CN111440809B - 结构基因在改变枸杞果色中的应用 - Google Patents

结构基因在改变枸杞果色中的应用 Download PDF

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CN111440809B
CN111440809B CN202010282780.9A CN202010282780A CN111440809B CN 111440809 B CN111440809 B CN 111440809B CN 202010282780 A CN202010282780 A CN 202010282780A CN 111440809 B CN111440809 B CN 111440809B
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祁银燕
刘小利
顾文毅
魏海斌
史文君
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Qinghai Academy of Agricultural and Forestry Sciences
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Abstract

本发明提供了结构基因在改变枸杞果色中的应用，涉及枸杞果色转变技术领域，所述结构基因包括F3′5′H、UFGT、ANS、DFR和CHS；所述F3′5′H的核苷酸序列如SEQ ID NO.1所示，所述ANS的核苷酸序列如SEQ ID NO.2所示；所述UFGT的核苷酸序列如SEQ ID NO.3所示；所述CHS的核苷酸序列如SEQ ID NO.4所示；所述DFR的核苷酸序列如SEQ ID NO.5所示。本发明实施例中，以黑果枸杞和白色变异枸杞为原材料进行对比，发现所述结构基因的表达和花青素的堆积速率显著正相关，最高的转录水平是在开花后25d和/或开花后35d时期，并且在黑果中高表达，白果中低表达。

Description

结构基因在改变枸杞果色中的应用
技术领域
本发明属于枸杞果色转变技术领域，具体涉及结构基因在改变枸杞果色中的应用。
背景技术
青海省柴达木盆地是我国枸杞的原生地之一。盆地独特的自然环境和高原气候，孕育出珍稀的植物资源---黑果枸杞(Lycium ruthenicum Murr.)。强烈紫外线和恶劣土壤环境诱导黑果枸杞的浆果内特异性地积累了大量花青素，这种类黄酮物质不仅能增强植株自身抗逆性而使其在逆境中正常生长发育，还对人体有重要的医疗保健作用。黑果枸杞黑色鲜果中，矮牵牛素是最主要的一类色素，占总花青素含量的95％，是其呈现黑色表型的物质基础。但是在2013和2014年对青海全省黑果枸杞野生资源进行清查时，发现颜色变异白色浆果的存在，花青素的消失很可能会降低植株抗逆性使之不能适应柴达木盆地的恶劣环境，而且对黑果枸杞浆果品质的影响也是致命的。所以研究如何转变枸杞果色则成为一个新的技术难题。
发明内容
有鉴于此，本发明的目的在于提供结构基因在改变枸杞果色中的应用，揭示白色浆果颜色的变异机制，为珍惜资源特异种质的合理推广和可持续发展奠定重要理论基础。
为了实现上述发明目的，本发明提供以下技术方案：
本发明提供了结构基因在改变枸杞果色中的应用，所述结构基因包括F3'5'H、UFGT、ANS、DFR和CHS；所述F3′5′H的核苷酸序列如SEQ ID NO.1所示，所述ANS的核苷酸序列如SEQ ID NO.2所示；所述UFGT的核苷酸序列如SEQ ID NO.3所示；所述CHS的核苷酸序列如SEQ ID NO.4所示；所述DFR的核苷酸序列如SEQ ID NO.5所示。
优选的，所述枸杞果色为黑色或白色。
优选的，所述枸杞果色的改变为由花青素缺失引起。
优选的，当所述F3′5′H、ANS、UFGT、DFR和CHS基因沉默后，黑色枸杞改变为白色枸杞；
当超表达所述F3′5′H、ANS、UFGT、DFR和CHS基因后，白色枸杞改变为黑色枸杞。
本发明提供了结构基因在改变枸杞果色中的应用，所述结构基因包括F3′5′H、ANS、UFGT、DFR和CHS。本发明所述结构基因均为花青素生物合成相关基因，参与枸杞中类黄酮生物合成。本发明实施例中，以黑果枸杞和白色变异枸杞为原材料进行对比，发现在开花后5d、15d、25d、35d和45d这5个时期中，在不断成熟的黑果中，结构基因CHS(c105048)、CHS(c89559)、CHI(c101670)、F3′5′H(c102345)、DFR(c92376)、ANS(c70865)、UFGT(c87418)、UFGT(c103977)的表达和花青素的堆积速率显著的正相关，最高的转录水平是在开花后25d和/或开花后35d时期，并且在黑果中高表达，白果中低表达，尤其是F3′5′H(c102345)的转录水平，在开花后35d时，黑果中是白果中的2391倍。就表达量看，黑白果中最大的差异基因为结构基因，依次为：依次为F3'5'H(c102345)、UFGT(c103977)、ANS(c70865)、DFR(c92376)、CHS(c105048)。
附图说明
图1为黑果枸杞样品和白色果实样品中着色表型和花青素含量，其中A表示在三个发育阶段的果实表面着色表型，其中Bar＝5mm；B表示三个发育阶段的花青素含量；
图2为在不同发育时期黑果和白果差异基因KEGG富集显著性最可靠的前20个通路，其中图2A为时期1的黑果和白果间的KEGG通路富集分析散点图；图2B为时期2的黑果和白果间的KEGG通路富集分析散点图；图2C为时期3的黑果和白果间的KEGG通路富集分析散点图；图2中Y轴代表KEGG通路，X轴代表富集因子；
图3为黑果和白果间差异表达基因的聚类分析；
图4为黑果和白果中花青素生物合成基因和转录因子的相对表达；
图5为转录组组装统计图，其中A：Transcript的长度分布图；B：Unigene长度分布图；
图6为定量验证的5个发育时期果实的颜色表型，其中A：5个时期黑果的颜色表型，B：5个时期白果的颜色表型。
具体实施方式
本发明提供了结构基因在改变枸杞果色中的应用，所述结构基因包括F3'5'H、UFGT、ANS、DFR和CHS；所述F3′5′H的核苷酸序列如SEQ ID NO.1所示，所述ANS的核苷酸序列如SEQ ID NO.2所示；所述UFGT的核苷酸序列如SEQ ID NO.3所示；所述CHS的核苷酸序列如SEQ ID NO.4所示；所述DFR的核苷酸序列如SEQ ID NO.5所示。
本发明所述枸杞果色优选为黑色或白色，所述枸杞果色的改变优选由花青素缺失引起。在本发明中，对枸杞果色进行改变，优选的包括：当所述F3′5′H、ANS、UFGT、DFR和CHS基因沉默后，黑色枸杞改变为白色枸杞；
当超表达所述F3′5′H、ANS、UFGT、DFR和CHS基因后，白色枸杞改变为黑色枸杞。本发明所述结构基因均为花青素生物合成相关基因，参与枸杞中类黄酮生物合成。
下面结合实施例对本发明提供的结构基因在改变枸杞果色中的应用进行详细的说明，但是不能把它们理解为对本发明保护范围的限定。
实施例1
1、植物材料
2015年9月，黑果枸杞样品和白色果实样品(正常光条件下的3个发育时期的健康果实)采于青海省柴达木盆地。3个发育时期的判断基于果实表皮的着色。如图1中A所示：黑果：(BlackFruit，缩写为BF)；白果：(White Fruit，缩写为WF)，
黑果和白果的第一时期(分别缩写为BS1和WS1，开花后的5天的果实)：二者表现为都未成熟，果实绿色；
黑果和白果的第二时期(分别缩写为BS2和WS2，开花后的15天的果实)：黑果轻微着色，紫红色，白果呈现黄白色；
黑果和白果的第三时期(分别缩写为BS3和WS3，开花后的35天的果实)：黑、白果果实膨大，黑果呈现黑紫色，白果显示白色。
果实采摘时用剪刀剪下，避免任何机械损伤。生理检测和生化分析时，都3重复。所有样品采下立即液氮冷冻后，存于-80℃冰柜。
2、三个发育阶段的黑果枸杞果实中花青素的含量
采用MeOH/HCl提取法，测定成熟果实的总花青素含量。根据黑果的着色表型(图1中A)，在三个发育时期对果实进行取样，其视觉上的果实色素沉着增加可能是花青素含量增加。
花青素测定结果表明，两种果实的花青素含量在第一时期均保持在极低水平，在黑果中花青素含量从第二时期开始增加，在第三时期达到显著水平，而白果中的花青素含量在第二到第三时期几乎没有变化(图1中B和表1)，仍保持在极低水平。因此，黑果枸杞果实颜色表型的变化主要是由花青素的含量引起的。
表1花青素含量变化
Figure BDA0002447346070000031
Figure BDA0002447346070000041
3、转录组测序和序列组装
通过高通量测序建立黑果枸杞和白果枸杞2个样品3个果实发育阶段样品的18个文库(每个样品3个生物重复)。对产生的高质量de novo组装，根据标准参数使用Trinity软件(http://trinityrnaseq.sourceforge.net/)进行de novo组装，结果共获得201581个N50为1826bp的转录本，unigene数据集包含101466个N50为1492bp的序列。Transcript和unigene的长度分布在图5中列出。利用unigenes进行注释分析。COG,GO,KEGG,KOG,Pfam,Swissprot,eggNOG和NR公共数据库中寻找同源序列。在这些数据库中，至少有一个数据库中发现了54986个unigenes(表2)。
表2 unigenes注释分析的统计数据
Figure BDA0002447346070000042
4、果实发育过程中差异表达基因(DEGs)的研究
为了研究不同发育时期黑果和白果中unigene的差异表达，首先分析黑果和白果文库中unigene的差异表达。比较unigene在黑果中的表达，发现BS1与BS2、BS2与BS3、BS1与BS3分别各有2260、5465、8212个表达差异的unigene，1342、2543、4126个unigene表达上调，918、2922、4086个unigene表达下调。对于白果样本中的unigene，分别在WS1对比WS2、WS2对比WS3、WS1对比WS3中共有2175、3585、7172个差异表达的unigene，1072、1231、2813个表达上调，1103、2354、4359个表达下调。白果和黑果样本之间的DEGs共有3185、3962、4910个unigene，在WS1对比BS1、WS2对比BS2以及WS3对比BS3中分别有1967、2952、3717个上调、1218、1010、1193个下调。
5、全部差异基因的KEGG富集分析
为了描述黑果枸杞黑果和白果发育阶段之间的功能差异，差异表达基因(DEGs)通过KEGG途径富集分析来探索相关的生物学功能(图2)。差异基因的KEGG富集分析列表的比较揭示，就不同发育期的黑果来说，BS1对比BS2，BS2对比BS3和BS1对比BS3中有5个相同的信号通路，都包含关于角质、软木脂和蜡质生物合成、类胡萝卜素生物合成、植物激素信号转导、苯丙素生物合成、淀粉和蔗糖代谢。BS2对比BS3和BS1对比BS3中出现了类黄酮生物合成，但是BS1对比BS2没有。说明黑果S1到S2时期，色素途径上的基因的表达差异不明显，这些结果与形态学变化吻合。白果中，WS1对比WS2，WS2对比WS3和WS1对比WS3中有11个相同的信号通路，且类黄酮生物合成在3组中都出现了，与形态学观察不符。黑白果的对比中，WS1对比BS1不同于WS2对比BS2和WS3对比BS3(图2)，3者中仅有一个相同的差异信号通路(类黄酮生物合成)。WS1对比BS1中还出现了花青素生物合成，说明黑白果中花青素生物合成基因表达的差异从早期就开始了。
6、黑果和白果发育阶段关于颜色发育相关的差异表达基因
利用上述18个文库，分析涉及颜色次生代谢途径(类黄酮、花青素、黄酮、黄酮醇、黄烷酮等生物合成)的基因。总共有261个unigene归到这些途径中。转录组数据表达分析显示，这261个unigene在黑果和白果间都差异表达。因此，选择这261个unigene进行后续试验。
7、花青素生物合成相关基因
分析261个果实着色相关差异表达基因，并对基因名字进行精细注释。重点研究261个基因中的参与类黄酮生物合成每个步骤的55个结构基因和10个调节基因(转录因子)：包括结构基因CHS,CHI,F3H,F3′H,f(F3′5′H,DFR,ANS，UFGT,5AT,5MaT1,FLS,ANR和调节基因MYB,bZIP,C2H2,HD-Zip。图3热图展示了基因的转录水平。
8、黑果和白果间花青素生物合成结构基因表达模式的比较
对比黑、白果中这55个结构基因的表达模式，差异表达的基因有26个。以白果中的表达为基准，分为4类(图3)。第1类：是最大的组，包含14个基因(54％)，它们在白果的整个发育期低表达，但在黑果中，上调表达的9个(c105048(CHS),c103053(CHI),c101670(CHI),c102345(F3′5′H),c92376(DFR),c40236(DFR),c70865(ANS),c103977(UFGT)，c79009(ANR))，下调的4个(c104723(DFR),c84984(UFGT),c97618(ANR),c105446(ANR))，整个发育期高表达的1个(c37514(5MaT1))。第2类：9个基因(34％)，它们在白果中随发育上调，但在黑果中上调，表达量高白果很多的3个(c89559(CHS),c86844(F3′H)，c87418(UFGT))，稳定高表达的2个(c101538(UFGT),c74476(5MaT1))，先下调再上调的2个(c54636(F3′H),c103688(UFGT))，下调的2个(c98294(F3′H),c91064(5AT))。第3类：2个基因(8％)，他们在白果中随发育下调，在黑果中也下调的1个(c83433(CHI))，整个发育期稳定高表达的1个(c82232(5MaT1))。第4类：1个基因(c107855(DFR),4％)，它在白果的整个发育期高表达，在黑果中呈现了相反的表达模式。
9、黑果和白果间花青素生物合成调节因子表达模式的比较
261个DEGs中包含10个转录因子，包含MYB(c75274,c94823)、bZIP(c93507)、C2H2(c71956,c84178)、HD-Zip(c101652,c13343,c39706,c67782,c88135)。在热图分析中，c101652，c75274,c71956,c39706的表达在样品间的重复性不好，剩余转录因子的表达在黑白果中的趋势都是一致的，但是13343,88135，94823,67782,93507的表达在黑果中高于白果中的(图3)。
10、定量验证
将21个从黑、白果3个着色时期的类黄酮生物合成途径中的差异结构基因和6个转录因子做实时定量PCR。EF1α作为内参基因。
定量体系为：
Figure BDA0002447346070000061
qRT-PCR反应在CFX Connect荧光定量PCR仪(Bio-Rad Laboratories,USA)上进行。反应程序为95℃预变性3min，95℃变性10s，60℃退火和延伸30s，40个循环。
其中涉及到的引物序列如SEQ ID NO.6～61所示。
在测转录组数据时，选择了转色的3个时期，为了更全面的反应整个发育期的表达水平，在第三时期的前后，各补充了1个时期做定量，分别为开花后25天(S3-before，缩写为S3-be)和开花后45天(S3-after，S3-af)，共5个时期(图6)。结果显示，在不断成熟的黑果中，结构基因CHS(c105048)、CHS(c89559)、CHI(c101670)、F3′5′H(c102345)、DFR(c92376)、ANS(c70865)、UFGT(c87418)、UFGT(c103977)的表达和花青素的堆积速率显著的正相关(图4)。
所有这些基因的表达分析都高度相似，最高的转录水平是在S3-be和/或第三时期，并且在黑果中高表达，但是在白果中低表达，尤其是F3′5′H(c102345)的转录水平，在第三时期，黑果中是白果中的2391倍。就表达量看，黑白果中最大的差异基因为结构基因，尤其是F3′5′H(c102345)、UFGT(c103977)、ANS(c70865)、DFR(c92376)、CHS(c105048)的转录水平，在S3时期，黑果中分别是白果中的2391倍，119倍，96倍，85倍，25倍。所以应该依次为：F3′5′H(c102345)、UFGT(c103977)、ANS(c70865)、DFR(c92376)、CHS(c105048)。所以，在黑果枸杞的白果中，这些基因的低表达可能解释了缺少花青素的原因。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
序列表
<110> 青海省农林科学院
<120> 结构基因在改变枸杞果色中的应用
<160> 61
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2750
<212> DNA
<213> Lycium ruthenicum Murr.
<400> 1
cgatatatca ttacaagctt catcttattt ttaacaatca tggtgctact tgctattgag 60
ctgagtatag cagctataat atttactata gcacatattt ttattacaaa aataaccact 120
agccggaggc ggcgactgcc gccaggtccc atggggtggc cggtgatcgg agcacttcca 180
cttctaggtg ctatgccaca tgtgtcactt gcaaaaatgg ccaaaaaata tggacctgtt 240
atgtatctca aagttggaac atgtggtatg gctgttgctt caacccctag tgctgctaaa 300
gcattcttga aaacacttga tatcaatttt tccaatcgtc cacccaatgc aggtgccaca 360
cacttggcct ataacgccca agacatggtt tttgcacatt atggaccccg ttggaagttg 420
ctaaggaaat tgagcaactt gcacatgcta ggtggaaaag ccttggatga ttgggcaaat 480
atccgtgtca atgagctagg tcacatgcta aaatcgatgt tcgatgcgag ccgggagggc 540
gagcgggtgg tggtggcgga tatgctgacg ttcgcgatgg caaacatgct cggacaagta 600
atattaagca agagagtgtt cgtggaccaa aagggggctg aggtcaacga atttaaggat 660
atggtggtgg aattaatgac tgtagcgggg tattttaaca ttggtgattt tattcctcaa 720
ttagcttgga tggatttaca aggaattgaa agagggatga aacgattgca caaaaagttt 780
gatgatttat tgacaaagat gtttgatgaa cacaaagcaa ctagctatga acgtgaaggg 840
aagcctgatt ttcttgatgt tgttatggaa aatagggata attctgaagg agaaaggctc 900
agcacaacca atatcaaagc agttttgctg gtatgtatcg tctaaatagt agtaacttat 960
gtcatttatt tacttgaaag gtatgttatt tggactctta aaaaaaatca ttgatatgtg 1020
ataactcgga ctcttaaaaa gaatcatcca tatgtgataa cctgaaaaat aggataatag 1080
tatgccgatt aaattgcatg tatagtttag aactcttgac atttcagtgt atgagttata 1140
aacttatttt tttttcatat attatgttat gcagtaatta ctttttcttt tttattctat 1200
aaccaggtca gtctcatttt ttcgcgttca aagaatattc cttcaaactt tgggataaaa 1260
caaatgaagg atcactttaa accaagtgtg tgttttattg ctaggccacg taacagtacc 1320
ctctctcatt aagtgaatga ttgtttcagt agttttccct atgttgctca ctctaccaaa 1380
atgatatcgt acttgtatcg gattctttaa aaatatgcta ttttaaatgg atttgatatg 1440
tatccgacaa tattttttaa agagttctct tttgcacaac ttcattcatt acccaaaaaa 1500
aaaatagatg gacaactttt gtcattaaac aacaaaattt tgttgctaat ccgatttagc 1560
gatacattag cgatgaatta tcaataatgt tattcatggt ctatttagcg acatattaac 1620
gacaaatttc gaagctaatt cttatttttt gtttgtcgat cggcatcatt gccataaatg 1680
gaaatgttct atatatatta accactactt ttttttctct ctcattaatt ttctgatgta 1740
tttttctttg tcaaaatttt gttgcagaat ttattcactg ctggtacgga cacttcatct 1800
agtgcaatag aatgggcact ggcagaaatg atgaaaaatc ccaacattct caaaaaagca 1860
caacaagaaa tggaccaagt cattgggaaa gatagacgtt taattgaatc tgacatccca 1920
aatctccctt atttacgtgc aatttgcaaa gaaacattcc gaaaacaccc ttcaacacca 1980
ttaaatctcc ctagggtatc aaacgaaccg tgtgtagtcg atggttatta cataccaaaa 2040
aatactaggc tcagcgtcaa catatgggca ataggacgag accccgacgt gtgggaaaat 2100
ccgctcaaat tcgatcctga gaggttcatg agcggaaaaa atgcgaagat tgatccgcgg 2160
ggcaatgatt ttgagttaat tccatttggt gcaggacgaa ggatttgtgc ggggacaaga 2220
atgggaatag tgatggtgga gtacatattg ggaactttgg ttcattcatt tgattggaaa 2280
ttaccaaatg atgttattga gattaatatg gaggaatctt ttggattggc tttgcaaaaa 2340
gctgttcctc ttgaagctat ggttactcca aggctacctt tggatgttta ttacgcaaat 2400
taaaacctta tttggtttta atttgcataa taatgaaaat ttgggagtcg atttggctat 2460
atatagatat tgataattct atattttgaa ggggtaattt gtcttgtaat ttgtttggct 2520
tcttctaatt ataattaatt cttatgcttg tggaaaattg tcttagtatt atttgtcgga 2580
tttaattgtt ttgtcatata gtttggtact ttgtttgtcc atgctagaca agaaaagaat 2640
aaaaagattg gaatttttat tttatctttt tgttctttta ttggaattcc ttgccttaga 2700
cctttgatct atttcataat attcagaaga tatttaagaa ttaataaaag 2750
<210> 2
<211> 1251
<212> DNA
<213> Lycium ruthenicum Murr.
<400> 2
atggtgagtg cagttgttcc aaccccttca agagttgaaa gcttggctaa aagtggaatc 60
caggccattc ctaaagagta tgtgaggcca caagaagagt taaatggaat gggaaacatc 120
ttcgaggaag agaagaaagg aggacctcaa gtaccgacga tagatcttaa acaaatcgac 180
tcagaagaca atgaaattcg cgagagatgc caccaagagt tgaagaaagc agccatggaa 240
tggggtgtga tgcacctcgt aaaccatggt atatcggatg agctaattgg tcgtgttaag 300
gttgccggag gtaccttctt tgatttacct gttgaagaaa aggagaagta tgctaatgat 360
caaacctctg gcaatgtcca aggctatggc agcaagctag caaatagtgc ttctggtcag 420
cttgagtggg aggattactt cttccattgt gttttccccg aggacaagca cgacttggcc 480
atctggccta aaacccctac cgactacatt ccagcaacaa gtgaatatgc caagcagata 540
aggaacctag caacaaagat tttagctgtg ctttctattg gactgggact ggaagaagga 600
agactagaga aggaagttgg aggcatggaa gacctgttgc ttcaaatgaa gattaattac 660
taccctaaat gcccccaacc agaactagca cttggcgtcg aagctcatac tgatgtgagt 720
gcactgactt tcatcctcca caatatggtg cccggcttgc aactcttcta tgaagggaag 780
tgggtaactg caaagtgtgt gcctaattcc ataatcatgc acattgggga tacccttgaa 840
atcctaagca atggaaagta caagagcatt cttcacagag gggttgtgaa caaagagaaa 900
gtaaggattt catgggcgat tttctgtgag ccgccaaagg ataagatcat ccttaagccc 960
ctacctgaaa ctgtcaccga ggctgatcca cctcgattcc cacctcgcac ctttgcacag 1020
catatggcgc acaagctctt caagaaggat gatcacgatg ctgctgctga ccacaaagta 1080
tataagaagg atgatcagga ttctgctact gaacagaaag tcttcaagaa ggacgatcag 1140
gattctgctg gtaaacagaa agccgccaaa aaggatgaac aggatgccaa tgctgagcac 1200
aaagtctccg agaaggataa tcaggatatt gctgctgaag aatctaaata g 1251
<210> 3
<211> 1961
<212> DNA
<213> Lycium ruthenicum Murr.
<400> 3
cactcactct ctcaatatta aactagagaa tcaattatgg agaatgagaa ctcaaatgat 60
gttcttcata tagttatgct cccttttttt gcttttggtc atatcagtcc atttgttcag 120
cttgctaaca agctttcctc tcatggtctc aaagtttctt ttttcactgc atctggcaat 180
gctggcagag tcaaatctat gctgaattct gctcccacta ctcatatagt ccctcttact 240
cttcctcaag ttgaaggtct acctcctggt gcagaaagta ctgcagaatt gacaccagta 300
actgctgaac ttctcaaagt tgctttagac caaatgcaac cacaaatcaa gtctctactt 360
tccaatctca aaccccattt tgttctcttt gattttgctc aagaatggct ccctaaaatg 420
gctgatgaat tagggatcaa gactgttttt tactctgttt ttgtagcact ttccactgct 480
tttcttactt gccctgctag agttcctcaa cccaagaaat atccaactct tgaagacatg 540
aagaaacctc cacctggatt tcctatcacc tctgtcacct cagtcaaaac ctttgaggct 600
caagattttc tatatatttt caagagcttc catggtggtc ctactgtata tgaccgtgta 660
ctctcaggac ttaagggttg ctcagctata ctagctaaga cttgttccca aatggagggg 720
ccttatatag aatacgtgaa atcgcagttc gataaacctg tttttctagt aggaccagta 780
gttcctgacc caccttcagg aaaattggaa gagagatggg ctagttggtt aaacaagttt 840
gaagctggaa cagttattta ctgttctttt ggaagtgaaa ctttcttgaa tgatgatcag 900
atcaaagaac tagctttagg tttggaacaa actgggctac ctttctttct ggtcttaaat 960
tttcctgcca atgtcgatgt cccagccgaa ctaaaccgag ctttaccaga aggttttctg 1020
gagagagtga aagacaaggg aatcattcat tcaggttggg tgcagcaaca gaacatactt 1080
gctcatgcca gtgtaggttg ctatgtatgc cattcagggt tcagttcagt gatagaggca 1140
ctagtgaatg actgtcaagt tgttatgttg cctcagaaag gtgatcagtt cttgaatgca 1200
aagctggtga gtggtgatat gaaagctggg gtggaggtaa ataggaggga tgaagatggc 1260
tattttggta aagaagatat taaaaaagct gtggagatgg tgatggtgga ggttgacaag 1320
cagccaggta aattaattag agcaaatcag aagaaatgga aggagttttt gttgaacaag 1380
gatatccaat gcaagtttat tgaggattta gttaatgaaa tgacagcaat ggctaaggtc 1440
tcaagtaact aggatatgtc aatggtcatc tcatctttca atagtagtat agtcggcatt 1500
attaccatca ctaaagcatt tcccaatgta cccgcttttt gtatttttat ttttattttc 1560
atcaacagaa tgtcaacttc tgacaatcag atcacttgac ataaaactat agattaactt 1620
tttactaaaa cattaattta aaatctgaaa aaagagagga gaaaattgtt acactccata 1680
ataatccatg ctaatcgaag taaaacaaga aagaatgagt taagaaggtt caaggaaagt 1740
taatcgagtt agtaagaata tagttatata tatatagttg ccttaagtat tccgaggtga 1800
tatggtaacc tagaggaatt gaaatcgcgt tatgagtata tgaggtaatg tgagcgacct 1860
tttaaagtat ttgatattat tagtaatgat atagaggatg gacaaaagat atgaatatac 1920
ttttacgatt ggagttttgt cgaagctttc atatttcggc c 1961
<210> 4
<211> 1562
<212> DNA
<213> Lycium ruthenicum Murr.
<400> 4
aaagaaaaaa ttggtttgac cttttgtttg ttatctgatc tattttttaa aatgttttct 60
tctcttaaaa attaataaat agtgtaagta tgtttcaatc cctttgggca ataaactgat 120
taaaaggttt acattggaat ggtcacattt tcctttataa ttagcttctc caagatgaag 180
tttagattct ttgaatgaaa caagattcga aggaagctga gcttttgttt tagttctttg 240
ttgatctttg caattagtag gatttcgatt atcataatta actttctgac ttttgattag 300
gcgcaatttg ttcattcaat gattttaagt ttaagcacta tacatactgg cagtttattg 360
atgactttag gttctctcat gtaatgcagg tgacaaatct atgattaaga agaggtacat 420
gcacttaact gaagaaatcc taaaagaaaa ccccaatatt tgtgaataca tggctccttc 480
tattgatgct aggcaagaca tagtggtggt tgaagtgcca aaacttggca aagaagcagc 540
ccaaaaggcc atcaaagaat ggggccaacc caagtccaag attacccatt tggtattttg 600
caccactagt ggggtggaca tgcctggggc cgactaccag ctcactaagc ttcttgggct 660
tcgaccctcc gttaagaggt tcatgatgta ccaacaaggt tgttttgctg gtggtactgt 720
tatcagattg gccaaggact tagccgaaaa caacaagggt gctcgagtcc ttgttgtttg 780
ctcagagatc actgcagtta cttttcgtgg cccaagtgac actcacttgg atagtatggt 840
tggacaagcc ttatttgggg atggggcagg tgcactcatt gtaggttctg atccattacc 900
tgaggttgaa aggcctttat tcgagcttgt ctcagcagcc caaactctgc tcccagacag 960
cgaaggtgct atcgatgggc accttcgtga agttgggcta acatttcact tactcaaaga 1020
tgttcctgga ttgatctcaa agaacattga gaagagcttg atggaagcat tccaaccatt 1080
gggcatttct gattggaact ctctcttttg gattgctcat ccaggtgggc cggcaattct 1140
ggaccaagtt gaactaaagt tggccttaaa gcccgaaaaa cttcgagcta caaggcaagt 1200
cttgagtgac tatggaaata tgtctagtgc ttgtgttttg tttattttgg atgaaatgag 1260
gaaggcctca gccaaagaag ggcttggtag cactggtgaa ggccttgatt ggggtgtact 1320
ctttggattt gggcctgggc taacagttga gactgttgtg ctccatagtg tctctactta 1380
gtatgctgcg tattgattta tattgtgggg gtgatcgaag tattaaattg attttctatg 1440
taatcttaaa tttcttttat atattgtact atgtatttga tttgttttca attgaacttt 1500
gcaagtaatg ttatttgtaa tgcatattat gtataaaaag aagtccattt ctccctctaa 1560
aa 1562
<210> 5
<211> 2129
<212> DNA
<213> Lycium ruthenicum Murr.
<400> 5
tagttgtttg tgtacaaatg aaaatttcaa ggctataaat gtcatttacc cacttaagtt 60
gaaatgtaga taaactcgat gattcactaa ctttaggtgt gatgaatctt aaatctgttc 120
ctctaatttg ttaaactatt ttgagaattt ggtcaaaatg aaggacaaac taaccaatat 180
tttcacaaca atcattcaat ttgcaaagcc acacatagaa atgtttcttc agcaaggtac 240
acttaattct atatgcagat gcgaaagtgg ctgccagatg gccagaaccc attttcttcc 300
tctctcacca accagtcata tttggttaat ccaatgaagt tacttaacgg tgcgttgagc 360
acgtgcttac catctaacac tacgtatcac tcctaacaca acctacgtgc aagcagtata 420
aagatagaca taaacataaa tgaagttcat cttgttgtaa tttagtccgg tcaattaaga 480
ttcattttcc gactcttttt actttttatt tctgaaaatg gcaagtgaag ctcatgcagc 540
tattaatgct ccggcggcgc cgactgtttg tgtcactgga gccgctggat ttattggttc 600
ttggcttgtc atgagactcc ttgaacgagg ttacaatgtc catgctaccg ttcgtgatcc 660
tgagaacaag aagaaggtga agcatctgct agaattgccg aaagctgata ccaacttaac 720
gctgtggaaa gcagacttga aagtggaagg aagctttgat gaagccattc aaggctgtca 780
aggagtattt catgtggcaa cacctatgga tttcgagtcg aaggaccccg agaatgaagt 840
aataaaacca acagtcaggg gaatgttaag tatcatagaa tcatgtgtca aggcaaacac 900
ggtaaggaga ctggttttca cttcatctgc tggaactctt gatgtccaag agcaccaaaa 960
actcttctat gacgaaacca gttggagtga cttggatttc atctatgcta agaagatgac 1020
aggatggatg tattttgttt ccaagatact ggcagagaag gctgcaatgg aagaagctaa 1080
gaagaacaac attgatttca ttagcatcat accaccactg gttgtgggtc cgttcatcac 1140
acctacgttc ccacctagct taatcactgc cctttcacta attaccggta tgatgtgatc 1200
acaatgtaca gaagacagtc gcttccttta atcccaacaa ttactttagt aaacagcttt 1260
ccacaacaca atcatgggtt taatcagatt gagatgtgca gggaatgaag ctcactactg 1320
catcattaaa caaggtcaat atgtgcattt agatgatctt tgtgaggctc atatattcct 1380
ttacgagcac cccaaggcag agggaagata catttgctct tcccatcatg caatcatata 1440
tgatgtggct aagattgttc gacaaaaatg gccagagtac tacgttccta cggagtaagc 1500
ctcctctgta tccctaagta taattgactc cttcgttgag tgaggcttgg taactcagtc 1560
tggttaaata acaggtttaa aggcatcgat aaggacttgc ccataatatc tttttcgtca 1620
aagaagctaa tggacatggg gtttcaattc aaatacactt tggaggatat gtataaagga 1680
gccattgaga cttgccgaca gaagcagttg cttcccttat ctacacgaag cactgcggac 1740
aatggacaag acaaagaagc cattccaatt tctgccgaaa actatgcaag tggcaaggag 1800
aatgcacgag ttgcaaatgg tacaggaaag ttaaccaatg atgaaatcta gaactgtaac 1860
cttacaaaat aaagaggcga gcttgcctag caatatgttt gcttcttggt tccctgatat 1920
ctgttttgag gttttccaat actaaatgcg taaaatgttc aacgactata tggaaattct 1980
ggtcttgcta agttacaggc agctgcacag taggatatca aataatccta ggtatatttt 2040
gcctagaaaa gctgtcttca aggctcttgc ataccatttg ccaaaaaaga aattatcatt 2100
tttcttaatc attggaggga aacgagaga 2129
<210> 6
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
ccatcgatag caccttcgct 20
<210> 7
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
acttttcgtg gcccaagtga 20
<210> 8
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
ctccggtttt agggccaact 20
<210> 9
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
cagcctctgg gcatttctga 20
<210> 10
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
tatgctgcag agctcgagtg 20
<210> 11
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
tgcgatgggg ttgagtttga 20
<210> 12
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
tcctgacact gggaatgctg 20
<210> 13
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
ttccgcgaga ctacacttgg 20
<210> 14
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
acggatcagc ccgttgtaag 20
<210> 15
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
tttggtgctg ggcgtagaat 20
<210> 16
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
tgttctggcc aatcacacga 20
<210> 17
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
tattgcagca ggcggatgat 20
<210> 18
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 18
aaccggccac gttacaatct 20
<210> 19
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 19
ttgacctggt tgtggggaag 20
<210> 20
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 20
tagcaacttc caacggggtc 20
<210> 21
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 21
tgcttcaacc cctagtgctg 20
<210> 22
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 22
acttgccgac agaagcagtt 20
<210> 23
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 23
tgcaactcgg tgcattctcc t 21
<210> 24
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 24
ggcggttacc acgttaaagc 20
<210> 25
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 25
gagcaagctt ggaggagtga 20
<210> 26
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 26
aggacctcaa gtaccgacga 20
<210> 27
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 27
ttacgaggtg catcacaccc 20
<210> 28
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 28
taccgtcaca gcattacccc 20
<210> 29
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 29
tcaaggaacc ctttcggcaa 20
<210> 30
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 30
ttgttcccaa atggaggggc 20
<210> 31
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 31
tcctgaaggt gggtcaggaa 20
<210> 32
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 32
cccgtgaaag ctcctccttt 20
<210> 33
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 33
atgtggttgt agcacctccc 20
<210> 34
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 34
tgatctcaac tgcggttccc 20
<210> 35
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 35
cagtcggggg atttgtgtca 20
<210> 36
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 36
ggcaattagg caagcaccaa a 21
<210> 37
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 37
tgggaccaac ccacacacta 20
<210> 38
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 38
ccacctgaca aggttcgagg 20
<210> 39
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 39
atatcttctc cgatcgcggc 20
<210> 40
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 40
aagctaagcc cgtgggtgaa 20
<210> 41
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 41
gggaaggcct gcgaagtta 19
<210> 42
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 42
aaggctttac atttgctgcg g 21
<210> 43
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 43
gcctccccca accaaaatca 20
<210> 44
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 44
aaggagtggc cacatggaaa a 21
<210> 45
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 45
atgccaccct caggaacttg 20
<210> 46
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 46
ggagtccgca acctaccatc 20
<210> 47
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 47
gccatagcat ttgtgtggct 20
<210> 48
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 48
tgacgagatt atgccggacg 20
<210> 49
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 49
tgttactgtg catggtcccg 20
<210> 50
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 50
acagttggct ccaccattca a 21
<210> 51
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 51
cagaagcatc gacagataac gg 22
<210> 52
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 52
tcacaacttt tgcgctggtg 20
<210> 53
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 53
ggttcaccag ggtcgctaat 20
<210> 54
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 54
tccttctggc tgtgttgtcc 20
<210> 55
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 55
tggtgattca cgccctcttc 20
<210> 56
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 56
gctgttgcaa tgggaaggtc 20
<210> 57
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 57
tacggggtgt gcctatggta 20
<210> 58
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 58
ggcaaaggag gcttggatca 20
<210> 59
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 59
atgagctact aacactgcgc t 21
<210> 60
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 60
gaagggtgtc cctcagatca 20
<210> 61
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 61
ccgtccatgt cgtctctttt 20

Claims (1)

1.结构基因在改变枸杞果色中的应用，其特征在于，所述结构基因包括F3'5'H、UFGT、ANS、DFR和CHS；所述F3′5′H的核苷酸序列如SEQ ID NO.1所示，所述ANS的核苷酸序列如SEQID NO.2所示；所述UFGT的核苷酸序列如SEQ ID NO.3所示；所述CHS的核苷酸序列如SEQ IDNO.4所示；所述DFR的核苷酸序列如SEQ ID NO.5所示；
所述枸杞果色为黑色或白色；
所述枸杞果色的改变为由花青素缺失引起；
当所述F3′5′H、ANS、UFGT、DFR和CHS基因沉默后，黑色枸杞改变为白色枸杞。
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CN101258245A (zh) * 2005-08-30 2008-09-03 新西兰园艺和食品研究院有限公司 调控植物中色素生产的组合物和方法
CN108503699A (zh) * 2018-06-12 2018-09-07 中国科学院西北高原生物研究所 枸杞基因以及其编码蛋白质、重组载体、及其用途

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CN101258245A (zh) * 2005-08-30 2008-09-03 新西兰园艺和食品研究院有限公司 调控植物中色素生产的组合物和方法
CN108503699A (zh) * 2018-06-12 2018-09-07 中国科学院西北高原生物研究所 枸杞基因以及其编码蛋白质、重组载体、及其用途

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黑果枸杞花青素生物合成关键基因的生物信息学分析与克隆;虎娟 等;《西北林学院学报》;20171231;摘要，第1-2页 *

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