CN111235167A - 编码鸡血藤花青素还原酶的基因及其应用 - Google Patents
编码鸡血藤花青素还原酶的基因及其应用 Download PDFInfo
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Abstract
本发明公开了一种编码鸡血藤花青素还原酶的基因及其应用，填补了我国传统中药材鸡血藤中分离克隆花青素还原酶基因的空白。本发明所提供的花青素还原酶基因具有SEQ ID NO.1所示的核苷酸序列或者添加、取代、插入或缺失一个或多个核苷酸的同源序列或其等位基因及其衍生的核苷酸序列。所述基因编码的蛋白质具有SEQ ID NO.3所示的氨基酸序列或者添加、取代、插入或缺失一个或多个氨基酸的同源序列。该基因在鸡血藤根、茎、叶、花以及果实中均有表达，其中在根和茎中表达量较高，在叶中含量很低。本发明提供的花青素还原酶有助于利用基因工程技术提高鸡血藤中活性成分表儿茶素的含量，对今后鸡血藤种质资源改良具有很好的应用前景。
Description
技术领域
本发明涉及生物技术领域，涉及鸡血藤活性成分表儿茶素生物合成途径中的关键酶及其编码基因，更具体地说，本发明涉及编码鸡血藤花青素还原酶的基因及其应用。
背景技术
鸡血藤来源于豆科植物密花豆Spatholobus suberectus Dunn的干燥藤茎，具有补血活血，调经止痛，舒筋活络的功效。用于治疗月经不调，痛经，经闭，风湿痹痛，麻木瘫痪，血虚萎黄等病症，广泛用于妇科、风湿痹痛等中成药中。研究结果表明，鸡血藤主要活性成分为黄酮类化合物，其中表儿茶素是主要活性成分之一。
表儿茶素合成途径中，花青素还原酶(Anthocyanidin reductase,ANR)催化花青素转变为表儿茶素。然而，目前尚未对鸡血藤ANR基因进行克隆，也未发现其作为提高植物表儿茶素含量的应用的研究和报道。
发明内容
本发明的一个目的是解决至少上述缺陷，并提供至少后面将说明的优点。
本发明的另一个目的是通过基因克隆，获得鸡血藤花青素还原酶(简称SsANR)基因的gDNA序列、cDNA序列及氨基酸序列，为后续利用生物技术手段提高鸡血藤有效部位表儿茶素的含量提供重要基础。
为了实现根据本发明的这些目的和其它优点，本发明提供编码鸡血藤花青素还原酶的gDNA，其中，为下列核苷酸序列之一：
1)具有SEQ ID No.1所示的序列；
2)能编码SEQ ID No.1经添加、取代、插入或缺失一个或多个氨基酸且具有鸡血藤花青素还原酶活性蛋白质的核苷酸序列。
编码鸡血藤花青素还原酶的cDNA，其中，为下列核苷酸序列之一：
1)具有SEQ ID No.2所示的序列；
2)在严格条件下与SEQ ID No.2的cDNA序列杂交且编码具有鸡血藤花青素还原酶活性蛋白质的DNA分子；
3)编码SEQ ID No.3所示氨基酸序列的核苷酸序列；
4)编码SEQ ID No.3经添加、取代、插入或缺失一个或多个氨基酸且具有鸡血藤花青素还原酶活性蛋白质的核苷酸序列。
具有鸡血藤花青素还原酶活性的蛋白质，其中，为下列氨基酸序列之一：
1)具有SEQ ID No.3所示的氨基酸序列；
2)SEQ ID No.3经添加、取代、插入或缺失一个或多个氨基酸且具有鸡血藤花青素还原酶活性的氨基酸序列。
一种用于扩增gDNA的引物，其中，所述引物的序列如SEQ ID No.4和SEQ ID No.5所示。
一种用于扩增cDNA的引物，其中，所述引物的序列如SEQ ID No.6和SEQ ID No.7所示。
一种gDNA或cDNA或蛋白质或引物作为提高植物表儿茶素含量的应用。
具体来说，能够极大程度提高植物的根和茎中表儿茶素的含量。
含有本发明的SsANR基因全序列或部分序列的重组载体，如原核类载体、真核类载体及RNAi载体均属于本发明的保护范围。
含有本发明的SsANR基因全序列或部分序列的宿主细胞，如含有上述的重组载体的宿主细胞也属于本发明的保护范围。
本发明至少包括以下有益效果：
本发明针对鸡血藤表儿茶素生物合成研究方面的空白，提供了鸡血藤花青素还原酶(SsANR)蛋白质及其编码基因的gDNA序列、cDNA序列及氨基酸序列通过植物表达载体转化到植物中时，能够有效提高所得转基因植物中儿茶素的含量，同时对不同组织部分的基因表达分析，为今后利用生物技术手段提高鸡血藤有效部位表儿茶素含量提供重要理论依据，具有较高的应用价值。
本发明的其它优点、目标和特征将部分通过下面的说明体现，部分还将通过对本发明的研究和实践而为本领域的技术人员所理解。
附图说明
图1为本发明所述的鸡血藤花青素还原酶(SsANR)与大豆(Glycine max)、菜豆(Phaseolus vulgaris)、豇豆(Vigna unguiculata)、野生大豆(Glycine soja)、红豆草(Onobrychis viciifolia)、湿地百脉根(Lotus uliginosus)、百脉根(Lotuscorniculatus)的ANR基因构建的系统进化树(NJ法)；
图2为本发明所述的鸡血藤花青素还原酶(SsANR)在不同组织器官中的表达水平图。
具体实施方式
下面结合实施例对本发明做进一步的详细说明，以令本领域技术人员参照说明书文字能够据以实施。
实施例1
SsANR基因克隆
(1)RNA和DNA的提取：采用Trizol试剂(Invitrogen，美国)从鸡血藤新鲜幼叶中提取总RNA，并用RNase Free DNase(Promega，美国)进行预处理消除基因组DNA污染。用1.5％琼脂糖凝胶分析RNA完整性，用分光光度法测定RNA的纯度和浓度。
采用植物DNA试剂盒(TIANGEN)从鸡血藤新鲜幼叶中提取总DNA。
(2)基因克隆：根据鸡血藤基因组中的ANR序列设计引物，以DNA为模板，用Primer5.0软件设计基因特异性引物
F1:CGGATTTATGGCCTCTTG(SEQ ID NO：4)
R1:TGACTTGATTCTCGCACC(SEQ ID NO：5)
通过PCR扩增，获得长度为1996bp的gDNA序列，如SEQ ID NO：1。
实施例2
SsANR基因克隆
将实施例1提取的RNA进行逆转录(AMV first strand cDNA synthesis kit：Roche，瑞士)，以第一链cDNA为模板，利用引物：
F1:TCTTCCAGCTTGCTACACC(SEQ ID NO：6)
R1:CTTTGAGGGACAATCTTCG(SEQ ID NO：7)
通过PCR扩增，获得长度为644bp的cDNA序列SEQ ID NO：2。
实施例3
根据实施例2的cDNA序列翻译后获得编码具鸡血藤花青素还原酶(SsANR)的氨基酸序列SEQ ID NO：3。
实施例4
SsANR基因的生物信息学分析
将SsANR的开放阅读框序列及其编码蛋白的氨基酸序列在NCBI上进行同源性检索，发现该基因与其它物种有较高的同源性，其中与野生大豆、大豆、豇豆、菜豆的ANR基因同源性都很高(如图1所示)。
实施例5
SsANR基因在不同组织中的表达差异情况
(1)材料准备：采集新鲜鸡血藤的根、茎、叶、花、果实，将样品用自封袋包好，液氮速冻后，于-80℃超低温冰箱中储存。
(2)RNA提取：采用Trizol试剂(Invitrogen，美国)从鸡血藤的根、茎、叶、花和果实中提取总RNA，并用RNase Free DNase(Promega，美国)进行预处理消除基因组DNA污染。用1.5％琼脂糖凝胶分析RNA完整性，分光光度法测定RNA含量。
(3)cDNA制备：采用AMV第一链cDNA合成试剂盒(Roche，瑞士)合成cDNA。
(4)引物设计：选择18S为内源参考基因，利用Primer 5.0软件设计用于qRT-PCR的特异性引物，其中，
ANR-F:GGGGACAGGTCTGGTTATGGA(SEQ ID NO：8)
ANR-R:GCCTTTGAGGGACAATCTTCG(SEQ ID NO：9)
18S-F:CGTTCCCGCCAATATCTCAC(SEQ ID NO：10)
18S-R:TGTTCAATACCAGCCGCACC(SEQ ID NO：11)。
(5)qRT-PCR：根据SYBR Green Fast qPCR Master Mix(BBI，加拿大)的说明在StepOnereal-time PCR系统(ABI，美国)中进行聚合酶链式反应，用熔解曲线法分析目标基因的特异性扩增。
(6)采用2-ΔΔCT法计算SsANR基因在不同组织中的表达量。结果显示SsANR基因在根、茎、叶、花、果实中均有表达，其中在根中表达量最高，其次是茎，叶中表达量最低(如图2所示)。
实施例6
SsANR基因功能研究和应用
(1)植物表达载体的构建
以鸡血藤的cDNA为模板，利用引物SEQ ID NO：6和SEQ ID NO：7进行PCR扩增，用Bg1II和BstEII在37℃下酶切扩增产物2h，利用回收试剂盒(Takara公司，中国)纯化酶切产物。
同时利用Bg1II和BstEII在37℃酶切pCAMBIA1301载体2h，利用回收试剂盒(Takara公司，中国)纯化酶切产物。
将回收的两个片段混合，在DNA连接酶的作用下，于16℃反应12h，连接产物转化DH5α感受态，挑取阳性单克隆在LB培养基中培养，37℃下振荡培养8h，取1μL菌液进行PCR鉴定，将含有两个片段的菌株进行扩繁，提取质粒得到构建的植物表达载体，命名为131-35s-YFP。
(2)转化至本氏烟草
将本氏烟草叶片切割为约1×1cm的小块，将叶块正面向下放置在MS预培养基上，预培养2d后放入含有上述植物表达载体的农杆菌悬浮液中浸泡5～10min，无菌纸吸干多余菌液，叶块正面朝下接种到MS固体培养基，暗培养2d。
将培养后的烟草叶块移至MS筛选培养基，每隔15d更换一次培养基，至有绿色芽生成。待芽长至2cm左右时转移至MS生根培养基上进行生根诱导。幼苗长至5～7cm时炼苗出土，提取叶片DNA进行PCR鉴定，筛选得到阳性植株即为转基因烟草。
(3)转基因烟草代谢产物分析
采用UPLC-ESI-MS/MS法，测定转基因烟草叶片中表儿茶素的含量。结果表明，转基因烟草中的表儿茶素含量为非转基因烟草的3.1倍，可见本发明的基因用于提高植物中儿茶素含量能够取得显著的效果。
花青素还原酶作为催化无色花青素转变为表儿茶素的最后一个限速酶。其表达活性直接影响表儿茶素的合成。本发明通过基因克隆，获得SsANR基因的gDNA序列、cDNA序列及氨基酸序列，为后续利用生物技术手段提高鸡血藤有效部位表儿茶素的含量提供重要基础。
尽管本发明的实施方案已公开如上，但其并不仅仅限于说明书和实施方式中所列运用。它完全可以被适用于各种适合本发明的领域。对于熟悉本领域的人员而言，可容易地实现另外的修改。
<110> 广西壮族自治区药用植物园
<120> 编码鸡血藤花青素还原酶的基因及其应用
<160> 11
<170> PatentIn version 3.3
<210> 1
<211> 1996
<212> DNA
<213> 鸡血藤（Spatholobus suberectus Dunn）
<400> 1
cggatttatg gcctcttgct tgatcaagca gttgcttgaa aagggttatg ctgtcaatac 60
taccgttaga gacccaggtc tctctctttc tctctctatg aaatatgtca ttgtaaaatt 120
gtttatttat tttgttaaca aaaaactgtt tatttatttt gtaccaacca aaaacacaat 180
ctctctgaag aatgaaaaca aaacctaaga aaaggtactt ggagactagt ttgttatagc 240
ccataacaag aaacactagg ccgatcgccc ttctgaagtt gtattaaaga atagagaaac 300
caaataagat tttattatga acaaaagttc ttatgctggt gttttgtgca gataatacta 360
aaaaaatatc tcaccttttg gcactacaaa gtttgggcga attgaaaata tttggagcag 420
atttaacagg tgcaaaagat tttgatgccc ctatagcagg ctgtgaactt gtcttccagc 480
ttgctacacc tgtgaacttt gcttctgaag atcctgaggt agattgaaca ctgaactaaa 540
aactgcatta tacatttaac aaccaaattg aatgctctta tgcgtacgtg cgtgtgagtg 600
ttgaagaaaa agataatgag gaaattcttt tgcaattact attcagaatg acatgatcaa 660
gcctgcaata tcgggtgtct tgaatgtgtt gaaagcatgt gctcgagcaa aaggagtcaa 720
acgagtcatc ttaacatctt cagcagctgc tgtaaccata aaccaactcc aggggacagg 780
tctggttatg gatgaaagca actggactga tgttgagttc ttgaacactg caaagccacc 840
cacttgggta aaagtcaaac cttatttagt gtgtgtttaa gtcagccttt gcactttgca 900
aaattgattt cggtttaaat tgattttgaa attaagttat ttatgtttag atgattttat 960
tctgaaacaa cttagtagta aaactcaata taaaagtttt tattcaacac aaaatctact 1020
taaagttgtt tgatttttca tctcaaaatc agttttagac ccagacttaa tttctcaatg 1080
tgggactaaa tgtgtgaacg tttatctaaa atcacattag ctagtatttc aacatgattt 1140
tggacgatcc aacatgaatc caaacatgta cttagaactt acgatgggga tgaggaatgg 1200
ctcttcacac tgccttgcct cattatcatt cctagttgga aaagactact actcactgga 1260
tagactctga agtagagtgt tcacccaaac atgcaggggt atcctgtctc caaaacatta 1320
gcagagaagg cggcatggaa atttgctgaa gaaaatcaca ttgatctcat cactgtgata 1380
ccttctctca caactggtcc ttctctcact ccagacattc cttcaagtgt tggcctcgcc 1440
acgtccctta taacaggttc gaatctgcca aatctactgg tttaaggaca ttttagacgt 1500
gtcatgtaat tgcacttttt tgtatacctt gcaggcaatg atttcctcat aaacgctatg 1560
aaaggtatgc agtttctgtc aggttcaata tccatcactc atgtggagga tatttgccga 1620
gcacatatat ttgtggcgga gaaagaatca gcttctggtc gatacatttg ctctgctcac 1680
aatactagtg tccctgagct tgcaaagttt cttagcaaac gatatcctca gtataaaatt 1740
ccaaccgagt aagcttctaa gttctaacca tacatttagt ctgaaatgtt taaccaaagg 1800
ccctttcctt tttcattttc tttgtcattc aagcaatccc tttccttggt tatagataaa 1860
ttcttttttt cttgctgtac aaatgaaggc aacgaggttt gaacttaaaa cttcttacaa 1920
actatccaaa cctcccacta ttaggccgac gctagtaggt tatatagata aattctttgg 1980
tgcgagaatc aagtca 1996
<210> 2
<211> 644
<212> DNA
<213> 鸡血藤（Spatholobus suberectus Dunn）
<400> 2
tcttccagct tgctacacct gtgaactttg cttctgaaga tcctgagaat gacatgatca 60
agcctgcaat atcgggtgtc ttgaatgtgt tgaaagcatg tgctcgagca aaaggagtca 120
aacgagtcat cttaacatct tcagcagctg ctgtaaccat aaaccaactc caggggacag 180
gtctggttat ggatgaaagc aactggactg atgttgagtt cttgaacact gcaaagccac 240
ccacttgggg gtatcctgtc tccaaaacat tagcagagaa ggcggcatgg aaatttgctg 300
aagaaaatca cattgatctc atcactgtga taccttctct cacaactggt ccttctctca 360
ctccagacat tccttcaagt gttggcctcg ccacgtccct tataacaggc aatgatttcc 420
tcataaacgc tatgaaaggt atgcagtttc tgtcaggttc aatatccatc actcatgtgg 480
aggatatttg ccgagcacat atatttgtgg cggagaaaga atcagcttct ggtcgataca 540
tttgctctgc tcacaatact agtgtccctg agcttgcaaa gtttcttagc aaacgatatc 600
ctcagtataa aattccaacc gaattcgaag attgtccctc aaag 644
<210> 3
<211> 644
<212> PRT
<213> 鸡血藤（Spatholobus suberectus Dunn）
<400> 3
Thr Cys Thr Thr Cys Cys Ala Gly Cys Thr Thr Gly Cys Thr Ala Cys
1 5 10 15
Ala Cys Cys Thr Gly Thr Gly Ala Ala Cys Thr Thr Thr Gly Cys Thr
20 25 30
Thr Cys Thr Gly Ala Ala Gly Ala Thr Cys Cys Thr Gly Ala Gly Ala
35 40 45
Ala Thr Gly Ala Cys Ala Thr Gly Ala Thr Cys Ala Ala Gly Cys Cys
50 55 60
Thr Gly Cys Ala Ala Thr Ala Thr Cys Gly Gly Gly Thr Gly Thr Cys
65 70 75 80
Thr Thr Gly Ala Ala Thr Gly Thr Gly Thr Thr Gly Ala Ala Ala Gly
85 90 95
Cys Ala Thr Gly Thr Gly Cys Thr Cys Gly Ala Gly Cys Ala Ala Ala
100 105 110
Ala Gly Gly Ala Gly Thr Cys Ala Ala Ala Cys Gly Ala Gly Thr Cys
115 120 125
Ala Thr Cys Thr Thr Ala Ala Cys Ala Thr Cys Thr Thr Cys Ala Gly
130 135 140
Cys Ala Gly Cys Thr Gly Cys Thr Gly Thr Ala Ala Cys Cys Ala Thr
145 150 155 160
Ala Ala Ala Cys Cys Ala Ala Cys Thr Cys Cys Ala Gly Gly Gly Gly
165 170 175
Ala Cys Ala Gly Gly Thr Cys Thr Gly Gly Thr Thr Ala Thr Gly Gly
180 185 190
Ala Thr Gly Ala Ala Ala Gly Cys Ala Ala Cys Thr Gly Gly Ala Cys
195 200 205
Thr Gly Ala Thr Gly Thr Thr Gly Ala Gly Thr Thr Cys Thr Thr Gly
210 215 220
Ala Ala Cys Ala Cys Thr Gly Cys Ala Ala Ala Gly Cys Cys Ala Cys
225 230 235 240
Cys Cys Ala Cys Thr Thr Gly Gly Gly Gly Gly Thr Ala Thr Cys Cys
245 250 255
Thr Gly Thr Cys Thr Cys Cys Ala Ala Ala Ala Cys Ala Thr Thr Ala
260 265 270
Gly Cys Ala Gly Ala Gly Ala Ala Gly Gly Cys Gly Gly Cys Ala Thr
275 280 285
Gly Gly Ala Ala Ala Thr Thr Thr Gly Cys Thr Gly Ala Ala Gly Ala
290 295 300
Ala Ala Ala Thr Cys Ala Cys Ala Thr Thr Gly Ala Thr Cys Thr Cys
305 310 315 320
Ala Thr Cys Ala Cys Thr Gly Thr Gly Ala Thr Ala Cys Cys Thr Thr
325 330 335
Cys Thr Cys Thr Cys Ala Cys Ala Ala Cys Thr Gly Gly Thr Cys Cys
340 345 350
Thr Thr Cys Thr Cys Thr Cys Ala Cys Thr Cys Cys Ala Gly Ala Cys
355 360 365
Ala Thr Thr Cys Cys Thr Thr Cys Ala Ala Gly Thr Gly Thr Thr Gly
370 375 380
Gly Cys Cys Thr Cys Gly Cys Cys Ala Cys Gly Thr Cys Cys Cys Thr
385 390 395 400
Thr Ala Thr Ala Ala Cys Ala Gly Gly Cys Ala Ala Thr Gly Ala Thr
405 410 415
Thr Thr Cys Cys Thr Cys Ala Thr Ala Ala Ala Cys Gly Cys Thr Ala
420 425 430
Thr Gly Ala Ala Ala Gly Gly Thr Ala Thr Gly Cys Ala Gly Thr Thr
435 440 445
Thr Cys Thr Gly Thr Cys Ala Gly Gly Thr Thr Cys Ala Ala Thr Ala
450 455 460
Thr Cys Cys Ala Thr Cys Ala Cys Thr Cys Ala Thr Gly Thr Gly Gly
465 470 475 480
Ala Gly Gly Ala Thr Ala Thr Thr Thr Gly Cys Cys Gly Ala Gly Cys
485 490 495
Ala Cys Ala Thr Ala Thr Ala Thr Thr Thr Gly Thr Gly Gly Cys Gly
500 505 510
Gly Ala Gly Ala Ala Ala Gly Ala Ala Thr Cys Ala Gly Cys Thr Thr
515 520 525
Cys Thr Gly Gly Thr Cys Gly Ala Thr Ala Cys Ala Thr Thr Thr Gly
530 535 540
Cys Thr Cys Thr Gly Cys Thr Cys Ala Cys Ala Ala Thr Ala Cys Thr
545 550 555 560
Ala Gly Thr Gly Thr Cys Cys Cys Thr Gly Ala Gly Cys Thr Thr Gly
565 570 575
Cys Ala Ala Ala Gly Thr Thr Thr Cys Thr Thr Ala Gly Cys Ala Ala
580 585 590
Ala Cys Gly Ala Thr Ala Thr Cys Cys Thr Cys Ala Gly Thr Ala Thr
595 600 605
Ala Ala Ala Ala Thr Thr Cys Cys Ala Ala Cys Cys Gly Ala Ala Thr
610 615 620
Thr Cys Gly Ala Ala Gly Ala Thr Thr Gly Thr Cys Cys Cys Thr Cys
625 630 635 640
Ala Ala Ala Gly
<210> 4
<211> 18
<212> DNA
<213> 鸡血藤（Spatholobus suberectus Dunn）
<400> 4
cggatttatg gcctcttg 18
<210> 5
<211> 18
<212> DNA
<213> 鸡血藤（Spatholobus suberectus Dunn）
<400> 5
tgacttgatt ctcgcacc 18
<210> 6
<211> 19
<212> DNA
<213> 鸡血藤（Spatholobus suberectus Dunn）
<400> 6
tcttccagct tgctacacc 19
<210> 7
<211> 19
<212> DNA
<213> 鸡血藤（Spatholobus suberectus Dunn）
<400> 7
ctttgaggga caatcttcg 19
<210> 8
<211> 21
<212> DNA
<213> 鸡血藤（Spatholobus suberectus Dunn）
<400> 8
ggggacaggt ctggttatgg a 21
<210> 9
<211> 21
<212> DNA
<213> 鸡血藤（Spatholobus suberectus Dunn）
<400> 9
gcctttgagg gacaatcttc g 21
<210> 10
<211> 20
<212> DNA
<213> 鸡血藤（Spatholobus suberectus Dunn）
<400> 10
cgttcccgcc aatatctcac 20
<210> 11
<211> 20
<212> DNA
<213> 鸡血藤（Spatholobus suberectus Dunn）
<400> 11
tgttcaatac cagccgcacc 20
Claims (6)
1.编码鸡血藤花青素还原酶的gDNA，其特征在于，为下列核苷酸序列之一：
1)具有SEQ ID No.1所示的序列；
2)能编码SEQ ID No.1经添加、取代、插入或缺失一个或多个氨基酸且具有鸡血藤花青素还原酶活性蛋白质的核苷酸序列。
2.编码鸡血藤花青素还原酶的cDNA，其特征在于，为下列核苷酸序列之一：
1)具有SEQ ID No.2所示的序列；
2)在严格条件下与SEQ ID No.2的cDNA序列杂交且编码具有鸡血藤花青素还原酶活性蛋白质的DNA分子；
3)编码SEQ ID No.3所示氨基酸序列的核苷酸序列；
4)编码SEQ ID No.3经添加、取代、插入或缺失一个或多个氨基酸且具有鸡血藤花青素还原酶活性蛋白质的核苷酸序列。
3.具有鸡血藤花青素还原酶活性的蛋白质，其特征在于，为下列氨基酸序列之一：
1)具有SEQ ID No.3所示的氨基酸序列；
2)SEQ ID No.3经添加、取代、插入或缺失一个或多个氨基酸且具有鸡血藤花青素还原酶活性的氨基酸序列。
4.一种用于扩增权利要求1所述的gDNA的引物，其特征在于，所述引物的序列如SEQ IDNo.4和SEQ ID No.5所示。
5.一种用于扩增权利要求2所述的cDNA的引物，其特征在于，所述引物的序列如SEQ IDNo.6和SEQ ID No.7所示。
6.一种如权利要求1所述的gDNA或权利要求2所述的cDNA或权利要求3所述的蛋白质或权利要求4所述的引物或权利要求5所述的引物作为提高植物表儿茶素含量的应用。
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103031282A (zh) * | 2011-09-30 | 2013-04-10 | 中国农业科学院北京畜牧兽医研究所 | 紫花苜蓿花青素还原酶基因及其编码的蛋白和应用 |
| CN103146719A (zh) * | 2013-02-21 | 2013-06-12 | 吉首大学 | 华南美丽葡萄花青素还原酶基因及其编码的蛋白与应用 |
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2020
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103031282A (zh) * | 2011-09-30 | 2013-04-10 | 中国农业科学院北京畜牧兽医研究所 | 紫花苜蓿花青素还原酶基因及其编码的蛋白和应用 |
| CN103146719A (zh) * | 2013-02-21 | 2013-06-12 | 吉首大学 | 华南美丽葡萄花青素还原酶基因及其编码的蛋白与应用 |
Non-Patent Citations (2)
| Title |
|---|
| QIN,S.: ""TKY59382.1"", 《GENBANK》 * |
| 无: ""XM_028387217.1"", 《GENBANK》 * |
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