CN116497038B - 一种黄花苜蓿抗低温基因MfJAZ1及其应用 - Google Patents
一种黄花苜蓿抗低温基因MfJAZ1及其应用 Download PDFInfo
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Abstract
本发明公开了一种黄花苜蓿抗低温基因MfJAZ1及其应用,属于植物基因工程技术领域,所述抗低温基因MfJAZ1其核苷酸序列如序列1所示,所述MfJAZ1全长1176bp,分子式为C1181H1885N325O382S12,分子量为27.13kDa,还公开了黄花苜蓿抗低温基因MfJAZ1在提高植物对环境抗寒或抗低温性能中的应用,为培育出优良的抗寒植物做出贡献。
Description
技术领域
本发明涉及植物基因工程技术领域,尤其涉及一种黄花苜蓿抗低温基因MfJAZ1及其应用。
背景技术
温度是影响植物生长发育和地理分布的主要环境因子。低温胁迫(冷害和冻害)是限制作物生长、产量和品质的主要因素之一。因此,探索植物在低温胁迫下的应答机制及耐受机理,对改善作物低温耐受性,提高作物产量,具有十分重要的经济和社会价值。自然条件下,低温胁迫分为冷害和冻害,冻害对植物的影响比冷害大,会导致植物细胞内形成冰晶,促使细胞脱水,对膜脂结构造成机械损伤。低温胁迫下,植物会产生一系列的应激变化以低于低温损伤,如光合作用下降、呼吸作用上升、细胞代谢物质变化等。冷驯化是利用植物遇低温会产生体内物质变化,将植物暴露于不致死的低温下一定时间,从而提高植物对低温胁迫的耐受性。
苜蓿产业是我国草产业的支柱产业,但北方地区的冬季低温一直是限制苜蓿生长和越冬的主要环境因子。改良苜蓿品种的抗低温能力是保证其安全越冬的关键,是苜蓿育种领域亟需解决的关键科学问题。黄花苜蓿是豆科苜蓿属多年生牧草,其适应能力强,抗低温能力优于紫花苜蓿,并含有很多紫花苜蓿所不具备的抗性基因。发掘黄花苜蓿的优良抗逆基因来改良紫花苜蓿,培育出优良的栽培品种,可以提高苜蓿的产量和质量。因此研究黄花苜蓿的抗低温性能,对改善作物低温耐受性,提高作物产量,具有十分重要的经济和社会价值。
发明内容
鉴于此,本发明的目的是提供一种黄花苜蓿的抗低温基因MfJAZ1及其应用,进而培育出优良苜蓿品种。
本发明同过以下技术方案解决上述问题:
一种黄花苜蓿抗低温基因MfJAZ1,所述抗低温基因MfJAZ1其核苷酸序列如序列1所示,具体的核苷酸序列如下:
序列1:
AAGCAGTGGTATCAACGCAGAGTACGGGGGATCCAAAACAACAACACAAAGAATCATTCACACTTTCATTCTTATTACAACGCGTTTTGTTTTCTCTCTCTCTCTTGAGTCTTGAATATCATCATCTCTAACTATGTCTACCTCATCGGAATATTCAGAAGTTTCCGGCAACAAACCACCGGCGAAGTCACCGGAGAAAACAACTTTCTCTCAAACATGTAGTTTATTGAGTCAATATATTAAGGAAAAGGGTTGCTTCAAAGATCTTTCTCTTGGTATCACATGCAACGCAGACCCTTCTGGGTCTTCTGAGACTTCTTCTCAATCTGCAACAACCATGAACTTGTTTCCAACCATGGAAAACAATTTGACACAAAAGAACCTTACAACTATGGATTTGCTCACTCCACAAGCTTCTTTGAACAATTCCAATGCTATCAAGGGACCTAAAGCTGCACAATTGACAATGTTTTATAATGGTCAAGTTATTGTATTTGACGATTTTCCTGCCGACAAAGCACATGAGCTCATGGCTTTTGCTAATAAAGGAATCTCTCAAAGTCAGAACAATTCTGTGTACACTTACACACAGAGCCAGCCTTCATTTCCTCCTAATTTGGTCAGAACTTCGGTTAACACAACCGCTCCAATCGTTCCTACTGTGAACATCATTCCTAGTACTGGCACCGGCACCGGCTCGATTAATGAACACCTTCAAGTGCCTTCCAGACCTAATCTTTGCGATCTGCCAATTATGAGGAAAGCCTCGCTTCATCGGTTTCTGGAGAAGAGAAAGGATAGAATTGCTGCCAATGCACCATATCAAGTTAATAAGCCAGCTGAGTCCATGTCATGGCTTGTGGGTGCAAAATCAACTCAAATTTGATCTCAATTCTCAGCTATAATTTTAATGTAATTTTGAGCATTTTTGTTACAAAAAAACAGATGCTACTTAGTCAGATTTATTTTTGCCGGAATTTTTTAGACAAAGAAGTTTCTGGTTATTTTTTCCTTGACTAGATAGCATTATTAATATTATCATTATTTTTCTTGTTTAATGCACATGGTTAAGATGTAATATGTTATTGGTTTTTTGTTGGCCAAAAAAAAAAAAAAAAAAAAAAAAAAAGTACTCTGCGTTGATACCACTGCTTGCCCTATAGTGAGTCGTATTAG。
采用Protparam分析表明MfJAZ1编码蛋白含有20种基本氨基酸,Ser(12.4%)含量最高,Trp(0.4%)含量最低,22个带正电荷氨基酸残基(Arg、Lys),和18个带负电荷氨基酸残基(Asp、Glu)。
所述的MfJAZ1基因分子式为C1181H1885N325O382S12,分子量为27.13kDa,理论等电点为8.83;消光系数为14440-14690;不稳定系数是44.96,为不稳定蛋白;脂肪系数是68.32,平均亲水系数是-0.444,为亲水性蛋白。
利用SOPMA预测MfJAZ1基因蛋白二级结构,该蛋白含37个α-螺旋占比14.8%,7个β-转角占比2.8%,24个延伸链占比9.6%,182个无规卷曲占比72.8%。
本发明还公开了黄花苜蓿抗低温基因MfJAZ1的应用,黄花苜蓿抗低温基因MfJAZ1在提高植物对环境抗寒或抗低温性能中的应用,所述植物包括黄花苜蓿、拟南芥、烟草等。
有益效果:
MfJAZ1可以通过调节ROS清除、渗透平衡和KIN、ERD10C、COR15A、GSTF6、GSTF7、LEA5、和CAT基因的表达来降低植物抗低温能力,该结果为苜蓿耐寒育种提供了新的负调节基因,丰富了黄花苜蓿抗低温分子机制的研究,对提高苜蓿的产量和质量有重要意义。除此之外,也为将该基因应用于其他植物上开发抗低温作物奠定基础。
附图说明
图1:电泳检测结果图,其中A:总RNA电泳;B:cDNA质量评估;C:MfJAZ1 CDS序列电泳条带;M:2000bp DNA marker;1-3:电泳条带;
图2:大肠杆菌中MfJAZ1 5’及3’序列检测电泳图,其中第1、2、3泳道为5’序列,第4泳道为3’序列;
图3:MfJAZ1蛋白的三维模型;
图4:Genome Walking Kit特异性引物设计示意图;
图5:总DNA电泳(使用15000bp DNA Maker),B:染色体步移电泳(使用2000bp DNAMaker);
图6:黄花苜蓿MfJAZ1基因时空表达模式;A:组织特异性表达;B:低温胁迫下MfJAZ1基因的相对表达量变化;C:干旱胁迫下MfJAZ1基因的相对表达量变化;D:盐胁迫下MfJAZ1基因的相对表达量变化;E:碱胁迫下MfJAZ1基因的相对表达量变化;F:ABA胁迫下MfJAZ1基因的相对表达量变化;
图7:瞬时表达载体中MfJAZ1片段验证电泳图;
图8:MfJAZ1基因在烟草中的亚细胞定位;将MfAJZ1-GFP重组载体与35S:GFP空载体转入本氏烟烟草表皮细胞,比例尺,25μm;
图9:转基因拟南芥株系的叶片图;从左到右依次为WT(A)、过表达(B)、突变体(C)以及互补(D)植株叶片,比例尺,0.2cm
图10:转基因拟南芥在4℃(A)及-5℃(B)胁迫下相对电导率、MDA变化;
图11:转基因拟南芥在4℃(A)及-5℃(B)胁迫下O2·-、SOD活性变化;
图12:转基因拟南芥在4℃(A)及-5℃(B)胁迫下POD、CAT、GSH活性变化;
图13:转基因拟南芥在4℃(A)及-5℃(B)胁迫下可溶性糖含量、可溶性蛋白含量、脯氨酸含量变化。
图14:4℃和-5℃胁迫下转基因拟南芥中CAT基因、POD基因表达水平;
图15:4℃和-5℃胁迫下转基因拟南芥中KIN基因、LEA5基因表达水平;
图16:4℃和-5℃胁迫下转基因拟南芥中ERD10C基因表达水平;
图17:4℃和-5℃胁迫下转基因拟南芥中COR15A基因、GSTF6基因表达水平
图18:4℃和-5℃胁迫下转基因拟南芥中GSTF7基因表达水平。
具体实施方式
以下将结合具体实施例和附图对本发明进行详细说明:
本发明实施例中所使用的试验方法如无特殊说明,均为常规方法。本发明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。
本发明采用的黄花苜蓿、拟南芥和本氏烟种子由东北农业大学黑龙江省牧草种质资源与育种重点试验室提供。拟南芥jaz1突变体种子购自福州拜尔森特科技有限公司。
实施例1、植物培养
选取色泽明亮且饱满的黄花苜蓿种子,用5%NaClO溶液消毒5min,再用70%酒精溶液消毒30s,然后用蒸馏水冲洗5次。将清洗后的种子置于装有平铺的湿润滤纸的培养皿中,置于温度25℃和湿度70%的培养箱中培养。5d后将萌发的幼苗转移至装满蛭石的穴盘后置于人工温室中,昼夜温度及相对湿度分别设定为25℃/55%和20℃/70%。幼苗每2d用营养液喷洒1次。
实施例2、引物设计
qPCR引物利用IntegratedDNATechnologies设计,用Oligoo6.0对引物进行检测。3′和5’RACE扩增特异性引物利用诺唯赞公司云平台设计,其中5’RACE包括两条引物5’GSP和5’NGSP;3’RACE的特异性引物为3’GSP。其余引物由PrimerExpress5.0软件设计。具体的引物序列详情见表1。
表1引物序列
注:引物序列中的小写字母为同源臂序列,下划线处为酶切位点序列。
实施例3:黄花苜蓿MfJAZ1基因克隆
1、总RNA提取与反转录
选用正常生长条件下的健康黄花苜蓿植株,根据超纯RNA试剂盒(CWBIO,China)说明书对黄花苜蓿叶片进行研磨、裂解和纯化提取其总RNA,通过测定浓度以及电泳试验验证RNA完整性,验证无误后将RNA保存于-80℃冰箱中。根据HiScript ll ReverseTranscriptase反转录试剂盒(Vazyme,China)说明书计算RNA使用量,反转录成cDNA。
2、MfJAZ1基因CDS区的克隆
以黄花苜蓿cDNA为模板,使用2×Taq Master Mix进行PCR扩增,Taq酶PCR反应体系见表2,反应程序如下。琼脂糖凝胶电泳验证,在紫外光下观察片段长度与NCBI中蒺藜苜蓿MtJAZ1片段长度相似后,使用2×Max Master Mix再次进行PCR。高保真PCR反应体系见表3,反应程序如下。将PCR产物与上样缓冲液混合后进行1%的琼脂糖凝胶电泳,使用试剂盒进行切胶回收,测量回收产物浓度,标记后置于-20℃保存。
表2Taq酶PCR反应体系
Taq酶PCR反应程序如下:
表3高保真PCR反应体系
高保真PCR反应程序如下:
结果分析:
对提取的黄花苜蓿幼苗总RNA进行电泳检测,得到的结果如图1-A所示,28S和18S条带清晰。利用反转录试剂盒将RNA进行反转录,以MfActin基因为内参引物评估cDNA质量,得到的结果如图1-B所示。以豆科模式植物蒺藜苜蓿的MtJAZ1基因序列为模板设计基因特异性引物(F/R),对反转的黄花苜蓿DNA通过PCR扩增获得黄花苜蓿中JAZ1的CDS序列,命名为MfJAZ1,并进行琼脂糖凝胶电泳验证DNA质量,得到的结果如图1-C所示。
3、3’cDNA和5’cDNA的克隆
根据得到的中间片段,在上下游设计特异性引物,利用HiScript-TS 5’/3’RACEKit试剂盒的参考说明书进行该序列的3’端扩增和5’端扩增。首先提取RNA,之后分别进行3’端扩增和5’端扩增,得到的结果如图2所示,切胶回收、转入T载、转化DH5α大肠杆菌感受态、测序得到的序列长度去掉引物部分分别为291bp和133bp。利用DNAMAN5.0进行三段序列的拼接,获得全长为1176bp的cDNA序列如下:
AAGCAGTGGTATCAACGCAGAGTACGGGGGATCCAAAACAACAACACAAAGAATCATTCACACTTTCATTCTTATTACAACGCGTTTTGTTTTCTCTCTCTCTCTTGAGTCTTGAATATCATCATCTCTAACTATGTCTACCTCATCGGAATATTCAGAAGTTTCCGGCAACAAACCACCGGCGAAGTCACCGGAGAAAACAACTTTCTCTCAAACATGTAGTTTATTGAGTCAATATATTAAGGAAAAGGGTTGCTTCAAAGATCTTTCTCTTGGTATCACATGCAACGCAGACCCTTCTGGGTCTTCTGAGACTTCTTCTCAATCTGCAACAACCATGAACTTGTTTCCAACCATGGAAAACAATTTGACACAAAAGAACCTTACAACTATGGATTTGCTCACTCCACAAGCTTCTTTGAACAATTCCAATGCTATCAAGGGACCTAAAGCTGCACAATTGACAATGTTTTATAATGGTCAAGTTATTGTATTTGACGATTTTCCTGCCGACAAAGCACATGAGCTCATGGCTTTTGCTAATAAAGGAATCTCTCAAAGTCAGAACAATTCTGTGTACACTTACACACAGAGCCAGCCTTCATTTCCTCCTAATTTGGTCAGAACTTCGGTTAACACAACCGCTCCAATCGTTCCTACTGTGAACATCATTCCTAGTACTGGCACCGGCACCGGCTCGATTAATGAACACCTTCAAGTGCCTTCCAGACCTAATCTTTGCGATCTGCCAATTATGAGGAAAGCCTCGCTTCATCGGTTTCTGGAGAAGAGAAAGGATAGAATTGCTGCCAATGCACCATATCAAGTTAATAAGCCAGCTGAGTCCATGTCATGGCTTGTGGGTGCAAAATCAACTCAAATTTGATCTCAATTCTCAGCTATAATTTTAATGTAATTTTGAGCATTTTTGTTACAAAAAAACAGATGCTACTTAGTCAGATTTATTTTTGCCGGAATTTTTTAGACAAAGAAGTTTCTGGTTATTTTTTCCTTGACTAGATAGCATTATTAATATTATCATTATTTTTCTTGTTTAATGCACATGGTTAAGATGTAATATGTTATTGGTTTTTTGTTGGCCAAAAAAAAAAAAAAAAAAAAAAAAAAAGTACTCTGCGTTGATACCACTGCTTGCCCTATAGTGAGTCGTATTAG。
采用Protparam分析表明MfJAZ1编码蛋白含有20种基本氨基酸,Ser(12.4%)含量最高,Trp(0.4%)含量最低,22个带正电荷氨基酸残基(Arg、Lys),和18个带负电荷氨基酸残基(Asp、Glu)。MfJAZ1分子式为C1181H1885N325O382S12,分子量为27.13kDa,理论等电点为8.83;消光系数为14440-14690;不稳定系数是44.96,为不稳定蛋白;脂肪系数是68.32,平均亲水系数是-0.444,为亲水性蛋白。
利用SOPMA预测MfJAZ1基因蛋白二级结构,该蛋白含37个α-螺旋占比14.8%,7个β-转角占比2.8%,24个延伸链占比9.6%,182个无规卷曲占比72.8%。SWISS-MODEL预测蛋白三维结构如图3所示。
实施例4:MfJAZ1启动子克隆
利用Takara公司的Universal Genomic DNA Extraction Kit试剂盒对总DNA进行提取,具体研究方式按照说明书方法进行,随后根据已经获得的MfJAZ1的cDNA5’端上游序列设计3条特异性引物,设计的引物为反向互补引物,分别为SP1、SP2和SP3,详情见表1,用于巢式PCR,引具体位置如图4所示。
根据DNA提取试剂盒的相关要求对黄花苜蓿提取基因组DNA并进行测序,得到的结果如图5-A所示。根据得到的cDNA片段,在上游设计三条特异性引物SP1、SP2、SP3,以黄花苜蓿基因组DNA为模板,利用染色体步移试剂盒进行三次步移反应,得到的结果如图5-B所示。对后两次次产物进行胶回收后转入T载、转化DH5α感受态测序对比拼接后得到序列长度为933bp。
采用PlantCARE对启动子序列进行分析,分析结果如下:
MfJAZ1启动子序列中包含有GATA-motif、TCT-motif以及G-box三种光响应元件,一个水杨酸响应元件(TCA),2个MYB结合位点(MBS),4个MYB相关元件(一个MYB-like还有3个MYB),一个MYBHv1结合位点,一个厌氧诱导调节元件(ARE),还有2个AT~TATA-box、13个TATA-box以及8个CAAT-box,具体的MfJAZ1基因启动子序列如序列表2所示:
序列表2:
GTCGAGAGAGAAGAAGGAGGAAATGCACCAAAAGGACGTTGCTGCTCCAGGTCACACACAACCGATGTGGTGGGGTGCGCAACCTGTGCAACTGTTTCAGACACGCCTCGCCAGAGCTTTAAGTTTCTATGTTTCCACAAGCTCCACAAAAGGGCAACAAAACGTTGGCTCTCATCGTGTGAAAGATCCTGCACCAAAGCAAATATAGAATCGACAGCACAAACGAAAGGAGTACATGCACGTTGCACTTTCTCCTAAAGCCCAACCCTAAGCCTCACCTGCAAAGCAAAAAAGCAATCAAAGAAAACATGATGAAGATTTTCGAGAGGTCCGTCGCATATAATCTTACTATATATAAGTACCTCCTCCCACATTTATAAACAAATACCTACATTTTAGATATATTAAACATTTAATGAAGATCAAATTCACCAAATATTCAACGGATCTAAAAAGTAGTTATTTATTTATAAATGTGATAGCTAGGAGAGAATAAATAATATCGTATTACATAAAAAATAATGTTTTTTTTTAAGTTGAGTTTGGACAAAACCCAAGAAACATAGGAGACAACTGCAAAGAAAAAAAATAATAAAATAAAAAAAGAAAAAGAGAAGAAGCATCCCTTAGACCAACTTTAAATAAAATCACACACATATTCCTCGTGCTTCAATTTCCCATCCCCAAAAAAGATAAAGACATTAATTTATTCCTCCAATTAAACTTTATACTAATATTAATTATTTCATTTCATTTTCTTATATATATAAGCTAACATAAGATTAACCATCCAAAACAACAACACAAAGAATCATTTCACACTTTCATTCTTATTACAACGCGTTTTGTTTTCTCTCTCTCGAGTCTTGAATATCATCATCTTCAACTATGTCTACCTCATCGGAATATTCAGAAGTTTCCGGCAACAAACCA
实施例5:表达模式分析
以培养5周的生长均一的黄花苜蓿幼苗为材料,进行MfJAZ1表达模式分析。首选,选取部分幼苗,根、茎、叶分组织取样,用以测定MfJAZ1在黄花苜蓿各组织特异性表达情况。然后,将幼苗分成5组,分别进行低温、干旱、盐、碱和ABA胁迫处理,处理时间为0、1、3、6、12、24h。其中低温胁迫处理温度为4℃,干旱、盐和碱胁迫处理分别使用浓度20% PEG6000、150mM NaCl和150mM NaHCO3水溶液进行胁迫,ABA胁迫处理通过外源喷施浓度100μM ABA进行胁迫。将各处理的黄花苜蓿幼苗分组织取样后-80℃超低温冰箱,以备后续的实时荧光定量PCR(qRT-PCR)分析。
qRT-PCR方法:提取总RNA,利用反转录试剂盒反转cDNA,PCR扩增并电泳验证后,对反转的cDNA进行稀释。以MfActin作为内参基因,对样品进行基因的定量检测。
结果分析:
如图6-A所示,黄花苜蓿叶片中的MfJAZ1表达量最高,而茎中表达量最低。与根相比,黄花苜蓿叶和茎中MfJAZ1的表达量显著具有显著性差异(P<0.05),分别为地下部MfJAZ1表达量的9.17倍和0.3倍。
由于MfJAZ1在黄花苜蓿的叶片上的表达量较高,所以选取黄花苜蓿的叶片来研究MfJAZ1对于逆境胁迫的响应情况。低温胁迫下,随着胁迫时间的延长,MfJAZ1表达量呈现先升高后降低的趋势,胁迫4h后,MfJAZ1表达量显著上调,表达量达到最大值,为0h的5.4倍;胁迫8h后,MfJAZ1表达量较胁迫4h显著下调(P<0.05),降至4h的37%(图6-B)。干旱胁迫下MfJAZ1表达量先略微降低,而后升高,胁迫24h后显著增加(P<0.05),达到最高值,为0h下表达量的7.66倍(图6-C)。盐胁迫6h后基因表达量显著降低(P<0.05)达到最低,为0h的0.024倍;之后在24h显著提高(P<0.05),为6h的10.04倍(图6-D)。在碱胁迫下MfJAZ1基因表达量持续下降,在48h达到最低值,为WT的0.18倍(图6-E)。而在ABA处理下,在1h MfJAZ1基因表达量显著降低(P<0.05),为WT的38.5%,之后较1h没有显著性差异变化(图6-F)。即,低温胁迫下,黄花苜蓿叶片中MfJAZ1的表达呈先升高后降低的趋势。
实施例6:过表达及瞬时表达载体的构建
以MfJAZ1的cDNA序列,用Primer5软件,分别设计过表达引物和瞬时表达引物,其中过表达及瞬时表达引物F端引物一致,而过表达引物R端含终止密码子,瞬时表达引物R端不含终止密码子。然后,按照质粒提取试剂盒说明书提取并纯化DH5α菌液的质粒,并以质粒为模板,使用含有保护碱基和酶切位点碱基的瞬时(TE)和过表达(OE)一步克隆引物进行PCR扩增,反应体系见表4。反应后进行1%琼脂糖凝胶电泳,观察条带无误后进行回收获得插入片段,并测定浓度,置入-20℃保存。
表4一步克隆PCR反应体系
将含有pCBMBIA1300-35S-sGFP空载体的大肠杆菌置于37℃、180rpm的摇床上,待其浑浊后提取质粒,测定浓度并标记。按照表5配制酶切反应体系,37℃酶切6h,加入7μLloading Buffer终止反应。对酶切载体片段进行0.8%琼脂糖凝胶电泳,回收较长片段,纯化后测定浓度。按照一步克隆试剂盒说明书将线性化载体与插入片段重组,反应体系见表6,反应程序:37℃、30min,冰上冷却5min,取5μL产物转化DH5α感受态。获取菌液后进行PCR及电泳验证,测序比对后提取质粒,将其转化至EHA105感受态,将菌液涂布在含50μg/mLKan和20μg/mL利福平(Rif)的YEB平板上,倒置培养48-72h,挑取单克隆菌落转入含相同浓度Kan和Rif的YEB液体培养基中,摇菌60h,PCR检测后将阳性菌液混合甘油后存于-80℃备用。
表5酶切体系
表6重组反应体系
实施例7:MfJAZ1亚细胞定位
选取籽粒饱满的本氏烟草种子,培养于装有蛭石与营养土1:1混合的盆中。待烟草生长出5-6片叶片时进行瞬时表达试验。
首先用BamH1和SacI限制性内切酶对pCAMBIA1300载体进行双酶切,回收载体片段,电泳检测后回收提纯。然后利用瞬时表达的一步克隆引物MfJAZ1-TE-F/R,将MfJAZ1插入片段与线性化载体重组。转入T载、转化DH5α感受态,获得菌液进行MfJAZ1基因片段PCR鉴定,如图7所示,通过琼脂糖凝胶电泳检测验证重组成功。
将构建的烟草瞬时表达载体35S-MfJAZ1-GFP转化至EHA105感受态中,经验证后将菌液转入培养基培养至对数生长期,离心收集菌体沉淀,以0.5mol/LMES、1mol/L MgCl2、100mmol/L乙酰丁香酮为工作液,调节OD600分别为0.2和0.4。用渗入法将其注射到本氏烟烟草叶面背部感染烟草细胞,暗培养48h,后在激光共聚焦显微镜下观察。
结果分析:如图8所示,MfJAZ1基因定位于细胞核中,说明该基因可能参与调节植物的生长发育、遗传代谢等重要生理过程。
实施例8:转基因拟南芥的验证
根据TAIR公布的突变体(SALK_011957C)信息,设计三条特异性引物JAZ1-F1、JAZ1-R1以及JAZ1-T1(具体的如表1所示)进行PCR反应;
采用农杆菌侵染拟南芥花序的转化方法得到侵染后的拟南芥植株;
将拟南芥植株收种后筛选疫苗,待苗长20d左右,提取拟南芥基因组DNA,进行PCR检测;
将阳性苗进行T1加代种植,然后进行T2加代种植,收取T2代种子以备后期使用。
根据实施例1的步骤培养WT、过表达、突变体、互补株系拟南芥幼苗。取培养3周的生长一致的植株分成两组进行低温胁迫处理。一组进行4℃胁迫处理,处理时间为1、3、6、12、24h,另一组进行-5℃胁迫处理,处理时间为0(培养箱温度降到-5℃时的处理时间)、0.5、1h,室温环境生长的拟南芥幼苗为对照(Control)。分别收取各处理下的拟南芥叶片存至-80℃冰箱,以备后续的生理指标测定。生理指标测定方法见表7,得到的结果见表8和图9-13。
表7生理指标测定方法汇总
表8转基因株系拟南芥叶面积、周长、叶长及叶宽对比表
分析图9-13的结果可知:
成功构建过表达载体pCAMBIA1300-MfJAZ1,并通过农杆菌遗传转化WT拟南芥和jaz1突变体,获得MfJAZ1过表达和MfJAZ1互补株系。形态观测表明:MfJAZ1过表达株系的叶面积要显著小于WT、jaz1突变体和MfJAZ1互补株系,而JAZ1缺失会使拟南芥叶片狭长。生理指标分析表明:4℃胁迫下,MfJAZ1的过表达显著提高了拟南芥的相对电导率,降低了CAT活性和MDA含量。MfJAZ1的过表达能够显著增加-5℃胁迫下拟南芥的相对电导率、MDA、可溶性糖及脯氨酸含量,显著降低GSH、CAT及POD含量。然而,jaz1突变体中响应指标则呈现出相反的结果。
实施例9:转基因拟南芥在低温胁迫下相应基因的定量表达
综合实施例8的测定结果,选取MfJAZ1转基因拟南芥中响应生理机制较为明显的4℃胁迫24h以及-5℃胁迫1h,分别对WT、MfJAZ1过表达、jaz1突变体及MfJAZ1互补株系拟南芥进行4℃胁迫24h和-5℃胁迫1h后对8个胁迫响应基因的定量表达进行研究。利用qPCR检测检测低温胁迫下转基因拟南芥中MfJAZ1以及8个胁迫响应基因的表达,胁迫响应基因具体功能见表9。以AtActin作为内参基因,对样品进行胁迫响应基因的定量检测。得到的结果如图14-18所示。
表9胁迫响应基因汇总
分析图14-18的结果可知:
通过分析低温胁迫下KIN、ERD10C、COR15A、GSTF6、GSTF7、LEA5、CAT和POD等8个胁迫响应基因的表达模式发现,MfJAZ1的过表达能够在低温胁迫下显著下调KIN、ERD10C、COR15A、GSTF6、GSTF7、LEA5、和CAT的表达,而jaz1突变体中KIN、ERD10C、COR15A、GSTF6、GSTF7、LEA5、和CAT的表达量显著上调。说明MfJAZ1可以通过负调控胁迫响应基因的表达从而降低植物对低温的耐受性,而敲除JAZ1基因可以提高植物的抗寒能力。
以上实施例仅用以说明本发明的技术方案而非限制,尽管参照较佳实施例对本发明进行了详细说明,本领域的普通技术人员应当理解,可以对本发明的技术方案进行修改或者等同替换,而不脱离本发明技术方案的宗旨和范围,其均应涵盖在本发明的权利要求范围当中。本发明未详细描述的技术、形状、构造部分均为公知技术。
Claims (3)
1.一种黄花苜蓿抗低温基因MfJAZ1,其特征在于,所述抗低温基因MfJAZ1其核苷酸序列如序列1所示。
2.根据权利要求1所述的一种黄花苜蓿抗低温基因MfJAZ1的应用,其特征在于,所述黄花苜蓿抗低温基因MfJAZ1在提高黄花苜蓿对环境抗寒或抗低温性能中的应用。
3.根据权利要求1所述的一种黄花苜蓿抗低温基因MfJAZ1的应用,其特征在于,所述黄花苜蓿抗低温基因MfJAZ1在提高拟南芥对环境抗寒或抗低温性能中的应用。
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