CN108864264A - 玉米OXS2a基因、其编码蛋白及应用 - Google Patents
玉米OXS2a基因、其编码蛋白及应用 Download PDFInfo
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- CN108864264A CN108864264A CN201710330006.9A CN201710330006A CN108864264A CN 108864264 A CN108864264 A CN 108864264A CN 201710330006 A CN201710330006 A CN 201710330006A CN 108864264 A CN108864264 A CN 108864264A
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Abstract
本发明公开了玉米OXS2家族中的OXS2a基因、其编码蛋白及应用。玉米OXS2a的氨基酸序列如SED ID NO.2所示,其编码基因如SEQ ID NO.1所示。本发明所克隆到的玉米OXS2a可以被高温所诱导。酵母抗性检测表明,这个基因的异源表达可增强酵母抗氧化剂二酰胺的能力。当该基因在拟南芥中大量表达时,可以增强拟南芥抗高温的能力。因此,该基因具有极大的潜力应用于培育具有抗高温的转基因作物中,从而提高粮食作物的产量。
Description
技术领域
本发明属于分子生物学领域,具体涉及玉米OXS2a基因、其编码蛋白及应用。
背景技术
全球气候变暖,导致我国许多作物产区气温逐年升高。在干燥的季节夜间气温每升高1℃将导致水稻籽粒产量下降10%。小麦、玉米和大麦等主要农作物的产量也受全球气候变暖的严重影响。气温的升高影响作物的育性及结实率,导致作物产量下降;同时影响物质的积累,导致有机物积累比例发生变化,影响到作物品质。不同作物的不同生长阶段对气温有不同需求。气温的升高,一方面可能导致作物生长期变长,尤其在营养充足的条件下增加灌浆期,减缓衰老并增加作物产量;但另一方面,气温上升也可能在作物生长关键时期造成热胁迫并同时加重干旱胁迫,从而导致作物减产。植物在高温胁迫下生物膜会遭到破坏,蛋白质发生变性,进而引起植物饥饿、氨毒害等间接危害。
玉米(学名:Zea mays)为重要的粮食和工业原料作物,近年其播种面积约1亿多亩,是全世界总产量最高的粮食作物。我国是世界第二大玉米生产国,也是第二大玉米消费国。随着科技的进步,玉米深加工被广泛应用于食品工业,医药工业和化学工业,具有广阔的应用前景。然而,随着全球气候变暖,环境污染问题的日益凸显,玉米等农作物在生长发育过程中所受到的各种生物胁迫及非生物胁迫不断增多,其品质、产量等倍受影响。所以,研究玉米中的抗性基因及其功能,对于改善玉米以及其他粮食作物的品质和产量具有重大意义。
发明内容
本发明的目的在于公开了玉米OXS2a基因、其编码蛋白及应用。
本发明所采取的技术方案是:本发明所采取的技术方案是:
玉米OXS2a蛋白,其含有如下(1)和(2)任一项所示的氨基酸序列:
(1)SEQ ID NO.2所示的氨基酸序列
(2)SEQ ID NO.2所示的氨基酸序列经取代、缺失和/或增加一个或多个氨基酸和/或末端修饰后且具有提高植物抗逆功能的序列。
编码权利要求1所述玉米OXS2a蛋白的基因。
含有权利要求2所述基因序列的重组载体、重组菌或转基因细胞系、转基因植物株系。
玉米OXS2a蛋白、其编码基因在培育抗逆植物中的应用。
一种抗逆植物的培育方法,包括将OXS2a蛋白的基因转入受体植物中,得到转基因植物;转基因植物与受体植物相比,其对高温胁迫的抗性提高。
本发明的有益效果是:本发明在玉米种克隆了OXS2家族中的OXS2a基因。这个基因的表达受高温胁迫的诱导。当它在拟南芥中大量表达时,可以特异性地增强拟南芥抗高温的能力。因此,这个基因将具有极大的潜力应用于培育具有抗高温的转基因作物中,从而提高粮食作物的产量和品质。
附图说明
图1为ZmOXS2a基因PCR产物的1%的琼脂糖凝胶电泳图。
图2为玉米种ZmOXS2a的基因结构示意图;其中,268~373之间长方形为ANKYRIN重复序列;820~958之间长方形为锌指结构域;黑色直线部分为AT3片段;蛋白编码区中并不存在内含子;数字表示从转录起始位点开始的DNA基因组位置。
图3为滴板检测ZmOXS2a在粟酒裂殖酵母中的组成型表达,其中用转化空载体的酵母作为负对照,三角形表示,浓度依次十倍稀释。
图4拟南芥种子种植在1/2MS培养基正常条件培养10天,转移至37℃高温处理6天,接着正常生长条件恢复6天后的表型,其中line1、line2和line3为三个独立的转基因株系。
图5拟南芥种子种植在1/2MS培养基正常条件培养10天,转移至37℃高温处理6天,接着正常生长条件恢复6天后统计的存活率;
图6为拟南芥种子种植在1/2MS培养基正常条件培养10天,转移至37℃高温处理6天,接着正常生长条件恢复6天后地上部分的叶绿素a和b含量。
图7为拟南芥种子种植在1/2MS培养基正常条件培养10天,转移至37℃高温处理6天,接着正常生长条件恢复6天后地上部分的类胡萝卜素的含量。
图8拟南芥种子种植在1/2MS培养基正常条件培养10天,转移至37℃高温处理6天,接着正常生长条件恢复6天后通过测定地上部分离子外泄情况后所得到的膜系统损伤比率。
具体实施方式
下面结合实施例对本发明作进一步的说明,但并不局限于此。
以下实施例中所采用的分子生物学实验技术包括PCR扩增、质粒提取、质粒转化、DNA片段连接、酶切、凝胶电泳等,如无特殊说明,通常按照常规方法操作,具体可参见《分子克隆实验指南》(第三版)(Sambrook J,Russell DW,Janssen K,Argentine J.黄培堂等译,2002,北京:科学出版社),或按照制造厂商建议的条件。
一、材料与方法
1、生物材料与处理:
本实验采用的玉米品种为基因组序列已测通的科研品种B73。野生型拟南芥(Arabidopsis thaliana)采用Columbia(Col-0)生态型。
本实验基因的克隆采用B73。玉米种子用1比10的次氯酸钠消毒两次共10分钟,再用无菌水洗5次共五分钟,种于湿润的珍珠岩中进行萌发。生长条件:22℃,16/8光周期。5天后玉米种子萌发成3-5cm左右的小苗,可取幼苗用于提取DNA,作为克隆基因的模板。
2、转基因拟南芥的获得
拟南芥种子用1比13的次氯酸钠消毒两次共8分钟,再用无菌水洗6次共六分钟,种于1/2MS培养基。含有种子的1/2MS培养基放于4℃冰箱低温处理3天,然后置于光照培养箱中以20℃16小时光照、20℃8小时黑暗为一光周期培养10天后将幼苗移至营养土:蛭石:珍珠岩=4:3:3的塑料盒中定期浇水培养至开花,然后用于基因转化。
运用电转法将带有ZmOXS2a基因的表达载体pCambia3300导入根癌农杆菌GV3101中。筛选抗利福平和卡那霉素的农杆菌进行菌落PCR。挑选阳性的菌落摇菌,扩培,用浸花法侵染拟南芥。在含100μg/mL除草剂(ppt)的1/2MS培养基上筛选T0的种子(T1代幼苗),取T1代和Col野生型植株的叶片提取DNA,以野生型为阴性对照进行PCR鉴定,选取阳性株。每种转基因拟南芥得到大约十株T1代植株,通过分离比,筛选转基因拟南芥的单位点插入纯合体种子保存,并从中随机挑选3个株系作为后续研究的材料进行抗性检测。发明人对作为研究材料所有独立株系进行RT-PCR验证,发现转基因均有较高的表达。
3、酵母的抗性检测
在长有酵母的EMM固体筛选培养基上挑选菌落,加入到含有3-5mlEMM液体筛选培养基的50ml离心管中,充分分散、混匀。250rpm,30℃,摇床上摇12-18小时(OD为2-4)。稀释母液至OD为0.15-0.2,250rpm,30℃,摇床上摇4-5小时,使OD值等于或略大于0.3,稀释调节OD至0.3。以0.3OD的酵母液为母液,分别稀释到1/10,1/100,1/1000倍不同浓度梯度,各取3μl溶液,依次滴至不同浓度diamide,H2O2,CdCl2的EMM固体筛选培养基上。30℃倒置培养5-7天。
二、实验结果
1、玉米OXS2a基因的克隆
以已完成测序的科研品种B73为材料,提取其基因组,并以其基因组DNA为模板,设计引物克隆B73中的ZmOXS2a基因,引物序列如表1所述。利用高保真DNA聚合酶(High-Fidelity DNA Polymerase)PCR反应体系。取50μl PCR产物进行1%的琼脂糖凝胶电泳分析,如图1所述。取目标条带进行测序,所得ZmOXS2a基因的核苷酸序列如SEQ ID NO.1所示,其所编码的蛋白序列如SEQ ID NO.2所示。
表1PCR引物
Primer name | Sequence | 编号 |
ZmOXS2aF | 5’-ATGAACGGCATGCCGATCTC-3’ | SEQ ID NO.3 |
ZmOXS2aR | 5’-CTATGTTGCTAGACTACCAATCTGCTG-3’ | SEQ ID NO.4 |
2、玉米OXS2a基因信息
如图2所示,ZmOXS2a蛋白含有两个锚蛋白重复序列和两个锌指结构域。
3、玉米OXS2a基因在酵母中的抗性检测
发明人克隆了ZmOXS2a基因全长并连接到酵母表达载体pART1中,转化酵母。如图3所示,在酵母中过量表达ZmOXS2a可以增强酵母对二酰胺的抗性。因为化学物质二酰胺能够直接引起氧化反应,所以ZmOXS2a有可能在氧化胁迫响应机制上起作用。
4、玉米OXS2a基因在拟南芥中的抗性检测
为了进一步在植物中验证ZmOXS2a基因在植物中的功能,发明人将其转化拟南芥以进行抗性检测。通过花序侵染法,得到大约10株以Col为背景的ZmOXS2a高表达转基因拟南芥的独立株系。通过分离比,筛选该转基因拟南芥的单位点插入纯合体种子保存,并从中随机挑选3个株系作为后续研究的材料。发明人对作为研究材料所有独立株系进行RT-PCR验证,发现转基因均有较高的表达。
随后,发明人对该转基因拟南芥WT(ZmOXS2a)和它的野生型对照进行了胁迫的处理。首先将转基因拟南芥WT(ZmOXS2a)和它的野生型对照在正常条件下培养10天,然后37℃高温处理6天,接着转移回正常条件恢复培养6天。如图4所示,在正常的生长条件下,野生型转基因株系生长状况一致。当经高温处理和恢复培养后,以野生型为背景过量表达ZmOXS2a的转基因拟南芥生长状况明显好于野生型(图4),存活率(图5)、叶绿素含量(图6)和类胡萝卜素含量(图7)均高于野生型,且损伤率(图8)明显低于野生型。
以上实验结果表明,在拟南芥中大量表达ZmOXS2a基因,可以特异性地增强拟南芥抗高温。因此,该基因具有极大的潜力应用于培育具有抗逆性状的转基因作物中,从而提高粮食作物的产量。
SEQUENCE LISTING
<110> 中国科学院华南植物园
<120> 玉米OXS2a基因、其编码蛋白及应用
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<170> PatentIn version 3.5
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atgaacggca tgccgatctc tgcgtcctct gcggcaagcg tcgacagtgg cgcggtggcg 60
gccggcacgc ccatgaagaa tgcaacggcg gcggacgctc tcgccgagat ggcgagacat 120
cttaccgtcg acacggaaga caccttcgcg ggcctgctgg agctcgccgc tgacgacgac 180
gcagaggggc tgcgcctcgc gctggagcgc gcgccacctg ccgccgcgga cgaggctggc 240
ctctggtacg gccgccggaa ggtcctggag caccgcaccc cgctgatggt cgctgccacc 300
tatgggagcc ttgccgcgct ccgcctgcta gtgtccatct cgtatgtgga cgtcaaccgc 360
cgctcgggca cggacggcac cactgccctc cactgcgccg cctctggtgg ctcacggacg 420
gctgttgagt cggtcaagct gcttctggga gccggggcag acgctaacac catggatgac 480
gccgggcgtc gtccggctga tgtgatctct gtgccaccga agatgtttga tgccaagttc 540
gcccttcaag atcttcttgg attccccaag tctgagcatg gcatgctccg ggtggtgaca 600
aggtccacca actcgatctc atcgcctgtt tcatccccca ctgcagagga tgcacggtca 660
ccgtctgctt ctgtgatgat gatgacaaag ttcgctgact tgcctagggt tgcgacatcg 720
gagaagaagg aatatccagt ggatccatcc cttccagaca tcaagaacag catatatgct 780
tccgatgagt tccgcatgta ctcgttcaag atccgccctt gctcacgggc atactcgcat 840
gactggactg agtgcccatt tgtccaccca ggagagaatg ctcggcgccg agacccccgc 900
aagtaccact acagttgtgt gccatgccca gatttcagga agggcgtatg ccggcgcgga 960
gacatgtgtg aatatgctca tggagtgttt gagtgctggc tccatccagc acagtaccgg 1020
acgcgcctat gcaaggacgg cacaagttgc aatcgtcgcg tctgtttctt tgcccacaca 1080
actgatgaac tccgtccatt gtatgtctcg actggatcag cggtgccatc tccaagagct 1140
tcagcaacag ctgctatgga gatggctgca gcaatgggct tgatgcccgg ttccccatca 1200
tcggtttccg cggtaatgtc cccgtttacc ccaccaatgt ccccttcggg caatgggatg 1260
cccccttcat tgggctggca gcagccaaat gttccaactt tacatcttcc tggcagcagc 1320
cttcagtcaa gcaggctccg aacatcactt agtgcaaggg atatgcctgc tgatgactac 1380
tccatgatgc gggatcttga ttcccaactt atgaatgatc tttgctattc ccgtctcagt 1440
tcatccacag ggaaccactc tgctagaacc aagtccctga atccatcaaa cctggatgac 1500
ctcttctctg ctgaaatgtt atcatcccca aggtatagta atgctgacca gggtgcaatg 1560
ttttcacctt ctcacaaggc tgctatcctg aatcagttcc agcagcagca gcaagcactg 1620
ctgtctccaa tcaacacagg agtttactct ccaaagggcg tggacaacca gcagttacct 1680
tcacactcat cgctgctgca agcatcactc gggatgccct cccctggccg gatgtctcca 1740
cgatgtgtcg aatcgggctc cccaatgagc tcccacctag ccgctgctct tgtccagcgg 1800
gagaagcagc agcagacaat gagaagtctc agctctcgtg accttggtcc tagcgcggca 1860
cgagcttcgg ctcttggctc ccctctaagc tcatcatggt ctaagtgggg atctccttcg 1920
gggacaccag attggggcat aaatggtgaa gaattgggta agctccggcg ctcatcgtcc 1980
ttcgagctga ggtctggtgg tgacgaccct gatctctctt gggtgcatac actggtcaag 2040
gaatctccac cggagaagca agttactacg gctgaatcaa ttagttccgt tggaccctca 2100
ccactaatgc ctcccggcat gaacaatggc gacggttctg gcctgagcac ccagctggat 2160
ggacacgacc aagccgcggt tataggagcg ttgcttgaac agatgcagct tgaccagcag 2220
attggtagtc tagcaacata g 2241
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Claims (8)
1.玉米OXS2a蛋白,其含有如下(1)-(2)任一项所示的氨基酸序列:
(1)SEQ ID NO.2所示的氨基酸序列;
(2)SEQ ID NO.2所示的氨基酸序列经取代、缺失和/或增加一个或多个氨基酸和/或修饰后且具有提高植物抗高温胁迫功能的序列。
2.编码权利要求1所述玉米OXS2a蛋白的基因。
3.含有权利要求2所述基因序列的重组载体、重组菌或转基因细胞系、转基因植物株系。
4.玉米OXS2a蛋白、其编码基因在培育抗逆植物中的应用,其中,所述玉米OXS2a蛋白如权利要求1所示。
5.根据权利要求4所述的应用,其特征在于,所述抗逆植物的抗逆性状为抗高温胁迫。
6.一种抗逆植物的培育方法,包括将权利要求2所述的基因转入受体植物中,得到转基因植物。
7.根据权利要求6所述的培养方法,其特征在于,所述抗逆植物的抗逆效果为,抗高温胁迫。
8.根据权利要求6所述的培养方法,其特征在于,所述抗逆植物包括拟南芥、玉米、水稻、小麦、棉花中的任意一种。
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CN112175056A (zh) * | 2020-09-14 | 2021-01-05 | 中国科学院华南植物园 | OsABCG48基因及其在提高单细胞生物和植物对镉胁迫抗性中的应用 |
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CN111893134B (zh) * | 2020-07-30 | 2022-07-19 | 中国科学院华南植物园 | Oxs2基因及其编码蛋白在提高植物抗盐胁迫性中的应用 |
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