CN112746060B - 水稻类异戊二烯合成相关蛋白dxr在调控水稻叶色中的应用 - Google Patents

水稻类异戊二烯合成相关蛋白dxr在调控水稻叶色中的应用 Download PDF

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CN112746060B
CN112746060B CN202110100699.9A CN202110100699A CN112746060B CN 112746060 B CN112746060 B CN 112746060B CN 202110100699 A CN202110100699 A CN 202110100699A CN 112746060 B CN112746060 B CN 112746060B
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刘喜
王迪
许子怡
杨颜榕
程行
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Hefei Jinglong Environmental Protection Technology Co ltd
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Abstract

本发明属于植物基因工程技术领域，具体涉及水稻类异戊二烯合成相关蛋白DXR在调控水稻叶色中的应用。本发明要解决的技术问题是对水稻DXR蛋白开展功能研究，为水稻的后续分子设计育种奠定基础。本发明的技术方案是水稻类异戊二烯合成相关蛋白DXR在调控水稻叶色中的应用，氨基酸序列如SEQ ID No.2所示。本发明还提供了调控水稻叶色的方法，包括如下步骤：将调控DXR蛋白表达的重组载体、含有该重组载体的转基因细胞系或重组菌转化水稻，培育筛选，获得目标叶色的水稻。本发明通过实验验证了水稻类异戊二烯合成相关蛋白DXR的作用，明确了该蛋白参与了水稻的叶绿素合成，可用于调控水稻叶色，对水稻遗传改良具有重要的意义。

Description

水稻类异戊二烯合成相关蛋白DXR在调控水稻叶色中的应用
技术领域
本发明属于植物基因工程技术领域，具体涉及水稻类异戊二烯合成相关蛋白DXR在调控水稻叶色中的应用。
背景技术
叶绿素是一种植物的主要光合色素，可捕获光能并产生活性氧。在拟南芥中鉴定出15种参与叶绿素合成的酶。这些叶绿素合成基因突变均导致拟南芥呈现出色素缺乏的表型。叶绿素含量直接影响光合效率，最终影响作物产量。
水稻是重要的主食作物，是植物遗传学研究的理想模式材料。通过物理和化学诱变、T-DNA插入和基因编辑技术创制鉴定了许多水稻叶色变异突变体。在这些水稻叶色突变体中，有3个涉及类异戊二烯生物合成，即OsHMBPP、IspE与IspF。类异戊二烯对植物生长和发育至关重要，从古细菌、细菌和真核生物中分离出数以万计的类异戊二烯化合物。异戊烯基二磷酸(IPP)和二甲基烯丙基二磷酸(DMAPP)是类异戊二烯生物合成所需的两种基本的五碳分子。在高等植物中，类异戊二烯的合成途径有两条，分别位于质体和细胞质中，即细胞质戊酸甲酯(MVA)途径和2-c-甲基-脱三醇-4-磷酸(MEP)途径。DOXP还原异构酶(DXR) 是MEP途径的主要限速酶。DXR可以催化1-脱氧-D-木酮糖-5-磷酸(DOXP)转化为2-C- 甲基-D-赤藓糖醇4-磷酸(MEP)。在薄荷中，DXR的超量表达提高精油的产量。然而，DXR 在作物尤其是水稻中的作用尚未报道。
发明内容
本发明要解决的技术问题是对水稻DXR蛋白开展功能研究，为水稻的后续分子设计育种奠定基础。
本发明的技术方案是水稻类异戊二烯合成相关蛋白DXR在调控水稻叶色中的应用，氨基酸序列如SEQ ID No.2所示。
进一步的，水稻类异戊二烯合成相关蛋白DXR的编码基因的核苷酸序列如SEQ IDNo.1 所示。
本发明还提供了调控水稻叶色的方法，包括如下步骤：将调控DXR蛋白表达的重组载体、含有该重组载体的转基因细胞系或重组菌转化水稻，培育筛选，获得目标叶色的水稻；所述 DXR蛋白的氨基酸序列如SEQ ID No.2所示。
具体的，所述调控DXR蛋白表达的重组载体为抑制DXR蛋白表达的重组载体。
进一步的，所述抑制DXR蛋白表达的重组载体为sgRNA表达载体表达载体，载体的靶标位点的核苷酸序列如SEQ ID NO.1的第49位～68位所示。
具体的，所述方法的具体操作如下：构建具有靶标序列的sgRNA表达载体，转入根癌农杆菌EHA105，用含有靶标序列的sgRNA表达载体的根癌农杆菌转化水稻细胞中，培育筛选，得到叶片白化的转基因水稻。
类异戊二烯合成途径涉及到很多酶，在水稻中，目前仅克隆到相关三个基因与水稻叶色相关，且三个基因突变体叶色表型也是各不相同的。因此，为了验证水稻类异戊二烯合成相关蛋白DXR的编码基因功能，申请人进行了相关研究。
本发明的有益效果：
本发明通过实验验证了水稻类异戊二烯合成相关蛋白DXR的作用，明确了该蛋白参与了水稻的叶绿素合成，可用于调控水稻叶色，对水稻遗传改良具有重要的意义。基于该功能，可对利用该基因来调控水稻的叶色，在观光农业中获得白化观赏水稻。同时，DXR参与了叶绿素的合成，叶绿素又会影响光合作用，涉及营养的积累，调控类异戊二烯合成相关蛋白DXR 表达，可进一步控制水稻类异戊二烯相关产物的含量，从而达到调控稻谷品质的目的。
附图说明
图1、野生型日本晴与突变体dxr-1、dxr-2的表型(b、c)；a、CRISPR/Cas9基因编辑鉴定图；ATG与TAG分别代表起始密码子与终止密码子。
图2、野生型和突变体叶绿素含量测定图；纵坐标为叶绿素含量，横坐标代表叶绿素a (Chla)和叶绿素b(Chlb)。
图3、野生型和突变体叶绿体透射电镜观察图；a、b是野生型水稻叶绿体结构图；c、d 是突变体的叶绿体结构图；a、c标尺为2μm；b、d标尺为500nm。
图4、DXR的组织表达(a)与亚细胞定位图(b)。
图5、叶绿素合成与叶绿体发育相关基因的表达水平分析图；纵坐标为相对表达，横坐标为叶绿素合成(HEMA、URO-D、CHLM、CHLH、DVR、CRD、CHLG、CAO、PORA) 与叶绿体发育(V1、V4、rbcL、psaA、NDHB、rpl20)相关基因。
具体实施方式
下面结合具体实施例对本发明进一步进行描述。
实施例1水稻叶色相关突变体dxr的表型与基因编辑位点鉴定
为了探究水稻类异戊二烯合成相关蛋白DXR(Gramene，http://www.gramene.org/)在水稻叶色调控中的作用，在DXR基因编码区设计一个CRISPR/Cas9基因编辑靶点，靶向第1外显子，如图1A所示，靶位点为SEQ ID NO.1所示序列的第49位～68位核苷酸序列。合成基于CRISPR/Cas9系统的靶序列引物，序列如下：
DXR-F：5'-GGCATTCCTCGACTCCAACAG-3'(SEQ ID No.3)，
DXR-R：5'-AAACCTGTTGGAGTCGAGGAA-3'(SEQ ID No.4)；
对DXR-F和DXR-R在95℃保温5min，在室温下自然退火，形成双链DNA。用限制性内切酶AarI(Thermo Scientific，ER1582)在37℃酶切CRISPR/Cas9双元载体(载体的构建参考Lu Y,Ye X,Guo R,Huang J,Wang W,Tang JY,Tan LT,Zhu JK,Chu CC,Qian YW(2017)Genome-wide targeted mutagenesis in rice using the CRISPR/Cas9 system.MolPlant 10:1242-1245.)，得到载体片段。用T4连接酶(Takara)将上述双链DNA与载体进行连接，将重组表达载体转入大肠杆菌，将阳性重组表达质粒转入根癌农杆菌EHA105中，侵染日本晴愈伤组织进行水稻遗传转化。
上述重组载体遗传转化获得了15株T0代转基因水稻植株。采用CTAB法提取T0代转基因水稻植株叶片DNA，利用KOD DNA聚合酶(TOYOBO)和基因组引物对含靶标位点的DNA片段进行扩增测序鉴定。所述引物序列为：
DXR-Cas9F：5'-GAGTCTCAGATTCCCATCTCGTC-3'(SEQ ID No.5)；
DXR-Cas9R：5'-CTGCGGATTATCTTGAAACAGG-3'(SEQ ID No.6)。
测序结果表明，有8株转基因植株带有目标突变，其中纯合突变体2株、杂合突变体6 株。2株纯合突变体dxr-1与dxr-2表现出白化的表型，如图1B、C所示。
称取约～0.2g野生型和突变体叶片剪碎，加入5mL 95％无水乙醇，室温黑暗静置48小时。色素溶液离心2min，吸取2mL上清液放入比色皿中，利用双通道紫外分光光度计(TU1901，北京)测定663nm和645nm处的吸光值，计算出单位质量的叶绿素含量。结果表明，相比野生型，突变体叶绿素a和叶绿素b含量极显著降低(图2)。
取野生型和突变苗期叶片进行透射电镜观察叶绿体形态结构。首先，将野生型和突变体叶片固定于2.5％的戊二醛的固定液中，再用1％锇酸固定4小时，而后经不同浓度梯度的无水乙醇脱水，再进行包埋、切片，醋酸铀染色后，利用透射电子显微镜(JEM-1200EX，日本日立)进行观察拍照。透射电镜结果表明，野生型中叶绿体形态正常，类囊体和基粒排列有序(图3a和b)，而突变体缺乏成熟的叶绿体，类囊体和基粒排列无序，甚至出现空泡，导致植物不能进行正常的光合作用(图3c和d)。
综上所述，类异戊二烯合成相关蛋白DXR正向调控水稻叶色，影响水稻叶绿体发育和叶绿素合成。
实施例2DXR的组织表达与亚细胞定位分析
1、DXR的组织表达
利用Primer Premier 5.0软件设计DXR的组织表达分析引物。所述引物序列如下：
DXR-qRT-F：5'-AAACGAGGGACAGAAGAGCA-3'(SEQ ID No.7)
DXR-qRT-R：5'-GAACCGGTTGAGCCAACAAT-3'(SEQ ID No.8)
取野生型根、茎、叶以及穗子等组织研磨，利用天根生化科技有限公司的RNA EasyFast
植物组织RNA快速提取试剂盒(DP452，北京)提取总RNA。使用Nano Drop与琼脂糖凝胶电泳检测所得RNA的浓度和质量。选用大连宝生物公司的反转录试剂盒(PrimeScriptReverse Transcriptase kit)对总RNA进行反转cDNA。以cDNA为模板，选用SYBR premix ExTaqTM(TaKaRa,Japan)试剂盒进行Real-time PCR，在伯乐荧光定量PCR仪CFX96上扩增，采用2–ΔΔCT方法进行DXR组织表达分析。结果表明，DXR在水稻中呈组成型表达，以叶片中表达水平最高(图4a)。
2、DXR的亚细胞定位
为了明确DXR的亚细胞定位，设计了引物：
pAN580-DXR-F：CGGAGCTAGCTCTAGAATGGCGCTCAAGGTCGTCTC(SEQ ID No.9)
pAN580-DXR-R：TGCTCACCATGGATCCACAGGTACAGGGCTGA(SEQ ID No.10)
扩增DXR全长编码区(去除终止子)，融合到pAN580-GFP载体上形成 pAN580-DXR-GFP，瞬时转化水稻原生质体，利用蔡司激光共聚焦显微镜观察DXR蛋白定位，以叶绿素自发荧光为对照。结果表明，pAN580-DXR-GFP绿色荧光信号与叶绿素自发荧光完全重叠，说明DXR蛋白定位于叶绿体，这与其调控水稻叶色形成是一致的(图4b)。
实施例3叶绿素合成与叶绿体发育相关基因的表达水平分析
植物形成正常的叶绿体和正常的进行叶绿素合成受细胞核编码的基因与质体编码的基因共同调控。为了明确DXR是否调控水稻叶绿体发育与叶绿素合成相关基因的表达，提取了野生型和突变体苗期的总RNA，进而进行荧光定量PCR分析上述相关基因的表达水平。结果表明，相比野生型，突变体中叶绿素合成与叶绿体发育相关基因的表达水平发生显著变化，如叶绿素生物合成基因HEMA、DVR、CHLM、PORA与叶绿体发育相关基因rbcL、NDHB 和V1的表达水平显著降低(图5)。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明的保护范围之内。
序列表
<110> 淮阴师范学院
<120> 水稻类异戊二烯合成相关蛋白DXR在调控水稻叶色中的应用
<160> 10
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1422
<212> DNA
<213> Oryza sativa
<400> 1
atggcgctca aggtcgtctc tttccccggg gacttggccg cggtctcatt cctcgactcc 60
aacagaggag gagctttcaa ccagctcaaa gtggacctcc cgtttcaaac gagggacaga 120
agagcagttt ccctgagaag gacttgctgt tcaatgcaac aggctccacc accagcatgg 180
cctggtcgag ccgttgttga acctgggagg aggtcatggg atggccccaa gcctatctca 240
attgttggct caaccggttc tattggcaca cagacattgg acatagttgc ggagaatcca 300
gataaattcc gggttgttgc tcttgctgct ggctccaatg tgactcttct agctgatcag 360
gtgaaaacat tcaaaccaaa gcttgttgct gtaagaaatg agtcattagt tgatgagcta 420
aaggaagcct tagctgattg tgattggaag ccagaaatta ttcctggtga gcaaggtgtc 480
atagaggttg ctcgccaccc agatgcagtt acagttgtta ctgggatagt agggtgtgca 540
ggactgaagc ctacagttgc tgcaattgaa gctgggaaag atatagcatt ggcgaacaaa 600
gagacactta ttgcaggtgg tccttttgtg cttccccttg cacaaaagca caaagtgaaa 660
atacttcctg ctgattctga gcactctgct atatttcagt gtatacaagg cttgcccgaa 720
ggagcacttc gccgcattat tttgactgca tcaggtggtg ctttcaggga ctggccagtt 780
gacaagttga aagaagtaaa agttgctgat gctttaaagc acccgaactg gaatatgggg 840
aagaagatta ctgtagattc tgctacatta ttcaacaagg gtttagaagt tattgaagca 900
cattatttat ttggtgctga atacgatgac attgaaattg tgatccaccc acaatctatc 960
atacactcta tgattgaaac ccaggattca tctgtgttgg ctcaactggg atggccagat 1020
atgcggatac caatcttata caccatgtct tggccagaca gaatctattg ctcagaggtc 1080
acctggcccc gactagatct ttgcaagctg ggttcactga cattcaaagc tcctgacaat 1140
gtgaaatacc cgtcgatgga tctcgcctat gcagctggaa gagctggggg caccatgaca 1200
ggagttctga gtgctgctaa tgagaaggct gtggagttgt tcatcgatga aaagatcggg 1260
tacctggaca tcttcaaggt ggtggagctg acatgcgacg ctcatcggaa tgagctagta 1320
acaaggccat cactggagga gatcatacat tatgatctgt gggcgaggga gtatgctgcc 1380
agcctacagc catccactgg cctcagccct gtacctgtct ag 1422
<210> 2
<211> 473
<212> PRT
<213> Oryza sativa
<400> 2
Met Ala Leu Lys Val Val Ser Phe Pro Gly Asp Leu Ala Ala Val Ser
1 5 10 15
Phe Leu Asp Ser Asn Arg Gly Gly Ala Phe Asn Gln Leu Lys Val Asp
20 25 30
Leu Pro Phe Gln Thr Arg Asp Arg Arg Ala Val Ser Leu Arg Arg Thr
35 40 45
Cys Cys Ser Met Gln Gln Ala Pro Pro Pro Ala Trp Pro Gly Arg Ala
50 55 60
Val Val Glu Pro Gly Arg Arg Ser Trp Asp Gly Pro Lys Pro Ile Ser
65 70 75 80
Ile Val Gly Ser Thr Gly Ser Ile Gly Thr Gln Thr Leu Asp Ile Val
85 90 95
Ala Glu Asn Pro Asp Lys Phe Arg Val Val Ala Leu Ala Ala Gly Ser
100 105 110
Asn Val Thr Leu Leu Ala Asp Gln Val Lys Thr Phe Lys Pro Lys Leu
115 120 125
Val Ala Val Arg Asn Glu Ser Leu Val Asp Glu Leu Lys Glu Ala Leu
130 135 140
Ala Asp Cys Asp Trp Lys Pro Glu Ile Ile Pro Gly Glu Gln Gly Val
145 150 155 160
Ile Glu Val Ala Arg His Pro Asp Ala Val Thr Val Val Thr Gly Ile
165 170 175
Val Gly Cys Ala Gly Leu Lys Pro Thr Val Ala Ala Ile Glu Ala Gly
180 185 190
Lys Asp Ile Ala Leu Ala Asn Lys Glu Thr Leu Ile Ala Gly Gly Pro
195 200 205
Phe Val Leu Pro Leu Ala Gln Lys His Lys Val Lys Ile Leu Pro Ala
210 215 220
Asp Ser Glu His Ser Ala Ile Phe Gln Cys Ile Gln Gly Leu Pro Glu
225 230 235 240
Gly Ala Leu Arg Arg Ile Ile Leu Thr Ala Ser Gly Gly Ala Phe Arg
245 250 255
Asp Trp Pro Val Asp Lys Leu Lys Glu Val Lys Val Ala Asp Ala Leu
260 265 270
Lys His Pro Asn Trp Asn Met Gly Lys Lys Ile Thr Val Asp Ser Ala
275 280 285
Thr Leu Phe Asn Lys Gly Leu Glu Val Ile Glu Ala His Tyr Leu Phe
290 295 300
Gly Ala Glu Tyr Asp Asp Ile Glu Ile Val Ile His Pro Gln Ser Ile
305 310 315 320
Ile His Ser Met Ile Glu Thr Gln Asp Ser Ser Val Leu Ala Gln Leu
325 330 335
Gly Trp Pro Asp Met Arg Ile Pro Ile Leu Tyr Thr Met Ser Trp Pro
340 345 350
Asp Arg Ile Tyr Cys Ser Glu Val Thr Trp Pro Arg Leu Asp Leu Cys
355 360 365
Lys Leu Gly Ser Leu Thr Phe Lys Ala Pro Asp Asn Val Lys Tyr Pro
370 375 380
Ser Met Asp Leu Ala Tyr Ala Ala Gly Arg Ala Gly Gly Thr Met Thr
385 390 395 400
Gly Val Leu Ser Ala Ala Asn Glu Lys Ala Val Glu Leu Phe Ile Asp
405 410 415
Glu Lys Ile Gly Tyr Leu Asp Ile Phe Lys Val Val Glu Leu Thr Cys
420 425 430
Asp Ala His Arg Asn Glu Leu Val Thr Arg Pro Ser Leu Glu Glu Ile
435 440 445
Ile His Tyr Asp Leu Trp Ala Arg Glu Tyr Ala Ala Ser Leu Gln Pro
450 455 460
Ser Thr Gly Leu Ser Pro Val Pro Val
465 470
<210> 3
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
ggcattcctc gactccaaca g 21
<210> 4
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
aaacctgttg gagtcgagga a 21
<210> 5
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
gagtctcaga ttcccatctc gtc 23
<210> 6
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
ctgcggatta tcttgaaaca gg 22
<210> 7
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
aaacgaggga cagaagagca 20
<210> 8
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
gaaccggttg agccaacaat 20
<210> 9
<211> 36
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
cggagctagc tctagaatgg cgctcaaggt cgtctc 36
<210> 10
<211> 32
<212> DNA
<213> 人工序列(Artificial Sequence)
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tgctcaccat ggatccacag gtacagggct ga 32

Claims (2)

1.调控水稻叶色的方法，其特征在于，包括如下步骤：将抑制水稻类异戊二烯合成相关蛋白DXR表达的sgRNA表达载体转化水稻，培育筛选，获得目标叶色的水稻；所述DXR蛋白的氨基酸序列如SEQ ID No.2所示；所述DXR蛋白的编码基因的核苷酸序列如SEQ ID No.1所示；载体的靶标位点的核苷酸序列如SEQ ID NO.1的第49位～68位所示。
2.如权利要求1所述的方法，其特征在于，所述方法的具体操作如下：构建具有靶标序列的sgRNA表达载体，转入根癌农杆菌EHA105，用含有靶标序列的sgRNA表达载体的根癌农杆菌转化水稻细胞中，培育筛选，得到叶片白化的转基因水稻。
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