CN117551659A - AcMT2a基因及其应用 - Google Patents
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Abstract
本发明提供了一种如SEQ ID NO:1所示的AcMT2a基因及其编码基因与应用。本发明首次从猕猴桃中克隆了AcMT2a基因的全长cDNA,该基因在树皮、雌花、根、雄花和果实中表达丰度高,参与对丁香假单胞菌胁迫应答过程,且能影响猕猴桃果实的发育和后熟软化,可作为猕猴桃转基因育种的重要靶基因。将该基因转入原核表达菌株,转基因菌株在重金属离子、NaCl胁迫、过氧化氢胁迫以及高温胁迫下的生长得到明显提高,在低温胁迫下的生长得到明显抑制,也说明该基因编码的蛋白可通过清除活性氧提高植物对逆境的耐受性。所以,该基因可作为重要的基因资源,可以在猕猴桃以及其他植物或微生物的抗逆和抗重金属胁迫基因工程中得到应用。
Description
技术领域
本发明属于基因工程技术领域,具体涉及一种AcMT2a基因及其应用。
背景技术
猕猴桃属猕猴桃科猕猴桃属植物,营养丰富,VC含量甚高,鲜食与加工皆可,经济价值高,素有水果之王的美誉。近年来,猕猴桃产业尽管取得了较快发展,但受土壤重金属污染和非生物胁迫,植物的生长发育受到严重的影响(Huang Wei,Wan Mingchang,QiaoRong.Development status and countermeasures of kiwifruit industry inGuizhou.Guizhou Agricultural Sciences,2012,40(4):184-186.)。
我们从猕猴桃中鉴定到一个AcMT2a基因,其与猕猴桃果实的发育和后熟软化密切相关,且参与了猕猴桃对丁香假单胞菌胁迫应答过程,可作为猕猴桃转基因育种的重要靶基因,而且该基因还可以提高原核菌株在重金属胁迫、氯化钠胁迫、过氧化氢胁迫和高温胁迫下的生长,可作为重要的基因资源,可以在猕猴桃以及其他植物或微生物的抗逆和抗重金属胁迫基因工程中得到应用。
发明内容
本发明的目的在于克服现有技术中的不足,提供一种AcMT2a基因及其应用。
本发明的第一个方面是提供一种猕猴桃AcMT2a基因,其核苷酸序列如SEQ ID NO:1所示。
本发明的第二个方面是提供本发明第一个方面所述的猕猴桃AcMT2a基因编码的蛋白质,其氨基酸序列如SEQ ID NO:2所示。
本发明的第三个方面是提供含有本发明第一个方面所述的猕猴桃AcMT2a基因编码区的重组载体或宿主菌。
其中,所述原始载体可以采用基因重组领域中常用的载体,例如病毒、质粒等。本发明对此不进行限定。在本发明的一个具体实施方式中,所述原始载体采用PET28a载体质粒或pMD18-T载体质粒,但应当理解的是,本发明还可以采用其他质粒、或者病毒等。
优选地,所述原始载体为PET28a表达载体,猕猴桃AcMT2a基因编码区位于PET28a表达载体的EcoR I和SalI两限制性内切酶位点之间。
本发明的第四个方面是提供如本发明第一个方面所述的AcMT2a基因、或者本发明第二个方面所述的蛋白质、或者本发明第三个方面所述的重组表达载体在提高原核表达菌株抗铜金属胁迫、和/或锌金属胁迫、和/或NaCl胁迫、和/或过氧化氢胁迫、和/或高温胁迫中的应用;或者如本发明第一个方面所述的AcMT2a基因、或者本发明第二个方面所述的蛋白质、或者本发明第三个方面所述的重组表达载体在降低原核表达菌株抗低温胁迫中的应用。
其中,所述原核表达菌株可以是基因重组领域中常用的原核表达菌株,本发明对此不进行限定,比如E.coli BL21(DE3)、E.coli Rosetta和E.coli OrigamiB(DE3)等。在本发明的一个具体实施方式中,所述原核表达菌株为大肠杆菌E.coli BL21(DE3)。
本发明的第五个方面是提供如本发明第一个方面所述的AcMT2a基因、或者本发明第二个方面所述的蛋白质、或者本发明第三个方面所述的重组表达载体或宿主菌在调控猕猴桃果实的发育、和/或后熟软化中的应用。
本发明的第六个方面是提供如本发明第一个方面所述的AcMT2a基因、或者本发明第二个方面所述的蛋白质、或者本发明第三个方面所述的重组表达载体或宿主菌在调控猕猴桃对丁香假单胞菌胁迫应答中的应用。
本发明的第七个方面是提供如本发明第一个方面所述的AcMT2a基因、或者本发明第二个方面所述的蛋白质、或者本发明第三个方面所述的重组表达载体或宿主菌在通过清除活性氧提高植物对逆境的耐受性中的应用。
本发明的第八个方面是提供一种引物对,所述引物对为:5′-TCTCTCACAACTCTCTCTCTCTC-3′和5′-TGCTTTGTAGAAGGCTGGTAATTAG-3′;或者所述引物对为:5'-GAAAAATGTCTTGCTGCGGAG-3'和R:5'-TACATCTGGCACCACAACGAC-3';或者所述引物对为:5'-CCGGAATTCATGTCTTGCTGCGGAGG-3'和5'-ACGCGTCGACTCATTTGCAGGTGCAAG-3'。
本发明首次从猕猴桃中克隆了AcMT2a基因的全长cDNA,基因表达分析显示AcMT2a基因在树皮、雌花、根、雄花和果实中表达丰度高,丁香假单胞菌侵染猕猴桃能够调控AcMT2a基因的表达,且AcMT2a基因表达变化与猕猴桃果实的发育和后熟软化密切相关,表明该基因参与对丁香假单胞菌胁迫应答过程,且能影响猕猴桃果实的发育和后熟软化,可作为猕猴桃转基因育种的重要靶基因。将该基因转入原核表达菌株,转基因菌株在重金属离子(Cu2+、Zn2+)、NaCl胁迫、过氧化氢胁迫以及高温胁迫下的生长得到明显提高,在低温胁迫下的生长得到明显抑制,说明AcMT2a基因编码的蛋白可通过清除活性氧提高植物(比如猕猴桃等)对逆境的耐受性。所以,该基因可作为重要的基因资源,可以在猕猴桃以及其他植物或微生物的抗逆和抗重金属胁迫基因工程中得到应用。
附图说明
图1为AcMT2a基因的组织特异性表达分析(叶子、种子、树皮、果实、雌花、雄花、根)。
图2为AcMT2a基因在不同处理猕猴桃组织样品中的转录组数据分析。
图3为AcMT2a的原核表达分析(箭头所指条带为受IPTG诱导后所产生的特异表达的目标蛋白)。
图4A-4F为转基因大肠杆菌在培养基上生长情况(A:在重金属铜离子胁迫下的OD600测定;B:在过氧化氢胁迫下的OD600测定;C:在氯化钠胁迫下的OD600测定;D:在重金属锌离子胁迫下的OD600测定;E:在20℃胁迫下的OD600测定;F:在45℃胁迫下的OD600测定)。
具体实施方式
下面参照附图,结合具体的实施例对本发明作进一步的说明,以更好地理解本发明。实施例中未注明具体技术或条件者,按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者,均为可以通过市购获得的常规产品。
实施例1.猕猴桃AcMT2a基因的克隆
特异性引物设计如下:
F(5′端):5′-TCTCTCACAACTCTCTCTCTCTC-3′;
R(3′端):5′-TGCTTTGTAGAAGGCTGGTAATTAG-3′。
以“红阳”猕猴桃树皮的cDNA为模板(以随机引物反转录获得),F和R为引物,进行PCR扩增。反应体系为:cDNA模板1μL、10×PCRBuffer 2μL、Taq plus DNAPolymerase(5U·μL-1)0.2μL、dNTP(2.5mmol·L-1)1.6μL、上下游引物(10μmol·L-1)各1μL、ddH2O 13.2μL,共20μL。扩增程序为:94℃预变性5min;94℃变性30s,58℃退火30s,72℃延伸60s,30次循环;72℃延伸10min。
将该获得的一段320bp左右的核苷酸片段连接到PMD18-T载体(TAKARA公司)进行测序,该片段序列如SEQ ID No:1所示,全长为325个核苷酸,包含一个长度为255个核苷酸的开放阅读框(ORF,自序列1的5′端第35-289位核苷酸序列)、34个核苷酸的5′-UTR(自序列1的5′端第1-34位核苷酸序列)和36个核苷酸的3′-UTR(自序列1的5′端第290-325位核苷酸序列),编码一个长度为84个氨基酸(序列表中SEQ ID No:2)、分子量约为8.5kDa的蛋白。将该基因命名为AcMT2a基因,将上述含有SEQ ID No:1的核苷酸的PMD18-T重组载体命名为PMD18-AcMT2a。
实施例2.猕猴桃AcMT2a基因表达模式分析
<1>猕猴桃AcMT2a基因组织特异性表达
以“红阳”猕猴桃树皮、雄花、根、雌花、果实、种子、叶片总RNA逆转录得到的cDNA为模板,用AcMT2a基因特异引物F:5'-GAAAAATGTCTTGCTGCGGAG-3'和R:5'-TACATCTGGCACCACAACGAC-3'进行实时荧光定量PCR。反应体系为2μL模板、10μL2×SYBRPremix和10μmol·L-1的上下游引物各0.3μL;扩增程序为95℃预变性30s;94℃5s,60℃20s,72℃20s,45个循环。利用LightCycler 4.05软件分别计算引物扩增效率和相应Qr值。结果显示该基因在“红阳”猕猴桃的7个组织中均有表达,在“红阳”猕猴桃的树皮中表达量最高,雄花其次,叶子中最低(图1)。
<2>对猕猴桃进行不同处理对AcMT2a基因的表达影响
通过高通量测序技术可获得物种在不同发育时段、逆境胁迫下基因时空表达模式的变化。本实验利用Kiwifruit Genome Database(http://kiwifruitgenome.org/)中的转录组数据,获取AcMT2在不同处理下猕猴桃组织样品中的RPKM值,分析其表达模式。
样品HY0、HY12、HY24、HY48和HY96指在‘红阳’猕猴桃的树皮上接种能引起猕猴桃细菌性溃疡病的丁香假单胞菌,分别在接种后0小时、12小时、24小时、48小时和96小时后采集树皮。
样品Aa_0DPI、Aa_2DPI、Aa_14DPI‘软枣’猕猴桃叶片接种丁香假单胞菌0天、2天和14天后采集叶片。
样品Ae_0DPI、Ae_2DPI、Ae_14DPI‘毛花’猕猴桃叶片接种丁香假单胞菌0天、2天和14天后采集叶片。
样品AH_0DPI、AH_2DPI、AH_14DPI‘红阳’猕猴桃叶片接种丁香假单胞菌0天、2天和14天后采集叶片。
样品AJ_0DPI、AJ_2DPI、AJ_14DPI‘金艳’猕猴桃叶片接种丁香假单胞菌0天、2天和14天后采集叶片。
样品HealtyControl、I3、I24、I48未接种丁香假单胞菌、接种丁香假单胞菌3小时、24小时和48小时的中华猕猴桃,采集整株猕猴桃苗。
样品ASM、ASMI3、ASMI24、ASMI48指对植物激活剂苯丙噻重氮(ASM)处理15天后的中华猕猴桃幼苗,未处理、接种丁香假单胞菌3小时、24小时和48小时,采集整株猕猴桃苗。
样品DAP20_immature、DAP120_matureGreen、DAP127_ripe采集授粉后20天、120天和127天后红阳猕猴桃的果实。
样品147DAFB、168DAFB、175DAFB、223DAFB采集花后147天、168天、175天和223天的‘Hort16A’猕猴桃果实。
样品231DAFB_CT、231DAFB_1DAT、231DAFB_2DAT、231DAFB_4DAT采集花后231天的‘Hort16A’猕猴桃果实未处理、乙烯处理1天、乙烯处理2天和乙烯处理4天后的果实。
样品Sanuki_Gold_Control、Sanuki_Gold_5CW4、Sanuki_Gold_20CW4指采集成熟的‘Sanuki_Gold’猕猴桃、5度储藏4个星期、20度储藏4个星期的果实。
AH_leaf、AH_root、AH_stem红阳猕猴桃的叶、根、茎。
结果如图2所示。接种了丁香假单胞菌后,“红阳”猕猴桃树皮和中华猕猴桃组培苗中的AcMT2的表达量在前期有明显的上调表达,之后下降恢复至初始水平;而AcMT2a基因在“金艳”猕猴桃的叶片中明显上调表达,在“红阳”猕猴桃和“毛花”猕猴桃叶片中呈下调表达,在“软枣”猕猴桃叶片中变化不大;且植物抗病性化学诱导剂苯丙噻重氮(ASM)处理后,其表达水平呈明显下降。而随着果实的发育,AcMT2a在“红阳”和“Hort16A”两个品种的果实中都呈现明显的下调表达;但在猕猴桃果实后熟软化阶段,乙烯和低温两种促进果实后熟的调控手段能够上调AcMT2a基因的表达。结果表明,AcMT2a基因与猕猴桃果实的发育和后熟软化密切相关,且参与了猕猴桃对丁香假单胞菌胁迫应答过程。
实施例3.原核表达与AcMT2a基因的功能验证
利用PET28a表达载体构建了AcMT2a基因的原核表达载体(本实施例中表达载体仅为举例,本发明也可以采用其他表达质粒和病毒载体等),同时采用大肠杆菌表达菌株E.coli BL21(DE3)诱导重组蛋白,测定重组蛋白对BL21菌株生长的影响,具体方法如下:
<1>含AcMT2a基因编码区重组载体的获得
设计AcMT2a基因编码区引物
F:5'-CCGGAATTC(EcoRI酶切位点)ATGTCTTGCTGCGGAGG-3',
R:5'-ACGCGTCGAC(SalI酶切位点)TCATTTGCAGGTGCAAG-3',
以pMD18-AcMT2a为模版,进行PCR扩增,反应体系为cDNA模板1μL、10×PCRBuffer2μL、Taqplus DNAPolymerase(5U·μL-1)0.2μL、dNTP(2.5mmol·L-1)1.6μL、上下游引物(10μmol·L-1)各1μL、ddH2O 13.2μL,共20μL。扩增程序为:95℃预变性4min;94℃变性45s,68℃退火30s,共30次循环;72℃延伸8min。将扩增产物与PET28a表达载体通过EcoR I和SalI双酶切连接,得到重组载体。重组载体进行测序鉴定,将鉴定表明正确的含有序列表中序列1的第35-289位核苷酸序列、读框准确的重组表达载体命名为PET28a-AcMT2a。
<2>AcMT2a基因的原核表达
将获得的重组载体PET28a-AcMT2a导入E.coli BL21(DE3)中,得到重组表达菌,将鉴定正确的重组菌在含50μg/mL氨苄青霉素的LB培养基中培养至OD600=0.4~0.6,添加IPTG(异丙基-β-D-硫代半乳糖苷)至终浓度为1mM,30℃下诱导培养4h,以空载质粒PET28a为对照,离心收集菌体,菌体蛋白进行15%SDS-PAGE电泳检测。结果表明,在IPTG诱导下AcMT2a这个基因实现了高效异源表达,重组蛋白包含了目的蛋白且蛋白的分子量与理论分子量相近,大约16kDa(图3)。
<3>AcMT2a重组蛋白对菌株生长的影响
将含有PET28a-AcMT2a和PET28a的菌株培养至相同OD600,添加IPTG至终浓度1mM,测定含有PET28a-AcMT2a和PET28a的菌株在重金属离子(Cu2+,500μM;Zn2+,500μM)、盐(NaCl,500mM)、过氧化氢(H2O2,1mM)、低温(20℃)和高温(45℃)胁迫下培养0h、0.5h、1h、1.5h、2h、2.5h、3h和3.5h后分别测定其OD600值。结果如图4显示:除了低温胁迫外,在其他胁迫下含有PET28a-AcMT2a菌株的增长高于含有PET28a的菌株,说明AcMT2a基因编码的蛋白可以提高菌株在重金属铜离子、锌离子、氯化钠、过氧化氢胁迫和高温迫下的生长,也说明AcMT3a基因编码的蛋白可通过清除活性氧提高植物(比如猕猴桃等)对逆境的耐受性。将AcMT2a基因导入其他原核表达菌(E.coli Rosetta和E.coli OrigamiB(DE3)等)进行上述试验,结果同大肠杆菌,AcMT2a基因编码的蛋白可以提高其他原核菌株在重金属胁迫、氯化钠胁迫、过氧化氢胁迫和高低温胁迫下的生长。
以上对本发明的具体实施例进行了详细描述,但其只是作为范例,本发明并不限制于以上描述的具体实施例。对于本领域技术人员而言,任何对本发明进行的等同修改和替代也都在本发明的范畴之中。因此,在不脱离本发明的精神和范围下所作的均等变换和修改,都应涵盖在本发明的范围内。
Claims (10)
1.一种猕猴桃AcMT2a基因,其特征在于,其核苷酸序列如SEQ ID NO:1所示。
2.权利要求1所述猕猴桃AcMT2a基因编码的蛋白质,其氨基酸序列如SEQ ID NO:2所示。
3.含有权利要求1所述猕猴桃AcMT2a基因编码区的重组载体或宿主菌。
4.根据权利要求3所述的重组表达载体,其特征在于,所述原始载体为PET28a表达载体,猕猴桃AcMT2a基因编码区位于PET28a表达载体的EcoR I和SalI两限制性内切酶位点之间。
5.如权利要求1所述的猕猴桃AcMT2a基因、或者权利要求2所述的蛋白质、或者权利要求3或4所述的重组表达载体在提高原核表达菌株抗铜金属胁迫、和/或锌金属胁迫、和/或NaCl胁迫、和/或过氧化氢胁迫、和/或高温胁迫中的应用;或者
如本发明第一个方面所述的AcMT2a基因、或者本发明第二个方面所述的蛋白质、或者本发明第三个方面所述的重组表达载体在降低原核表达菌株抗低温胁迫中的应用。
6.根据权利要求5所述的应用,其特征在于,所述原核表达菌株为大肠杆菌E.coliBL21(DE3)、E.coli Rosetta或E.coli OrigamiB(DE3)。
7.如权利要求1所述的猕猴桃AcMT2a基因、或者权利要求2所述的蛋白质、或者权利要求3或4所述的重组表达载体或宿主菌在调控猕猴桃果实的发育、和/或后熟软化中的应用。
8.如权利要求1所述的猕猴桃AcMT2a基因、或者权利要求2所述的蛋白质、或者权利要求3或4所述的重组表达载体或宿主菌在调控猕猴桃对丁香假单胞菌胁迫应答中的应用。
9.如权利要求1所述的猕猴桃AcMT2a基因、或者权利要求2所述的蛋白质、或者权利要求3或4所述的重组表达载体或宿主菌在通过清除活性氧提高植物对逆境的耐受性中的应用。
10.一种引物对,其特征在于,所述引物对为:5′-TCTCTCACAACTCTCTCTCTCTC-3′和5′-TGCTTTGTAGAAGGCTGGTAATTAG-3′;或者
所述引物对为:5'-GAAAAATGTCTTGCTGCGGAG-3'和R:5'-TACATCTGGCACCACAACGAC-3';或者
所述引物对为:5'-CCGGAATTCATGTCTTGCTGCGGAGG-3'和5'-ACGCGTCGACTCATTTGCAGGTGCAAG-3'。
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