CN108977414A - 一种β-胡萝卜素酮化酶的人工合成突变体及其编码序列和应用 - Google Patents
一种β-胡萝卜素酮化酶的人工合成突变体及其编码序列和应用 Download PDFInfo
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Abstract
本发明提供了一种β‑胡萝卜素酮化酶的人工合成突变体及其编码序列和应用，属于基因工程技术领域。本发明提供了一种β‑胡萝卜素酮化酶的人工合成突变体，所述人工合成突变体的氨基酸序列如SEQ ID No.1所示。本发明提供了上述人工合成突变体的编码序列，所述编码序列的核苷酸序列如SEQ ID NO.2所示。利用上述编码序列转化苔藓，可显著提高被转化苔藓的色素合成能力以及抗逆能力。
Description
技术领域
本发明属于基因工程技术领域，具体涉及一种β-胡萝卜素酮化酶的人工合成突变体及其编码序列和应用。
背景技术
类胡萝卜素是自然界普遍存在的一种天然色素，目前，已发现600多种天然类胡萝卜素，β-胡萝卜素、叶黄素、玉米黄质和虾青素等都属于类胡萝卜素。类胡萝卜素是维生素A前体，能预防癌症、延缓衰老、提高免疫力等，对人类健康起着关键作用。β-胡萝卜素酮化酶(BKT)是类胡萝卜素生物合成途径中的关键调节酶，是玉米黄素和β-胡萝卜素等类胡萝卜素合成虾青素等酮式类胡萝卜素的过程中的关键酶。通常，从β-胡萝卜素生物合成虾青素的过程中，需分别在β-胡萝卜素羟化酶(BHY)和酮化酶(BKT)的催化作用下将末端β-紫罗兰酮环的3号和4号位C原子合成羰基和羟基。多数高等植物中因缺乏β-胡萝卜素酮化酶导致植株体内类胡萝卜素含量偏低。
已有许多研究显示，高等植物中过表达类胡萝卜素合成途径中的关键基因能引起色素水平的变化。目前，在马铃薯、烟草和番茄中都成功异源表达了类胡萝卜素合成关键酶基因，获得了虾青素、叶黄素等含量升高且类胡萝卜素总含量升高的转基因植株。
苔藓植物因其植株矮小，枝形精细优美，持绿性强在立体绿化市场深受欢迎，然而缺乏抗逆性限制了其大面积推广应用。目前，对类胡萝卜素异源表达的研究多集中在粮食蔬菜类作物，针对苔藓等园艺绿化植物的研究还未见报道，极大地限制了苔藓产业的发展。
发明内容
有鉴于此，本发明的目的在于提供一种能提高苔藓色素合成能力及抗逆性的β-胡萝卜素酮化酶的人工合成突变体及其编码序列和应用。
本发明提供了一种β-胡萝卜素酮化酶的人工合成突变体，所述人工合成突变体的氨基酸序列如SEQ ID No.1所示。
本发明提供了上述人工合成突变体的编码序列，所述编码序列的核苷酸序列如SEQ ID NO.2所示。
本发明提供了一种含有上述编码序列的重组载体，所述重组载体包括重组质粒或重组植物表达载体。
本发明还提供了一种包含上述重组载体的表达系统，所述表达系统包括重组载体和宿主细胞，所述宿主细胞包括大肠杆菌细胞、农杆菌细胞或植物细胞。
本发明提供了上述人工合成突变体、编码序列、重组载体和/或表达系统在促进植物色素合成和/或抗逆改良中的应用。
优选的，所述促进植物色素合成和/或抗逆改良的方法包括如下步骤：
(1)将所述编码序列连接于植物表达调控序列，形成植物表达载体；
(2)将所述植物表达载体转入植物细胞，筛选得到转化细胞；
(3)将所述转化细胞进行植株再生，得到改良植物。
优选的，所述植物为苔藓。
优选的，所述抗逆的性状包括抗高温、抗干旱和抗高盐。
有益效果：本发明提供了一种β-胡萝卜素酮化酶的人工合成突变体，所述人工合成突变体由385个氨基酸组成，其氨基酸序列如SEQ ID No.1所示。本发明还提供了上述人工合成突变体的编码序列，所述编码序列的核苷酸序列如SEQ ID NO.2所示。所述编码序列不仅具有控制苔藓色素合成的功能，还能提高苔藓抗逆性，以此为基础可用来进行苔藓品种改良。本发明利用上述编码序列构建重组载体，然后利用PEG介导的方式转化苔藓，可在苔藓植株体内表达得到β-胡萝卜素酮化酶的人工合成突变体，促进β-胡萝卜素在苔藓植株体内的酮化，进而提高被转化苔藓植株体内酮式类胡萝卜素的合成能力以及被转化苔藓植株的抗逆能力。
附图说明
图1为本发明实施例1所述CrBKT过表达植株及野生型色素水平分析结果统计；
图2为本发明实施例1所述CrBKTm过表达植株在DNA水平鉴定下的PCR扩增产物电泳结果；
图3为本发明实施例1所述CrBKTm过表达植株在RNA水平鉴定下的PCR扩增产物电泳结果；
图4为本发明实施例1所述高温处理前后及恢复阶段CrBKT过表达植株和野生型植株叶绿素荧光参数Fv/Fm的变化结果统计。
具体实施方式
本发明提供了一种β-胡萝卜素酮化酶的人工合成突变体，所述人工合成突变体的氨基酸序列如SEQ ID No.1所示，总共包含385个氨基酸。所述人工合成突变体通过包含所述突变体的编码序列的重组载体转入宿主菌或植物细胞中表达获得。
本发明提供了上述人工合成突变体的编码序列，所述编码序列的核苷酸序列如SEQ ID NO.2所示。所述编码序列是在莱茵衣藻的β-胡萝卜素酮化酶编码序列(CrBKT)的基础上，通过基因优化获得。所述优化的方法是针对苔藓植物，通过在原始CrBKT基因的上游增加了一段突变序列，从而使其具有在苔藓中表达的能力。所述突变序列的核苷酸序列如SEQ ID NO.3所示。
本发明提供了一种含有上述编码序列的重组载体，所述重组载体包括重组质粒或重组植物表达载体。所述重组质粒能导入大肠杆菌或农杆菌，通过细菌的增殖实现编码序列的扩增，或利用农杆菌对植物体进行转化；所述重组植物表达载体能转入植物细胞中获得表达，所述植物细胞能通过再生手段得到具有表达上述编码序列功能的改良植物。在本发明中，所述重组载体的构建方法参考NIBB实验室的方法(http://moss.nibb.ac.jp/protocol.html)。本发明提供了扩增该编码序列的引物对，其中，上游引物的核苷酸序列如SEQ IDNo.4所示，下游引物的核苷酸序列如SEQ ID No.5所示。
本发明还提供了一种用于表达上述重组载体的表达系统。所述表达系统包括重组载体和宿主细胞，所述宿主细胞包括大肠杆菌细胞、农杆菌细胞或植物细胞。本发明对上述宿主细胞的来源不作特别限定，本领域常规用于重组载体表达的宿主细胞均可。
本发明还提供了上述β-胡萝卜素酮化酶的人工合成突变体、编码序列、DNA水平鉴定用引物对、RNA水平鉴定用引物对、重组载体和/或宿主细胞在促进植物色素合成和抗逆改良中的应用。
在本发明中，所述促进植物色素合成和/或抗逆改良的方法优选包括如下步骤：
(1)将所述编码序列连接于植物表达调控序列，形成重组植物表达载体；
(2)将所述植物表达载体转入植物细胞，筛选得到转化细胞；
(3)将所述转化细胞进行植株再生，得到改良植物。
在本发明中，所述植物优选为苔藓。所述抗逆的性状优选包括抗高温、抗干旱和抗高盐。
得到改良植物后，本发明还优选包括对改良植物的转化及表达情况的鉴定方法。所述鉴定方法包括DNA水平鉴定和RNA水平鉴定。所述DNA水平鉴定用引物对的上游引物的核苷酸序列如SEQ ID No.6所示，下游引物的核苷酸序列如SEQ ID No.7所示。在含有所述编码序列的扩增体系中，使用上述DNA水平鉴定用引物对能扩增得到长度为569bp的片段。所述RNA水平鉴定用引物对的上游引物的核苷酸序列如SEQ ID No.8所示，下游引物的核苷酸序列如SEQ ID No.9所示。在含有所述编码序列转录的mRNA的扩增体系中，使用上述RNA水平鉴定用引物对能扩增得到长度为153bp的片段。
下面结合实施例对本发明提供的一种β-胡萝卜素酮化酶的人工合成突变体及其编码序列和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
苔藓β-胡萝卜素酮化酶基因人工合成突变体(CrBKTm)的制备及功能验证
(1)苔藓的培养方法
使用BCDAT培养基培养苔藓，从培养5～6天的原丝体材料打磨继代后于14小时光照/10小时黑暗的光周期，500umol·m-2·s-1的光照强度，25℃条件下培养40天，进入均一的配子体世代。
(2)转化载体构建
将莱茵衣藻的β-胡萝卜素酮化酶基因(CrBKT)的编码序列通过同源重组方法克隆在质粒pPOG1的Not1和Sal1酶切位点之间。该载体信息来自于网站http://moss.nibb.ac.jp/protocol.html。
(3)苔藓的转化
选用聚乙二醇(PEG)介导的DNA引入原生质体的方法。PEG介导转化法是最高效的同源重组与基因打靶的方法。本实验所涉及的苔藓PEG介导转化法，该方法参考日本NIBB实验室进行(http://moss.nibb.ac.jp/protocol.html)。
(4)色素的提取及含量的测定
本实验中色素含量的测定用分光光度法进行，用N，N-二甲基甲酰胺(DMF)作为提取液进行色素的提取，提取液4℃过夜浸泡样品后取样分别测定664nm、647nm、480nm出的吸光度，并将侵泡后样品烘干测定其干重，按Moran的方法(Moran et al.,1980)计算各色素成分的含量，CrBKT过表达植株及野生型色素水平分析结果统计见图1。图1分别表示了过表达植株CrBKT和野生型WT植株的叶绿素a、叶绿素b、叶绿素总量及类胡萝卜素总量水平。结果显示：与WT相比，CrBKT过表达植株中的叶绿素a、叶绿素b、叶绿素总量显著降低，而类胡萝卜素总量显著升高。表明CrBKT基因的过表达引起了苔藓植株色素含量的变化。
(5)DNA水平鉴定方法
本实验中苔藓总DNA的提取采用CTAB法，并使用南京诺唯赞公司的高保真聚合酶：PhataMaster(p505)进行PCR扩增，体系(50μl)：F(10μM)1ul，R(10μM)1ul，PhataMaster 1μl，2×buffer25μl，dNTP 1μl，H2O21μl。使用伯乐T100PCR仪，程序：95℃预变性5min，95℃变性20s，59℃退火30s，72℃延伸1min(扩增效率1kb/min)，35个循环，循环后72℃延伸5min，4℃保温。DNA水平鉴定用引物对如表1所示。PCR扩增结果见图2。其中，左起第一泳道M为DNAMarker，第二泳道WT为野生型，第三泳道CrBKT为测试阳性植物样本，第四泳道P为阳性对照。PCR检测可以得到预期大小的片段567bp，说明目标基因已经存在于苔藓基因组中。
表1.DNA水平鉴定引物
(6)RNA水平鉴定方法
本实验中总RNA的提取采用Trizol法，并使用购自北京全式金生物公司的反转录试剂盒One-Step gDNA Removal and cDNA Synthesis SuperMix进行cDNA合成，之后用南京诺唯赞公司的高保真聚合酶：PhataMaster(p505)进行RNA水平的检测。体系(50μl)：F(10μM)1ul，R(10μM)1ul，PhataMaster1μl，2×buffer 25μl，dNTP 1μl，H2O21μl。使用伯乐T100PCR仪，程序：95℃预变性5min，95℃变性20s，56℃退火30s，72℃延伸30s(扩增效率1kb/min)，27个循环，循环后72℃延伸5min，4℃保温。RNA水平鉴定用引物对如表2所示。PCR扩增结果见图3。其中，左起第一泳道M1为DNAMarker，第二泳道CrBKT为测试阳性植物样本，第六泳道WT为野生型。PCR检测可以得到预期大小的片段153bp，说明目标基因在过表达植株中表达。
表2.RNA水平鉴定引物：
(7)高温胁迫处理
将培养有5周的均一的配子体世代的植物材料的培养基，在45℃恒温培养箱中处理4小时，然后将培养基及材料转移到正常培养条件下恢复培养。高温处理前后及恢复阶段CrBKT过表达植株和野生型植株叶绿素荧光参数Fv/Fm的变化结果统计见图4。图4分别表示过表达植株CrBKT和野生型WT植株在正常条件下和45℃，4h处理后及恢复培养7d后Fv/Fm的变化情况。结果显示：WT和CrBKT在处理前Fv/Fm无差异，经45℃，4h处理后，WT和CrBKT的Fv/Fm值都急剧下降，但CrBKT的值显著高于WT；在7天恢复培养后，植株的Fv/Fm值都发生了部分恢复，但CrBKT的值显著高于WT，这表明：与野生型相比，CrBKT过表达植株具有更强的高温胁迫耐受力。
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> 中国科学院昆明植物研究所
<120> 一种β-胡萝卜素酮化酶的人工合成突变体及其编码序列和应用
<160> 9
<170> SIPOSequenceListing 1.0
<210> 1
<211> 385
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Met Ala Ser Met Ile Ser Ser Ser Ala Val Thr Thr Val Ser Arg Ala
1 5 10 15
Ser Arg Gly Gln Ser Ala Ala Val Ala Pro Phe Gly Gly Leu Lys Ser
20 25 30
Met Thr Gly Phe Pro Val Lys Lys Val Asn Thr Asp Ile Thr Ser Ile
35 40 45
Thr Ser Asn Gly Gly Arg Val Lys Cys Met Gly Pro Gly Ile Gln Pro
50 55 60
Thr Ser Ala Arg Pro Cys Ser Arg Thr Lys His Ser Arg Phe Ala Leu
65 70 75 80
Leu Ala Ala Ala Leu Thr Ala Arg Arg Val Lys Gln Phe Thr Lys Gln
85 90 95
Phe Arg Ser Arg Arg Met Ala Glu Asp Ile Leu Lys Leu Trp Gln Arg
100 105 110
Gln Tyr His Leu Pro Arg Glu Asp Ser Asp Lys Arg Thr Leu Arg Glu
115 120 125
Arg Val His Leu Tyr Arg Pro Pro Arg Ser Asp Leu Gly Gly Ile Ala
130 135 140
Val Ala Val Thr Val Ile Ala Leu Trp Ala Thr Leu Phe Val Tyr Gly
145 150 155 160
Leu Trp Phe Val Lys Leu Pro Trp Ala Leu Lys Val Gly Glu Thr Ala
165 170 175
Thr Ser Trp Ala Thr Ile Ala Ala Val Phe Phe Ser Leu Glu Phe Leu
180 185 190
Tyr Thr Gly Leu Phe Ile Thr Thr His Asp Ala Met His Gly Thr Ile
195 200 205
Ala Leu Arg Asn Arg Arg Leu Asn Asp Phe Leu Gly Gln Leu Ala Ile
210 215 220
Ser Leu Tyr Ala Trp Phe Asp Tyr Ser Val Leu His Arg Lys His Trp
225 230 235 240
Glu His His Asn His Thr Gly Glu Pro Arg Val Asp Pro Asp Phe His
245 250 255
Arg Gly Asn Pro Asn Leu Ala Val Trp Phe Ala Gln Phe Met Val Ser
260 265 270
Tyr Met Thr Leu Ser Gln Phe Leu Lys Ile Ala Val Trp Ser Asn Leu
275 280 285
Leu Leu Leu Ala Gly Ala Pro Leu Ala Asn Gln Leu Leu Phe Met Thr
290 295 300
Ala Ala Pro Ile Leu Ser Ala Phe Arg Leu Phe Tyr Tyr Gly Thr Tyr
305 310 315 320
Val Pro His His Pro Glu Lys Gly His Thr Gly Ala Met Pro Trp Gln
325 330 335
Val Ser Arg Thr Ser Ser Ala Ser Arg Leu Gln Ser Phe Leu Thr Cys
340 345 350
Tyr His Phe Asp Leu His Trp Glu His His Arg Trp Pro Tyr Ala Pro
355 360 365
Trp Trp Glu Leu Pro Lys Cys Arg Gln Ile Ala Arg Gly Ala Ala Leu
370 375 380
Ala
385
<210> 2
<211> 1155
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
atggcttcta tgatatcctc ttccgctgtg acaacagtca gccgtgcctc tagggggcaa 60
tccgccgcag tggctccatt cggaggcctg aaatccatga ctggattccc agtgaagaag 120
gtcaacactg acattacttc cattacaagc aatggtggaa gagtaaagtg catgggccct 180
gggatacaac ccacttccgc gcgaccgtgt tctaggacca aacacagtcg atttgcgcta 240
cttgccgcag cgctgaccgc acgacgcgtc aagcagttca cgaagcagtt ccgctcgcgt 300
aggatggcgg aggacatact gaagctgtgg cagcgccaat atcacctgcc gcgcgaggat 360
tctgacaagc gcacgctgcg cgagcgcgtt cacctgtacc gcccgccgcg ttcagaccta 420
ggtggcattg cggtcgctgt gacagtcatc gcgctgtggg cgacgctgtt tgtctacggg 480
ctgtggttcg tcaagctgcc atgggcgctc aaagtgggcg agacagccac gtcctgggca 540
accattgctg ctgtattctt tagcctggaa ttcctttaca ccgggctctt catcaccacg 600
cacgacgcga tgcatggcac catcgcgctg cgcaaccggc gcctgaacga ctttctgggc 660
cagctggcaa tcagcctata cgcctggttt gactactccg tcctgcaccg caagcactgg 720
gagcaccaca accacaccgg ggagccgcgt gtggatccgg acttccaccg cggcaacccc 780
aacctggcgg tgtggttcgc gcagttcatg gtgtcgtaca tgaccctcag ccagttcctc 840
aagatcgcgg tctggtccaa cctgctgctg ctggcgggtg cgccgctggc caaccagctg 900
ctgttcatga cggcggcgcc catcctgtcc gccttccgcc tgttctacta cggcacctac 960
gtgccgcacc acccggagaa ggggcacacc ggcgccatgc cctggcaggt atcccgcacc 1020
agctccgcct cccggctgca gtcgttcctc acctgctacc acttcgacct gcactgggag 1080
caccaccgct ggccctacgc gccctggtgg gagctgccca agtgccgcca gattgcccgc 1140
ggcgcagccc tggcg 1155
<210> 3
<211> 171
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
atggcttcta tgatatcctc ttccgctgtg acaacagtca gccgtgcctc tagggggcaa 60
tccgccgcag tggctccatt cggaggcctg aaatccatga ctggattccc agtgaagaag 120
gtcaacactg acattacttc cattacaagc aatggtggaa gagtaaagtg c 171
<210> 4
<211> 18
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
atggcttcta tgatatcc 18
<210> 5
<211> 14
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
cgccagggct gcgc 14
<210> 6
<211> 26
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
cgcatggctt ctatgatatc ctcttc 26
<210> 7
<211> 26
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
gctaaagaat acagcagcaa tggttg 26
<210> 8
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
caatcagcct atacgcctgg 20
<210> 9
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
tgtacgacac catgaactgc 20
Claims (8)
1.一种β-胡萝卜素酮化酶的人工合成突变体，所述人工合成突变体的氨基酸序列如SEQID No.1所示。
2.权利要求1所述人工合成突变体的编码序列，所述编码序列的核苷酸序列如SEQ IDNO.2所示。
3.一种含有权利要求2所述编码序列的重组载体，所述重组载体包括重组质粒或重组植物表达载体。
4.一种包含表达权利要求3所述重组载体的表达系统，包括重组载体和宿主细胞，其特征在于，所述宿主细胞包括大肠杆菌细胞、农杆菌细胞或植物细胞。
5.权利要求1所述人工合成突变体、权利要求2所述编码序列、权利要求3所述重组载体和权利要求4所述表达系统中的任一项或任几项在促进植物色素合成和/或抗逆改良中的应用。
6.根据权利要求5所述的应用，其特征在于，所述促进植物色素合成和/或抗逆改良的方法包括如下步骤：
(1)将权利要求2所述编码序列连接于植物表达调控序列，形成植物表达载体；
(2)将所述植物表达载体转入植物细胞，筛选得到转化细胞；
(3)将所述转化细胞进行植株再生，得到改良植物。
7.根据权利要求5所述的应用，其特征在于，所述植物为苔藓。
8.根据权利要求5所述的应用，其特征在于，所述抗逆的性状包括抗高温、抗干旱和抗高盐。
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