CN108948164A - 甘薯耐盐抗旱相关蛋白IbbZIP1及其编码基因与应用 - Google Patents
甘薯耐盐抗旱相关蛋白IbbZIP1及其编码基因与应用 Download PDFInfo
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Abstract
本发明公开了甘薯耐盐抗旱相关蛋白IbbZIP1及其编码基因与应用。本发明提供了一种蛋白质,为如下1)或2)或3):1)氨基酸序列是序列表中序列1所示的蛋白质;2)在序列表中序列1所示的蛋白质的N端或/和C端连接标签得到的融合蛋白质;3)将1)或2)所示的蛋白质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抗逆性相关的蛋白质。实验证明,在拟南芥中过表达IbbZIP1基因可提高拟南芥叶片和种子中的类胡萝卜素含量和拟南芥植株的耐盐和抗旱性。因此,类胡萝卜素合成和耐盐抗旱相关蛋白IbbZIP1及其编码基因在调控植物类胡萝卜素合成以及耐盐抗旱性中具有重要的理论意义和实用价值。
Description
技术领域
本发明属于生物技术领域,具体涉及甘薯耐盐抗旱相关蛋白IbbZIP1及其编码基因与应用。
背景技术
当今世界耕地面积有限,且盐碱、干旱等边际土地面积巨大。据不完全统计,世界范围内存在8亿hm2盐渍化土地,约占总陆地面积的6%,约20%灌溉农业用地的土壤盐渍化。而中国更是世界上盐碱地分布最多的国家之一。世界人口的增加和可耕土地面积的减少,严重的威胁到粮食的安全,而中国面临的挑战则更加严峻。此外,随着世界人口急剧膨胀与全球气候变暖的影响,水资源的短缺也已成为农业面临的主要挑战之一。耕地的盐渍化和水资源的严重缺乏影响了农业的可持续发展,研究植物耐盐抗旱机理对农业生产和生态建设具有重要的意义。
为了抵抗外界不利环境因素的影响,植株在进化过程中逐步形成了一系列抗性机制。植物体内的抗逆机制十分复杂,是一个涉及多基因、多抗性的复杂过程。主要包括:一,渗透调节机制,逆境胁迫下,植物自身合成的有机渗透调节小分子物质,氨基酸及其衍生物(甜菜碱、甘氨酸、脯氨酸等),多元醇和糖类等具有较厚的水化层,溶解度高,极性电荷少,可以起到稳定细胞质中酶分子活性结构的作用。二,ROS清除***是植物体在长期的进化中为了减少氧化损伤衍生出来的为了降低氧化损伤的***,包括超氧化物歧化酶SOD、谷胱甘肽过氧化物酶GPX、过氧化氢酶CAT、过氧化物酶POD和抗坏血酸过氧化物酶APX等。三,信号转导机制,植物对外部刺激作出反应是通过信号级联作用完成的。近年来,越来越多的信号转导相关的基因以及信号通路被报道,与信号转导相关的基因及信号通路,基因在信号通路中所处的位置、发挥的作用以及不同基因间编码的蛋白间的相互作用逐渐被广泛研究。四,逆境胁迫激素信号转导机制,植物激素作为植物体内合成的微量有机物质,在植物生长发育的各个阶段和植物对生物及非生物胁迫适应中发挥重要功能。目前研究较多的是ABA响应途径,以JA和MeJA为代表的茉莉酸类物质(Jasmonic acids,JAs),油菜素甾醇类(Brassinosteroids,BRs)响应途径等。五,基因表达的转录调控,植物的抗逆胁迫是被认为涉及多条信号转导途径、多个基因及多种基因产物的复杂过程。当植物受到逆境胁迫时,植物体内会产生一系列信号传导物质,这些物质能够激活转录因子(Transcription factor,TF)的表达。植物中已克隆了大量植物抗逆性相关的转录因子。
发明内容
本发明的一个目的是提供甘薯耐盐抗旱相关蛋白IbbZIP1及其编码基因。
本发明所提供的甘薯耐盐抗旱相关蛋白,名称为IbbZIP1,来源于甘薯(Ipomoeabatatas),是如下(a)或(b)的蛋白质:
(a)由序列表中序列1所示的氨基酸序列组成的蛋白质;
(b)将序列表中序列1所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且与植物抗逆性相关的由序列1衍生的蛋白质。
所述序列1由359个氨基酸残基组成。
所述编码上述蛋白的核酸分子也属于本发明的保护范围。
所述核酸分子如下(a1)或(a2)或(a3)或(a4)所示的DNA分子:
(a1)编码区包括序列表中序列2的DNA分子;
(a2)核苷酸序列是序列表中序列2的DNA分子;
(a3)与(a1)或(a2)限定的核苷酸序列具有75%或75%以上同一性,且编码上述蛋白质的DNA分子;
(a4)在严格条件下与(a1)或(a2)限定的核苷酸序列杂交,且编码上述蛋白质的DNA分子。
所述序列2由1080个碱基组成,其开放阅读框(ORF)为自5′末端起第1位-第1080位碱基,编码氨基酸序列是序列表中序列1所示的蛋白。
上述严格条件可为用6×SSC,0.5%SDS的溶液,在65℃下杂交,然后用2×SSC,0.1%SDS和1×SSC,0.1%SDS各洗膜一次。
含有编码上述蛋白的核酸分子的表达盒、重组表达载体、转基因细胞系或重组微生物也属于本发明的保护范围。
所述重组表达载体是在载体pCBGUS的多克隆位点间***编码上述蛋白的DNA分子得到的重组表达载体;
所述载体pCBGUS是通过包括如下步骤的方法得到的:
(1)将pCAMBIA3301载体经过HindIII和EcoRI双酶切,回收载体大片段;
(2)将pBI121载体经过HindIII和EcoRI双酶切,回收包含gusA基因的片段;
(3)将步骤(1)中回收的载体大片段与步骤(2)中回收的包含gusA基因的片段连接,得到重组载体pCBGUS。
所述pCAMBIA3301载体购自CAMBIA公司;所述pBI121载体购自Clontech公司。
扩增上述DNA分子全长或其任一片段的引物对也属于本发明的保护范围。
所述引物对为如下(1)或(2)所示:
1)由序列表中序列3所示的DNA分子和序列表中序列4所示的DNA分子组成的引物对;
2)由序列表中序列5所示的DNA分子和序列表中序列6所示的DNA分子组成的引物对。
如下b1)或b2)的应用也是本发明保护的范围:
b1)上述蛋白质,或,上述核酸分子,或,含有上述核酸分子的表达盒、重组载体、重组微生物或转基因细胞系,在调控植物抗逆性中的应用;
b2)上述蛋白质,或,上述核酸分子,或,含有上述核酸分子的表达盒、重组载体、重组微生物或转基因细胞系,在培育抗逆性改变的转基因植物中的应用。
上述抗逆性改变具体为抗逆性提高;
上述调控植物抗逆性具体为提高植物抗逆性。
本发明的另一个目的是提供一种培育抗逆性高转基因植物的方法。
本发明所提供的培育抗逆性高转基因植物的方法,为提高目的植物中上述蛋白质的含量或活性,得到转基因植物;
所述转基因植物的抗逆性高于所述目的植物。
上述提高目的植物中上述蛋白质的含量或活性为提高目的植物中编码上述蛋白质的DNA分子的表达,具体为将编码上述蛋白质的DNA分子导入目的植物中,得到转基因植物;上述导入方式为通过重组表达载体导入。
本发明第3个目的是提供一种培育抗逆性高的植物的方法。
本发明提供的方法,为培育上述方法得到的转基因植物。
上述中,所述抗逆性为耐盐性和/或抗旱性和/或抗氧化性和/或抗病性。
上述中,所述植物是如下c1)至c4)中的任一种:
c1)双子叶植物;
c2)单子叶植物;
c3)十字花科植物;
c4)拟南芥(Arabidopsis thaliana)或甘薯(Ipomoea batatas)。。
上述耐盐性通过增加植物根长和鲜重、增加ABA含量、增加SOD活性、增加脯氨酸含量和/或降低过氧化氢(H2O2)含量体现。
上述抗旱性通过增加植物株高、增加ABA含量、增加SOD活性、增加脯氨酸含量和/或降低过氧化氢(H2O2)含量体现。
本发明的实验证明,本发明发现了IbbZIP1蛋白及其编码基因,将IbbZIP1蛋白编码基因导入拟南芥中,得到IbbZIP1的转基因拟南芥植株。将转基因拟南芥植株进行盐、旱胁迫处理,与野生型拟南芥相比,转基因株系耐盐性和抗旱性增强,具体体现在增加植物株高,增加ABA含量、SOD活性、脯氨酸含量和降低H2O2含量。结果表明,IbbZIP1基因及其所编码的蛋白在植物对抗高盐和抗干旱的过程中起着重要的作用。本发明所提供的IbbZIP1蛋白及其编码基因在提高植物耐盐抗旱研究中具有重要的应用价值。本发明将在农业领域具有广阔的应用空间和市场前景。
附图说明
图1为拟南芥转基因植株的PCR扩增结果。
图2为IbbZIP1基因在过表达拟南芥植株和野生型拟南芥植株(WT)中的表达。
图3为IbbZIP1拟南芥转基因植株耐盐和抗旱性离体鉴定。
图4为IbbZIP1拟南芥转基因植株耐盐和抗旱性盆栽鉴定。
具体实施方式
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。
下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
下面结合具体实施方式对本发明进行进一步的详细描述,给出的实施例仅为了阐明本发明,而不是为了限制本发明的范围。
甘薯品系HVB-3记载于如下文献中:李瑞杰.橘红肉色甘薯的转录组分析及类胡萝卜素生物合成相关基因的克隆与功能分析,博士学位论文,中国农业大学,2017。公众可从中国农业大学甘薯遗传育种研究室获得,以重复本实验。
拟南芥哥伦比亚野生型col-0(能够从商业途径获得)。
克隆载体pMD19-T为宝生物工程(大连)公司产品,产品目录号为6013。载体pCAMBIA3301为Cambia公司产品。载体pBI121为Clontech公司产品。植物总RNA提取试剂盒全式金(TransGen Biotech,北京)的Transzol Up植物总RNA提取试剂盒(目录号:ET111)。pEASY-Blunt simple载体为北京全式金生物技术有限公司的产品。QuantScript RT KitQuant cDNA第一链合成试剂盒为天根(TIANGEN,北京)限公司的产品(目录号:KR103)。
1/2霍格兰营养液记载于如下文献中:刘德高.过表达IbP5CR、IbERD3、IbELT、IbNFU1基因的甘薯植株的获得及耐盐性鉴定.北京.2014年.中国农业大学博士毕业论文。
实施例1、IbbZIP1基因的获得
1、cDNA模板的获得
用植物总RNA提取试剂盒提取甘薯品系HVB-3的幼嫩叶片的总RNA,将该总RNA用PQuantScript RT Kit Quant cDNA Kit反转录出第一链cDNA。
2、以步骤1获得的cDNA为模板,根据EST序列设计并人工合成引物GSP-1和GSP-2,利用RACE方法扩增获得约700bp的3′-RACE片段,将3′-RACE片段和克隆载体pMD19-T连接,得到重组质粒2。将重组质粒2进行测序,获得3′-RACE片段的核苷酸序列。引物序列如下:
GSP-1:CTTCTTCTGGAACCGACAAAAAG
GSP-2:CATGAACATGGAGAATAAAGCGC
3、根据EST序列设计并人工合成引物GSP-3和GSP-4,以步骤1获得的cDNA为模板,利用RACE方法扩增获得约700bp的5′-RACE片段,将5′-RACE片段和克隆载体pMD19-T连接,得到重组质粒3。将重组质粒3进行测序,获得5′-RACE片段的核苷酸序列。引物序列如下:
GSP-3:ATGCCGTGTGGATGCATAAT
GSP-4:CGGGATAGGCATCATGTTTCT
4、将获得的3′-RACE片段的核苷酸序列和5′-RACE片段的核苷酸序列,利用DNAMAN软件拼接候选的IbbZIP1基因。依据拼接候选的IbbZIP1基因序列进一步设计并人工合成引物O-F(序列表序列3)和O-R(序列表序列4),以步骤1获得的cDNA为模板,进行PCR扩增,获得约1080bp的PCR扩增产物并测序。
结果表明,PCR扩增产物的核苷酸序列如序列表中序列2自5′末端起第1至1080位所示,将该序列所示的基因命名为IbbZIP1基因,其编码的蛋白命名为IbbZIP1蛋白或蛋白质IbbZIP1,氨基酸序列如序列表中序列1所示。
实施例2、IbbZIP1蛋白在提高植物耐盐抗旱中的应用
一、重组质粒pCB-IbbZIP1的构建
1、人工合成序列表的序列1自5′末端起第1至1080位所示的双链DNA分子。以该双链DNA分子为模板,以OE-F-BamHI:5′-CGGGATCCATGGGTTCTGTTGAAGAAAAAG-3′(序列5)(下划线为限制性内切酶BamHI的识别序列)和OE-R-SacI:5′-CGAGCTCTCAGTGACAACCATCGGTCG-3′(序列6)(下划线为限制性内切酶SacI的识别序列)为引物进行PCR扩增,得到N端含有限制性内切酶XbaI和C端含有限制性内切酶SacI的双链DNA分子。
2、将N端含有限制性内切酶XbaI和C端含有限制性内切酶SacI的双链DNA分子连接至pEASY-Blunt simple载体,得到重组质粒pEASY-IbbZIP1。
3、完成步骤2后,用限制性内切酶BamHI和SacI双酶切重组质粒pEASY-IbbZIP1,回收约1100bp的片段1。
4、用限制性内切酶HindIII和EcoRI双酶切载体pCAMBIA3301,回收约11256bp的载体骨架1。
5、用限制性内切酶HindIII和EcoRI双酶切载体pBI121,回收包含约3412bp的片段2。
6、将片段2与载体骨架1连接,得到重组质粒pCBGUS。
7、用限制性内切酶BamHI和SacI双酶切重组质粒pCBGUS,回收约12388bp的载体骨架2。
8、将片段1与载体骨架2连接,得到重组质粒pCB-IbbZIP1。
根据测序结果,对重组质粒pCB-IbbZIP1进行结构描述如下:将重组质粒pCBGUS的限制性内切酶BamHI和SacI识别序列间的小片段替换为序列表中序列1自5′末端起第1至1080位所示的DNA分子。重组质粒pCB-IbbZIP1表达序列表中序列2所示的IbbZIP1蛋白。
重组质粒pCBGUS为将3032bp的片段2(即pBI121载体上35S启动子加GUS基因序列)替换pCAMBIA3301载体的HindIII和EcoRI双酶切位点间得到的载体。
二、转IbbZIP1拟南芥植株的获得
拟南芥转基因阳性植株的再生
1、将重组质粒pCB-IbbZIP1转化根癌农杆菌GV3101,得到重组农杆菌甲,将重组农杆菌甲命名为GV3101/pCB-IbbZIP1。
2、种子消毒处理:取适量拟南芥哥伦比亚野生型(Col-0;以下简称为野生型拟南芥)种子装入2.0mL离心管中,用2%次氯酸钠溶液消毒5min,上下颠倒充分混匀,低速离心后倒掉次氯酸钠,加入无菌蒸馏水充分洗涤5-6次;将处理好的种子均匀地铺在1/2MS的平板上。
3、春化:将铺好种子的1/2MS平板密封置于4℃环境中3-4天。
4、将春化后的平板放置在22±1℃的光照培养室(光照强度:5500lx±300lx光照时间:12h)中培养,待幼苗长出3-5片真叶后,用镊子小心的将幼苗从培养基中取出,尽量除净幼苗上的培养基,然后移栽至营养土和蛭石(1:1)中,用塑料薄膜盖住保湿1周左右。
5、农杆菌的培养:在抗性平板上活化农杆菌菌液,挑取单菌落接种于5mL的已加入对应抗生素的LB液体培养基中,28℃下200rpm振荡培养,直到OD600值在0.8~1.0范围内。5000rpm离心并去上清液,用等体积的含有3%的蔗糖的1/2MS溶液重悬菌体,在菌液中加入0.02%的SilwetL-77混匀,重悬液OD600约为0.8。
6、花序侵染:将上述菌悬液放入口径约为9cm的玻璃培养皿中,用于侵染。将带花序的拟南芥植株倒转,使花序全部侵润在农杆菌重悬液中30s左右。侵染过的植株放置于黑暗条件下24h,此时保持土壤湿润,每周重复侵染一次,一共侵染3次。
7、抗性种子的筛选:T1种子表面消毒并统一发芽后,将种子播种于含12.5mg/L的PPT的1/2MS固体培养基上筛选阳性植株,将阳性植株移栽到土中继续生长,单株收种获得T2代种子。之后将T2代种子消毒后播种于含12.5mg/L的PPT的1/2MS固体培养基上继续筛选阳性植株,统计分离比,在筛选培养基上阳性植株和非阳性植株符合3:1比例的株系即为单拷贝***的株系,繁种,获得T3代种子。将T3代种子消毒后播种于含12.5mg/L的PPT的1/2MS固体培养基上,所有种子均能正常生长的株系即为单拷贝***纯合植株,保存种子,记作T3代转IbbZIP1拟南芥。
8、转基因植株的鉴定与再生:
T3代转IbbZIP1拟南芥的鉴定使用GUS染色和PCR检测相结合的方法。
1)GUS染色
T3代转IbbZIP1拟南芥经过GUS染色,植株组织变为蓝色即为GUS检测阳性,可初步认定为过表达植株,并进一步对GUS阳性T3代转IbbZIP1拟南芥进行PCR检测。
2)PCR检测
GUS阳性T3代转IbbZIP1拟南芥提取叶片基因组DNA,用下面的引物对其进行PCR检测,同时以pCB-IbbZIP1载体质粒DNA为阳性对照,水和野生型拟南芥植株DNA为阴性对照。引物序列:
35S-F:TCAGAAAGAATGCTAACCCACA
IbbZIP1-R:TTATTTTTCAAAAGAGAGGTTTTTA
将扩增得到的PCR产物在1%(w/v)的琼脂糖凝胶中进行电泳分离,PCR阳性植株应该具有一条特异的1080bp的电泳条带,记录下PCR阳性植株的株系号。
结果如图1所示,M为DNA分子Marker,W为阴性对照,P为阳性对照,WT为野生型拟南芥植株的基因组DNA,L1、L2、L3、L4、L5、L6、L7和L8均为GUS阳性T3代转IbbZIP1拟南芥;结果表明,L1、L2、L3、L4、L5、L6、L7和L8均为甘薯转基因阳性植株。
GUS检测与PCR检测均为阳性的株系即确定为过表达IbbZIP1基因的转基因株系,即为阳性T3代转IbbZIP1拟南芥。将阳性T3代转IbbZIP1拟南芥株系的种子保存。
3)QRT-PCR
将阳性T3代转IbbZIP1拟南芥株系提取RNA,反转录得到cDNA,进行QRT-PCR,以未转化的野生型为对照。
Atactin基因为内参:
Atactin-F:GCACCCTGTTCTTCTTACCGA
Atactin-R:AGTAAGGTCACGTCCAGCAAGG
IbbZIP1引物序列为:
IbbZIP1-F:ACCCCTTCTTTCGGTCACAA
IbbZIP1-R:GCCGATGACCTCTACGTACA
结果如图2所示,WT为野生型拟南芥植株的基因组DNA,L1、L2、L3、L4、L5、L6、L7和L8均为阳性T3代转IbbZIP1拟南芥,表明IbbZIP1在转基因拟南芥植株中有不同程度的表达。
三、转IbbZIP1拟南芥植株抗逆性鉴定
1、转基因植株耐盐性和抗旱性离体鉴定
转基因拟南芥为阳性T3代转IbbZIP1拟南芥L1的植株、阳性T3代转IbbZIP1拟南芥L6的植株、阳性T3代转IbbZIP1拟南芥L5的植株、阳性T3代转IbbZIP1拟南芥L8的植株。
具体步骤:转基因拟南芥与野生型拟南芥(WT)种子消毒后点播于1/2MS培养基中,待子叶完全展开后,选取长势一致的转基因植株和野生型拟南芥植株转移到含有200mMNaCl、300mM甘露醇或正常的1/2MS培养基(对照)上,每个株系4株苗,直立放置生长2周后观察植株生长情况,并测量其根长和鲜重。
结果如图3所示,
在含有200mM NaCl或300mM甘露醇的1/2MS培养基上直立放置生长2周后,WT长势较差,根系较短;阳性T3代转IbbZIP1拟南芥株系的生长状态和生根情况不同程度的优于WT,主要体现在根长和鲜重。在正常的1/2MS培养基(对照)上,阳性T3代转IbbZIP1拟南芥株系和WT在无显著差异。
离体鉴定结果初步说明IbbZIP1基因的过表达提高了拟南芥植株的耐盐性和抗旱性。
2、转基因植株耐盐性和抗旱性盆栽鉴定
转基因拟南芥为阳性T3代转IbbZIP1拟南芥L1的植株、阳性T3代转IbbZIP1拟南芥L3的植株、阳性T3代转IbbZIP1拟南芥L6的植株、阳性T3代转IbbZIP1拟南芥L8的植株。
过表达IbbZIP1基因拟南芥植株耐盐抗旱性盆栽鉴定:转基因拟南芥与野生型拟南芥(WT)种子消毒后点播于1/2MS培养基中,正常生长7d后移栽至7cm的营养钵中继续进行生长,2周后开始进行盐、旱处理。
a无胁迫(对照):正常条件生长4周,观察植株生长情况;
b盐处理(NaCl):用含有200mM NaCl的1/2霍格兰营养液每2天灌溉1次,处理2周,观察植株生长情况;
c旱处理(自然干旱):自然干旱胁迫处理后,处理组植株不再浇水,干旱胁迫4周,然后复水处理2天后观察植株生长情况。
结果如图4A所示,可以看出,用含200mM NaCl的1/2霍格兰营养液每2天灌溉1次,处理2周后,野生型拟南芥植株叶片几乎全部退绿死亡,而阳性T3代转IbbZIP1拟南芥株系叶片绿色部分还比较多;干旱胁迫4周,然后复水处理2天后,阳性T3代转IbbZIP1拟南芥能回复吸水能力并保持健康生长,而野生型拟南芥植株几乎丧失生长活力。
该实验结果表明,过表达IbbZIP1基因能显著提高拟南芥植株对高盐和干旱胁迫的抵抗能力。
3、生理生化指标的测定
(1)脯氨酸含量测定
植物在正常条件下,游离脯氨酸含量很低,但遇到干旱、低温、盐等胁迫时,游离的氨基酸便会大量积累,并且积累指数和植物的抗逆性有关。因此,脯氨酸可以作为植物抗逆性的一项生化指标。
用脯氨酸(PRO)含量试剂盒(苏州科铭生物,目录号:PRO-1-Y)来检测拟南芥植株的脯氨酸含量。拟南芥植株为上述2中a无胁迫处理2周的拟南芥植株、上述2中b盐处理1周的拟南芥植株和上述2中c旱处理2周的拟南芥植株。实验需重复三次,结果取平均值。
拟南芥植株株系为阳性T3代转IbbZIP1拟南芥L1的植株、阳性T3代转IbbZIP1拟南芥L3的植株、阳性T3代转IbbZIP1拟南芥L6的植株、阳性T3代转IbbZIP1拟南芥L8的植株和野生型拟南芥(WT)。
结果如图4B所示,对照为无胁迫,NaCl为盐胁迫,干旱为自然干旱胁迫,结果表明,L1的植株、L3的植株、L6的植株和L8的植株的脯氨酸含量显著高于野生型拟南芥植株。
(2)SOD活性测定
SOD活性可以作为植物抗逆性的一项生化指标。SOD的活性越低,植物遭受逆境伤害的程度越大。
用超氧化物歧化酶(SOD)试剂盒(苏州科铭生物,目录号:SOD-1-Y)来检测拟南芥植株的SOD活性。拟南芥植株为上述2中a无胁迫处理2周的拟南芥植株、上述2中b盐处理1周的拟南芥植株和上述2中c旱处理2周的拟南芥植株。实验需重复三次,结果取平均值。
拟南芥植株株系为阳性T3代转IbbZIP1拟南芥L1的植株、阳性T3代转IbbZIP1拟南芥L3的植株、阳性T3代转IbbZIP1拟南芥L6的植株、阳性T3代转IbbZIP1拟南芥L8的植株和野生型拟南芥(WT)。
结果如图4B所示,对照为无胁迫,NaCl为盐胁迫,干旱为自然干旱胁迫,结果表明,L1的植株、L3的植株、L6的植株和L8的植株的SOD活性显著高于野生型拟南芥植株。
(3)H2O2含量测定
植物在逆境下或衰老时,由于体内活性氧代谢加强而使H2O2发生累积。H2O2可以直接或间接地氧化细胞内核酸,蛋白质等生物大分子,并使细胞膜遭受损害,从而加速细胞的衰老和解体。因此,H2O2的含量越高,植物遭受逆境伤害的程度越大。
过氧化氢(H2O2)试剂盒(苏州科铭生物,目录号:H2O2-2-Y)来检测拟南芥植株的H2O2积累量。实验需重复三次,结果取平均值。拟南芥植株为上述2中a无胁迫处理2周的拟南芥植株、上述2中b盐处理1周的拟南芥植株和上述2中c旱处理2周的拟南芥植株。实验需重复三次,结果取平均值。
拟南芥植株株系为阳性T3代转IbbZIP1拟南芥L1的植株、阳性T3代转IbbZIP1拟南芥L3的植株、阳性T3代转IbbZIP1拟南芥L6的植株、阳性T3代转IbbZIP1拟南芥L8的植株和野生型拟南芥(WT)。
结果如图4B所示,对照为无胁迫,NaCl为盐胁迫,干旱为自然干旱胁迫,结果表明,L1的植株、L3的植株、L6的植株和L8的植株的H2O2积累量显著低于野生型拟南芥植株。
(4)ABA含量测定
ABA在植物逆境胁迫反应中具有重要作用。ABA可以提高植物的耐盐性,缓解盐分过多造成的渗透胁迫和离子胁迫,维持水分平衡,诱导植物渗透调剂物质脯氨酸大量积累,维持细胞膜结构的稳定性,提高保护性酶的活性。旱害胁迫时,ABA能明显减少叶片水分蒸发,降低叶片细胞膜透性,增加叶片细胞可溶性蛋白质含量,诱导生物膜***保护酶形成,降低膜脂过氧化程度,增强抗氧化能力,提高植物的抗旱性。
测定方法参照参考文献(Zhai H,Wang F,Si Z,et al.A myo-inositol-1-phosphate synthase gene,IbMIPS1,enhances salt and drought tolerance and stemnematode resistance in transgenic sweet potato[J].Plant BiotechnologyJournal,2016,14(2):592.)拟南芥植株为上述2中a无胁迫处理2周的拟南芥植株、上述2中b盐处理1周的拟南芥植株和上述2中c旱处理2周的拟南芥植株。实验需重复三次,结果取平均值。
拟南芥植株株系为阳性T3代转IbbZIP1拟南芥L1的植株、阳性T3代转IbbZIP1拟南芥L3的植株、阳性T3代转IbbZIP1拟南芥L6的植株、阳性T3代转IbbZIP1拟南芥L8的植株和野生型拟南芥(WT)。
结果如图4B所示,对照为无胁迫,NaCl为盐胁迫,干旱为自然干旱胁迫,结果表明,L1的植株、L3的植株、L6的植株和L8的植株的ABA含量显著高于野生型拟南芥植株。
上述结果表明,过表达IbbZIP1基因可提高拟南芥的抗逆性;抗逆性可为耐盐性和抗旱性。
序列表
<110> 中国农业大学
<120> 甘薯耐盐抗旱相关蛋白IbbZIP1及其编码基因与应用
<160> 6
<210> 1
<211> 359
<212> PRT
<213> 甘薯(Ipomoea batatas)
<400> 1
Met Ala Asn Phe Glu Gly Gln Ser Ser Ile Arg Asn Val Met Phe Asn
1 5 10 15
Gly Lys Ser Ser Leu Leu Pro Pro Lys Ser Pro Phe Pro Ser Leu Ser
20 25 30
Pro Ser Tyr Ile Glu Tyr Thr Pro Ser Phe Gly His Lys Gly Ile Pro
35 40 45
Lys Pro Arg Glu Gly Asn Ser His His Gln Arg Thr Ser Ser Glu Ser
50 55 60
Phe Leu Ile Glu Glu Gln Pro Ser Trp Leu Asp Asp Leu Leu Asn Glu
65 70 75 80
Pro Asp Thr Pro Val Arg Arg Gly His Arg Arg Ser Ser Ser Asp Ser
85 90 95
Phe Thr Tyr Tyr Asp Ala Pro Asn Val Ala Asn Leu Asp Phe Ile Val
100 105 110
Gln Asp His Asn Asn Phe Arg Asn Met Met Pro Ile Arg Ser Trp Gly
115 120 125
Ser Gln Glu Phe Asp Tyr His Gly Asp Ala His His Thr Ala Phe Tyr
130 135 140
Gly Asp His Asn Ser Ser Asn Arg His Lys Asn Arg Thr Arg Asp Ala
145 150 155 160
Ser Pro Asn Lys Ile Met His Pro His Gly Ile Pro Ser Pro Lys Glu
165 170 175
Asn Leu Ile Val Gln Ser Ser Gly Ser Pro Cys Pro Pro Gln Gly Asp
180 185 190
Arg Ala Gln Ser Pro Ala Ile Asp Lys Gln Asp Leu Leu Glu Ser Gly
195 200 205
Pro Pro Asp Pro Asn Ser Ser Ala Glu Lys Arg Asp Ser Leu Val Lys
210 215 220
Asn Ser Ser Ser Gly Thr Asp Lys Lys Tyr Ser Arg Gln Gln Phe Ala
225 230 235 240
Gln Arg Ser Arg Val Arg Lys Leu Gln Tyr Ile Ala Glu Leu Glu Arg
245 250 255
Asn Val Gln Ala Leu Gln Ala Glu Gly Ser Glu Val Ser Ala Glu Leu
260 265 270
Glu Phe Leu Asn Gln Gln Asn Leu Ile Met Asn Met Glu Asn Lys Ala
275 280 285
Leu Lys Gln Arg Leu Glu Ser Leu Thr Gln Glu Lys Leu Leu Lys Tyr
290 295 300
Val Glu His Glu Leu Leu Glu Lys Glu Arg Glu Arg Leu Gln Ala Leu
305 310 315 320
Tyr Gln Gln Gln Gln Gln Pro Gln Pro Gln Gln Gln Gln Lys Gln Gln
325 330 335
Ser His Gly His Arg Arg Thr Thr Ser Arg Asp Leu Glu Gln Gln Phe
340 345 350
Lys Asn Leu Ser Phe Glu Lys
355
<210> 2
<211> 1080
<212> DNA
<213> 甘薯(Ipomoea batatas)
<400> 2
atggcaaact ttgagggaca atctagcatc aggaatgtga tgttcaatgg gaagagttct 60
ttactgcctc ctaaaagtcc atttcctagc ttatctccat catatattga gtatacccct 120
tctttcggtc acaaaggcat tccaaaaccg agggaaggta attcacacca tcaacgtacc 180
tcctctgaaa gctttctgat agaggagcag ccatcttggc ttgatgatct tcttaatgag 240
ccagacaccc ctgtacgtag aggtcatcgg cgctcatcaa gtgactcctt tacatattac 300
gatgctccta atgttgcaaa cttagatttc atagttcaag atcataacaa ctttagaaac 360
atgatgccta tccgttcgtg gggatcccaa gaatttgatt accacgggga tgctcaccac 420
actgctttct atggggatca taactcttca aatagacaca agaacaggac aagagatgca 480
tctccaaata aaattatgca tccacacggc atcccttctc caaaagaaaa tcttattgtt 540
caaagctcag gatcaccatg tcccccacaa ggagacaggg ctcaatctcc agcaattgac 600
aagcaggatc tactcgaatc tggcccacct gatccaaaca gctctgcaga gaagagggat 660
tcacttgtca agaattcttc ttctggaacc gacaaaaagt actcaaggca gcaatttgct 720
cagcgttcaa gagtccggaa gcttcaatac atagctgagc tggaaaggaa tgttcaagct 780
ctacaggctg aaggctctga agtttccgct gagcttgaat tccttaacca gcaaaatctt 840
atcatgaaca tggagaataa agcgctaaag cagcgtttag aaagtttaac tcaagaaaag 900
cttttaaagt atgtggaaca tgaactttta gagaaagaaa gagaaagact acaagctttg 960
tatcaacaac agcaacaacc acaaccgcaa cagcaacaga agcaacaatc ccatggtcat 1020
cgccgcacca caagtagaga tcttgaacaa caatttaaaa acctctcttt tgaaaaataa 1080
<210> 3
<211> 25
<212> DNA
<213> 人工序列
<220>
<223>
<400> 3
atggcaaact ttgagggaca atcta 25
<210> 4
<211> 25
<212> DNA
<213> 人工序列
<220>
<223>
<400> 4
ttatttttca aaagagaggt tttta 25
<210> 5
<211> 30
<212> DNA
<213> 人工序列
<220>
<223>
<400> 5
cgggatccat gggttctgtt gaagaaaaag 30
<210> 6
<211> 27
<212> DNA
<213> 人工序列
<220>
<223>
<400> 6
cgagctctca gtgacaacca tcggtcg 27
Claims (9)
1.蛋白质,为如下1)或2)或3):
1)氨基酸序列是序列表中序列1所示的蛋白质;
2)在序列表中序列1所示的蛋白质的N端或/和C端连接标签得到的融合蛋白质;
3)将1)或2)所示的蛋白质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抗逆性相关的蛋白质。
2.编码权利要求1所述蛋白质的核酸分子。
3.如权利要求2所述的核酸分子,其特征在于:所述核酸分子为如下(a1)或(a2)或(a3)或(a4)所示的DNA分子:
(a1)编码区包括序列表中序列2的DNA分子;
(a2)核苷酸序列是序列表中序列2的DNA分子;
(a3)与(a1)或(a2)限定的核苷酸序列具有75%或75%以上同一性,且编码权利要求1所述蛋白质的DNA分子;
(a4)在严格条件下与(a1)或(a2)限定的核苷酸序列杂交,且编码权利要求1所述蛋白质的DNA分子。
4.含有权利要求2或3所述核酸分子的表达盒、重组载体、重组微生物或转基因细胞系。
5.b1)或b2)的应用:
b1)权利要求1所述蛋白质,或,权利要求2或3所述核酸分子,或,含有权利要求2或3所述核酸分子的表达盒、重组载体、重组微生物或转基因细胞系,在调控植物抗逆性中的应用;
b2)权利要求1所述蛋白质,或,权利要求2或3所述核酸分子,或,含有权利要求2或3所述核酸分子的表达盒、重组载体、重组微生物或转基因细胞系,在培育抗逆性改变的转基因植物中的应用。
6.一种培育抗逆性高转基因植物的方法,为提高目的植物中权利要求1所述蛋白质的含量或活性,得到转基因植物;
所述转基因植物的抗逆性高于所述目的植物。
7.一种培育抗逆性高的植物的方法,为培育权利要求6所述方法得到的转基因植物。
8.如权利要求1所述蛋白质,或,权利要求5所述的应用,或,权利要求6或7所述的方法,其特征在于:所述抗逆性为耐盐性和/或抗旱性和/或抗氧化性和/或抗病性。
9.如权利要求1或8所述蛋白质,或,权利要求5或8所述的应用,或,权利要求6至8任一所述的方法,其特征在于:所述植物是如下c1)至c4)中的任一种:
c1)双子叶植物;
c2)单子叶植物;
c3)十字花科植物;
c4)拟南芥。
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