CN113249387B - OsPIN9基因在调控水稻抗冷胁迫中的应用 - Google Patents
OsPIN9基因在调控水稻抗冷胁迫中的应用 Download PDFInfo
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Abstract
本发明涉及OsPIN9基因在调控水稻抗冷胁迫中的应用，属于植物基因工程技术领域，本发明采用CRISPR/Cas9基因编辑敲除系统，对OsPIN9基因进行定点编辑，研究发现，OsPIN9敲除后获得的阳性纯合植株，表现出抗低温的特性，为培育强抗冷性转基因水稻品种、提高水稻抗冷性方面提供了依据和可能。
Description
技术领域
本发明属于植物基因工程技术领域，具体地，涉及OsPIN9基因在调控水稻抗冷胁迫中的应用。
背景技术
水稻（Oryza sativa）是世界近一半人口的主食，为了解决困扰全球的粮食安全危机，水稻育种计划的一个关键重点就是通过优化粮食产量的组成和植物结构来提高作物生产力。然而，水稻的生长发育经常受到各种生物或非生物胁迫的严重影响。霜冻、寒潮和低温冷害是在晚春和早秋经常出现的主要气象灾害。在世界范围内，由低温灾害的频繁发生所造成的农林生产的损失达数千亿美元。因此，研究水稻抗冷性相关基因，提高水稻对低温胁迫的耐受力具有十分重要的意义。
水稻一般在5月份进行育苗，而水稻属于温度敏感型作物，在2 叶期时其胚乳物质基本消耗光，此时正是水稻抗性最弱的时期，遇到低温极易发生低温冷害，影响水稻幼苗的正常生长，且对水稻后期的生长发育带来不利的影响。苗期低温冷害是影响水稻成苗和秧苗生长的重要限制因素之一，遭遇冷害，易导致生产上出现烂芽和死苗。提高水稻苗期的抗低温能力，是确保水稻高产丰收的前提条件，而水稻抗低温能力的提高离不开植物激素的调控。生长素是第一个发现的植物激素，在许多植物发育过程中发挥重要作用，包括胚胎发生、根的分化、维管组织分化、顶端优势、向光性、向重性和其他生理过程。生长素作为一个具有极性运输特性的植物激素，其在细胞水平上的极性运输主要通过几个生长素转运蛋白家族完成，包括输入载体蛋白家族AUX/LAX (Auxin Resistant1/Like Aux)，输出载体蛋白家族PIN(PIN-FORMED)，以及ABCB/ PGP (Multidrug-Resistant ABCB/P-glycoprotein)载体蛋白家族。PIN是一种具有高度可调节和极性定位特点的生长素输出蛋白，植物组织中生长素的极性运输在很大程度上依赖于PIN蛋白。水稻基因组中有12个PIN基因，包括OsPIN1a、OsPIN1b、OsPIN1c、OsPIN1d、OsPIN2、OsPIN5a、OsPIN5b、OsPIN5c、OsPIN8、OsPIN9、OsPIN10a和OsPIN10b，其中部分基因的功能已有报道，如OsPIN1家族、OsPIN2、OsPIN5b等。OsPIN9作为单子叶植物特有的生长素输出蛋白，目前研究表明其在调控根系构型以及分蘖方面发挥功能，但其他方面的功能仍待进一步深入研究。因此，通过研究OsPIN9在水稻抗冷性方面的功能，有助于加深我们对水稻抗冷性分子机制的认识，并有助于提升水稻的抗冷性。
发明内容
针对上述问题，本发明的目的一在于提供生长素输出蛋白OsPIN9基因在调控水稻抗冷性方面的应用，目的二在于提供一种提高水稻抗冷性的方法，目的三在于提供一种抗冷性水稻的育种方法。
为了实现上述目的，本发明采用的技术方案为：
采用CRISPR/Cas9基因编辑敲除系统，对OsPIN9基因进行编辑，研究发现，OsPIN9敲除后获得的阳性纯合植株，表现出抗低温的特性，表明OsPIN9基因的重新编辑在改良水稻抗冷性中具有一定的应用价值。
本发明请求保护生长素输出蛋白OsPIN9基因在调控水稻抗冷胁迫中的应用。进一步地，所述OsPIN9基因的核苷酸序列如SEQ ID NO. 1所示。更进一步地，利用CRISPR/Cas9基因编辑技术突变水稻OsPIN9基因，进而获得抗低温的水稻突变体；所述OsPIN9基因突变后编码区核苷酸序列如SEQ ID NO. 2所示，编码的氨基酸序列如SEQ ID NO. 3所示。
本发明还请求保护一种提高水稻抗冷性的方法，利用CRISPR/Cas9基因编辑技术定点编辑核苷酸序列如SEQ ID NO. 1所示的水稻OsPIN9基因的靶位点，所述靶位点的序列为：5’- TTCTCCAACGAGCAGTGCGC-3’。
具体地，将所述水稻OsPIN9基因的靶位点连入CRISPR/Cas9表达载体CRISPR-RICE，构建重组表达载体；通过农杆菌介导法将所述重组表达载体转化到受体植物材料中，获得抗冷性提高的基因编辑植株。
本发明另外请求保护一种抗冷性水稻的培育方法，包括以下步骤：
步骤一、根据OsPIN9基因设计并得到靶位点序列，针对所述靶位点序列设计引物，将引物变性、退火后形成双链DNA，然后连接到经Bsa酶切后的CRISPR-RICE载体，构建得到CRISPR/Cas9重组表达载体；
步骤一中，所述引物为：
PIN9-crispr-F: 5’-TGTGTTTCTCCAACGAGCAGTGCGC-3’，
PIN9-crispr-R: 5’-AAACGCGCACTGCTCGTTGGAGAAA-3’；
步骤二、将步骤一所得CRISPR/Cas9重组表达载体转入农杆菌；
步骤三、通过农杆菌介导法将CRISPR/Cas9重组表达载体转入待基因编辑的受体水稻中，通过组织培养获得T0代阳性苗，通过PCR扩增获得含有靶位点的OsPIN9片段，测序确认获得ospin9突变体T0代植株；
步骤四、将步骤三所得T0代植株自交获得T1代植株，进一步通过PCR筛选获得ospin9突变的纯合植株；
步骤三和步骤四中，所述PCR采用的引物为：
PIN9-assay-F: 5’-CGACCTGGCTTACGAACGAA-3’，
PIN9-assay-R: 5’-CCATGTCGAAGATGAGCACC-3’。
有益效果：
本发明通过利用CRISPR/Cas9系统构建水稻OsPIN9基因靶位点特异性敲除的植物重组表达载体，利用基因编辑获得OsPIN9纯合稳定的突变体，可用于分析OsPIN9基因在水稻中的生物学功能；本发明提供的水稻ospin9突变体植株可以为增强水稻抗冷性提供更多理论依据，可用于培育强抗冷性转基因水稻品种，在提高水稻抗冷性方面具有潜在的应用价值。
附图说明
图1是应用CRISPR/Cas9技术突变OsPIN9基因靶位点示意图；
图2是应用CRISPR/Cas9技术突变OsPIN9基因突变序列示意图；
图3是野生型OsPIN9蛋白和应用CRISPR/Cas9技术突变的OsPIN9蛋白的跨膜结构对比图；
图4是野生型OsPIN9蛋白和应用CRISPR/Cas9技术突变的OsPIN9蛋白的结构对比图；其中，（A）为野生型OsPIN9的蛋白结构，（B）为突变体OsPIN9蛋白的结构；
图5是野生型和ospin9-1突变体处理前、处理6d后及恢复6d后的表型对比图；
图6是野生型和ospin9-1突变体存活率对比图；
图7是野生型和ospin9-1突变体膜透性分析对比图。
具体实施方式
本发明提供了OsPIN9基因在调控水稻抗冷性中的新用途，为培育具有抗冷特性的水稻品种提供依据。具体技术方案如下：
本发明采用CRISPR/Cas9基因编辑敲除系统，对OsPIN9基因（其编码区核苷酸序列如SEQ ID NO. 1所示）进行编辑，研究发现，OsPIN9敲除后获得的阳性纯合植株，表现出抗低温的特性，表明OsPIN9基因的重新编辑在改良水稻抗冷性中具有一定的应用价值。
因此，本发明提供了核苷酸序列如SEQ ID NO.1所示的水稻OsPIN9基因在调控水稻抗冷性方面的应用。
本发明利用CRISPR/Cas9技术突变水稻OsPIN9基因，进而获得抗低温的水稻突变体。其中，OsPIN9基因突变后编码区核苷酸序列如SEQ ID NO. 2所示，编辑后编码的氨基酸序列如SEQ ID NO. 3所示。
本发明利用CRISPR/Cas9技术定点编辑核苷酸序列如SEQ ID NO. 1所示的水稻OsPIN9基因的靶位点，所述靶位点的序列为：5’- TTCTCCAACGAGCAGTGCGC-3’。具体地，将所述水稻OsPIN9基因的靶位点连入CRISPR/Cas9表达载体CRISPR-RICE，构建重组表达载体；通过农杆菌介导法将重组表达载体转化到受体植物材料中，获得抗冷性提高的基因编辑植株。
所述利用CRISPR/Cas9技术定点编辑的方法，包括以下步骤：
（1）针对靶位点设计引物，将引物变性、退火后形成双链DNA，将其连接到经Bsa 酶切后的CRISPR-RICE载体，构建得到CRISPR/Cas9重组表达载体；
（2）将重组表达载体转入农杆菌；
（3）通过农杆菌介导法将CRISPR/Cas9重组表达载体转入待基因编辑的受体水稻中，通过组织培养获得T0代阳性苗，通过PCR扩增获得含有靶位点的OsPIN9片段，测序确认获得ospin9突变体；
（4）T0代植株自交获得T1代植株，进一步通过PCR筛选获得OsPIN9突变的纯合植株；
步骤（1）中，所述引物为：
PIN9-crispr-F: 5’-TGTGTTTCTCCAACGAGCAGTGCGC-3’，
PIN9-crispr-R: 5’-AAACGCGCACTGCTCGTTGGAGAAA-3’；
步骤（3）和（4）中，采用的PCR引物为：
PIN9-assay-F: 5’-CGACCTGGCTTACGAACGAA-3’，
PIN9-assay-R: 5’-CCATGTCGAAGATGAGCACC-3’。
下面将结合本发明实施例，对本发明实施例中的技术方案进行清楚、完整地描述。
以下实施例中没有注明具体实验条件的试验方法，通常遵循常规实验条件。
所使用的材料试剂等，如无特殊说明，均为从商业途径得到的试剂及材料。
实施例中所使用的引物序列均由生工生物工程（上海）股份有限公司合成。
日本晴 (Nipponbare) 水稻品种，属粳稻亚种，已经完成全基因组测序。
实施例1：水稻OsPIN9的序列分析
基因来源：从美国国家生物信息中心 (NCBI) 获得OsPIN9基因 (LOC_Os01g58860) 序列，其编码区核苷酸序列如序列表SEQ ID NO. 1所示，序列长度为1281bp。
实施例2：构建OsPIN9的CRISPR/Cas9基因编辑载体及转基因植株的获得
根据靶点设计原则，使用在线CRISPR设计工具CRISPR-GE (http://skl.scau.edu.cn/) 导入OsPIN9基因组序列，设计sgRNA，得到最终的靶点序列为TTCTCCAACGAGCAGTGCGC。由公司直接合成PIN9-crispr-F和PIN9-crispr-R，通过退火将两条引物结合到一起。
将得到的片段与Bsa酶切后的CRISPR-RICE基因编辑载体连接。连接产物转化大肠杆菌DH10B，通过PCR筛选阳性克隆，所用引物为M13F和PIN9-crispr-R。
测序正确的重组载体通过冻融法转化农杆菌EHA105，利用农杆菌介导法侵染水稻日本晴的愈伤组织，经过抗性愈伤的预分化、分化及幼苗的生根壮苗后得到转基因植株。采用CTAB法提取T0代转基因植株的叶片基因组DNA，通过PCR扩增获得含有靶位点的基因组片段，测序后分析突变情况，测序引物为PIN9-assay-F和PIN9-assay-R。
实施例3：基因编辑 ospin9突变体的抗冷性鉴定
将实施例2得到的转基因植株T0代进行繁种，继续采用CTAB法提取T1代转基因植株的叶片基因组DNA，PCR扩增基因组片段，测序分析突变情况，并进一步获得纯合突变体。
对于获得的测序图谱，使用华南农业大学刘耀光院士课题组开发的网络在线工具DSDecodeM (http://skl.scau.edu.cn/) 进行突变分析，可以得出所鉴定植株的突变位点和突变类型，相比野生型，纯合突变体OsPIN9基因的靶位点序列中插入了一个碱基T，OsPIN9基因突变位点见图2。使用在线跨膜结构域预测软件TMHMM (http://www.cbs.dtu.dk/services/TMHMM/) 分析此突变对OsPIN9蛋白的跨膜结构域的影响，结果显示野生型OsPIN9蛋白有10个跨膜结构域和1个核心疏水区，为膜蛋白（图3），而突变体中OsPIN9的突变导致转录提前终止，所编码蛋白跨膜结构域消失（图3）。通过SWISS-MODEL预测了野生型和ospin9-1突变体的OsPIN9蛋白三级结构（图4），由于OsPIN9编码区的提前终止导致OsPIN9蛋白的分子量仅为18.84kD，远远小于野生型蛋白的45.56kD，以上结果表明OsPIN9基因在核酸水平和蛋白水平都发生了有效突变。
以ospin9-1纯合突变体和野生型水稻进行抗冷性实验。抗冷性实验过程为：经消毒的种子在适温 (25℃) 下浸种48h，待其露白后移入底部开口的96孔板，放入垫有两层湿润滤纸的不透光容器中，置于28℃的生长箱中进行萌发。观察到主根从下部开口露出后即可移至木村B营养液中，于温度28℃，相对湿度50％，光周期为12h/12h的光照培养箱中培养。每份材料30粒种子，3次重复。培养至14日龄移至温度4℃，光周期为12h/12h的光照培养箱中进行低温处理6d，6d后移至正常培养条件下进行恢复，恢复6d后观察植株表型（如图5所示），调查植株存活率并统计结果。结果表明，如图6所示，纯合突变体ospin9-1的低温处理存活率显著高于野生型，表现出较强的抗冷性 。
低温胁迫会对植物细胞膜造成损伤，导致膜渗透性上升，膜渗透性的大小可以通过测定相对电导率来反映，进而得知植物细胞受损伤的程度。分别测定冷胁迫过程中不同时间点突变体ospin9-1和野生型的叶片相对电导率。叶片相对电导率测定过程为：用纯净水冲洗两遍离心管、管盖及叶片压（自制），每个离心管加20ml纯净水（多准备一管用来测温），打开水浴锅，设定温度为99.9℃。取相同部位，相近状态的叶片，剪去叶尖，将叶片剪成长2cm左右的小段。称重后混匀，分成重量相近的三份，用超纯水清洗两遍，用干净滤纸吸干表面水分后移入离心管，用叶片压将叶片压到液面以下，不用盖盖子。打开真空干燥器盖子，在盖沿和连接口处涂抹真空脂，将离心管放入真空干燥器，盖好盖子，打开玻璃气阀，用抽气管将真空干燥器和真空泵连接好。打开真空泵，抽真空至真空表读数-0.1 Mpa后停止，关闭电源和玻璃气阀，保持负压30min。打开电导率仪电源，确认并校正电极常数，记录纯净水电导率，记为本底值。20min结束后，拔下抽气管，慢慢打开气阀，缓慢放气。放气完毕后打开盖子，拿出离心管，盖上盖子，至水平摇床上摇匀30min。结束后取出离心管，取出叶片压，测定温度后在电导率仪上更改设置。开始测定样品电导率，待读数稳定后记录，记为初值。将离心管盖上管盖，拧至1/2或1/3，不要拧紧。转入沸水浴加热30min。结束后用自来水降温10min，水平摇床摇匀10min。测定温度并校正电导率仪，测定样品电导率，记为终值。结果计算：相对电导率 =（初值-本底值）/（终值-本底值）。结果表明，ospin9-1突变体在低温处理后叶片相对电导率显著低于野生型，进一步证明ospin9-1突变体具有较强的抗冷性 (如图7所示)。
上述实施例为本发明一种具体的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化等优化改进行为，均应为等效的置换方式，都包含在本发明的权利要求保护范围之内。
SEQUENCE LISTING
<110> 河南科技大学
<120> OsPIN9基因在调控水稻抗冷胁迫中的应用
<130> 1
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 1281
<212> DNA
<213> 水稻
<400> 1
atgattacgg gttcggaggt gtaccaggtg gtggaggcga tggcgccgct gtacacggcg 60
gcggcgctgg ggtacgggtc ggtgcggtgg ctgaaggcgt tctccaacga gcagtgcgcc 120
gggatcaacc acttcgtggc gctctacgcc gtgccggtgc tcatcttcga catggtgtcc 180
accaacaacg tgtacaagat gaacggccgc ctcatcgccg ccgacacgct gcagaaggcc 240
gtgctgctgc tgggcctcat ggcgtgggcg ctctgggagc ggtcgcgcgc gcgcggcgcc 300
ggggccaagg ccaaggcggc ggtgtcgtcg ccgctgcagt gggtcatcac ctgcttctcc 360
gtcgcgtcgc tgcccaacac catcatcatg ggcgtcccgc tcctcaacgg catgtacggg 420
cccgtgtcca aggacctcat gaagcagatc gtcgtcatgc agttctgcat ctggtacaac 480
gtcatcatct tcctctacga gtacatggcg gcgcgtagat cggcctcggc gccgccgccg 540
gcgtcgtcgg agggcagcgc caagatcagc ccttcgtcgc cggtgaaagc tgctgcggcg 600
gcggcggaca caaacggcaa tgctgtcgcg gccgaccggc cgcaagaagt ggcggtgaac 660
atcgaaatca cggagatggc ggcgtccacg gcacgagacg gcgtgtccgg cgagacgacg 720
gcggccgcca aggaggtgag ctctggtgaa gttgctccgg tggaagagga ggaggcgtct 780
gcgccagcgc cgtcgatgaa gcacgtcatc tggatggcgg tgaagaagct gctacagatt 840
ccgaatacct atgcaagctt ccttggcctc atctggtctc taatcgcatt caagtgtgga 900
ttctcgatgc caaaaatcgt cgaggactct ctgttcacca ttcgtaccac cgctgtaggc 960
ctaagcatgt tttcttcagg gacgttcata gcgcggcagt cgcggttcgt gccgtgcgga 1020
tacaagatag cgtcgttctc catggtcatc aagtttctga taggcccggt tgtgatgctg 1080
ttcgcctcgc tcgtcatcgg catgcacggc acacttctgc acatcgctgt tgtgcaggcg 1140
gctctccccc tggcagtgac atcatttgtg tacgctgaag agtacaaggt ccacgcagac 1200
atcatgagca caggggttat tcttgggata tttatatcac ttcctgtgac aattgtttac 1260
tatattctgt tggggctgtg a 1281
<210> 2
<211> 519
<212> DNA
<213> 水稻
<400> 2
atgattacgg gttcggaggt gtaccaggtg gtggaggcga tggcgccgct gtacacggcg 60
gcggcgctgg ggtacgggtc ggtgcggtgg ctgaaggcgt tctccaacga gcagtgtcgc 120
cgggatcaac cacttcgtgg cgctctacgc cgtgccggtg ctcatcttcg acatggtgtc 180
caccaacaac gtgtacaaga tgaacggccg cctcatcgcc gccgacacgc tgcagaaggc 240
cgtgctgctg ctgggcctca tggcgtgggc gctctgggag cggtcgcgcg cgcgcggcgc 300
cggggccaag gccaaggcgg cggtgtcgtc gccgctgcag tgggtcatca cctgcttctc 360
cgtcgcgtcg ctgcccaaca ccatcatcat gggcgtcccg ctcctcaacg gcatgtacgg 420
gcccgtgtcc aaggacctca tgaagcagat cgtcgtcatg cagttctgca tctggtacaa 480
cgtcatcatc ttcctctacg agtacatggc ggcgcgtag 519
<210> 3
<211> 172
<212> PRT
<213> 水稻
<400> 3
Met Ile Thr Gly Ser Glu Val Tyr Gln Val Val Glu Ala Met Ala Pro
1 5 10 15
Leu Tyr Thr Ala Ala Ala Leu Gly Tyr Gly Ser Val Arg Trp Leu Lys
20 25 30
Ala Phe Ser Asn Glu Gln Cys Arg Arg Asp Gln Pro Leu Arg Gly Ala
35 40 45
Leu Arg Arg Ala Gly Ala His Leu Arg His Gly Val His Gln Gln Arg
50 55 60
Val Gln Asp Glu Arg Pro Pro His Arg Arg Arg His Ala Ala Glu Gly
65 70 75 80
Arg Ala Ala Ala Gly Pro His Gly Val Gly Ala Leu Gly Ala Val Ala
85 90 95
Arg Ala Arg Arg Arg Gly Gln Gly Gln Gly Gly Gly Val Val Ala Ala
100 105 110
Ala Val Gly His His Leu Leu Leu Arg Arg Val Ala Ala Gln His His
115 120 125
His His Gly Arg Pro Ala Pro Gln Arg His Val Arg Ala Arg Val Gln
130 135 140
Gly Pro His Glu Ala Asp Arg Arg His Ala Val Leu His Leu Val Gln
145 150 155 160
Arg His His Leu Pro Leu Arg Val His Gly Gly Ala
165 170
Claims (6)
1.OsPIN9突变基因在调控水稻抗冷胁迫中的应用，其特征在于：利用CRISPR/Cas9基因编辑技术突变核苷酸序列如SEQ ID NO：1所示的水稻OsPIN9基因，进而获得抗低温的水稻突变体；所述OsPIN9基因突变后编码区核苷酸序列如SEQ ID NO：2所示，编码的氨基酸序列如SEQ ID NO：3所示。
2.一种提高水稻抗冷性的方法，其特征在于：利用CRISPR/Cas9基因编辑技术定点编辑水稻OsPIN9基因的靶位点；所述OsPIN9基因的核苷酸序列如SEQ ID NO：1所示，所述靶位点的序列为：5’- TTCTCCAACGAGCAGTGCGC-3’；编辑后的靶位点序列为：5’-TTCTCCAACGAGCAGTGTCGC-3’。
3.根据权利要求2所述的方法，其特征在于：将所述水稻OsPIN9基因的靶位点连入CRISPR/Cas9表达载体CRISPR-RICE，构建重组表达载体；通过农杆菌介导法将所述重组表达载体转化到受体植物材料中，获得抗冷性提高的基因编辑植株。
4.一种抗冷性水稻的培育方法，其特征在于：包括以下步骤：
步骤一、根据OsPIN9基因设计并得到靶位点序列，针对所述靶位点序列设计引物，将引物变性、退火后形成双链DNA，然后连接到经Bsa酶切后的CRISPR-RICE载体，构建得到CRISPR/Cas9重组表达载体；所述OsPIN9基因的核苷酸序列如SEQ ID NO. 1所示，所述靶位点的核苷酸序列为5’- TTCTCCAACGAGCAGTGCGC-3’；
步骤二、将步骤一所得CRISPR/Cas9重组表达载体转入农杆菌；
步骤三、通过农杆菌介导法将CRISPR/Cas9重组表达载体转入待基因编辑的受体水稻中，通过组织培养获得T0代阳性苗，通过PCR扩增获得含有靶位点的OsPIN9片段，测序确认获得OsPIN9突变体T0代植株；
步骤四、将步骤三所得T0代植株自交获得T1代植株，进一步通过PCR筛选获得OsPIN9突变的纯合植株；纯合突变体OsPIN9基因的靶位点序列突变为：5’-TTCTCCAACGAGCAGTGTCGC-3’，突变后OsPIN9基因编码的氨基酸序列如SEQ ID NO：3所示。
5.根据权利要求4所述的培育方法，其特征在于：步骤一中，所述引物为：
PIN9-crispr-F: 5’-TGTGTTTCTCCAACGAGCAGTGCGC-3’，
PIN9-crispr-R: 5’-AAACGCGCACTGCTCGTTGGAGAAA-3’。
6.根据权利要求4所述的培育方法，其特征在于：步骤三和步骤四中，所述PCR采用的引物为：
PIN9-assay-F: 5’-CGACCTGGCTTACGAACGAA-3’，
PIN9-assay-R: 5’-CCATGTCGAAGATGAGCACC-3’。
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