CN109628485B - OsDBP1蛋白在调控水稻耐冷性中的应用 - Google Patents
OsDBP1蛋白在调控水稻耐冷性中的应用 Download PDFInfo
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Abstract
本发明公开了OsDBP1蛋白在调控水稻耐冷性中的应用。本发明提供了一种培育转基因植物的方法,包括如下步骤:抑制出发植物中osdbp1基因的表达,得到耐冷性增强的转基因植物。本发明还保护一种植物育种方法,包括如下步骤:降低目的植物中OsDBP1蛋白的含量和/或活性,从而增加目的植物的耐冷性。本发明提供的方法对于以培育耐冷水稻为目的的水稻育种,具有重要的应用推广价值。
Description
技术领域
本发明属于生物技术领域,具体涉及OsDBP1蛋白在调控水稻耐冷性中的应用。
背景技术
水稻(Oryza sativa L.)起源于热带或亚热带地区,属喜温性作物。现今推广的高产栽培品种对低温耐受能力有限。易遭受低温天气引起的冷害,导致产量减少,尤其是苗期遇冷可能会导致绝收。所以提高水稻的耐冷能力具有重要的经济和现实意义。
目前关于水稻低温信号通路的研究取得了一定进展。水稻中的低温响应途径主要包括ABA依赖的信号途径,DREB-CRT/DRE途径和ROS激活的激酶级联放大途径。
发明内容
本发明的目的是提供OsDBP1蛋白在调控水稻耐冷性中的应用。
本发明提供了一种培育转基因植物的方法,包括如下步骤:抑制出发植物中osdbp1基因的表达,得到耐冷性增强的转基因植物。所述出发植物为禾本科植物。所述出发植物为水稻。所述出发植物为粳稻。所述出发植物为水稻Dongjin或水稻中花11号。所述抑制出发植物中osdbp1基因的表达是通过导入干扰载体实现的。所述干扰载体为RNAi干扰载体。所述干扰载体具体可为OsDBP1-RNAi载体。OsDBP1-RNAi载体:以载体pTCK303为出发载体,插入了正向片段和反向片段;正向片段如序列表的序列4所示;正向片段和反向片段为反向互补关系。OsDBP1-RNAi载体:以载体pTCK303为出发载体,在SpeI和SacI酶切位点之间插入了正向片段,在BamHI和KpnI酶切位点之间插入了反向片段;正向片段如序列表的序列4所示;正向片段和反向片段为反向互补关系。
本发明还保护一种植物育种方法,包括如下步骤:降低目的植物中OsDBP1蛋白的含量和/或活性,从而增加目的植物的耐冷性。所述目的植物为禾本科植物。所述目的植物为水稻。所述目的植物为粳稻。所述目的植物为水稻Dongjin或水稻中花11号。
本发明还保护用于抑制OsDBP1蛋白的物质在培育耐冷植物中的应用。所述植物为禾本科植物。所述植物为水稻。所述植物为粳稻。所述植物为水稻Dongjin或水稻中花11号。
本发明还保护用于抑制osdbp1基因表达的物质在培育耐冷植物中的应用。所述植物为禾本科植物。所述植物为水稻。所述植物为粳稻。所述植物为水稻Dongjin或水稻中花11号。所述抑制osdbp1基因表达的物质为干扰载体。所述干扰载体为RNAi干扰载体。所述干扰载体具体可为OsDBP1-RNAi载体。OsDBP1-RNAi载体:以载体pTCK303为出发载体,插入了正向片段和反向片段;正向片段如序列表的序列4所示;正向片段和反向片段为反向互补关系。OsDBP1-RNAi载体:以载体pTCK303为出发载体,在SpeI和SacI酶切位点之间插入了正向片段,在BamHI和KpnI酶切位点之间插入了反向片段;正向片段如序列表的序列4所示;正向片段和反向片段为反向互补关系。
本发明还保护OsDBP1蛋白在调控植物耐冷性中的应用。所述植物为禾本科植物。所述植物为水稻。所述植物为粳稻。所述植物为水稻Dongjin或水稻中花11号。所述调控为负调控。
本发明还保护以上任一所述方法在植物育种中的应用。所述植物育种的目的为提高植物的耐冷性。所述植物为禾本科植物。所述植物为水稻。所述植物为粳稻。所述植物为水稻Dongjin或水稻中花11号。
所述osdbp1基因为编码OsDBP1蛋白的基因。
所述OsDBP1蛋白为如下(a1)或(a2)或(a3):
(a1)序列表中序列1所示的蛋白质;
(a2)将(a1)经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的与植物耐冷性相关的蛋白质;
(a3)来源于水稻且与(a1)具有98%以上同一性且与植物耐冷性相关的蛋白质。
所述osdbp1基因为如下(b1)或(b2)或(b3)或(b4):
(b1)编码区如序列表中序列2所示的DNA分子;
(b2)序列表中序列3所示的DNA分子;
(b3)来源于水稻且与(b1)或(b2)具有95%以上同一性且编码所述蛋白质的DNA分子;
(b4)在严格条件下与(b1)或(b2)限定的核苷酸序列杂交且编码所述蛋白质的DNA分子。
所述水稻为粳稻。所述水稻为水稻Dongjin或水稻中花11号。
以上任一所述耐冷可为苗期耐冷。
本发明提供的方法对于以培育耐冷水稻为目的的水稻育种,具有重要的应用推广价值。
附图说明
图1为实施例1中LP、RP、RB在基因组上的位置。
图2为实施例1中T-DNA插入osdbp1基因中的纯合植株以及水稻Dongjin进行PCR扩增后的0.8%的琼脂糖凝胶电泳图。
图3为实施例1中幼苗中OsDBP1基因的相对表达水平。
图4为实施例1中表型鉴定中的植株照片。
图5为实施例1中的存活率结果。
图6为实施例2中OsDBP1-RNAi载体的元件示意图。
图7为实施例2中幼苗中OsDBP1基因的相对表达水平。
图8为实施例2中表型鉴定中的植株照片。
图9为实施例2中的存活率结果。
具体实施方式
以下的实施例便于更好地理解本发明,但并不限定本发明。下述实施例中的实验方法,如无特殊说明,均为常规方法。下述实施例中所用的试验材料,如无特殊说明,均为自常规生化试剂商店购买得到的。以下实施例中的定量试验,均设置三次重复实验,结果取平均值。
粳稻(包括水稻Dongjin和水稻中花11号)中,OsDBP1蛋白如序列表的序列1所示。粳稻的cDNA中,编码OsDBP1蛋白的开放阅读框如序列表的序列2所示。粳稻的基因组DNA中,编码OsDBP1蛋白的基因如序列表的序列3所示。
A液母液(1L):含(NH4)2SO4 9.64g、KH2PO4 4.96g、KNO3 3.7g、K2SO4 3.18g、MgSO4·7H2O 29.965g,余量为水。B液母液(1L):Ca(NO3)2·4H2O 17.235g,余量为水。EDTA-Fe母液(1L):溶解5.57g FeSO4·7H2O于200mL蒸馏水中,溶解7.45g Na2-EDTA于200mL蒸馏水中,加热Na2-EDTA溶液,加入FeSO4·7H2O溶液,不断搅拌,冷却后定容至1L。微量元素母液(1L):含H3BO4 2.86g、CuSO4·H2O 0.08g、ZnSO4·7H2O 0.22g、MnCl2·4H2O 1.81g、H2MO4·H2O0.09g,余量为水。取5mL A液母液、5ml B液母液、1mL EDTA-Fe母液、1mL微量元素母液、200mg硅酸钠混合加蒸馏水稀释至1L,用1mol/L HCl调pH值为5.8,得到1L木村B培养液。
N6D2培养基:含300mg/L水解酪蛋白、500mg/L脯氨酸、500mg/L谷氨酰胺、30g/L蔗糖和2mg/L 2,4-D的固体MS培养基。
N6D2S1培养基:含25mg/L潮霉素和600mg/L头孢霉素的N6D2培养基。
N6D2S2培养基:含50mg/L潮霉素和300mg/L头孢霉素的N6D2培养基。
分化培养基A:含300mg/L水解酪蛋白、50mg/L潮霉素、1mg/L 6-BA、0.5mg/L KT、0.2mg/L ZT、0.25mg/L NAA、30g/L蔗糖和30g/L山梨醇的N6D2培养基。
分化培养基B:含300mg/L水解酪蛋白、50mg/L潮霉素、1mg/L 6-BA、0.5mg/L KT、0.2mg/L ZT、0.5mg/L NAA、30g/L蔗糖和20g/L山梨醇的N6D2培养基。
生根壮苗培养基:含1mg/L多效唑和0.5mg/L NAA的固体1/2MS培养基。
实施例1、
一、OsDBP1突变体材料osdbp1的获得和鉴定
从韩国突变体库RISD(http://cbi.khu.ac.kr/RISD_DB.html)获得水稻osdbp1(PFG_1E-01523,T-DNA插入突变体)及其对应的野生型-水稻Dongjin。水稻Dongjin的拉丁名为Oryza.sativa L.spp.japonica cv.Dongjin。获得的水稻均为种子形式。
将水稻种子去壳,然后用70%酒精水溶液浸泡30s,然后用2.5%NaClO水溶液浸泡10-15min,然后用无菌水清洗4-5遍(每次10min),然后接种于1/2MS培养基平板上,放置于光照培养箱中(光强为10000μmol/m2/s,光照时间为16h/d,温度为30℃)培养三周。取幼苗,提取基因组DNA,以基因组DNA为模板,分别采用LP/RP引物对和RB/RP引物对进行PCR扩增。LP:5′-TCTTCCCATTCACACCTTCC-3′;RP:5′-TCGAGCACGAGTATGACTCG-3′;RB:5′-TGGCAGGATATATTGTGGTGTAAAC-3′。LP、RP、RB在基因组上的位置如图1所示(黑色方块为外显子,灰色方块为非翻译区UTR,黑色方块之间的线段为内含子)。采用RB/RP引物对具有扩增产物且采用LP/RP引物对无扩增产物的植株即为T-DNA插入osdbp1基因中的纯合植株。
T-DNA插入osdbp1基因中的纯合植株以及水稻Dongjin进行上述PCR扩增后的0.8%的琼脂糖凝胶电泳图见图2。图2中:左图为采用LP/RP引物对,右图为采用RB/RP引物对;1对应Marker,2对应水稻Dongjin的种子,3对应T-DNA插入osdbp1基因中的纯合植株的种子。
培养T-DNA插入osdbp1基因中的纯合植株,自交收获种子,用于步骤二和步骤三。
培养水稻Dongjin,收获种子,用于步骤二和步骤三。
二、荧光实时定量PCR鉴定
1、取种子,置于阳光下晾晒一周,37℃浸种两天,然后将发芽的种子置于切底的96孔板中,置于木村B培养液里,在光照培养箱中(光强为10000μmol/m2/s,光照时间为16h/d,温度为30℃)培养至3叶期。
2、取3叶期幼苗,提取总RNA,反转录得到cDNA,进行荧光实时定量PCR。用于检测OsDBP1基因的引物:5′-CTTTGATGGGCATGGTG-3′;5′-CAAATGACCTCCTGACAACT-3′。用于定量分析的试剂为SYBR Green Realtime PCR Master Mix(TOYOBO)。所用仪器为美国Stratagene公司实时荧光定量PCR仪Mx3000P。Ubqtin基因作为内参。
幼苗中OsDBP1基因的相对表达水平见图3(进行三次重复试验,每次重复试验中设置6个生物学重复,结果取平均值)。图3中,2对应水稻Dongjin的种子,1对应T-DNA插入osdbp1基因中的纯合植株的种子。
三、表型鉴定
1、取种子,置于阳光下晾晒一周,37℃浸种两天,然后将发芽的种子置于切底的96孔板中,置于木村B培养液里,在光照培养箱中(光强为10000μmol/m2/s,光照时间为16h/d,温度为30℃)培养至3叶期,拍照(处理前)。
2、将3叶期幼苗置于4℃环境中放置72h,然后放回所述光照培养箱恢复培养3周,拍照(处理后恢复)并统计存活率。
进行三次重复试验,每次重复试验设置30-40个生物学重复。用t-Test进行差异显著性分析。
照片见图4。图4中,WT表示水稻Dongjin的种子,osdbp1表示T-DNA插入osdbp1基因中的纯合植株的种子,标尺为6厘米。低温胁迫后,T-DNA插入osdbp1基因中的纯合植株的生长状况显著优于水稻Dongjin。
存活率结果见图5。图5中,WT表示水稻Dongjin的种子,osdbp1表示T-DNA插入osdbp1基因中的纯合植株的种子。低温胁迫后,osdbp1存活率显著高于WT。
实施例2、通过RNAi干扰抑制OsDBP1基因表达从而提高水稻苗期耐冷能力
水稻中花11号,简称中花11,用ZH11表示。
植物RNAi表达载体pTCK303,简称载体pTCK303,即申请号为03160092.1(授权公告日为2007年6月20日;授权公告号为CN1322131C)的专利中实施例1制备的质粒pTCK303。
一、构建重组质粒
1、利用水稻中花11号3叶期幼苗,提取总RNA,反转录得到cDNA。
2、以步骤1得到的cDNA为模板,采用OsDBP1-RNAiF和OsDBP1-RNAiR组成的引物对进行PCR扩增,回收PCR扩增产物。
OsDBP1-RNAiF:5’-GGGGTACCACTAGTCGCCTTCGCTTAATAATCTCCT-3’;
OsDBP1-RNAiR:5’-CGGGATCCGAGCTCCCTGATCGGATAACTGGGACAA-3’。
3、取步骤2得到的PCR扩增产物,采用限制性内切酶SpeI和SacI进行双酶切,回收酶切产物。
4、取步骤2得到的PCR扩增产物,采用限制性内切酶BamHI和KpnI进行双酶切,回收酶切产物。
5、取载体pTCK303,采用限制性内切酶SpeI和SacI进行双酶切,回收载体骨架。
6、将步骤3得到的酶切产物和步骤5得到的载体骨架连接,得到重组质粒。
7、取步骤6得到的重组质粒,采用限制性内切酶BamHI和KpnI进行双酶切,回收载体骨架。
8、将步骤4得到的酶切产物和步骤7得到的载体骨架连接,得到OsDBP1-RNAi载体。
根据测序结果,对OsDBP1-RNAi载体进行结构描述如下:以载体pTCK303为出发载体,在SpeI和SacI酶切位点之间插入了正向片段,在BamHI和KpnI酶切位点之间插入了反向片段。正向片段如序列表的序列4所示(391bp)。正向片段和反向片段为反向互补关系。
OsDBP1-RNAi载体的元件示意图见图6。
二、制备转基因植株
1、将OsDBP1-RNAi载体导入根癌农杆菌EHA105,得到重组农杆菌。
2、通过农杆菌介导法将步骤1得到的重组农杆菌对中花11的胚性愈伤组织进行遗传转化,然后将愈伤组织转移到N6D2S1培养基上,培养2周。
3、完成步骤2后,将愈伤组织转移到N6D2S2培养基上,培养2周。
4、完成步骤3后,将愈伤组织转移到N6D2S2培养基上,培养2周。
5、完成步骤4后,将愈伤组织转移到分化培养基A上,培养7天。培养条件:12小时光照/12小时黑暗,光照强度为8000lux;光照时28℃,黑暗时25℃。
6、完成步骤5后,将愈伤组织转移到分化培养基B上,培养至长出幼苗。培养条件:12小时光照/12小时黑暗,光照强度为8000lux;光照时28℃,黑暗时25℃。
7、完成步骤6后,将苗高为2cm左右的幼苗转移至生根壮苗培养基上,培养至苗高为10cm左右。培养条件:12小时光照/12小时黑暗,光照强度为8000lux;光照时28℃,黑暗时25℃。
8、完成步骤7后,炼苗2-3d,然后转移到人工气候室,正常培养,即为TO代再生植株。
9、TO代再生植株进行GUS组织化学染色。
取TO代再生植株,截取2-3mm长的根段,进行GUS染色。GUS染色液(pH7.0):含100mMNa3PO4、0.1%(体积比)Triton X-100、10mM EDTA、0.5mM亚铁氰化钾、0.5mM铁氰化钾、1mg/ml X-Gluc。染色步骤:先在GUS染色液中37℃孵育过夜,然后用70%乙醇脱色。如果根段GUS染色显示为蓝色,该植株为转基因植株。
10、取TO代转基因植株,自交并收获种子,将种子培育为植株,即为T1代植株。
11、取T1代转基因植株,自交并收获种子,将种子培育为植株,即为T2代植株。
将T1代植株和抽样的T2代植株进行进行GUS组织化学染色,方法同步骤9。
对于某一T1代转基因植株来说,如果其抽样得到的T2代植株均为转基因植株,该T1代转基因植株为纯合的转基因植株,该T1代转基因植株及其自交后代为一个纯合的转基因株系。
三、制备转空载体植株
用载体pTCK303代替OsDBP1-RNAi载体,其他同步骤二,得到TO代再生植株。
四、植株的鉴定
1、定量PCR鉴定
从步骤二得到的纯合的转基因株系中随机取两个株系,RNAi2株系和RNAi5株系。
供试植株:RNAi2株系的T2代植株、RNAi5株系的T2代植株、中花11植株。
取供试植株的3叶期幼苗,提取总RNA,反转录得到cDNA,进行荧光实时定量PCR。用于检测OsDBP1基因的引物:5′-CTTTGATGGGCATGGTG-3′;5′-CAAATGACCTCCTGACAACT-3′。用于定量分析的试剂为SYBR Green Realtime PCR Master Mix(TOYOBO)。所用仪器为美国Stratagene公司实时荧光定量PCR仪Mx3000P。Ubqtin基因作为内参。
幼苗中OsDBP1基因的相对表达水平见图7(进行三次重复试验,每次重复试验中设置6个生物学重复,结果取平均值)。
2、表型鉴定
从步骤二得到的纯合的转基因株系中随机取两个株系,RNAi2株系和RNAi5株系。
供试植株:RNAi2株系的T2代种子、RNAi5株系的T2代种子、转空载体株系的T2代种子、中花11种子。
(1)取种子,置于阳光下晾晒一周,37℃浸种两天,然后将发芽的种子置于切底的96孔板中,置于木村B培养液里,在光照培养箱中(光强为10000μmol/m2/s,光照时间为16h/d,温度为30℃)培养至3叶期,拍照(处理前)。
(2)将3叶期幼苗置于4℃环境中放置72h,然后放回所述光照培养箱恢复培养3周,拍照(处理后恢复)并统计存活率。
进行三次重复试验,每次重复试验设置30-40个生物学重复。用t-Test进行差异显著性分析。
照片见图8。图8中,标尺为6厘米。低温胁迫后,RNAi2株系植株和RNAi5株系植株的生长状况均显著优于中花11,转空载体株系植株的生长状况和中花11无显著差异。
存活率结果见图9。低温胁迫后,RNAi2株系植株和RNAi5株系植株的存活率均显著高于中花11。转空载体株系植株的存活率为6%,和中花11无显著差异。
SEQUENCE LISTING
<110> 中国科学院植物研究所
<120> OsDBP1蛋白在调控水稻耐冷性中的应用
<130> GNCYX190427
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 354
<212> PRT
<213> Oryza sativa Japonica Group
<400> 1
Met Cys Val Glu Glu Leu Glu Gly Ala Glu Arg Leu Asp Phe Gly Gly
1 5 10 15
Val Ala Glu Leu Glu Thr Thr Pro Ala Asp Phe Glu Met Glu Lys Val
20 25 30
Cys Glu Asn Thr Val Ser Leu Asp Phe Lys Gln Ala Arg Ser Ser Ser
35 40 45
Phe Val Pro Val Ile Arg Ser Gly Asp Trp Ser Asp Ile Gly Gly Arg
50 55 60
Asp Tyr Met Glu Asp Ala His Val Cys Ile Ser Asp Leu Ala Asn Asn
65 70 75 80
Phe Gly His Asn Ser Val Asp Asp Glu Ile Ile Ser Phe Tyr Gly Val
85 90 95
Phe Asp Gly His Gly Gly Lys Asp Ala Ala His Tyr Val Arg Asp Asn
100 105 110
Leu Pro Arg Val Ile Val Glu Asp Ala Asp Phe Pro Leu Glu Leu Glu
115 120 125
Lys Val Val Arg Arg Ser Phe Val Gln Thr Asp Ser Gln Phe Ala Glu
130 135 140
Arg Cys Ser His Gln Asn Ala Leu Ser Ser Gly Thr Thr Ala Leu Thr
145 150 155 160
Ala Met Ile Phe Gly Arg Ser Leu Leu Val Ala Asn Ala Gly Asp Cys
165 170 175
Arg Ala Val Leu Ser Arg Arg Gly Thr Ala Ile Glu Met Ser Lys Asp
180 185 190
His Arg Thr Cys Cys Leu Asn Glu Arg Lys Arg Ile Glu Ser Leu Gly
195 200 205
Gly Tyr Val Asp Asp Gly Tyr Leu Asn Gly Gln Leu Ala Val Thr Arg
210 215 220
Ala Leu Gly Asp Trp His Leu Glu Gly Leu Lys Glu Val Gly Glu Pro
225 230 235 240
Gly Gly Pro Leu Ser Ala Glu Pro Glu Leu Lys Met Ile Thr Leu Thr
245 250 255
Lys Glu Asp Glu Phe Leu Ile Ile Gly Ser Asp Gly Ile Trp Asp Phe
260 265 270
Phe Ser Asn Gln Asn Ala Val Asp Phe Thr Arg Lys Arg Leu Gln Glu
275 280 285
His Asn Asp Leu Arg Leu Cys Cys Lys Gln Ile Val Glu Glu Ala Ile
290 295 300
Arg Arg Gly Ala Ser Asp Asn Leu Thr Ala Val Met Val Ser Phe His
305 310 315 320
Gln Glu Ala Pro Pro Gln Leu Arg Val Asn Arg Thr Gly Arg Val Glu
325 330 335
Arg Ser Ile Ser Ala Glu Gly Leu His Ser Leu Arg Val Leu Leu Glu
340 345 350
Gly Gln
<210> 2
<211> 1065
<212> DNA
<213> Oryza sativa Japonica Group
<400> 2
atgtgcgtgg aggaactcga aggcgcagag aggctcgatt ttggtggggt tgcggagctc 60
gagacaacac cggcagactt cgagatggag aaagtttgtg agaacacagt atctcttgat 120
ttcaagcagg ctaggtcgag cagttttgtc ccagttatcc gatcagggga ctggtcggat 180
attggaggtc gcgattacat ggaagatgct catgtctgca tctcagatct agctaataat 240
tttggtcata attcagtgga tgatgagatt atttcctttt atggggtctt tgatgggcat 300
ggtggaaaag atgcagctca ttatgtgcgt gataacttgc cacgggttat cgtggaagat 360
gctgattttc ctcttgagct agagaaagtt gtcaggaggt catttgtgca aactgatagt 420
caatttgcag agaggtgctc tcatcagaat gcactttctt ctggaacgac agcgcttaca 480
gcaatgattt ttggaaggtc tcttctggtt gctaatgctg gtgattgccg agcagttctt 540
tcaaggcgtg gtaccgcaat tgaaatgtcc aaggaccaca ggacttgctg cctcaacgaa 600
agaaagcgta tagaatcact tggcggctat gttgatgatg gctatttgaa tggtcaatta 660
gcagtcacta gagcattggg tgactggcat ctcgagggtc tgaaagaagt gggtgagcca 720
ggtggcccct tgagcgcgga accagagctc aagatgatca cactgacgaa ggaagacgag 780
ttcttgataa taggaagcga cggcatctgg gacttcttct cgaaccagaa tgccgtggat 840
ttcaccagga agaggctcca agagcacaat gacttgaggt tgtgctgcaa gcagatcgtc 900
gaggaggcaa taaggcgagg ggcctcagac aacctaacgg cggtgatggt ctcattccac 960
caggaggccc ctcctcagct cagagtgaac aggacgggga gggtcgagcg gagcatatcg 1020
gctgaagggc ttcacagcct cagggtgctc ctggaaggcc aatga 1065
<210> 3
<211> 4030
<212> DNA
<213> Oryza sativa Japonica Group
<400> 3
catcaaaatc gccttcgctt aataatctcc tcctccgtat taaactcggt ttttttagat 60
attcccaaga atctctcctc tcctttccat tgttgctccg ctcggattcc gctcttcttc 120
ttcttctccg tctccgattc ggtctcctcc tcctctgctc cgaccaggat tctgcagcgg 180
cagcggcagc tgctgcggtt ggtttttcag gctcggtggc ggcaggcagc ggcatgtgcg 240
tggaggaact cgaaggcgca gagaggctcg attttggtgg ggttgcggag ctcgagacaa 300
caccggcaga cttcgaggtg agtagagtaa actcccggat ttcggaagct ccctgttctt 360
tcttgctttc tttctttctc gtgttcttga tgcattcgtt tgtgatttga tttcgcaacc 420
ggatgctttt ctttgggtgt tgggatgatc ggttgagggg aaatccaact gaggtggttg 480
cgttgggtat actcttcgcg tgcccgaatg cttctatttt cttattttct tttcttgtct 540
ggaaatctgg agaaagtagg agagattagt tagtttatta gtaccactga aataaatgaa 600
ctcccttatc aattaatata atataatatt tcaacgtttt tttagtatag taacaactag 660
cagcaaataa tttaggaaat tgtttgttct tgaacggaag gtgtgaatgg gaagatgcgc 720
tttgaattgt tcagcggcaa gaggaagttt ctaggaggag agggattcag attgcgtgaa 780
aaacacaatc tttttatttt ttggggtgcc ttgtcgtagc ttgtgggtga cttgtctgct 840
tgcgcattgc gttgttctcc tgtacttttg attctgtggc gagaagtcgt gtgggatttt 900
cagttgatgt gttcgtacta ctgcatcttg tgcgcatgcg tatctgctct gattctgata 960
tgctgtgata agatttttat gcatgtgagt gacagtgagt actacctgaa aactcttcgt 1020
accaagcaga aatgaactct cttaatactg tactgcagaa ttcaagaaag tagtgtactt 1080
tgtttctctt caaatcaaga aagtagagca ccaaatggat attagaaaga atgtcctttg 1140
tgatgttatg cctttagaag tcaaatgtta tttacatgca gaactgtcaa tcaataaatc 1200
agtactggta aagttcagtg gtacttctct ctcagttgag gccaagaaaa atgttctttt 1260
cttaaaatgc tggcctcctg gttaagactg ttgtttggac aatgcttagt gacccttgtg 1320
atttgtagct ctagaagtac tacataatat atcttctgat gaactgtgaa ccccgtcgtt 1380
agaccttgat gggatatctt tgtcgcttta tcttgtaatg atggttgcaa gcaggaaatg 1440
gattatggag tgttgcggaa gaatgaacac ctggtggatt ttcatggctc tagtactaat 1500
tagtttaaca gggattaaca gttggagtgg ctgtagtcct atcatattgt cacaactgtt 1560
gtgccttttt tttgtcaagt catatgctcc tgtgtctgtt ttgcctgaaa aatactaagc 1620
tgtactgcac ttttgtgcat tcttgggtgt tttgtttact aacgccctct ctagaaatgg 1680
ccactcgtag gcttgtccaa taagggtttg cctcttgtca cctagttgac tgaattgggg 1740
cttctgtttc tgtgtcgctt tcaactaggt ggattagtat ttaccgtaag tagttcattt 1800
tcgatgtgat gaatgatgat gtcgacagca tcccttaaag caatttgtgc tcttggcaga 1860
gttgaagtgc tagtatattc atttgaagtt ttgaactagc atcatgttct gccaaagttg 1920
gcattgatct gttatttttt ataaggctga cgtcctctgc caatgcttgt tactatcctt 1980
aaggcattta gaacttgtgt tgtaattatg cttcatcaga gttgcttctt gtttcttatt 2040
tttgccatgc tctaatcttt tacctttctt ggtaatgtgc agatggagaa agtttgtgag 2100
aacacagtat ctcttgattt caagcaggct aggtcgagca gttttgtccc agttatccga 2160
tcaggggact ggtcggatat tggaggtcgc gattacatgg aagatgctca tgtctgcatc 2220
tcagatctag ctaataattt tggtcataat tcagtggatg atgagattat ttccttttat 2280
ggggtaactt cttgttttta ctctctttat tttgattctt cagttcccat ttgatatgga 2340
aatcaatacc tagttgagca ttccagagtc ctaaaacgtg cctaatccat tgtaaagtaa 2400
ttaaaaaaat gtattatctt taagtgcagt gaactcataa cagcagcagt ttagagagat 2460
tgcatctacc aaatcagata catggatcat gaaattgtac atgcttgtct ttttctaaga 2520
tcatcttgtc cttaaatttt tcgcaggtct ttgatgggca tggtggaaaa gatgcagctc 2580
attatgtgcg tgataacttg ccacgggtta tcgtggaaga tgctgatttt cctcttgagc 2640
tagagaaagt tgtcaggagg tcatttgtgc aaactgatag tcaatttgca gagaggtgct 2700
ctcatcagaa tgcactttct tctggaacga cagcgcttac agcaatgatt tttggaaggt 2760
actattccta ctgcaggcta gaagtagaat ttagcctagg tcaatctgct tcttagcatg 2820
ctatgtttga ttcttaagtc tggcaaacta ctcccaagct tgttctttgg ttggttattc 2880
cgcattttct acgtgtgcct tgcaaatatt ttctgaaagc ttatccaata gcataaaatt 2940
cttaggaatg ttccacttaa ggagttgagg tgttaggagt ttcccctgaa cctccgtgtt 3000
gtcactacag agccctttgt ctacttgttc agagagattc aggcgatttc atattctaat 3060
cattaggatg tactgtgcag gtctcttctg gttgctaatg ctggtgattg ccgagcagtt 3120
ctttcaaggc gtggtaccgc aattgaaatg tccaaggacc acaggacttg ctgcctcaac 3180
gaaagaaagc gtatagaatc acttggcggc tatgttgatg atggctattt gaatggtcaa 3240
ttagcagtca ctagagcatt gggtgactgg catctcgagg gtctgaaaga agtgggtgag 3300
ccaggtggcc ccttgagcgc ggaaccagag ctcaagatga tcacactgac gaaggaagac 3360
gagttcttga taataggaag cgacggcatc tgggacttct tctcgaacca gaatgccgtg 3420
gatttcacca ggaagaggct ccaagagcac aatgacttga ggttgtgctg caagcagatc 3480
gtcgaggagg caataaggcg aggggcctca gacaacctaa cggcggtgat ggtctcattc 3540
caccaggagg cccctcctca gctcagagtg aacaggacgg ggagggtcga gcggagcata 3600
tcggctgaag ggcttcacag cctcagggtg ctcctggaag gccaatgata gctccactgc 3660
tatcatcagc cttgaagttg tgcagaattg aagagaaacc ttatcttgat gttgtgcaga 3720
attgaagagc aactgtattt agtgattttc ggcatcttgc tgtttacccc caatccattg 3780
tcgacattcc agtgtgtgtt attgtgtgag aataaaacca acgaacctga gatctcacct 3840
tgcattgtga ggcccaggca ttgtaaataa gttgggagat agtaggtaag gagtgtgagt 3900
agtgtgcatc cagggggctt tgaggctcat tatgcgatgt attcccctgt catctgtaaa 3960
aaaataaata ccgggattgt tagtgtgatt aaaagttcta tcgtgtgatg aatacctctc 4020
tgactgttca 4030
<210> 4
<211> 391
<212> DNA
<213> Oryza sativa Japonica Group
<400> 4
cgccttcgct taataatctc ctcctccgta ttaaactcgg tttttttaga tattcccaag 60
aatctctcct ctcctttcca ttgttgctcc gctcggattc cgctcttctt cttcttctcc 120
gtctccgatt cggtctcctc ctcctctgct ccgaccagga ttctgcagcg gcagcggcag 180
ctgctgcggt tggtttttca ggctcggtgg cggcaggcag cggcatgtgc gtggaggaac 240
tcgaaggcgc agagaggctc gattttggtg gggttgcgga gctcgagaca acaccggcag 300
acttcgagat ggagaaagtt tgtgagaaca cagtatctct tgatttcaag caggctaggt 360
cgagcagttt tgtcccagtt atccgatcag g 391
Claims (3)
1.一种培育转基因植物的方法,包括如下步骤:抑制出发植物中osdbp1基因的表达,得到耐冷性增强的转基因植物;所述osdbp1基因为编码OsDBP1蛋白的基因;所述OsDBP1蛋白为序列表中序列1所示的蛋白质;所述出发植物为水稻。
2.用于抑制osdbp1基因表达的物质在培育耐冷植物中的应用;所述osdbp1基因为编码OsDBP1蛋白的基因;所述OsDBP1蛋白为序列表中序列1所示的蛋白质;所述植物为水稻。
3.OsDBP1蛋白在调控植物耐冷性中的应用;所述OsDBP1蛋白为序列表中序列1所示的蛋白质;所述植物为水稻。
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