专利名称：白桦苯丙氨酸解氨酶cDNA序列及其编码的氨基酸序列和制备过程中设计的引物的制作方法
技术领域：
本发明涉及一种苯丙氨酸解氨酶cDNA序列及其编码的氨基酸序列和制备过程中设计的引物。
背景技术：
白桦苯丙氨酸解氨酶(PAL)基因编码的苯丙酸解氨酶是植物体内多种次生代谢途径的关键酶。白桦苯丙氨酸解氨酶在木质素单体生物合成中催化苯丙氨酸脱氨形成肉桂酸，肉桂酸再经羟基化、甲基化、还原等反应生成香豆醇、松柏醇和芥子醇3种主要单体；白桦苯丙氨酸解氨酶可调节木质素的生物合成，但白桦苯丙氨酸解氨酶并非特异地参与木质素单体合成，它也是非木质素酚类物质合成的中间环节，研究发现，经苯丙酸途径合成的其它物质比木质素对白桦苯丙氨酸解氨酶(PAL)敏感得多。因此，白桦苯丙氨酸解氨酶的基因研究对降低植物中木质素的含量或改变木质素的组分及控制多种物质的代谢方面都有着重要的意义。
发明内容
鉴于白桦苯丙氨酸解氨酶在降低植物中木质素的含量或改变木质素的组分及控制多种物质的代谢方面都有着重要的意义，本发明旨在利用RT-PCR技术和RACE技术获得白桦苯丙氨酸解氨酶(PAL)基因的全长编码(cDNA)序列。白桦苯丙氨酸解氨酶基因的cDNA序列全长为2322bp，其中T、C、G、A分别为572bp(25％)、544bp(23％)、590bp(25％)、616bp(27％)。白桦苯丙氨酸解氨酶基因的cDNA序列编码白桦苯丙氨酸解氨酶(PAL)773个氨基酸。本发明为制备白桦苯丙氨酸解氨酶设计了P1～P6六个引物。
本发明成功的制备出白桦苯丙氨酸解氨酶(PAL)基因的全长编码(cDNA)序列，并可通过该基因合成白桦苯丙氨酸解氨酶(PAL)。白桦苯丙氨酸解氨酶(PAL)可降低植物中木质素的含量或改变木质素的组分，用以培育优良的牧草或林木纸浆木材新品种；还可用来调控多种代谢途径，为更好的应用、推广白桦苯丙氨酸解氨酶奠定了物质基础。
具体实施例方式
具体实施方式
一本实施方式通过以下步骤实现白桦苯丙氨酸解氨酶基因cDNA的制备(一)白桦次生木质部总RNA的提取1、研钵在180℃条件下烘烤8～10h，以灭活、除去RNase、DNA和蛋白质等杂质；2、待研钵冷却至室温后，将液氮倒入研钵，进行预冷处理；3、将0.5～1g白桦(Betula platyphylla)次生木质部材料放入经预冷处理的研钵中，再倒入液氮并快速研磨粉碎白桦次生木质部材料；4、研磨产物快速放入装有700μL 2×CTAB，60～100μL 5％的巯基乙醇的离心管中；5、离心管在65±1℃条件下水浴5min，并在水浴过程中摇动离心管2～3次；6、加入体积比为1∶1，浓度为98.5％的氯仿和Tris饱和酚共650～750μL，振荡3～5min；7、在4℃、12500r/min的条件下离心8min，取上清液，加入体积比为1∶1，浓度为98.5％的氯仿和Tris饱和酚共650～750μL，振荡3～5min，再在4℃、12500r/min的条件下离心8min，取上清液；8、加入与上清液等体积的浓度为98.5％的氯仿550～650μL，振荡3～5min，再在4℃、12000r/min的条件下离心10min；9、取上清液加入上清液1/2体积的无水乙醇和与上清液等体积的4M LiCl，混匀后冰浴10min；10、冰浴后离心管在4℃、12800r/min的条件下离心10min，弃去上清液，沉淀物用浓度为70％的乙醇洗涤两次；11、室温干燥5～10min后用50～100μL DEPC水溶解沉淀物，置于-70℃的环境中保存。
(二)逆转录多聚酶链式反应(RT-PCR)获得白桦苯丙氨酸解氨酶(PAL)的部分cDNA片段1、总RNA为模板，用Invitrogen公司的ThermoScriptTM RT-PCR System反转录试剂盒反转录合成cDNA(RT-PCR反转录合成步骤参见RT-PCR试剂盒使用操作手册)；2、利用设计的RT-PCR反应简并引物P1和P2进行RT-PCR扩增，简并引物P1和P2的序列见表1，扩增程序见表2，RT-CR扩增产物后可放于4℃的环境中保存；3、行琼脂糖凝胶电泳，将特异条带回收克隆并测序；4、应用美国国家生物信息中心提供的BLASTn程序(NCBI BLASTN)对测序结果进行比对，确定该条DNA片段为PAL的部分cDNA片段，片段序列长为894bp，其中T、C、G、A分别为218bp(24％)、222bp(25％)、219bp(24％)、235bp(26％)。
表1
K＝G或T；D＝A、G或T；Y＝T或C；W＝A或T；V＝A、G或C；M＝A或C。
表2
(三)利用RACE技术获得白桦苯丙氨酸解氨酶(PAL)基因的全长cDNA序列及白桦苯丙氨酸解氨酶(PAL)基因cDNA编码的氨基酸序列1、在已得到的部分cDNA片段基础上设计5’RACE特异引物P3、P4和3’RACE特异引物P5、P6，特异引物P3、P4、P5和P6见表3；表3
2、用RACE特异引物和Invitrogen公司的SuperScriptTMII RACE试剂盒进行cDNA末端PCR扩增(操作步骤参见RACE试剂盒使用操作手册)，扩增程序见表4，PCR扩增产物后可放于4℃的环境中保存；表4
3、扩增产物进行琼脂糖凝胶电泳，并用V-gene凝胶回收试剂盒回收特异片段，然后将特异片段与pEGM T-easy载体连接，连接体系为10μL，如表5所示；表5
4、连接产物转化感受态细胞大肠杆菌DH5α，挑取初步认定为阳性克隆的白色菌落进行细菌质粒的PCR反应；5、利用上海博亚生物技术有限公司出品的通用引物T7和SP6对PCR反应产物进行测序，测序结果用美国国家生物信息中心(NCBI)提供的BLASTN和BLASTP软件或DNAMAN、DNASTAR生物分析软件进行比对、分析；6、确认为白桦苯丙氨酸解氨酶基因cDNA片段后，用DNASTAR序列分析软件将三段cDNA核苷酸序列拼接到一起，得到白桦苯丙氨酸解氨酶(PAL)基因的全长cDNA序列，共2322bp，其中T、C、G、A分别为572bp(25％)、544bp(23％)、590bp(25％)、616bp(27％)；7、通过白桦苯丙氨酸解氨酶(PAL)基因的全长cDNA序列得到白桦苯丙氨酸解氨酶(PAL)的氨基酸序列，共773个氨基酸。
序列表<110>东北林业大学 杨传平 刘雪梅<120>白桦苯丙氨酸解氨酶cDNA序列及其编码的氨基酸序列和制备过程中设计的引物<160>9<210>1<211>2322<212>DNA<213>白桦种(Betula platyphylla Suk.)<220>
<221>CDS<222>(1)...(2322)<223>cDNA<400>1atg gca acc gag gga gga ctt tcg gcc cgc caa tac aga aat gcc cgt 48Met Ala Thr Glu Gly Gly Leu Ser Ala Arg Gln Tyr Arg Asn Ala Arg1 5 10 15cct cca acc ctc cct ctc cca aaa gac agc gta aac ctc att tgc gta 96Pro Pro Thr Leu Pro Leu Pro Lys Asp Ser Val Asn Leu Ile Cys Val20 25 30tca gaa aat tct cac ttg tca gcc cca ctg cac tgg aag aaa gct gca 144Ser Glu Asn Ser His Leu Ser Ala Pro Leu His Trp Lys Lys Ala Ala35 40 45gag gcc ctc cag tgc acc cat ttt gat gaa gtc tgc cgc atg gtt tcc 192Glu Ala Leu Gln Cys Thr His Phe Asp Glu Val Cys Arg Met Val Ser50 55 60caa ttt gca aag gtt caa agc gtt gat atc cag gga acc tcc ctc acg 240Gln Phe Ala Lys Val Gln Ser Val Asp Ile Gln Gly Thr Ser Leu Thr65 70 75 80gtt gct cag gtc act gca att gca cgc cgc agt gaa gta aaa gtt aat 288Val Ala Gln Val Thr Ala Ile Ala Arg Arg Ser Glu Val Lys Val Asn85 90 95ctt gat gag gct gcg gcg cgt gaa cgt gtg gcc aaa agt gcc aac tgg 336Leu Asp Glu Ala Ala Ala Arg Glu Arg Val Ala Lys Ser Ala Asn Trp100 105 110gtt gct gaa aat atc tcc cgt gga aca gac acg tat ggt gtt acc acc 384Val Ala Glu Asn Ile Ser Arg Gly Thr Asp Thr Tyr Gly Val Thr Thr115 120 125ggc ttc gga gcc acg tca cac cgg cgc acc aac aaa act gtg gac ctc 432Gly Phe Gly Ala Thr Ser His Arg Arg Thr Asn Lys Thr Val Asp Leu130 135 140cag aca gag ctt ata agg ttc ctt aat gcc gga gtg atc ggg aaa gag 480Gln Thr Glu Leu Ile Arg Phe Leu Asn Ala Gly Val Ile Gly Lys Glu
145 150 155 160agt ctt ccg tcc act tat tca aag gtg gca atg ctc gtt cgc acg aac 528Ser Leu Pro Ser Thr Tyr Ser Lys Val Ala Met Leu Val Arg Thr Asn165 170 175gaa cac act cat gca agg ggt ttt tcg ggc att cga tgg gag ata ctg 576Glu His Thr His Ala Arg Gly Phe Ser Gly Ile Arg Trp Glu Ile Leu180 185 190gaa gcc atg gcc aat ctg atg aac aag aat ttg atc cct aaa ttg cct 624Glu Ala Met Ala Asn Leu Met Asn Lys Asn Leu Ile Pro Lys Leu Pro195 200 205cta agg ggg act atc act gcc tct ggg gat ctt gta cca ctc tct tat 672Leu Arg Gly Thr Ile Thr Ala Ser Gly Asp Leu Val Pro Leu Ser Tyr210 215 220att gcc ggt ttg cta act ggc cgc cat aat tcc aaa gtg gtc acc cct 720Ile Ala Gly Leu Leu Thr Gly Arg His Asn Ser Lys Val Val Thr Pro225 230 235 240gaa gat gaa gaa atc aca gcc ttg gaa gct ctc aag agg gca gga att 768Glu Asp Glu Glu Ile Thr Ala Leu Glu Ala Leu Lys Arg Ala Gly Ile245 250 255cca gct cct ttc gag ctt caa gca aag gag ggt cta gct ctt gta aac 826Pro Ala Pro Phe Glu Leu Gln Ala Lys Glu Gly Leu Ala Leu Val Asn260 265 270ggg aca gct gtt ggc tcg gct gtg gct gca acg gtt tgt ttt gat gcc 864Gly Thr Ala Val Gly Ser Ala Val Ala Ala Thr Val Cys Phe Asp Ala275 280 285aac att ttg gct ctt ttt gca gaa att ctc tct gct ctg ttt tgt gaa 912Asn Ile Leu Ala Leu Phe Ala Glu Ile Leu Ser Ala Leu Phe Cys Glu290 295 300gtc atg ctt gga aat gcc gag ttc aca gac cca ctg acg cat gag ctc 960Val Met Leu Gly Asn Ala Glu Phe Thr Asp Pro Leu Thr His Glu Leu305 310 315 320aag cac cac cct ggg cag att gag gca gcc gct acc atg gag ttc ctt 1008Lys His His Pro Gly Gln Ile Glu Ala Ala Ala Thr Met Glu Phe Leu325 330 335ttg gat gaa agc gat tac atg aaa gaa gca aag ctt cgt tac gag aaa 1056Leu Asp Glu Ser Asp Tyr Met Lys Glu Ala Lys Leu Arg Tyr Glu Lys340 345 350gac cca ttg aca aag cca aaa cag gac agg tat gct ctc aga acc tcc 1104Asp Pro Leu Thr Lys Pro Lys Gln Asp Arg Tyr Ala Leu Arg Thr Ser355 360 365cct caa tgg ctc gga cct caa att gaa gtt att cgc atg gca aca cat 1152Pro Gln Trp Leu Gly Pro Gln Ile Glu Val Ile Arg Met Ala Thr His370 375 380tcg att gag agg gaa atc aac tct gtg aac gac aac ccg ttg atc gat 1200Ser Ile Glu Arg Glu Ile Asn Ser Val Asn Asp Asn Pro Leu Ile Asp385 390 395 400
gtt gct cga gat att gct ctc cat gga ggc aat ttc cag ggt acg cca 1248Val Ala Arg Asp Ile Ala Leu His Gly Gly Asn Phe Gln Gly Thr Pro405 410 415atc ggt gtc tcc atg gac aat ctc cgc att gcc att gca gca att ggg 1296Ile Gly Val Ser Met Asp Asn Leu Arg Ile Ala Ile Ala Ala Ile Gly420 425 430aag ctc atg ttt gct cag ttc tct gaa ctc gta tgt gat tac tac aac 1344Lys Leu Met Phe Ala Gln Phe Ser Glu Leu Val Cys Asp Tyr Tyr Ash435 440 445aat ggg ttg cct tcc agt ttg agt ggc ggg cct aat ccc agc ttg gat 1392Asn Gly Leu Pro Ser Ser Leu Ser Gly Gly Pro Asn Pro Ser Leu Asp450 455 460tat ggc ttc aaa ggg gca gag ata gcc atg gct tcc tac tgc tct gag 1440Tyr Gly Phe Lys Gly Ala Glu Ile Ala Met Ala Ser Tyr Cys Ser Glu465 470 475 480ctt cag tac ctg gcc aac cct gtc aca acg cat gta cag agt gct gaa 1488Leu Gln Tyr Leu Ala Asn Pro Val Thr Thr His Val Gln Ser Ala Glu485 490 495caa cac aat caa gac gta aat tct ctt ggt ttg att tca gct agg aag 1536Gln His Asn Gln Asp Val Asn Ser Leu Gly Leu Ile Ser Ala Arg Lys500 505 510agt gca gaa gcc att gag atc cta aag ctc atg gtt gcc aca tat atg 1584Ser Ala Glu Ala Ile Glu Ile Leu Lys Leu Met Val Ala Thr Tyr Met515 520 525gtg gga ctt tgc cag gca att gat ctg aga cat ttg gag gag aac atg 1632Val Gly Leu Cys Gln Ala Ile Asp Leu Arg His Leu Glu Glu Asn Met530 535 540aag gag gtt gtt aaa cat gtc gtc ctc cag gtt gtg agg aag acc ctt 1680Lys Glu Val Val Lys His Val Val Leu Gln Val Val Arg Lys Thr Leu545 550 555 560tac act gcc gaa gat gga tca ttg ctt gaa tct cgt ttc tgc gag aaa 1728Tyr Thr Ala Glu Asp Gly Ser Leu Leu Glu Ser Arg Phe Cys Glu Lys565 570 575gaa ctg ttg cag gtt atc gag cac cag cct gtt ttc aat tat ctc gac 1776Glu Leu Leu Gln Val Ile Glu His Gln Pro Val Phe Asn Tyr Leu Asp580 585 590gac ccc act agt tcc tct tat gcc ctc ttg cct aaa ttg cga gaa gta 1824Asp Pro Thr Ser Ser Ser Tyr Ala Leu Leu Pro Lys Leu Arg Glu Val595 600 605ctt gta gag aaa gca ctc aaa gag cat ccc aag act gaa aat ggc cac 1872Leu Val Glu Lys Ala Leu Lys Glu His Pro Lys Thr Glu Asn Gly His610 615 620gac gag ggc ttc tca att ttc aag cgg att cca gta ttc cta gac gaa 1920Asp Glu Gly Phe Ser Ile Phe Lys Arg Ile Pro Val Phe Leu Asp Glu625 630 635 640ctg aaa gca agg ctg gga gaa gag gta gtg aaa gcg cgg gat aga ttc 1968
Leu Lys Ala Arg Leu Gly Glu Glu Val Val Lys Ala Arg Asp Arg Phe645 650 655gag aag gga gat ttt ccg atg cca aac aga atc aag aaa tgc agg act 2016Glu Lys Gly Asp Phe Pro Met Pro Asn Arg Ile Lys Lys Cys Arg Thr660 665 670tat ccg ata tat cgg ttt gtg agg act gaa gtg ggg act acg ctt ctc 2064Tyr Pro Ile Tyr Arg Phe Val Arg Thr Glu Val Gly Thr Thr Leu Leu675 680 685act gga gca aag atg gtg agc cca ggt gaa gat ata gag aag gtt tat 2112Thr Gly Ala Lys Met Val Ser Pro Gly Glu Asp Ile Glu Lys Val Tyr690 695 700gag gcc atc aat gga ggc aaa ttg gga gat gtg gtg gtg aag tgc ttg 2160Glu Ala Ile Asn Gly Gly Lys Leu Gly Asp Val Val Val Lys Cys Leu705 710 715 720aca gat tgg aaa ggg tgt gca ggt cca ttt aca cca agg cct ccg acg 2208Thr Asp Trp Lys Gly Cys Ala Gly Pro Phe Thr Pro Arg Pro Pro Thr725 730 735gta ccg ttt tct cca atg cag tgt aat cct gag tat tgg ggt tgg ttt 2256Val Pro Phe Ser Pro Met Gln Cys Asn Pro Glu Tyr Trp Gly Trp Phe740 745 750gag att gag agt gta agg tct cct tct caa gtt cca cat gct cca att 2304Glu Ile Glu Ser Val Arg Ser Pro Ser Gln Val Pro His Ala Pro Ile755 760 765ggg gga gga acc ttt tga 2322Gly Gly Gly Thr Phe770<210>2<211>773<212>PRT<213>白桦种(Betula platyphylla Suk.)<400>2Met Ala Thr Glu Gly Gly Leu Ser Ala Arg Gln Tyr Arg Asn Ala Arg1 5 10 15Pro Pro Thr Leu Pro Leu Pro Lys Asp Ser Val Asn Leu Ile Cys Val20 25 30Ser Glu Asn Ser His Leu Ser Ala Pro Leu His Trp Lys Lys Ala Ala35 40 45Glu Ala Leu Gln Cys Thr His Phe Asp Glu Val Cys Arg Met Val Ser50 55 60Gln Phe Ala Lys Val Gln Ser Val Asp Ile Gln Gly Thr Ser Leu Thr65 70 75 80Val Ala Gln Val Thr Ala Ile Ala Arg Arg Ser Glu Val Lys Val Asn85 90 95Leu Asp Glu Ala Ala Ala Arg Glu Arg Val Ala Lys Ser Ala Asn Trp
100 105 110Val Ala Glu Asn Ile Ser Arg Gly Thr Asp Thr Tyr Gly Val Thr Thr115 120 125Gly Phe Gly Ala Thr Ser His Arg Arg Thr Asn Lys Thr Val Asp Leu130 135 140Gln Thr Glu Leu Ile Arg Phe Leu Asn Ala Gly Val Ile Gly Lys Glu145 150 155 160Ser Leu Pro Ser Thr Tyr Ser Lys Val Ala Met Leu Val Arg Thr Asn165 170 175Glu His Thr His Ala Arg Gly Phe Ser Gly Ile Arg Trp Glu Ile Leu180 185 190Glu Ala Met Ala Asn Leu Met Asn Lys Asn Leu Ile Pro Lys Leu Pro195 200 205Leu Arg Gly Thr Ile Thr Ala Ser Gly Asp Leu Val Pro Leu Ser Tyr210 215 220Ile Ala Gly Leu Leu Thr Gly Arg His Asn Ser Lys Val Val Thr Pro225 230 235 240Glu Asp Glu Glu Ile Thr Ala Leu Glu Ala Leu Lys Arg Ala Gly Ile245 250 255Pro Ala Pro Phe Glu Leu Gln Ala Lys Glu Gly Leu Ala Leu Val Asn260 265 270Gly Thr Ala Val Gly Ser Ala Val Ala Ala Thr Val Cys Phe Asp Ala275 280 285Asn Ile Leu Ala Leu Phe Ala Glu Ile Leu Ser Ala Leu Phe Cys Glu290 295 300Val Met Leu Gly Asn Ala Glu Phe Thr Asp Pro Leu Thr His Glu Leu305 310 315 320Lys His His Pro Gly Gln Ile Glu Ala Ala Ala Thr Met Glu Phe Leu325 330 335Leu Asp Glu Ser Asp Tyr Met Lys Glu Ala Lys Leu Arg Tyr Glu Lys340 345 350Asp Pro Leu Thr Lys Pro Lys Gln Asp Arg Tyr Ala Leu Arg Thr Ser355 360 365Pro Gln Trp Leu Gly Pro Gln Ile Glu Val Ile Arg Met Ala Thr His370 375 380Ser Ile Glu Arg Glu Ile Asn Ser Val Asn Asp Asn Pro Leu Ile Asp385 390 395 400Val Ala Arg Asp Ile Ala Leu His Gly Gly Asn Phe Gln Gly Thr Pro405 410 415Ile Gly Val Ser Met Asp Asn Leu Arg Ile Ala Ile Ala Ala Ile Gly420 425 430Lys Leu Met Phe Ala Gln Phe Ser Glu Leu Val Cys Asp Tyr Tyr Asn435 440 445Asn Gly Leu Pro Ser Ser Leu Ser Gly Gly Pro Asn Pro Ser Leu Asp450 455 460Tyr Gly Phe Lys Gly Ala Glu Ile Ala Met Ala Ser Tyr Cys Ser Glu465 470 475 480
Leu Gln Tyr Leu Ala Asn Pro Val Thr Thr His Val Gln Ser Ala Glu485 490 495Gln His Asn Gln Asp Val Asn Ser Leu Gly Leu Ile Ser Ala Arg Lys500 505 510Ser Ala Glu Ala Ile Glu Ile Leu Lys Leu Met Val Ala Thr Tyr Met515 520 525Val Gly Leu Cys Gln Ala Ile Asp Leu Arg His Leu Glu Glu Asn Met530 535 540Lys Glu Val Val Lys His Val Val Leu Gln Val Val Arg Lys Thr Leu545 550 555 560Tyr Thr Ala Glu Asp Gly Ser Leu Leu Glu Ser Arg Phe Cys Glu Lys565 570 575Glu Leu Leu Gln Val Ile Glu His Gln Pro Val Phe Asn Tyr Leu Asp580 585 590Asp Pro Thr Ser Ser Ser Tyr Ala Leu Leu Pro Lys Leu Arg Glu Val595 600 605Leu Val Glu Lys Ala Leu Lys Glu His Pro Lys Thr Glu Asn Gly His610 615 620Asp Glu Gly Phe Ser Ile Phe Lys Arg Ile Pro Val Phe Leu Asp Glu625 630 635 640Leu Lys Ala Arg Leu Gly Glu Glu Val Val Lys Ala Arg Asp Arg Phe645 650 655Glu Lys Gly Asp Phe Pro Met Pro Asn Arg Ile Lys Lys Cys Arg Thr660 665 670Tyr Pro Ile Tyr Arg Phe Val Arg Thr Glu Val Gly Thr Thr Leu Leu675 680 685Thr Gly Ala Lys Met Val Ser Pro Gly Glu Asp Ile Glu Lys Val Tyr690 695 700Glu Ala Ile Asn Gly Gly Lys Leu Gly Asp Val Val Val Lys Cys Leu705 710 715 720Thr Asp Trp Lys Gly Cys Ala Gly Pro Phe Thr Pro Arg Pro Pro Thr725 730 735Val Pro Phe Ser Pro Met Gln Cys Asn Pro Glu Tyr Trp Gly Trp Phe740 745 750Glu Ile Glu Ser Val Arg Ser Pro Ser Gln Val Pro His Ala Pro Ile755 760 765Gly Gly Gly Thr Phe770<210>3<211>894<212>DNA<213>白桦种(Betula platyphylla Suk.)<220>
<223>白桦苯丙氨酸解氨酶PAL的部分cDNA片段
<400>3tgttaccacc ggcttcggag ccacgtcaca ccggcgcacc aacaaaactg tggacctcca 60gacagagctt ataaggttcc ttaatgccgg agtgatcggg aaagagagtc ttccgtccac 120ttattcaaag gtggcaatgc tcgttcgcac gaacgaacac actcatgcaa ggggtttttc 180gggcattcga tgggagatac tggaagccat ggccaatctg atgaacaaga atttgatccc 240taaattgcct ctaaggggga ctatcactgc ctctggggat cttgtaccac tctcttatat 300tgccggtttg ctaactggcc gccataattc caaagtggtc acccctgaag atgaagaaat 360cacagccttg gaagctctca agagggcagg aattccagct cctttcgagc ttcaagcaaa 420ggagggtcta gctcttgtaa acgggacagc tgttggctcg gctgtggctg caacggtttg 480ttttgatgcc aacattttgg ctctttttgc agaaattctc tctgctctgt tttgtgaagt 540catgcttgga aatgccgagt tcacagaccc actgacgcat gagctcaagc accaccctgg 600gcagattgag gcagccgcta ccatggagtt ccttttggat gaaagcgatt acatgaaaga 660agcaaagctt cgttacgaga aagacccatt gacaaagcca aaacaggaca ggtatgctct 720cagaacctcc cctcaatggc tcggacctca aattgaagtt attcgcatgg caacacattc 780gattgagagg gaaatcaact ctgtgaacga caacccgttg atcgatgttg ctcgagatat 840tgctctccat ggaggcaatt tccagggtac gccaatcggt gtctccatgg acaa 894<210>4<211>18<212>DNA<213>人工序列<220>
<221>misc_feature<223>k＝g或t；d＝a或g或t；y＝t或c；w＝a或t<223>(3，5，7，10，13，15)<220>
<223>RT-PCR反转录合成cDNA的简并引物P1<400>4tgkcdcyacy ggdtwcgg 18<210>5<211>20<212>DNA<213>人工序列<220>
<221>misc_feature<223>d＝a或g或t；y＝t或c；w＝a或t；v＝a或g或c；m＝a或c<223>(2，6，10，15，18)<220>
<223>RT-PCR反转录合成cDNA的简并引物P2
<400>5tyctcvatgd aaacwccmat 20<210>6<211>22<212>DNA<213>人工序列<220>
<223>根据已得到的部分cDNA片段而设计的5’RACE特异引物P3<400>6gggggactat cactgcctct gg 22<210>7<211>20<212>DNA<213>人工序列<220>
<223>根据已得到的部分cDNA片段而设计的5’RACE特异引物P4<400>7atgcttggaa atgccgagtt 20<210>8<211>22<212>DNA<213>人工序列<220>
<223>根据已得到的部分cDNA片段而设计的3’RACE特异引物P5<400>8gctagaccct cctttgcttg aa 22<210>9<211>21<212>DNA<213>人工序列<220>
<223>根据已得到的部分cDNA片段而设计的3’RACE特异引物P6<400>9gcttccaagg ctgtgatttc t 2权利要求
1.白桦苯丙氨酸解氨酶cDNA序列，其特征是白桦苯丙氨酸解氨酶基因的cDNA序列如下所示ATGGCAACCGAGGGAGGACTTTCGGCCCGCCAATACAGAAATGCCCGTCCTCCAACCCTCCCTCTCCCAAAAGACAGCGTAAACCTCATTTGCGTATCAGAAAATTCTCACTTGTCAGCCCCACTGCACTGGAAGAAAGCTGCAGAGGCCCTCCAGTGCACCCATTTTGATGAAGTCTGCCGCATGGTTTCCCAATTTGCAAAGGTTCAAAGCGTTGATATCCAGGGAACCTCCCTCACGGTTGCTCAGGTCACTGCAATTGCACGCCGCAGTGAAGTAAAAGTTAATCTTGATGAGGCTGCGGCGCGTGAACGTGTGGCCAAAAGTGCCAACTGGGTTGCTGAAAATATCTCCCGTGGAACAGACACGTATGGTGTTACCACCGGCTTCGGAGCCACGTCACACCGGCGCACCAACAAAACTGTGGACCTCCAGACAGAGCTTATAAGGTTCCTTAATGCCGGAGTGATCGGGAAAGAGAGTCTTCCGTCCACTTATTCAAAGGTGGCAATGCTCGTTCGCACGAACGAACACACTCATGCAAGGGGTTTTTCGGGCATTCGATGGGAGATACTGGAAGCCATGGCCAATCTGATGAACAAGAATTTGATCCCTAAATTGCCTCTAAGGGGGACTATCACTGCCTCTGGGGATCTTGTACCACTCTCTTATATTGCCGGTTTGCTAACTGGCCGCCATAATTCCAAAGTGGTCACCCCTGAAGATGAAGAAATCACAGCCTTGGAAGCTCTCAAGAGGGCAGGAATTCCAGCTCCTTTCGAGCTTCAAGCAAAGGAGGGTCTAGCTCTTGTAAACGGGACAGCTGTTGGCTCGGCTGTGGCTGCAACGGTTTGTTTTGATGCCAACATTTTGGCTCTTTTTGCAGAAATTCTCTCTGCTCTGTTTTGTGAAGTCATGCTTGGAAATGCCGAGTTCACAGACCCACTGACGCATGAGCTCAAGCACCACCCTGGGCAGATTGAGGCAGCCGCTACCATGGAGTTCCTTTTGGATGAAAGCGATTACATGAAAGAAGCAAAGCTTCGTTACGAGAAAGACCCATTGACAAAGCCAAAACAGGACAGGTATGCTCTCAGAACCTCCCCTCAATGGCTCGGACCTCAAATTGAAGTTATTCGCATGGCAACACATTCGATTGAGAGGGAAATCAACTCTGTGAACGACAACCCGTTGATCGATGTTGCTCGAGATATTGCTCTCCATGGAGGCAATTTCCAGGGTACGCCAATCGGTGTCTCCATGGACAATCTCCGCATTGCCATTGCAGCAATTGGGAAGCTCATGTTTGCTCAGTTCTCTGAACTCGTATGTGATTACTACAACAATGGGTTGCCTTCCAGTTTGAGTGGCGGGCCTAATCCCAGCTTGGATTATGGCTTCAAAGGGGCAGAGATAGCCATGGCTTCCTACTGCTCTGAGCTTCAGTACCTGGCCAACCCTGTCACAACGCATGTACAGAGTGCTGAACAACACAATCAAGACGTAAATTCTCTTGGTTTGATTTCAGCTAGGAAGAGTGCAGAAGCCATTGAGATCCTAAAGCTCATGGTTGCCACATATATGGTGGGACTTTGCCAGGCAATTGATCTGAGACATTTGGAGGAGAACATGAAGGAGGTTGTTAAACATGTCGTCCTCCAGGTTGTGAGGAAGACCCTTTACACTGCCGAAGATGGATCATTGCTTGAATCTCGTTTCTGCGAGAAAGAACTGTTGCAGGTTATCGAGCACCAGCCTGTTTTCAATTATCTCGACGACCCCACTAGTTCCTCTTATGCCCTCTTGCCTAAATTGCGAGAAGTACTTGTAGAGAAAGCACTCAAAGAGCATCCCAAGACTGAAAATGGCCACGACGAGGGCTTCTCAATTTTCAAGCGGATTCCAGTATTCCTAGACGAACTGAAAGCAAGGCTGGGAGAAGAGGTAGTGAAAGCGCGGGATAGATTCGAGAAGGGAGATTTTCCGATGCCAAACAGAATCAAGAAATGCAGGACTTATCCGATATATCGGTTTGTGAGGACTGAAGTGGGGACTACGCTTCTCACTGGAGCAAAGATGGTGAGCCCAGGTGAAGATATAGAGAAGGTTTATGAGGCCATCAATGGAGGCAAATTGGGAGATGTGGTGGTGAAGTGCTTGACAGATTGGAAAGGGTGTGCAGGTCCATTTACACCAAGGCCTCCGACGGTACCGTTTTCTCCAATGCAGTGTAATCCTGAGTATTGGGGTTGGTTTGAGATTGAGAGTGTAAGGTCTCCTTCTCAAGTTCCACATGCTCCAATTGGGGGAGGAACCTTTTGA。
2.白桦苯丙氨酸解氨酶，其特征是白桦苯丙氨酸解氨酶基因cDNA编码的氨基酸序列如下所示MetAlaThrGluGlyGlyLeuSerAlaArgGlnTyrArgAsnAlaArgProProThrLeuProLeuProLysAspSerValAsnLeuIleCysValSerGluAsnSerHisLeuSerAlaProLeuHisTrpLysLysAlaAlaGluAlaLeuGlnCysThrHisPheAspGluValCysArgMetValSerGlnPheAlaLysValGlnSerValAspIleGlnGlyThrSerLeuThrValAlaGlnValThrAlaIleAlaArgArgSerGluValLysValAsnLeuAspGluAlaAlaAlaArgGluArgValAlaLysSerAlaAsnTrpValAlaGluAsnIleSerArgGlyThrAspThrTyrGlyValThrThrGlyPheGlyAlaThrSerHisArgArgThrAsnLysThrValAspLeuGlnThrGluLeuIleArgPheLeuAsnAlaGlyValIleGlyLysGluSerLeuProSerThrTyrSerLysValAlaMetLeuValArgThrAsnGluHisThrHisAlaArgGlyPheSerGlyIleArgTrpGluIleLeuGluAlaMetAlaAsnLeuMetAsnLysAsnLeuIleProLysLeuProLeuArgGlyThrIleThrAlaSerGlyAspLeuValProLeuSerTyrIleAlaGlyLeuLeuThrGlyArgHisAsnSerLysValValThrProGluAspGluGluIleThrAlaLeuGluAlaLeuLysArgAlaGlyIleProAlaProPheGluLeuGlnAlaLysGluGlyLeuAlaLeuValAsnGlyThrAlaValGlySerAlaValAlaAlaThrValCysPheAspAlaAsnIleLeuAlaLeuPheAlaGluIleLeuSerAlaLeuPheCysGluValMetLeuGlyAsnAlaGluPheThrAspProLeuThrHisGluLeuLysHisHisProGlyGlnIleGluAlaAlaAlaThrMetGluPheLeuLeuAspGluSerAspTyrMetLysGluAlaLysLeuArgTyrGluLysAspProLeuThrLysProLysGlnAspArgTyrAlaLeuArgThrSerProGlnTrpLeuGlyProGlnIleGluValIleArgMetAlaThrHisSerIleGluArgGluIleAsnSerValAsnAspAsnProLeuIleAspValAlaArgAspIleAlaLeuHisGlyGlyAsnPheGlnGlyThrProIleGlyValSerMetAspAsnLeuArgIleAlaIleAlaAlaIleGlyLysLeuMetPheAlaGlnPheSerGluLeuValCysAspTyrTyrAsnAsnGlyLeuProSerSerLeuSerGlyGlyProAsnProSerLeuAspTyrGlyPheLysGlyAlaGluIleAlaMetAlaSerTyrCysSerGluLeuGlnTyrLeuAlaAsnProValThrThrHisValGlnSerAlaGluGlnHisAsnGlnAspValAsnSerLeuGlyLeuIleSerAlaArgLysSerAlaGluAlaIleGluIleLeuLysLeuMetValAlaThrTyrMetValGlyLeuCysGlnAlaIleAspLeuArgHisLeuGluGluAsnMetLysGluValValLysHisValValLeuGlnValValArgLysThrLeuTyrThrAlaGluAspGlySerLeuLeuGluSerArgPheCysGluLysGluLeuLeuGlnValIleGluHisGlnProValPheAsnTyrLeuAspAspProThrSerSerSerTyrAlaLeuLeuProLysLeuArgGluValLeuValGluLysAlaLeuLysGluHisProLysThrGluAsnGlyHisAspGluGlyPheSerIlePheLysArgIleProValPheLeuAspGluLeuLysAlaArgLeuGlyGluGluValValLysAlaArgAspArgPheGluLysGlyAspPheProMetProAsnArgIleLysLysCysArgThrTyrProIleTyrArgPheValArgThrGluValGlyThrThrLeuLeuThrGlyAlaLysMetValSerProGlyGluAspIleGluLysValTyrGluAlaIleAsnGlyGlyLysLeuGlyAspValValValLysCysLeuThrAspTrpLysGlyCysAlaGlyProPheThrProArgProProThrValProPheSerProMetGlnCysAsnProGluTyrTrpGlyTrpPheGluIleGluSerValArgSerProSerGlnValProHisAlaProIleGlyGlyGlyThrPhe。
3.白桦苯丙氨酸解氨酶基因制备过程中设计的引物，其特征是RT-PCR反转录合成cDNA的简并引物基因序列为P15’-TGKCDCYACYGGDTWCGG-3’P25’-TYCTCVATGDAAACWCCMAT-3’。
4.白桦苯丙氨酸解氨酶基因制备过程中设计的引物，其特征在于设计的RACE特异引物的基因序列为5’RACE特异引物P35’-GGGGGACTATCACTGCCTCTGG-3’5’RACE特异引物P45’-ATGCTTGGAAATGCCGAGTT-3’3’RACE特异引物P55’-GCTAGACCCTCCTTTGCTTGAA-3’3’RACE特异引物P65’-GCTTCCAAGGCTGTGATTTCT-3’。
全文摘要
白桦苯丙氨酸解氨酶cDNA序列及其编码的氨基酸序列和制备过程中设计的引物，它涉及一种苯丙氨酸解氨酶cDNA序列及其编码的氨基酸序列和制备过程中设计的引物。白桦苯丙氨酸解氨酶在降低植物中木质素的含量或改变木质素的组分及控制多种物质的代谢方面都有着重要的意义，本发明利用RT－PCR和RACE技术获得PAL基因的cDNA序列。白桦苯丙氨酸解氨酶基因的cDNA序列全长为2322bp，其中T、C、G、A分别为572bp、544bp、590bp、616bp。白桦苯丙氨酸解氨酶基因的cDNA序列编码白桦苯丙氨酸解氨酶PAL773个氨基酸。本发明为制备白桦苯丙氨酸解氨酶设计了P1～P6六个引物。本发明为更好的应用、推广白桦苯丙氨酸解氨酶奠定了物质基础。
文档编号C07H21/00GK1807642SQ20061000971
公开日2006年7月26日 申请日期2006年2月17日 优先权日2006年2月17日
发明者杨传平, 刘雪梅, 王秋玉, 梁慧燕, 宋福南 申请人:东北林业大学, 杨传平, 刘雪梅