CN112852833A - 一种唐古特白刺NtCBL1基因及其表达蛋白和应用 - Google Patents
一种唐古特白刺NtCBL1基因及其表达蛋白和应用 Download PDFInfo
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Abstract
本发明公开了一种唐古特白刺NtCBL1基因及其表达蛋白和应用,属于植物基因工程技术领域。本发明公开的唐古特白刺NtCBL1基因,其核苷酸序列如SEQID NO.1所示,其表达蛋白的氨基酸序列如SEQ ID NO.2所示。本发明根据唐古特白刺的耐盐和抗旱等抗逆特性,利用唐古特白刺的叶片组织,在已有的部分转录组数据的基础上,同源克隆了唐古特白刺抗逆相关的CBL基因全长,依据同源基因而命名为NtCBL1。通过纯合NtCBL1基因拟南芥植株的耐盐和抗旱分析,证明了唐古特白刺NtCBL1基因转化植物具备耐盐性和抗旱性,为植物抗逆基因库增加了资源。
Description
技术领域
本发明属于植物基因工程技术领域,更具体地说,涉及一种唐古特白刺NtCBL1基因及其表达蛋白和应用。
背景技术
植物在整个生命周期会遇到各种不利的非生物逆境胁迫,如高盐、干旱、高温、低温、高pH等。植物感受到非生物胁迫后会产生一些机制来避免伤害发生,而其中的关键在于对非生物胁迫的检测。钙离子作为重要的第二信使,普遍存在于真核生物的细胞中,在植物生长发育及响应外界环境胁迫中起关键作用。外界环境刺激,如土壤盐分、干旱、低温、激素、高pH等都能引起细胞中游离的Ca2+浓度在时空上出现特异性的变化。这种Ca2+信号变化会被钙结合蛋白感知并传递给下游的靶蛋白,靶蛋白通过调控下游目的基因从而激活相应的应答途径。到目前为止,在高等植物中主要有钙调蛋白(Calmodulins,CaM)、类钙调蛋白(Calmodulin-like proteins,CML)、钙依赖蛋白激酶(Calcium-dependent proteinkinases,CDPK),类钙调磷酸酶B蛋白(Calcineurin B-like proteins,CBL)这四种Ca2+感受器蛋白家族。而盐胁迫作为最常见的环境胁迫因子之一,引起植物的氧化胁迫、离子胁迫、渗透胁迫,抑制植物的生长发育,甚至可以导致植物的死亡。盐碱化问题已经全球性的生态环境与资源问题,日益受到人们的重视。
1998年CBL首次在拟南芥中被克隆出来,由于与动物的钙调神经磷酸酶B(CNB)和神经元钙传感器(NCS)同源,由此被命名为钙调神经磷酸酶B亚基蛋白。类钙调磷酸酶B亚基蛋白CBL是一个多基因家族钙离子感受器,可以检测到钙离子的时空变化。研究表明,CBL基因家族在不同植物中的数目是不等的,拟南芥、水稻均有10个,黄瓜有6个,茶树有7个。CBL通过其特异性靶蛋白丝氨酸/苏氨酸蛋白激酶CIPK互作构成CBL-CIPK信号系统在植物应答外界环境刺激中起重要作用。
唐古特白刺是蒺藜科(Zygophyllaceae)白刺属(Nitraria L.)的一种盐生植物,为我国所特有,主要分布在河西走廊干旱盐渍化地区,具有耐干旱、耐盐碱、防风固沙等特点,在治理土壤荒漠化和盐碱化中发挥着重要作用。唐古特白刺含有大量的营养成分和药用成分,具有药用价值和经济价值。但是唐古特白刺的研究集中在生理生化分析、成分的提取、优良家系的选择上,对于从分子水平上阐述耐盐机制的研究较少。
发明内容
针对现有技术存在的上述问题,本发明所要解决的技术问题在于提供一种唐古特白刺NtCBL1基因。本发明所要解决的另一技术问题在于提供所述唐古特白刺NtCBL1基因在提高植物耐盐性或抗旱性中的应用。
为了解决上述技术问题,本发明所采用的技术方案如下:
一种唐古特白刺NtCBL1基因,其核苷酸序列如SEQ ID NO.1所示。
所述的唐古特白刺NtCBL1基因的表达蛋白,其氨基酸序列如SEQ ID NO.2所示。
含有所述的唐古特白刺NtCBL1基因的载体、重组菌或宿主细胞。
进一步地,所述的载体为植物重组表达载体。
进一步地,所述的载体为pBI121+NtCBL1。
进一步地,NtCBL1基因的启动子是35S。
所述的唐古特白刺NtCBL1基因在提高植物耐盐性或抗旱性中的应用。
所述的唐古特白刺NtCBL1基因在提高植物耐盐性或抗旱性中的应用,包括以下步骤:
1)构建唐古特白刺NtCBL1基因的重组载体;
2)将所构建的唐古特白刺NtCBL1基因的重组载体转化到植物细胞中;
3)培育筛选,得到具有耐盐性或抗旱性的植株。
相比于现有技术,本发明的有益效果为:
本发明在已有的部分转录组数据的基础上,同源克隆了唐古特白刺抗逆相关的CBL基因全长,依据同源基因命名为NtCBL1,其核苷酸序列如SEQ ID NO.1所示,其表达蛋白的氨基酸序列如SEQ ID NO.2所示。在正常培养环境中,唐古特白刺NtCBL1基因过表达的拟南芥T3代纯合植株的生长发育与野生型无明显差异。但通过纯合NtCBL1基因拟南芥植株的耐盐和抗旱分析,显示盐胁迫和干旱胁迫下,过表达NtCBL1植株比WT植株在同一时期萌发率更高,根更长,叶片数量更多,根的数量也更多,证明了唐古特白刺NtCBL1基因转化植物具备良好的耐盐性和抗旱性,NtCBL1基因的公开为植物抗逆基因库增加了资源,对于提高植物的耐盐和耐寒的研究具有重要意义和价值。
附图说明
图1为唐古特白刺NtCBL1基因的PCR扩增电泳结果图;注:M:DL2000DNA marker;1:PCR扩增产物;
图2为NtCBL1蛋白结构示意图;
图3为NtCBL1与其它植物CBL1蛋白的多序列比对分析图;
图4为CBL进化树分析图;
图5为NtCBL1基因转化拟南芥原生质的亚细胞定位结果图;
图6为在400mmol/L NaCl盐胁迫条件下NtCBL1基因表达分析结果图;
图7为200mm NaCl盐胁迫处理下过表达转基因拟南芥与野生型拟南芥表型图。
具体实施方式
下面结合具体实施例对本发明进一步进行描述。
实施例1:
(一)材料与处理
将唐古特白刺种子用沙土4℃藏2至6个月,取出后或用发芽盒萌发,待种子长出两片子叶时将其种到土壤中。种植土壤以营养土:珍珠岩:蛭石为5∶1∶1进行配制,每隔四天浇一次水。种植白刺六周后,选择长势一致、生长良好的唐古特白刺并随机分成三组,每组6株。0mmol/L NaCl为对照,设置400mmol/L NaCl进行盐胁迫处理。对照组正常浇水,处理组浇灌盐溶液,漏于托盘中的水或盐溶液倒回盆中。分别在盐胁迫时间为0、1、3、6h剪取白刺叶片,用液氮速冻,于-80℃超低温冰箱内储存备用。
(二)唐古特白刺总RNA的提取和反转录
根据Bioteke RNA多糖多酚型植物试剂盒提取唐古特白刺的总RNA,再参照 III 1st Strand cDNA反转试剂盒(Vazyme)进行RNA反转录为cDNA。为了尽量减少RNA酶的污染,进行试验所需的研钵、枪头、离心管等均用1%的DEPC水浸泡且高压灭菌后使用,总RNA的提取和反转录均在超净台内操作。
(三)唐古特白刺NtCBL1基因的克隆
根据本实验室已有的部分唐古特白刺转录组数据(未发表),进行同源基因比对后找出目的基因,使用Oligo7.0设计特异性引物,将引物命名为NtCBL1-F、NtCBL1-R,如表1所示。将反转得到的cDNA为模板,对目的CBL1片段进行PCR反应扩增。1%琼脂糖凝胶电泳检测目的片段,切胶回收后的纯化产物与pMD19-T Vector连接,在转化到DH5α感受态细胞中,筛选得到的阳性克隆菌液送到公司测序。
表1实验所用引物序列
引物名称 | 引物序列(5′-3′) |
NtCBL1-F | 5′-ATGGGGTGTTTTCAGTCAAA-3′ |
NtCBL1-R | 5′-TTATGTGGCAAGCTCATCCA-3′ |
qCBL1-F | 5′-AGCGTGAAGAGGTCAAGCAA-3 |
qCBL1-R | 5′-ACCTGGTTTGCATCGGCTT-3′ |
β-Actin-F | 5′-CTTATAACGCTATCATAGGGCGTCTGAGC-3′ |
β-Actin-R | 5′-TCGGTCAATATCTAGCCATGCGGG-3′ |
gNtCBL1-F | 5′-ttccaccatggcgtgcaggtcgactctagaATGGGGTGGTTTTCAGTCAAAGGT-3′ |
gNtCBL1-R | 5′-cagctcctcgcccttgctcaccatggatccTGTGGCAAGCTCATCCACTTCT-3′ |
NtC1-121-F | 5′-gaacacgggggactctagaggatccccggggATGGGGTGTTTTCAGTCAAAGG-3′ |
NtC1-121-R | 5′-ttgaacgatcggggaaattcgagctcTGTGGCAAGCTCATCCACTTCT-3′ |
35S-F | 5′-GACGCACAATCCCACTATCC-3′ |
以唐古特白刺叶片cDNA为模板,设计特异性引物NtCBL1-F、NtCBL1-R进行PCR扩增,获得与预期条带大小一致的扩增条带(图1)。测序得到的NtCBL1基因的最大开放阅读框长642bp,具体序列如SEQ ID NO.1所示,所表达的蛋白序列如SEQ ID NO.2所示。
利用http://web.expasy.org/网址中的ProtParam工具对编码蛋白质的理化性质进行预测,得出NtCBL1蛋白质相对分子量(MW)为52.77kD;理论等电点(pI)5.21,分子式为C1971H3302N642O836S106。脂肪系数为30.37,平均亲水系数为0.657,预测其为疏水蛋白。蛋白质不稳定指数为32.78,被认定为稳定性蛋白。
利用SMART(http://smart.embl-heidelberg.de/)在线软件分析,结果如图2所示显示,NtCB上1蛋白具有3个EF手型结构域,含有Ca2+binding位点,是钙离子结合蛋白,可进行钙信号传导。
(四)CBL基因的生物信息学分析
在NCBI上下载其他CBL1蛋白序列(拟南芥AAC26008.1,毛果杨ABO43660.1,木薯AXM42381.1,大麻KAF4362542.1,番茄NP_001239047.1,胡杨ABC67906.1),再用DNAMAN软件对NtCBL1蛋白与其他植物的CBL1蛋白的氨基酸序列进行比对,如图3显示,NtCBL1蛋白与其他蛋白的同源相似性在80%至90%之间。其中,NtCBL1蛋白与毛果杨PtCBL1蛋白的同源相似性最高,达到88.26%,与拟南芥的则为83.1%。这些结果表明,NtCBL1所编码的蛋白序列与其他植物的同源性较高且它们均含有豆蔻酰化结构域(MGXXXS/T)。
本申请用MEGA-X软件,采用邻接法(neighbor-joining,NJ)对氨基酸序列构建系统发生树,Bootstrap设置为1000,如图4所示,进化树主要聚类成两大支,唐古特白刺NtCBL1和拟南芥AtCBL1、AtCBL9的同源性最高,它们的亲缘关系最近。综上表明,所克隆得到的唐古特白刺NtCBL1基因为CBL基因家族,编码唐古特白刺类钙调磷酸酶B亚基蛋白。
(五)唐古特白刺NtCBL1基因的亚细胞定位
利用XbaI与BamhI酶切位点对pJIT166质粒对进行双酶切,根据NtCBL1的ORF设计引物gNtCBL1-F、gNtCBL1-R(表1)。利用同源重组的方法构建pJIT166-NtCBL1-GFP融合表达载体,以哥伦比亚col拟南芥叶片制备原生质体,参照PEG介导法(Yoo S,Cho Y H,SheenJ.Arabidopsis mesophyll protoplasts:a versatile cell system for transientgene expression analysis[J].Nature Protocols,2007,2(7):1565-1572.)进行目的基因的亚细胞定位。
亚细胞定是确定某种植物蛋白作用位置的一种重要方法。ProtComp 9.0在线软件对NtCBL1蛋白进行亚细胞定位预测,结果显示其定位在质膜上。通过PEG介导法对唐古特白刺的NtCBL1基因进行亚细胞定位分析,如图5所示,对照组PJIT166的GFP分布于整个原生质体中,经过明场和荧光场叠加一起表明,空载PJIT166在细胞核、细胞质、质膜都有表达。而NtCBL1主要在质膜在发出荧光,说明这NtCBL1基因翻译的蛋白可能在质膜上发挥作用。
(六)唐古特白刺NtCBL1基因的表达分析
以不同盐胁迫条件下不同时间处理的唐古特白刺叶为材料,提取RNA并将反转的cDNA为实时荧光定量PCR的模板。在NBCI在线设计荧光引物qCBL1F、qCBL1R,内参引物为β-Actin-F、β-Actin-R(表1)。采用2-ΔΔCT(J L K,D S T.Analysis of relative geneexpression data using real-time quantitative PCR and the 2(-Delta Delta C(T))Method.[J].Methods,2001,25(4):402-408)计算基因的相对表达量,数据结果利用Excel进行分析。
为了研究NtCBL1在不同盐浓度胁迫下的表达情况,以β-Actin为内参基因,根据NtCBL1基因序列设计qCBL1-F/R荧光引物,利用实时荧光定量PCR技术进行检测(图6)。结果显示,400mmol/L盐胁迫处理下NtCBL1基因的表达发生变化,基因表达量在1h、3h、6h均表现上调。在400mmol/L NaC1盐胁迫下,NtCBL1基因的表达量随着处理时间先增高加后下降,在3h时表达量最高,为ck组的3.55倍,1h、6h分别上调了1.68、1.56倍。
(七)唐古特白刺NtCBL1基因功能验证
构建唐古特白刺NtCBL1过表达载体,并将其转入农杆菌菌株,通过农杆菌介导法转化拟南芥花序,获得过表达NtCBL1基因的阳性转基因植株,观察表型,推测NtCBL1功能。
(1)质粒的获取
取出用甘油保存于-80℃的pBI121大肠杆菌,将其划板活化,倒置37℃培养12h,挑取单菌落加入kan的LB液体培养基中扩大培养。通过菌液PCR确认为阳性后,再进行质粒的提取。按照高纯度质粒大提试剂盒(天根)提取。
(2)基因片段添加末端同源序列
设计带有Xma1、Sac1酶切位点的引物,所用引物序列见表1,以带有NtCBL1基因的克隆载体pMD-19T质粒为模板,通过PCR反应取得添加粘性末端的的基因片段,PCR产物通过1%琼脂糖凝胶电泳检测正确后,对正确条带进行切胶回收。
(3)pBI121质粒双酶切
通过紫外线分光光度计和琼脂糖凝胶电泳对提取的pBI121质粒进行浓度和大小的检测,使用NEB在线软件查询XmaI、SacI双酶切反应体系,具体连接反应体系为:pBI1212g,10×CutSmart Buffer 5μL,XmaI 1μL,SacI 1μL,加ddH2O至50μL。反应程序为:37℃15min,4℃∞。
(4)NtCBL1目的基因与pBI121载体的连接
将已添加末端同源序列的NtCBL1目的基因片段进行切胶回收,再与同样切胶回收得到的pBI121双酶切产物进行连接,按照NEB Gibson assembly master mix试剂进行同源重组反应,具体反应体系为:Gibson Assembly Master Mix 10μL,NtCBL1 2μL,PBI121 2μL,ddH2O补足至20μL。反应条件为:50℃50min,4℃∞。
将连接产物转化至DH5α大肠杆菌感受态细胞(天根)中,然后均匀的涂布于含有Kan抗性的LB培养基上,37℃倒置过夜培养。挑取单克隆菌落培养后用2×T5进行菌液PCR验证,验证完后将菌液送至擎科生物进行测序,最后将测序正确的pBI121-NtCBL1重组质体保存在-80℃备用。
(5)重组载体转化农杆菌
从含有pBI121-NtCBL1重组载体的大肠杆菌中提取质粒,将质粒转化至GV3101农杆菌(FCNCS)中,转化步骤如下:
1、取出GV3101农杆菌置于冰上融化,加入pBI121-NtCBL1重组质粒后轻弹混匀,于冰上静置5min,迅速转移至液氮中速冻5min。
2、37℃热水浴中热激40~70s,立刻在冰上静置2min。
3、加入500μL无抗LB液体培养基,于28℃200rpm摇床中复苏4h。
4、1200rpm离心30s,吸走500 2μL上清液,余下的100μL用移液枪吹打重悬,加到Kan抗性的LB平板上,待平板上的液体完全被吸收后,28℃倒置培养24~48h。
(6)拟南芥的培养
拟南芥种子消毒操作步骤如下:
1、取适量拟南芥种子放入灭过菌的1.5mL离心管中,配制好的1%NaClO灭菌大约12min。
2、倒掉液体,用无菌水清洗种子3~5次,彻底清洗次氯酸钠溶液,倒掉无菌水以1mL 0.1%无菌琼脂糖使种子重悬。
3、将拟南芥种子均匀的点播到添加了卡那霉素的1/2MS固体培养基上,置于4℃冰箱避光春化2~3天。
从4℃冰箱中取出种子,放至23℃16h光照8h黑暗的培养箱中培养,培养10~18天后移栽至营养土中继续生长,剪去主枝,诱导更多的侧枝生长。当生长出多个花序时,用于转化实验。
(7)农杆菌侵染液的制备
从-80℃冰箱中取出用甘油保存的农杆菌,用烧好的细铁丝蘸取少量菌液划在Kan抗性的LB平板上,进行活化。28℃倒置培养24~36h,挑取单克隆菌落于已经加过800μL Kan抗性的LB液体培养基的1.5mL离心管,在28℃250rpm摇床上震荡培养12~16h。吸取60μL菌液与3mL LB液体培养基于10mL离心管中继续培养12~16h,摇好后直接将10mL离心管中的菌液和量好的50mL LB液体培养基倒入250mL三角瓶内,震荡培养4~6h,使得OD600的吸收光度值在0.6~0.8之间。将浓度适宜的农杆菌菌液在5000rpm下室温离心10min,倒掉上清液,菌体重悬于5%蔗糖的1/2MS液体培养基,之后添加0.05%silwet-77,充分混匀使其产生丰富的泡沫。
(8)拟南芥的侵染转化
侵染前一天将拟南芥浇透水,使其充分吸水。侵染当天,选取健壮的拟南芥用于侵染实验。将选取的拟南芥荚果和已开放的花朵剪掉,然后将花序浸没在农杆菌侵染液中,EHA105农杆菌感受态(唯地)侵染时间为15~25s,GV3101农杆菌感受态(FCNCS)侵染时间为30~45s。全部浸泡完成后,将拟南芥平放于托盘中,用黑色塑料袋罩住并封口,在塑料袋上扎几个小口使拟南芥透气,在常温下黑暗培养1~2天。取下塑料袋,将拟南芥扶正放至光照培养箱中培养,直至收获到侵染的种子。收获种子后于37℃烘箱干燥,短期保存烘3天,长期保存烘7~10天。
(9)阳性转基因植株的筛选与鉴定
将干燥好的种子经过灭菌(方法同上文)后,点播在含50mg/L Kan的1/2MS培养基上,4℃黑暗春化2~3天,转移到光照培养箱中培养,非转基因植株在筛选压下无法正常生长直至死亡,而能够正常生长出根茎叶的植株长出4片叶子后,转移到营养土中,置于光照培养箱中培养,每星期浇水2~3次。拟南芥长大后剪取叶片,用CTAB法提取DNA,具体步骤如下:
1、取叶片装入2mL的离心管中,加入一个大两个小的瓷珠,用液氮速冻。球磨仪把叶片彻底打碎,加入600mL CTAB,涡旋混匀。
2、65℃水浴或空气浴裂解15~20min,每隔5min震荡混匀。
3、在通风橱中加入等体积的24∶1(或者25∶24∶1也就是tris平衡酚:三氯甲烷:异戊醇),大约为700μL。震荡混匀,12,000rpm室温离心5min。(注意:25∶24∶1需要现配现用,推荐使用24∶1,可以配制好后存放。)
4、吸取上清液(大约500μL)于1.5mL灭菌离心管中,加入1.5倍体积的冰乙酸,大约为750mL(或者2/3异丙醇),置于4℃冰箱中30min,彻底沉淀。
5、12,000rpm室温离心5min,弃上清液。75%乙醇清洗两次,混匀离心1min,弃液,倒扣在面纸上使其吸干,DNA不溶于酒精。
6、加入50~100μL ddH2O溶解DNA,-20℃保存备用。
(10)转基因植株检验
以提取的基因组DNA模板,使用35S-F为上游引物,NtCBL1-121-R或者NtCBL1-R为下游引物进行PCR扩增反应,电泳检测拟南芥是否为转基因植株。
(11)T2代NtCBL1转基因拟南芥抗盐实验
用拟南芥种子萌发9天后,将野生型拟南芥和转基因植株转移到营养土中,放在23℃16h光照8h黑暗的培养箱中培养,每星期浇2~3次水。在土壤中培养10天后,用200mmol/LNaCl进行浇灌,一共处理7天。NaCl处理第一天,野生型拟南芥叶片开始表现出轻微的萎蔫,转基因植株没有明显的变化;第三天,野生型拟南芥叶片开始变黄,而转基因植株开始出现萎蔫状态;第五天后,野生型拟南芥叶片萎蔫严重且叶片大面积变黄,而转基因植株叶片颜色开始变黄;第七天后,野生型拟南芥枯死,转基因植株叶片部分变黄。拟南芥的盐胁迫试验结果进一步证明了NtCBL1增强植株耐盐性的功能。
序列表
<110> 南京林业大学
<120> 一种唐古特白刺NtCBL1基因及其表达蛋白和应用
<130> 100
<160> 13
<170> SIPOSequenceListing 1.0
<210> 1
<211> 642
<212> DNA
<213> Nitraria tangutorum
<400> 1
atggggtgtt ttcagtcaaa ggtagcaagg cagcatcctg ggtatgagga cccagttgtt 60
ctggcttcgc aaactgcgtt tagtgttagt gaagttgaag ccttgtttga gctattcaag 120
agcattagcg gttctctcat tgacgatggg ttaataagca aggaagagtt tcagttggct 180
ctcttcaaaa acagaaagaa ggaaaatcta tttgccaaca ggctctttga actttttgat 240
gtgaagcgaa aggggctcat tgattttagt gattttgtta gatcactaaa tgtttttcat 300
ccaaatgctt ctcaagagga caagattgac ttctcattta ggctatatga tcaagataat 360
acaggatata tcgagcgtga agaggtcaag caaatgctga ttgcacttct atgtgaatct 420
gaaatgaagc tggctgatga aacgattgaa gtaatactcg ataagacttt cttggaagcc 480
gatgcaaacc aggatggcaa aatagataaa acagaatggc agaactttgt ttctaaaaac 540
ccatcgttgt tgaagatcat gacccttcct tatttgaggg acataacgac gacttttccc 600
agttttgttt ttaattcaga agtggatgag cttgccacat aa 642
<210> 2
<211> 213
<212> PRT
<213> Nitraria tangutorum
<400> 2
Met Gly Cys Phe Gln Ser Lys Val Ala Arg Gln His Pro Gly Tyr Glu
1 5 10 15
Asp Pro Val Val Leu Ala Ser Gln Thr Ala Phe Ser Val Ser Glu Val
20 25 30
Glu Ala Leu Phe Glu Leu Phe Lys Ser Ile Ser Gly Ser Leu Ile Asp
35 40 45
Asp Gly Leu Ile Ser Lys Glu Glu Phe Gln Leu Ala Leu Phe Lys Asn
50 55 60
Arg Lys Lys Glu Asn Leu Phe Ala Asn Arg Leu Phe Glu Leu Phe Asp
65 70 75 80
Val Lys Arg Lys Gly Leu Ile Asp Phe Ser Asp Phe Val Arg Ser Leu
85 90 95
Asn Val Phe His Pro Asn Ala Ser Gln Glu Asp Lys Ile Asp Phe Ser
100 105 110
Phe Arg Leu Tyr Asp Gln Asp Asn Thr Gly Tyr Ile Glu Arg Glu Glu
115 120 125
Val Lys Gln Met Leu Ile Ala Leu Leu Cys Glu Ser Glu Met Lys Leu
130 135 140
Ala Asp Glu Thr Ile Glu Val Ile Leu Asp Lys Thr Phe Leu Glu Ala
145 150 155 160
Asp Ala Asn Gln Asp Gly Lys Ile Asp Lys Thr Glu Trp Gln Asn Phe
165 170 175
Val Ser Lys Asn Pro Ser Leu Leu Lys Ile Met Thr Leu Pro Tyr Leu
180 185 190
Arg Asp Ile Thr Thr Thr Phe Pro Ser Phe Val Phe Asn Ser Glu Val
195 200 205
Asp Glu Leu Ala Thr
210
<210> 3
<211> 20
<212> DNA
<213> NtCBL1-F(Artificial)
<400> 3
atggggtgtt ttcagtcaaa 20
<210> 4
<211> 20
<212> DNA
<213> NtCBL1-R(Artificial)
<400> 4
ttatgtggca agctcatcca 20
<210> 5
<211> 20
<212> DNA
<213> qCBL1-F(Artificial)
<400> 5
agcgtgaaga ggtcaagcaa 20
<210> 6
<211> 19
<212> DNA
<213> qCBL1-R(Artificial)
<400> 6
acctggtttg catcggctt 19
<210> 7
<211> 29
<212> DNA
<213> β-Actin-F(Artificial)
<400> 7
cttataacgc tatcataggg cgtctgagc 29
<210> 8
<211> 24
<212> DNA
<213> β-Actin-R(Artificial)
<400> 8
tcggtcaata tctagccatg cggg 24
<210> 9
<211> 54
<212> DNA
<213> gNtCBL1-F(Artificial)
<400> 9
ttccaccatg gcgtgcaggt cgactctaga atggggtggt tttcagtcaa aggt 54
<210> 10
<211> 52
<212> DNA
<213> gNtCBL1-R(Artificial)
<400> 10
cagctcctcg cccttgctca ccatggatcc tgtggcaagc tcatccactt ct 52
<210> 11
<211> 53
<212> DNA
<213> NtC1-121-F(Artificial)
<400> 11
gaacacgggg gactctagag gatccccggg gatggggtgt tttcagtcaa agg 53
<210> 12
<211> 48
<212> DNA
<213> NtC1-121-R(Artificial)
<400> 12
ttgaacgatc ggggaaattc gagctctgtg gcaagctcat ccacttct 48
<210> 13
<211> 20
<212> DNA
<213> 35S-F(Artificial)
<400> 13
gacgcacaat cccactatcc 20
Claims (8)
1.一种唐古特白刺NtCBL1基因,其核苷酸序列如SEQ ID NO.1所示。
2.权利要求1所述的唐古特白刺NtCBL1基因的表达蛋白,其氨基酸序列如SEQ ID NO.2所示。
3.含有权利要求1所述的唐古特白刺NtCBL1基因的载体、重组菌或宿主细胞。
4.根据权利要求3所述的唐古特白刺NtCBL1基因的载体,其特征在于,所述的载体为植物重组表达载体。
5.根据权利要求4所述的植物重组表达载体,其特征在于,所述的载体为pBI121+NtCBL1。
6.根据权利要求5所述的pBI121+NtCBL1,其特征在于,NtCBL1基因的启动子是35S。
7.权利要求1所述的唐古特白刺NtCBL1基因在提高植物耐盐性或抗旱性中的应用。
8.根据权利要求7所述的唐古特白刺NtCBL1基因在提高植物耐盐性或抗旱性中的应用,其特征在于,包括以下步骤:
1)构建唐古特白刺NtCBL1基因的重组载体;
2)将所构建的唐古特白刺NtCBL1基因的重组载体转化到植物细胞中;
3)培育筛选,得到具有耐盐性或抗旱性的植株。
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Non-Patent Citations (2)
Title |
---|
NCBI REFERENCE SEQUENCE: XM_006477006.3: "PREDICTED: Citrus sinensis calcineurin Blike protein 1 (LOC102622664), transcript variant X1, mRNA", 《NCBI》 * |
YONG HWA CHEONG: "CBL1, a Calcium Sensor That Differentially Regulates Salt, Drought, and Cold Responses in Arabidopsis", 《THE PLANT CELL》 * |
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