTGTTAGACCTTTCAGGAGCAGACAGGCTGCTCACAATTTGGAAAGCTGACCTCGGAGAGGAAGGAAGTTTCGACGAGGTGGTACAAGGATGCACTGCTGTTTTCCATGTCGCAACTCCGATGGATTTCGAGTCACAAGACCCAGAAAATGAAGTGATCAAGCCGGCGATAAATGGTATGCTGAGCATCATGAGGTCCTGTAAAAGGGCTGGCAGCGTCGAGCGAGTAATTTTCACATCATCTGCGGGGACCGTGAACGTCGAAGAACATCAACGAGCAGAGTACGATGAGAATTGTTGGAGTGACGTCGAGTTCTGCAGACGTGTGAAGATGACTGGCTGGATGTATTTTGTGTCGAAGACACTCGCGGAGAAAGCCGCTTGGGATTTCGCACTAGAGAATGGAATTCATCTTATAACGATCATTCCAACATTAGTGGTCGGTCCTTTCATCACCACAACTATGCCACCAAGTATGATCACTGCATTATCATTGATCACAGGAAATGAAGGTCACTATTCGATAATAAAGCAAGCGCAACTTGTTCACCTAGATGACTTGTGTGCTGCTCACATTTACCTCCTGAACCATCCTGAAGCAAAAGGGAGGTACATATGCTCTTCTCATGATGCTACCATCTATGATCTGGCAAGGATGCTCAGAGAGAGGCATCCTTGGTGTTCCATTCCTCAAAAGTTCAAAGGGATTAATGAGGGTATTGAAACTGTGCACTTCTCTTCAAAGAAGCTGATGGACCTTGGATTCAGGTTTAAGTACTCGATGGAGGAGATGTTCGATGAGGCCATCCAGACATGCATCGAGAAGAAGCTCATAACACTACCTCAGAGTGGTGATCGCAAAGTCGAGAATGGCTGATTGGATGCGGTCGGAGAGGAAGTAGCTATGGTTAAAGTGTAGTTCACGGTCTTCATATGGAGACTAGCTGGAAGGTGAAATAAGGGC
其中2：由FhDFR2基因核苷酸序列推测得到的蛋白质序列（SEQ ID NO. 2）
MGTVVVTGASGYVGSWLLMKLLQNGYAVRATVRDPTNLRKTKPLLDLSGADRLLTIWKADLGEEGSFDEVVQGCTAVFHVATPMDFESQDPENEVIKPAINGMLSIMRSCKRAGSVERVIFTSSAGTVNVEEHQRAEYDENCWSDVEFCRRVKMTGWMYFVSKTLAEKAAWDFALENGIHLITIIPTLVVGPFITTTMPPSMITALSLITGNEGHYSIIKQAQLVHLDDLCAAHIYLLNHPEAKGRYICSSHDATIYDLARMLRERHPWCSIPQKFKGINEGIETVHFSSKKLMDLGFRFKYSMEEMFDEAIQTCIEKKLITLPQSGDRKVENG-
其中3：FhDFR3基因的全长核苷酸序列（SEQ ID NO.3）
ATGGGGAAACGAGAGTTGAGCTTAATTAGCCTTTACTAATTACTTGATTAGGATACTATCATCAATGGCCATTACAGCCGACACAGAGAGTAAGGGGCCAGTCGTCGTCACCGGAGCTAGCGGTTACGTCGGTTCATGGCTGGTTATGCTGCTTCTCAAGGATGGTTATGCTGTCAGGGCAACAGTTAGAGATCCAACTGACAAGAGGAAGACGAAGCCGCTGCTTGACCTTCCGGGGGCTGACACGCTGCTTACGCTGTGGAAAGCAGACCTCAGCGAGGATGGAAGTTTCGATGAGGCAATTGCAGGATGCACTGCAGTTTTCCATGTTGCAACCCCAATGGATTTTGAGTCCAAAGACCCTGAAAATGAAGTGATCAAGCCTACGGTAGATGGCGTGTTAAGTATAATGAGGTCTTGTAAGAATTCCGGTACTGTCAAGCGCGTGATCTTCACGTCATCTGCCGGGACCGTCGACGTCCAAGAACATCAGCTGGCAGAGTACGACGAGAACTCGTGGAGTGACATCGAGTTTTGCAGACGTGTGAAGATGACTGGCTGGATGTATTTTGTATCGAAGACTCTAGCCGAGCAAGCCGCATCGGGTTTCGCTAGGGAGAATGACATTGATCTAATAAGCATCATTCCTACCTTAGTTGTGGGTCCTTTCATCACCACAAGCATGCCACCAAGCATGATCACTGCATTGTCATTGATCACAGGAAATGAAGACCATTATCATATAATTAAGCAAGCCCAACTTGTTCACTTGGATGACTTGTGTGCTGCTCACATTTACCTCCTAAACCATCCTGAAGCCAAAGGAAGGTACATATGCTCTTCCCATGAAGTTACCATCTTCGAGTTGGCGAGGATGATCAAAGAGAGACATCCCTGGTGTTCCGTCCCGGAGAAGTTCGAAGGCATTGATGAGAATATTAAACCTGTGCACTTCTCTTCGAAGAAGCTGACGGACCTGGGATTCAAGTTTCAACACACGATGGAGGAAATGTTCGACGAAGCCATCGAGTCGTGCATTCAGAAGAAGCTTATAGAACTCCCTCAAACTGAAAAATCAGTACCTGAAAATGTGATTCAGGGAGCAAAGGAACAAGCCACAGTTGCTGCTGAAAACTGAGATTAGTGCCCTGGAGTG
其中4：由FhDFR3基因核苷酸序列推测得到的蛋白质序列（SEQ ID NO. 4）
MAITADTESKGPVVVTGASGYVGSWLVMLLLKDGYAVRATVRDPTDKRKTKPLLDLPGADTLLTLWKADLSEDGSFDEAIAGCTAVFHVATPMDFESKDPENEVIKPTVDGVLSIMRSCKNSGTVKRVIFTSSAGTVDVQEHQLAEYDENSWSDIEFCRRVKMTGWMYFVSKTLAEQAASGFARENDIDLISIIPTLVVGPFITTSMPPSMITALSLITGNEDHYHIIKQAQLVHLDDLCAAHIYLLNHPEAKGRYICSSHEVTIFELARMIKERHPWCSVPEKFEGIDENIKPVHFSSKKLTDLGFKFQHTMEEMFDEAIESCIQKKLIELPQTEKSVPENVIQGAKEQATVAAEN
其中5：FhDFR6基因的全长核苷酸序列（SEQ ID NO.5）GGGGCCTAGGAAAGAACGATGGGGACGGTTGTCGTGACCGGAGCTAGCGGATACGTCGGCTCATGGCTCCTCATGAAGCTTCTGCAGAATGGCTACGCCGTCAGGGCCACCGTCAGAGACCCCACCAACCTGAGAAAGACGAAGCCTCTGTTAGACCTTTCAGGAGCAGACAGGCTGCTCACAATTTGGAAAGCTGACCTCGGAGAGGAAGGAAGTTTCGACGAGGTGGTACAAGGATGCACTGCTGTTTTCCATGTCGCAACTCCGATGGATTTCGAGTCACAAGACCCAGAAAATGAAGTGATCAAGCCGACGATAAATGGTATGCTAAGCATCATGAGGTCCTGTAAAAGGGCTGGCAGCGTCGAGCGAGTAATTTTCACATCATCTGCGGGGACCGTGAACGTCGAAGAACATCAACGAGCAGAGTACGATGAGAATTGTTGGAGTGACGTCGAGTTCTGCAGACGTGTGAAGATGACTGGCTGGATGTATTTTGTGTCGAAGACACTCGCGGAGAAAGCCGCTTGGGATTTCGCACGTGAGAATGGAATTCATCTTATAACGATCATTCCAACATTAGTTGTCGGTCCTTTCATCACCACAACTATGCCACCAAGTATGATCACTGCATTATCATTGATCACAGGAAATGAAGCTCACTATTCGATAATAAAGCAAGCGCAACTTGTTCACCTAGATGACTTGTGTGCTGCTCACATTTACCTCCTGAACCATCCTGAAGCAAAAGGGAGGTACATATGCTCTTCTCATGATGCTACCATCTATGATCTGGCAAGGATGCTCAGAGAGAGGCATCCTTGGTGTTCCATTCCTCAAAAGTTCAAAGGGATTAATGAGGGTATTGAAATTGTGCACTTCTCTTCAAAGAAGCTGATGGACCTTGGATTCCGGTTTAAGTACTCGATGGAGGAGATGTTCGATGAGGCCATCCAGACATGCATCGAGAAGAAGCTCATAACACTACCTCAGAGTGGTGATCGCAAAGTCGAGAATGGCCGATTGGATGCGGTCGGAGAGGAAGTAGCTATGGTTAAAGTGTAGTTCATGGTCTT
其中6：由FhDFR6基因核苷酸序列推测得到的蛋白质序列（SEQ ID NO. 6）
MGTVVVTGASGYVGSWLLMKLLQNGYAVRATVRDPTNLRKTKPLLDLSGADRLLTIWKADLGEEGSFDEVVQGCTAVFHVATPMDFESQDPENEVIKPTINGMLSIMRSCKRAGSVERVIFTSSAGTVNVEEHQRAEYDENCWSDVEFCRRVKMTGWMYFVSKTLAEKAAWDFARENGIHLITIIPTLVVGPFITTTMPPSMITALSLITGNEAHYSIIKQAQLVHLDDLCAAHIYLLNHPEAKGRYICSSHDATIYDLARMLRERHPWCSIPQKFKGINEGIEIVHFSSKKLMDLGFRFKYSMEEMFDEAIQTCIEKKLITLPQSGDRKVENGRLDAVGEEVAMVKV
附图说明
[0014] 图1：三条香雪兰 FhDFR基因PCR扩增产物电泳图M：DL2000 DNA Marker；1-3分别为FhDFR2、FhDFR 3、FhDFR 6 全长cDNA；该图说明已经克隆得到了FhDFR2、FhDFR 3、FhDFR 6 全长cDNA。
[0015] 图2：香雪兰FhDFR蛋白保守结构域分析三条序列均属于NADPH依赖性短链还原酶（SDR）家族成员
图3：同源序列的氨基酸比对结果
氨基酸序列比对：FhDFR2、 FhDFR3 、FhDFR6都具有NADPH和底物结合区域。
图3：同源序列的氨基酸比对结果
氨基酸序列比对：FhDFR2、 FhDFR3 、FhDFR6都具有NADPH和底物结合区域。
[0016]图4：FhDFRs基因对拟南芥 tt3突变体种皮及幼苗的影响
通过与野生型和突变体对比发现，转基因植株的种子和幼苗在表型上均具有一定的恢复，初步说明FhDFR2、 FhDFR3 、FhDFR6参与香雪兰花瓣中花色苷的合成。
通过与野生型和突变体对比发现，转基因植株的种子和幼苗在表型上均具有一定的恢复，初步说明FhDFR2、 FhDFR3 、FhDFR6参与香雪兰花瓣中花色苷的合成。
[0017] 图5：FhDFRs基因在香雪兰红花不同时期的表达电泳图及模式分析在香雪兰红花各时期具有相似的表达趋势，可能具有影响花色的功能。
[0018] 图6：FhDFRs基因与花色苷相关性分析分析显示FhDFR2、 FhDFR3 、FhDFR6基因表达与香雪兰总花色苷合成具有显著的相关性。
具体实施方式
[0019] 在下面的具体实施例子中进一步说明了本发明，这并不限制本发明的范围。
[0020] 实施例1 ：FhDFRs基因全长的克隆实施例1-1：FhDFRs 3’末端的获得：根据筛选得到的中间片段，设计引物进行
3’RACE-PCR扩增，引物组合分别是：
反应体系如下：
PCR反映程序如下：95 ℃ 5 min；94 ℃ 30 s，X ℃ 30 s，72 ℃ 1 min，30 Cycle。
3’RACE-PCR扩增，引物组合分别是：
反应体系如下：
PCR反映程序如下：95 ℃ 5 min；94 ℃ 30 s，X ℃ 30 s，72 ℃ 1 min，30 Cycle。
[0022] PCR产物胶回收后连接到T载体上，转化到大肠杆菌JM109感受态细胞中，用Amp抗性的LB平板培养基进行筛选。随机挑取单克隆，摇菌后进行菌液PCR验证，鉴定出的阳性克隆进行测序。
[0023]实施例1-2： FhDFRs 3’末端的获得：拼接筛选得到的中间片段和扩增得到的3’末端片段，然后以此为基础设计引物，使用Clontech公司的RACE试剂盒(SMART RACEcDNA Amplification Kit)进行5’RACE实验。
[0024] 引物组合如下：反应体系如下：
①FhDFR2的降落PCR反应
反应体系如下：
反映程序如下：
②FhDFR3的巢式PCR反应
A.以DFR3-GSP2作下游引物PCR，反应体系体系如下：
反应程序如下：
B.将上述PCR产物稀释50倍后作模板,以DFR3-GSP1与NUP为巢式引物进行PCR
扩增。反应体系体系如下：
反应程序如下：
③FhDFR6的正常PCR反应
反应体系如下：
反映程序如下：
回收PCR产物，连接T载后转化到大肠杆菌JM109感受态细胞中，用Amp抗性的LB平板培养基进行筛选。随机挑取单克隆，摇菌后进行菌液PCR验证，鉴定出的阳性克隆进行测序。
①FhDFR2的降落PCR反应
反应体系如下：
反映程序如下：
②FhDFR3的巢式PCR反应
A.以DFR3-GSP2作下游引物PCR，反应体系体系如下：
反应程序如下：
B.将上述PCR产物稀释50倍后作模板,以DFR3-GSP1与NUP为巢式引物进行PCR
扩增。反应体系体系如下：
反应程序如下：
③FhDFR6的正常PCR反应
反应体系如下：
反映程序如下：
回收PCR产物，连接T载后转化到大肠杆菌JM109感受态细胞中，用Amp抗性的LB平板培养基进行筛选。随机挑取单克隆，摇菌后进行菌液PCR验证，鉴定出的阳性克隆进行测序。
[0025]实施例1-3： FhDFRs 全长cDNA的获得：拼接上述3’和5’RACE-PCR扩增产物测序结果，获得全长cDNA序列，然后设计引物扩增全长，多次测序确定核苷酸序列的准确性。
[0026] 引物组合如下：反应体系如下：
PCR反映程序如下：95 ℃ 5 min；94 ℃ 30 s，X ℃ 60 s，72 ℃ 1min，30 Cycles。
PCR反映程序如下：95 ℃ 5 min；94 ℃ 30 s，X ℃ 60 s，72 ℃ 1min，30 Cycles。
[0027] 其中X ℃表示退火温度，其中FhDFR2(58℃)，FhDFR3(58℃)，FhDFR6(58℃)。
[0028] 实施例2：FhDFR多基因家族cDNA以及DNA序列的生物信息学分析应用NCBI中的BLAST程序（blastn、blastp、blastx）进行核酸和氨基酸序列的同源性分析；应用DNAMAN软件推导氨基酸序列；应用Clustalx1.83、MEGA5.0软件进行系统进化树分析；应用NCBI-ORF Finder寻找和翻译FhDFR的开放阅读框；应用ExPASy（http://www.expasy.org/tools/）网站的相关生物信息学分析软件对FhDFR蛋白序列的基本性质进行分析，其中Protparam分析蛋白质分子量、等电点等理化性质。
[0029] 实施例3:FhDFR基因功能的验证以pGEM-FhDFR2，pGEM-FhDFR3，pGEM-FhDFR6质粒为模板，分别利用DFR2-PF，DFR2-PR；
DFR3-PF，DFR3-PR；DFR6-PF，DFR6-PR 为引物，扩增得到含有BamH I和Xba I酶切位点的FhDFR基因，然后与克隆载体pGEM-T连接，转入到大肠杆菌 JM109中，将PCR验证为阳性的克隆送去测序，分别命名为pGEM-FhDFR2 （带酶切位点）pGEM-FhDFR3（带酶切位点），pGEM-FhDFR6（带酶切位点）。
DFR3-PF，DFR3-PR；DFR6-PF，DFR6-PR 为引物，扩增得到含有BamH I和Xba I酶切位点的FhDFR基因，然后与克隆载体pGEM-T连接，转入到大肠杆菌 JM109中，将PCR验证为阳性的克隆送去测序，分别命名为pGEM-FhDFR2 （带酶切位点）pGEM-FhDFR3（带酶切位点），pGEM-FhDFR6（带酶切位点）。
[0030] 引物组合如下：提取测序正确的克隆质粒，同时用BamH I和Xba I双酶切pGEM-FhDFR2 （带酶切位点）pGEM-FhDFR3（带酶切位点），pGEM-FhDFR6（带酶切位点）质粒以及pBI121空白质粒，回收大小正确的目的片段后，T4连接酶过夜连接，然后转化到大肠杆菌JM109感受态细胞中，随机挑取单克隆，摇菌，将PCR和双酶切鉴定正确的单克隆送去测序，-20 ℃保存测序正确的菌种，分别命名为pBI121-FhDFR2，pBI121-FhDFR3，pBI121-FhDFR6。
[0031] 将上述载体导入农杆菌GV3101细胞中，并将含有所述基因的农杆菌侵染拟南芥未授粉的花苞，再筛选出阳性植株，观察其表型变化是否发生补偿性改变。
[0032] 实施例4:FhDFR基因基因表达与花色苷合成相关性分析分别提取红花香兰雪花朵绿苞、红绿间、红苞、初开、盛开五个不同时期的RNA，逆转后得到总cDNA，再以它为模板，用3条基因各自的引物进行半定量RT-PCR，分析其表达量情况。再用SPSS软件，将基因表达量和这五个时期的花色苷积累含量一起进行相关性分析。
[0033] SEQUENCE LISTING<110> 东北师范大学
东北师范大学 , 高翔
<120> 三个与香雪兰花色苷合成相关的二氢黄酮醇-4还原酶基因的cDNA
<130> -
<140> -
<141> 2015-06-18
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 1110
<212> DNA
<213> Freesia alba
<220>
<221> misc_feature
<223> 从香雪兰花瓣中克隆得到二氢黄酮醇-4还原酶的cDNA
<220>
<221> 5'UTR
<222> (1)..(18)
<220>
<221> CDS
<222> (19)..(1023)
<220>
<221> 3'UTR
<222> (1024)..(1110)
<400> 1
ggggcctagg aaagaacg atg ggg acg gtt gtc gtg acc gga gct agc gga 51Met Gly Thr Val Val Val Thr Gly Ala Ser Gly
1 5 10
tac gtc ggc tca tgg ctc ctc atg aag ctt ctg cag aat ggc tac gcc 99Tyr Val Gly Ser Trp Leu Leu Met Lys Leu Leu Gln Asn Gly Tyr Ala
15 20 25
gtc agg gcc acc gtc aga gac ccc acc aac ctg aga aag acg aag cct 147Val Arg Ala Thr Val Arg Asp Pro Thr Asn Leu Arg Lys Thr Lys Pro
30 35 40
ctg tta gac ctt tca gga gca gac agg ctg ctc aca att tgg aaa gct 195Leu Leu Asp Leu Ser Gly Ala Asp Arg Leu Leu Thr Ile Trp Lys Ala
45 50 55
gac ctc gga gag gaa gga agt ttc gac gag gtg gta caa gga tgc act 243Asp Leu Gly Glu Glu Gly Ser Phe Asp Glu Val Val Gln Gly Cys Thr
60 65 70 75
gct gtt ttc cat gtc gca act ccg atg gat ttc gag tca caa gac cca 291Ala Val Phe His Val Ala Thr Pro Met Asp Phe Glu Ser Gln Asp Pro
80 85 90
gaa aat gaa gtg atc aag ccg gcg ata aat ggt atg ctg agc atc atg 339Glu Asn Glu Val Ile Lys Pro Ala Ile Asn Gly Met Leu Ser Ile Met
95 100 105
agg tcc tgt aaa agg gct ggc agc gtc gag cga gta att ttc aca tca 387Arg Ser Cys Lys Arg Ala Gly Ser Val Glu Arg Val Ile Phe Thr Ser
110 115 120
tct gcg ggg acc gtg aac gtc gaa gaa cat caa cga gca gag tac gat 435Ser Ala Gly Thr Val Asn Val Glu Glu His Gln Arg Ala Glu Tyr Asp
125 130 135
gag aat tgt tgg agt gac gtc gag ttc tgc aga cgt gtg aag atg act 483Glu Asn Cys Trp Ser Asp Val Glu Phe Cys Arg Arg Val Lys Met Thr
140 145 150 155
ggc tgg atg tat ttt gtg tcg aag aca ctc gcg gag aaa gcc gct tgg 531Gly Trp Met Tyr Phe Val Ser Lys Thr Leu Ala Glu Lys Ala Ala Trp
160 165 170
gat ttc gca cta gag aat gga att cat ctt ata acg atc att cca aca 579Asp Phe Ala Leu Glu Asn Gly Ile His Leu Ile Thr Ile Ile Pro Thr
175 180 185
tta gtg gtc ggt cct ttc atc acc aca act atg cca cca agt atg atc 627Leu Val Val Gly Pro Phe Ile Thr Thr Thr Met Pro Pro Ser Met Ile
190 195 200
act gca tta tca ttg atc aca gga aat gaa ggt cac tat tcg ata ata 675Thr Ala Leu Ser Leu Ile Thr Gly Asn Glu Gly His Tyr Ser Ile Ile
205 210 215
aag caa gcg caa ctt gtt cac cta gat gac ttg tgt gct gct cac att 723Lys Gln Ala Gln Leu Val His Leu Asp Asp Leu Cys Ala Ala His Ile
220 225 230 235
tac ctc ctg aac cat cct gaa gca aaa ggg agg tac ata tgc tct tct 771Tyr Leu Leu Asn His Pro Glu Ala Lys Gly Arg Tyr Ile Cys Ser Ser
240 245 250
cat gat gct acc atc tat gat ctg gca agg atg ctc aga gag agg cat 819His Asp Ala Thr Ile Tyr Asp Leu Ala Arg Met Leu Arg Glu Arg His
255 260 265
cct tgg tgt tcc att cct caa aag ttc aaa ggg att aat gag ggt att 867Pro Trp Cys Ser Ile Pro Gln Lys Phe Lys Gly Ile Asn Glu Gly Ile
270 275 280
gaa act gtg cac ttc tct tca aag aag ctg atg gac ctt gga ttc agg 915Glu Thr Val His Phe Ser Ser Lys Lys Leu Met Asp Leu Gly Phe Arg
285 290 295
ttt aag tac tcg atg gag gag atg ttc gat gag gcc atc cag aca tgc 963Phe Lys Tyr Ser Met Glu Glu Met Phe Asp Glu Ala Ile Gln Thr Cys
300 305 310 315
atc gag aag aag ctc ata aca cta cct cag agt ggt gat cgc aaa gtc 1011Ile Glu Lys Lys Leu Ile Thr Leu Pro Gln Ser Gly Asp Arg Lys Val
320 325 330
gag aat ggc tga ttggatgcgg tcggagagga agtagctatg gttaaagtgt 1063Glu Asn Gly
agttcacggt cttcatatgg agactagctg gaaggtgaaa taagggc 1110<210> 2
<211> 334
<212> PRT
<213> Freesia alba
<400> 2
Met Gly Thr Val Val Val Thr Gly Ala Ser Gly Tyr Val Gly Ser Trp
1 5 10 15
Leu Leu Met Lys Leu Leu Gln Asn Gly Tyr Ala Val Arg Ala Thr Val
20 25 30
Arg Asp Pro Thr Asn Leu Arg Lys Thr Lys Pro Leu Leu Asp Leu Ser
35 40 45
Gly Ala Asp Arg Leu Leu Thr Ile Trp Lys Ala Asp Leu Gly Glu Glu
50 55 60
Gly Ser Phe Asp Glu Val Val Gln Gly Cys Thr Ala Val Phe His Val
65 70 75 80
Ala Thr Pro Met Asp Phe Glu Ser Gln Asp Pro Glu Asn Glu Val Ile
85 90 95
Lys Pro Ala Ile Asn Gly Met Leu Ser Ile Met Arg Ser Cys Lys Arg
100 105 110
Ala Gly Ser Val Glu Arg Val Ile Phe Thr Ser Ser Ala Gly Thr Val
115 120 125
Asn Val Glu Glu His Gln Arg Ala Glu Tyr Asp Glu Asn Cys Trp Ser
130 135 140
Asp Val Glu Phe Cys Arg Arg Val Lys Met Thr Gly Trp Met Tyr Phe
145 150 155 160
Val Ser Lys Thr Leu Ala Glu Lys Ala Ala Trp Asp Phe Ala Leu Glu
165 170 175
Asn Gly Ile His Leu Ile Thr Ile Ile Pro Thr Leu Val Val Gly Pro
180 185 190
Phe Ile Thr Thr Thr Met Pro Pro Ser Met Ile Thr Ala Leu Ser Leu
195 200 205
Ile Thr Gly Asn Glu Gly His Tyr Ser Ile Ile Lys Gln Ala Gln Leu
210 215 220
Val His Leu Asp Asp Leu Cys Ala Ala His Ile Tyr Leu Leu Asn His
225 230 235 240
Pro Glu Ala Lys Gly Arg Tyr Ile Cys Ser Ser His Asp Ala Thr Ile
245 250 255
Tyr Asp Leu Ala Arg Met Leu Arg Glu Arg His Pro Trp Cys Ser Ile
260 265 270
Pro Gln Lys Phe Lys Gly Ile Asn Glu Gly Ile Glu Thr Val His Phe
275 280 285
Ser Ser Lys Lys Leu Met Asp Leu Gly Phe Arg Phe Lys Tyr Ser Met
290 295 300
Glu Glu Met Phe Asp Glu Ala Ile Gln Thr Cys Ile Glu Lys Lys Leu
305 310 315 320
Ile Thr Leu Pro Gln Ser Gly Asp Arg Lys Val Glu Asn Gly
325 330
东北师范大学 , 高翔
<120> 三个与香雪兰花色苷合成相关的二氢黄酮醇-4还原酶基因的cDNA
<130> -
<140> -
<141> 2015-06-18
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 1110
<212> DNA
<213> Freesia alba
<220>
<221> misc_feature
<223> 从香雪兰花瓣中克隆得到二氢黄酮醇-4还原酶的cDNA
<220>
<221> 5'UTR
<222> (1)..(18)
<220>
<221> CDS
<222> (19)..(1023)
<220>
<221> 3'UTR
<222> (1024)..(1110)
<400> 1
ggggcctagg aaagaacg atg ggg acg gtt gtc gtg acc gga gct agc gga 51Met Gly Thr Val Val Val Thr Gly Ala Ser Gly
1 5 10
tac gtc ggc tca tgg ctc ctc atg aag ctt ctg cag aat ggc tac gcc 99Tyr Val Gly Ser Trp Leu Leu Met Lys Leu Leu Gln Asn Gly Tyr Ala
15 20 25
gtc agg gcc acc gtc aga gac ccc acc aac ctg aga aag acg aag cct 147Val Arg Ala Thr Val Arg Asp Pro Thr Asn Leu Arg Lys Thr Lys Pro
30 35 40
ctg tta gac ctt tca gga gca gac agg ctg ctc aca att tgg aaa gct 195Leu Leu Asp Leu Ser Gly Ala Asp Arg Leu Leu Thr Ile Trp Lys Ala
45 50 55
gac ctc gga gag gaa gga agt ttc gac gag gtg gta caa gga tgc act 243Asp Leu Gly Glu Glu Gly Ser Phe Asp Glu Val Val Gln Gly Cys Thr
60 65 70 75
gct gtt ttc cat gtc gca act ccg atg gat ttc gag tca caa gac cca 291Ala Val Phe His Val Ala Thr Pro Met Asp Phe Glu Ser Gln Asp Pro
80 85 90
gaa aat gaa gtg atc aag ccg gcg ata aat ggt atg ctg agc atc atg 339Glu Asn Glu Val Ile Lys Pro Ala Ile Asn Gly Met Leu Ser Ile Met
95 100 105
agg tcc tgt aaa agg gct ggc agc gtc gag cga gta att ttc aca tca 387Arg Ser Cys Lys Arg Ala Gly Ser Val Glu Arg Val Ile Phe Thr Ser
110 115 120
tct gcg ggg acc gtg aac gtc gaa gaa cat caa cga gca gag tac gat 435Ser Ala Gly Thr Val Asn Val Glu Glu His Gln Arg Ala Glu Tyr Asp
125 130 135
gag aat tgt tgg agt gac gtc gag ttc tgc aga cgt gtg aag atg act 483Glu Asn Cys Trp Ser Asp Val Glu Phe Cys Arg Arg Val Lys Met Thr
140 145 150 155
ggc tgg atg tat ttt gtg tcg aag aca ctc gcg gag aaa gcc gct tgg 531Gly Trp Met Tyr Phe Val Ser Lys Thr Leu Ala Glu Lys Ala Ala Trp
160 165 170
gat ttc gca cta gag aat gga att cat ctt ata acg atc att cca aca 579Asp Phe Ala Leu Glu Asn Gly Ile His Leu Ile Thr Ile Ile Pro Thr
175 180 185
tta gtg gtc ggt cct ttc atc acc aca act atg cca cca agt atg atc 627Leu Val Val Gly Pro Phe Ile Thr Thr Thr Met Pro Pro Ser Met Ile
190 195 200
act gca tta tca ttg atc aca gga aat gaa ggt cac tat tcg ata ata 675Thr Ala Leu Ser Leu Ile Thr Gly Asn Glu Gly His Tyr Ser Ile Ile
205 210 215
aag caa gcg caa ctt gtt cac cta gat gac ttg tgt gct gct cac att 723Lys Gln Ala Gln Leu Val His Leu Asp Asp Leu Cys Ala Ala His Ile
220 225 230 235
tac ctc ctg aac cat cct gaa gca aaa ggg agg tac ata tgc tct tct 771Tyr Leu Leu Asn His Pro Glu Ala Lys Gly Arg Tyr Ile Cys Ser Ser
240 245 250
cat gat gct acc atc tat gat ctg gca agg atg ctc aga gag agg cat 819His Asp Ala Thr Ile Tyr Asp Leu Ala Arg Met Leu Arg Glu Arg His
255 260 265
cct tgg tgt tcc att cct caa aag ttc aaa ggg att aat gag ggt att 867Pro Trp Cys Ser Ile Pro Gln Lys Phe Lys Gly Ile Asn Glu Gly Ile
270 275 280
gaa act gtg cac ttc tct tca aag aag ctg atg gac ctt gga ttc agg 915Glu Thr Val His Phe Ser Ser Lys Lys Leu Met Asp Leu Gly Phe Arg
285 290 295
ttt aag tac tcg atg gag gag atg ttc gat gag gcc atc cag aca tgc 963Phe Lys Tyr Ser Met Glu Glu Met Phe Asp Glu Ala Ile Gln Thr Cys
300 305 310 315
atc gag aag aag ctc ata aca cta cct cag agt ggt gat cgc aaa gtc 1011Ile Glu Lys Lys Leu Ile Thr Leu Pro Gln Ser Gly Asp Arg Lys Val
320 325 330
gag aat ggc tga ttggatgcgg tcggagagga agtagctatg gttaaagtgt 1063Glu Asn Gly
agttcacggt cttcatatgg agactagctg gaaggtgaaa taagggc 1110<210> 2
<211> 334
<212> PRT
<213> Freesia alba
<400> 2
Met Gly Thr Val Val Val Thr Gly Ala Ser Gly Tyr Val Gly Ser Trp
1 5 10 15
Leu Leu Met Lys Leu Leu Gln Asn Gly Tyr Ala Val Arg Ala Thr Val
20 25 30
Arg Asp Pro Thr Asn Leu Arg Lys Thr Lys Pro Leu Leu Asp Leu Ser
35 40 45
Gly Ala Asp Arg Leu Leu Thr Ile Trp Lys Ala Asp Leu Gly Glu Glu
50 55 60
Gly Ser Phe Asp Glu Val Val Gln Gly Cys Thr Ala Val Phe His Val
65 70 75 80
Ala Thr Pro Met Asp Phe Glu Ser Gln Asp Pro Glu Asn Glu Val Ile
85 90 95
Lys Pro Ala Ile Asn Gly Met Leu Ser Ile Met Arg Ser Cys Lys Arg
100 105 110
Ala Gly Ser Val Glu Arg Val Ile Phe Thr Ser Ser Ala Gly Thr Val
115 120 125
Asn Val Glu Glu His Gln Arg Ala Glu Tyr Asp Glu Asn Cys Trp Ser
130 135 140
Asp Val Glu Phe Cys Arg Arg Val Lys Met Thr Gly Trp Met Tyr Phe
145 150 155 160
Val Ser Lys Thr Leu Ala Glu Lys Ala Ala Trp Asp Phe Ala Leu Glu
165 170 175
Asn Gly Ile His Leu Ile Thr Ile Ile Pro Thr Leu Val Val Gly Pro
180 185 190
Phe Ile Thr Thr Thr Met Pro Pro Ser Met Ile Thr Ala Leu Ser Leu
195 200 205
Ile Thr Gly Asn Glu Gly His Tyr Ser Ile Ile Lys Gln Ala Gln Leu
210 215 220
Val His Leu Asp Asp Leu Cys Ala Ala His Ile Tyr Leu Leu Asn His
225 230 235 240
Pro Glu Ala Lys Gly Arg Tyr Ile Cys Ser Ser His Asp Ala Thr Ile
245 250 255
Tyr Asp Leu Ala Arg Met Leu Arg Glu Arg His Pro Trp Cys Ser Ile
260 265 270
Pro Gln Lys Phe Lys Gly Ile Asn Glu Gly Ile Glu Thr Val His Phe
275 280 285
Ser Ser Lys Lys Leu Met Asp Leu Gly Phe Arg Phe Lys Tyr Ser Met
290 295 300
Glu Glu Met Phe Asp Glu Ala Ile Gln Thr Cys Ile Glu Lys Lys Leu
305 310 315 320
Ile Thr Leu Pro Gln Ser Gly Asp Arg Lys Val Glu Asn Gly
325 330
[0034]SEQUENCE LISTING
<110> 东北师范大学
东北师范大学, 高翔
<120> 三个与香雪兰花色苷合成相关的二氢黄酮醇-4还原酶基因的cDNA
<130> -
<140> -
<141> 2015-06-18
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 1156
<212> DNA
<213> Freesia alba
<220>
<221> misc_feature
<223> 从香雪兰中克隆得到二羟基黄酮醇-4还原酶的cDNA
<220>
<221> 5'UTR
<222> (1)..(64)
<220>
<221> CDS
<222> (65)..(1138)
<220>
<221> 3'UTR
<222> (1139)..(1156)
<400> 1
atggggaaac gagagttgag cttaattagc ctttactaat tacttgatta ggatactatc 60atca atg gcc att aca gcc gac aca gag agt aag ggg cca gtc gtc gtc 109Met Ala Ile Thr Ala Asp Thr Glu Ser Lys Gly Pro Val Val Val
1 5 10 15
acc gga gct agc ggt tac gtc ggt tca tgg ctg gtt atg ctg ctt ctc 157Thr Gly Ala Ser Gly Tyr Val Gly Ser Trp Leu Val Met Leu Leu Leu
20 25 30
aag gat ggt tat gct gtc agg gca aca gtt aga gat cca act gac aag 205Lys Asp Gly Tyr Ala Val Arg Ala Thr Val Arg Asp Pro Thr Asp Lys
35 40 45
agg aag acg aag ccg ctg ctt gac ctt ccg ggg gct gac acg ctg ctt 253Arg Lys Thr Lys Pro Leu Leu Asp Leu Pro Gly Ala Asp Thr Leu Leu
50 55 60
acg ctg tgg aaa gca gac ctc agc gag gat gga agt ttc gat gag gca 301Thr Leu Trp Lys Ala Asp Leu Ser Glu Asp Gly Ser Phe Asp Glu Ala
65 70 75
att gca gga tgc act gca gtt ttc cat gtt gca acc cca atg gat ttt 349Ile Ala Gly Cys Thr Ala Val Phe His Val Ala Thr Pro Met Asp Phe
80 85 90 95
gag tcc aaa gac cct gaa aat gaa gtg atc aag cct acg gta gat ggc 397Glu Ser Lys Asp Pro Glu Asn Glu Val Ile Lys Pro Thr Val Asp Gly
100 105 110
gtg tta agt ata atg agg tct tgt aag aat tcc ggt act gtc aag cgc 445Val Leu Ser Ile Met Arg Ser Cys Lys Asn Ser Gly Thr Val Lys Arg
115 120 125
gtg atc ttc acg tca tct gcc ggg acc gtc gac gtc caa gaa cat cag 493Val Ile Phe Thr Ser Ser Ala Gly Thr Val Asp Val Gln Glu His Gln
130 135 140
ctg gca gag tac gac gag aac tcg tgg agt gac atc gag ttt tgc aga 541Leu Ala Glu Tyr Asp Glu Asn Ser Trp Ser Asp Ile Glu Phe Cys Arg
145 150 155
cgt gtg aag atg act ggc tgg atg tat ttt gta tcg aag act cta gcc 589Arg Val Lys Met Thr Gly Trp Met Tyr Phe Val Ser Lys Thr Leu Ala
160 165 170 175
gag caa gcc gca tcg ggt ttc gct agg gag aat gac att gat cta ata 637Glu Gln Ala Ala Ser Gly Phe Ala Arg Glu Asn Asp Ile Asp Leu Ile
180 185 190
agc atc att cct acc tta gtt gtg ggt cct ttc atc acc aca agc atg 685Ser Ile Ile Pro Thr Leu Val Val Gly Pro Phe Ile Thr Thr Ser Met
195 200 205
cca cca agc atg atc act gca ttg tca ttg atc aca gga aat gaa gac 733Pro Pro Ser Met Ile Thr Ala Leu Ser Leu Ile Thr Gly Asn Glu Asp
210 215 220
cat tat cat ata att aag caa gcc caa ctt gtt cac ttg gat gac ttg 781His Tyr His Ile Ile Lys Gln Ala Gln Leu Val His Leu Asp Asp Leu
225 230 235
tgt gct gct cac att tac ctc cta aac cat cct gaa gcc aaa gga agg 829Cys Ala Ala His Ile Tyr Leu Leu Asn His Pro Glu Ala Lys Gly Arg
240 245 250 255
tac ata tgc tct tcc cat gaa gtt acc atc ttc gag ttg gcg agg atg 877Tyr Ile Cys Ser Ser His Glu Val Thr Ile Phe Glu Leu Ala Arg Met
260 265 270
atc aaa gag aga cat ccc tgg tgt tcc gtc ccg gag aag ttc gaa ggc 925Ile Lys Glu Arg His Pro Trp Cys Ser Val Pro Glu Lys Phe Glu Gly
275 280 285
att gat gag aat att aaa cct gtg cac ttc tct tcg aag aag ctg acg 973Ile Asp Glu Asn Ile Lys Pro Val His Phe Ser Ser Lys Lys Leu Thr
290 295 300
gac ctg gga ttc aag ttt caa cac acg atg gag gaa atg ttc gac gaa 1021Asp Leu Gly Phe Lys Phe Gln His Thr Met Glu Glu Met Phe Asp Glu
305 310 315
gcc atc gag tcg tgc att cag aag aag ctt ata gaa ctc cct caa act 1069Ala Ile Glu Ser Cys Ile Gln Lys Lys Leu Ile Glu Leu Pro Gln Thr
320 325 330 335
gaa aaa tca gta cct gaa aat gtg att cag gga gca aag gaa caa gcc 1117Glu Lys Ser Val Pro Glu Asn Val Ile Gln Gly Ala Lys Glu Gln Ala
340 345 350
aca gtt gct gct gaa aac tga gattagtgcc ctggagtg 1156Thr Val Ala Ala Glu Asn
355
<210> 2
<211> 357
<212> PRT
<213> Freesia alba
<400> 2
Met Ala Ile Thr Ala Asp Thr Glu Ser Lys Gly Pro Val Val Val Thr
1 5 10 15
Gly Ala Ser Gly Tyr Val Gly Ser Trp Leu Val Met Leu Leu Leu Lys
20 25 30
Asp Gly Tyr Ala Val Arg Ala Thr Val Arg Asp Pro Thr Asp Lys Arg
35 40 45
Lys Thr Lys Pro Leu Leu Asp Leu Pro Gly Ala Asp Thr Leu Leu Thr
50 55 60
Leu Trp Lys Ala Asp Leu Ser Glu Asp Gly Ser Phe Asp Glu Ala Ile
65 70 75 80
Ala Gly Cys Thr Ala Val Phe His Val Ala Thr Pro Met Asp Phe Glu
85 90 95
Ser Lys Asp Pro Glu Asn Glu Val Ile Lys Pro Thr Val Asp Gly Val
100 105 110
Leu Ser Ile Met Arg Ser Cys Lys Asn Ser Gly Thr Val Lys Arg Val
115 120 125
Ile Phe Thr Ser Ser Ala Gly Thr Val Asp Val Gln Glu His Gln Leu
130 135 140
Ala Glu Tyr Asp Glu Asn Ser Trp Ser Asp Ile Glu Phe Cys Arg Arg
145 150 155 160
Val Lys Met Thr Gly Trp Met Tyr Phe Val Ser Lys Thr Leu Ala Glu
165 170 175
Gln Ala Ala Ser Gly Phe Ala Arg Glu Asn Asp Ile Asp Leu Ile Ser
180 185 190
Ile Ile Pro Thr Leu Val Val Gly Pro Phe Ile Thr Thr Ser Met Pro
195 200 205
Pro Ser Met Ile Thr Ala Leu Ser Leu Ile Thr Gly Asn Glu Asp His
210 215 220
Tyr His Ile Ile Lys Gln Ala Gln Leu Val His Leu Asp Asp Leu Cys
225 230 235 240
Ala Ala His Ile Tyr Leu Leu Asn His Pro Glu Ala Lys Gly Arg Tyr
245 250 255
Ile Cys Ser Ser His Glu Val Thr Ile Phe Glu Leu Ala Arg Met Ile
260 265 270
Lys Glu Arg His Pro Trp Cys Ser Val Pro Glu Lys Phe Glu Gly Ile
275 280 285
Asp Glu Asn Ile Lys Pro Val His Phe Ser Ser Lys Lys Leu Thr Asp
290 295 300
Leu Gly Phe Lys Phe Gln His Thr Met Glu Glu Met Phe Asp Glu Ala
305 310 315 320
Ile Glu Ser Cys Ile Gln Lys Lys Leu Ile Glu Leu Pro Gln Thr Glu
325 330 335
Lys Ser Val Pro Glu Asn Val Ile Gln Gly Ala Lys Glu Gln Ala Thr
340 345 350
Val Ala Ala Glu Asn
355
<110> 东北师范大学
东北师范大学, 高翔
<120> 三个与香雪兰花色苷合成相关的二氢黄酮醇-4还原酶基因的cDNA
<130> -
<140> -
<141> 2015-06-18
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 1156
<212> DNA
<213> Freesia alba
<220>
<221> misc_feature
<223> 从香雪兰中克隆得到二羟基黄酮醇-4还原酶的cDNA
<220>
<221> 5'UTR
<222> (1)..(64)
<220>
<221> CDS
<222> (65)..(1138)
<220>
<221> 3'UTR
<222> (1139)..(1156)
<400> 1
atggggaaac gagagttgag cttaattagc ctttactaat tacttgatta ggatactatc 60atca atg gcc att aca gcc gac aca gag agt aag ggg cca gtc gtc gtc 109Met Ala Ile Thr Ala Asp Thr Glu Ser Lys Gly Pro Val Val Val
1 5 10 15
acc gga gct agc ggt tac gtc ggt tca tgg ctg gtt atg ctg ctt ctc 157Thr Gly Ala Ser Gly Tyr Val Gly Ser Trp Leu Val Met Leu Leu Leu
20 25 30
aag gat ggt tat gct gtc agg gca aca gtt aga gat cca act gac aag 205Lys Asp Gly Tyr Ala Val Arg Ala Thr Val Arg Asp Pro Thr Asp Lys
35 40 45
agg aag acg aag ccg ctg ctt gac ctt ccg ggg gct gac acg ctg ctt 253Arg Lys Thr Lys Pro Leu Leu Asp Leu Pro Gly Ala Asp Thr Leu Leu
50 55 60
acg ctg tgg aaa gca gac ctc agc gag gat gga agt ttc gat gag gca 301Thr Leu Trp Lys Ala Asp Leu Ser Glu Asp Gly Ser Phe Asp Glu Ala
65 70 75
att gca gga tgc act gca gtt ttc cat gtt gca acc cca atg gat ttt 349Ile Ala Gly Cys Thr Ala Val Phe His Val Ala Thr Pro Met Asp Phe
80 85 90 95
gag tcc aaa gac cct gaa aat gaa gtg atc aag cct acg gta gat ggc 397Glu Ser Lys Asp Pro Glu Asn Glu Val Ile Lys Pro Thr Val Asp Gly
100 105 110
gtg tta agt ata atg agg tct tgt aag aat tcc ggt act gtc aag cgc 445Val Leu Ser Ile Met Arg Ser Cys Lys Asn Ser Gly Thr Val Lys Arg
115 120 125
gtg atc ttc acg tca tct gcc ggg acc gtc gac gtc caa gaa cat cag 493Val Ile Phe Thr Ser Ser Ala Gly Thr Val Asp Val Gln Glu His Gln
130 135 140
ctg gca gag tac gac gag aac tcg tgg agt gac atc gag ttt tgc aga 541Leu Ala Glu Tyr Asp Glu Asn Ser Trp Ser Asp Ile Glu Phe Cys Arg
145 150 155
cgt gtg aag atg act ggc tgg atg tat ttt gta tcg aag act cta gcc 589Arg Val Lys Met Thr Gly Trp Met Tyr Phe Val Ser Lys Thr Leu Ala
160 165 170 175
gag caa gcc gca tcg ggt ttc gct agg gag aat gac att gat cta ata 637Glu Gln Ala Ala Ser Gly Phe Ala Arg Glu Asn Asp Ile Asp Leu Ile
180 185 190
agc atc att cct acc tta gtt gtg ggt cct ttc atc acc aca agc atg 685Ser Ile Ile Pro Thr Leu Val Val Gly Pro Phe Ile Thr Thr Ser Met
195 200 205
cca cca agc atg atc act gca ttg tca ttg atc aca gga aat gaa gac 733Pro Pro Ser Met Ile Thr Ala Leu Ser Leu Ile Thr Gly Asn Glu Asp
210 215 220
cat tat cat ata att aag caa gcc caa ctt gtt cac ttg gat gac ttg 781His Tyr His Ile Ile Lys Gln Ala Gln Leu Val His Leu Asp Asp Leu
225 230 235
tgt gct gct cac att tac ctc cta aac cat cct gaa gcc aaa gga agg 829Cys Ala Ala His Ile Tyr Leu Leu Asn His Pro Glu Ala Lys Gly Arg
240 245 250 255
tac ata tgc tct tcc cat gaa gtt acc atc ttc gag ttg gcg agg atg 877Tyr Ile Cys Ser Ser His Glu Val Thr Ile Phe Glu Leu Ala Arg Met
260 265 270
atc aaa gag aga cat ccc tgg tgt tcc gtc ccg gag aag ttc gaa ggc 925Ile Lys Glu Arg His Pro Trp Cys Ser Val Pro Glu Lys Phe Glu Gly
275 280 285
att gat gag aat att aaa cct gtg cac ttc tct tcg aag aag ctg acg 973Ile Asp Glu Asn Ile Lys Pro Val His Phe Ser Ser Lys Lys Leu Thr
290 295 300
gac ctg gga ttc aag ttt caa cac acg atg gag gaa atg ttc gac gaa 1021Asp Leu Gly Phe Lys Phe Gln His Thr Met Glu Glu Met Phe Asp Glu
305 310 315
gcc atc gag tcg tgc att cag aag aag ctt ata gaa ctc cct caa act 1069Ala Ile Glu Ser Cys Ile Gln Lys Lys Leu Ile Glu Leu Pro Gln Thr
320 325 330 335
gaa aaa tca gta cct gaa aat gtg att cag gga gca aag gaa caa gcc 1117Glu Lys Ser Val Pro Glu Asn Val Ile Gln Gly Ala Lys Glu Gln Ala
340 345 350
aca gtt gct gct gaa aac tga gattagtgcc ctggagtg 1156Thr Val Ala Ala Glu Asn
355
<210> 2
<211> 357
<212> PRT
<213> Freesia alba
<400> 2
Met Ala Ile Thr Ala Asp Thr Glu Ser Lys Gly Pro Val Val Val Thr
1 5 10 15
Gly Ala Ser Gly Tyr Val Gly Ser Trp Leu Val Met Leu Leu Leu Lys
20 25 30
Asp Gly Tyr Ala Val Arg Ala Thr Val Arg Asp Pro Thr Asp Lys Arg
35 40 45
Lys Thr Lys Pro Leu Leu Asp Leu Pro Gly Ala Asp Thr Leu Leu Thr
50 55 60
Leu Trp Lys Ala Asp Leu Ser Glu Asp Gly Ser Phe Asp Glu Ala Ile
65 70 75 80
Ala Gly Cys Thr Ala Val Phe His Val Ala Thr Pro Met Asp Phe Glu
85 90 95
Ser Lys Asp Pro Glu Asn Glu Val Ile Lys Pro Thr Val Asp Gly Val
100 105 110
Leu Ser Ile Met Arg Ser Cys Lys Asn Ser Gly Thr Val Lys Arg Val
115 120 125
Ile Phe Thr Ser Ser Ala Gly Thr Val Asp Val Gln Glu His Gln Leu
130 135 140
Ala Glu Tyr Asp Glu Asn Ser Trp Ser Asp Ile Glu Phe Cys Arg Arg
145 150 155 160
Val Lys Met Thr Gly Trp Met Tyr Phe Val Ser Lys Thr Leu Ala Glu
165 170 175
Gln Ala Ala Ser Gly Phe Ala Arg Glu Asn Asp Ile Asp Leu Ile Ser
180 185 190
Ile Ile Pro Thr Leu Val Val Gly Pro Phe Ile Thr Thr Ser Met Pro
195 200 205
Pro Ser Met Ile Thr Ala Leu Ser Leu Ile Thr Gly Asn Glu Asp His
210 215 220
Tyr His Ile Ile Lys Gln Ala Gln Leu Val His Leu Asp Asp Leu Cys
225 230 235 240
Ala Ala His Ile Tyr Leu Leu Asn His Pro Glu Ala Lys Gly Arg Tyr
245 250 255
Ile Cys Ser Ser His Glu Val Thr Ile Phe Glu Leu Ala Arg Met Ile
260 265 270
Lys Glu Arg His Pro Trp Cys Ser Val Pro Glu Lys Phe Glu Gly Ile
275 280 285
Asp Glu Asn Ile Lys Pro Val His Phe Ser Ser Lys Lys Leu Thr Asp
290 295 300
Leu Gly Phe Lys Phe Gln His Thr Met Glu Glu Met Phe Asp Glu Ala
305 310 315 320
Ile Glu Ser Cys Ile Gln Lys Lys Leu Ile Glu Leu Pro Gln Thr Glu
325 330 335
Lys Ser Val Pro Glu Asn Val Ile Gln Gly Ala Lys Glu Gln Ala Thr
340 345 350
Val Ala Ala Glu Asn
355
[0035]SEQUENCE LISTING
<110> 东北师范大学
东北师范大学 高翔
<120> 三个与香雪兰花色苷合成相关的二氢黄酮醇-4还原酶基因的cDNA
<130> -
<140> -
<141> 2015-06-18
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 1076
<212> DNA
<213> Freesia alba
<220>
<221> misc_feature
<223> 从香雪兰中克隆得到二羟基黄酮醇-4还原酶的cDNA
<220>
<221> 5'UTR
<222> (1)..(18)
<220>
<221> CDS
<222> (19)..(1065)
<220>
<221> 3'UTR
<222> (1065)..(1076)
<400> 1
ggggcctagg aaagaacg atg ggg acg gtt gtc gtg acc gga gct agc gga 51Met Gly Thr Val Val Val Thr Gly Ala Ser Gly
1 5 10
tac gtc ggc tca tgg ctc ctc atg aag ctt ctg cag aat ggc tac gcc 99Tyr Val Gly Ser Trp Leu Leu Met Lys Leu Leu Gln Asn Gly Tyr Ala
15 20 25
gtc agg gcc acc gtc aga gac ccc acc aac ctg aga aag acg aag cct 147Val Arg Ala Thr Val Arg Asp Pro Thr Asn Leu Arg Lys Thr Lys Pro
30 35 40
ctg tta gac ctt tca gga gca gac agg ctg ctc aca att tgg aaa gct 195Leu Leu Asp Leu Ser Gly Ala Asp Arg Leu Leu Thr Ile Trp Lys Ala
45 50 55
gac ctc gga gag gaa gga agt ttc gac gag gtg gta caa gga tgc act 243Asp Leu Gly Glu Glu Gly Ser Phe Asp Glu Val Val Gln Gly Cys Thr
60 65 70 75
gct gtt ttc cat gtc gca act ccg atg gat ttc gag tca caa gac cca 291Ala Val Phe His Val Ala Thr Pro Met Asp Phe Glu Ser Gln Asp Pro
80 85 90
gaa aat gaa gtg atc aag ccg acg ata aat ggt atg cta agc atc atg 339Glu Asn Glu Val Ile Lys Pro Thr Ile Asn Gly Met Leu Ser Ile Met
95 100 105
agg tcc tgt aaa agg gct ggc agc gtc gag cga gta att ttc aca tca 387Arg Ser Cys Lys Arg Ala Gly Ser Val Glu Arg Val Ile Phe Thr Ser
110 115 120
tct gcg ggg acc gtg aac gtc gaa gaa cat caa cga gca gag tac gat 435Ser Ala Gly Thr Val Asn Val Glu Glu His Gln Arg Ala Glu Tyr Asp
125 130 135
gag aat tgt tgg agt gac gtc gag ttc tgc aga cgt gtg aag atg act 483Glu Asn Cys Trp Ser Asp Val Glu Phe Cys Arg Arg Val Lys Met Thr
140 145 150 155
ggc tgg atg tat ttt gtg tcg aag aca ctc gcg gag aaa gcc gct tgg 531Gly Trp Met Tyr Phe Val Ser Lys Thr Leu Ala Glu Lys Ala Ala Trp
160 165 170
gat ttc gca cgt gag aat gga att cat ctt ata acg atc att cca aca 579Asp Phe Ala Arg Glu Asn Gly Ile His Leu Ile Thr Ile Ile Pro Thr
175 180 185
tta gtt gtc ggt cct ttc atc acc aca act atg cca cca agt atg atc 627Leu Val Val Gly Pro Phe Ile Thr Thr Thr Met Pro Pro Ser Met Ile
190 195 200
act gca tta tca ttg atc aca gga aat gaa gct cac tat tcg ata ata 675Thr Ala Leu Ser Leu Ile Thr Gly Asn Glu Ala His Tyr Ser Ile Ile
205 210 215
aag caa gcg caa ctt gtt cac cta gat gac ttg tgt gct gct cac att 723Lys Gln Ala Gln Leu Val His Leu Asp Asp Leu Cys Ala Ala His Ile
220 225 230 235
tac ctc ctg aac cat cct gaa gca aaa ggg agg tac ata tgc tct tct 771Tyr Leu Leu Asn His Pro Glu Ala Lys Gly Arg Tyr Ile Cys Ser Ser
240 245 250
cat gat gct acc atc tat gat ctg gca agg atg ctc aga gag agg cat 819His Asp Ala Thr Ile Tyr Asp Leu Ala Arg Met Leu Arg Glu Arg His
255 260 265
cct tgg tgt tcc att cct caa aag ttc aaa ggg att aat gag ggt att 867Pro Trp Cys Ser Ile Pro Gln Lys Phe Lys Gly Ile Asn Glu Gly Ile
270 275 280
gaa att gtg cac ttc tct tca aag aag ctg atg gac ctt gga ttc cgg 915Glu Ile Val His Phe Ser Ser Lys Lys Leu Met Asp Leu Gly Phe Arg
285 290 295
ttt aag tac tcg atg gag gag atg ttc gat gag gcc atc cag aca tgc 963Phe Lys Tyr Ser Met Glu Glu Met Phe Asp Glu Ala Ile Gln Thr Cys
300 305 310 315
atc gag aag aag ctc ata aca cta cct cag agt ggt gat cgc aaa gtc 1011Ile Glu Lys Lys Leu Ile Thr Leu Pro Gln Ser Gly Asp Arg Lys Val
320 325 330
gag aat ggc cga ttg gat gcg gtc gga gag gaa gta gct atg gtt aaa 1059Glu Asn Gly Arg Leu Asp Ala Val Gly Glu Glu Val Ala Met Val Lys
335 340 345
gtg tag ttcatggtct t 1076Val
<210> 2
<211> 348
<212> PRT
<213> Freesia alba
<400> 2
Met Gly Thr Val Val Val Thr Gly Ala Ser Gly Tyr Val Gly Ser Trp
1 5 10 15
Leu Leu Met Lys Leu Leu Gln Asn Gly Tyr Ala Val Arg Ala Thr Val
20 25 30
Arg Asp Pro Thr Asn Leu Arg Lys Thr Lys Pro Leu Leu Asp Leu Ser
35 40 45
Gly Ala Asp Arg Leu Leu Thr Ile Trp Lys Ala Asp Leu Gly Glu Glu
50 55 60
Gly Ser Phe Asp Glu Val Val Gln Gly Cys Thr Ala Val Phe His Val
65 70 75 80
Ala Thr Pro Met Asp Phe Glu Ser Gln Asp Pro Glu Asn Glu Val Ile
85 90 95
Lys Pro Thr Ile Asn Gly Met Leu Ser Ile Met Arg Ser Cys Lys Arg
100 105 110
Ala Gly Ser Val Glu Arg Val Ile Phe Thr Ser Ser Ala Gly Thr Val
115 120 125
Asn Val Glu Glu His Gln Arg Ala Glu Tyr Asp Glu Asn Cys Trp Ser
130 135 140
Asp Val Glu Phe Cys Arg Arg Val Lys Met Thr Gly Trp Met Tyr Phe
145 150 155 160
Val Ser Lys Thr Leu Ala Glu Lys Ala Ala Trp Asp Phe Ala Arg Glu
165 170 175
Asn Gly Ile His Leu Ile Thr Ile Ile Pro Thr Leu Val Val Gly Pro
180 185 190
Phe Ile Thr Thr Thr Met Pro Pro Ser Met Ile Thr Ala Leu Ser Leu
195 200 205
Ile Thr Gly Asn Glu Ala His Tyr Ser Ile Ile Lys Gln Ala Gln Leu
210 215 220
Val His Leu Asp Asp Leu Cys Ala Ala His Ile Tyr Leu Leu Asn His
225 230 235 240
Pro Glu Ala Lys Gly Arg Tyr Ile Cys Ser Ser His Asp Ala Thr Ile
245 250 255
Tyr Asp Leu Ala Arg Met Leu Arg Glu Arg His Pro Trp Cys Ser Ile
260 265 270
Pro Gln Lys Phe Lys Gly Ile Asn Glu Gly Ile Glu Ile Val His Phe
275 280 285
Ser Ser Lys Lys Leu Met Asp Leu Gly Phe Arg Phe Lys Tyr Ser Met
290 295 300
Glu Glu Met Phe Asp Glu Ala Ile Gln Thr Cys Ile Glu Lys Lys Leu
305 310 315 320
Ile Thr Leu Pro Gln Ser Gly Asp Arg Lys Val Glu Asn Gly Arg Leu
325 330 335
Asp Ala Val Gly Glu Glu Val Ala Met Val Lys Val
340 345
<110> 东北师范大学
东北师范大学 高翔
<120> 三个与香雪兰花色苷合成相关的二氢黄酮醇-4还原酶基因的cDNA
<130> -
<140> -
<141> 2015-06-18
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 1076
<212> DNA
<213> Freesia alba
<220>
<221> misc_feature
<223> 从香雪兰中克隆得到二羟基黄酮醇-4还原酶的cDNA
<220>
<221> 5'UTR
<222> (1)..(18)
<220>
<221> CDS
<222> (19)..(1065)
<220>
<221> 3'UTR
<222> (1065)..(1076)
<400> 1
ggggcctagg aaagaacg atg ggg acg gtt gtc gtg acc gga gct agc gga 51Met Gly Thr Val Val Val Thr Gly Ala Ser Gly
1 5 10
tac gtc ggc tca tgg ctc ctc atg aag ctt ctg cag aat ggc tac gcc 99Tyr Val Gly Ser Trp Leu Leu Met Lys Leu Leu Gln Asn Gly Tyr Ala
15 20 25
gtc agg gcc acc gtc aga gac ccc acc aac ctg aga aag acg aag cct 147Val Arg Ala Thr Val Arg Asp Pro Thr Asn Leu Arg Lys Thr Lys Pro
30 35 40
ctg tta gac ctt tca gga gca gac agg ctg ctc aca att tgg aaa gct 195Leu Leu Asp Leu Ser Gly Ala Asp Arg Leu Leu Thr Ile Trp Lys Ala
45 50 55
gac ctc gga gag gaa gga agt ttc gac gag gtg gta caa gga tgc act 243Asp Leu Gly Glu Glu Gly Ser Phe Asp Glu Val Val Gln Gly Cys Thr
60 65 70 75
gct gtt ttc cat gtc gca act ccg atg gat ttc gag tca caa gac cca 291Ala Val Phe His Val Ala Thr Pro Met Asp Phe Glu Ser Gln Asp Pro
80 85 90
gaa aat gaa gtg atc aag ccg acg ata aat ggt atg cta agc atc atg 339Glu Asn Glu Val Ile Lys Pro Thr Ile Asn Gly Met Leu Ser Ile Met
95 100 105
agg tcc tgt aaa agg gct ggc agc gtc gag cga gta att ttc aca tca 387Arg Ser Cys Lys Arg Ala Gly Ser Val Glu Arg Val Ile Phe Thr Ser
110 115 120
tct gcg ggg acc gtg aac gtc gaa gaa cat caa cga gca gag tac gat 435Ser Ala Gly Thr Val Asn Val Glu Glu His Gln Arg Ala Glu Tyr Asp
125 130 135
gag aat tgt tgg agt gac gtc gag ttc tgc aga cgt gtg aag atg act 483Glu Asn Cys Trp Ser Asp Val Glu Phe Cys Arg Arg Val Lys Met Thr
140 145 150 155
ggc tgg atg tat ttt gtg tcg aag aca ctc gcg gag aaa gcc gct tgg 531Gly Trp Met Tyr Phe Val Ser Lys Thr Leu Ala Glu Lys Ala Ala Trp
160 165 170
gat ttc gca cgt gag aat gga att cat ctt ata acg atc att cca aca 579Asp Phe Ala Arg Glu Asn Gly Ile His Leu Ile Thr Ile Ile Pro Thr
175 180 185
tta gtt gtc ggt cct ttc atc acc aca act atg cca cca agt atg atc 627Leu Val Val Gly Pro Phe Ile Thr Thr Thr Met Pro Pro Ser Met Ile
190 195 200
act gca tta tca ttg atc aca gga aat gaa gct cac tat tcg ata ata 675Thr Ala Leu Ser Leu Ile Thr Gly Asn Glu Ala His Tyr Ser Ile Ile
205 210 215
aag caa gcg caa ctt gtt cac cta gat gac ttg tgt gct gct cac att 723Lys Gln Ala Gln Leu Val His Leu Asp Asp Leu Cys Ala Ala His Ile
220 225 230 235
tac ctc ctg aac cat cct gaa gca aaa ggg agg tac ata tgc tct tct 771Tyr Leu Leu Asn His Pro Glu Ala Lys Gly Arg Tyr Ile Cys Ser Ser
240 245 250
cat gat gct acc atc tat gat ctg gca agg atg ctc aga gag agg cat 819His Asp Ala Thr Ile Tyr Asp Leu Ala Arg Met Leu Arg Glu Arg His
255 260 265
cct tgg tgt tcc att cct caa aag ttc aaa ggg att aat gag ggt att 867Pro Trp Cys Ser Ile Pro Gln Lys Phe Lys Gly Ile Asn Glu Gly Ile
270 275 280
gaa att gtg cac ttc tct tca aag aag ctg atg gac ctt gga ttc cgg 915Glu Ile Val His Phe Ser Ser Lys Lys Leu Met Asp Leu Gly Phe Arg
285 290 295
ttt aag tac tcg atg gag gag atg ttc gat gag gcc atc cag aca tgc 963Phe Lys Tyr Ser Met Glu Glu Met Phe Asp Glu Ala Ile Gln Thr Cys
300 305 310 315
atc gag aag aag ctc ata aca cta cct cag agt ggt gat cgc aaa gtc 1011Ile Glu Lys Lys Leu Ile Thr Leu Pro Gln Ser Gly Asp Arg Lys Val
320 325 330
gag aat ggc cga ttg gat gcg gtc gga gag gaa gta gct atg gtt aaa 1059Glu Asn Gly Arg Leu Asp Ala Val Gly Glu Glu Val Ala Met Val Lys
335 340 345
gtg tag ttcatggtct t 1076Val
<210> 2
<211> 348
<212> PRT
<213> Freesia alba
<400> 2
Met Gly Thr Val Val Val Thr Gly Ala Ser Gly Tyr Val Gly Ser Trp
1 5 10 15
Leu Leu Met Lys Leu Leu Gln Asn Gly Tyr Ala Val Arg Ala Thr Val
20 25 30
Arg Asp Pro Thr Asn Leu Arg Lys Thr Lys Pro Leu Leu Asp Leu Ser
35 40 45
Gly Ala Asp Arg Leu Leu Thr Ile Trp Lys Ala Asp Leu Gly Glu Glu
50 55 60
Gly Ser Phe Asp Glu Val Val Gln Gly Cys Thr Ala Val Phe His Val
65 70 75 80
Ala Thr Pro Met Asp Phe Glu Ser Gln Asp Pro Glu Asn Glu Val Ile
85 90 95
Lys Pro Thr Ile Asn Gly Met Leu Ser Ile Met Arg Ser Cys Lys Arg
100 105 110
Ala Gly Ser Val Glu Arg Val Ile Phe Thr Ser Ser Ala Gly Thr Val
115 120 125
Asn Val Glu Glu His Gln Arg Ala Glu Tyr Asp Glu Asn Cys Trp Ser
130 135 140
Asp Val Glu Phe Cys Arg Arg Val Lys Met Thr Gly Trp Met Tyr Phe
145 150 155 160
Val Ser Lys Thr Leu Ala Glu Lys Ala Ala Trp Asp Phe Ala Arg Glu
165 170 175
Asn Gly Ile His Leu Ile Thr Ile Ile Pro Thr Leu Val Val Gly Pro
180 185 190
Phe Ile Thr Thr Thr Met Pro Pro Ser Met Ile Thr Ala Leu Ser Leu
195 200 205
Ile Thr Gly Asn Glu Ala His Tyr Ser Ile Ile Lys Gln Ala Gln Leu
210 215 220
Val His Leu Asp Asp Leu Cys Ala Ala His Ile Tyr Leu Leu Asn His
225 230 235 240
Pro Glu Ala Lys Gly Arg Tyr Ile Cys Ser Ser His Asp Ala Thr Ile
245 250 255
Tyr Asp Leu Ala Arg Met Leu Arg Glu Arg His Pro Trp Cys Ser Ile
260 265 270
Pro Gln Lys Phe Lys Gly Ile Asn Glu Gly Ile Glu Ile Val His Phe
275 280 285
Ser Ser Lys Lys Leu Met Asp Leu Gly Phe Arg Phe Lys Tyr Ser Met
290 295 300
Glu Glu Met Phe Asp Glu Ala Ile Gln Thr Cys Ile Glu Lys Lys Leu
305 310 315 320
Ile Thr Leu Pro Gln Ser Gly Asp Arg Lys Val Glu Asn Gly Arg Leu
325 330 335
Asp Ala Val Gly Glu Glu Val Ala Met Val Lys Val
340 345
| 标题 | 发布/更新时间 | 阅读量 |
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