CN116200411A - 水稻耐盐基因OsINH2和OsINH3及其应用 - Google Patents
水稻耐盐基因OsINH2和OsINH3及其应用 Download PDFInfo
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Abstract
本发明提供了水稻耐盐基因OsINH2和OsINH3及其应用。在挖掘抗逆基因时，发现CRISPR‑Cas敲除这两个基因后的水稻突变体对盐敏感，而超表达的株系在盐胁迫下不仅存活高，且生长也未受到明显抑制。基于这些表型结果，我们认为通过对OsINH2和OsINH3基因研究对与改良水稻抗盐抗逆、扩充抗逆基因库有积极作用。进一步研究发现OsINH2和OsINH3基因在调节植物育性、调节植物花粉活力、清除活性氧(ROS)中也具有功能。
Description
技术领域
本发明属于水稻抗性基因挖掘领域，具体涉及水稻耐盐基因OsINH2和OsINH3及其应用。
背景技术
土壤盐渍过度严重损害植物生长、发育以及农作物产量，而全世界约有20％的耕地及将近一半的灌溉土地遭受过度盐渍的影响，且工业污染以及农业的不合理灌溉正在加剧这一现状；伴随着人口的增长、对生活物质水平的需求提高，我们迫切的需要寻找一些在植物中广泛存在且保守的逆境胁迫相关的基因，来改善生态环境、提高作物在盐渍土壤中的产量。因此，发掘盐响应通路中关键调节蛋白对阐明植物响应盐的分子机理，以及在农作物中进行抗盐品质提升尤为重要。水稻是最重要的谷类作物之一，是所有谷类作物中对盐最敏感的一种，特别是在幼苗和生殖阶段，所以深入挖掘水稻中抗逆耐盐基因、解析其生理机制是目前生物技术育种的首要任务。
植物在生长发育过程中面临着各种生物和非生物胁迫，为了应对不断变化的环境，植物必须精准的调节生长发育和逆境响应的平衡。在合适的环境下，植物萌发生长、开会结果，正常地完成生命周期；而在胁迫环境下，植物促进胁迫响应基因表达，增强植株抗逆，但同时抑制生长相关基因的表达，导致生长延缓甚至停滞，这种生长-防御的权衡广泛存在于作物中，如小麦、玉米和水稻等，协调作物的生长发育和抗逆。关于水稻育种的抗逆响应或产量提升基因的挖掘，往往由于生长-防御的权衡而导致对另一方造成损害，目前仅有少量文献报道了一些参与生长和抗逆调控节点的基因，包括一些参与生长发育与逆境响应的植物激素ABA以及BR等信号通路节点。因此，解决生长-防御权衡仍是目前挖掘水稻中抗逆耐盐基因的难点。现有技术存在的问题：现有技术未见OsINH2和OsINH3具有抗盐性的报道。
发明内容
本发明要解决的关键技术问题在于OsINH2和OsINH3具有抗盐性的应用。为解决上述技术问题，本发明采用如下技术方案：
实施例1
1.水稻基因OsINH2，所述水稻基因OsINH2具有如SEQ ID NO.1所示的核苷酸序列，全长分别为4741bp；上述水稻基因OsINH2的转录本(mRNA)具有如SEQ ID NO.2所示的核苷酸序列，其中239bp-736bp为CDS区，共498bp的序列。
2.水稻基因OsINH3，包括：水稻基因OsINH3具有如SEQ ID NO.3所示的核苷酸序列，全长分别为1243bp；上述水稻基因OsINH3的转录本(mRNA)具有如SEQ ID NO.4所示的核苷酸序列，其中88bp-465bp为CDS区，共378bp的序列。
3.水稻基因OsINH2和OsINH3的克隆和互作蛋白验证方法，具体包括：(1)水稻基因OsINH2和OsINH3的克隆，(2)OsINH2和OsINH3与OsTOPPs互作。
4.水稻基因OsINH2和OsINH3的基因功能验证方法，具体包括：(1)OsINH2和OsINH3的亚细胞定位分析，(2)OsINH2和OsINH3表达模式分析，(3)水稻超表达OsINH2和OsINH3载体的构建，(4)水稻突变体osinh2、osinh3的构建，(5)水稻遗传转化和阳性株筛选，(6)ABA响应分析，(7)渗透调节分析。
5.OsINH2和OsINH3调节植物育性的应用。
6.OsINH2和OsINH3调节植物花粉活力的应用。
7.OsINH2和OsINH3清除活性氧(ROS)的应用。
8.OsINH3与OsSnRK互作的应用。
9.OsINH2和OsINH3抗盐性的应用。
10.一种包含水稻OsINH2或OsINH3的载体，其特征在于所述OsINH2具有如SEQ IDNO.1所示的核苷酸序列；所述OsINH3具有如SEQ ID NO.3所示的核苷酸序列。
有益效果：在挖掘抗逆基因时，发现CRISPR-Cas敲除这两个基因后的水稻突变体对盐敏感，而超表达株系在盐胁迫下不仅存活高，且生长也未受到明显抑制。此外，OsINH2和OsINH3敲除株系的育性较低、花粉活力降低、活性氧显著积累，而超表达株系的育性和花粉活力与野生型相似、活性氧显著降低。基于这些表型结果，我们认为通过对OsINH2和OsINH3基因研究对于改良水稻清除活性氧及抗盐抗逆能力、扩充抗逆基因库有积极作用。进一步研究该基因作用机理、在其他作物中的保守性，从而在作物中推广。
附图说明
图1为OsINH2和OsINH3克隆琼脂糖凝胶电泳图。图中(a)OsINH2基因扩增大小为495bp，(b)OsINH3基因扩增大小为375bp。
图2为OsINH2和OsINH3与OsTOPPs相互作用及细胞内共定位。图中(a)显示OsINH2和OsINH3与OsTOPPs在酵母双杂交系统中的相互作用。OsINH2-AD和OsSAPK1-BD或OsINH3-AD和OsSAPK1-BD用作阳性对照。与空AD融合的OsINH2和OsINH3以及与空BD融合的OsTOPPs用作阴性对照。DDO、SD/-Leu/-Trp；QDO/X、SD/-Leu/-Trp/-His/-Ade辅以X-α-Gal。图中(b)代表OsINH2和OsINH3与OsTOPPs相互作用的BiFC分析。OsINH2-YC和OsSAPK1-YN和OsINH3-YC和OsSAPK1-YN用作阳性对照。OsINH2-YC和OsSAPK4-YN或OsINH3-YC和OsSAPK4-YN用作阴性对照。图中(c)为瞬时表达分析表明OsINH2在体内与OsTOPPs共定位，OsSAPK1-RFP和YFP-OsINH2用作阳性对照。图中(d)为瞬时表达分析表明OsINH3在体内与OsTOPPs共定位，OsSAPK1-RFP和YFP-OsINH3用作阳性对照。
图3为OsINH2和OsINH3的组织特异性表达及其蛋白质亚细胞定位。图中(a)为OsINH2的表达模式，(i)幼苗，(ii)初生根，(iii)冠根，(iv)叶，(v)茎，(vi)小花，(vii)花，和(viii)花粉粒。图中(b)为OsINH3的表达模式，(i)幼苗，(ii)初生根，(iii)冠根，(iv)叶，(v)茎，(vi)小花，(vii)花，和(viii)花粉粒。图中(c)为OsINH2的亚细胞定位。图中(d)为图中为OsINH3的亚细胞定位。
图4为定量PCR鉴定OsINH2-OE和OsINH3-OE表达水平。图中(a)OsINH2-OE株系#1、#3中OsINH2的基因表达水平与野生型中花11相比显著上调。(b)OsINH3-OE株系#1、#4中OsINH3的基因表达水平与中花11相比显著上调。
图5为osinh2和osinh3突变体对ABA敏感。图中(a)和(b)分别为对照和ABA处理下OsINH2突变体和超表达株系种子萌发率。图中(c)和(d)为统计OsINH2突变体和超表达株系幼苗的根和芽长度。图中(e)和(f)为对照和ABA处理下OsINH3突变体和超表达株系种子萌发率。图中(g)和(h)为统计OsINH3突变体和超表达株系幼苗的根和芽长度。上图中幼苗在0μM和5μM ABA的1/2MS培养基上生长，最多记录7天的发芽率(％)。
图6为OsINH2和OsINH3参与了ABA响应的ROS清除和渗透调节。图中(a)为OsINH2和OsINH3突变体和超表达株系在正常以及ABA处理中的ROS积累，种子在1/2MS培养基上生长，将一周龄的幼苗转移到营养液中，在三叶期喷洒100μM ABA，叶子样品在NBT溶液中孵育过夜，用75％乙醇脱色后观察ROS。图中(b)和(c)为OsINH2和OsINH3突变体和超表达株系中的脯氨酸含量。图中(d)和(e)为OsINH2和OsINH3突变体和超表达株系中OsP5CS1的表达谱。
图7为OsINH2和OsINH3的突变体和超表达株系的成熟种子和花药表型。图中(a)为野生型(WT)和OsINH2突变体和超表达株系的小穗生育力的表型。图中(b)为统计WT和OsINH2突变体和超表达株系中每一个穗的种子数量。图中(c)为WT和OsINH2突变体和超表达株系花粉粒的形态学观察。比例尺，30μm。图中(d)表示WT和OsINH2突变体和超表达株系活性和非活性花粉的百分比。图中(e)为WT和OsINH3突变体和超表达株系中小穗生育力的表型。图中(f)为统计WT和OsINH3突变体和超表达株系中每一个穗的种子数量。图中(g)为WT和OsINH3突变体和超表达株系花粉粒的形态学观察。图中(h)代表WT和OsINH3突变体和超表达株系中活性和非活性花粉的百分比。
图8为OsINH2和OsINH3参与了ABA响应的ROS清除。图中(a)和(b)为OsINH2和OsINH3的突变体和超表达株系中的MDA含量。图中(c)和(d)为OsINH2和OsINH3的突变体和超表达株系中的POD活性，种子在1/2MS培养基上生长，将一周龄的幼苗转移到营养液中，在三叶期喷洒100μMABA，从叶片中检测到MDA含量和POD活性。图中(e)和(h)为OsLEA3和OsLIP19在OsINH2和OsINH3的突变体和超表达株系中的表达谱。
图9为OsINH2和OsINH3与OsSAPKs相互作用。图中(a)酵母双杂交证明OsINH2和OsINH3与OsSAPKs的相互作用。OsINH2-AD和OsTOPP4-BD或OsINH3-AD和OsSAPK4-BD用作阳性对照。空AD融合的OsINH2和OsINH3以及与空BD融合的OsSAPKs用作阴性对照。DDO：SD/-Leu/-Trp；QDO/X：SD/-Leu/-Trp/-His/-Ade辅以X-α-Gal。(b)BiFC结果显示OsINH2-YC和OsINH3-YC与OsSAPK1/2/3与OsSAPK8/9相互作用。OsINH2-YC和OsTOPP4-YN、OsINH3-YC和OsTOPP4-YN用作阳性对照。OsINH2-YC和OsSAPK5-YN、OsINH3-YC和OsSAPK5-YN用作阴性对照。BF，明场。比例尺，50μm。
图10为OsINH2和OsINH3突变体和超表达株系在盐胁迫下发芽期的表型。
图11为野生型中花11与OsINH2和OsINH3突变体和超表达株系在盐胁迫下的茎长统计。图中(a)0mM NaCl处理时，野生型中花11与osinh2、OsINH2-OE茎长无差异；而在盐处理时，与中花11相比，osinh2茎长显著变小，而OsINH2-OE茎长无差异。(b)0mM NaCl处理时，野生型中花11与osinh3、OsINH3-OE茎长无差异；而在盐处理时，与中花11相比，osinh3茎长显著变小，而OsINH3-OE茎长无差异。
图12为野生型中花11与OsINH2和OsINH3突变体和超表达株系在盐胁迫下的根长统计。图中(a)0mM NaCl处理时，中花11与osinh2、OsINH2-OE根长无差异；而在盐处理时，osinh2相比中花11根长显著变小，而OsINH2-OE根长无差异。(b)0mM NaCl处理时，osinh3、OsINH3-OE的根长与中花11无差异；而在盐处理时，与中花11相比，osinh3根长显著变小，而OsINH3-OE根长无差异。
图13为OsINH2-OE的耐盐表型。(a)在盐胁迫处理前，野生型中花11与osinh2生长发育没有差异，而在150mM NaCl处理7天后，osinh2相比中花11明显更矮，且叶片枯黄程度更严重；在正常水稻营养液中恢复14天后，与中花11相比osinh2存活率更低，株高也没有恢复。(b)在盐胁迫处理前，中花11与OsINH2-OE生长发育没有差异，而150mM NaCl处理7天后，OsINH2-OE相比中花11受到的盐损害较小，株高更高且叶片枯黄程度更轻；在正常水稻营养液恢复14天后，OsINH2-OE存活率以及株高与中花11相比显著更高。
图14为OsINH3-OE的耐盐表型。(a)在盐胁迫处理前，野生型中花11与osinh3生长发育没有差异，而在150mM NaCl处理7天后，osinh3株高相比中花11明显更矮，且叶片枯黄程度更严重；在正常水稻营养液中恢复14天后，与中花11相比osinh3存活率更低，株高也没有恢复。(b)在盐胁迫处理前，中花11与OsINH3-OE生长发育没有差异，而150mM NaCl处理7天后，OsINH3-OE相比中花11受到的盐损害较小，株高更高且叶片枯黄程度更轻；在正常水稻营养液恢复14天后，OsINH3-OE存活率以及株高与中花11相比显著更高。
图15为盐胁迫处理后野生型中花11与OsINH2-OE、osinh2和OsINH3-OE、osinh3的存活统计。图中(a)和野生型中花11相比，osinh2和osinh3在盐处理后，存活率显著降低。(b)和中花11相比，OsINH2-OE和OsINH3-OE在盐处理后，存活率显著升高。
具体实施方法
本发明专利下述实施例中使用方法和装置，如无特殊说明，均为常规方法和装置；所用器材、试剂均为试剂公司购买的常规器材和试剂。为使本发明专利的目的、技术方案和优点更加清楚，下面结合具体实施例对本发明专利的具体实施方式进行详细说明。这些优选实施方式的示例在具体实施例中进行了例示。在此，还需要说明的是，为了避免因不必要的细节而模糊了本发明专利的技术方案，在实施例中仅仅示出了与根据本发明专利的方案密切相关的技术方案和/或处理步骤，而省略了关系不大的其他细节。
实施例1
本实施例提供水稻基因OsINH2，包括：水稻基因OsINH2具有如SEQ ID NO.1所示的核苷酸序列，全长分别为4741bp；上述水稻基因OsINH2的mRNA具有如SEQ ID NO.2所示的核苷酸序列，其中239bp-736bp为CDS区，共498bp的序列(为方便分析U替换成了T)。
实施例2
本实施例提供水稻基因OsINH3，包括：水稻基因OsINH3具有如SEQ ID NO.3所示的核苷酸序列，全长分别为1243bp；上述水稻基因OsINH3的mRNA具有如SEQ ID NO.4所示的核苷酸序列，其中88bp-465bp为CDS区，共378bp的序列(为方便分析U替换成了T)。
实施例3
本实施例提供水稻基因OsINH2和OsINH3的克隆和互作蛋白验证，具体包括：
1.水稻基因OsINH2和OsINH3的克隆
在哺乳动物细胞及酵母中率先报道了inhibitor2和inhibitor3基因，其作为蛋白磷酸酶PP1的调节亚基，通过调节PP1功能调控了细胞分裂等多个方面。而后在拟南芥中发现其同源基因AtI2和Atinh3，我们通过蛋白序列比对，发现与拟南芥同源性很高的两个基因OsINH2和OsINH3。
从phytozome网站获得目的基因的CDS序列，设计引物克隆水稻OsINH2和OsINH3基因：OsINH2-F-1:GGGGTACCCCATGAGCTCTCGTCGTGTGAAG，OsINH2-R-1:GGACTAGTCCTGTTTGCGGTGGGGGTG。OsINH3-F-1:GGGGTACCCCATGGCAACGCGCGCGCCGGCGA，OsINH3-R-1:GGACTAGTCC ATGGTCGTGGCCGTGGCC。
分别以OsINH2-F-1/OsINH2-R-1和OsINH3-F-1/OsINH3-R-1为上下游引物，水稻中花11幼苗叶片cDNA为模板，进行PCR扩增，获得目的基因。PCR扩增反应在BIO-RADS1000Thermal Cycler PCR仪中进行，程序为：95℃预变性4min；94℃变性30s、58℃退火30s、72℃延伸1min，循环35次；72℃延伸5min；15℃保存。PCR产物经琼脂糖凝胶电泳验证，如附图1所示。
2.OsINH2和OsINH3与OsTOPPs互作
在酵母双杂交系统中检测OsINH2和OsINH3与OsTOPP1-5是否直接互作，方法参考(Zhang et al.,2020)。OsINH2和OsINH3与Gal4激活结构域(AD)融合，OsTOPP1-5与Gal4DNA结合结构域(BD)连接。将含AD和BD质粒的酵母涂抹在DDO(SD/-Leu/-Trp)培养基板上，28℃孵育3-4天。孵育结束后，每个样品接种于QDO(SD/-Leu/-Trp/-His/-Ade/+X-a-Gal琼脂平板上，孵育3-7天。然后观察菌落形态。酵母双杂交检测如附图2所示，OsINH2和OsINH3与OsTOPP1-5可以直接互作。
同时在烟草叶片中进行了双分子荧光互补(BiFc)实验，方法参考(Hu et.al.，2018)。将OsINH2-cYFP或OsINH3-cYFP与OsTOPPs-NYFP质粒在烟草中共表达，并通过激光共聚焦显微镜观察荧光信号。结果如附图2所示，OsINH2和OsINH3与OsTOPP1-5在植物体内直接互作。
实施例4
本实施例提供水稻基因OsINH2和OsINH3的基因功能验证方法，具体包括：
1.OsINH2和OsINH3的亚细胞定位分析
设计引物如下：OsINH2-F-2：GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGAGCTCTCGTCGTGTGAAG，OsINH2-R-2：GGGGACCACTTTGTACAAGAAAGCTGGGTTTCATGTTTGCGGTGGGGGTG，OsINH3-F-2：GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGGCAACGCGCGCGCCGGCGA，OsINH3-R-2：GGGGACCACTTTGTACAAGAAAGCTGGGTTTCAATGGTCGTGGCCGTGGCC。
分别以OsINH2-F-2/OsINH2-R-2和OsINH3-F-2/OsINH3-R-2为引物，以中花11幼苗叶片cDNA为模板，进行PCR扩增，获得目的基因。PCR扩增反应在BIO-RAD S1000 ThermalCycler PCR仪中进行，程序为：95℃预变性4min；94℃变性30s、58℃退火30s、72℃延伸1min，循环35次；72℃延伸5min；15℃保存。将PCR产物回收纯化后用BP酶将目的基因重组到中间载体pDONR-Zeo上，将连接产物转化到大肠杆菌感受态DH5α细胞中，涂布与含Zeo抗生素的LB培养皿37℃过夜培养，挑取菌斑以通用引物M13F、M13R进行菌落PCR，并送测验证。将测序无误的菌斑用含Zeo的LB悬浮培养，提取质粒。将含目的基因的pDONR-Zeo载体用LR酶重组到pEarleyGate 104(N-YFP)表达载体上，将连接产物转化到大肠杆菌感受态DH5α细胞中，涂布于含卡那霉素抗生素的LB培养基37℃过夜培养，挑取菌斑用含卡那霉素的LB悬浮培养，提取质粒转化农杆菌GV3101。在烟草叶片中瞬时表达，结果如附图3所示，检测到OsINH2在细胞质和细胞核中分布，而OsINH3定位于细胞核中。
2.OsINH2和OsINH3表达模式分析
将OsINH2和OsINH3启动子序列分别插入到pCAMBIA1301 GUS质粒中，序列大小分别为3.3Kb和3Kb。将转化至中花11后的GUS转基因植株不同组织(ProOsINH2:GUS/WT和ProOsINH2:GUS/WT)在37℃下用GUS工作液染色1晚，然后用70％的乙醇脱色，方法参考(Qinet al.,2014)。使用LEICA(M205A)立体显微镜成像。结果如附图3所示，OsINH2和OsINH3的启动子在萌发期和萌发后期高度激活，在幼苗、主根、冠根、叶片和地上部分表达较高。在生殖阶段的穗、花药和花粉粒中均有表达。
3.水稻超表达OsINH2和OsINH3载体的构建
分别以OsINH2-F-1/OsINH2-R-1和OsINH3-F-1/OsINH3-R-1引物扩增基因并胶回收。用BamHI和KpnI双酶切POX载体得到线性载体，与上述PCR回收纯化产物用infusion酶在15℃水浴中反应10min将目的基因重组到POX上，将连接产物转化到大肠杆菌感受态DH5α细胞中，挑取菌斑以OsINH2-F-1/OsINH2-R-1或OsINH3-F-1/OsINH3-R-1为引物进行PCR扩增，并送测验证，将测序无误的菌斑用含卡那霉素的LB悬浮培养，提取质粒。
4.水稻突变体osinh2、osinh3的构建
在https://crispr.dbcls.jp/输入OsINH2和OsINH3基因序列，获得CRISPR编辑特异靶序列GTTAGGCAGAAGATAACTGA和CATCCACGACCGTGACGGTG，设计接头引物，用CRISPR-Cas9构建突变体。
5.水稻遗传转化和阳性株筛选
(1)诱导愈伤组织：剥去水稻种子颖壳，放在70％的乙醇中浸泡1min，并用无菌水清洗3次。然后放入有效氯5％的次氯酸钠溶液中浸泡15min，并用无菌水清洗5次。放置在N6D培养基上32℃培养两周左右，诱导愈伤组织的分化。
(2)农杆菌共转化：将转化用的农杆菌加入200mL的LB培养基中，28℃培养16h左右。摇好的菌液5000rpm离心15min，倒掉上清液，用加入乙酰丁香酮(AS)的AAM培养基悬浮菌体，28℃摇菌2h。切下水稻愈伤组织放入灭菌的三角瓶中，倒入摇好的菌液浸泡10min。倒掉菌液，将愈伤组织置于无菌滤纸上吸干表面菌液，放在N6 AS培养基上25℃培养48～72h。
(3)去除农杆菌：用无菌水配置3瓶500ml的头孢噻肟钠(cef)溶液，浓度分别为500mg/L、1000mg/L和2000mg/L。将农杆菌共培养的愈伤组织放入三角瓶中，按照浓度梯度从低到高依次用cef溶液清洗愈伤组织。
(4)分化培养：用无菌滤纸吸干除过菌的愈伤组织表面液体，将愈伤组织放在N6DS培养基上32℃培养两周，再将新长出的愈伤组织转接到RE培养基上继续培养，这时愈伤组织会逐渐分化出成熟的幼苗。
(5)生根培养：待RE培养基上分化出的水稻幼苗长到3cm左右时，将幼苗转到HF培养基上28℃培养两周左右，之后移栽至无菌珍珠岩上炼苗一周，即可移栽至温室。
对水稻超表达OsINH2和OsINH3转化株系，我们进行了实时荧光PCR检测如附图4所示。分别筛选到两个表达水平显著高于野生型的株系OsINH2OE-1、OsINH2OE-3，OsINH3OE-1、OsINH3OE-4。
对CRISPR-Cas技术敲除构建的转化株系，我们在靶点上下游设计引物，通过基因测序分别鉴定到两个碱基插入或缺失导致的移码突变体osinh2-6、osinh2-11，osinh3-4、osinh3-9。
6.ABA响应分析
正常条件下，OsINH2和OsINH3敲除和超表达株系的种子发芽率与野生型相同。ABA处理后，敲除株系osinh2-6、osinh2-11，osinh3-4、osinh3-9的种子发芽率显著降低，幼苗生长也受到抑制，与野生型相比，敲除株系的根和芽生长明显变慢，如附图5所示。虽然OsINH2和OsINH3过表达系的种子萌发和幼苗生长与野生型相似，但与各自的敲除系相比，它们对ABA胁迫表现出了耐受力，如附图5所示。
7.渗透调节分析
脯氨酸是一种兼容的渗透性物质，通过稳定亚细胞结构和促进应激损伤后的细胞恢复发挥作用，可以作为影响适应性反应的应激指标。游离脯氨酸含量计算参考(Lou etal.，2017)。用l-脯氨酸计算脯氨酸的标准浓度，用分光光度计在波长530nm处测定吸光度。如附图6所示，ABA作用下，OsINH2和OsINH3敲除株系积累的游离脯氨酸含量明显减少，而过表达株系的游离脯氨酸含量较野生型显著增加。此外，在ABA处理后，脯氨酸生物合成基因OsP5CS1在OsINH2和OsINH3敲除株系中的表达水平显著降低，而过表达株系中明显升高。
实施例5
本实施例提供水稻基因OsINH2和OsINH3的应用，具体包括：
1.OsINH2和OsINH3调节植物育性的应用
如附图7所示，在生殖发育时期，OsINH2和OsINH3敲除株系的育性较低、种子数量显著减少，而OsINH2和OsINH3过表达株系的育性与野生型相同，种子数量没有显著差异。因此,OsINH2和OsINH3可能在水稻育性调控中发挥重要作用。
2.OsINH2和OsINH3调节植物花粉活力的应用
花粉粒染色方法参考(Xiang et al.,2019)。选择发育完全且花药包在小花内的花，在载玻片上爆破一个花药，加入1-2滴碘化钾溶液，在光镜下观察。有活性的花粉变成深蓝色，没有活性或发育不良的花粉变成棕色。花药在OsINH2和OsINH3敲除株系中变干、呈棕色，没有活性的花粉粒与野生型相比增多。而OsINH2和OsINH3过表达株系中活性花粉比例与野生型相似，如附图7所示。因此,OsINH2和OsINH3可能在水稻花粉发育中至关重要。
3.OsINH2和OsINH3清除活性氧(ROS)的应用
检测ROS的方法参考(He et al.，2012)。用硝基蓝四氮唑(NBT)对叶片进行染色。结果如附图6所示，ROS在OsINH2和OsINH3敲除株系中积累，而在超表达株系中明显减少。根据商业试剂盒说明书(Solarbio,Cat No:BC0020,Solarbio Cat No:BC0200)评估MDA含量和POD酶活性。结果如附图8所示，ABA处理后，OsINH2和OsINH3敲除株系中MDA形成量增加，而过表达株系的MDA含量显著降低，表明OsINH2和OsINH3可以通过降低脂质过氧化形成来保护膜损伤。与野生型相比，ABA处理后，OsINH2和OsINH3敲除株系中POD活性较低，而超表达株系POD活性升高。ABA处理后，OsINH2和OsINH3敲除株系中OsLEA3和OsLIP9基因表达显著下调。相反，过表达株系中这些基因的表达被诱导上调。这些结果表明，OsINH2和OsINH3可能通过刺激抗氧化酶的活性和触发应激反应基因来调节ROS，促进水稻逆境下生长。
4.OsINH3与OsSnRK互作的应用
结果如附图9所示，酵母双杂交检测证实OsINH3都与ABA信号通路重要调控因子OsSAPK1、OsSAPK2、OsSAPK3、OsSAPK8和OsSAPK9相互作用。因此OsINH3可能通过OsSAPKs参与ABA信号转导过程
5.OsINH2和OsINH3抗盐性的应用
幼苗期OsINH2-OE、OsINH3-OE、osinh2、osinh3耐盐性分析的方法如下所述：水稻种子置于45℃烘箱处理一周打破休眠，然后将种子经70％酒精的消毒60s，再用50％次氯酸钠溶液浸泡10min,灭菌水清洗5次，种植于1/2MS培养基中，培养条件为：14小时光照(28℃)/10小时黑暗(24℃)，光照强度800μmol·m-2·s-1，相对湿度70％。待种子萌发后，转移至含150mM NaCl的1/2MS培养基，生长5天，统计株高、根长。结果如附图10，附图11，附图12所示，种子萌发期OsINH2和OsINH3敲除株系对盐处理更敏感，而超表达株系反应与野生型相似。
当幼苗生长至四叶期时进行盐胁迫处理，在水稻营养液中加入150mM NaCl,每3天更换一次。盐处理7天后，将幼苗重新置于不添加NaCl的Yoshida营养液中，7天后观察表型并统计幼苗存活率。结果如附图13和14所示，敲除OsINH2和OsINH3基因降低了水稻耐盐性，而超表达OsINH2和OsINH3基因则增强了水稻耐盐能力。
以上所述仅是本申请的具体实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本申请原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本申请的保护范围。
参考文献：
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Xiang,X.,Zhang,P.,Yu,P.,Zhang,Y.,Yang,Z.,Sun,L.,Wu,W.,Khan,R.M.,Abbas,A.,Cheng,S.,Cao,L.,2019.LSSR1 facilitates seed setting rate bypromoting fertilization in rice.Rice(New York,N.Y.)12,31.
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Claims (10)
1.水稻基因OsINH2，其特征在于所述水稻基因OsINH2具有如SEQ ID NO.1所示的核苷酸序列，全长为4741bp；其转录本具有如SEQ ID NO.2所示的核苷酸序列，其中239bp-736bp为CDS区，共498bp的序列。
2.水稻基因OsINH3，其特征在于所述水稻基因OsINH3具有如SEQ ID NO.3所示的核苷酸序列，全长为1243bp；其转录本具有如SEQ ID NO.4所示的核苷酸序列，其中88bp-465bp为CDS区，共378bp的序列。
3.水稻基因OsINH2和OsINH3的克隆和互作蛋白验证方法，其特征在于所述方法包括如下步骤：(1)水稻基因OsINH2和OsINH3的克隆，(2)OsINH2和OsINH3与OsTOPPs互作。
4.水稻基因OsINH2和OsINH3的基因功能验证方法，其特征在于所述方法包括如下步骤：(1)OsINH2和OsINH3的亚细胞定位分析，(2)OsINH2和OsINH3表达模式分析，(3)水稻超表达OsINH2和OsINH3载体的构建，(4)水稻突变体osinh2、osinh3的构建，(5)水稻遗传转化和阳性株筛选，(6)ABA响应分析，(7)渗透调节分析。
5.水稻OsINH2和OsINH3调节植物育性的应用，其特征在于所述OsINH2具有如SEQ IDNO.1所示的核苷酸序列；所述OsINH3具有如SEQ ID NO.3所示的核苷酸序列。
6.水稻OsINH2和OsINH3调节植物花粉活力的应用，其特征在于所述OsINH2具有如SEQID NO.1所示的核苷酸序列；所述OsINH3具有如SEQ ID NO.3所示的核苷酸序列。
7.水稻OsINH2和OsINH3清除活性氧的应用，其特征在于所述OsINH2具有如SEQ IDNO.1所示的核苷酸序列；所述OsINH3具有如SEQ ID NO.3所示的核苷酸序列。
8.水稻OsINH3与OsSnRK互作的应用，其特征在于所述OsINH3具有如SEQ ID NO.3所示的核苷酸序列。
9.水稻OsINH2和OsINH3抗盐性的应用，其特征在于所述OsINH2具有如SEQ ID NO.1所示的核苷酸序列；所述OsINH3具有如SEQ ID NO.3所示的核苷酸序列。
10.一种包含水稻OsINH2或OsINH3的载体，其特征在于所述OsINH2具有如SEQ ID NO.1所示的核苷酸序列；所述OsINH3具有如SEQ ID NO.3所示的核苷酸序列。
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