CN104725494A - 水稻基因OsARC促进水稻抗干旱与抗盐胁迫中的应用 - Google Patents
水稻基因OsARC促进水稻抗干旱与抗盐胁迫中的应用 Download PDFInfo
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- C12N15/8273—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for drought, cold, salt resistance
Abstract
本发明涉及植物基因工程技术领域。具体涉及一种水稻DNA片断的分离克隆、氨基酸序列优化以及功能验证及其应用。所述的DNA片断包含与水稻抗干旱与抗盐胁迫相关基因OsARC，它赋予水稻植株的抗干旱与抗盐胁迫的能力。将该DNA片段与外源启动子序列连接后直接转入植物体，转基因植物植株的抗干旱与抗盐胁迫得到相应的提高。
Description
技术领域
本发明涉及植物生物技术领域。具体涉及一种水稻DNA片断的分离克隆、氨基酸序列优化以及功能验证及其应用。
背景技术
水稻原产亚洲热带，在中国广为栽种后，逐渐传播到世界各地。按照不同的方法，水稻可以分为籼稻和粳稻、早稻和中晚稻、糯稻和非糯稻。我国科学家袁隆平对杂交水稻的研究作出了巨大贡献，被誉为“杂交水稻之父”。水稻所结子实即稻谷，稻谷(粒)去壳后称大米、香米、稻米.世界上近一半人口，都以大米为食。大米的食用方法多种多样，有米饭、米粥、米饼、米糕、米酒等。水稻除可食用外，还可以酿酒、制糖作工业原料，稻壳、稻秆，可以作为饲料。我国水稻主产区主要是东北地区、长江流域、珠江流域。属于直接经济作物。还是世界上三分之一人类的主食。
锚蛋白重复序列(ankyrin repeat)是普遍存在于真核、原核及病毒中的一种蛋白质序列模体，其功能主要是参与蛋白质与蛋白质的相互作用。1987年，Breeden和Nasmy th在两个酵母细胞周期调控蛋白Sw i6和Cdc10中，首次发现了ANK模体，随后在细胞骨架锚蛋白(ankyrin)中发现了含有24个此重复序列模体的结构，将其命名为锚蛋白重复序列模体(ANK repeat s mot if)。典型的ANK重复序列一般含有33个残基，数目不等的ANK单元串联起来，形成许多大的ANK结构域(ANKdomain)，行使生物功能。由于该蛋白质家族成员中ANK在数目、一级序列以及空间结构上都存在差异，使得AN K模体能够与多种配体结合，实现纷繁复杂的生物功能。有关动物中锚蛋白重复序列的研究报道很多，
其中主要包括细胞骨架形成体(cytoskeletal org an-izers)，CDK(cyclin dependent kinase)抑制剂，转录调控因子，发育调控子，膜蛋白等。拟南芥和水稻等模式植物基因组测序发现锚蛋白重复序列也存在于许多植物蛋白中，但植物锚蛋白的研究还相对滞后，主要集中在有关信号转导中的自身防御、发育调控、物质转运及蛋白磷酸化等。因此，研究ANK模体对于认识这类植物蛋白质家族成员的结构和功能具有十分重要的作用，也必定会给植物信号传导的研究带来新的切入点。植物依靠敏感的识别机制，识别各种入侵的病原微生物，从而激活防卫反应、保护自己。遗传方法发现有很多抗性基因参与防卫反应的信号传导，它们能被独立地激活。锚蛋白作为重要的调节因子，广泛参与植物防卫反应。
应用突变体分析和基因克隆技术，已分离和鉴定了一些参与防卫反应的锚蛋白基因，例如：At-P hos43、A K P 2、A N K 1、A CD 6、DRE B2和NPR1等。Scott等用二维电泳结合质谱分析在拟南芥中分离得到一个43KD的蛋白质AtPho s43，细菌或真菌感染后几分钟内AtPhos43迅速被磷酸化，其C-末端含2个锚蛋白单元，可能介导蛋白质的识别或相互作用。有趣的是表达分析发现许多植物如水稻、棉花、苜蓿等均存在A tPhos43的同源蛋白，而酵母、动物和人类均没找到其同源蛋白质，表明A tP hos43是一个植物特有的锚蛋白基因。Yan等应用酵母双杂交分离和鉴定了一个锚蛋白基因AK R2，A K R 2蛋白C-末端有4个锚蛋白单元，而N-末端包含PEST序列，类似动物的IkB蛋白及植物N PR 1/N IM1蛋白。用反义RNA抑制AKR2表达导致叶片小块坏死，并伴随H2 O2产生，类似过敏反应。AK R2可能是PR-1的负调节子，还可能参与抗病和抗胁迫反应中的抗氧代谢，锚蛋白重复序列在其中的具体作用还有待进一步研究。
Hua Lu等2003年在拟南芥中分离得到一个新的锚蛋白基因ACD6，它影响和调节拟南芥防卫反应的SA(水杨酸)信号途径。ACD6是一个必需且剂量依赖的激活因子，在对病原体防卫反应和SA依赖的细胞程序死亡中起作用，它属于一个大的家族，编码的蛋白质含9个锚蛋白单元，N-末端有5个跨膜结构域。
因此，提高植株的整体抗病或抗逆能力对于提高水稻的产量具有积极的意义。提高水稻的产量一直是科学家孜孜不倦的追求，而增加植株的抗病和抗逆能力对于提高水稻的产量具有重要的意义。本发明克隆并优化得到了Os ARC基因，利用该基因进行水稻遗传转化并鉴定了转基因植株的抗逆及抗病能力。
发明内容
本发明的目的是从水稻中分离克隆一个植株抗逆及抗病相关的完整编码区段的DNA片段，利用这个基因促进植物的抗病及抗逆能力。这个基因被命名为Os ARC。
本发明涉及分离和应用一种包含OsARC基因的DNA片段，该片段赋予水稻植株的抗逆及抗病能力。为了增强所述基因的效果，申请人对所述的基因片段进行基因的定点突变，从120个突变体中通过转基因实验证实有10个蛋白具有较好的实验效果，因此选择这10个具有较好效果的突变体蛋白。所述氨基酸片段如序列表SEQ ID NO：1-11所示。
可以采用已经克隆的OsARC基因作探针，从cDNA和基因组文库中筛选得到本发明的基因或同源基因。同样，也可以采用PCR(polymerase chain reaction)技术，从基因组、mRNA和eDNA中扩增得到本发明的OsARC基因以及任何感兴趣的一段DNA或与其同源的一段DNA。采用以上技术，可以分离得到包含OsARC基因的序列，将这一序列与任何一种可以引导外源基因在植物中表达的表达载体转化植株，可获得植株繁殖能力增强以及分蘖能力提高的转基因植株。本发明的基因在构建到植物表达载体中时，在其转录起始核苷酸前加上任何一种强启动子或诱导型启动子。本发明的基因在构建到植物表达载体中时，也可使用增强子，这些增强子区域可以是ATG起始密码子和邻接区域起始密码子等，但必需与编码序列的阅读框相同，以保证整个序列的翻译。
携带有本发明OsARC基因的表达载体可通过使用Ti质粒，植物病毒载体，直接DNA转化，微注射，电穿孔等常规生物技术方法导入植物细胞(Weissbach，1998.Method for Plant Molecular Biology VIII，Academy Press，New York，pp.411-463；Geiserson and Corey，1998，Plant MolecularBiology(2ndEdition)。
可使用包括本发明的OsARC基因的表达载体转化宿主是包括水稻在内多种植物，培育增加植物量植物品种。
下面结合实施例对本发明做进一步说明。
具体实施方式
实施例1：分离克隆包含有OsARC基因区段的DNA片段
以水稻品种“珍汕97”作为模板，并设计序列特异引物外加接头BamHI位点和XbaI位点。将扩增获得的PCR产物连入pGEM-T载体(购自Promega公司)，筛选阳性克隆并测序(ABI3730测序仪)，获得所需的全长基因cDNA，并得到了氨基酸序列为SEQ ID NO：1所示。该克隆被命名为pGEM-OsARC。
实施例2OsARC蛋白定点突变
将SEQ ID NO：1的蛋白质序列，通过设计引物，实现蛋白质序列中基因的定点突变。突变了120个突变体，并获得了相应的核苷酸序列并构建相应的pGEM-OsARC突变载体。
实施例3OsARC基因抑制表达、超量表达以及启动子表达载体的构建和转化
为了能更好地阐明此基因的功能，申请人将其在水稻中抑制表达和超量表达，从转基因植株的表型来验证。
抑制表达(RNAi)载体构建方法如下：首先根据“slqdliq”靶点通过常规的设计方法设计了siRNA，合成所述的siRNA并构建RNAi载体。
超量表达载体构建方法如下：首先将实施例1中以及实施例2得到的阳性克隆pGEM-OsARC质粒用BamHI和XbaI双酶切，回收外源片段；同时，用同样的方法酶切携带不同启动子的遗传转化载体pC1301S。酶切完毕，用氯仿：异戊醇(按以及比：24∶1)抽提，纯化酶切产物。用包含OsARC基因的酶切片段和酶切后的载体做连接反应，转化大肠杆菌DH10β(菌株购自Invitrogen公司)。通过酶切筛选阳性克隆，获得转化载体。遗传转化的载体pC1301S是在国际上常用的植物遗传转化载体pCAMBIA1301基础上，在酶切位点EcoRI和SacI处引入广泛使用的组成型表达的烟草花叶病毒CaMV 35S启动子而得到的。
通过农杆菌介导的水稻遗传转化方法将其导入到水稻品种“中花11”(中国水稻研究所提供的一个公开使用的一个水稻品种)中(其它任意的水稻品种也能够达到相同的效果，再次不一一列举)，经过预培养、侵染、共培养、筛选具有潮霉素抗性的愈伤、分化、生根、练苗移栽，得到转基因植株。农杆菌介导的水稻(粳稻亚种)遗传转化方法应用Hiei等人报道的方法(Hiei等，Efficienttransformation of rice，Oryza sativa L.，mediated by Agrobacterium andsequence analysis of the boundaries of the T-DNA，Plant J，6：271-282，1994)进行。
上述农杆菌介导的遗传转化具体步骤的培养基如下所述：
试剂配方：(1)试剂和溶液缩写：本发明中培养基所用到的植物激素的缩写表示如下：6-BA(6-苄基腺嘌呤)；CN(羧苄青霉素)；KT(激动素或称细胞分裂素)；NAA(萘乙酸)；IAA(吲哚乙酸)；2，4-D(2、4-二氯苯氧乙酸)；AS(Acetosringone，乙酰丁香酮)；CH(水解酪蛋白)；HN(Hygromycin B，潮霉素)；DMSO(二甲基亚砜)；N6max(N6大量成分溶液)；N6mix(N6微量成分溶液)；MSmax(MS大量成分溶液)；Msmix(MS微量成分溶液)(2)主要溶液配方：
1)N6培养基大量元素母液[10倍浓缩液(10X)]的配制：
逐一溶解，然后室温下定容至1000ml。
2)N6培养基微量元素母液[100倍浓缩液(100X)]的配制
室温下溶解并定容至1000ml。
3)铁盐(Fe2EDTA)贮存液(100X)的配制
准备800ml双蒸水并加热至70℃，加入乙二铵四乙酸二钠(Na2EDTA·2H2O)3.73克，充分溶解后在70℃水浴中保持2小时，定容至1000ml，4℃保存备用。
4)维生素贮存液(100X)配制
加水定容至1000ml，4℃保存备用。
5)MS培养基大量元素母液(10X)的配制
室温下溶解并定容至1000ml。
6)MS培养基微量元素母液(100X)的配制
室温下溶解并定容至1000ml。
7)2，4-D贮存液，6-BA贮存液，萘乙酸(NAA)贮存液，吲哚乙酸(IAA)贮存液：1均为mg/ml。
8)葡萄糖贮存液：0.5g/ml。
9)AS贮存液的配制：秤取AS 0.392g，DMSO 10ml。
(3)用于水稻遗传转化的培养基配方
1)诱导培养基
加蒸馏水至900ml，1N氢氧化钾调节pH值到5.9，煮沸并定容至1000ml，分装到50ml三角瓶(25ml/瓶)，封口灭菌。
2)继代培养基
加蒸馏水至900ml，1N氢氧化钾调节pH值到5.9，煮沸并定容至1000ml，分装到50ml三角瓶(25ml/瓶)，封口灭菌。
3)预培养基
加蒸馏水至250ml，1N氢氧化钾调节pH值到5.6，封口灭菌。使用前加热溶解培养基并加入5ml葡萄糖贮存液和250μl AS贮存液，分装倒入培养皿中(25ml/皿)。
4)共培养基
加蒸馏水至250ml，1N氢氧化钾调节pH值到5.6，封口灭菌。使用前加热溶解培养基并加入5ml葡萄糖贮存液和250μl AS贮存液，分装倒入培养皿中(25ml/每皿)。
5)悬浮培养基
加蒸馏水至100ml，调节pH值到5.4，分装到两个100ml的三角瓶中，封口灭菌。使用前加入1ml葡萄糖贮存液和100μl AS贮存液。
6)选择培养基
加蒸馏水至250ml，调节pH值到6.0，封口灭菌。使用前溶解培养基，加入250μl HN和400ppm CN，分装倒入培养皿中(25ml/皿)。
7)预分化培养基
加蒸馏水至250ml，1N氢氧化钾调节pH值到5.9，封口灭菌。使用前溶解培养基，加入250μl HN和200ppm CN，分装倒入培养皿中(25ml/皿)。
8)分化培养基
加蒸馏水至900ml，1N氢氧化钾调节pH值到6.0。煮沸并定容至1000ml，分装到50ml三角瓶(50ml/瓶)，封口灭菌。
9)生根培养基
加蒸馏水至900ml，1N氢氧化钾调节pH值到5.8。煮沸并定容至1000ml，分装到生根管中(25ml/管)，封口灭菌
将OsARC抑制表达的转基因水稻植株分别命名为OsARC-Y；将OsARC超量表达的转基因水稻植株分别命名为OsARC-C。每个转化载体获得了至少10株独立的转基因水稻植株。
实施例4：检测水稻内源基因OsARC的表达水平以及植株的生物量
以转基因的和未转基因的水稻品种““中花11””为材料，提取不同生长时期的组织的RNA用RT-PCR检测OsARC基因的表达水平。将扩增出的OsARC基因及Actin1基因的特异带用GelImager软件(GenRes公司)进行处理，将带的亮度数量化，并以Actin1基因的特异带的亮度为标准进行均一化后比较不同组织中OsARC基因的表达量。结果如下表1所示：OsARC基因在整个植株中都有表达，其中在转基因植株中的表达量要远远大于未转基因的植株。
表1OsARC的RNA表达量(相对表达量)
解释：蛋白质名称中287N/T表示将SEQ ID NO：1氨基酸序列的第287的N修饰为T；其余修饰的表示都参考相同的定义。
实施例4：OsARC提高转基因T1家系耐盐胁迫能力
为评价OsARC是否能使转基因植株耐盐性增强，首先以200mM NaCl处理水稻转基因系和空载转化系及野生型离体苗。转基因系和野生型处理24小时后，在表型上没有明显差异。但是48小时后，野生型及空白对照叶片明显呈现出水渍坏死，而转基因植株水渍程度明显较低。以基本相似部位的叶片的平均表面积，作为耐盐指标，表面积越大，说明耐盐性能越好。得到结果如表2所示，从结果可以看出，经过改造的基因在培育转基因耐盐植物上具有较好的效果。
实施例5OsARC提高转基因T1家系耐旱用途
转基因和空白对照苗在MS培养基上培养2周且生长状况基本一致的无菌苗移至甘露醇含量为200mM的MS培养基中，光照培养箱培养30天，观察叶片萎缩情况。结果如表3所示。
表3叶片萎缩比率
从以上的数据可以看出，OsARC基因对于调节水稻耐盐和耐干旱胁迫具有正向的积极意义，并且本发明进一步对OsARC基因进行了一系列点突变，突变后的基因在调节水稻耐盐和耐干旱胁迫能力上相对与野生型OsARC基因也有了明显的提高。
Claims (5)
1.OsARC基因的蛋白质序列，如序列表SEQ ID NO：1-11任一序列所示。
2.编码权利要求1所述的蛋白质的DNA序列。
3.权利要求1-2任一项所述的蛋白质序列或DNA序列在增加植物抗干旱能力中的应用。
4.权利要求1-2任一项所述的蛋白质序列或DNA序列在增加植物抗盐胁迫中的应用。
5.如权利要求3-4任一项所述的应用，其特征在于：所述植物为水稻。
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