CN109797158A - 基因OsNTL3在改良水稻高温抗性方面的应用及获得的水稻高温抗性基因 - Google Patents
基因OsNTL3在改良水稻高温抗性方面的应用及获得的水稻高温抗性基因 Download PDFInfo
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- CN109797158A CN109797158A CN201910033160.9A CN201910033160A CN109797158A CN 109797158 A CN109797158 A CN 109797158A CN 201910033160 A CN201910033160 A CN 201910033160A CN 109797158 A CN109797158 A CN 109797158A
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Abstract
本发明提供一种基因OsNTL3在改良水稻高温抗性方面的应用及获得的水稻高温抗性基因。基因OsNTL3其碱基序列如SEQIDNO.1所示。本发明利用高温诱导型启动子过表达去除跨膜域的OsNTL3基因，该转基因水稻材料在高温抗性方面显著增强。所述的高温诱导型启动子为BiP2和BiP4，BiP2的碱基序列如SEQIDNO.4所示，BiP4的碱基序列如SEQIDNO.5所示。水稻高温抗性基因，其碱基序列如SEQIDNO.6所示。水稻高温抗性基因编码的蛋白质，其氨基酸序列如SEQIDNO.7所示。此转基因水稻材料在苗期的高温抗性方面表现出耐受性状，能够明显提高农作物对高温等胁迫的抗性。
Description
技术领域
本发明涉及基因工程技术领域，具体涉及一种基因OsNTL3在改良水稻高温抗性方面的应用及获得的水稻高温抗性基因。
背景技术
高温、干旱、盐碱、冷害、冻害等环境胁迫是世界范围内粮食减产、品质降低的主要原因之一。为此，植物在长期的进化过程中形成了响应各种生物胁迫和非生物胁迫，提高抗性的适应机制和调控网络。植物对环境胁迫的应答过程涉及基因表达的调控，而转录因子是植物响应环境胁迫的主要调节因子，当环境条件发生变化时，植物通过一系列的信号感知和加工过程，激活转录因子，转录因子与相应的顺式作用元件结合后，启动或抑制下游基因的转录和翻译，最终使植物对内、外界的变化作出相应的调节作用。很多转录因子家族成员都参与植物对非生物环境胁迫的响应，比较大的基因家族有：bZIP、AP2/ERF、NAC、WRKY、MYB、ZincFinger和bHLH等(S.Lindemose,et al.Structure,functionand networksoftranscription factors involvedin abiotic stress responses.Int.J.Mol.Sci.14(2013)5842-5878)。大量的研究表明，通过基因工程调控这些参与植物抗逆过程的转录因子的表达，可以显著提高植物对高温、干旱、盐害、低温等非生物胁迫和生物胁迫的抗性，说明对转录因子的调控是植物抵御环境胁迫的关键环节。本课题组利用蛋白错误折叠诱导剂诱导水稻材料进行基因芯片分析时，发现OsNTL3是受衣霉素TM(Tunicamycin，抑制内质网中蛋白的N-糖基化从而造成蛋白折叠错误)和氮杂环丁烷-2羧酸AZC(Azetidine-2-carboxylicacid，一种脯氨酸类似物，可取代脯氨酸整合到蛋白中从而造成蛋白折叠错误)上调表达的水稻膜结合NAC转录因子，由此推测它在水稻响应特定环境胁迫引起蛋白错误折叠的调控途径中起着至关重要的作用。
水稻基因组中含有大约140个NAC转录因子，其中一些转录因子的功能也被报道(Y.J.Fang,et al.Systematicsequenceanalysisandidentificationoftissue-specificorstress-responsivegenesofNACtranscriptionfactorfamilyinrice,Mol.Genet.Genomics280(2008)547-563.)。SNAC1和SNAC2能够响应非生物胁迫(H.H.Hu,etal.OverexpressingaNAM,ATAF,andCUC(NAC)transcriptionfactorenhancesdroughtresistanceandsalttoleranceinrice,Proc.Natl.Acad.Sci.U.S.A.103(2006)12987-12992.)。SNAC2参与了植物耐盐的过程。而OsNAC6能够响应许多的生物胁迫和非生物胁迫，过表达OsNAC6的水稻材料表现出明显的耐旱，耐盐的性状(K.Nakashima,etal.FunctionalanalysisofaNAC-typetranscriptionfactorOsNAC6involvedinabioticandbioticstressresponsivegeneexpressioninrice,PlantJ.51(2007)617-630.)。分析NAC转录因子的蛋白结构发现水稻中含有5个膜结合NAC转录因子。膜结合NAC转录因子也称为NTL(NTM1-Like)。拟南芥中的NTL大多受非生物胁迫的诱导，他们参与了植物对非生物胁迫的响应。Chung-MoPark组初步研究水稻膜结合NAC转录因子的表达模式，发现它们与拟南芥NTLs相似，水稻膜结合NAC转录因子大多也可被非生物胁迫诱导，因此我们推测膜结合转录因子的调控是基因调节网络的重要组成部分，在植物应对不良环境过程中发挥着重要作用。
发明内容
本发明提供一种基因OsNTL3在改良水稻高温抗性方面的应用及获得的水稻高温抗性基因，通过基因手段获得基因组改变的水稻植株。
本发明利用水稻膜结合NAC转录因子OsNTL3影响水稻高温抗性方面的应用。本发明提供的OsNTL3基因是水稻第1号染色体上的一个膜结合NAC转录因子基因，基因编号为Os01g0261200(RAP编号)或LOC_Os01g15640(MSU编号)。本发明中使用的启动子序列来自水稻第3号染色体和第5号染色体的BiP2和BiP4基因，基因编码分别为Os03g0710500、Os05g0428600(RAP编号)或LOC_Os03g50250、LOC_Os05g35400(MSU编号)。
OsNTL3的基因组DNA全长4718bp，共含有4个外显子，序列如SEQ ID NO.1所示，其结构图见图1。OsNTL3基因编码区CDS全长为1470bp，序列如SEQ ID NO.2所示；OsNTL3基因编码489个氨基酸，具体序列如SEQ ID NO.3所示。OsNTL3蛋白含有一个NAC结构域(NACdomain)，一个跨膜结构域(transmembranedomain)，具体见图2所示。
本发明提供一种利用OsNTL3基因改变水稻高温性状方面的应用，具体为：设计针对OsNTL3基因的sgRNA，采用CRISPR-CAS9基因组编辑技术，在OsNTL3基因组特定位置造成碱基突变、缺失或插入，从而获得基因组改变的水稻植株；设计针对BiP2和BiP4的启动子DNA的引物序列，针对去除跨膜域OsNTL3基因的引物序列，构建过表达载体，通过农杆菌转化实验获得OSNTL3过表达的转基因材料。BiP2和BiP4启动子DNA全长分别为1642bp和2232bp，序列如SEQ ID NO.4和SEQ ID NO.5。编码去除跨膜域的OsNTL3蛋白(OsNTL3deltaC)的基因序列和蛋白序列如SEQ ID NO.6和SEQ ID NO.7。
本发明提供设计并合成sgRNA所需的前导序列(GuideSequence)，其序列碱基为：
CC-NTL3-F1 5-GTGTGTATATACAAGCATGAACCA-324mer(SEQ ID NO.8)
CC-NTL3-F2 5-GTGTCAGGCTGTTCGGATGCTCGA-324mer(SEQ ID NO.9)
CC-NTL3-R1 5-AAACTGGTTCATGCTTGTATATAC-324mer(SEQ ID NO.10)
CC-NTL3-R2 5-AAACTCGAGCATCCGAACAGCCTG-324mer(SEQ ID NO.11)
将CC-NTL3-F1和CC-NTL3-R1，CC-NTL3-F2和CC-NTL3-R2等量混合后，合成双链。具体为：在94℃加热5min，在60℃保持30min。将此合成的双链插入到CRISPR-CAS9载体中。
本发明提供设计并合成BiP2/4启动子所需的正反向引物序列，其序列碱基为：
Pro:BiP2-F 5-CCGGAATTCATATTCGGCCTCGTCGCCTCGC-3 31mer(SEQ ID NO.12)
Pro:BiP2-R 5-CGGGGTACCGTTTCTTGCTCTGCTCATGCA-3 30mer(SEQ ID NO.13)
Pro:BiP4-F 5-GAATTCGACATCAGGCAGGCCGTGAAAC-3 28mer(SEQ IDNO.14)
Pro:BiP4-R 5-GGTACCGATCGTCGTCTTCGCTTTTGG-327mer(SEQ ID NO.15)
分别利用Pro:BiP2-F和Pro:BiP2-R，Pro:BiP2-F和Pro:BiP2-R，以日本晴基因组DNA为模板扩增启动子，将扩增的启动子插入pCAMBIA1300载体。具体为：在95℃预变性5min；在95℃变性30sec，60℃退火30sec，72℃延伸2min，在第2-第4步循环35cycle。将此合成的双链插入到pCAMBIA1300载体中，获得pCAMBIA-proBiP2/4中间载体。
本发明提供设计并合成去除跨膜域的OsNTL3基因的正反向引物序列，其序列碱基为：
OsNTL3-F 5-CGGGTCGACATGGAGTCTTTACGTGACATGGTC-333m(SEQ ID NO.16)
OsNTL3ΔC-R5-GCCTGCAGCTACTGAATTCCACTCCAAACATCG-333me(SEQ ID NO.17)
利用OsNTL3-F和OsNTL3ΔC-R以日本晴cDNA为模板扩增去除跨膜域的OsNTL3，将扩增的片段插入pCAMBIA-proBiP2/4中间载体。具体为：在95℃预变性5min；在95℃变性30sec，60℃退火30sec，72℃延伸1min，在第2-第4步循环35cycle。将此合成的双链插入到pCAMBIA-proBiP2/4中间载体中，获得pCAMBIA-proBiP2/4::OsNTL3deltaC载体。
本发明提供含有以上设计的CRISPR-CAS9载体、pCAMBIA1300载体。
本发明提供含有以上CRISPR-CAS9载体、pCAMBIA1300载体的大肠杆菌和农杆菌工程菌。
本发明提供利用农杆菌将设计的CRISPR-CAS9载体、pCAMBIA1300载体转化到水稻品种日本晴中，并筛选得到基因改良水稻植株。具体方法如下：
(1)构建工程菌：将构建的CRISPR-CAS9载体、pCAMBIA-proBiP2/4::OsNTL3deltaC载体通过冻融法转化到农杆菌菌株EHA105中，通过卡那霉素和利福平筛选，获得含有此CRISPR-CAS9载体、pCAMBIA-proBiP2/4::OsNTL3deltaC载体的基因工程菌。
(2)CRISPR-CAS9载体转化水稻愈伤并获得水稻再生苗：用含有CRISPR-CAS9载体、pCAMBIA-proBiP2/4::OsNTL3deltaC载体的EHA105侵染水稻愈伤组织，并于22℃培养室中共培养3天，再用液体培养基洗去农杆菌后，将水稻愈伤放置在含有合适抗生素的筛选培养基上培养。经过3-4周培养后，可获得抗性愈伤，将抗性愈伤分化成小苗，种植于水稻田。
(3)鉴定位点突变情况：设计引物将CRISPR-CAS9的编辑位点区域扩增出来，引物序列为：
1564-fl-F9 5-CCATGGAGTCTTTACGTGACA-321mer(SEQ ID NO.18)
CC1564-F10 5-CGAATGAAGCTGCTGATAATGG-321mer(SEQ ID NO.19)
CC1564-R105-GTGCGATGTTCCAACTCAGG-321mer(SEQ ID NO.20)
1564-fl-R9 5-AGATCAGCAGCATACCTAGC-321mer(SEQ ID NO.21)
PCR产物分别为571bp和429bp。将571bp和429bp的PCR产物委托公司测序，确定突变类型；并在后代中筛选突变类型稳定遗传的植株。确定突变类型，其中ntl3-1的突变为在目标区域增加了一个A，引起移码突变，突变后的碱基序列如SEQIDNO.22所示(ntl3-1的DNA序列)，其编码的氨基酸序列如SEQIDNO.23所示；ntl3-2的突变为在目标区域减少了GC，也引起移码突变，突变后的碱基序列如SEQIDNO.24所示，其编码的氨基酸序列如SEQIDNO.25所示。
OsNTL3deltaC过表达水稻中高温抗性基因，其碱基序列如SEQIDNO.6所示。
OsNTL3deltaC过表达水稻高温抗性基因编码的蛋白质，其氨基酸序列如SEQIDNO.7所示。
此转基因水稻材料在苗期的高温抗性方面表现出耐受性状，能够明显提高农作物对高温等胁迫的抗性。
鉴定过表达材料：设计引物扩增转基因材料的基因组DNA，引物序列为：
Pro:BiP2-F 5-CCGGAATTCATATTCGGCCTCGTCGCCTCGC-3 31me(SEQ ID NO.26)
Pro:BiP4-F 5-GAATTCGACATCAGGCAGGCCGTGAAAC-3 28mer(SEQ ID NO.27)
CC1564-R10 5-GTGCGATGTTCCAACTCAGG-321mer(SEQ ID NO.28)
利用BiP2/4启动子的正向引物序列和OsNTL3的反向引物序列扩增，条带分别含有1832bp和1692bp，以确定过表达的转基因材料。
本发明提供OsNTL3基因改良水稻在高温抗性方面的应用。敲除水稻OsNTL3基因后，其对高温抗性比对照敏感，具体见图5。OsNTL3基因突变体和对照日本晴高温抗性存活率的统计比较见图6，OsNTL3基因过表达材料比对照日本晴高温抗性增强，具体见图7，OsNTL3基因过表达材料比对照日本晴高温下存活率高的统计比较见图8。本发明显示OsNTL3基因在高温抗性方面发挥重要作用，具有很大的应用价值。
与现有技术相比，本发明具有如下优点：
本发明在水稻中过表达去除跨膜域的膜结合NAC转录因子编码基因OsNTL3，以及利用基因工程技术突变OsNTL3，探究水稻抵抗高温的性状。本发明通过设计针对OsNTL3基因编码区的sgRNA，利用CRISPR-CAS9技术破坏OsNTL3基因编码区，此基因编辑水稻材料在苗期的高温抗性方面表现出敏感性状；通过利用高温诱导型启动子过表达去除跨膜域的OsNTL3基因，此转基因水稻材料在苗期的高温抗性方面表现出耐受性状。本发明提供的基因及操作技术在提高水稻高温抗性方面具有明显的作用，具有很高的应用价值，能够明显提高农作物对高温等胁迫的抗性。
附图说明
图1为OsNTL3基因组结构图，其中，外显子用黑框表示，内含子用粗线条表示；
图2为OsNTL3蛋白结构图，其中，NAC domain为DNA结合域，TM为跨膜域；
图3为定量PCR实验验证OsNTL3能够响应包括高温在内的非生物胁迫；
图4为双荧光报告实验验证bZIP74能够调控OsNTL3的表达；
图5为对照日本晴和突变体ntl3-1、ntl3-2的高温表型；
图6为对照日本晴和突变体ntl3-1、ntl3-2的存活率比较；
图7为对照日本晴和OsNTL3过表达(去除跨膜域)材料的高温表型；
图8为对照日本晴和OsNTL3过表达(去除跨膜域)材料存活率比较；
图9为对照日本晴和突变体ntl3-1、ntl3-2的离子泄漏率实验；
图10为OsNTL3亚细胞定位实验验证其定位于内质网上，高温处理后定位于细胞核；
图11为OsNTL3在高温处理下会产生一条较小的蛋白形式；
图12为酵母自激活验证OsNTL3具有转录激活活性；
图13为转录组测序实验验证OsNTL3能够调控下游基因的表达。
图14为凝胶迁移实验验证OsNTL3能够结合bZIP74的启动子序列。
具体实施方式
以下实施例用于说明本发明，但不用来限制本发明的范围。
以下实施例中未注明具体条件的实验方法，均按照常规步骤进行，所用材料和试剂均为市售商品。
实施例1：用CRISPR-CAS9敲除OsNTL3基因探究其在高温抗性方面的应用
以下实施案例采用水稻粳稻品种日本晴(OryzasativaL.japonica.cv.Nipponbare)为例。利用农杆菌将设计的CRISPR-CAS9载体转化到日本晴基因组中，并筛选得到基因改良水稻植株。具体方法如下：
(1)CRISPR-CAS9载体构建
设计并合成sgRNA所需的前导序列(GuideSequence)，其序列碱基为：
CC-NTL3-F15-GTGTGTATATACAAGCATGAACCA-324mer(SEQIDNO.12)
CC-NTL3-F25-GTGTCAGGCTGTTCGGATGCTCGA-324mer(SEQIDNO.13)
CC-NTL3-R15-AAACTGGTTCATGCTTGTATATAC-324mer(SEQIDNO.14)
CC-NTL3-R25-AAACTCGAGCATCCGAACAGCCTG-324mer(SEQIDNO.15)
将CC-NTL3-F1和CC-NTL3-R1，CC-NTL3-F2和CC-NTL3-R2等量混合后，合成双链。具体为：在94℃加热5min，在60℃保持30min。将此合成的双链插入到CRISPR-CAS9中间载体中(Z.Feng,etal.EfficientgenomeeditinginplantsusingaCRISPR/Cassystem.CellRes.23,(2013)1229-1232.)，并通过酶切连接到双元载体pCAMBIA1300上。
(2)CRISPR-CAS9载体转化农杆菌
将构建的CRISPR-CAS9载体通过冻融法转化到农杆菌菌株EHA105中，在LB平板上通过卡那霉素和利福平筛选，获得含有此CRISPR-CAS9载体的基因工程菌。
(3)水稻转基因
A：培养基配方：
N6D固体培养基：N6基本培养基+0.1g/L肌醇+2g/L水解酪蛋白+30g/L蔗糖+3g/LPhytagel+2mg/L2,4-D，pH5.8
N6I固体培养基：N6基本培养基+0.1g/L肌醇+2g/L水解酪蛋白+40g/L蔗糖+10g/L葡萄糖+3g/LPhytagel+2mg/L2,4-D，pH5.2
N6CH固体培养基：N6基本培养基+0.1g/L肌醇+2g/L水解酪蛋白+30g/L蔗糖+3g/LPhytagel+2mg/L2,4-D+50mg/L潮霉素B+300mg/L头孢霉素，pH5.8
N6R固体培养基：N6基本培养基+0.1g/L肌醇+2g/L水解酪蛋白+30g/L蔗糖+3g/LPhytagel+2mg/L6-BA+0.5mg/LNAA+50mg/L潮霉素B+300mg/L头孢霉素，pH5.8
B：水稻愈伤组织诱导：
去壳的水稻成熟种子先用体积百分数70％乙醇浸泡1-2min，然后用15％NaClO(v/v)浸泡并摇晃30min，进行表面灭菌(可在摇床上进行)，然后在超净工作台上用无菌水冲洗3-4次，再将种子放在无菌滤纸上吸干水分后，放在成熟胚愈伤诱导培养基N6D上，28℃暗培养。约10-15天后，可见到从胚的位置生长出愈伤组织。
C：水稻愈伤组织的继代培养
剥下从成熟胚上长出的愈伤组织，转入同样的N6D固体培养基上，在28℃下继代培养。以后每两周继代培养一次。
D：农杆菌介导的水稻转基因方法
农杆菌的培养：在转化前一天将含有目的载体的农杆菌加入到50mLLB+50mg/L卡那霉素+25mg/L利福平的液体培养基中，28℃，200rpm震荡培养12-16h至OD600＝0.4-0.6。
水稻愈伤的收集：将培养的水稻愈伤组织在超净工作台上收集到一个灭过菌的容器中待用。
侵染菌液的准备：将新鲜培养的农杆菌加入到50mL离心管中，5000rpm，10min收集农杆菌。用10mMMgSO4洗一次。沉淀悬浮于N6I液体培养基(成分同N6I固体培养基，但不加phytagel)中，调整菌体浓度至OD600为0.5，加入AS(乙酰丁香酮)，使AS终浓度为100mΜ。
侵染和共培养：在收集的水稻愈伤组织中加入适量体积的农杆菌悬浮液，使菌液没过水稻愈伤组织，室温放置20min，并不时晃动。倒掉菌液，将愈伤组织放在无菌滤纸上吸去多余菌液，随即转移到铺有一层无菌滤纸的固体共培养基，固体培养基为加入100mMAS的N6I固体培养基，24℃黑暗培养2-3天。
除菌：将共培养后的愈伤转移至50mL的灭菌离心管，用无菌水清洗3次以上，直至洗脱液体比较清亮。倒出洗脱液后，用加入300mg/L头孢霉素的N6D液体培养基再洗一次，倒去洗脱液后，将洗净后的水稻愈伤组织倒在一张无菌滤纸上，吸干多余的水分。
筛选：将洗过的水稻愈伤转移至N6CH固体培养基上，在28℃暗室中培养。每2周继代一次。约4周后，可看到新鲜长出来的抗性愈伤，将这些抗性愈伤转移到新鲜的N6CH培养基上继代。
分化：将抗性愈伤转移至N6R固体培养基上，在光照培养室16h光/8h暗培养。一般经过7-10天左右有绿点出现。30-40天后绿点进一步分化出小苗。将小苗种植于水稻田。
(4)鉴定目的位置突变情况
设计引物将CRISPR-CAS9的编辑位点区域扩增出来，引物序列为：
1564-fl-F9 5-CCATGGAGTCTTTACGTGACA-321mer(SEQIDNO.22)
CC1564-F10 5-CGAATGAAGCTGCTGATAATGG-321mer(SEQIDNO.23)
CC1564-R10 5-GTGCGATGTTCCAACTCAGG-321mer(SEQIDNO.24)
1564-fl-R9 5-AGATCAGCAGCATACCTAGC-321mer(SEQIDNO.25)
PCR产物分别为571bp和429bp。将571bp和429bp的PCR产物测序，确定突变类型，其中ntl3-1的突变为在目标区域增加了一个A，引起移码突变，突变后的碱基序列如SEQIDNO.8所示(ntl3-1的DNA序列)，其编码的氨基酸序列如SEQIDNO.9所示；ntl3-2的突变为在目标区域减少了GC，也引起移码突变，突变后的碱基序列如SEQIDNO.10所示，其编码的氨基酸序列如SEQIDNO.11所示。
(5)鉴定过表达材料：设计引物扩增转基因材料的基因组DNA，引物序列为：
Pro:BiP2-F 5-CCGGAATTCATATTCGGCCTCGTCGCCTCGC-3 31me(SEQ ID NO.26)
Pro:BiP4-F 5-GAATTCGACATCAGGCAGGCCGTGAAAC-3 28mer(SEQ ID NO.27)
CC1564-R10 5-GTGCGATGTTCCAACTCAGG-321mer(SEQ ID NO.28)
利用BiP2/4启动子的正向引物序列和OsNTL3的反向引物序列扩增，条带分别含有1832bp和1692bp，以确定过表达的转基因材料。OsNTL3deltaC过表达水稻中高温抗性基因，其碱基序列如SEQIDNO.6所示。OsNTL3deltaC过表达水稻高温抗性基因编码的蛋白质，其氨基酸序列如SEQIDNO.7所示。此转基因水稻材料在苗期的高温抗性方面表现出耐受性状，能够明显提高农作物对高温等胁迫的抗性。
(6)基因突变水稻在高温抗性方面的考察
本发明提供的OsNTL3基因在改良水稻高温抗性方面具有很高的应用价值。敲除水稻OsNTL3基因后，其高温抗性比对照敏感；去除跨膜域的NAC转录因子的过表达OsNTL3材料在高温抗性方面显著强于日本晴。具体见图5，图6，图7，图8所示。
如图3所示，为定量PCR实验验证OsNTL3能够响应包括高温在内的非生物胁迫。图例：8天大小的水稻日本晴幼苗进行各种处理(control,water；TM,5μg/ml；DTT,2mM；NaCl,250mM；ABA,0.1mM；PEG 4000,15％；4℃,4小时；45℃，4小时，收样取根部样品进行NTL3表达分析。相对表达量(Relative gene expression)是处理样品中NTL3的表达量与未处理样品中NTL3的表达量的比值，ACTIN的表达作为内参进行均一化处理。误差线表示3个重复样品的标准误差。
如图4所示，为双荧光报告实验验证bZIP74能够调控OsNTL3的表达。图例：不同片段大小的NTL3启动子序列连接到35S最小启动子(M35S)和报告基因荧火虫荧光素酶上。转录起始位点(TSS)的相对位置也已标示。在双报告基因系统中，活化形式的bZIP16(bZIP16ΔC)或bZIP17(bZIP17ΔC)或bZIP74(bZIP74A)作为效应基因与报告基因共转于烟草细胞中。空载体作为实验对照(control)。相对荧光素酶活为萤火虫荧光素酶活性与组成型表达的海肾荧光素酶活性的比值。误差线表示3个重复样品的标准误差。
图5为对照WT和突变体ntl3-1、ntl3-2的高温表型。图例：29℃正常生长8天左右的水稻野生型(WT)和NTL3的基因编辑水稻突变体材料(ntl3-1和ntl3-2)转移到45℃培养4-5天，然后转回29℃培养箱7天恢复生长后观察表型照相。
图6为对照日本晴和突变体ntl3-1、ntl3-2的存活率比较。图例：29℃正常生长8天左右的水稻野生型(WT)和NTL3的基因编辑水稻突变体材料(ntl3-1和ntl3-2)转移到45℃培养4-5天，然后转回29℃培养箱7天恢复生长后统计存活率。绿色新叶出现计为存活苗，未出现绿色新叶计为死亡苗。误差线表示6个重复样品的标准误差。每一种温度条件每一种材料观察了141颗苗。
图7为对照日本晴和OsNTL3deltaC过表达(去除跨膜域)材料的高温表型。图例：29℃正常生长8天左右的水稻野生型(WT)和NTL3deltaC的诱导表达水稻突变体材料(NTL3OE-1、NTL3OE-2、NTL3OE-3)转移到45℃培养3-4天，然后转回29℃培养箱7天恢复生长后观察表型照相。
图8为对照日本晴和OsNTL3deltaC过表达(去除跨膜域)材料存活率比较。图例：29℃正常生长8天左右的水稻野生型(WT)和NTL3deltaC的诱导表达水稻突变体材料(NTL3OE-1、NTL3OE-2、NTL3OE-3)转移到45℃培养3-4天，然后转回29℃培养箱7天恢复生长后统计存活率。绿色新叶出现计为存活苗，未出现绿色新叶计为死亡苗。误差线表示3个重复样品的标准误差。NTL3OE-1为BiP2启动子驱动，NTL3OE-2和NTL3OE-3为BiP4启动子驱动。每一种温度条件每一种材料观察了84颗苗。
图9对照日本晴和突变体ntl3-1、ntl3-2的离子泄漏率实验。图例：29℃正常生长8天左右的水稻野生型(WT)和NTL3的基因编辑水稻突变体材料(ntl3-1and ntl3-2)转移到45℃培养5小时后取样进行离子泄露率实验。误差线表示3个重复样品的标准误差。
图10为烟草叶片中OsNTL3亚细胞定位实验验证其定位于内质网上，高温处理后定位于细胞核。图例：YFP-NTL3ΔC为去掉跨膜域的截短形式，DAPI染色显示细胞核。
图11为OsNTL3在高温处理下会产生一条较小的蛋白形式。图例：诱导型启动子驱动的MYC–NTL3水稻幼苗先进行β雌激素(10μM)处理16小时，然后进行高温胁迫处理和western blot分析。
图12为酵母自激活验证OsNTL3具有转录激活活性。图例：不同片段的NTL3与酵母GAL4DNA结合域融合后在酵母中检测报告基因激活情况。NAC结构域与跨膜域(TM)分别用长方形表示。
图13为转录组测序实验验证OsNTL3能够调控下游基因的表达。图例：29℃正常生长8天左右的水稻野生型(WT)和NTL3的基因编辑水稻突变体材料(ntl3-1)转移到45℃培养3小时后取样进行RNA-Seq分析。维恩图显示的是各材料间上调(Up-regulated)和下调(Down-regulated)的基因数目。差异表达的标准为上调时P≤0.05,fold change(FC)≥2，下调时P≤0.05,FC≤0.5。
图14为凝胶迁移实验验证OsNTL3能够结合bZIP74的启动子序列。图例：MBP标签融合的NTL3ΔC体外表达后与标记的bZIP74启动子序列孵育后跑胶检查探针迁移情况。不同浓度的非标记探针进行竞争实验。
序列表
<110> 浙江大学
<120> 基因OsNTL3在改良水稻高温抗性方面的应用及获得的水稻高温抗性基因
<130> 1
<160> 28
<170> SIPOSequenceListing 1.0
<210> 1
<211> 4718
<212> DNA
<213> Oryza sativa
<400> 1
ctcctaccca tccatcccta gcttcctcgc tgcttcgccc gccacgtcac cctcctcctc 60
ctcccctttt ttttctctcg ctcgcctcgc catccatggc gtcgatggcg aaccagcagt 120
agtagtagtc caccccaacg acgcgcgcga gccagctccg gggtacccgc cgctcgtgcc 180
cgggggggaa gggggtgcac cggggagccg tcgcgccgcg cggctctgat tcttcccgtt 240
cccggaggtg agtgggttga ctgactcctc cccgctccgc gccgcgccgc gactggcctc 300
gactctgcgt gtgtgtttgg ttaggtgatt tggattgttc cgcgcttgac gacgtccccg 360
ctcgttgttg gggggtttcg tcttggtgcg ggggattttt ttgttgttta gcgttcgttc 420
atgttagggg tagttgtttc gtctcgtctc gggaggtggg gggttttagg gtgattcttc 480
gatcgagatc ttcgtttagg ggttgcgatc actcgccgat tgcggatctt tgccgcggaa 540
tttttcttat tctttttttt ttttttgcct cctggggttt tcgattcggg gggcagattc 600
ttgggggatt cgagacgcgc gtaatgggga ttggttgcga ccccgtcgaa atccgagtgg 660
atcggtcggt tggttggttg tgcgaagaaa tggtgttctt gaagtagttc ggtatttggg 720
gaattaggat agctgctctt ccttcagtgc ttcaatagga tgataaatca gtgaattccc 780
gattgttttt tcttttccca ttttagctag cctataatag gatatcaccc cacttttcag 840
gtggtttaat gtgaaatttg gagttattac catattccta ctaatcattt tagaatttcc 900
tcccctgctt gctttgaacc tgaaattgtc aataattaga aaggaggaag catcattgtt 960
catatggatt tttgttagtt tgcctccatg tttatcttct gtaaaatgct tggggatgtt 1020
actccatgag catactaaaa tcgccaacgt ttgtgttgag aatctttcta gaaagattta 1080
taagatcgtc acttatgttc tttagttttt aggcagtaag tcgttgtgct aaatacccat 1140
gccgttttag attctccctg atcatgtttg cttttcttga ttcatgcaca gatttgaacc 1200
atggagtctt tacgtgacat ggtcctccca ccaggatttg gattccaccc aaaagatact 1260
gagctcattt ctcactatct gaaaaagaaa atacatggcc aaaagattga atatgagatc 1320
attccagagg tggatatata caagcatgaa ccatgggatt tacctggtaa gttgaacatt 1380
attccatgac aacctatatt tgtttcgttc catagagttt gctgatgaga aaagagtcaa 1440
tatttgacca tcaatttaaa ctagataatg ctggtagatg gaaacattca tgtttgcaag 1500
aaccactggg gaatgcacca gaatgttttc tgtattctta gtactgttgt catttcttag 1560
tactgttgca tcctttgcct ttttaaatat tatgcaactg attgccaatt atgtatcttc 1620
tttgtccaat tctctgccct gttattttga tagatgagtt tcttttttct actttttggc 1680
ttcctttgtt tgtatgccgc ttggaatcta attatgtaaa ctgctgatga tatttcacat 1740
tacagcaaag tgcgatgttc caactcagga taataagtgg catttcttcg ctgctcgtga 1800
cagaaagtat cctaacggtt ctcggtcaaa tagggcaaca gttgctgggt attggaaatc 1860
cactggaaag gatcgagcca taaaaatggg caagcaaact ataggaacga agaaaacttt 1920
agtttttcat gaaggccgcc cacccactgg gagacgtact gagtggatca tgcatgagta 1980
ctatatagat gagcgtgaat gtcaagcctg ccctgatatg aaggtattga actaattatt 2040
aagattttaa agtggcttat ttagaaaaga gagaactata ttgtggcttt acatgatact 2100
ccctccgttt caaaatattt gacaccgttg actttttagt acgtgtttga ctattcgtct 2160
tattcaaaaa atttaagtaa ttatttattc ttttcatatc atttgattca ttgttaaata 2220
tactttcatg tacacatata gttttacata tttcacaaat tttttttaat aagacgaacg 2280
gttaaacatg tgctaaaaaa tcaacggtgt caaacatttt gaaacggagg gagtatataa 2340
attaatgtgc actgtacctt cttgatgtca ctcttcaaac aacttgctgg tttagatgat 2400
ataatcatat gatttgctga ctaagggcga attccagtat ctaatgtagg gcttttattt 2460
tattgtccct caggatgcat atgtactttg tcgtattacc aaaagaaatg actggatccc 2520
agggaatggc aatgagttgg acaacagtga tcctcatcca gaaccatatg atgctcctcc 2580
atcagttatc agtactgaac agctaaatcc tgctgctgaa ccagttgtcg gtgttgaagc 2640
tgcaccagtt accgttgctg aacctgacgg tgttactacg tcagctatca ctgctaatat 2700
accatctcct agtgatgaca ttaatctgga tgattggttg aatgaactat ttgatccttt 2760
ctttgaccct gaacagagtc tggcttctgc tgatctgtct ccagatgaac aaaatgttga 2820
atcatcggtg agtttcttca cagtcactgt gtttgcaggt taaataattc aatggataaa 2880
ttattagaat gttgtagtga cattattaga atgttgtagt gactccagct ccctatgaga 2940
acgggctatt gcatgaacaa gaatctgttg gacaaatcta acatgcttgt tactcataat 3000
aaattgatct tcactggtcc acttgtgcat actgattgct atttaatata tctgcataat 3060
gcatttctga tagacaaaat actgaataac ttcacgaaca tatagaaacc aaacacaaca 3120
ttgcattata gtaatgtttg tgagcctagt acagcactgg ttactgcttg ggaattgata 3180
ctaagcctag tacagcactg gttactgctt gggaattgat actaatatga agcatgtgac 3240
tggtaatgac ctttagagat acctagacat aggcaaatca caatgttgtg accatttttt 3300
tttgtgactt agtatggctg cccttttatt ttcttttgct ggttgacaaa gaagatttgc 3360
ttgtaatttt tttaggaaca ccggagtaat gcattctcat ggataatatt tgaaactcat 3420
tgaaaattat tatgatttgt ttggattgac tttttaaaaa atagccatga tttgcatata 3480
ccatggactg aagctatctt atgtgttacg gtttgttcca cctcttctga gatttctggt 3540
tattgccaag tatctggtct gttatattat ggggaaacgt tgactgtagt tgtgttttca 3600
taccatgttc tttctttaac acggaaccta ggagtcaatg tattttctgc aaatcatgaa 3660
ttgtacttgt cattgtgctg tgcattctgc cattctacta tgttttgatt gcagttctta 3720
gcctgtgcat acttgctcat cgcgctatct tccttcttaa tttacagaac gtaggtgctt 3780
tggctccaaa ggtggaacaa gactattcta gtcctaacga gaacgtagta gatgatacgg 3840
agtacttgtt gcctgaagat gtttacaaca tactgcaccc tggtacagat gacttcaata 3900
tgctccagaa tcctttggat cagtacccca ttcaatatgc aaccgatgtt tggagtggaa 3960
ttcagaagga agaactgtgg tcgccccagg ccaatgctga gcctagtcag tcgaatgaag 4020
ctgctgataa tggaattata agacgttatc gtagtatgaa aacacctgaa actagtgtgc 4080
cgcagttcaa gggtaaaact caggctaaaa tgcgggttgg gatcaacaag atggctacga 4140
gtagttctga atctatcaac cagactatca agtttgagaa cagtggtcgt cttgttgagc 4200
accagaagaa ccaagcgcat gatgtagctt ctaccaagcg gtcagacgca gggaagccaa 4260
gtacagagct cagcagcaat cgaggatttc tcaggggtat ccgaaatgca tttgcaggct 4320
gttcggatgc tcgatggaac atgatacttg ttgcgggttt cgctattgga gtcgctgtgg 4380
tagcgcttca tataggccaa cgccttggat taagccagag agatcagcag catacctagc 4440
ctttaggttt gcaggttact gggtttctat gctttttgca atctggagaa acgtaaacaa 4500
actccgatct ttggttggtg cagaacattt gatgtatgcg ccgttttttt tgtgttggtt 4560
ttgtacatat tattatcttc ggttttggtc gacttttgtt ctcgccagct agtagtacta 4620
tgttgacgaa tgtagaagaa aacctcaaaa aaaaaagaac aaatgaaaaa aaattgacga 4680
tgaaaattta tataattaac cgcttgcatt gtactata 4718
<210> 2
<211> 1470
<212> DNA
<213> Oryza sativa
<400> 2
atggagtctt tacgtgacat ggtcctccca ccaggatttg gattccaccc aaaagatact 60
gagctcattt ctcactatct gaaaaagaaa atacatggcc aaaagattga atatgagatc 120
attccagagg tggatatata caagcatgaa ccatgggatt tacctgcaaa gtgcgatgtt 180
ccaactcagg ataataagtg gcatttcttc gctgctcgtg acagaaagta tcctaacggt 240
tctcggtcaa atagggcaac agttgctggg tattggaaat ccactggaaa ggatcgagcc 300
ataaaaatgg gcaagcaaac tataggaacg aagaaaactt tagtttttca tgaaggccgc 360
ccacccactg ggagacgtac tgagtggatc atgcatgagt actatataga tgagcgtgaa 420
tgtcaagcct gccctgatat gaaggatgca tatgtacttt gtcgtattac caaaagaaat 480
gactggatcc cagggaatgg caatgagttg gacaacagtg atcctcatcc agaaccatat 540
gatgctcctc catcagttat cagtactgaa cagctaaatc ctgctgctga accagttgtc 600
ggtgttgaag ctgcaccagt taccgttgct gaacctgacg gtgttactac gtcagctatc 660
actgctaata taccatctcc tagtgatgac attaatctgg atgattggtt gaatgaacta 720
tttgatcctt tctttgaccc tgaacagagt ctggcttctg ctgatctgtc tccagatgaa 780
caaaatgttg aatcatcgaa cgtaggtgct ttggctccaa aggtggaaca agactattct 840
agtcctaacg agaacgtagt agatgatacg gagtacttgt tgcctgaaga tgtttacaac 900
atactgcacc ctggtacaga tgacttcaat atgctccaga atcctttgga tcagtacccc 960
attcaatatg caaccgatgt ttggagtgga attcagaagg aagaactgtg gtcgccccag 1020
gccaatgctg agcctagtca gtcgaatgaa gctgctgata atggaattat aagacgttat 1080
cgtagtatga aaacacctga aactagtgtg ccgcagttca agggtaaaac tcaggctaaa 1140
atgcgggttg ggatcaacaa gatggctacg agtagttctg aatctatcaa ccagactatc 1200
aagtttgaga acagtggtcg tcttgttgag caccagaaga accaagcgca tgatgtagct 1260
tctaccaagc ggtcagacgc agggaagcca agtacagagc tcagcagcaa tcgaggattt 1320
ctcaggggta tccgaaatgc atttgcaggc tgttcggatg ctcgatggaa catgatactt 1380
gttgcgggtt tcgctattgg agtcgctgtg gtagcgcttc atataggcca acgccttgga 1440
ttaagccaga gagatcagca gcatacctag 1470
<210> 3
<211> 489
<212> PRT
<213> Oryza sativa
<400> 3
Met Glu Ser Leu Arg Asp Met Val Leu Pro Pro Gly Phe Gly Phe His
1 5 10 15
Pro Lys Asp Thr Glu Leu Ile Ser His Tyr Leu Lys Lys Lys Ile His
20 25 30
Gly Gln Lys Ile Glu Tyr Glu Ile Ile Pro Glu Val Asp Ile Tyr Lys
35 40 45
His Glu Pro Trp Asp Leu Pro Ala Lys Cys Asp Val Pro Thr Gln Asp
50 55 60
Asn Lys Trp His Phe Phe Ala Ala Arg Asp Arg Lys Tyr Pro Asn Gly
65 70 75 80
Ser Arg Ser Asn Arg Ala Thr Val Ala Gly Tyr Trp Lys Ser Thr Gly
85 90 95
Lys Asp Arg Ala Ile Lys Met Gly Lys Gln Thr Ile Gly Thr Lys Lys
100 105 110
Thr Leu Val Phe His Glu Gly Arg Pro Pro Thr Gly Arg Arg Thr Glu
115 120 125
Trp Ile Met His Glu Tyr Tyr Ile Asp Glu Arg Glu Cys Gln Ala Cys
130 135 140
Pro Asp Met Lys Asp Ala Tyr Val Leu Cys Arg Ile Thr Lys Arg Asn
145 150 155 160
Asp Trp Ile Pro Gly Asn Gly Asn Glu Leu Asp Asn Ser Asp Pro His
165 170 175
Pro Glu Pro Tyr Asp Ala Pro Pro Ser Val Ile Ser Thr Glu Gln Leu
180 185 190
Asn Pro Ala Ala Glu Pro Val Val Gly Val Glu Ala Ala Pro Val Thr
195 200 205
Val Ala Glu Pro Asp Gly Val Thr Thr Ser Ala Ile Thr Ala Asn Ile
210 215 220
Pro Ser Pro Ser Asp Asp Ile Asn Leu Asp Asp Trp Leu Asn Glu Leu
225 230 235 240
Phe Asp Pro Phe Phe Asp Pro Glu Gln Ser Leu Ala Ser Ala Asp Leu
245 250 255
Ser Pro Asp Glu Gln Asn Val Glu Ser Ser Asn Val Gly Ala Leu Ala
260 265 270
Pro Lys Val Glu Gln Asp Tyr Ser Ser Pro Asn Glu Asn Val Val Asp
275 280 285
Asp Thr Glu Tyr Leu Leu Pro Glu Asp Val Tyr Asn Ile Leu His Pro
290 295 300
Gly Thr Asp Asp Phe Asn Met Leu Gln Asn Pro Leu Asp Gln Tyr Pro
305 310 315 320
Ile Gln Tyr Ala Thr Asp Val Trp Ser Gly Ile Gln Lys Glu Glu Leu
325 330 335
Trp Ser Pro Gln Ala Asn Ala Glu Pro Ser Gln Ser Asn Glu Ala Ala
340 345 350
Asp Asn Gly Ile Ile Arg Arg Tyr Arg Ser Met Lys Thr Pro Glu Thr
355 360 365
Ser Val Pro Gln Phe Lys Gly Lys Thr Gln Ala Lys Met Arg Val Gly
370 375 380
Ile Asn Lys Met Ala Thr Ser Ser Ser Glu Ser Ile Asn Gln Thr Ile
385 390 395 400
Lys Phe Glu Asn Ser Gly Arg Leu Val Glu His Gln Lys Asn Gln Ala
405 410 415
His Asp Val Ala Ser Thr Lys Arg Ser Asp Ala Gly Lys Pro Ser Thr
420 425 430
Glu Leu Ser Ser Asn Arg Gly Phe Leu Arg Gly Ile Arg Asn Ala Phe
435 440 445
Ala Gly Cys Ser Asp Ala Arg Trp Asn Met Ile Leu Val Ala Gly Phe
450 455 460
Ala Ile Gly Val Ala Val Val Ala Leu His Ile Gly Gln Arg Leu Gly
465 470 475 480
Leu Ser Gln Arg Asp Gln Gln His Thr
485
<210> 4
<211> 1642
<212> DNA
<213> Oryza sativa
<400> 4
atattcggcc tcgtcgcctc gctttcgtgc agtccagcga cgacgaggag gaagacttcc 60
ttgttgggtc cgccccgggc gctgccgcgg cccgccgcca ccttcttctc cggcgcagtt 120
ctcgctatcc ccttctctct atccctctct ctctatcctc tctccttcct cttctctcta 180
tcccccctct ctctccctga gaatggtgag tggcgacgga aggcgcacag gggcggcgac 240
ggagctgaga cttgagaggc ggcaggtagc ttgtggagcg gcggctggag gacgacgagc 300
gcggcgtaga ggcactcggc gatgccgtcc cggtgcgcgg agggtgtctg ggtggtcggc 360
ggcggcgaca cggatagtgg cgaagcgacg gagaagaagg tggcggagaa gaggagagag 420
agagagagga aggagagagg atatagagag agagggtagt gaggatgaca ggtggggtcc 480
acgtgggccc caccattttt tttaattatt gtgtgtgaaa ctgtcatgtg gatcccatag 540
tttttaatat ttttcggatc aaattgccac ataagcgtca cgtcaatgcc acgtcagatg 600
aagaccgagt caaattagcc acgtaggtgc cacctcagcc aaaaccgggg tcaaattctt 660
ccgagggaac ttatttaaac ggtttcgtaa gttgggggac ctgttgtacc cggttttgcg 720
atcaagggac gaaaatcaga ctgggtgaca aatagaggga cccaaagtga acttatccga 780
catgcctagg cgtcattcgg gtgtcatttc ttaaaatagg cgtaactttc tcatacggac 840
tcggaatcag gcaaatgata tatccacgga catctacaga aaatgttaca tccgattctc 900
ctcagctctc tcccaagggg cgacacaaca cgagggacgc agtggcgtga gtcaaaagag 960
agagagagag acaaaaagag attccttcct ttccacggtc taaaatcctt ggcccagtta 1020
ggcagaccat ccagccaacc cactgaacag cccatgccat gacggagctg agacttgtcg 1080
gtgactcgcg acacgaacgt tctggctaga tcgctggacc tgaccaaacc aagaccaaga 1140
ccaccccgct acgtgtccac ccacgattgg acccctcacg tggaccgttt tcacggtgcc 1200
ttgtgatgtg gagctcgtac agggccagaa cactccggcg agtcctactc cgatctcctc 1260
accataatcc agaacctatt taagcccccc gcatgtacag caagcatcga ccgatcacga 1320
tcattctcca agcctaaggc tgtgttcgct atgggttgtt cccaatagca cggaaaacgg 1380
agcgatccat tagcacgtga ttaattaaat attagctatt tttttcaaaa atggattaat 1440
ttgatttttt taagcaactt ttgtatagaa actttttgca aaaaaaacac cgtttagcag 1500
tttaaaaagc gtgcgcgcgg aaaacgaggg agataagttg ggaaaatgga cgtccgaaca 1560
cagaccgact agatactcca ttacacagct tcaccaaatc gcacatcgta gagacaagtg 1620
ttgcatgagc agagcaagaa ac 1642
<210> 5
<211> 2232
<212> DNA
<213> Oryza sativa
<400> 5
gacatcaggc aggccgtgaa acaaatgagt aaggtggttg ccgggacttc tagaaagtaa 60
aaaaaaaaac ctcattgata atcatattcg attgttaaaa tcttaatgac aataaaaagg 120
agaagcagca gacggggtgc ataagagtat tgttgcagag ccgccgacgg ctgacgggac 180
ttctagaaaa taaaaaataa attccaacga tagttatgtt cgatatttag aatcacaatg 240
acaattaaag agtaggcggc ggacgggcta cagaggggca tagtggcatt gtttgatggg 300
acttctaaaa attataaaaa atgaaactac aagtccaatt tttcaaggtt caaaatttct 360
aaaaagtaaa aaaaataatg ataatcatgt tcgattttaa aatctcaaag acaataaaga 420
agggagacag cgggcaagcc gtagaggagt acaatggcaa agccaatgac ggtttggtgg 480
gacttctaga aagtaaaaaa tgaacctaga caataattat gttcgatttt taaaatctca 540
atgtcaataa agagaagaga cagtggacag gccatagagg agtataatgt caacgtttga 600
cgggacattt agaaattata aaaataaaac ccaacatgac aataaactct aaaaactata 660
aaatccagtt tttaaaggtt ccaagaaaaa tgaaaagaaa tagtggtaga tcaagcaagc 720
aaataaagaa ggagatgaca taaggggtag caactggtat gacttttaaa aactctataa 780
ttagaaacac gaggatgata aggtttggtc tttcaaagtc ttaagacaat aagatagcta 840
tttaataaat tttaagtaaa atcatataaa aaatatgatt ttgttcgggt gctagccgcg 900
caattacgcg ggccactcag ctagtttgat ctaaagtttg atttcaaaaa taatcaaagt 960
ggcatataat atgaaataga gggattaaca atcaacagag aagctgatca ttgacattta 1020
cttttctttg tttcgctatg atgctacaag attttgtata gtagtagttg ttacacatgt 1080
ctttcgatat actcccttca tccaaagatg taagacattt cttgtgtcac caagaacaag 1140
aagcaattaa ctcatctctc atgtaacata ttttatcacc caaaaccaaa aaggaattaa 1200
ctcatctctc atgtaacaaa tcaaggaagt atatgcacgc atacaagtat taagatgact 1260
ccttaagttt cggtcaacaa ttaatttagc aatctgaaca ctaaaaatat gactatctct 1320
tttgaaaaaa aaaacagatg cctaatattt ttgaatgaag gatgtattaa gtgggatatg 1380
gttaatacaa gttcgtaaca aactagactt tttatcttag ttcttattag tatttaaaac 1440
gtaatttcga gatccaatcc acacctattg agtaagtact cattccatct atttttgata 1500
gatatatatc taaatctgaa aaaattattt ttaataacat atttcaatct aaccacctat 1560
catcttaatg actttctcgg atttaatgcg taactctcca ttctttcatt ggctacatgg 1620
gcattgagaa atgtaaatat taatgaacca cttgtttacg aggaataact agtggcatgt 1680
ttaaatggat gataagtaga attacttatc cttattcagt gtggcaagat gaaatattac 1740
tatcaaaaat agatgaaggg actataaagt ttttttttcg cgagaggagg gactataaag 1800
ttgaagccat atgggactag tttttaataa aaaaagacac catatgggac tctgcgttta 1860
gtactaattg gagtagtatt ttgttagtgt aaacctcaat gacacacgat ccaactctgt 1920
aaaccgttga gtccgtaccg atccgtgtac gaagatgaaa ccgattcggc accgatacgc 1980
tacgcgcgca cggccaccgg cacacgtcat taccacgtct cacaccctac cctgaccgtc 2040
cgatcggatc aaccaacggc ccagatcacg ccactgacgt gtacgccctg ccgtgagcgc 2100
ctccgattgg gcggcacagc gcacacacca gatcggagtc ggcctcggcc tcggccgccg 2160
cacgctatat atatatcgcg acgctccccc gcctcgtata tcgacacaaa accaaaagcg 2220
aagacgacga tc 2232
<210> 6
<211> 999
<212> DNA
<213> Oryza sativa
<400> 6
atggagtctt tacgtgacat ggtcctccca ccaggatttg gattccaccc aaaagatact 60
gagctcattt ctcactatct gaaaaagaaa atacatggcc aaaagattga atatgagatc 120
attccagagg tggatatata caagcatgaa ccatgggatt tacctgcaaa gtgcgatgtt 180
ccaactcagg ataataagtg gcatttcttc gctgctcgtg acagaaagta tcctaacggt 240
tctcggtcaa atagggcaac agttgctggg tattggaaat ccactggaaa ggatcgagcc 300
ataaaaatgg gcaagcaaac tataggaacg aagaaaactt tagtttttca tgaaggccgc 360
ccacccactg ggagacgtac tgagtggatc atgcatgagt actatataga tgagcgtgaa 420
tgtcaagcct gccctgatat gaaggatgca tatgtacttt gtcgtattac caaaagaaat 480
gactggatcc cagggaatgg caatgagttg gacaacagtg atcctcatcc agaaccatat 540
gatgctcctc catcagttat cagtactgaa cagctaaatc ctgctgctga accagttgtc 600
ggtgttgaag ctgcaccagt taccgttgct gaacctgacg gtgttactac gtcagctatc 660
actgctaata taccatctcc tagtgatgac attaatctgg atgattggtt gaatgaacta 720
tttgatcctt tctttgaccc tgaacagagt ctggcttctg ctgatctgtc tccagatgaa 780
caaaatgttg aatcatcgaa cgtaggtgct ttggctccaa aggtggaaca agactattct 840
agtcctaacg agaacgtagt agatgatacg gagtacttgt tgcctgaaga tgtttacaac 900
atactgcacc ctggtacaga tgacttcaat atgctccaga atcctttgga tcagtacccc 960
attcaatatg caaccgatgt ttggagtgga attcagtaa 999
<210> 7
<211> 332
<212> PRT
<213> Oryza sativa
<400> 7
Met Glu Ser Leu Arg Asp Met Val Leu Pro Pro Gly Phe Gly Phe His
1 5 10 15
Pro Lys Asp Thr Glu Leu Ile Ser His Tyr Leu Lys Lys Lys Ile His
20 25 30
Gly Gln Lys Ile Glu Tyr Glu Ile Ile Pro Glu Val Asp Ile Tyr Lys
35 40 45
His Glu Pro Trp Asp Leu Pro Ala Lys Cys Asp Val Pro Thr Gln Asp
50 55 60
Asn Lys Trp His Phe Phe Ala Ala Arg Asp Arg Lys Tyr Pro Asn Gly
65 70 75 80
Ser Arg Ser Asn Arg Ala Thr Val Ala Gly Tyr Trp Lys Ser Thr Gly
85 90 95
Lys Asp Arg Ala Ile Lys Met Gly Lys Gln Thr Ile Gly Thr Lys Lys
100 105 110
Thr Leu Val Phe His Glu Gly Arg Pro Pro Thr Gly Arg Arg Thr Glu
115 120 125
Trp Ile Met His Glu Tyr Tyr Ile Asp Glu Arg Glu Cys Gln Ala Cys
130 135 140
Pro Asp Met Lys Asp Ala Tyr Val Leu Cys Arg Ile Thr Lys Arg Asn
145 150 155 160
Asp Trp Ile Pro Gly Asn Gly Asn Glu Leu Asp Asn Ser Asp Pro His
165 170 175
Pro Glu Pro Tyr Asp Ala Pro Pro Ser Val Ile Ser Thr Glu Gln Leu
180 185 190
Asn Pro Ala Ala Glu Pro Val Val Gly Val Glu Ala Ala Pro Val Thr
195 200 205
Val Ala Glu Pro Asp Gly Val Thr Thr Ser Ala Ile Thr Ala Asn Ile
210 215 220
Pro Ser Pro Ser Asp Asp Ile Asn Leu Asp Asp Trp Leu Asn Glu Leu
225 230 235 240
Phe Asp Pro Phe Phe Asp Pro Glu Gln Ser Leu Ala Ser Ala Asp Leu
245 250 255
Ser Pro Asp Glu Gln Asn Val Glu Ser Ser Asn Val Gly Ala Leu Ala
260 265 270
Pro Lys Val Glu Gln Asp Tyr Ser Ser Pro Asn Glu Asn Val Val Asp
275 280 285
Asp Thr Glu Tyr Leu Leu Pro Glu Asp Val Tyr Asn Ile Leu His Pro
290 295 300
Gly Thr Asp Asp Phe Asn Met Leu Gln Asn Pro Leu Asp Gln Tyr Pro
305 310 315 320
Ile Gln Tyr Ala Thr Asp Val Trp Ser Gly Ile Gln
325 330
<210> 8
<211> 24
<212> DNA
<213> Oryza sativa
<400> 8
gtgtgtatat acaagcatga acca 24
<210> 9
<211> 24
<212> DNA
<213> Oryza sativa
<400> 9
gtgtcaggct gttcggatgc tcga 24
<210> 10
<211> 24
<212> DNA
<213> Oryza sativa
<400> 10
aaactggttc atgcttgtat atac 24
<210> 11
<211> 24
<212> DNA
<213> Oryza sativa
<400> 11
aaactcgagc atccgaacag cctg 24
<210> 12
<211> 31
<212> DNA
<213> Oryza sativa
<400> 12
ccggaattca tattcggcct cgtcgcctcg c 31
<210> 13
<211> 30
<212> DNA
<213> Oryza sativa
<400> 13
cggggtaccg tttcttgctc tgctcatgca 30
<210> 14
<211> 28
<212> DNA
<213> Oryza sativa
<400> 14
gaattcgaca tcaggcaggc cgtgaaac 28
<210> 15
<211> 27
<212> DNA
<213> Oryza sativa
<400> 15
ggtaccgatc gtcgtcttcg cttttgg 27
<210> 16
<211> 33
<212> DNA
<213> Oryza sativa
<400> 16
cgggtcgaca tggagtcttt acgtgacatg gtc 33
<210> 17
<211> 33
<212> DNA
<213> Oryza sativa
<400> 17
gcctgcagct actgaattcc actccaaaca tcg 33
<210> 18
<211> 21
<212> DNA
<213> Oryza sativa
<400> 18
ccatggagtc tttacgtgac a 21
<210> 19
<211> 22
<212> DNA
<213> Oryza sativa
<400> 19
cgaatgaagc tgctgataat gg 22
<210> 20
<211> 20
<212> DNA
<213> Oryza sativa
<400> 20
gtgcgatgtt ccaactcagg 20
<210> 21
<211> 20
<212> DNA
<213> Oryza sativa
<400> 21
agatcagcag catacctagc 20
<210> 22
<211> 4719
<212> DNA
<213> Oryza sativa
<400> 22
ctcctaccca tccatcccta gcttcctcgc tgcttcgccc gccacgtcac cctcctcctc 60
ctcccctttt ttttctctcg ctcgcctcgc catccatggc gtcgatggcg aaccagcagt 120
agtagtagtc caccccaacg acgcgcgcga gccagctccg gggtacccgc cgctcgtgcc 180
cgggggggaa gggggtgcac cggggagccg tcgcgccgcg cggctctgat tcttcccgtt 240
cccggaggtg agtgggttga ctgactcctc cccgctccgc gccgcgccgc gactggcctc 300
gactctgcgt gtgtgtttgg ttaggtgatt tggattgttc cgcgcttgac gacgtccccg 360
ctcgttgttg gggggtttcg tcttggtgcg ggggattttt ttgttgttta gcgttcgttc 420
atgttagggg tagttgtttc gtctcgtctc gggaggtggg gggttttagg gtgattcttc 480
gatcgagatc ttcgtttagg ggttgcgatc actcgccgat tgcggatctt tgccgcggaa 540
tttttcttat tctttttttt ttttttgcct cctggggttt tcgattcggg gggcagattc 600
ttgggggatt cgagacgcgc gtaatgggga ttggttgcga ccccgtcgaa atccgagtgg 660
atcggtcggt tggttggttg tgcgaagaaa tggtgttctt gaagtagttc ggtatttggg 720
gaattaggat agctgctctt ccttcagtgc ttcaatagga tgataaatca gtgaattccc 780
gattgttttt tcttttccca ttttagctag cctataatag gatatcaccc cacttttcag 840
gtggtttaat gtgaaatttg gagttattac catattccta ctaatcattt tagaatttcc 900
tcccctgctt gctttgaacc tgaaattgtc aataattaga aaggaggaag catcattgtt 960
catatggatt tttgttagtt tgcctccatg tttatcttct gtaaaatgct tggggatgtt 1020
actccatgag catactaaaa tcgccaacgt ttgtgttgag aatctttcta gaaagattta 1080
taagatcgtc acttatgttc tttagttttt aggcagtaag tcgttgtgct aaatacccat 1140
gccgttttag attctccctg atcatgtttg cttttcttga ttcatgcaca gatttgaacc 1200
atggagtctt tacgtgacat ggtcctccca ccaggatttg gattccaccc aaaagatact 1260
gagctcattt ctcactatct gaaaaagaaa atacatggcc aaaagattga atatgagatc 1320
attccagagg tggatatata caagcatgaa accatgggat ttacctggta agttgaacat 1380
tattccatga caacctatat ttgtttcgtt ccatagagtt tgctgatgag aaaagagtca 1440
atatttgacc atcaatttaa actagataat gctggtagat ggaaacattc atgtttgcaa 1500
gaaccactgg ggaatgcacc agaatgtttt ctgtattctt agtactgttg tcatttctta 1560
gtactgttgc atcctttgcc tttttaaata ttatgcaact gattgccaat tatgtatctt 1620
ctttgtccaa ttctctgccc tgttattttg atagatgagt ttcttttttc tactttttgg 1680
cttcctttgt ttgtatgccg cttggaatct aattatgtaa actgctgatg atatttcaca 1740
ttacagcaaa gtgcgatgtt ccaactcagg ataataagtg gcatttcttc gctgctcgtg 1800
acagaaagta tcctaacggt tctcggtcaa atagggcaac agttgctggg tattggaaat 1860
ccactggaaa ggatcgagcc ataaaaatgg gcaagcaaac tataggaacg aagaaaactt 1920
tagtttttca tgaaggccgc ccacccactg ggagacgtac tgagtggatc atgcatgagt 1980
actatataga tgagcgtgaa tgtcaagcct gccctgatat gaaggtattg aactaattat 2040
taagatttta aagtggctta tttagaaaag agagaactat attgtggctt tacatgatac 2100
tccctccgtt tcaaaatatt tgacaccgtt gactttttag tacgtgtttg actattcgtc 2160
ttattcaaaa aatttaagta attatttatt cttttcatat catttgattc attgttaaat 2220
atactttcat gtacacatat agttttacat atttcacaaa ttttttttaa taagacgaac 2280
ggttaaacat gtgctaaaaa atcaacggtg tcaaacattt tgaaacggag ggagtatata 2340
aattaatgtg cactgtacct tcttgatgtc actcttcaaa caacttgctg gtttagatga 2400
tataatcata tgatttgctg actaagggcg aattccagta tctaatgtag ggcttttatt 2460
ttattgtccc tcaggatgca tatgtacttt gtcgtattac caaaagaaat gactggatcc 2520
cagggaatgg caatgagttg gacaacagtg atcctcatcc agaaccatat gatgctcctc 2580
catcagttat cagtactgaa cagctaaatc ctgctgctga accagttgtc ggtgttgaag 2640
ctgcaccagt taccgttgct gaacctgacg gtgttactac gtcagctatc actgctaata 2700
taccatctcc tagtgatgac attaatctgg atgattggtt gaatgaacta tttgatcctt 2760
tctttgaccc tgaacagagt ctggcttctg ctgatctgtc tccagatgaa caaaatgttg 2820
aatcatcggt gagtttcttc acagtcactg tgtttgcagg ttaaataatt caatggataa 2880
attattagaa tgttgtagtg acattattag aatgttgtag tgactccagc tccctatgag 2940
aacgggctat tgcatgaaca agaatctgtt ggacaaatct aacatgcttg ttactcataa 3000
taaattgatc ttcactggtc cacttgtgca tactgattgc tatttaatat atctgcataa 3060
tgcatttctg atagacaaaa tactgaataa cttcacgaac atatagaaac caaacacaac 3120
attgcattat agtaatgttt gtgagcctag tacagcactg gttactgctt gggaattgat 3180
actaagccta gtacagcact ggttactgct tgggaattga tactaatatg aagcatgtga 3240
ctggtaatga cctttagaga tacctagaca taggcaaatc acaatgttgt gaccattttt 3300
ttttgtgact tagtatggct gcccttttat tttcttttgc tggttgacaa agaagatttg 3360
cttgtaattt ttttaggaac accggagtaa tgcattctca tggataatat ttgaaactca 3420
ttgaaaatta ttatgatttg tttggattga ctttttaaaa aatagccatg atttgcatat 3480
accatggact gaagctatct tatgtgttac ggtttgttcc acctcttctg agatttctgg 3540
ttattgccaa gtatctggtc tgttatatta tggggaaacg ttgactgtag ttgtgttttc 3600
ataccatgtt ctttctttaa cacggaacct aggagtcaat gtattttctg caaatcatga 3660
attgtacttg tcattgtgct gtgcattctg ccattctact atgttttgat tgcagttctt 3720
agcctgtgca tacttgctca tcgcgctatc ttccttctta atttacagaa cgtaggtgct 3780
ttggctccaa aggtggaaca agactattct agtcctaacg agaacgtagt agatgatacg 3840
gagtacttgt tgcctgaaga tgtttacaac atactgcacc ctggtacaga tgacttcaat 3900
atgctccaga atcctttgga tcagtacccc attcaatatg caaccgatgt ttggagtgga 3960
attcagaagg aagaactgtg gtcgccccag gccaatgctg agcctagtca gtcgaatgaa 4020
gctgctgata atggaattat aagacgttat cgtagtatga aaacacctga aactagtgtg 4080
ccgcagttca agggtaaaac tcaggctaaa atgcgggttg ggatcaacaa gatggctacg 4140
agtagttctg aatctatcaa ccagactatc aagtttgaga acagtggtcg tcttgttgag 4200
caccagaaga accaagcgca tgatgtagct tctaccaagc ggtcagacgc agggaagcca 4260
agtacagagc tcagcagcaa tcgaggattt ctcaggggta tccgaaatgc atttgcaggc 4320
tgttcggatg ctcgatggaa catgatactt gttgcgggtt tcgctattgg agtcgctgtg 4380
gtagcgcttc atataggcca acgccttgga ttaagccaga gagatcagca gcatacctag 4440
cctttaggtt tgcaggttac tgggtttcta tgctttttgc aatctggaga aacgtaaaca 4500
aactccgatc tttggttggt gcagaacatt tgatgtatgc gccgtttttt ttgtgttggt 4560
tttgtacata ttattatctt cggttttggt cgacttttgt tctcgccagc tagtagtact 4620
atgttgacga atgtagaaga aaacctcaaa aaaaaaagaa caaatgaaaa aaaattgacg 4680
atgaaaattt atataattaa ccgcttgcat tgtactata 4719
<210> 23
<211> 64
<212> PRT
<213> Oryza sativa
<400> 23
Met Glu Ser Leu Arg Asp Met Val Leu Pro Pro Gly Phe Gly Phe His
1 5 10 15
Pro Lys Asp Thr Glu Leu Ile Ser His Tyr Leu Lys Lys Lys Ile His
20 25 30
Gly Gln Lys Ile Glu Tyr Glu Ile Ile Pro Glu Val Asp Ile Tyr Lys
35 40 45
His Glu Thr Met Gly Phe Thr Cys Lys Val Arg Cys Ser Asn Ser Gly
50 55 60
<210> 24
<211> 4716
<212> DNA
<213> Oryza sativa
<400> 24
ctcctaccca tccatcccta gcttcctcgc tgcttcgccc gccacgtcac cctcctcctc 60
ctcccctttt ttttctctcg ctcgcctcgc catccatggc gtcgatggcg aaccagcagt 120
agtagtagtc caccccaacg acgcgcgcga gccagctccg gggtacccgc cgctcgtgcc 180
cgggggggaa gggggtgcac cggggagccg tcgcgccgcg cggctctgat tcttcccgtt 240
cccggaggtg agtgggttga ctgactcctc cccgctccgc gccgcgccgc gactggcctc 300
gactctgcgt gtgtgtttgg ttaggtgatt tggattgttc cgcgcttgac gacgtccccg 360
ctcgttgttg gggggtttcg tcttggtgcg ggggattttt ttgttgttta gcgttcgttc 420
atgttagggg tagttgtttc gtctcgtctc gggaggtggg gggttttagg gtgattcttc 480
gatcgagatc ttcgtttagg ggttgcgatc actcgccgat tgcggatctt tgccgcggaa 540
tttttcttat tctttttttt ttttttgcct cctggggttt tcgattcggg gggcagattc 600
ttgggggatt cgagacgcgc gtaatgggga ttggttgcga ccccgtcgaa atccgagtgg 660
atcggtcggt tggttggttg tgcgaagaaa tggtgttctt gaagtagttc ggtatttggg 720
gaattaggat agctgctctt ccttcagtgc ttcaatagga tgataaatca gtgaattccc 780
gattgttttt tcttttccca ttttagctag cctataatag gatatcaccc cacttttcag 840
gtggtttaat gtgaaatttg gagttattac catattccta ctaatcattt tagaatttcc 900
tcccctgctt gctttgaacc tgaaattgtc aataattaga aaggaggaag catcattgtt 960
catatggatt tttgttagtt tgcctccatg tttatcttct gtaaaatgct tggggatgtt 1020
actccatgag catactaaaa tcgccaacgt ttgtgttgag aatctttcta gaaagattta 1080
taagatcgtc acttatgttc tttagttttt aggcagtaag tcgttgtgct aaatacccat 1140
gccgttttag attctccctg atcatgtttg cttttcttga ttcatgcaca gatttgaacc 1200
atggagtctt tacgtgacat ggtcctccca ccaggatttg gattccaccc aaaagatact 1260
gagctcattt ctcactatct gaaaaagaaa atacatggcc aaaagattga atatgagatc 1320
attccagagg tggatatata caagcatgaa ccatgggatt tacctggtaa gttgaacatt 1380
attccatgac aacctatatt tgtttcgttc catagagttt gctgatgaga aaagagtcaa 1440
tatttgacca tcaatttaaa ctagataatg ctggtagatg gaaacattca tgtttgcaag 1500
aaccactggg gaatgcacca gaatgttttc tgtattctta gtactgttgt catttcttag 1560
tactgttgca tcctttgcct ttttaaatat tatgcaactg attgccaatt atgtatcttc 1620
tttgtccaat tctctgccct gttattttga tagatgagtt tcttttttct actttttggc 1680
ttcctttgtt tgtatgccgc ttggaatcta attatgtaaa ctgctgatga tatttcacat 1740
tacagcaaag tgcgatgttc caactcagga taataagtgg catttcttcg ctgctcgtga 1800
cagaaagtat cctaacggtt ctcggtcaaa tagggcaaca gttgctgggt attggaaatc 1860
cactggaaag gatcgagcca taaaaatggg caagcaaact ataggaacga agaaaacttt 1920
agtttttcat gaaggccgcc cacccactgg gagacgtact gagtggatca tgcatgagta 1980
ctatatagat gagcgtgaat gtcaagcctg ccctgatatg aaggtattga actaattatt 2040
aagattttaa agtggcttat ttagaaaaga gagaactata ttgtggcttt acatgatact 2100
ccctccgttt caaaatattt gacaccgttg actttttagt acgtgtttga ctattcgtct 2160
tattcaaaaa atttaagtaa ttatttattc ttttcatatc atttgattca ttgttaaata 2220
tactttcatg tacacatata gttttacata tttcacaaat tttttttaat aagacgaacg 2280
gttaaacatg tgctaaaaaa tcaacggtgt caaacatttt gaaacggagg gagtatataa 2340
attaatgtgc actgtacctt cttgatgtca ctcttcaaac aacttgctgg tttagatgat 2400
ataatcatat gatttgctga ctaagggcga attccagtat ctaatgtagg gcttttattt 2460
tattgtccct caggatgcat atgtactttg tcgtattacc aaaagaaatg actggatccc 2520
agggaatggc aatgagttgg acaacagtga tcctcatcca gaaccatatg atgctcctcc 2580
atcagttatc agtactgaac agctaaatcc tgctgctgaa ccagttgtcg gtgttgaagc 2640
tgcaccagtt accgttgctg aacctgacgg tgttactacg tcagctatca ctgctaatat 2700
accatctcct agtgatgaca ttaatctgga tgattggttg aatgaactat ttgatccttt 2760
ctttgaccct gaacagagtc tggcttctgc tgatctgtct ccagatgaac aaaatgttga 2820
atcatcggtg agtttcttca cagtcactgt gtttgcaggt taaataattc aatggataaa 2880
ttattagaat gttgtagtga cattattaga atgttgtagt gactccagct ccctatgaga 2940
acgggctatt gcatgaacaa gaatctgttg gacaaatcta acatgcttgt tactcataat 3000
aaattgatct tcactggtcc acttgtgcat actgattgct atttaatata tctgcataat 3060
gcatttctga tagacaaaat actgaataac ttcacgaaca tatagaaacc aaacacaaca 3120
ttgcattata gtaatgtttg tgagcctagt acagcactgg ttactgcttg ggaattgata 3180
ctaagcctag tacagcactg gttactgctt gggaattgat actaatatga agcatgtgac 3240
tggtaatgac ctttagagat acctagacat aggcaaatca caatgttgtg accatttttt 3300
tttgtgactt agtatggctg cccttttatt ttcttttgct ggttgacaaa gaagatttgc 3360
ttgtaatttt tttaggaaca ccggagtaat gcattctcat ggataatatt tgaaactcat 3420
tgaaaattat tatgatttgt ttggattgac tttttaaaaa atagccatga tttgcatata 3480
ccatggactg aagctatctt atgtgttacg gtttgttcca cctcttctga gatttctggt 3540
tattgccaag tatctggtct gttatattat ggggaaacgt tgactgtagt tgtgttttca 3600
taccatgttc tttctttaac acggaaccta ggagtcaatg tattttctgc aaatcatgaa 3660
ttgtacttgt cattgtgctg tgcattctgc cattctacta tgttttgatt gcagttctta 3720
gcctgtgcat acttgctcat cgcgctatct tccttcttaa tttacagaac gtaggtgctt 3780
tggctccaaa ggtggaacaa gactattcta gtcctaacga gaacgtagta gatgatacgg 3840
agtacttgtt gcctgaagat gtttacaaca tactgcaccc tggtacagat gacttcaata 3900
tgctccagaa tcctttggat cagtacccca ttcaatatgc aaccgatgtt tggagtggaa 3960
ttcagaagga agaactgtgg tcgccccagg ccaatgctga gcctagtcag tcgaatgaag 4020
ctgctgataa tggaattata agacgttatc gtagtatgaa aacacctgaa actagtgtgc 4080
cgcagttcaa gggtaaaact caggctaaaa tgcgggttgg gatcaacaag atggctacga 4140
gtagttctga atctatcaac cagactatca agtttgagaa cagtggtcgt cttgttgagc 4200
accagaagaa ccaagcgcat gatgtagctt ctaccaagcg gtcagacgca gggaagccaa 4260
gtacagagct cagcagcaat cgaggatttc tcaggggtat ccgaaatgca tttgcaggct 4320
gttcggattc gatggaacat gatacttgtt gcgggtttcg ctattggagt cgctgtggta 4380
gcgcttcata taggccaacg ccttggatta agccagagag atcagcagca tacctagcct 4440
ttaggtttgc aggttactgg gtttctatgc tttttgcaat ctggagaaac gtaaacaaac 4500
tccgatcttt ggttggtgca gaacatttga tgtatgcgcc gttttttttg tgttggtttt 4560
gtacatatta ttatcttcgg ttttggtcga cttttgttct cgccagctag tagtactatg 4620
ttgacgaatg tagaagaaaa cctcaaaaaa aaaagaacaa atgaaaaaaa attgacgatg 4680
aaaatttata taattaaccg cttgcattgt actata 4716
<210> 25
<211> 546
<212> PRT
<213> Oryza sativa
<400> 25
Met Glu Ser Leu Arg Asp Met Val Leu Pro Pro Gly Phe Gly Phe His
1 5 10 15
Pro Lys Asp Thr Glu Leu Ile Ser His Tyr Leu Lys Lys Lys Ile His
20 25 30
Gly Gln Lys Ile Glu Tyr Glu Ile Ile Pro Glu Val Asp Ile Tyr Lys
35 40 45
His Glu Pro Trp Asp Leu Pro Ala Lys Cys Asp Val Pro Thr Gln Asp
50 55 60
Asn Lys Trp His Phe Phe Ala Ala Arg Asp Arg Lys Tyr Pro Asn Gly
65 70 75 80
Ser Arg Ser Asn Arg Ala Thr Val Ala Gly Tyr Trp Lys Ser Thr Gly
85 90 95
Lys Asp Arg Ala Ile Lys Met Gly Lys Gln Thr Ile Gly Thr Lys Lys
100 105 110
Thr Leu Val Phe His Glu Gly Arg Pro Pro Thr Gly Arg Arg Thr Glu
115 120 125
Trp Ile Met His Glu Tyr Tyr Ile Asp Glu Arg Glu Cys Gln Ala Cys
130 135 140
Pro Asp Met Lys Asp Ala Tyr Val Leu Cys Arg Ile Thr Lys Arg Asn
145 150 155 160
Asp Trp Ile Pro Gly Asn Gly Asn Glu Leu Asp Asn Ser Asp Pro His
165 170 175
Pro Glu Pro Tyr Asp Ala Pro Pro Ser Val Ile Ser Thr Glu Gln Leu
180 185 190
Asn Pro Ala Ala Glu Pro Val Val Gly Val Glu Ala Ala Pro Val Thr
195 200 205
Val Ala Glu Pro Asp Gly Val Thr Thr Ser Ala Ile Thr Ala Asn Ile
210 215 220
Pro Ser Pro Ser Asp Asp Ile Asn Leu Asp Asp Trp Leu Asn Glu Leu
225 230 235 240
Phe Asp Pro Phe Phe Asp Pro Glu Gln Ser Leu Ala Ser Ala Asp Leu
245 250 255
Ser Pro Asp Glu Gln Asn Val Glu Ser Ser Asn Val Gly Ala Leu Ala
260 265 270
Pro Lys Val Glu Gln Asp Tyr Ser Ser Pro Asn Glu Asn Val Val Asp
275 280 285
Asp Thr Glu Tyr Leu Leu Pro Glu Asp Val Tyr Asn Ile Leu His Pro
290 295 300
Gly Thr Asp Asp Phe Asn Met Leu Gln Asn Pro Leu Asp Gln Tyr Pro
305 310 315 320
Ile Gln Tyr Ala Thr Asp Val Trp Ser Gly Ile Gln Lys Glu Glu Leu
325 330 335
Trp Ser Pro Gln Ala Asn Ala Glu Pro Ser Gln Ser Asn Glu Ala Ala
340 345 350
Asp Asn Gly Ile Ile Arg Arg Tyr Arg Ser Met Lys Thr Pro Glu Thr
355 360 365
Ser Val Pro Gln Phe Lys Gly Lys Thr Gln Ala Lys Met Arg Val Gly
370 375 380
Ile Asn Lys Met Ala Thr Ser Ser Ser Glu Ser Ile Asn Gln Thr Ile
385 390 395 400
Lys Phe Glu Asn Ser Gly Arg Leu Val Glu His Gln Lys Asn Gln Ala
405 410 415
His Asp Val Ala Ser Thr Lys Arg Ser Asp Ala Gly Lys Pro Ser Thr
420 425 430
Glu Leu Ser Ser Asn Arg Gly Phe Leu Arg Gly Ile Arg Asn Ala Phe
435 440 445
Ala Gly Cys Ser Asp Ser Met Glu His Asp Thr Cys Cys Gly Phe Arg
450 455 460
Tyr Trp Ser Arg Cys Gly Ser Ala Ser Tyr Arg Pro Thr Pro Trp Ile
465 470 475 480
Lys Pro Glu Arg Ser Ala Ala Tyr Leu Ala Phe Arg Phe Ala Gly Tyr
485 490 495
Trp Val Ser Met Leu Phe Ala Ile Trp Arg Asn Val Asn Lys Leu Arg
500 505 510
Ser Leu Val Gly Ala Glu His Leu Met Tyr Ala Pro Phe Phe Leu Cys
515 520 525
Trp Phe Cys Thr Tyr Tyr Tyr Leu Arg Phe Trp Ser Thr Phe Val Leu
530 535 540
Ala Ser
545
<210> 26
<211> 31
<212> DNA
<213> Oryza sativa
<400> 26
ccggaattca tattcggcct cgtcgcctcg c 31
<210> 27
<211> 28
<212> DNA
<213> Oryza sativa
<400> 27
gaattcgaca tcaggcaggc cgtgaaac 28
<210> 28
<211> 20
<212> DNA
<213> Oryza sativa
<400> 28
gtgcgatgtt ccaactcagg 20
Claims (6)
1.基因OsNTL3在改良水稻高温抗性方面的应用，其特征在于，基因OsNTL3的碱基序列如SEQ ID NO.1所示。
2.根据权利要求1所述的应用，其特征在于，利用高温诱导型启动子过表达去除跨膜域的OsNTL3基因，该转基因水稻材料在高温抗性方面显著增强。
3.根据权利要求2所述的应用，其特征在于，所述的高温诱导型启动子其中一个为BiP2，BiP2的碱基序列如SEQIDNO.4所示。
4.根据权利要求3所述的应用，其特征在于，所述的高温诱导型启动子其中一个为BiP4，BiP4的碱基序列如SEQIDNO.5所示。
5.一种水稻高温抗性基因，其特征在于，其碱基序列如SEQ ID NO.6所示。
6.一种水稻高温抗性基因编码的蛋白质，其特征在于，其氨基酸序列如SEQ ID NO.7所示。
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111690650A (zh) * | 2020-06-30 | 2020-09-22 | 安徽省农业科学院水稻研究所 | 一种植物热诱导表达启动子Posheat5及其应用 |
| CN115947808A (zh) * | 2022-09-29 | 2023-04-11 | 浙江大学 | 通过基因编辑突变膜结合转录因子制备高温抗性增强的水稻材料的应用 |
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2019
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111690650A (zh) * | 2020-06-30 | 2020-09-22 | 安徽省农业科学院水稻研究所 | 一种植物热诱导表达启动子Posheat5及其应用 |
| CN111690650B (zh) * | 2020-06-30 | 2022-03-25 | 安徽省农业科学院水稻研究所 | 一种植物热诱导表达启动子Posheat5及其应用 |
| CN115947808A (zh) * | 2022-09-29 | 2023-04-11 | 浙江大学 | 通过基因编辑突变膜结合转录因子制备高温抗性增强的水稻材料的应用 |
| CN115947808B (zh) * | 2022-09-29 | 2023-12-12 | 浙江大学 | 通过基因编辑突变膜结合转录因子制备高温抗性增强的水稻材料的应用 |
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