CN114634940B - 一种玉米类钙调磷酸酶b蛋白基因及其在提高植物耐旱性和耐盐性的应用 - Google Patents
一种玉米类钙调磷酸酶b蛋白基因及其在提高植物耐旱性和耐盐性的应用 Download PDFInfo
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Abstract
本发明提供一种玉米类钙调磷酸酶B蛋白基因、其编码的蛋白及用途，所述玉米类钙调磷酸酶B蛋白基因的序列如SEQIDNO.1所示，将所述的基因转入其他植物中，得到转基因植株，可提高植物的耐旱性和耐盐性。本发明提供的玉米类钙调磷酸酶B蛋白基因是一种新的提高植物耐旱性和耐盐性的基因，为改良耐旱性和耐盐性较差的玉米品种提供了新的思路，也为其他作物利用异源基因技术提高耐旱性和耐盐性提供了理论支持，可用于植物分子育种，能够解决传统选育的效率低和周期长的难题。
Description
技术领域
本发明属于分子生物学技术领域，涉及一种玉米类钙调磷酸酶B蛋白基因及其在提高植物耐旱性和耐盐性的应用。
背景技术
干旱是影响植物生产的主要环境因素。如何提高植物抗旱并促进相关的分子育种是植物抗逆研究领域的关键问题。作为分子定向育种，功能基因是其关键，由于抗旱机制的复杂性，不断挖掘新的功能基因就成为利用转基因开展分子育种的前提。
玉米是世界重要的粮食作物之一，是一种耐旱作物，因此，研究玉米的耐旱、耐盐基因，有助于从中挖掘新的功能基因，为未来的转基因分子育种提供可用的储备基因。
发明内容
本发明的目的是提供一种玉米类钙调磷酸酶B蛋白基因及其在提高植物耐旱性和耐盐性的应用，该基因具有提高植物耐旱性和耐盐性的功能。
上述玉米类钙调磷酸酶B蛋白基因，所述基因的序列如SEQ ID NO.1所示，具有585个碱基，其编码的氨基酸序列如SEQ ID NO.2所示。
本发明还提供了一种表达载体，其含有所述的玉米类钙调磷酸酶B蛋白基因；所述表达载体如pET-28a、pCAMBIA2301、pSP72、pROKII、pBin438、pCAMBIA1302、pCAMBIA1301、pCAMBIA1300、pBI121、pCAMBIA1391-Xa或pCAMBIA1391-Xb等。
本发明还提供一种宿主细胞，其含有所述的表达载体转化的原核细胞或真核细胞。
本发明的另一个目的是提供上述玉米类钙调磷酸酶B蛋白基因在提高植物耐旱性和耐盐性中的用途。
本发明还提供了一种提高植物耐旱性和耐盐性的方法，将SEQ ID No.1所示的玉米类钙调磷酸酶B蛋白基因构建重组表达载体导入受体植物中，获得表达玉米类钙调磷酸酶B蛋白基因的转基因植物。
其中，所述重组表达载体为载体pCAMBIA1301，重组表达载体pCAMBIA1301的构建方法是将NocⅠ和PmlⅠ识别位点间的序列替换为SEQ ID No.1所示的DNA序列。
其中，所述重组表达载体可通过使用农杆菌介导、Ti质粒、植物病毒载体、直接DNA转化、微注射、电穿孔等常规生物技术方法导入植物细胞或组织。
其中，所述方法还包括从导入SEQ ID No.1所示的基因的受体植物中筛选所述玉米类钙调磷酸酶B蛋白基因表达的植物，得到转基因植物的步骤。
其中，所述转基因植物理解为不仅包含将所述基因转化受体植物得到的第一代转基因植物，也包括其子代。对于转基因植物，可以在该物种中繁殖该基因，也可用常规育种技术将该基因转移进入相同物种的其它品种，特别包括商业品种中。所述转基因植物包括种子、愈伤组织、完整植株和细胞。
本发明的有益效果
本发明提供了一种玉米类钙调磷酸酶B蛋白基因、该基因编码的蛋白及其用于提高植物耐旱性和耐盐性的用途，通过将基因导入受体植物中，获得转基因植物，转基因植物的耐旱性和耐盐性都得到了提高。本发明提供的玉米类钙调磷酸酶B蛋白基因是一种新的提高植物耐旱性和耐盐性的基因，为改良耐旱性和耐盐性较差的玉米品种提供了新的思路，也为其他作物利用异源基因技术提高耐旱性和耐盐性提供了理论支持，可用于植物分子育种，能够解决传统选育的效率低和周期长的难题。
附图说明
图1为筛选抗性拟南芥的实验图；
在图1中，A为在含霉素MS培养基上筛选抗性拟南芥转化植株，B为PCR扩增鉴定转基因株的PCR产物电泳图，C为荧光定量PCR检测抗性拟南芥转基因株中的玉米类钙调磷酸酶B蛋白基因的表达。
图2为基因功能鉴定结果图；
在图2中，A为表达玉米类钙调磷酸酶B蛋白基因的转基因拟南芥植株在含有甘露醇的MS固体培养基上的表型，B为表达玉米类钙调磷酸酶B蛋白基因的转基因拟南芥植株在添加3％PEG6000的盆栽上的表型，C为表达玉米类钙调磷酸酶B蛋白基因的转基因拟南芥植株在含有NaCl的MS固体培养基上的表型。
具体实施方式
以下实施例进一步说明本发明的内容，但不应理解为对本发明的限制。在不背离本发明精神和实质的情况下，对本发明方法、步骤或条件所作的修改或替换，均属于本发明的范围。
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。下述实施例中所使用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1玉米类钙调磷酸酶B蛋白基因序列的克隆
1、根据拟南芥类钙调磷酸酶B蛋白基因信息在玉米基因组中筛选同源玉米类钙调磷酸酶B蛋白基因
从拟南芥数据库(https://www.arabidopsis.org/)下载拟南芥类钙调磷酸酶B蛋白基因(AT5G24270.1)氨基酸序列。从Phytozome 13(https://phytozome-next.jgi.doe.gov/)网站下载的玉米V4版参考基因组(Zea mays RefGen_V4)中所有蛋白序列。利用TBtools软件将AT5G24270.1比对至V4版参考基因组中所有的蛋白的氨基酸序列，获得玉米基因组中AtCBL4的同源序列，再利用PfamScan(https://www.ebi.ac.uk/Tools/pfa/pfamscan/)在线网站筛选出具有3个EF-hand(PF00036)保守基序的同源基因。在Zea mays RefGen_V4基因组注释文件中检索AT5G24270.1进一步校验，最后得获得玉米类钙调磷酸酶B蛋白基因(Zm00001d053293_T001)信息。
2、合成克隆玉米类钙调磷酸酶B蛋白基因
从maizeGDB(https://www.maizegdb.org/)上下载玉米类钙调磷酸酶B蛋白基因(Zm00001d053293_T001)的CDS序列如SEQ ID NO.1所示，进行人工合成克隆。该基因序列具有585个碱基，编码的蛋白长为194个氨基酸残基，其序列如SEQ ID NO.2所示，具有3个EF-hand(PF00036)保守基序。
实施例2基因功能鉴定
1、玉米类钙调磷酸酶B蛋白基因转入拟南芥植株
将玉米类钙调磷酸酶B蛋白基因的cDNA的完整放阅读框序列克隆到植物转基因表达载体Pcambia-1301Vector的NocⅠ和PmlⅠ酶切位点之间，得到重组表达载体。
利用热激法将重组表达载体导入根癌农杆菌菌株LB4404，获得携带玉米类钙调磷酸酶B蛋白基因CDS的转化菌株。
以蘸花转化法将玉米类钙调磷酸酶B蛋白基因导入哥伦比亚(Columbia)拟南芥，将收获的拟南芥种子在含有25mg/L潮霉素MS培养基上、16h光照/8h黑暗光周期条件下筛选具有潮霉素抗性的T0转化株(图1-A)，然后利用盆栽方法放置于常规条件的温室中培养至结种，收获T1代转化株种子。
以具有潮霉素抗性的T0代拟南芥植株的基因组DNA为模板，利用玉米类钙调磷酸酶B蛋白基因特异性引物进行PCR扩增验证(图1-B)，特异性引物的上游引物序列为：3’-acacgggggactcttgaccatATGGGGTGTGTGTCCTCCAA-5’(SEQ ID NO.3)，下游引物序列为：3’-gtcacctgtaattcacacgtgTTACTTGAGGTATGGGAGCGTCA-5’(SEQ ID NO.4)。
利用商业化试剂盒，提取潮霉素抗性拟南芥植株的RNA,再反转录合成第一链cDNA，以第一链cDNA为模板，利用荧光定量PCR分析验证转基因植株中的玉米类钙调磷酸酶B蛋白基因的表达(图1-C)。从图1-C中可以看出，转基因拟南芥中的玉米类钙调磷酸酶B蛋白基因表达量远远高于野生型拟南芥植株和转入空表达载体的拟南芥植株。
经过上述检测分析后，获得异源表达玉米类钙调磷酸酶B蛋白基因的拟南芥转基因植株。
2、功能鉴定和结果
在含有50mM、70mM和150mM的甘露醇(Mannitol)的MS固体培养基上，分别培养T2代表达玉米类钙调磷酸酶B蛋白基因的转基因拟南芥10天，以野生型拟南芥和转入空载体(Pcambia-1301)的拟南芥作为对照。结果表明，与野生型和空载体转化的拟南芥植株相比，表达玉米类钙调磷酸酶B蛋白基因的转基因拟南芥植株的上部生长生长势更好，生物量增加，根系长度也更长(图2-A)。
将T2代表达玉米类钙调磷酸酶B蛋白基因的转基因拟南芥植株种植在含有蛭石的盆中，向盆中浇灌含3％PEG6000的MS液体培养基，培养14天，以野生型拟南芥和转入空载体(Pcambia-1301)的拟南芥作为对照。14天后，表达玉米类钙调磷酸酶B蛋白基因的转基因拟南芥植株比野生型和空载体转化株长势更好(图2-B)。
将T2代表达玉米类钙调磷酸酶B蛋白基因的转基因拟南芥种植在含有50mM、75mM和100mM的NaCl的MS固体培养基上，分别培养10天，以野生型拟南芥和转入空载体(Pcambia-1301)的拟南芥作为对照。与野生型拟南芥和空载体转化的拟南芥植株相比，表达玉米类钙调磷酸酶B蛋白基因的转基因拟南芥植株的根系长度显著增加，特别是在100mM的NaCl条件下更为显著(图2-C)。
这些试验说明了在拟南芥中表达玉米类钙调磷酸酶B蛋白基因后能显著提高拟南芥的耐旱和耐盐能力。
序列表
<110> 广西大学
<120> 一种玉米类钙调磷酸酶B蛋白基因及其在提高植物耐旱性和耐盐性的应用
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Claims (5)
1.一种玉米类钙调磷酸酶B蛋白基因在提高植物耐旱性和耐盐性中的用途，其特征在于，所述玉米类钙调磷酸酶B蛋白基因的序列如SEQ ID NO.1所示，所述植物为玉米或拟南芥。
2.一种利用玉米类钙调磷酸酶B蛋白基因培育耐旱和耐盐植物的方法，其特征在于：将SEQ ID No.1所示的玉米类钙调磷酸酶B蛋白基因构建重组表达载体导入受体植物中，获得表达玉米类钙调磷酸酶B蛋白基因的转基因植物，所述植物为玉米或拟南芥。
3.根据权利要求2所述的方法，其特征在于：所述重组表达载体为载体pCAMBIA1301，重组表达载体pCAMBIA1301的构建方法是将Noc Ⅰ 和Pml Ⅰ 识别位点间的序列替换为SEQ IDNo.1所示的DNA 序列。
4.根据权利要求2所述的方法，其特征在于：所述重组表达载体通过使用农杆菌介导、植物病毒载体、直接 DNA 转化的常规生物技术方法导入植物细胞或组织。
5.根据权利要求2所述的方法，其特征在于：所述方法还包括从导入SEQ ID No.1所示基因的受体植物中筛选玉米类钙调磷酸酶B蛋白基因表达的植物，得到转基因植物的步骤。
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