CN111154795A - 于特定植物组织中沉默八氢蕃茄红素合成酶基因表现之dna构筑体及其方法 - Google Patents
于特定植物组织中沉默八氢蕃茄红素合成酶基因表现之dna构筑体及其方法 Download PDFInfo
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Abstract
本发明揭示一种可以沉默或降低八氢番茄红素合成酶基因于特定组织表现的方法与DNA卡匣，此DNA卡匣包含一核苷酸序列，该核苷酸序列转录后的RNA片段可用以调控特定组织中八氢番茄红素合成酶基因之讯息RNA(messenger RNA,mRNA)的含量，以达到调控花色、气味、或其他需要类胡萝卜素产物之植物生理机制。
Description
【技术领域】
本发明系关于沉默八氢蕃茄红素合成酶(phytoene synthase,PSY)基因表现,以控制文心兰花色领域及用于调节PSY基因表现之核苷酸片段组合物及方法，且更特定言之系关于藉由基因转殖一段DNA卡匣而在特定组织中产生RNA干扰片段,，因此下调PSY基因于植株特定组织中的表现。
【先前技术】
文心蘭(Oncidium)是台湾主要的外销切花之一，台湾文心蘭切花的生产有85％是外销至其他国家，而日本是台湾文心蘭切花主要的外销市场，台湾文心蘭切花亦占日本文心蘭切花总输入量的第一位。然而，品种单一是目前切花文心兰产业中很严重的困难之一，因此本发明的愿景为创造多样化、具差异性的品种，例如开发多样化花色以满足不同外销国家及外销市场之需求，以开创新的广大市场。台湾文心兰切花以黄花文心兰为主，目前主流品系皆是由南西文心兰(Oncidesa Gower Ramsey)经由组织培养变异而得。然而，南西文心兰因其来自远缘交配(distant hybridization)，经由远缘交配所获得的第一代杂交种(hybrid)之基因组发生重大改变，同源染色体在配对过程中不平衡甚至发生部分缺失(chromosome deletion)，导致南西文心兰无法进行有性繁殖，均需以组织培养的方式进行幼苗生产，也难以用传统杂交方式产生多样化的新品系，为了突破此现象，本发明利用RNA干扰技术藉由基因转殖之方法，开发不同花色品系文心兰。
颜色是植物性商品中重要的特性之一，构成高等植物的花色主要由甜菜素(betalains)、类黄酮(flavonoids)与类胡萝卜素(carotenoids)等色素产生而成。南西品系文心兰花朵之黄色唇瓣部位是由类胡萝卜素所组成，主要成分为violaxanthin(堇菜黄质)、9-cis-violaxanthin、neoxanthin(新黄素)以及少量之lutein，而花朵上方之红褐色斑点是由花青素(anthocyanins)所组成，主要成分为cyanidin(矢车菊色素)、delphinidin(花翠素)、及少量之peonidin(芍药色素)。其中植物的类胡萝卜素生合成机制中，八氢蕃茄红素合成酶(phytoene synthase,PSY)为关键速率决定酵素，可将两个geranylgeranyldiphosphate合成为一个phytoene(八氢蕃茄红素)，是类胡萝卜素生合成路径中最上游之化合物，后续phytoene再经由phytoene desaturase、ζ-carotene desaturase去饱和化形成lycopene(茄红素)，并经由下游酵素进一步催化形成各种不同的产物。因此八氢蕃茄红素合成酶是类胡萝卜素生成途径中重要的酶之一，经由抑制八氢蕃茄红素合成酶基因(OgPSY)之表现，可抑制相关类胡萝卜素生成，进而调控花色。
RNA干扰原理或RNAi最初由Fire等人所发现,彼等以双股RNA(dsRNA)注射进入线虫(Caenorhabidites elegan)体中,发现虫体中某些基因表现会被阻断。在植物方面，利用反义股RNA改变花色生成途径而创造出不同颜色花朵之案例，如利用反义股RNA抑制矮牵牛花(Petunia hybrid)中chalcone synthase(PhCHS)的基因表达而产生白色矮牵牛(Vander Krol et al.,1990)；如利用反义股RNA抑制非洲菊(Gerbera hybrida)之chalconesynthase(GCHS)基因表达而创造出橙色非洲菊。目前为止，利用RNAi干扰技术于调整类胡萝卜素以改变花色之案例仅出现于基因转殖菊花中，藉由减弱(knockdown)CCD4使白色菊花转变成鲜明黄色之花色。另外，由于类胡萝卜素为植物生长之重要元素，若严重缺乏则会使植物面临许多生长与发育的问题，例如光合作用，因此选择适当之启动子来驱动调控类胡萝卜素色素生成机制中的催化酵素之基因，如phytoene desaturase、Lycopeneε-cyclase、或Lycopeneβ-cyclase等酵素之基因，能使特定基因于特定组织中专一性表达，仅达到改变花色之目的，但仍然维持植株正常生长之生理机能,此为技术之重要关键。
本发明之目的即在于利用特定启动子产生干扰性RNA片段，以减弱或抑制文心兰花朵中八氢蕃茄红素合成酶基因表现，因此减少类胡萝卜素之生成,进而达到改变花色之目的，此等利用干扰性RNA技术于观赏植物上的应用，与前述案例用于调控不同的基因与植物种类完全不同。
【发明内容】
本发明较佳实施例之主要目的系提供一种可以沉默或降低文心兰八氢蕃茄红素合成酶基因表现的方法，其提供一种可以沉默文心兰八氢蕃茄红素合成酶基因的DNA卡匣，此DNA卡匣为一核苷酸序列，该核苷酸序列转录后的RNA片段可用以调控文心兰中八氢蕃茄红素合成酶基因之讯息RNA(messenger RNA,mRNA)的含量，因此达到调控花色、气味、或其他需要类胡萝卜素产物之植物生理机制。
为了达成上述目的，本发明且该核苷酸序列所转录产生的mRNA系以八氢蕃茄红素合成酶mRNA为互补结合的目标，因为其与八氢蕃茄红素合成酶mRNA结合成双股的RNA后，干扰八氢蕃茄红素合成酶基因之mRNA，使之无法进一步转译成酶，因此，类胡萝卜素的生合成反应受到影响。该核苷酸序列为一启动子与一段可转录出干扰性RNA片段的DNA卡匣。该启动子包含SEQ ID NO：1之核苷酸序列。
本发明较佳实施例提出一DNA卡匣，此卡匣包含一个启动子(P)，之后在3’端接续一段SEQ ID NO：3之DNA片段，再接续一段含有GUS基因的内含子片段(intronGUS核苷酸序列)，及一段方向倒置的SEQ ID NO：3之DNA片段,最后接上一段NOS基因的终止片段(T-NOS核苷酸序列)。其中上述之重组质体或载体，包括但不限于特定之启动子、内含子、或终结片段，或其他适用本发明之重组质体或载体。其中，一段核苷酸序列，置于一段启动子Pchrc之后,此一段核苷酸序列,包括两小段各约150个核苷酸之SEQ ID NO：3的核苷酸片段,两者呈反向排列,中间夹着一段GUS-intron核苷酸片段，此一核苷酸序列片段在启动子Pchrc的驱动之下可以转录产生发夹结构的RNA分子。此RNA片段具有与目标mRNA，如八氢蕃茄红素合成酶基因之mRNA，因核苷酸序列互补而形成双股RNA结构。
编码SEQ ID NO：3为编码SEQ ID NO：2中自第51个核苷酸起之150个核苷酸长度之核苷酸序列。
本发明较佳实施例提供一核苷酸序列，包含编码SEQ ID NO：1、SEQ ID NO：2、SEQID NO：3；及与SEQ ID NO：1、SEQ ID NO：2、SEQ ID NO：3之序列互补的核苷酸序列。
本发明较佳实施例之该核苷酸序列提供于一载体或一穿梭载体。
本发明较佳实施例之该核苷酸序列提供于一拟原球体
本发明较佳实施例之该载体可经转殖导入至一植物细胞或拟原球体(PLB,Protocorm-like body)。
本发明较佳实施例之该核苷酸序列提供于一植物组织。
为了达成上述目的，本发明较佳实施例之文心兰八氢蕃茄红素合成酶基因之调降及沉默方法包含：提供至少一DNA卡匣,此DNA卡匣的核苷酸序列包含一特定启动子与一段可转录成发夹型干扰RNA片段的特定核苷酸片段；将该核苷酸序列导入至一植物细胞(或PLB)，以获得一转殖植物细胞(或PLB)；及利用该已转殖植物细胞再生发育成为植株,并达到改变植株花色之目的。
本发明较佳实施例之核苷酸序列经由基因选殖技术(gene cloning)而获得。
本发明较佳实施例之该已转殖植物细胞用以改变一植物体内之八氢蕃茄红素合成酶表现量。
本发明较佳实施例利用该已转殖植物细胞再生制造一已转殖植物。
本发明较佳实施例之该植物细胞选自一兰科植物细胞。
【图式简单说明】
图1、沉默八氢蕃茄红素合成酶基因的DNA卡匣构筑示意图。
图2、沉默八氢蕃茄红素合成酶基因的DNA卡匣构筑图。
LB/RB：T-DNA的左/右边界、P：启动子
图3、花朵照片，由左至右分别显示为野生型(WF)Oncidesa Honey Angel的花朵、已转殖植物Oncidesa MF-1、与已转殖植物Oncidesa MF-5。
图4、花朵组织中类胡萝卜素含量图，黑色图示为野生型(WF)Oncidesa HoneyAngel、灰色图示为已转殖植物Oncidesa MF-1、与白色图式为已转殖植物Oncidesa MF-5。横轴所显示之四种类胡萝卜素种类为9-顺式菫菜黄质(9-cis-violaxanthin)、新黄素(neoxanthin)、菫菜黄质(violaxanthin)及β-胡萝卜素(β-carotene)。
图5，利用半定量实时聚合酶连锁反应(Semi-quantitative real-time PCRanalysis)分析野生型(WF)Oncidesa Honey Angel(a)、已转殖植物Oncidesa MF-1(b)、与已转殖植物Oncidesa MF-5(c)植株中各部位之八氢蕃茄红素聚合酶调控基因(OgPSY)表现量。简称分别为：花朵(F)、HPTII(hygromycin phosphotransferase II gene)。
图6，利用北方墨点法(Northern blot analysis)分析各植物组织之小RNA分子(small RNA,SiRNA)之表现量，由左而右分别为已转殖植物Oncidesa MF-5花朵组织、已转殖植物Oncidesa MF-1花朵组织、已转殖植物Oncidesa MF-5叶组织，与野生种(WF)Oncidesa Honey Angel花朵组织。
【实施方式】
为了充分了解本发明，于下文将举例较佳实施例并配合所附图式作详细说明，且其并非用以限定本发明。
本发明较佳实施例之文心兰八氢蕃茄红素合成酶基因适用于提供其基因产物(gene product)或其编码蛋白质，但其并非用以限定本发明之范围。再者，本发明较佳实施例之文心兰八氢蕃茄红素合成酶基因及其调控方法适用于以各种基因工程进行基因转殖(gene transformation)于各种植物细胞或拟原球体，但其并非用以限定本发明之范围。
举例而言，本发明较佳实施例所采用之文心兰为黄花品种(如：柠檬绿文心兰；Oncidesa Honey Angel)，但本发明适用其他各种文心兰品种，其并非用以限定本发明之范围。
本发明较佳实施例之一种沉默文心兰八氢蕃茄红素合成酶基因表现之技术，系一种产生干扰性RNA的DNA卡匣，其结构示意图如第2图揭示，建构一可转录出嵌合发夹RNA之转移DNA(T－DNA)卡匣，然后组合于载体中，此DNA卡匣包含一个启动子，在其3’端接续包含SEQ ID NO：3之正向置放的核苷酸序列，与intronGUS内含子片段(intronGUS核苷酸序列)，再接续一段SEQ ID NO：3之方向倒置的核苷酸序列，再接续NOS基因之终止片段之T-NOS核苷酸序列。其中上述之重组质体或载体，包括但不限于特定之启动子或内含子片段，或其他适用本发明之重组质体或载体。
1.自柠檬绿文心兰(Oncidesa Honey Angel)钓取启动子。其方法是将柠檬绿文心兰的总RNA利用Oligotex mRNA Kit(Qiagen)纯化出Poly(A)+RNA。根据Clontech厂商提供的操作方法，利用快速扩增技术(Rapid Amplification of cDNA Ends,RACE),自cDNA之5’端与3’端末端扩增出全长的DNA,取得柠檬绿文心兰chromoplast specific carotenoidassociated protein(CHRC)基因之全长cDNA核苷酸序列。
本发明所使用之启动子选殖方式，为利用分子生物学的基因步移技术(GenomeWalkerUniversal Kit,Clontech)自CHRC cDNA扩增出此基因之启动子片段Pchrc,共1704bp(图1)。
2.RNA干扰目标选择。
对照NCBI数据库中的四种单子叶植物种(水稻，小麦，玉米，蝴蝶兰)的八氢蕃茄红素合成酶核苷酸序列进行同源性分析,发现150bp八氢蕃茄红素合成酶调控基因的cDNA区域(开放阅读框(open reading frame)的+51bp～+200bp)，含有22-bp 100％同源性的保守序列在相应的互补DNA(cDNA)序列中，选择成为RNA干扰之目标核苷酸序列。
3.基因转殖与栽培方法。
将含本发明之DNA卡匣构筑成载体，利用农杆菌携带此载体感染文心兰(Onc.HoneyAngel)的拟原球体(Protocorm-like body,PLB)，并利用含适当浓度抗生素之培养基筛选转殖后的拟原球体，再以出芽(shooting)及生根(rooting)之培养基加以诱导成完整植株，植株以盆栽种植两年,以观察其花色。上述之转殖的方法包括但不限于：农杆菌媒介法、基因重组病毒感染法、跳跃子载体转殖法、基因枪转殖法、电穿孔、显微注射法、花粉管法、脂质体媒介转殖法、超音波媒介转殖法、碳化硅纤维媒介转殖法等为本发明所属技术领域人士熟悉的；兰花组织培养之方法亦为本发明所属技术领域人士熟悉的，故不再赘述。
《实例1：文心兰花色调控》
如图3所示，由左至右分别显示为野生型(WF)Oncidesa Honey Angel的花朵、经植入本发明DNA卡匣核苷酸序列之已转殖株Oncidesa MF-1、与经植入本发明DNA卡匣核苷酸序列之已转殖植株Oncidesa MF-5，可看出MF-1为淡白色花色，MF-5为雪白色花色。
为维持产品的表型稳定，本实例是利用包含30ppm潮霉素(hygromycin)之半浓度量(1/2)的Murashige&Skoog(MS)培养基培养已转殖植株Oncidesa MF-1与Oncidesa MF-5的P0阶段微小的分生组织芽。经过拟原球体分化和植株分蘗生长作用，两株系(line)于培植2年后各产出两千株P1幼苗于温室种植。幼苗生长温度为30℃/25℃(白天/夜晚)，光强度控制在光合光子通量密度(PPFD)约为300μmolm-2s-1之环境。
《实例2：文心兰花朵中类胡萝卜素含量》
如图4所示，已转殖植物Oncidesa MF-1与Oncidesa MF-5之类胡萝卜素都大幅降低，前述两者的9-cis-violaxanthin含量皆为野生型的40％，而Oncidesa MF-1的新黄素(neoxanthin)、菫菜黄质(violaxanthin)及β-胡萝卜素(β-carotene)皆为野生型的30％；呈现纯白色花色之Oncidesa MF-5中，此三种色素的含量皆仅为野生型的1-3％，此结果显示经由转殖入本发明之DNA卡匣核苷酸序列能有效控制类胡萝卜素生成，以达控制花色之效果。
《实例3：文心兰组织中之基因表现量》
图5显示八氢蕃茄红素合成酶基因(OgPSY)表现量，可见野生型文心兰的OgPSY表现量高，已转殖植株Oncidesa MF-1与Oncidesa MF-5看不到条带，显示其表现量明显降低,而野生型依然表现OgPSY。从图6亦可显示已转殖植株Oncidesa MF-1与Oncidesa MF-5之siRNA(小分子的干扰RNA)表现强烈,从基因表现量可以判断结果。此亦显示本发明能成功干扰OgPSY表现,达到控制色素与花色之目的。
序列表
<110> 叶开温(YEH KAI-WUN)
<120> 于特定植物组织中沉默八氢蕃茄红素合成酶基因表现之DNA构筑体及其方法
<150> TW 108132149
<151> 2019-09-06
<160> 3
<170> SIPOSequenceListing 1.0
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<213> Oncidium
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<223> 调节功能序列 putative repeated elements (ATAGAA)
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<223> 调节功能序列 putative repeated elements (ATAGAA)
<220>
<221> misc_feature
<222> (1598)..(1601)
<223> CAAT box
<220>
<221> misc_feature
<222> (1672)..(1678)
<223> TATA box
<400> 1
gtcgtgagat attctcattt gctgctcaca tacattgctt atgacatcta cgtgaaaatt 60
tcaagaaagt agtcaagaaa atagggattg gagatgtgaa gttcatgtgt cagaagatat 120
acatggcggg gaacagtgat gatatgatca ccttcaatcg gttgatgact gatataatta 180
acataaaggt agacgcttat aattggctga ttgaatgaga tgtgacaaag tggagcctac 240
tgaatgacgg tggaatgcat agtggagcaa tgacaaccaa tgcttcagaa tgcttcaatt 300
cagtcttgaa gcgagctaga tgtttgccca ttcaggcatt aataataaca gtttattaca 360
atattatcac aatttttttg aagcgttata agagtgcttc agaacagctt gaggccaacg 420
aatcgacatt cgcacaacgc acattgcgtc aactccaaca gtatggacga gaagttagga 480
attttccttc gccaattaac atcaatagaa gcgagttaca agtgtttgac aaagactcaa 540
gatctttcca agttcagaat attctcctgc tgaaatatgt agttgcactt gtgatagatg 600
tgagatgtat cacatgcctt gtgctcatgc catcaaagct cttgcaagta ggcgtatggt 660
ttataccaca tatgtctcca gatattattc agtggacttc tatagaaata cctacacaga 720
tgaattccac actctacctg atcgctcata ctggccagat tgcaacgaag aaatgagatt 780
gattcagcta ctaccaccac caaacaggaa gcgttcaggc cgaccacgct caaatcgatt 840
tcgtaacacg atggacgagg gtccaagcaa atgtagatga gggatgtcca tttcaatacc 900
tgctttgtaa tattaaacat ctatttggtt aataaattgt tctagtgtga tgttttaata 960
tttttgtaat attgaacatg atgaattttc atttcaattt acttcatctt cgtttctcta 1020
ttttcatata tttctgtcat tttacatgtt ccaaatgaat tgctacttac acattaaact 1080
acatttatgt atatttttca atatttttaa ttgataaaag acatttttat cttcaattcc 1140
atataaaatt ttctcatttc aatgcaaaac ttaaaatatt atggattttc taacctaaaa 1200
ccccgagagc aaaatggaag agactaaaaa aaaaaaatag gaagttagaa atcgacgacc 1260
gtgtcatcga tttcacgaga aatcgattat acgatcgtcg atttcttata aataagtaaa 1320
atcgttttac ctgtcatcga tttctataaa acacttttct attactttcc tgccaacgtg 1380
gcatctgagc tagaatgaaa tcggactatt agagtccgat tatatctatt tttaaatttt 1440
ttttaatgat acttattttg ctaattaaat tattaatcat acttatttta aaaatttgac 1500
ccaaaaaact gcaatccaaa catccaaaat ttttagtcaa ttcgttacct aacaccccta 1560
aatatacaaa aattaaaaca atgcccatcc acacaaacaa taaaaaaatc tcggccctcg 1620
cactcggtaa ctcggtattc ttttaagcac gtaaaaattc ctcataacat ctataaaata 1680
gctccccaac tcccaagttc aaac 1704
<210> 2
<211> 1383
<212> DNA
<213> Oncidium
<400> 2
ctccaattaa ctgtagcgca tcttcttctt cctttccgag ccacaatcga aggattgaat 60
ggcagcttcg cagttgtgct ttgttgggtt tttggaggga ataagaggag gagatcggtt 120
atggaacgcg aagaatgatt gcaggttttc gcagaggaag aagatgagat ggagtttcta 180
ctgtctgttt acgaacttta actatgcttg tgttagtcag gaaccagaga aggatttgaa 240
gtttcctatt tattcgagtt tggttgtgaa tccagtggga gaggtcgcca tttcttcgga 300
gcagaaggtt tatgatgttg ttttgaagca ggcggctctt gtggagcagc agctgaggaa 360
caggacagtt ctggaagaga agacaggaac tacttttttg ttgaacgaag cttatgatcg 420
atgtgggcaa atctgcgagg agtatgctaa gactttttac ttgggaacgc tgctgatgac 480
acctgagagg agaagagcga tctgggcaat ctatgtgtgg tgcaggagaa cagatgaact 540
tgtggatggt cccaacgcgt cccatatcac tccatcagcc ctcgatcggt gggaggcaag 600
gctggaggat ctgtttgctg gtcgaccata tgacatgctt gatgcttccc tctcagatac 660
tgttgttaat ttccctgttg acatccagcc attcaaggac atgattgaag gaatgagaat 720
ggatttgaag aaatccagat acaagaattt cgacgagctc tatctctatt gctactatgt 780
tgctggtacc gtgggattaa tgagtgttcc tgtgatggga attgatccag aatctgatgc 840
tacaactgag agtgtctaca atgctgcatt gtctctagga attgcgaacc agcttacaaa 900
cattctcagg gatgtgggtg aagatacaag aagaggaagg gtgtatcttc cacaggatga 960
gcttgccgag gccgggcttt cagatgagga tatatttaat gggaaagtaa ctgatagatg 1020
gaggaatttc atgaaaaatc aaattaaaag ggctcggacc ttcttccagg aggcagagaa 1080
ggggattgct gagcttaacc aagctagtag gtggccggtg ttggcttctt tacttctcta 1140
ccggcaaata ctggacgaga tcgaagcaaa tgactacaat aatttcacaa aaagagcata 1200
tgtgagcaaa gcaaagaagc tgatggcagt gcccgtagct tatggaagat ctctcattag 1260
gccatcaatg aagaaaccat ctcttgtaaa accatgaaat tttcttataa gttcatacat 1320
tctcaataag tttgtaatgc atatttgata catgtgctta gtagtttgta aatttaaagc 1380
aaa 1383
<210> 3
<211> 150
<212> DNA
<213> Oncidium
<400> 3
aggattgaat ggcagcttcg cagttgtgct ttgttgggtt tttggaggga ataagaggag 60
gagatcggtt atggaacgcg aagaatgatt gcaggttttc gcagaggaag aagatgagat 120
ggagtttcta ctgtctgttt acgaacttta 150
Claims (10)
1.一种调控文心兰花色之DNA卡匣构筑体，其包含：由几段核苷酸序列所组合而成，该DNA卡匣可于植物特定组织中，干扰八氢蕃茄红素合成酶基因之mRNA，以达花色调控之目的，该DNA卡匣包含一启动子与一段可产生发夹型干扰性RNA的DNA核苷酸序列。
2.如申请专利范围第1项所述之DNA卡匣，其中该启动子包含SEQ ID NO：1核苷酸序列，且该启动子能使特定基因于特定组织中专一性表达，仅达到改变花色之目的，但仍维持植株正常生长之生理机能。
3.如申请专利范围第1项所述之DNA卡匣，其中一段可产生发夹型干扰性RNA的DNA核苷酸序列，可经由如申请专利范围第2项所述之启动子之驱动下，于特定组织中转录产生发夹型干扰性RNA序列以干扰八氢蕃茄红素合成酶基因之mRNA，使之无法进一步转译成酶，并由下列核苷酸序列所组成：正向置放的PSY核苷酸序列、intronGUS核苷酸序列、方向倒置的PSY核苷酸序列、及NOS基因之终止片段(T-NOS核苷酸序列)，其中该PSY核苷酸序列包含SEQID NO：3核苷酸序列。
4.如申请专利范围第1项所述之DNA卡匣，其中该核苷酸序列提供于一载体或一穿梭载体或一拟原球体，其中该载体可经转殖导入至一植物细胞，或一拟原球体。
5.如申请专利范围3所述之SEQ ID NO：3核苷酸序列为SEQ ID NO：2核苷酸序列中自第51个核苷酸起之150个核苷酸长度之核苷酸序列。
6.一种调控文心兰花色之DNA卡匣构筑方法，其包含：提供至少一DNA卡匣，此DNA卡匣的核苷酸序列包含一特定启动子与一段可转录成发夹型干扰RNA片段的特定核苷酸片段，将该核苷酸序列导入(或转殖)至一植物细胞，以获得一转殖植物细胞，及利用该已转殖植物细胞再生发育成为植株，并达到改变植株花色之目的。
7.依申请专利范围第6项所述之一种调控文心兰花色之DNA卡匣构筑方法，其中该核苷酸序列经由基因选殖方式获得。
8.依申请专利范围第6项所述之一种调控文心兰花色之DNA卡匣构筑方法，其中该已转殖植物细胞用以改变一植物体内控制类胡萝卜色素之一种八氢蕃茄红素合成酶表现量。
9.依申请专利范围第6项所述之一种调控文心兰花色之DNA卡匣构筑方法，利用该已转殖兰科植物细胞再生制造一已转殖植物。
10.本发明较佳实施例提供一核苷酸序列，包含SEQ ID NO：1、SEQ ID NO：2、SEQ IDNO：3及与SEQ ID NO：1、SEQ ID NO：2、SEQ ID NO：3之序列互补的核苷酸序列。
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| CHIOU ET AL.: "Characterization and promoter activity of chromoplast specific carotenoid associated gene (CHRC) from Oncidium Gower Ramsey", 《BIOTECHNOL LETT》 * |
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