CN117625627A - 一种诱导型ProPgJOX4启动子及其在人参皂苷生物合成中的应用 - Google Patents
一种诱导型ProPgJOX4启动子及其在人参皂苷生物合成中的应用 Download PDFInfo
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- CN117625627A CN117625627A CN202410112622.7A CN202410112622A CN117625627A CN 117625627 A CN117625627 A CN 117625627A CN 202410112622 A CN202410112622 A CN 202410112622A CN 117625627 A CN117625627 A CN 117625627A
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Abstract
本发明公开了一种诱导型ProPgJOX4启动子及其在人参皂苷生物合成中的应用,属于植物基因工程技术领域,ProPgJOX4启动子来源于人参根部,受茉莉酸甲酯、冠菌素、脱落酸和干旱胁迫诱导;ProPgJOX4启动子与植物表达载体pBI121组成重组载体pBI121‑ProPgJOX4::GUS,在ProPgJOX4启动子的下游连接GUS报告基因;利用基因重组ProPgJOX4启动子可以提高人参细胞中特定基因的表达水平,并对逆境具有较强的适应能力,在提高细胞对逆境适应的同时,诱导的人参皂苷等次生代谢产物的生物合成与积累,因此,人参皂苷等次生代谢产物的合成与细胞生长达到较好的平衡。
Description
技术领域
本发明涉及植物基因工程技术领域,具体涉及一种诱导型ProPgJOX4启动子及其在人参皂苷生物合成中的应用。
背景技术
启动子是基因DNA序列的一部分,作为基因转录起始的核心区域,位于结构基因的5端上游区域,对于基因的表达调控具有至关重要的作用。基因启动子也是RNA聚合酶识别和结合的位点,没有启动子序列,RNA聚合酶就无法找到转录起始位点,也就无法进行基因转录,进而无法发挥其应有的功能。通常情况下,启动子区域无需编码蛋白质,但能够被转录因子识别,与RNA聚合酶共同协作,以起始转录过程。特别是在植物中,启动子的功能多样性以及其与环境因素的相互作用,为植物适应性、生长发育调控等研究带来挑战。
植物基因启动子可以分为组成型启动子、组织特异性启动子和诱导型启动子。其中组成型启动子在大多数组织中都持续表达,不受环境变化的影响,能够保持基因表达的稳定性。组织特异性启动子能够调控基因在某些特定的器官或组织部位中精准表达,具有发育调节的特性,其最大的优点是所启动的外源基因在受体中仅在需要的部位特异表达。诱导型启动子则会在特定环境刺激下表达,如生物胁迫、非生物胁迫等。许多植物基因的启动子含有与环境因子相关的顺式作用元件,这些元件可以与转录因子结合,调控基因的表达。例如,在光照、温度、湿度、营养等环境因子发生变化时,启动子可以通过与相关转录因子的结合,调控下游基因的表达,以适应环境变化。这些启动子的研究不仅有助于深入了解基因表达调控的机制,也为植物基因工程提供了重要的工具和应用前景。因此,具有特定功能的启动子在植物基因工程中具有广泛的应用。通过选择和利用特定功能的启动子,可以实现对植物基因的高效、精准调控。例如,利用组成型启动子可以实现基因的稳定表达,而利用诱导型启动子则可以在特定环境下激活基因的表达,如抗虫、抗病、抗逆和合成次生代谢产物等方面的应用。
植物次生代谢产物是一类对植物生长发育和适应环境至关重要的化合物,利用特定的启动子调控这些化合物生物合成是次生代谢调控的重要手段之一。研究发现在植物的根、茎、叶等不同组织中,次生代谢产物的合成和积累存在显著的差异,而这些差异与组织特异性启动子的调控密切相关。通过利用组织特异性启动子,可以实现在特定的组织或器官中合成次生代谢产物,从而提高其产量和品质。一些启动子可以在环境刺激下调控基因的表达,进而影响次生代谢产物的合成。例如,一些病原菌的侵染、激素和外界化学物质等能够诱导植物中抗病等次生代谢产物的合成,而光、温、水等环境因素也可以通过调节启动子的活性来影响次生代谢产物的合成。研究这些环境刺激诱导的启动子及其作用机制,可以为提高植物次生代谢产物的产量和品质提供新的途径。总之,启动子在植物次生代谢产物的生物合成调控中发挥重要的作用。
然而,目前尚未有研究人员发现人参ProPgJOX4启动子,仍然不清楚ProPgJOX4启动子的活性及其作用,ProPgJOX4启动子的克隆及其应用于提高适应环境胁迫和人参皂苷生物合成调控的研究更未见报道。如何通过ProPgJOX4调节特定基因的表达进而对细胞中次生代谢产物合成与积累调控,对于培养植物具有逆境适应能力、提高高价值的次生代谢产物的积累具有重要的应用价值。
发明内容
为解决上述问题,本方案还提供了一种诱导型ProPgJOX4启动子及其在人参皂苷生物合成中的应用,ProPgJOX4启动子受MeJA(茉莉酸甲酯)、COR(冠菌素)、ABA(脱落酸)和干旱胁迫诱导,利用该启动子可以提高人参对逆境的适应能力并能提高人参皂苷的含量。
为达到以上目的,本方案首先提供了一种诱导型ProPgJOX4启动子及其在人参皂苷生物合成中的应用,所述ProPgJOX4启动子序列如SEQ ID NO.1所示。
作为优选,所述ProPgJOX4启动子克隆的引物序列如SEQ ID NO.2和SEQ ID NO.3所示。
基于一个总的发明构思,本方案还提供了一种诱导型ProPgJOX4启动子的重组载体,所述ProPgJOX4启动子与植物表达载体pBI121组成所述重组载体pBI121-ProPgJOX4::GUS,在ProPgJOX4启动子的下游连接 GUS 报告基因。
作为优选,所述重组载体的构建步骤为:用Bam HI/Hind I双酶切pBI121和ProPgJOX4,回收pBI121载体和ProPgJOX4启动子片段,将两片段连接并转化TOP10,通过筛选和鉴定成功后获得重组质粒。
作为优选,所述Bam HI/Hind III酶切位点的特异引物如SEQ ID NO.4和SEQ IDNO.5所示。
基于一个总的发明构思,本方案还提供了一种诱导型ProPgJOX4启动子或诱导型ProPgJOX4启动子的重组载体在受MeJA、COR、ABA和干旱胁迫诱导的人参皂苷合成与积累中的应用方法。
下面对本发明作进一步说明:
本发明提供的ProPgJOX4启动子属于人参JOX4基因的启动子,该启动子来源于人参(Panax ginseng C.A. Meyer)并命名为ProPgJOX4,基因序列如SEQ ID NO.1所示。
通过基因重组ProPgJOX4启动子,启动人参皂苷生物合成调控基因,适度调节细胞中人参皂苷生物合成调控基因的表达水平,在提高细胞对环境适应能力的情况下,激活人参皂苷的生物合成与积累,本发明所用的人参ProPgJOX4启动子是DNA序列(SEQ ID NO.1),或者是与ProPgJOX4启动子序列高度同源,且具有相同功能的DNA序列。
人参ProPgJOX4启动子植物表达载体是ProPgJOX4启动子DNA序列替换pBI121载体GUS基因上游的CaMV 35S启动子构成的pBI121-ProPgJOX4::GUS重组载体。
总之,本发明利用人参ProPgJOX4启动子具有较高的启动活性,且受MeJA、COR、ABA和干旱胁迫的诱导,通过基因重组所述人参ProPgJOX4启动子,控制人参细胞、组织和植株中特定基因的表达,实现促进次生代谢产物的合成与生长的平衡,是一种比较有效的生产高价值人参皂苷等次生代谢产物的方法。
与现有技术相比,本发明具有以下有益效果:
(1)本发明基于转录组和基因组测序等技术发现人参中多个JOX家族基因及其启动子,通过农杆菌A4介导ProPgJOX4启动子在人参愈伤组织中的表达,发现ProPgJOX4启动子具有较高的活性,且受MeJA、COR、ABA和干旱胁迫的诱导;基于这些研究结果可知,利用基因重组ProPgJOX4启动子可以提高人参细胞中特定基因的表达水平,并对逆境具有较强的适应能力。
(2)本发明利用人参ProPgJOX4启动子调节在人参细胞内人参皂苷等次生代谢产物含量,具体体现在ProPgJOX4启动人参皂苷生物合成调控基因例如DDS基因但不限于该基因的表达进而实现对人参皂苷含量的调节,在提高细胞对逆境适应的同时,诱导的人参皂苷等次生代谢产物的生物合成与积累;因此,该方法能实现人参皂苷等次生代谢产物的合成与细胞生长达到较好的平衡。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1是本发明实施例1扩增的人参ProPgJOX4启动子片段电泳结果;
图2是本发明实施例1扩增的人参ProPgJOX4启动子顺式作用元件分析图;
图3是本发明实施例2中ProPgJOX4启动子驱动GUS基因表达载体表达框示意图;
图4是本发明实施例2中荧光定量PCR(qRT-PCR)检测转化人参愈伤组织中ProPgJOX4启动子启动的GUS基因的表达水平;
图5是本发明实施例2中转化人参愈伤组织中ProPgJOX4启动子启动的GUS基因的表达的酶活性;
图6是发明实施例3中ProPgJOX4启动子启动人参DDS基因在人参愈伤组织中瞬时表达后人参皂苷含量测定结果。
具体实施方式
为使本发明要解决的技术问题、技术方案和优点更加清楚,下面将结合附图及具体实施例进行详细描述。
以下实施例用于说明本发明,但不用来限制本发明的范围。在不背离本发明精神和实质的情况下,对本发明方法、步骤或条件所作的修改或替换,均属于本发明的范围。
若未特别指明,实施例中所用的技术手段为本领域技术人员所熟知的常规手段;若未特别指明,实施例中所用试剂均为市售。
实施例1 ProPgJOX4启动子的克隆
1、人参根基因组DNA的提取
取四年生新鲜人参根,自来水冲洗干净后用75%乙醇清洗30s,最后以无菌水冲洗3次,以CTAB(十六烷基三甲基溴化铵)法或植物基因组DNA提取试剂盒提取基因组DNA,-20℃保存备用。
2、PCR扩增克隆人参ProPgJOX4启动子
对COR诱导的人参细胞转录组测序结果进行分析,结合前期人参基因组测序结果,根据筛选得到的人参PgJMT基因序列信息设计ProPgJOX4启动子体外扩增引物,ProPgJOX4启动子扩增所用的引物序列如下。
ProPgJOX4-F1:5′-TCTAATCACATATATTTATGT-3′;(SEQ ID NO.2)
ProPgJOX4-R1:5′-AATTCCTCTTTTATAGGTAGAC-3′;(SEQ ID NO.3)
以人参基因组DNA为模板,利用ProPgJOX4-F1与ProPgJOX4-R1引物进行PCR扩增。PCR反应条件为:94℃预变性10min;94℃变性30s,58℃退火40s,72℃延伸2min,30个循环;72℃延伸10min。
PCR扩增产物以1.5%琼脂糖凝胶电泳分析PCR产物,电泳结果见图1,图中1表示PCR扩增产物,M表示Marker;结果显示获得大小约1000bp的条带,然后对候选的电泳条带进行胶回收,再对胶回收产物进行测序分析,测序后获得序列长度为998bp的基因片段。
对克隆的序列采用PlantCARE在线分析工具对所克隆的启动子片段进行顺式元件分析,分析结果见图2,结果显示克隆启动子序列中含有应有的基本元件(TATA-Box和CAAT-Box)和多种顺式作用元件(MYB、ABRE和 TGACG-motif等),其序列表中SEQ ID NO.1所示。
实施例2 植物表达载体的构建及转化
1、植物表达载体的构建
(1)根据启动子序列设计片段相对应的包含Bam HI/Hind III酶切位点的特异引物,上下游引物分别命名为ProPgJOX4-F2和ProPgJOX4-R2。
ProPgJOX4-F2:5′-CGCGGATCCTCTAATCACATATATTTATGT-3′;(SEQ ID NO.4)
ProPgJOX4-R2:5′-CCCAAGCTTAATTCCTCTTTTATAGGTAGAC-3′。(SEQ ID NO.5)
以实施例1所扩增的启动子为模板,引物ProPgJOX4-F2和ProPgJOX4-R2进行PCR扩增,扩增条件为:预变性94℃ 5min,变性94℃ 30s,退火58℃ 30s,72℃ 30s,35个循环;终延伸72℃ 5min。经过PCR扩增后的产物用1.5%琼脂糖凝胶电泳进行分离,回收目标条带。
然后使用Bam HI和Hind III对pBI121载体进行双酶切以获得切除GUS基因CaMV35S启动子的载体片段,以及Bam HI和Hind III双酶切ProPgJOX4启动子片段,其中胶回收和质粒提取步骤分别参见全式金公司质粒提取试剂盒和胶回收说明书。pBI121载体和ProPgJOX4启动子双酶切反应体系如下表1所示:
反应条件:37℃酶切10h,通过琼脂糖凝胶电泳分析后回收片段,将切除GUS基因CaMV 35S启动子的pBI121载体片段和ProPgJOX4启动子片段以T4 DNA连接酶连接并转化TOP10(感受态细胞),通过卡那霉素筛选和Bam HI/Hind III双酶切鉴定成功后获得重组质粒,重组质粒命名为pBI121-ProPgJOX4::GUS,ProPgJOX4启动子驱动GUS基因表达载体表达框示意图见图3,图3中RB:T-DNA右边界;LB:T-DNA左边界;NOS-pro:NOS 启动子;NPTⅡ:卡那霉素抗性基因;NOS-ter:NOS 终止子;
连接体系及条件如下表2所示:
(2)重组载体转化农杆菌
将获得的pBI121-ProPgJOX4::GUS重组载体以冻融法转化农杆菌A4,转化后阳性克隆以PCR进行筛选,再经测序鉴定成功后获得含pBI121-ProPgJOX4::GUS的农杆菌。
2、农杆菌介导基因转化
(1)含pBI121-ProPgJOX4::GUS质粒的根癌农杆菌培养
28℃划线培养含有pBI121-ProPgJOX4::GUS质粒的农杆菌约2d,挑取单菌落接种于含有50mg/L卡那霉素的YEB(发根农杆菌)液体培养基中,28℃振荡培养过夜,至OD600(在600nm波长处的吸光值)约为0.6时收集菌液,4000rpm离心10min,沉淀重悬于1/2MS(无机盐为MS培养基的一半)液体培养基中。
(2)农杆菌介导pBI121-ProPgJOX4::GUS基因转化人参愈伤组织
取4年生人参新鲜根诱导的愈伤组织,分别放入含有pBI121-ProPgJOX4::GUS和pBI121的农杆菌A4侵染液中5min,用无菌滤纸吸干菌液,置于1/2MS+2,4D(2,4-二氯苯氧乙酸)的培养基上避光共培养5d。
(3)qRT-PCR分析GUS基因表达水平
取农杆菌与愈伤组织共培养5d后的人参愈伤组织,用无菌去离子水清洗吸干表面水分后,提取总RNA,以oligod(T)18为引物,反转录合成cDNA,qRT-PCR分析基因表达水平,GUS基因所用引物为SEQ ID NO.6和SEQ ID NO.7,内参基因β-actin所用引物为SEQ IDNO.8和 SEQ ID NO.9。
图4是荧光定量PCR(qRT-PCR)检测转化人参愈伤组织中ProPgJOX4启动子启动的GUS基因的表达水平,其中CK为野生型人参愈伤组织,作为对照;TE为ProPgJOX4启动GUS基因瞬时过表达的人参愈伤组织;MeJA、COR、ABA和PEG分别表示ProPgJOX4启动子启动GUS报告基因在100μM MeJA、1μM COR、10μM ABA和10%PEG6000(平均分子量为6000的聚乙二醇)(模拟干旱胁迫)诱导下表达水平,以β-actin作为内参,对照CK中没有检测到GUS基因表达,TE中ProPgJOX4启动子具有启动GUS基因表达的活性,且GUS基因的表达受MeJA、COR、ABA和PEG的诱导。
GUS-F:5′-ATACCGAAAGGTTGGGCAGG-3′;(SEQ ID NO.6)
GUS-R:5′-CGGCAATAACATACGGCGTG-3′。(SEQ ID NO.7)
β-actin F:5′-TGCCCCAGAAGAGCACCCTGT-3′;(SEQ ID NO.8)
β-actin R:5′-AGCATACAGGGAAAGATCGGCTTGA-3′。(SEQ ID NO.9)
(4)ProPgJOX4启动GUS表达及其活性检测
取新鲜的适量人参愈伤组织,用液氮将其迅速冷冻,然后在液氮中研磨成细粉,并立即加入GUS提取缓冲液,充分混匀,12,000rpm,4℃离心5min,取上清至冰上,以考马斯亮蓝测定蛋白浓度。
参考GUS活性测定试剂盒说明,取提取的蛋白上清液,加入适量37℃预热的GUS提取缓冲液里,再加入MUG(4-甲基伞形酮葡糖苷酸)底物,37℃温浴,在不同时间的混合反应物加入反应终止液,测定样品液的荧光强度值。
图5是本发明实施例2中转化人参愈伤组织中ProPgJOX4启动子启动的GUS基因的表达的酶活性,其中CK为野生型人参愈伤组织,作为对照;CaMV 35S为CaMV 35S启动GUS基因瞬时过表达的人参愈伤组织;MeJA、COR、ABA和PEG分别表示ProPgJOX4启动子启动GUS报告基因在100μM MeJA、1μM COR、10μM ABA和10%PEG6000(模拟干旱胁迫)处理后GUS酶活性,结果显示ProPgJOX4启动子具有启动GUS基因表达的活性,且GUS的活性受MeJA、COR、ABA和PEG的诱导。
实施例3 ProPgJOX4启动子启动人参DDS基因在人参愈伤组织中瞬时表达
将pBI121-ProPgJOX4::GUS载体中的GUS基因序列替代为人参皂苷生物合成途径中关键酶达玛烯二醇合成酶(dammarenediol-II synthase,DDS)基因(KJ939266),参考实施例2中方法转化人参愈伤组织,取上述共培养后的新鲜的人参愈伤组织,ddH2O清洗干净后以60℃干燥,研磨成细粉,用80%甲醇60℃浸提(1g:40mL),超声波提取3次,每次10min;60℃蒸干甲醇,用水饱和正丁醇萃取,收集正丁醇层,60℃水浴蒸干正丁醇,再用适量的甲醇溶解后待测。
人参皂苷含量测定采用HPLC法,LC-MS 8050高效液相色谱仪;色谱柱为ACQUITYUPLC BEH Shield RP18柱(1.7μm,2.1mm×50mm);流动相为乙腈:1%甲酸,流速为1.0mL/min,柱温为35℃,进样量为3μL,检测波长为202nm。
以人参皂苷Rb1、Rb2、Rc、Rd、Re、Rg1、Rg2和Rg3作为标准品测定样品中的各皂苷单体的含量,再以各皂苷单体含量之和代表人参细胞中总皂苷的含量。
图6是ProPgJOX4启动子启动人参DDS基因在人参愈伤组织中瞬时表达后人参皂苷含量测定结果;其中CK为转空载体pBI121后的人参愈伤组织,作为对照;ProPgJOX4::DDS为ProPgJOX4启动子驱动的DDS基因转化后的人参愈伤组织,为实验组;与对照CK相比,ProPgJOX4::DDS中人参皂苷含量明显提高,增加1.69倍;与ProPgJOX4::DDS相比,ProPgJOX4::DDS+MeJA、ProPgJOX4::DDS+COR、ProPgJOX4::DDS+PEG和ProPgJOX4::DDS+ABA中人参皂苷含量均明显提高,分别提高2.53、2.74、2.22和1.88倍。这表明ProPgJOX4启动子可以启动DDS基因表达的同时,MeJA、COR、ABA和干旱胁迫均能有效促进ProPgJOX4驱动的人参皂苷合成与积累,为利用ProPgJOX4启动子在提高人参对环境适应能力的同时增加人参中人参皂苷的含量方面具有重要应用价值。
以上所述仅是本发明专利的优选实施方式,本发明专利的保护范围并不仅局限于上述实施例。对于本领域的技术人员来说,在不脱离本发明专利的技术构思前提下所得到的改进和变换也应视为本发明专利的保护范围。
Claims (6)
1.一种诱导型ProPgJOX4启动子,其特征在于,所述ProPgJOX4启动子序列如SEQ IDNO.1所示。
2.根据权利要求1所述的诱导型ProPgJOX4启动子,其特征在于,所述ProPgJOX4启动子克隆的引物序列如SEQ ID NO.2和SEQ ID NO.3所示。
3.一种如权利要求1所述的诱导型ProPgJOX4启动子的重组载体,其特征在于,所述的ProPgJOX4启动子与植物表达载体pBI121组成所述重组载体pBI121-ProPgJOX4::GUS,在ProPgJOX4启动子的下游连接 GUS 报告基因。
4.根据权利要求3所述的诱导型ProPgJOX4启动子的重组载体,其特征在于,所述重组载体的构建步骤为:用Bam HI/Hind I双酶切pBI121和ProPgJOX4,回收pBI121载体和ProPgJOX4启动子片段,将两片段连接并转化TOP10,通过筛选和鉴定成功后获得重组质粒。
5.根据权利要求4所述的诱导型ProPgJOX4启动子的重组载体,其特征在于,所述BamHI/Hind III酶切位点的特异引物如SEQ ID NO.4和SEQ ID NO.5所示。
6.一种如权利要求1 -2任一项所述的诱导型ProPgJOX4启动子或如权利要求3-5任一项所述的诱导型ProPgJOX4启动子的重组载体在受MeJA、COR、ABA和干旱胁迫诱导的人参皂苷合成与积累中的应用方法。
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