CN1944648B - 一种提高植物耐盐性的水稻Cyp2基因 - Google Patents
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Abstract
本发明公开了一种提高植物耐盐性的水稻Cyp2基因,具有序列表中SEQID NO.1所述的碱基序列。本发明利用以2-D电泳和质谱分析为基础的蛋白质组技术,快速分离与鉴定了一个水稻耐盐性相关蛋白质,然后通过功能基因组学和生化等手段证实Cyp2基因具有提高植物耐盐性的功能。将本发明耐盐基因Cyp2转化水稻或草莓等蔬菜、花卉等植物,可以提高植物耐盐性,一方面,可以增加水稻或露地蔬菜、花卉在盐碱地上的产量,提高我省滨海地区的盐碱地的利用;另一方面,可以克服因土壤返盐引起的连作障碍,提高蔬菜、花卉等植物的产量和品质。
Description
技术领域
本发明涉及功能基因组学,尤其是一种提高植物耐盐性的水稻Cyp2基因。
背景技术
土壤盐渍化对农业的威胁是一个全球性的问题。全世界共有10亿公顷的盐碱地,约占世界陆地面积7.6%,我国盐碱地近1亿多公顷,农业耕地因盐渍化引起的减产、弃耕地就近5亿亩。近年来,随着我国设施农业的快速发展,特别是蔬菜和花卉大棚生产面积的扩大,这些保护地因化肥用量过大,土壤表层水分不断蒸发,使深层水分不断通过毛细管作用上移,导致深层土壤盐分聚积到表层,加剧了保护地的次生盐渍化进程。对于盐渍化土壤的利用主要采取两种措施,一是用化学或物理方法改良土壤;二是通过常规杂交或生物技术培育耐盐作物品种。但前者不仅投入成本高,且随着大量化学物质的施用加重了土壤的次生盐渍化,因此,挖掘植物耐盐基因资源,培育耐盐的作物品种就显得十分重要。
盐分对植物胁迫分为渗透胁迫、离子伤害、离子不平衡或营养缺乏三类,渗透胁迫和离子伤害目前被认为是对植物危害的两个主要过程。植物的耐盐性总的来说可分为形态耐盐和生理耐盐。在盐胁迫下,由于外界渗透压较低,植物吸收水分困难,细胞会发生水分亏缺现象。植物为了避免这种伤害,会主动积累一些可溶性物质,降低细胞的渗透势,从而使水分顺利地进入植物体内,保证植物正常生理活动的进行。在盐胁迫条件下,植物耐盐性受多种生理机制调节。一方面,小分子相容性溶质如:脯氨酸、可溶性糖、果糖、蔗糖、多胺等在植物体内大量积累对植物耐盐性起着渗透调节作用。另一方面,一些功能性蛋白如:渗调蛋白、脯氨酸合成酶(P5CS)、通道蛋白(水通道蛋白和钾通道蛋白)、Lea蛋白(后期胚胎发生富集蛋白)和跨膜运输蛋白(质膜H+-ATPase和液泡膜H+-ATPase)等受盐诱导在植物体内高表达可以减缓植物盐害。植物体内Na+离子平衡是植物自身耐盐调节的重要机制。例如拟南芥SOS系列基因和水稻耐盐相关的数量性状基因SKCl的调控信号是植物自身调节Na+离子平衡或维持钠和钾平衡的重要途径。
近年来,随着高通量蛋白质组技术如双向电泳、质谱技术、蛋白质芯片技术和酵母双杂交体系的建立,加速了植物蛋白质组学的发展。蛋白质组是指一个生物体基因组表达的全部蛋白质。要研究生物体中“全部蛋白质”是一件非常困难的事情。目前生物学家提出了功能蛋白组学的概念,从局部着手,注重研究那些可能涉及到特定功能机理的蛋白质全体因此,从一个生物体的具有特定功能的器官、组织、细胞着手,在不同生理条件下研究表达蛋白质的功能。
许多研究结果表明,利用基于2-D和生物质谱技术可以成功地分离与鉴定了水稻幼苗耐盐性相关的蛋白质。这些蛋白质主要参与碳水化合物、氮和能量代谢调节、活性氧种类清除、mRNA和蛋白加工和细胞骨架稳定性。但是,上述蛋白质仅仅在蛋白表达谱水平和种类上加以分离与鉴定,并未提供其具有改变植物耐盐性的功能证据。
发明内容
本发明提供一种提高植物耐盐性的水稻Cyp2基因。
一种提高植物耐盐性的水稻Cyp2基因,具有序列表中SEQ ID NO.1所述的碱基序列。
所述的提高植物耐盐性的水稻Cyp2基因编码的蛋白质,具有序列表中SEQ ID NO.3所述的氨基酸序列。
含有权利要求1所述的提高植物耐盐性的水稻Cyp2基因的表达载体。
含有权利要求1所述的提高植物耐盐性的水稻Cyp2基因的转基因细胞系。
本发明利用以2-D电泳和质谱分析为基础的蛋白质组技术,分离与鉴定水稻耐盐性相关蛋白质,根据Expasy、NCBI、TIGER等蛋白质和核酸数据库查询结果,获得水稻耐盐相关基因(或蛋白质)序列信息,然后通过功能基因组学和生化等手段证实基因功能。
基因来源:
以杂交水稻耐盐组合汕优10号和盐敏感组合两优培九为材料,将种子播在含NaCl溶液浸湿滤纸培养皿中,置于培养箱中发芽,每天更换盐溶液,以保持盐浓度基本一致。待幼苗生长至10d,分别收集研究汕优10号和两优培九幼苗的叶片,用冷丙酮/三氯乙酸沉淀法快速提取叶总蛋白,经双向电泳分离,用PDQUEST软件分析胶图匹配情况,结果发现在汕优10号幼苗叶片中一个高表达蛋白点。在胶上切下该蛋白点,用胰蛋白酶(Trypsin)进行胶内消化(In-gel digest),提取酶切产物,真空干燥,然后用三氟乙酸溶解,将溶解物用基质辅助激光解吸离子化飞行质谱(MALDI-TOF-MS)分析,获得肽质量指纹(Peptide Mass Fingerprint,PMF)图谱,查询Mascot数据库,以高分值比对到水稻细胞免疫素cyclophilin 2蛋白。然后用串联电喷雾质谱(ESI-MS/MS)分析上述溶解物中分离到3个肽段的氨基酸序列,结果3个肽段序列均包含于水稻细胞免疫素蛋白中,进一步证实该蛋白为水稻细胞免疫素蛋白。
根据已有的水稻细胞免疫素cyclophilin 2的氨基酸序列,通过NCBI和TIGER数据库搜索,比对到编码水稻细胞免疫素蛋白peptidylprolylisomerase Cyp2的基因,基因号为Os02g02890(或OSJNBb0088N06.23)。该基因的ORF(开放阅读框)为519bp,mRNA长度为885bp。
基因克隆与转化:
以汕优10号幼苗(播后10天)总mRNA为模板,利用RT-PCR方法扩增到peptidylprolyl isomerase Cyp2基因的编码序列,然后将Cyp2基因装入PMD18-T载体中,经测序正确,用EcoRI和HindIII酶切,回收基因片段,然后连入Super1300载体中,再转入GV3101农杆菌,鉴定正确后,通过农杆菌介导花浸法转化模式植物拟南芥,在含潮霉素的MS固体培养基上筛选转基因拟南芥阳性植株,繁种并鉴定至T3代,获得纯合的转基因12个株系。
基因耐盐功能鉴定:
T3代纯合转基因株系和野生型((ecotype Columbia))拟南芥种子处理后培养7d,观察幼苗生长情况。待幼苗子叶完全展开后,将转基因株系和野生型的幼苗分别移入含NaCl和正常的MS培养基上,然后置于相同光温条件的培养箱中培养。培养12-15d后,观察转基因株系与野生型幼苗在高盐胁迫下的表型。结果发现在含170mM NaCl MS培养上,野生型幼苗基部叶片出现白化死亡,而转Cyp2基因株系仍保持绿色;而在200mM NaCl下,野生型幼苗全部白化死亡,转Cyp2基因株系仅在幼苗基部1-2老叶出现白化死亡,而在幼苗的生长点和其余叶片仍保持绿色,说明Cyp2基因在植物中超量表达可以缓解植物盐害。
本发明有益效果:将耐盐基因Cyp2转化模式植物拟南芥,可以提高拟南芥幼苗耐盐性。若将耐盐基因Cyp2转化水稻或草莓等蔬菜、花卉等植物,有可能提高植物耐盐性,一方面,可以增加水稻或露地蔬菜、花卉在盐碱地上的产量,提高我省滨海地区的盐碱地的利用;另一方面,可以克服设施条件下因土壤返盐引起的连作障碍,提高蔬菜、花卉等植物的产量和品质,增加农民收入。
具体实施方式
以下内容涉及浓度、含量的如果没有特殊标明单位即为质量百分比浓度或含量
基因的获得
以杂交水稻耐盐组合汕优10号和盐敏感组合两优培九为材料,将种子播在含100mM NaCl溶液浸湿滤纸培养皿中,置于30℃培养箱中发芽,每天更换盐溶液,以保持盐浓度基本一致。待幼苗生长至10d,分别收集汕优10号和两优培九幼苗的叶片。
用冷丙酮/三氯乙酸沉淀法(按Salekdeh等的改进方法:Salekdeh G H,Siopongco J,Wade L J,Ghareyazie B,Bennett J.A proteomic approach toanalyzing drought-and salt-responsiveness in rice.Field Crop Res,2002,76(2-3):199~219)快速提取叶总蛋白,方法如下:
水稻叶片用液氮研磨成细粉,分装入1.5ml离心管中,加入1ml蛋白提取液I(含10%三氯乙酸和0.07%β-巯基乙醇的丙酮溶液)在-20℃沉淀粗蛋白1h,在4℃下13000rpm离心20min,取沉淀,弃上清,然后再加入1ml蛋白提取液II(含0.07%β-巯基乙醇的丙酮溶液)在-20℃悬浮粗蛋白丸1h,在4℃下13000rpm离心20min,取沉淀,弃上清,再重复用蛋白提取液II在相同条件下悬浮清洗3次,真空抽干得粗蛋白干粉,用裂解液溶解沉淀,并在室温下放置1h,裂解期间不断涡旋5-6次/小时。裂解液中含7mol/L尿素、2mol/L硫脲、4%Chaps(美国Ameresco公司)、50mmol/L DTT(美国Promega公司)和0.5%两性电解质pH3-10,裂解液用量为25μl裂解液/mg沉淀。
根据Bradford法(Bradford M M.A rapid and sensitive method for thequantitation of microgram quantities of protein utilizing the principle ofprotein-dye binding.Anal Biochem,1976,72:248-54)用考马斯亮兰G-250(Sigma公司)测定蛋白含量,经双向电泳分离(变性/SDS-2D-PAGE,采用17cm pH7-10的IPGs干胶条(Bio-Rad公司),第一向等电聚焦分四步进行,第一步,电压250V 15min;第二步,电压10000V 5h;第三步,电压10000V,60000Vh;第四步,电压500V直到结束。在第一向等电聚焦向第二向转换时,需要平衡胶条,分两步进行,第一步,在平衡液I(含6.0mol/L尿素、2%SDS(美国Promega公司)、0.375mol/L Tris-HClpH 8.8(美国Promega公司)、20%甘油和130mmol/L DTT(美国Promega公司))中平衡10min,第二步,在平衡液II(含6.0mol/L尿素、2%SDS(美国Promega公司)、0.375mol/L Tris-HCl pH 8.8(美国Promega公司)、20%甘油和135mmol/L碘乙酰胺)中平衡10min,然后转移到第二向SDS-2D-PAGE胶,跑胶采用恒流,每块胶的电流24mA,运行5-6h。),用银染显色(用500ml固定液(40%甲醇+10%乙酸)固定30min,然后用500ml硝酸银染色液(500ml中含有3.6%氢氧化钠10.5ml、20%硝酸银9ml和5ml氨水)染色32-33min,用双蒸水冲洗4次,然后用500ml显色液(500ml中含有1%柠檬酸2.5ml和甲醛250μl)显色5-12min,最后用500ml 5%醋酸终止反应5min),扫描,用PDQUEST(Bio-Rad公司)软件分析胶图匹配情况,结果发现在汕优10号幼苗叶片中一个高表达蛋白点,估测其等电点和分子量分别为pI 9.0和19 KD左右。
在胶上切下该蛋白点,用8μl 10ng/μl胰蛋白酶(Trypsin,Roche,美国)(25mM碳酸氢铵溶液,pH 8.0)进行胶内消化(In-gel digest):置于4℃冰箱放置40min使胶片完全吸收酶液,再补加10μl 25mM碳酸氢铵缓冲液,于37℃温育12h。
提取酶切产物:用30-50μl 5%TFA(Merk公司,德国)于40℃提取酶切肽段1小时一次,再用相同体积的50%ACN/2.5%TFA(Merk公司,德国) 溶液于30℃提取1小时一次,最后用25μl CAN(Fischer公司,美国)超声提取一次,合并3次提取液。真空干燥,然后用4μl 0.5%三氟乙酸溶解,将0.6μl溶解物用基质辅助激光解吸离子化飞行质谱(MALDI-TOF-MS)分析,获得肽质量指纹(Peptide Mass Fingerprint,PMF)图谱,查询Mascot数据库,以较高分值(115)显著(比对分高于65)比对到水稻细胞免疫素cyclophilin 2蛋白,匹配序列占总氨基酸序列38%。然后用串联电喷雾质谱(ESI-MS/MS)分析上述溶解物中分离到3个肽段的氨基酸序列,结果发现3个肽段序列均包含于水稻细胞免疫素蛋白中,进一步证实该蛋白为水稻细胞免疫素蛋白。
根据已有的水稻细胞免疫素cyclophilin 2(Cyp2)的氨基酸序列,通过NCBI和TIGER数据库搜索,比对到编码水稻细胞免疫素蛋白peptidylprolyl isomerase Cyp2的基因,基因号为Os02g02890(或OSJNBb0088N06.23)。该基因的ORF(开放阅读框)为519bp,mRNA长度为885bp。
基因克隆与转化
以汕优10号幼苗(播后10天)总mRNA为模板,利用RT-PCR方法扩增到peptidylprolyl isomerase Cyp2基因的编码序列。
具体操作如下:首先,将mRNA反转录成第一链cDNA,所用反转录试剂盒为GIBCOBRL公司的SUPERSCRIPTTM III,反应体系20μl,依次加入1μl oligo dT、1μl 10mM dNTP、5μg RNA和DEPC水至13μl,在65℃变性5分钟,迅速在冰上冷却至少1分钟,稍微离心,然后依次加入4μl 5×First-Strand buffer、1μl 0.1M DTT、1μl RNase OUTTMRecombinant RNase inhibitor和1μl SuperScriptTM III RT。轻微混合均匀,50℃反应60分钟,70℃ 15分钟使酶失活。为了去掉与cDNA互补的RNA链,加入1μl RNase H在37℃温育20min,-20℃保存。然后以第一链cDNA为摸板扩增目的基因Cyp2,所用扩增配对引物:
Cyp2-F,5’-GAATTCATGTCGAACACGAGGGTGTT-3’,
Cyp2-R,AAGCTTCTAGGAGAGCTGGCCGCAGT,
PCR反应体系为50μl,依次加入10×PCR buffer 5μl、10mM dNTPs1μl、反转录产物5μl、10μM正向引物(Cyp2-F)1μl、10μM反向引物(Cyp2-R)1μl、5U/μl Tag DNA聚合酶0.5μl,最后加水至50μl。PCR反应条件:预变性95℃,变性95℃30s,退火58℃30s,延伸72℃ 1min,30个循环,最后延伸72℃ 10min,4℃保存。
扩增后将Cyp2基因装入pMD18-T载体中:pMD18-T载体由TakaRa公司生产。将回收纯化的Cyp2基因的DNA与pMD18-T载体进行连接反应,连接体系10μl,各组分分别为0.5μl pMD18-T载体、4.5μl纯化的Cyp2基因的DNA、5μl Solution I。在14℃~16℃下连接8~12小时,然后将连接产物转化到大肠杆菌DH5α感受态细胞中。
将Cyp2基因装入pMD18-T载体后经测序正确,用TakaRa公司生产的EcoRI和HindIII酶切,操作如下:酶切体系40μl,包括4μl10×buffer、8μl已***Cyp2基因的pMD18-T载体、1μl EcoRI、1μl HindIII和26μl水,在37℃水浴中温育6h。
用北京博大泰克生物技术公司生产的Glassmilk kit回收基因片段,操作如下:从琼脂糖凝胶上切下所需DNA片段,放在1.5ml的Eppendorf管中。加入3倍体积的溶胶液,室温下放置5min,期间轻摇Eppendorf管几次使胶完全溶化。加入10μl玻璃奶,颠倒混匀,冰浴下放置10min。每隔2-3min混匀1次,12000rpm离心30s,吸弃上清。加入250μl漂洗液,用移液器吹打漂洗液,轻柔地将玻璃奶悬浮混匀,12000rpm离心30s,吸弃上清。重复漂洗1次。用枪头将剩余的漂洗液吸干净。然后,放置于37℃温箱干燥15-20min。加入20μl的无菌蒸馏水,混匀,60℃水浴5min,12000rpm离心1min,回收上清液。
将回收的基因片段连入Super1300载体中,操作如下:连接体系10μl,包括2μl Super1300载体、6μl纯化的Cyp2基因的DNA、1μl 10×T4连接酶buffer和1μl T4连接酶,在4-10℃下连接12h,然后将连接产物转化到大肠杆菌DH5α感受态细胞中,提取质粒进行鉴定。
基因片段连入Super1300载体后再转入GV3101农杆菌中,操作如下:取200μl农杆菌感受态细胞,加入5~10μl构建好的质粒DNA,30℃冰浴30min,液氮中速冻1min,37℃水浴5min,然后加入1ml YEB培养基(1升YEB培养基含1g酵母提取物、5g牛肉浸膏、5g蛋白胨、5g蔗糖和0.5g MgSO4·7H2O,pH7.0),28℃恢复培养4h;10000g离心30s,弃上清,加入0.1ml YEB培养基重新悬浮细胞,涂布于含有100μg/ml卡那霉素、25μg/ml庆大霉素和125μg/ml利福平的YEB平板(1升YEB培养基含1g酵母提取物、5g牛肉浸膏、5g蛋白胨、5g蔗糖、0.5gMgSO4·7H2O和12g琼脂,pH 7.0)上,28℃培养约48h。
经鉴定正确后(挑取阳性克隆作为模板,用菌落PCR方法进行鉴定),通过农杆菌介导浸花法转化模式植物拟南芥,操作如下:接种含有目的质粒的农杆菌菌落于10ml YEB培养基(含0.1%酵母提取物、0.5%牛肉浸膏、0.5%蛋白胨、0.5%蔗糖、0.05%MgSO4·7H2O、1.2%琼脂、100μg/ml卡那霉素、25μg/ml庆大霉素和125μg/ml利福平)中28℃、200rpm震荡培养过夜,转化前一天按1∶50接种于200ml含相同抗生素的YEB培养液中扩大培养至OD600为1.2~1.6,约6h,5000g离心15min集菌,重悬于渗透缓冲液,使OD600为0.8,200ml重悬液可使用3次。转化所用浸泡液含有0.5×MS大量元素、0.5×MS微量元素、0.5mg/L VB5、5%蔗糖、44nM 6-BA(美国Sigma公司)和0.03%Silwet L-77(美国LEHLESEEDS公司)。将200ml含目的农杆菌的渗透转化液置于一容器中,翻转种有拟南芥的花盆,使植株浸入含有待转化农杆菌的渗透缓冲液中,浸5分钟,缓慢取出花盆,侧放于托盘中,盖上黑塑料布避光24小时,第二天取下塑料布,直立放置花盆。
制备MS筛选平板(MS培养基外加80g/ml潮霉素和50g/ml氨苄青霉素),转化收获的T1代种子经消毒后播种于筛选平板,每15cm的平板上可以筛选100μg左右的拟南芥种子。4℃春化3天,平放在生长箱中培养(22℃恒温,24h光照),7~10天后挑选在筛选培养基上根系和地上部生长正常的阳性植株,移入正常MS培养基缓苗3~5天后移植入土壤,单株收获T2代种子。繁种并鉴定至T3代,获得纯合的转基因12个株系。
基因耐盐功能鉴定
T3代纯合转基因株系和野生型(ecotype Columbia)拟南芥种子用1%次氯酸钠消毒,在4℃冰箱中春化3天,然后置于温度22℃、湿度50%、连续24h光照的培养箱中培养。培养7d后,观察幼苗生长情况。待幼苗子叶完全展开后,将转基因株系和野生型幼苗分别移入含170mM NaCl、200mMNaCl和正常的MS固体培养基(含1×大量元素、1×微量元素、1×铁盐、3%蔗糖和0.8%琼脂)上,然后置于相同光温条件(22℃、湿度50%、连续24h光照)的培养箱中培养。培养12-15d后,观察转基因株系与野生型幼苗在高盐胁迫下的表型。结果发现在含170mM NaCl MS培养上,野生型幼苗基部叶片出现白化死亡,而转Cyp2基因株系仍保持绿色;而在200mM NaCl下,野生型幼苗全部白化死亡,转Cyp2基因株系仅在幼苗基部1-2老叶出现白化死亡,而在幼苗的生长点和其余叶片仍保持绿色,说明Cyp2基因在植物中超量表达可以缓解植物盐害。
SEQUENCE LISTING
<110>杭州市农业科学研究院
<120>一种提高植物耐盐性的水稻Cyp2基因
<130>
<160>3
<170>PatentIn version 3.3
<210>1
<211>519
<212>DNA
<213>水稻
<400>1
atgtcgaaca cgagggtgtt cttcgacatg accgtcggcg gagctccggc ggggcggatc 60
gtgatggagc tgtacgcgaa ggacgtgccg cggacggcgg agaacttccg cgcgctctgc 120
accggcgaga agggcgtggg caagagcggc aagccgctgc actacaaggg gagcaccttc 180
caccgcgtga tcccggagtt catgtgccag ggcggcgact tcacccgcgg caacggcacg 240
ggaggggagt cgatctacgg cgagaagttc gccgacgagg tgttcaagtt caagcacgac 300
agccccggca tcctgtccat ggcgaacgcc gggcccaaca ctaacgggtc ccagttcttc 360
atctgcaccg tgccctgcag ctggctggac gggaagcacg tcgtgttcgg ccgcgtcgtc 420
gagggcatgg acgtcgtcaa ggccatcgag aaggtgggat cccgcggcgg gagcaccgcc 480
aagccggtcg tcatcgccga ctgcggccag ctctcctag 519
<210>2
<211>885
<212>mRNA
<213>水稻
<400>2
cgcagcgatc tgaagtgaaa cagcaaaaaa aatcaaacaa aaagaaaaaa tattccccat 60
ctgtgaaatt cgcaaaaccc tagcgcggcg gcgatgtcga acacgagggt gttcttcgac 120
atgaccgtcg gcggagctcc ggcggggcgg atcgtgatgg agctgtacgc gaaggacgtg 180
ccgcggacgg cggagaactt ccgcgcgctc tgcaccggcg agaagggcgt gggcaagagc 240
ggcaagccgc tgcactacaa ggggagcacc ttccaccgcg tgatcccgga gttcatgtgc 300
cagggcggcg acttcacccg cggcaacggc acgggagggg agtcgatcta cggcgagaag 360
ttcgccgacg aggtgttcaa gttcaagcac gacagccccg gcatcctgtc catggcgaac 420
gccgggccca acactaacgg gtcccagttc ttcatctgca ccgtgccctg cagctggctg 480
gacgggaagc acgtcgtgtt cggccgcgtc gtcgagggca tggacgtcgt caaggccatc 540
gagaaggtgg gatcccgcgg cgggagcacc gccaagccgg tcgtcatcgc cgactgcggc 600
cagctctcct agatctgtgc tgttcccctt cgcctttcgc cagtatcagt cgtcttgagt 660
cgtcgagtcc ctaaataagg aggaggtggt ggtggtgtta gtctttttat gagttcgtgt 720
cgtgttggtg agatgagatc gcccatggtt tggttggatt aggcggagtt cttggatcga 780
ttcggtggag ttggatctgc gatccttctt ggggttggtt ttaaatctta attcgtgtcg 840
ctgcttctat gatatcgcta tcaatcaatg agaacatttg ggatc 885
<210>3
<211>172
<212>PRT
<213>水稻
<400>3
Met Ser Asn Thr Arg Val Phe Phe Asp Met Thr Val Gly Gly Ala Pro
1 5 10 15
Ala Gly Arg Ile Val Met Glu Leu Tyr Ala Lys Asp Val Pro Arg Thr
20 25 30
Ala Glu Asn Phe Arg Ala Leu Cys Thr Gly Glu Lys Gly Val Gly Lys
35 40 45
Ser Gly Lys Pro Leu His Tyr Lys Gly Ser Thr Phe His Arg Val Ile
50 55 60
Pro Glu Phe Met Cys Gln Gly Gly Asp Phe Thr Arg Gly Asn Gly Thr
65 70 75 80
Gly Gly Glu Ser Ile Tyr Gly Glu Lys Phe Ala Asp Glu Val Phe Lys
85 90 95
Phe Lys His Asp Ser Pro Gly Ile Leu Ser Met Ala Asn Ala Gly Pro
100 105 110
Asn Thr Asn Gly Ser Gln Phe Phe Ile Cys Thr Val Pro Cys Ser Trp
115 120 125
Leu Asp Gly Lys His Val Val Phe Gly Arg Val Val Glu Gly Met Asp
130 135 140
Val Val Lys Ala Ile Glu Lys Val Gly Ser Arg Gly Gly Ser Thr Ala
145 150 155 160
Lys Pro Val Val Ile Ala Asp Cys Gly Gln Leu Ser
165 170
Claims (1)
1.水稻Cyp2基因在提高拟南芥耐盐性能中的应用,所述的水稻Cyp2基因的碱基序列如SEQ ID NO:1所示。
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