CN105566467B - 水稻细胞周期蛋白OsCYCP4；2的应用及提高水稻耐低磷胁迫的方法 - Google Patents
水稻细胞周期蛋白OsCYCP4；2的应用及提高水稻耐低磷胁迫的方法 Download PDFInfo
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- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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- C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明公开了一种水稻细胞周期蛋白OsCYCP4；2的应用及提高水稻耐低磷胁迫的方法，OsCYCP4；2的表达在转录水平和翻译水平上均受磷饥饿胁迫的诱导。过量表达OsCYCP4；2基因抑制水稻生长，证明其负调控水稻生长。在正常培养条件下，OsCYCP4；2功能缺失突变体与野生型相比没有明显的表型差异，但突变体磷含量明显高于野生型。在缺磷条件下，OsCYCP4；2功能缺失突变体降低低磷抑制地上部生长的敏感性。本发明为提高植物对低磷耐受性和培育适用于磷贫瘠土壤的水稻新品种提供了保障。
Description
技术领域
本发明涉及植物基因工程技术领域，特别涉及一种水稻细胞周期蛋白OsCYCP4；2的应用及提高水稻耐低磷胁迫的方法。
背景技术
磷是一切生命所必需的大量元素之一。磷元素不仅构成了细胞内包括DNA，RNA，ATP和磷脂等在内的重要大分子，而且还参与调节信号转导，能量代谢和光合作用等重要的生理生化过程(Abel S(2011)Phosphate sensing in root development.Curr OpinPlant Biol 14:303-309)。随着分子生物学研究水平的不断提高，我们对植物体磷吸收和体内代谢的复杂生理生化过程有了一定的了解。植物中参与响应低磷胁迫的基因不断被发现，这些基因包括编码受磷饥饿诱导的可使植物特异性高效吸收和利用磷的磷酸盐转运体和磷酸酶，而其他基因编码的转录因子参与调控这些磷饥饿诱导基因的表达(Wu P(2014)SPX4 Negatively Regulates Phosphate Signaling and Homeostasis through ItsInteraction with PHR2 in Rice.Plant Cell 26:1586-1597；Zhou J,Jiao F,Wu Z,LiY,Wang X,He X,Zhong W,Wu P(2008)OsPHR2 is involved in phosphate-starvationsignaling and excessive phosphate accumulation in shoots of plants.PlantPhysiol 146:1673-1686；Zhang Z,Liao H,Lucas WJ(2014)Molecular mechanismsunderlying phosphate sensing,signaling,and adaptation in plants.J IntegrPlant Biol 56:192-220)。
缺磷抑制作物地上部分生长，进而严重影响作物产量。但是到目前为止对于磷饥饿胁迫如何抑制植物生长的分子机制还不是很清楚。已有研究报道植物生长抑制不是由植物体内磷含量低直接造成的，而是由一系列受磷饥饿触发的基因调控网络协调后的结果(Rouached H,Stefanovic A,Secco D,Bulak AA,Gout E,Bligny R,Poirier Y(2011)Uncoupling phosphate deficiency from its major effects on growth andtranscriptome via PHO1 expression in Arabidopsis.Plant J 65:557-570)。因此，研究磷饥饿抑制植物地上部分生长的机理对培育磷高效利用作物非常重要。
细胞周期蛋白复合体PHO80/PHO85是酵母磷信号转导(PHO,Phosphate SignalTransduction)通路中转录因子PHO4的重要负调控因子(Gilliquet V,Legrain M,BerbenG,Hilger F(1990)Negative regulatory elements of the Saccharomyces cerevisiaePHO system:interaction between PHO80 and PHO85 proteins.Gene 96:181-188)，同时参与调控酵母细胞周期中G0期的起始(Wanke V,Pedruzzi I,Cameroni E,Dubouloz F,DeVirgilio C(2005)Regulation of G0 entry by the Pho80-Pho85 cyclin-CDKcomplex.EMBO J 24:4271-4278)。PHO80同源蛋白在拟南芥和水稻中各存在7个，被命名为一个植物细胞周期家族新成员P类型cyclin蛋白(CYCP)。Torres实验室通过互补实验发现，AtCYCP4；2可以部分恢复酵母pho80突变体中的磷信号；酵母双杂交实验显示AtCYCP与CDKA；1互作；但悬浮细胞中AtCYCPs的转录水平不受培养液磷浓度的影响(Torres AJ,deAlmeida EJ,Raes J,Magyar Z,De Groodt R,Inze D,De Veylder L(2004)Molecularcharacterization of Arabidopsis PHO80-like proteins,a novel class of CDKA；1-interacting cyclins.Cell Mol Life Sci 61:1485-1497)。但到目前为止没有发现拟南芥中CYCP家族与磷饥饿信号响应的联系。
发明内容
本发明的目的在于提供一种水稻细胞周期蛋白OsCYCP4；2的应用，水稻细胞周期蛋白OsCYCP4；2负调控水稻生长。OsCYCP4；2功能缺失突变体降低低磷抑制地上部生长的敏感性。
本发明还提供了一种提高水稻耐低磷胁迫的方法，通过将水稻细胞周期基因OsCYCP4；2从目的水稻上敲除，得到的转基因水稻在低磷状态下耐受性更高，为提高植物对低磷耐受性和培育适用于磷贫瘠土壤的水稻新品种提供了保障。
本发明解决其技术问题所采用的技术方案是：
一种水稻细胞周期蛋白OsCYCP4；2在调控低磷胁迫下水稻生长的应用。
作为优选，水稻细胞周期蛋白OsCYCP4；2的氨基酸序列见SEQ ID NO.1所示。
作为优选，水稻细胞周期蛋白OsCYCP4；2负调控水稻生长。
一种编码水稻细胞周期蛋白OsCYCP4；2的基因，该基因为水稻细胞周期基因OsCYCP4；2，水稻细胞周期基因OsCYCP4；2的核苷酸序列见SEQ ID NO.2所示。
发明人经过长期研究，首次发现低磷胁迫下与水稻生长调控直接相关的因子-水稻细胞周期蛋白OsCYCP4；2，水稻细胞周期基因OsCYCP4；2的表达在转录水平和翻译水平上均受磷饥饿胁迫的诱导。过量表达OsCYCP4；2基因抑制水稻生长，证明其负调控水稻生长。在正常培养条件下，OsCYCP4；2功能缺失突变体与野生型相比没有明显的表型差异，但突变体磷含量明显高于野生型。在缺磷条件下，OsCYCP4；2功能缺失突变体降低低磷抑制地上部生长的敏感性。利用发现的水稻细胞周期蛋白OsCYCP4；2在低磷胁迫下负调控水稻生长的特点，为提高植物对低磷耐受性和培育适用于磷贫瘠土壤的水稻新品种提供了保障。
一种提高水稻耐低磷胁迫的方法，通过将水稻细胞周期蛋白OsCYCP4；2的编码基因水稻细胞周期基因OsCYCP4；2从目的水稻上敲除，使得水稻耐低磷胁迫能力提高。水稻细胞周期基因OsCYCP4；2缺失后能提高植株磷含量，且降低植株对低磷抑制生长的敏感性。
作为优选，将水稻细胞周期蛋白OsCYCP4；2的编码基因水稻细胞周期基因OsCYCP4；2从目的水稻上敲除的方法为：采用农杆菌介导法将载体转入目的水稻的愈伤组织中，培养获得OsCYCP4；2基因敲除的突变体。
作为优选，水稻细胞周期基因OsCYCP4；2缺失后提高了水稻植株磷含量，且降低水稻植株对低磷抑制生长的敏感性。
作为优选，水稻细胞周期基因OsCYCP4；2的核苷酸序列见SEQ ID NO.2所示。
作为优选，所述载体为pYLCRISPR/Cas9-MH-OsCYCP4；2。
本发明的有益效果是：通过将水稻细胞周期基因OsCYCP4；2从目的水稻上敲除，得到的转基因水稻在低磷状态下耐受性更高，为提高植物对低磷耐受性和培育适用于磷贫瘠土壤的水稻新品种提供了保障。
附图说明
图1：OsCYCP4；2(水稻细胞周期基因)对低磷胁迫的响应。A,水稻正常供磷(200μM，HP)和低磷(10μM，LP)处理21天后地上(shoot)和地下(root)部分OsCYCP4；2基因表达水平的实时定量PCR检测结果；B，OsCYCP4；2基因组融合GUS的转基因植株在正常供磷(200μM，HP)和低磷(10μM，LP)处理21天后地上(shoot)和地下(root)部分GUS染色结果。
图2：OsCYCP4；2过表达转基因水稻的表型观察。A，7天苗表型；B，A中苗根(root)和地上部分(shoot)长度统计。苗数15颗。野生型：SSBM；OsCYCP4；2过表达植株：OsCYCP4；2OVER-1，OsCYCP4；2OVER-5和OsCYCP4；2OVER-7。
图3：突变体cycp4；2的磷含量：A，地上(shoot)和地下(root)部分无机磷含量；B，地上(shoot)和地下(root)部分总磷含量。
图4：突变体cycp4；2在低磷下的响应。A：突变体cycp4；2在正常供磷(200μM，HP)和低磷(10μM，LP)处理21天的表型观察；B，A中植株地上(shoot)和地下(root)部分长度的测量和统计；C，A中植株地上(shoot)和地下(root)部分相对干重的称量和统计。
具体实施方式
下面通过具体实施例，并结合附图，对本发明的技术方案作进一步的具体说明。
本发明中，若非特指，所采用的原料和设备等均可从市场购得或是本领域常用的。下述实施例中的方法，如无特别说明，均为本领域的常规方法。
实施例：
一种提高水稻耐低磷胁迫的方法，通过将水稻细胞周期蛋白OsCYCP4；2的编码基因水稻细胞周期基因OsCYCP4；2从目的水稻上敲除(具体步骤见试验部分第4节)，使得水稻耐低磷胁迫能力提高。
试验：
1、OsCYCP4；2在转录水平对低磷胁迫的响应
野生型水稻正常供磷(200μM)和低磷(10μM)条件下液体培养21天，分别提取地上和地下部分RNA样品，逆转录后进行实时定量PCR。结果显示OsCYCP4；2基因在地上和地下部分的表达均受低磷胁迫诱导(图1A)；OsCYCP4；2实时定量PCR引物：
P1：5’TAGTTAGTGTGGCAGTTGCTTTGA 3’(SEQ ID NO.4)
P2：5’CACCATTAGTACACACCGAAACAA 3’(SEQ ID NO.5)。
2、OsCYCP4；2在翻译水平对低磷胁迫的响应
根据NCBI网站提供的水稻基因组全序列，我们克隆了OsCYCP4；2的全长基因组序列SEQ ID NO.2，包括3000bp启动子，5’非翻译区，外显子和内含子。设计infusion扩增引物：
P3：5’TCTAGAGGATCCACGGTACCACGGACTGCCGCATGGTGAT 3’(SEQ ID NO.6)，
P4：
5’CTCAGATCTACCATGGTACCGCTTGCTTCCATGGCCGCTTCACG 3’(SEQ ID NO.7)。
提取水稻基因组DNA,取50ng DNA作为模板在50μl体系中进行目的片段的扩增。扩增体系为：DNA模板1μl，引物P3(10μM)0.2μl，引物P4(10μM)0.2μl,2×KOD缓冲液(购自TOYOBO公司)25μl，dNTP(2.5mM)2μl，KOD酶(购自TOYOBO公司)2μl加水至50μl。扩增条件为：94℃预变性5min，然后以94℃变性1min，62℃复性1min，72℃延伸4min，进行28个循环，最后72℃延伸10min。通过凝胶电泳回收扩增片段，通过infusion酶将目的片段融合入由pCAMBIA1300改造而来pCAMBIA1300-GUS(在pCAMBIA1300后面加入GUS序列和终止子Nos)载体中，转化大肠杆菌感受态细胞，挑选阳性克隆后进行测序获得重组克隆POsCYCP4；2:OsCYCP4；2-GUS。将表达载体POsCYCP4；2:OsCYCP4；2-GUS转化农杆菌，用于水稻转化。
通过农杆菌共培养的方法将表达载体POsCYCP4；2:OsCYCP4；2-GUS转至水稻。通过50mg/ml潮霉素筛选获得再生植株。对获得的转基因植株进行GUS检查，验证后，GUS表达模式一致的转基因植株用于收种和后续实验。对转基因幼苗进行正常供磷(200μM)和低磷(10μM)处理21天，分别对地上和地下部分进行GUS染色并观察。发现低磷处理后地上和地下部分的GUS染色均强于正常条件下的GUS染色(图1B)。证明OsCYCP4；2蛋白表达受低磷胁迫诱导，与mRNA水平一致。
3、OsCYCP4；2过表达转基因水稻的获得和表型观察
根据NCBI网站提供的水稻序列，我们克隆了OsCYCP4；2的cDNA序列SEQ ID NO.3。设计带有KpnI和BamH酶切位点和保护碱基的扩增引物：
P5：5’CGGGGTACCGGAGCGAGGCAAGGGAAGC 3’(SEQ ID NO.8)，
P6：5’CGCGGATCCCGTAGGACAGATCACATGTATGTACGC 3’(SEQ ID NO.9)。
提取水稻总RNA，将5μg总RNA进行逆转录，将逆转录产物-作为模板在50μl体系中进行目的片段的扩增。扩增体系为：逆转录产物1μl，引物P5(10μM)0.2μl，引物P6(10μM)0.2μl,2×KOD缓冲液25μl，dNTP(2.5mM)2μl，KOD酶2μl(购自TOYOBO公司)，加水至50μl。扩增条件为：94℃预变性5min，然后以94℃变性30s，60℃复性30s，72℃延伸45s，进行28个循环，最后72℃延伸10min。通过凝胶电泳回收扩增片段，通过酶切连接将目的片段融合入由pCAMBIA1300改造而来pCAMBIA1300-ubi-rbcs载体(在pCAMBIA1300中加入启动子ubi和终止子rbcs)中，转化大肠杆菌感受态细胞，挑选阳性克隆后进行测序获得重组克隆Pubi:OsCYCP4；2。将过表达载体Pubi:OsCYCP4；2转化农杆菌，用于农杆菌介导的水稻转化。获得转基因材料后，用定量PCR的方法检测OsCYCP4；2基因表达情况，将OsCYCP4；2基因超表达的转基因植株进行繁种，用于后续研究。观察发现OsCYCP4；2过表达转基因植株生长明显受到抑制，表现为地上部分只有野生型70-80％的生长，而地下部分只有野生型40％左右的生长。可见，OsCYCP4；2在水稻生长上起负调控作用(图2)。
4、水稻细胞周期基因OsCYCP4；2基因敲除和表型观察
根据植物CRISPR/Cas9载体系统靶位点的选择要求，我们将所选OsCYCP4；2的gRNA序列tgcacgctgtaggagtcga按文献：Ma X,Zhang Q,Zhu Q,Liu W,Chen Y,Qiu R,Wang B,Yang Z,Li H,Lin Y,Xie Y,Shen R,Chen S,Wang Z,Chen Y,Guo J,Chen L,Zhao X,DongZ,Liu YG(2015)A Robust CRISPR/Cas9System for Convenient,High-EfficiencyMultiplex Genome Editing in Monocot and Dicot Plants.Mol Plant 8:1274-1284，提供的方法构建载体pYLCRISPR/Cas9-MH-OsCYCP4；2。
选取成熟饱满的水稻种子脱壳后，经30％次氯酸钠消毒滤干后接种到愈伤诱导培养基上进行诱导培养。四周后选择外观颜色嫩黄、颗粒致密的状愈伤组织用于遗传转化。将pYLCRISPR/Cas9-MH-OsCYCP4；2载体导入农杆菌，采用农杆菌介导法将pYLCRISPR/Cas9-MH-OsCYCP4；2转入水稻愈伤组织中，用含有200μM乙酰丁香酮和OD600为0.02的农杆菌的AAM转化液进行浸泡，将浸泡过的愈伤组织转至共培养基上暗培养3天。洗涤后转移至含50mg/ml潮霉素和500mg/ml的头孢霉素选择培养基上培养，每15天继代一次。30天后将筛选出的抗性愈伤转至分化培养基上分化培养40天。将分化出的绿色小苗进行PCR鉴定，转化阳性苗再进行酶切鉴定，最后获得OsCYCP4；2碱基插入或缺失的突变体，繁种用于后续研究。我们将其中一个单碱基插入突变体命名为cycp4；2，该突变体在ATG后311位插入一个A，造成该基因翻译蛋白移码且提前终止。正常培养条件下，观察发现OsCYCP4；2基因缺失没有造成植株生长发育上的明显缺陷。磷含量的测定发现突变体植株的无机磷含量和总磷含量均高于野生型植株(图3A和B)。
对野生型植株和突变体cycp4；2植株进行长期的正常供磷和低磷胁迫处理，测定各项生长生理指标，如：根长，地上部分长度，生物量。统计结果发现，低磷对的cycp4；2根长诱导大于对野生植株根长的诱导。而且cycp4；2对低磷抑制地上部分生长的敏感性降低，表现为cycp4；2地上部分的相对干物重为51％，而野生型植株相对干物重为46％(图4)。
一、水稻转化培养基配方：
1)诱导培养基配方：
N6大量母液50mL/L，B5微量母液10mL/L，NB有机贮存液10mL/L，铁盐贮存液10mL/L，2,4-D 2.0mg/L，L-谷氨酰胺0.5g/L，L-脯氨酸2.8g/L，水解酪蛋白0.3g/L，蔗糖30g/L；
pH调至5.8后加入植物凝胶4g/L。
2)共培养基配方：
N6大量母液50mL/L，B5微量母液10mL/L，NB有机贮存液10mL/L，铁盐贮存液10mL/L，
2,4-D 2.0mg/L，L-谷氨酰胺0.5g/L，L-脯氨酸2.8g/L，水解酪蛋白0.6g/L，葡萄糖10g/L，蔗糖30g/L；
pH调至5.2后加入植物凝胶4g/L。
灭菌后，加入乙酰丁香酮200μmol/L。
3)选择培养基配方：
N6大量母液50mL/L，B5微量母液10mL/L，NB有机贮存液10mL/L，铁盐贮存液10mL/L，
2,4-D 2.0mg/L，L-谷氨酰胺0.5g/L，L-脯氨酸2.8g/L，水解酪蛋白0.6g/L，蔗糖30g/L；
pH调至5.8后加入植物凝胶4g/L。
灭菌后加入50mg/L潮霉素和500mg/L头孢。
4)分化培养基配方：
N6大量母液50mL/L，B5微量母液10mL/L，NB有机贮存液10mL/L，铁盐贮存液10mL/L，L-谷氨酰胺0.5g/L，L-脯氨酸0.5g/L，水解酪蛋白1g/L，6-BA 3.0mg/L，NAA 0.5mg/L，蔗糖30g/L，山梨醇20g/L；
pH调至5.8后加入植物凝胶4g/L。
5)生根培养基
N6大量母液50mL/L，B5微量母液10mL/L，NB有机贮存液10mL/L，铁盐贮存液10mL/L，
蔗糖20g/L；
pH调至5.8后加入植物凝胶3.5g/L。
6)AAM转化液
AA大量母液100mL/L，B5微量母液10mL/L，NB有机贮存液10mL/L，铁盐贮存液10mL/L，水解酪蛋白0.3g/L，麦芽糖30g/L；
pH调至5.5，灭菌后，加入乙酰丁香酮200μmol/L。
二、主要溶液配方：
1)N6大量元素母液(20倍浓缩液)：
硝酸钾56.6g，氯化钙3.32g，硫酸镁2.70g，磷酸二氢钾8.0g，硫酸铵9.26g，逐一溶解，室温下混合定容至1升。
2)B5微量元素母液(100倍浓缩液)：
碘化钾0.0750g，硼酸0.30g，硫酸锰1.0g，硫酸锌0.2g，硫酸铜0.0025g，逐一溶解，室温下混合定容至1升。
3)NB有机贮存液(100倍浓缩液)
烟酸1g，盐酸吡哆醇1g，盐酸硫胺素10g，肌醇10g，加水定容至1升。
4)铁盐贮存液(100倍浓缩液)
硫酸亚铁2.78g，乙二铵四乙酸二钠3.73g，室温下混合定容至1升。
5)AA大量母液
氯化钾2.95g，氯化钙0.15g，硫酸镁0.25g，磷酸二氢钾0.15g，室温下混合定容至1升。
综上所述，本发明首次报道了OsCYCP4；2在水稻基因工程技术领域的应用。水稻细胞周期蛋白OsCYCP4；2在水稻生长起负调控作用。而磷饥饿诱导该基因和蛋白的表达。水稻细胞周期基因OsCYCP4；2缺失后能提高植株磷含量，且降低植株对低磷抑制生长的敏感性，为培育适用于磷贫瘠土壤的水稻新品种提供了保障。
以上所述的实施例只是本发明的一种较佳的方案，并非对本发明作任何形式上的限制，在不超出权利要求所记载的技术方案的前提下还有其它的变体及改型。
Claims (4)
1.一种水稻细胞周期蛋白OsCYCP4；2在调控低磷胁迫下水稻生长中的应用，其特征在于：水稻细胞周期蛋白OsCYCP4；2的氨基酸序列如SEQ ID NO.1所示。
2.根据权利要求1所述的应用，其特征在于：水稻细胞周期蛋白OsCYCP4；2负调控水稻生长。
3.一种提高水稻耐低磷胁迫的方法，其特征在于：通过将水稻细胞周期蛋白OsCYCP4；2的编码基因水稻细胞周期基因OsCYCP4；2从目的水稻上敲除，使得水稻耐低磷胁迫能力提高；水稻细胞周期蛋白OsCYCP4；2的氨基酸序列如SEQ ID NO.1所示。
4.根据权利要求3所述的方法，其特征在于：水稻细胞周期基因OsCYCP4；2的核苷酸序列如SEQ ID NO.2所示。
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| CN102234328A (zh) * | 2010-04-29 | 2011-11-09 | 中国农业大学 | 植物耐低磷胁迫相关的蛋白AtLPT2及其编码基因与应用 |
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| CN102010464A (zh) * | 2010-08-26 | 2011-04-13 | 浙江大学 | 水稻磷吸收转运调控基因OsPHF1及其应用 |
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| GenBank: AK108827.1，Oryza sativa Japonica Group cDNA clone:002-151-F11, full insert sequence;Kikuchi,S.等;《Genbank Database》;20081204;DEFINITION、SOURCE、FEATURES及ORIGIN部分 * |
| OsCYCP1;1,a PHO80 homologous protein,negatively regulates;Minjuan Deng等;《Plant Mol Biol》;20141015;第86卷;第655-669页 * |
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