CN109825511B - 银杏GbBBX25基因及其表达蛋白和应用 - Google Patents
银杏GbBBX25基因及其表达蛋白和应用 Download PDFInfo
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- CN109825511B CN109825511B CN201910248136.7A CN201910248136A CN109825511B CN 109825511 B CN109825511 B CN 109825511B CN 201910248136 A CN201910248136 A CN 201910248136A CN 109825511 B CN109825511 B CN 109825511B
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Abstract
本申请公开了一种银杏GbBBX25基因及其表达蛋白和应用,该银杏GbBBX25基因的碱基序列如SEQ ID NO.1所示。本申请从银杏中分离得到GbBBX25基因,编码了273个氨基酸的819bp开放阅读框(ORF),包含两个b‑box结构域,但没有CCT结构域。GbBBX25定位于细胞核,具有转录因子重要的核定位特征。GbBBX25转录本主要在叶片中表达量最高,盐胁迫下诱导效果显著。GbBBX25在山新杨中过量表达,证实了在盐胁迫条件下,转基因杨树的可溶性糖、总蛋白含量和过氧化物酶(POD)活性均高于非转基因杨树,表明GbBBX25可以通过提高抗氧化系统的效率来提高耐盐性。GbBBX25在转基因杨树中过量表达可以提高植株的耐盐性,提高GbBBX25基因的表达水平可用于育种中来面对非生物胁迫,具有很好的实用性。
Description
技术领域
本发明属于基因工程技术领域,具体涉及一种银杏GbBBX25基因及其表达蛋白和应用。
背景技术
植物已进化出了一种复杂的网络,它以高度复杂的机制应对不稳定的非生物和生物的条件。干旱、高盐、低温等非生物胁迫是影响植物生长发育的重要因素,在育种工作中,不仅需培育高产、优质的优良品种,而且还要培育出适应多种逆境的高抗品种。锌指蛋白在调控植物生长发育和应对逆境过程中发挥着重要作用。B-box(BBX)蛋白质是锌指转录因子或转录调节因子,其中包含一个或两个B-box。1995年,在拟南芥中发现了第一个B-box蛋白(CO)。2009年,在拟南芥中发现了32个具有N端B-box结构域的蛋白,统一命名为BBX1-32。根据B-box域的数量和序列特征以及蛋白质是否包含CCT域,分为5个结构组。结构组I有6个成员(CO,也称为BBX1到6),每一个都包含两个b-box域和一个CCT域。第II组有7个成员(BBX7到13),结构类似于I组,但在第二个b-box域有差异。结构组III有4个成员(BBX14到17),其特征是只有一个b-box域和一个CCT域。BBX2到17都是共同的(COL)蛋白质。结构组IV有8个成员(BBX18到25),其中包含两个b-box框域,但没有CCT域。最后,结构组V有7个成员(BBX26到32),每个成员只有一个b-box域。
BBX蛋白在响应光、昼夜节律信号和油菜素类固醇的光串扰信号中起转录调节作用有研究表明BBX蛋白可以促进非生物应激反应。据报道,AtBBX24的过量表达被认为是提高了耐盐性。CmBBX24在菊花中的过量表达,提高了植物对低温和干旱的耐受水平。同时,AtBBX18通过控制一组热休克反应基因,参与热耐受。尽管取得了这些进展,BBX结构域在DNA结合中的确切生化作用及生物学功能仍在很大程度上不清楚。
银杏是一种生态经济树种,具有食品、医药、木材、景观和科研等价值-。银杏叶中含有丰富的活性成分,特别是黄酮类和萜内酯类化合物,能促进血液循环,抑制血栓形成。目前,由于其药用价值,它是世界上最受欢迎的功能性植物之一。银杏是银杏纲唯一存活的树种,可追溯到1.7亿年前侏罗纪时期。地质记录表明,银杏形态发生了微小的变化,表明其对环境变化的适应性强,对恶劣环境的耐受性强。
迄今为止,BBX基因已在一些物种中有报道,包括藻类、蕨类、针叶、单子叶和双子叶植物。但尚未有关于银杏BBX锌指类蛋白的功能研究。
发明内容
发明目的:针对现有技术中存在的不足,本发明的目的是提供一种银杏GbBBX25基因,满足耐盐使用需求。本发明的另一目的是提供一种上述银杏GbBBX25基因的表达蛋白。本发明还有一目的是提供一种上述银杏GbBBX25基因的应用。
技术方案:为了实现上述发明目的,本发明采用的技术方案为:
银杏GbBBX25基因,其碱基序列如SEQ ID NO.1所示。
所述的银杏GbBBX25基因的表达蛋白,其氨基酸序列如SEQ ID NO.2所示。
含有所述的银杏GbBBX25基因的载体或宿主菌。
所述的银杏GbBBX25基因在提高植物可溶性糖活性中的应用。
所述的银杏GbBBX25基因在提高植物总蛋白含量中的应用。
所述的银杏GbBBX25基因在提高植物过氧化物酶活性中的应用。
所述的银杏GbBBX25基因在提高植物耐盐性中的应用。
所述的银杏GbBBX25基因在植物育种中的应用。
所述的银杏GbBBX25基因在改善抗氧化系统效率中的应用。
本申请利用转录组数据SRP137637克隆了银杏GbBBX25基因的cDNA,它编码了一个具有两个b-box结构域的锌指蛋白,利用生物信息学工具推测其蛋白质的物化性质。采用实时定量技术,分析银杏不同组织、时间和处理条件下GbBBX25的表达规律。同时,为进一步了解GbBBX25的功能,本申请还利用农杆菌介导的叶盘转化方法,在山新杨中异源表达GbBBX25,并确定转基因杨树株系。最后测定非转基因杨树和转基因杨树在盐胁迫下的可溶性糖、总蛋白含量和过氧化物酶(POD)活性。总之,与非转基因杨树相比,GbBBX25的过量表达显著提高了转基因杨树的耐盐性,为GbBBX25在盐胁迫反应中的作用提供了参考。
有益效果:与现有技术相比,本申请从银杏中分离得到GbBBX25基因,编码了273个氨基酸的819bp开放阅读框(ORF),包含两个b-box结构域,但没有CCT结构域。GbBBX25定位于细胞核,具有转录因子重要的核定位特征。GbBBX25转录本主要在叶片中表达量最高,盐胁迫下诱导效果显著。GbBBX25在山新杨中过量表达,证实了在盐胁迫条件下,转基因杨树的可溶性糖、总蛋白含量和过氧化物酶(POD)活性均高于非转基因杨树,表明GbBBX25可以通过提高抗氧化系统的效率来提高耐盐性。GbBBX25在转基因杨树中过量表达可以提高植株的耐盐性,提高GbBBX25基因的表达水平可用于育种中来面对非生物胁迫,具有很好的实用性。
附图说明
图1是盐处理6h的未转基因和转基因杨树图;
图2是银杏GbBBX25的转录谱分析结果图;图中,A、B为GbBBX25在银杏不同组织和不同时期的时空表达分析,A为植物各器官间转录丰度的变化,R:根,S:茎,L:叶,K:种仁,B:芽,P:叶柄;将茎和6月的基因表达水平设为1;C为MJ和SA这2种激素处理下的GbBBX25转录水平变化;D为黑暗、干旱(20%PEG 6000)和NaCl胁迫(200mmol/L)下GbBBX25的表达量;
图3是银杏GbBBX25蛋白的亚细胞定位图;绿色荧光蛋白(GFP)、叶绿素自发荧光(Auto);采用35::GFP融合蛋白作为阳性对照,10μm比例尺;
图4是GbBBX25转基因植株和未转基因植株的各项结果图;图中,A.含有目的片段的非转基因和转基因杨树的DNA层面的PCR扩增图;B.qRT-PCR检测10个转基因株系和非转基因杨树中GbBBX25的相对表达水平;C.非转基因杨树和转基因杨树在30天的生长状况;M为Mark,NT为非转基因杨树,T1-10为转基因杨树株系;
图5是转基因和非转基因杨树叶片绒毛的变化结果图,包括叶片的正面和背面;
图6是盐胁迫处理下非转基因与转基因杨树T1株系生理指标比较的结果图;图中,A,可溶性糖含量;B,总蛋白含量;C,POD活性。
具体实施方式
下面结合具体实施例对本发明做进一步的说明。
实施例1
1、植物材料和处理Plant materials and multiple stress treatments
在南京林业大学温室栽培1年生银杏幼苗,取其根、茎、叶、叶柄和校内25年生的银杏树的种仁和芽为材料进行基因克隆和表达分析。此外,对银杏幼苗进行干旱、盐、黑暗、水杨酸(SA)和茉莉酸甲酯(MJ)处理。在不同的胁迫条件下,每个取样时间进行三次生物学复制试验。用20%(w/v)PEG(MW 6000)和盐度(200mmol/L NaCl)处理幼苗。暗处理是将植物放置在黑暗的室内。采用外源激素处理是在银杏幼苗叶片上分别喷施100μM/L SA和MJ。处理苗与对照苗的其他条件相似。在处理后0、3、6、12、24和48h收集处理和对照幼苗叶片。所有收集到的材料立即液氮速冻,并在-80℃冰箱储存直到提取总RNA。
此外,无性系山新杨(Populus davidiana×Populus bolleana)种植在组培室的MS培养基中,白天/黑夜温度和光照时间为25℃/18℃下16/8h。从6周的植株上摘取平展的幼叶进行原生质体分离试验。
2、分子克隆
在银杏转录组数据(NCBI Short Reads Archive(SRA)database under accessnumber SRP137637)挑选BBX序列片段,对片段序列进行PCR扩增测序。然后,在经过验证的片段测序的基础上,采用两步法获得全长序列。根据试剂盒使用手册,使用Oligo软件(6.0版)设计嵌套引物,使用SMATer RACE 5′/3试剂盒(Clontech,CA,Palo Alto,USA)克隆得到全长序列。通过PCR扩增,切胶回收纯化,克隆连接到-Blunt Zero载体上(TransGenBiotech),并转入大肠杆菌菌株细胞(Trans1-T1 Phage Resistant ChemicallyCompetent Cell),得到与预计产物大小相同的片段送去测序。通过对5′-RACE和3′-RACE序列的比对和拼接,得到了GbBBX25的全长cDNA序列。将预测的开放阅读框(open readingframe,ORF)进行PCR扩增,测序验证。利用相同引物对该基因组的DNA进行扩增,并进行测序验证。利用Takara PrimerSTAR Max DNA聚合酶扩增预测的ORF cDNA和GbBBX25的DNA基因组序列。所有引物序列如表1所示。
表1所有引物序列
3生物信息学分析
使用NCBI ORF finder软件(http://www.ncbi.nlm.nih.gov/gorf/gorf.html)预测GbBBX25的ORF。利用Gene Structure Display server(http://gsds.cbi.pku.edu.cn/)从cDNA和基因组序列比对中确定外显子和内含子的结构特征。利用ExPASy ProtParam对理论等电点(pI)、分子量、氨基酸组成进行预测和计算。利用DNAMAN v6.0软件(http://www,lynnon.com/)对拟南芥中BBX组IV蛋白和GbBBX25的氨基酸序列进行比对。
采用RACE法得到GbBBX25的全长cDNA序列,该序列含有1396个核苷酸(序列如SEQID NO.1所示),包含819bp的ORF,249bp的5′未翻译区域(UTR)和328bp的3′未翻译区域(UTR)。蛋白质中含有273个氨基酸(序列如SEQ ID NO.2所示),分子量为29.60kDa,等电点为5.05,脂肪指数为74.07,平均亲水系数为-0.32。利用SOPMA软件预测该蛋白的二级结构。此外,通过比对cDNA和基因组序列,确定GbBBX25的外显子与内含子结构,它包含2个内含子。
BLASTP搜索结果显示,GbBBX25多肽与其他植物编码的BBX蛋白具有较大的相似性;该序列在N端区存在高度保守的双个b-box域,但在C端缺少CCT域。
4实时定量进行表达量分析
根据GbBBX25的cDNA序列设计qPCR特异性引物(表1)。采用qRT-PCR检测GbBBX25的表达模式。QRT-PCR在ABI ViiA7 Real-Time PCR system(Applied Biosystems,Carlsbad,CA,USA)上进行,使用FastStart Universal SYBR Green Master with ROX RT-PCR试剂盒(Roche,Indianapolis,USA)。PCR过程是50℃和95℃反应各2min,然后45个循环下的95℃1s和60℃30s。通过对扩增的熔解曲线分析,证实了PCR反应的特异性。Glyceraldehyde-3-Phosphate Dehydrogenase(GAPDH)基因作为银杏的内参基因,Elongation Factor 1alpha(EF1a)作为山新杨的参考基因(表1)。所有样品(每个实验一式三份)计算使用2-ΔΔCt方法。
5亚细胞定位
本实施例采用Gateway技术(Invitrogen)按照制造商的说明构建载体。将缺失终止密码子的GbBBX25编码区域克隆到载体pCRTM8/GW/TOPOTM(Invitrogen,Carlsbad,CA,USA)中,进行简单的TOPO克隆反应。使用LR克隆酶混合(Invitrogen)将GbBBX25片段从入门载体转移到目的载体(p2GWF7),并在插入物的C端放置绿色荧光蛋白标记。生成的GFP融合载体(35S::GbBBX25-GFP)为双35S花椰菜花叶病毒(CaMV)启动子驱动的载体,以氨苄青霉素为筛选标记。原生质体分离和PEG介导转染参照Tan et al.(2013)所述。所有荧光实验均独立重复三次。
6过表达载体构建与山新杨转化
通过PCR扩增GbBBX25 cDNA的ORF,利用Gateway技术(Invitrogen,CA,Carlsbad)将位于CaMV 35S启动子下游的ccdB基因克隆到PBI121载体上。将含有Pro35S::GbBBX25的载体导入农杆菌菌株EHA105中进行转化。利用山新杨(Populus davidiana×Populusbolleana)稳定和高效的遗传转化体系(Han et al.,2013),将银杏的GbBBX25基因转基因到山新杨中。
7转基因植株和生理指标测定
采用卡那霉素(Kan)抗性筛选后,利用pH35GS载体35S基因的正向引物和GbBBX25ORF的反向引物,采用PCR方法对非转基因杨树和假定的转基因杨树株系进行测定。采用水培方法进行植物的耐盐性试验。其中,2个月非转基因杨树和转基因杨树分别在含有200mmol/L NaCl的MS液体培养基中培养。胁迫处理后,收集表达量最高的转基因株系T1和非转基因山新杨的叶片进行可溶性糖、总蛋白含量和POD抗氧化酶的检测。同时,对6周的幼苗观察胁迫处理后非转基因杨树与表达量最高的转基因株系T1之间的表型变化(图1)。最后,将非转基因苗和转基因株系T1的100mg叶片分别研磨成细粉(每个生理指标)。采用蒽酮比色法测定可溶性糖含量,利用植物可溶性糖含量试剂盒(A145);总蛋白含量采用总蛋白定量试剂盒(A045-2);POD活性的测定使用过氧化物酶试剂盒(A084-3),生理指标测定都按照制造商手册进行(南京建成生物工程有限公司)。
以非转基因苗为对照,每次处理进行三次重复。采用SPSS 22.0软件(SPSS Inc.,Chicago,IL,USA)进行统计分析。数据比较采用单因素方差分析(ANOVA),邓肯检验。
为了分析银杏GbBBX25基因的表达模式,本实施例采用qRT-PCR方法检测了GbBBX25基因在不同时期(包括4、5、6、7、8、9和10月)和不同组织(根、茎、叶、种仁、芽和叶柄)的表达水平。如图2A所示,GbBBX25基因除了根以外,其他各组织均有表达。在叶片中表达量最高,在种仁中几乎不表达。结果表明该基因在叶片中具有优先表达模式。此外,GbBBX25在6月高表达,随后是5月,在10月几乎不表达(图2B)。
此外,经MJ和SA(100μM)激素处理后,GbBBX25转录水平下降,12h达到最低值,然后从12h持续升高到48h(图2C)。在黑暗条件下,叶片中GbBBX25的表达在3h时显著增加,在24h时GbBBX25表达量最强,24h之后逐渐下降(图2D)。在盐胁迫下,GbBBX25表达量呈振荡趋势,在1h左右出现第一个高峰,随后是第二个高峰(6h)、24h时出现第三个高峰(图2D)。经20%PEG 6000模拟干旱处理后,GbBBX25的表达在1h时略有下降,6h时最低,6h后持续升高,在24h达高峰,随后持续下降(图2D)。
确定蛋白质的亚细胞定位对于研究基因的功能十分重要。为了探讨GbBBX25蛋白的亚细胞定位,在双35S CaMV启动子的调控下,将GFP融合载体(35S::GbBBX25-GFP)转化杨树原生质体。用共聚焦显微镜观察融合蛋白的细胞定位。GbBBX25-GFP融合蛋白仅位于杨树的细胞核内(图3),这表明该基因具有转录因子重要的核定位特征。在35S启动子的调控下,通过农杆菌介导转化成功获得了GbBBX25过量表达的转基因杨树。总共获得了38个无性系转基因苗,通过对基因组DNA的PCR分析初步筛选出32个株系的转基因杨树,其他转基因株系和非转基因株系均未检测到目的片段。挑选10个生长状况较好的转基因无性系在MS培养基中扩繁,从10个转基因株系获得了预期的扩增谱,说明GbBBX25基因已克隆到10个独立的转基因杨树的基因组中(图4A)。QRT-PCR结果显示,T1、T7、T10这3个无性系中GbBBX25表达量最高(图4B)。根据qRT-PCR结果选择表达量最高的T1进行进一步研究。从图4C看出,转基因苗和非转基因苗都能健康生长,可能由于转基因苗的培养基含有Kan抗性,导致其生长速度较非转基苗慢。此外,转基因苗的叶片绒毛较非转基因杨树要多,由于植物叶片绒毛相当于植物的传感器,能够挡住气孔,减少水分蒸发,同时可以减少阳光直射,利于光合作用,从而提高植株的耐旱和耐盐性(图5)。
由于GbBBX25在银杏叶中的表达量最高(图2A),所以采用叶片作为生理指标测定的材料。此外,对银杏叶片喷洒激素(MJ和SA),GBBBX25基因表达量不高(图2C),而非生物胁迫中的盐胁迫处理GbBBX25基因表达量显著升高(图2D),所以对转基因苗进行盐处理,进一步研究转基因杨树耐盐胁迫能力的机制。盐处理3h后,转基因杨树可溶性糖含量显著高于非转基因苗(图6A)。如图6B所示,在盐胁迫下,转基因和非转基因植株的总蛋白含量都是呈先增加再降低的趋势,在盐处理后的第3个小时转基因苗的蛋白含量约为非转基因苗的2倍。此外,为了测定盐胁迫下GbBBX25的抗氧化作用,在非转基因苗和转基因杨树T1中检测POD活性。与非转基因杨树相比,转基因杨树POD活性较高,在6h达到峰值(图6C)。
序列表
<110> 南京林业大学
<120> 银杏GbBBX25基因及其表达蛋白和应用
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Pro
Claims (8)
1.银杏GbBBX25基因,其碱基序列如SEQ ID NO.1所示。
2.权利要求1所述的银杏GbBBX25基因的表达蛋白,其氨基酸序列如SEQ ID NO.2所示。
3.含有权利要求1所述的银杏GbBBX25基因的载体或宿主菌。
4.权利要求1所述的银杏GbBBX25基因在提高植物可溶性糖活性中的应用。
5.权利要求1所述的银杏GbBBX25基因在提高植物总蛋白含量中的应用。
6.权利要求1所述的银杏GbBBX25基因在提高植物过氧化物酶活性中的应用。
7.权利要求1所述的银杏GbBBX25基因在提高植物耐盐性中的应用。
8.权利要求1所述的银杏GbBBX25基因在植物育种中的应用。
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