CN116926116A - gb-miR160—GbERF4模块在调控银杏萜内酯合成中的应用 - Google Patents
gb-miR160—GbERF4模块在调控银杏萜内酯合成中的应用 Download PDFInfo
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Abstract
本发明公开了gb‑miR160—GbERF4模块在调控银杏萜内酯合成中的应用，属于基因工程应用技术领域，本申请从银杏中分离了1个参与萜内酯合成的候选转录因子GbERF4，在烟草中过表达GbERF4显著提高了烟草萜类化合物的积累，同时上调烟草中萜类化合物途径中重要酶基因的表达，证明了GbERF4具有调控萜类化合物合成的功能。表达模式及前期miRNA测序表明gb‑miR160负调控银杏萜内酯合成。转基因实验显示gb‑miR160过表达可显著抑制萜类化合物的积累。降解组测序发现GbERF4是gb‑miR160的靶标，通过瞬时表达和双荧光素酶报告实验验证了gb‑miR160靶向并抑制GbERF4的表达。另外，酵母单杂和双荧光素酶报告分析结果显示GbERF4可与萜内酯合成途径中酶基因HMGS1，AACT1，DXS1，LPS2和GGPPS2的启动子结合，激活它们的表达进而参与银杏萜内酯的生物合成。
Description
技术领域
本发明属于基因工程应用技术领域，具体涉及一种gb-miR160—GbERF4模块在调控银杏萜内酯合成中的应用。
背景技术
萜内酯是一种仅存在于银杏中的天然次生代谢产物，包括银杏内酯和白果内酯，具有重要的药用价值。银杏萜内酯由两个独立的甲羟戊酸途径和5-磷酸脱氧木酮糖/2C-甲基4-磷酸-4D-赤藓糖醇途径合成。MVA途径主要负责植物倍半萜、甾醇和三萜的生物合成，其中AACT是MVA途径中的第一个酶，催化乙酰辅酶A生成乙酰乙酰辅酶A。3-羟基-3-甲基戊二酰辅酶A还原酶(HMGR)是萜类生物合成MVA途径中的限速酶之一，可催化3-羟基-3-甲基戊二酰辅酶A(HMG-CoA)形成甲羟戊酸。HMGS催化乙酰辅酶A缩合生成3-羟基-3-甲基戊二酰辅酶A。法尼基二磷酸合成酶(FPPS)是甲羟戊酸代谢途径分支点的关键酶，催化合成C15的FPP，为类胡萝卜素、脱落酸、赤霉素和甾醇等类异戊二烯物质的合成提供前提。MEP途径主要负责植物单萜和二萜的生物合成，其中DXS是MEP途径的第一个酶，负责催化丙酮酸和3-磷酸甘油醛生成DXP。GGPPS被认为是催化银杏内酯前体GGPP的生物合成的关键酶。LPS被认为是催化GGPP合成银杏内酯的第一步，能够将香叶二磷酸基转化为左旋吡喃二烯，然而，左旋吡喃二烯到银杏内酯间的催化酶还不明确。近年来，CYP450家族在银杏萜内酯合成中的功能逐渐被揭示，填补了左旋吡喃二烯到银杏内酯合成途径过程中的空白。这些基因的差异表达影响了萜内酯的生物合成速率和含量，然而，造成这些表达水平差异的原因仍然是未知的。
银杏萜内酯在各个组织中均有积累且在根中积累最显著。除了组织特异性外，银杏萜内酯的合成还受到多种非生物胁迫的诱导，如MeJA，SA，ABA，Eth，低温及黑暗等。转录组分析表明，外源激素诱导了银杏中转录因子和萜内酯合成途径中的结构基因的上调表达进而提高萜内酯含量。随着银杏基因组测序的完成，对于启动子的认识更加清晰和明确，提高了转录因子下游靶基因的筛选效率，但有关转录因子调控银杏萜内酯合成的功能验证与分子机制鲜有报道。Our previously多组学实验联合分析表明，miRNA通过介导AP2/ERF转录因子参与调控银杏萜内酯合成途径中的结构基因参与萜内酯的合成。然而，miRNA调控功能的验证需要更多的试验证据。
乙烯响应因子(ERF)是一类具有保守AP2/ERF结构域的转录因子家族，主要通过与乙烯反应序列基序结合而发挥转录调节剂的作用。ERF基因的全基因组分析已在多种植物中完成，如拟南芥(Nakano T,Suzuki K,Fujimura T,Shinshi H(2006)Genome-wideanalysis of the ERF gene family in Arabidopsis and rice.Plant Physiol 140:411–432.)、水稻(Nakano T,Suzuki K,Fujimura T,Shinshi H(2006)Genome-wideanalysis of the ERF gene family in Arabidopsis and rice.Plant Physiol 140:411–432.)、棉花(Zafar MM,Rehman A,Razzaq A,Parvaiz A,Mustafa G,Sharif F,Mo H,Youlu Y,Shakeel A,Ren M(2022)Genome-wide characterization and expressionanalysis of Erf gene family in cotton.BMC Plant Biol22:134.)、油菜(Ghorbani R,Zakipour Z,Alemzadeh A,Razi H(2020)Genome-wide analysis of AP2/ERFtranscription factors family in Brassica napus.Physiol Mol Biol Plants 26:1463–1476.)、板蓝根(Xiao L,Ren J,Li Q,Yang B,Liu Z,Chen R,Zhang L(2023)Genome-wide analysis of AP2/ERF superfamily in Isatis indigotica.J Integr Med 21:77–88.)、猕猴桃(Jiang Q,Wang Z,Hu G,Yao X(2022)Genome-wide identification andcharacterization of AP2/ERF gene superfamily during flower development inActinidia eriantha.BMC Genomics 23(1):650.)等。ERF TFs在植物生长发育和次生代谢产物的合成中发挥着关键作用，包括调节萜类化合物的生物合成。比如，先前的报道表明LcERF19正向调控山苍子柠檬醛(香叶醛和橙花醛)的生物合成(Wang M,Gao M,Zhao Y,Chen Y,Wu L,Yin H,Xiong S,Wang S,Wang J,Yang Y,Wang J,Wang Y(2022)LcERF19,anAP2/ERF transcription factor from Litsea cubeba,positively regulates geranialand neral biosynthesis.Hortic Res 9:uhac093.)。在丹参中，SmERF128通过激活SmCPS1、SmKSL1和SmCYP76AH1的表达促进丹参酮的生物合成(Zhang Y,Ji A,Xu Z,Luo H,Song J(2019)The AP2/ERF transcription factor SmERF128positively regulatesditerpenoid biosynthesis in Salvia miltiorrhiza.Plant Mol Biol100,83–93.)。在玉米中，EREB58通过直接结合启动子促进TPS10的表达，进而提高倍半萜(E)-β-法尼烯和(E)-α-佛手柑烯的含量(Li S,Wang H,Li F,Chen Z,Li X,Zhu L,Wang G,Yu J,Huang D,Lang Z(2015)The maize transcription factor EREB58 mediates the jasmonate-induced production of sesquiterpene volatiles.Plant J 84:296–308.)。PpERF61通过结合PpTPS1和PpTPS3启动子激活了PpTPS1和PpTPS3的转录来影响芳樟醇的积累。然而，关于ERFs参与银杏萜内酯的生物合成还未报道。
MicroRNAs(miRNAs)是由20-24个核苷酸组成的非编码RNA，它们在转录后水平调控特定靶基因的表达。研究表明，植物中的miRNA靶向多种TF基因，因此在植物生长发育的几乎所有过程中发挥重要作用。在水稻中，Os-miR1320通过靶向OsERF096介导JA信号的传导调节耐寒性(Sun M,Shen Y,Chen Y,Wang Y,Cai X,Yang J,Jia B,Dong W,Chen X,SunX(2022)Osa-miR1320 targets the ERF transcription factor OsERF096 to regulatecold tolerance via JA-mediated signaling.Plant Physiol 189:2500–2516.)。在青蒿中，ARF1是miR160的靶标，参与调控青蒿素的合成(Guo Z,Hao K,Lv Z,Yu L,Bu Q,Ren J,Zhang H,Chen R,Zhang L(2023)Profiling of phytohormone-specific microRNAs andcharacterization of the miR160-ARF1 module involved in glandular trichomedevelopment and artemisinin biosynthesis in Artemisia annua.Plant BiotechnolJ 21:591–605.)。在水稻中，miR2105通过介导OsbZIP86调控OsNCED3的表达来控制干旱胁迫下ABA的生物合成(Gao W,Li M,Yang S,Gao C,Su Y,Zeng X,Jiao Z,Xu W,Zhang M,XiaK(2022)miR2105 and the kinase OsSAPK10co-regulate OsbZIP86 to mediatedrought-induced ABA biosynthesis in rice.Plant Physiol 189:889–905.)。本实验室前期的多组学分析筛选了可能参与调控银杏萜内酯合成的miRNA(Ye J,Zhang X,Tan J,XuF,Cheng S,Chen Z,Zhang W,Liao Y(2020)Global identification of Ginkgo bilobamicroRNAs and insight into their role in metabolism regulatory network ofterpene trilactones by high-throughput sequencing and degradome analysis.IndCrop Prod 148,112289.)，但是这些miRNA的调控功能和机制尚没有验证与解析。
发明内容
本发明的目的是针对现有的问题，提供了一种gb-miR160—GbERF4模块在调控银杏萜内酯合成中的应用，揭示了gb-miR160-GbERF4模块调控银杏萜内酯合成的分子机制，为阐明银杏萜内酯合成的基因调控网络提供了理论模型，并为利用基因工程手段提高银杏萜内酯含量提供了技术支撑和靶标基因。
本发明是通过以下技术方案实现的：
本申请提供了gb-miR160—GbERF4模块在调控银杏萜内酯合成中的应用。
进一步地，包括gb-miR160—GbERF4调控组件的筛选鉴定。
进一步地，gb-miR160负向调控萜类化合物合成，GbERF4正向调控萜类化合物合成。
进一步地，GbERF4可与萜内酯合成途径中酶基因HMGS1，AACT1，DXS1，LPS2和GGPPS2的启动子结合，并激活它们的表达进而参与银杏萜内酯的生物合成。
进一步地，GbERF4是gb-miR160的靶标。
进一步地，gb-miR160靶向并切割转录因子GbERF4，抑制GbERF4的表达，从而抑制萜类化合物的积累。
银杏萜内酯具有保护神经、抗氧化、抗炎和保护心血管活性等功效，如何提高银杏萜内酯含量吸引了越来越多的研究人员的兴趣。目前，萜内酯只能从银杏提取物中获得，还没有有效获得萜内酯的方式。然而，银杏提取物中的含量极少，而提高银杏中萜内酯的含量是获得大量萜内酯的有效途径。因此，弄清银杏萜内酯合成的分子机制进而利用基因工程手段提高银杏萜内酯含量，具有重要意义。尽管有多项研究尝试通过基因组和生物信息学方法解析其中的调控机制，但关于调控银杏萜内酯合成的分子机制还没有阐明，全面解析银杏萜内酯合成的基因调控网络还有很多工作需要进行。
生物学功能分析表明，ERF广泛参与植物叶片生长，果实成熟，花青素合成，激素信号转导，萜类化合物合成和多种生物和非生物胁迫过程。在介导萜类化合物积累方面，ERF被认为是作为JA响应转录调控因子参与萜类化合物的合成代谢。迄今为止，参与调控银杏萜内酯生物合成的转录因子还鲜有报道。在申请研究中，系统进化分析结果表明GbERF4与TcERF15发挥着类似的功能(图2a)，同时GbERF4的表达能被MeJA诱导(图2d)，暗示了GbERF4具有调控萜内酯合成的功能。当GbERF4在烟草中过表达时，植醇、西柏烷三烯、香紫苏醇和角鲨烯等萜类化合物含量和豆甾醇含量显著提高(图5a图6a)，且萜类合成途径中的结构基因HMGR、HMGS、DXR、DXS、AACT和GGPPS均有不同程度的显著上调(图5b和图6b)，进一步证明了GbERF4参与了银杏萜内酯的合成调控。研究表明，ERF在不同物种内的相互调控功能具有保守性。例如，长春花中的ORCA3和ORCA5可以激活烟草中尼古丁合成途径中PMT(putrescine methyltransferase)和QPT(quinolinate phosphoribosyltransferase)基因的表达提高尼古丁含量，同样的，烟草ERF189在长春花中激活TIA(terpenoid indolealkaloids)合成通路中STR(Strictosidine synthase)基因的表达并诱导TIA积累(PaulP,Singh SK,Patra B,Liu X,Pattanaik S,Yuan L(2020)Mutually regulated AP2/ERFgene clusters modulate biosynthesis of specialized metabolites inplants.Plant Physiol 182(2):840–856.)。这些结果或许能解释ERF转录因子具有调控萜类化合物合成的保守功能，也为GbERF4作为银杏萜内酯合成的调节因子提供了证据。
MiR160是一类保守的miRNA，已在拟南芥、番茄、水稻、杨树等多种模式和非模式植物中发现，在植物的各种生物过程中起着至关重要的作用，包括次生代谢产物合成。降解组测序是一种高通量的检测方法，可以有效地全面鉴定miRNA的靶基因。本申请人所在的课题组先前的研究表明miR60与银杏萜内酯生物合成有关(Ye J,Zhang X,Tan J,Xu F,ChengS,Chen Z,Zhang W,Liao Y(2020)Global identification of Ginkgo biloba microRNAsand insight into their role in metabolism regulatory network of terpenetrilactones by high-throughput sequencing and degradome analysis.Ind CropProd 148,112289)，但未见具体的调控作用报道。在这项研究中，我们发现gb-miR160在萜内酯含量最高的根中表达水平最低，表明gb-miR160负向调控萜内酯积累(图8b)。此外，我们利用qRT-PCR分析了gb-miR160在银杏不同组织中的表达水平(图7)，这一结果验证了gb-miR160的真实性。转基因研究结果表明，miR160过表达降低了烟草中萜类化合物的合成(图11a和图6a)。本申请的研究表明银杏Gb-miR160与其他植物的miR160类似，参与了萜内酯的合成。
大量研究揭示了TF和miRNA之间普遍存在相互作用，miRNA会影响TFs的转录水平。尽管miR160-TF模块已被证明对调节植物生长发育多种过程至关重要，但参与植物次生代谢产物合成中的报道较少，尤其对于银杏萜内酯合成的调节机制尚未阐明。先前的研究表明，miR160在不同物种中主要通过靶向ARF家族不同成员参与了植物生长发育过程。本申请鉴定了一个miR160新的靶基因ERF4。我们在烟草叶片中观察到gb-miR160对GbERF4的靶向切割作用(图9b和d)，同时双荧光素酶基因报告实验也验证了gb-miR160对GbERF4的抑制作用(图9f)。这些结果为miR160-ERF调控模块的发现提供了直接的证据。
在本申请研究中，明确了银杏中miR160—GbERF4模块间的靶向关系，且提供了确凿的证据证明银杏中的miR160-ERF4模块调控萜内酯的生物合成。通过Y1H和双荧光素酶报告实验发现GbERF4可以与HMGS1、AACT1、DXS1、GGPPS2和LPS2的启动子在体外结合，并在体内激活它们(图11)。先前的研究表明，HMGS、AACT和DXS是萜类化合物合成途径中的关键基因，GGPPS在二萜产物合成中发挥了重要作用，LPS是银杏萜内酯合成的第一个酶基因，这些基因是萜内酯合成途径中的关键基因。因此，我们的研究结果表明GbERF4具有转录激活特异性，能激活多个萜内酯生物合成途径上游结构基因的表达，具有高效地调控银杏萜内酯合成的潜力。先前的多项研究表明MeJA能够诱导银杏萜内酯合成，但其中的原因一直未被揭示。本申请的研究结果从另一方面或许解答了这些疑惑，即GbERF4能够被MeJA诱导表达(图2c)，影响银杏萜内酯的积累。此外，随着基因组、高通量测序等技术的发展，银杏中的基因信息也逐渐清晰，因此还可能有潜在miR160或者GbERF4的靶基因未被鉴定，有待下一步研究。
综上所述，本申请的研究结果揭示了gb-miR160-GbERF4模块参与银杏萜内酯生物合成的分子机制(图12)。gb-miR160通过靶向剪切GbERF4来抑制GbERF4对银杏萜内酯合成途径中靶基因HMGS1、DXS1、AACT1、GGPPS2和LPS2的激活，进而控制着银杏萜内酯的合成和积累。gb-miR160-GbERF4模块同时对萜内酯合成途径中多个基因的调控是罕见且高效的，具有巨大的应用前景。这些发现拓展了我们对gb-miR160-GbERF4模块在银杏萜内酯生物合成调控作用中的理解，并为高萜内酯含量银杏的遗传改良提供了理论基础与技术支撑。
本发明相比现有技术具有以下优点：
本申请筛选鉴定了一个参与银杏萜内酯生物合成的调控组件gb-miR160—GbERF4。首先，通过表达模式分析鉴定了参与萜内酯合成的gb-miR160和GbERF4。转基因实验表明gb-miR160负向调控萜类化合物合成，GbERF4正向调控萜类化合物合成。降解组、瞬时表达和双荧光素酶报告实验验证了gb-miR160靶向并切割转录因子GbERF4。进一步的酵母单杂和双荧光素酶报告实验表明GbERF4通过结合HMGS1，AACT1，DXS1，LPS2和GGPPS2启动子并激活它们表达，进而参与了萜内酯的合成。综上所述，本研究揭示了gb-miR160-GbERF4模块调控银杏萜内酯合成的分子机制，为阐明银杏萜内酯合成的基因调控网络提供了理论模型，并为利用基因工程手段提高银杏萜内酯含量提供了技术支撑和靶标基因。
附图说明
图1为GbERF1/2/3/4在不同银杏组织中的表达模式图；
图2为参与银杏萜内酯生物合成的GbERF4基因筛选及表达模式分析图；
图3为过表达GbERF4转基因烟草的结果图；
图4为申请中的凝胶电泳验证图；
图5为GbERF4和gb-miR160过表达T1代烟草植株萜类化合物含量测定(a)及萜类合成途径中结构基因的表达水平检测对比图；
图6为GbERF4和gb-miR160过表达T0代烟草植株萜类化合物含量测定(a)及萜类合成途径中结构基因的表达水平检测对比图；
图7为gb-miR160在不同银杏组织中的表达模式图；
图8为从34年银杏树上采集的不同组织样品以及gb-miR160在不同组织中的表达模式及与萜内酯含量的相关性分析；
图9为GbERF4是gb-miR160的靶标分析图；
图10为gb-miR160的前体序列；
图11为GbERF4结合HMGS1，AACT1，DXS1，LPS2，GGPPS2的启动子并激活它们转录；
图12为gb-miR160-GbERF4模块参与调控银杏萜内酯生物合成的分子机制图。
具体实施方式
为了对本发明做更进一步的解释，下面结合下述具体实施例进行阐述。
实施例1
1.材料和方法
1.1植物材料
银杏不同组织采集于长江大学银杏园(30°21'18"E,112°8'20"N)中34年银杏树。2年生银杏幼苗和本氏烟草(Nicotiana benthamiana)在25℃的16h光照/8h黑暗条件下培养。用于转基因的大烟草(Nicotiana tabacum L.)生长在长江大学园艺园林学院日光温室中，选择3个独立的转基因株系进行生物学重复，采集现蕾期的烟草植株中部叶片用于萜类化合物的测定。
1.2 RNA提取和RT-qPCR分析
(1)按照TaKaRa MiniBEST Plant RNA Extraction Kit(TaKaRa,Beijing,China)说明书从烟草叶片和银杏不同组织中提取总RNA；
(2)使用HiScript IIQ RT SuperMix for qPCR试剂盒将上述总RNA(Vazyme,Nanjing,China)反转录为用于qRT-PCR反应的cDNA；
(3)qRT-PCR分析在Bioer LineGene 9600 Plus(BioRad)平台上使用ChamQUniversal SYBR qPCR Master Mix(Vazyme)完成；
(4)基因的相对表达水平使用2－△△Ct法(Livak and Schmittgen,2001)进行计算，银杏中和烟草中均使用甘油醛-3-磷酸脱氢酶基因(GAPDH)作为内参基因。
表1本申请所用引物序列
1.3miRNA的qRT-PCR分析
为了测定miRNA的表达水平，使用Mir-XTM miRNA First-Strand Synthesis kit(TaKaRa)将总RNA反转录以获得用于miRNA定量分析的cDNA。RT-PCR分析使用MicroRNAsqPCR Kit(Sangon Biotech,Shanghai,China)在Bioer LineGene 9600Plus上进行。正向引物为成熟的miR160序列，反向引物为试剂盒附带的mRQ 3’引物，使用U6核小RNA作为内参。相对表达水平使用2－△△Ct法进行计算。
1.4瞬时共表达实验
(1)通过Golden Gate克隆技术将gb-miR160的前体茎环序列插入到含CaMV 35S启动子的pICH86988质粒中；
(2)将GbERF4靶序列(ERF4TS)与GFP共同插入到pICH86988中的BsaI位点间以形成融合蛋白；
(3)将含有gb-miR160和ERF4TS的农杆菌用悬浮液(10mM2-(N-morpholine)-ethanesulphonic acid(MES),10mM MgCl2和150μM乙酰丁香酮，pH 5.8-6.0)重悬并混合后注射到本氏烟草叶片中；
(4)侵染后的烟草置于24℃，16h光照/8h黑暗条件下培养，3天后，使用手持式激发光源LUYOR-3280照射后观察荧光表达情况；
以gb-miR156和突变序列ERF4mTS组合作为阴性对照，为了将ERF4TS和ERF4mTS正确的插入质粒中，在目标序列的正向和反向引物两端分别加上AATG和CGAA酶切位点。
1.5烟草的遗传转化
(1)通过Nimble Cloning法将pre-miR160的前体茎环序列和GbERF4的CDS分别插入含有CaMV 35S启动子的pNC-Cam2304-MCS35S质粒中；
(2)用OD600为0.4的含有目标质粒的GV3101农杆菌侵染1cm×1cm的烟草叶片10-15分钟后转移到共培养基(MS+100μM AS+2mg/L 6-BA+0.5mg/L IAA)中在25℃环境下黑暗培养48小时；
(3)随后依次经过分化培养基(MS+2mg/L 6-BA+0.5mg/L IAA+100mg/L Kan+300mg/L cef)和生根培养基(0.1mg/L IBA+100mg/L Kan+300mg/L cef)诱导，以获得gb-miR160和GbERF4过表达转基因烟草植株，培养条件为25℃，16h光照/8h黑暗；
(4)使用目标基因的正向扩增引物和M13R进行PCR扩增以验证转基因烟草的阳性植株；同时，根据GUS染色试剂盒(华越洋生物，北京)对阳性植株染色以进一步确认并对染色情况拍照。
1.6银杏萜内酯含量和烟草中萜类化合物的测定
银杏萜内酯含量的测定方法如前所述(Zheng J,Zhang X,Fu M,Zeng H,Ye J,Zhang W,Liao Y,Xu F(2020)Effects of different stress treatments on the totalterpene trilactone content and expression levels of key genes in Ginkgobiloba leaves.Plant Mol Biol Rep 38:521–530.)；
烟草中萜类物质含量利用气相色谱质谱法(GC-MS)测定，具体为：
(1)采集现蕾期的过表达转基因(T0 generation和T1代)和野生型烟草植株的中部叶片，经液氮研磨后置于冷冻干燥仪中干燥；
(2)取50mg干燥后的样品，加入1.5mL提取液(含2.5mg/L十三烷酸)，涡旋6s后在30℃摇床中震荡提取60min，室温静置30s；
(3)12000rpm离心15min，取600～800μL上清液即为待测液：
色谱分析条件：色谱柱为DB-5MS气相色谱柱(规格30m×0.25mm i.d.×0.25μmd.f.)；进样方式：进样量1.0μL，进样口温度250℃，恒流模式，分流比20：1，载气：氦气(纯度99.999％)，载气流速1.0mL/min；升温程序：初始温度80℃2min，以15℃/min升至170℃并保持3min，再以15℃/min，升至330℃并保持3min。质谱条件设置：电离方式：电子轰击(EI)，电离能量70eV；溶剂延迟：7min；传输线温度：280℃；离子源温度：250℃；扫描方式：选择反应监测模式(SIM)。
1.7GbERFs的分离和表征
(1)将前期数据中筛选的GbERFs与TcERF15、AaERF01、AaERF02、AtERF03、TcERF12和AtERF04的系统发育进化树利用MEGA X软件(https://www.megasoftware.net)构建，采用邻居连接算法和1000次bootstrap重复构建系统发育树，其他参数选择默认参数，选择与TcERF15亲缘关系最近的Gb_36010作为候选基因；
(2)为了验证筛选的可靠性，随机选择Gb_26863、Gb_16683和Gb_36992一同进行后续研究；
(3)银杏叶片提取的RNA用于构建cDNA文库。根据基因组序列设计用于克隆启动子的引物；
1.8酵母单杂交实验
(1)利用Nimble Cloning将GbERF4基因的CDS克隆到pNC-GADT7中，将启动子序列插入到pNC-AbAi载体中；
(2)利用金担子素抑制pNC-AbAi质粒的自激活；
(3)将含有重组的pNC-GADT7和pNC-AbAi质粒的Y1H酵母菌在缺少Ura和Leu且含AbA的固体培养基上培养以确定他们之间能否结合。
1.9双荧光素酶(双LUC)报告基因测定
(1)通过Nimble Cloning法将GbERF4的CDS和gb-miR160的前体茎环序列插入pNC-Green-SK质粒中，将gb-miR160在靶基因GbERF4中的识别位点两端各延长200bp的序列和GbHMGS1、GbAACT1、GbDXS1、GbLPS2及GbGGPPS2启动子序列插入到pNC-Green-Luc中；
(2)重组质粒通过GV3101(psoup)农杆菌介导的瞬时转化法共转化烟草叶片，浸润48h后，使用Dual Luciferase Reporter Assay Kit(Vazyme,Nanjing,China)对样品预处理后，通过酶标仪(BioTek,SynergyTMH1)测定荧光值，以海参荧光素酶作为内参，靶序列活性的瞬时表达水平以LUC/REN比值表示。
本申请使用的所有引物均列于表1
2.结果
2.1参与银杏萜内酯合成的ERF转录因子的筛选与鉴定
为确定哪些ERF是银杏萜内酯合成的潜在调控因子，本申请从本课题组从前期转录组中初步筛选了与银杏萜内酯含量相关性较高的4个ERF转录因子(表2)，分别为Gb_26863(GbERF1)、Gb_16683(GbERF2)、Gb_36992(GbERF3)与Gb_36010(GbERF4)。实时荧光定量分析发现4个ERF基因在不同组织中的表达水平与转录组数据保持高度一致(图1)，且GbERF4表达量与萜内酯含量相关性最高，达到正显著相关水平(图2d)。通过与不同物种中萜类化合物合成相关的ERF TF基因进行系统发育树分析，发现GbERF4与TcERF15、AaERF01和AaERF02关系较近。因此，综合表达谱和进化树的分析结果预示GbERF4可能参与了萜类化合物的合成(图2a)。本申请人课题组前期研究表明MeJA能够诱导萜内酯的合成(Zheng J,Zhang X,Fu M,Zeng H,Ye J,Zhang W,Liao Y,Xu F(2020)Effects of different stresstreatments on the total terpene trilactone content and expression levels ofkey genes in Ginkgo biloba leaves.Plant Mol Biol Rep 38:521–530.)，进一步分析发现这4个ERF的启动子中均含有MeJA响应元件(图2b)。通过MeJA对2年生银杏幼苗处理，GbERF4的表达水平与萜内酯含量相关性最高，达到正显著相关水平(图2c)。综合上述结果表明GbERF4可能具有参与调控银杏萜内酯合成的功能。
图2中：(a)显示银杏乙烯响应因子(GbERFs)与其他ERF蛋白关系的系统发育树；(b)PlantCare分析发现4个候选银杏ERF基因启动子中含有响应JA信号的顺式元件；(c)MeJA诱导下的GbERF1/2/3/4表达水平与萜内酯含量的相关性分析，r为相关系数；(d)GbERF1/2/3/4表达水平在银杏不同组织中与萜内酯含量相关性分析，r为相关系数。
表2转录组中数据中ERFs在不同组织中的表达模式
2.2过表达GbERF4提高了萜类化合物的含量
为了进一步阐明GbERF4在萜类合成中的生物学功能，在35S启动子的驱动下，通过农杆菌介导获得了过表达GbERF4转基因烟草(图3a)，并选择3个T1代独立的现蕾期的GbERF4转基因株系(OEERF4#1、OEERF4#2、OEERF4#3)进行萜类含量和结构基因的表达水平测定分析(图3b和图4a)。采用PCR扩增和GUS染色验证了ERF4在转基因植株中超表达(图3c和d)。与野生型烟草相比，GbERF4过表达株系OEERF4#1、OEERF4#2、OEERF4#3中，被检测的几种萜类化合物的含量均升高，其中角鲨烯和西柏烷三烯的含量显著升高。实时荧光定量PCR结果表明，HMGR、HMGS、DXR、DXS、AACT和GGPPS的表达水平在GbERF4过表达株系中显著升高(图5b)。另外，本申请从T0代烟草植株中获得了一致的结果(图6)。
图3中：(a)根癌土壤杆菌感染烟草后的再生过程；(b)用烟草芽期植株测定了其萜类化合物的含量；(c)ERFs和gb-miR160过表达烟草的PCR扩增的电泳图谱；(d)GUS的报告基因表达。GUS用于通过与靶基因的融合来检测靶基因的表达。
图4中：(a)过表达载体构建；(b)烟草瞬时转化载体构建；(c-d)双荧光素酶基因报告载体的构建；(e)酵母单杂交载体构建。
2.3gb-miR160与银杏萜内酯含量呈负相关
本申请人的课题组之前miRNA测序结果表明gb-miR160的表达水平在不同组织中具有特异性(Ye J,Zhang X,Tan J,Xu F,Cheng S,Chen Z,Zhang W,Liao Y(2020)Globalidentification of Ginkgo biloba microRNAs and insight into their role inmetabolism regulatory network of terpene trilactones by high-throughputsequencing and degradome analysis.Ind Crop Prod 148,112289.)，通过qRT-PCR检测了gb-miR160在不同组织中的表达水平(图7)。为了进一步探究gb-miR160对萜内酯合成的影响，本申请研究了gb-miR160在银杏叶、茎、根、雄花、雌花和果实中的表达模式(图8a)。结果显示，gb-miR160在根中的表达量最低，且与TTLs的含量呈显著负相关(r＝-0.7374)(图8b)，暗示了gb-miR160可能参与银杏TTLs的合成。
2.4过表达gb-miR160降低了萜类化合物的含量
在35S启动子的驱动下，通过农杆菌介导获得了过表达gb-miR160转基因烟草(图3a)，并采用PCR扩增和GUS染色验证了gb-miR160在转基因植株中超表达(图3c和d)。选择3个T1代独立的现蕾期的miRNA转基因株系(OEmiR160#1、OEmiR160#2、OEmiR160#3)进行分析(图3b和图4a)。与野生型烟草相比，gb-miR160过表达株系OEmiR160#1、OEmiR160#2、OEmiR160#3中，被检测的几种萜类化合物的含量均下降，且植醇和角鲨烯的含量显著降低(图5a)。实时荧光定量PCR结果表明，HMGR、HMGS、DXR、DXS、AACT和GGPPS的表达水平在gb-miR160过表达株系中均降低，其中HMGR、HMGS、AACT和GGPPS的表达水平显著降低(图5b)。我们从T0代烟草植株中获得了一致的结果(图6)。
2.5gb-miR160靶向抑制GbERF4的活性
RNA二级结构分析证实，gb-miR160前体可以形成典型的茎环结构(图9a和图10，成熟gb-miR160的序列用下划线标注)。降解组结果显示发现GbERF4可能是gb-miR160的靶基因，GbERF4编码序列(CDS)含有与成熟的gb-miR160相匹配的靶结合序列(图9b)。为进一步探究gb-miR160对目标TF基因GbERF4的靶向切割结果，本申请验证了gb-miR160对GbERF4活性的影响。将pre-miR160(gb-miR160前体序列)和pre-miR156序列分别插入pICH86988载体中组成激活质粒，同时将GbERF4靶位点(ERF4TS)和其突变体(ERF4mTS)与GFP报告基因融合插入pICH86988载体中组成报告质粒(图9b和c)。瞬时表达实验结果表明pre-miR160与ERF4TS共表达观察不到GFP荧光，而替换为pre-miR156前体序列或者ERF4mTS则不影响GFP的表达(图9d)。以ERF4TS为中心前后延伸200bp的序列扩增后插入pGreenII 0800-LUC载体中与LUC报告基因融合，将gb-miR160序列插入pGreenII 62-SK载体中(图9e)。双荧光素酶基因报告实验结果表明，与gb-miR160或GbERF4单独表达相比，共表达gb-miR160和GbERF4显著降低了报告基因的活性(图9f)。上述结果表明，gb-miR160通过靶向切割抑制GbERF4的表达。
图9中：(a)RNA二级结构分析预测gb-miR160前体pre-miR160形成的茎环结构；(b)gb-miR160对GbERF4剪切的位点分析，ERF4mTS为ERF4TS的突变体，Gb-miR156作为阴性对照；(c-d)gb-miR160对GbERF4活性的影响，将gb-miR160或gb-miR156前体序列插入pICH86988载体中组成激活质粒，将GbERF4靶位点(ERF4TS)和其突变体(ERF4mTS)与GFP报告基因融合插入pICH86988载体中组成报告质粒；(e-f)瞬时表达实验验证了gb-miR160对GbERF4靶向剪切作用，并通过双荧光素酶报告试验在烟草叶片中得到证实。
Claims (6)
1.gb-miR160—GbERF4模块在调控银杏萜内酯合成中的应用。
2.根据权利要求1所述的应用，其特征在于，包括gb-miR160—GbERF4调控组件的筛选鉴定。
3.根据权利要求1所述的应用，其特征在于，gb-miR160负向调控萜类化合物合成，GbERF4正向调控萜类化合物合成。
4.根据权利要求1所述的应用，其特征在于，GbERF4可与萜内酯合成途径中酶基因HMGS1，AACT1，DXS1，LPS2和GGPPS2的启动子结合，并激活它们的表达进而参与银杏萜内酯的生物合成。
5.根据权利要求1所述的应用，其特征在于，GbERF4是gb-miR160的靶标。
6.根据权利要求1所述的应用，其特征在于，gb-miR160靶向并切割转录因子GbERF4，抑制GbERF4的表达，从而抑制萜类化合物的积累。
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