CN111218462B - 编码鸡血藤查尔酮合成酶的基因及其应用 - Google Patents
编码鸡血藤查尔酮合成酶的基因及其应用 Download PDFInfo
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- CN111218462B CN111218462B CN202010144945.6A CN202010144945A CN111218462B CN 111218462 B CN111218462 B CN 111218462B CN 202010144945 A CN202010144945 A CN 202010144945A CN 111218462 B CN111218462 B CN 111218462B
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Abstract
本发明公开了一种编码鸡血藤查尔酮合成酶的基因及其应用,该基因为首次从鸡血藤中获得,填补了我国传统中药材鸡血藤中分离克隆查尔酮合成酶基因的空白。本发明所提供的查尔酮合成酶基因具有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,其中在叶和花中表达量较高。本发明提供的查尔酮合成酶基因应用与提高鸡血藤中黄酮类化合物活性成分的含量,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,涉及鸡血藤黄酮类化合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查尔酮合成酶的基因及其应用。
背景技术
鸡血藤来源于豆科(Leguminosae)植物密花豆(Spatholobus suberectus Dunn)的干燥藤茎,为常用中药,味苦、甘,性温,归肝、肾经。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。目前市场上商品鸡血藤药材主要来源于野生资源,由于多年的采收导致中国的野生资源已濒临枯竭。
药用植物的次生代谢产物是中药材药效成分的主要来源,药效成分形成受其代谢途径中功能基因的调控。黄酮类化合物是植物次生代谢产物,常以游离态或与糖结合为苷的形式存在,数量种类繁多,结构类型复杂多样,在疾病的治疗过程中发挥重要的作用。黄酮类化合物是最早从鸡血藤药材中分离得到的化合物,且是鸡血藤发挥药效的主要成分,目前共分离鉴定了60个黄酮类化合物。
查尔酮合成酶是植物类黄酮物质生物合成途径中的第一个酶,也是黄酮类化合物合成的限速酶,对植物具有非常重要的生理意义。然而,目前尚未发现鸡血藤查尔酮合成酶(SsCHS)基因的克隆和作为提高植物中黄酮类化合物含量的应用研究。
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,获得鸡血藤查尔酮合成酶(简称SsCHS)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黄酮类化合物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查尔酮合成酶的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查尔酮合成酶活性蛋白质的核苷酸序列。
编码鸡血藤查尔酮合成酶的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查尔酮合成酶活性蛋白质的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查尔酮合成酶活性蛋白质的核苷酸序列。
具有鸡血藤查尔酮合成酶活性的蛋白质,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查尔酮合成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黄酮类化合物含量的应用。
具体的,能够极大程度提高植物叶和花部位中黄酮类化合物含量。
含有本发明的SsCHS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SsCHS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:
本发明针对鸡血藤黄酮类化合物生物合成研究方面的空白,提供了鸡血藤查尔酮合成酶(SsCHS)蛋白质及其编码基因的gDNA序列、cDNA序列及氨基酸序列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黄酮类化合物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SsCHS功能域预测分析;
图2为本发明所述的SsCHS与大豆、木豆、石榴、红掌、芸香、相思豆的CHS基因构建的系统进化树(NJ法);
图3为本发明所述的SsCHS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
SsCHS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CHS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:CCCGTCCCTCATACCTACTCGC(SEQ ID NO:4)
R1:AAGACATTCTCCTGCAACAC(SEQ ID NO:5)
通过PCR扩增,获得长度为2025bp的gDNA序列,如SEQ ID NO:1。
实施例2
SsCHS基因克隆
将实施例1提取的RNA进行逆转录(AMV first strand cDNAsynthesis kit:Roche,瑞士),以第一链cDNA为模板,利用引物
F1:ATGGTGACCGTGGAGGAAATCCG(SEQ ID NO:6)
R1:CCTTGGACCTCTTCCTCACC(SEQ ID NO:7)
通过PCR扩增,获得长度为1063bp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查尔酮合成酶(SsCHS)的氨基酸序列SEQ ID NO:3。
实施例4
SsCHS基因的生物信息学分析
本发明涉及的鸡血藤查尔酮合成酶基因经过ORF预测,序列中802-1857bp为ORF序列,长度为1056bp,编码氨基酸352个。将SsCHS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中的CHS有较高的同源性,其中与同科植物大豆同源性最高(如图1和2所示)。
实施例5
SsCHS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、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:
CHS-F:ACCGAAGTCAAAGATCACGC(SEQ ID NO:8)
CHS-R:TCCTCGACCTGGTCCAGAAT(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SsCHS基因在不同组织中的表达量。结果显示SsCHS基因在根、茎、叶、花、果实中均有表达,其中在花中表达量最高,其次是叶,果实中表达量最低(如图3所示)。
实施例6
SsCHS基因功能研究和应用
(1)植物表达载体的构建
以鸡血藤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)应用至本氏烟草
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。
将共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
采用紫外分光光度计法,以芦丁为对照品,测定转基因烟草叶片中总黄酮的含量。结果表明,转基因烟草中的总黄酮含量为非转基因烟草的2.6倍,效果显著。
查尔酮合成酶作为类黄酮生物合成途径中的第一个限速酶,其表达活性直接影响整个生物合成途径的进行。本发明通过基因克隆,获得SsCHS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黄酮类化合物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> 编码鸡血藤查尔酮合成酶的基因及其应用
<160> 11
<170> PatentIn version 3.5
<210> 1
<211> 2025
<212> DNA
<213> 鸡血藤(Spatholobus suberectus Dunn)
<400> 1
cccgtccctc atacctactc gccctcttca ctgcaccatc gaatctctct tctcagcgca 60
aaatatattt atttataacc ccttcccagt tctcccaatc cgtcaaaaat ctcacccact 120
tattcatctt ccaaattctt ccgctcctca cacctcagca aatctcactg tacccttttc 180
agcaccccgc gttgcctact gttaccaact aacccgagta aatggtgacc gtggaggaaa 240
tccgcaacgc ccagcgttcc catggccccg ccaccatctt ggccttcggc accgccactc 300
cctccaactg cgtcatccaa gccgattacc ccgactacta cttccgcata actaacagcg 360
aacacaagac cgacctcaaa gaaaaattca agcgcatgtg tatgtaatat gtaacattca 420
caataatagt gctccaccca atgtttttca atcgttgtcc cttggttagg ttgtcctagt 480
cgagaaaatt aggaaaattt aggattagtg ctgatggccc agctatttct tggtccgact 540
atgtgcatct gcagacaacc tgatccacgg agacggatat tagagcacgg attctccctg 600
cttgaaaaaa attgaaaagt gtgcagttca cacgtctaac catccatgca tgtatttttt 660
atatattcaa attattaaat attagaatta atcaatttaa gccgactcat tatcaaagtg 720
ctcggccctt tggactgaat tactaatgga atattcggcg tagttttggg gagggtccag 780
atgatgatac taatacaata ggttaagttt gggtttatta tagtcacggg actcactgaa 840
attaatttta attttgcagg tgagaagtcg atgataaaga agcggtacat gcacctgacg 900
gaggagtttc tgaaggagaa cccgaacatg tgcgcgtaca tggcaccgtc gctggacgcg 960
aggcaggaca tagtggtggt tgaagtgcca aagctcggga aggaggccgc gacaaaggcg 1020
atcaaggagt ggggtcaacc gaagtcaaag atcacgcacc tcgtgttctg caccacttca 1080
ggggtggaca tgcccggcgc agattaccag ctcacgaagc ttctgggcct gagaccctcc 1140
gtgaagcgcc tcatgatgta ccagcagggc tgcttcgccg gcggcaccgt gctccgcctc 1200
gcgaaggacc tcgccgagaa caacaagggc gccagggtgc tcgtcgtctg ctccgagatc 1260
accgccgtga cgttccgcgg cccgtcggac gcccacctcg actcgctcgt cgggcaggcg 1320
ctcttcggcg acggcgccgc ggccgtcatc gtcggcgcgg accccgaccc cgcggtcgag 1380
cggccggtct tcgagctcgt ctcggcggcg cagacgatcc tgccggactc cgagggcgcc 1440
atcgacggcc acctccgcga ggtggggctg acgtttcatc ttctcaagga cgttcccgga 1500
atcatctcga agaacatcga gaagagtctc gtggaagctt ttgcgccgat tgggatcagc 1560
gactggaact cgatcttttg ggtcgcgcac ccgggcggac cggcgattct ggaccaggtc 1620
gaggagaagc tccggctcaa ggaggagaag ctccggtcca cccggcacgt gctgagcgag 1680
tacgggaaca tgtccagcgc gtgcgttttg tttattctcg acgaggtgag gaagaggtcc 1740
aaggaggaag ggaagggaac cactggggaa gggttagagt ggggggtgct attcgggttc 1800
gggccgggtc tgaccgttga gaccgttgtg ctgcacagcg ttcccttgga gggttgatac 1860
atgaggtggg gcccactggt ttgtatggga agctaaagta gtactggtaa ttaataagga 1920
aacctcgaga aactggagga acagtgtgat gtgattttac tgttacggta gttttaaatt 1980
ttaattatgg gtttatcggg ccgtagtgtt gcaggagaat gtctt 2025
<210> 2
<211> 1063
<212> DNA
<213> 鸡血藤(Spatholobus suberectus Dunn)
<400> 2
atggtgaccg tggaggaaat ccgcaacgcc cagcgttccc atggccccgc caccatcttg 60
gccttcggca ccgccactcc ctccaactgc gtcatccaag ccgattaccc cgactactac 120
ttccgcataa ctaacagcga acacaagacc gacctcaaag aaaaattcaa gcgcatgtgt 180
gagaagtcga tgataaagaa gcggtacatg cacctgacgg aggagtttct gaaggagaac 240
ccgaacatgt gcgcgtacat ggcaccgtcg ctggacgcga ggcaggacat agtggtggtt 300
gaagtgccaa agctcgggaa ggaggccgcg acaaaggcga tcaaggagtg gggtcaaccg 360
aagtcaaaga tcacgcacct cgtgttctgc accacttcag gggtggacat gcccggcgca 420
gattaccagc tcacgaagct tctgggcctg agaccctccg tgaagcgcct catgatgtac 480
cagcagggct gcttcgccgg cggcaccgtg ctccgcctcg cgaaggacct cgccgagaac 540
aacaagggcg ccagggtgct cgtcgtctgc tccgagatca ccgccgtgac gttccgcggc 600
ccgtcggacg cccacctcga ctcgctcgtc gggcaggcgc tcttcggcga cggcgccgcg 660
gccgtcatcg tcggcgcgga ccccgacccc gcggtcgagc ggccggtctt cgagctcgtc 720
tcggcggcgc agacgatcct gccggactcc gagggcgcca tcgacggcca cctccgcgag 780
gtggggctga cgtttcatct tctcaaggac gttcccggaa tcatctcgaa gaacatcgag 840
aagagtctcg tggaagcttt tgcgccgatt gggatcagcg actggaactc gatcttttgg 900
gtcgcgcacc cgggcggacc ggcgattctg gaccaggtcg aggagaagct ccggctcaag 960
gaggagaagc tccggtccac ccggcacgtg ctgagcgagt acgggaacat gtccagcgcg 1020
tgcgttttgt ttattctcga cgaggtgagg aagaggtcca agg 1063
<210> 3
<211> 354
<212> PRT
<213> 鸡血藤(Spatholobus suberectus Dunn)
<400> 3
Met Val Thr Val Glu Glu Ile Arg Asn Ala Gln Arg Ser His Gly Pro
1 5 10 15
Ala Thr Ile Leu Ala Phe Gly Thr Ala Thr Pro Ser Asn Cys Val Ile
20 25 30
Gln Ala Asp Tyr Pro Asp Tyr Tyr Phe Arg Ile Thr Asn Ser Glu His
35 40 45
Lys Thr Asp Leu Lys Glu Lys Phe Lys Arg Met Cys Glu Lys Ser Met
50 55 60
Ile Lys Lys Arg Tyr Met His Leu Thr Glu Glu Phe Leu Lys Glu Asn
65 70 75 80
Pro Asn Met Cys Ala Tyr Met Ala Pro Ser Leu Asp Ala Arg Gln Asp
85 90 95
Ile Val Val Val Glu Val Pro Lys Leu Gly Lys Glu Ala Ala Thr Lys
100 105 110
Ala Ile Lys Glu Trp Gly Gln Pro Lys Ser Lys Ile Thr His Leu Val
115 120 125
Phe Cys Thr Thr Ser Gly Val Asp Met Pro Gly Ala Asp Tyr Gln Leu
130 135 140
Thr Lys Leu Leu Gly Leu Arg Pro Ser Val Lys Arg Leu Met Met Tyr
145 150 155 160
Gln Gln Gly Cys Phe Ala Gly Gly Thr Val Leu Arg Leu Ala Lys Asp
165 170 175
Leu Ala Glu Asn Asn Lys Gly Ala Arg Val Leu Val Val Cys Ser Glu
180 185 190
Ile Thr Ala Val Thr Phe Arg Gly Pro Ser Asp Ala His Leu Asp Ser
195 200 205
Leu Val Gly Gln Ala Leu Phe Gly Asp Gly Ala Ala Ala Val Ile Val
210 215 220
Gly Ala Asp Pro Asp Pro Ala Val Glu Arg Pro Val Phe Glu Leu Val
225 230 235 240
Ser Ala Ala Gln Thr Ile Leu Pro Asp Ser Glu Gly Ala Ile Asp Gly
245 250 255
His Leu Arg Glu Val Gly Leu Thr Phe His Leu Leu Lys Asp Val Pro
260 265 270
Gly Ile Ile Ser Lys Asn Ile Glu Lys Ser Leu Val Glu Ala Phe Ala
275 280 285
Pro Ile Gly Ile Ser Asp Trp Asn Ser Ile Phe Trp Val Ala His Pro
290 295 300
Gly Gly Pro Ala Ile Leu Asp Gln Val Glu Glu Lys Leu Arg Leu Lys
305 310 315 320
Glu Glu Lys Leu Arg Ser Thr Arg His Val Leu Ser Glu Tyr Gly Asn
325 330 335
Met Ser Ser Ala Cys Val Leu Phe Ile Leu Asp Glu Val Arg Lys Arg
340 345 350
Ser Lys
<210> 4
<211> 22
<212> DNA
<213> 鸡血藤(Spatholobus suberectus Dunn)
<400> 4
cccgtccctc atacctactc gc 22
<210> 5
<211> 20
<212> DNA
<213> 鸡血藤(Spatholobus suberectus Dunn)
<400> 5
aagacattct cctgcaacac 20
<210> 6
<211> 23
<212> DNA
<213> 鸡血藤(Spatholobus suberectus Dunn)
<400> 6
atggtgaccg tggaggaaat ccg 23
<210> 7
<211> 20
<212> DNA
<213> 鸡血藤(Spatholobus suberectus Dunn)
<400> 7
ccttggacct cttcctcacc 20
<210> 8
<211> 20
<212> DNA
<213> 鸡血藤(Spatholobus suberectus Dunn)
<400> 8
accgaagtca aagatcacgc 20
<210> 9
<211> 20
<212> DNA
<213> 鸡血藤(Spatholobus suberectus Dunn)
<400> 9
tcctcgacct ggtccagaat 20
<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 (2)
1.编码鸡血藤查尔酮合成酶的cDNA,其特征在于,为下列核苷酸序列之一:
1)如SEQ ID No.2所示的序列;
2)编码SEQ ID No.3所示氨基酸序列的核苷酸序列。
2.一种如权利要求1所述的cDNA作为提高植物黄酮类化合物含量的应用。
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