CN117448348A - 一个调控月季花香合成的转录因子基因RhMYB2及其应用 - Google Patents
一个调控月季花香合成的转录因子基因RhMYB2及其应用 Download PDFInfo
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- CN117448348A CN117448348A CN202311537253.8A CN202311537253A CN117448348A CN 117448348 A CN117448348 A CN 117448348A CN 202311537253 A CN202311537253 A CN 202311537253A CN 117448348 A CN117448348 A CN 117448348A
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Abstract
本发明公开了一个调控月季花香合成的转录因子基因RhMYB2及其应用。本发明首先利用分子生物学和生物信息学等技术手段,筛选到对月季花香具有调控作用的RhMYB2基因,通过在月季上的过表达分析及转录激活特性分析,验证RhMYB2调控萜烯类花香合成的功能;然后筛选出RhMYB2的下游调控基因RhHDR,验证了RhMYB2与其互作,并验证了RhHDR基因调控萜烯类花香合成的功能,最终证实了RhMYB2调控萜烯类花香合成的功能。本发明揭示了新型的MYB转录因子RhMYB2调控植物萜烯类花香合成,可以应用于月季花香品质改良中。
Description
技术领域
本发明属于植物基因工程领域,具体涉及一个调控月季花香合成的转录因子基因RhMYB2及其应用。
背景技术
月季是我国最重要的花卉经济作物,其种植面积、销售量和销售额均名列我国花卉生产的首位(农业部2016年统计资料)。花香是观赏花卉最重要的特征和经济性状之一,浓而不浊、醇正幽远的花香提高了月季的观赏价值和文化价值,受到人们的推崇。同时,月季的花香成分也是制备高级香水、化妆品、香料等的重要原料,具有极高的经济价值。
月季花香是一类由种类繁多的小分子化合物组成的混合物,目前已经分离出超过几百种,其中,萜烯类成分不但最多,而且是高级玫瑰精油的主要成分。然而,花香成分在月季花瓣中的含量并不高,且不少现代月季的品种都是无香或少香品种。虽然有些月季花香成分可以人工合成,但一方面难度较大(种类纷繁),另一方面品质和市场接受度也不高(中、高等级的化妆品只使用天然月季提取物)。
转录因子常常可以高效的驱动一条代谢途径上,甚至多条相关途径上的多个酶基因,显著影响代谢终产物的合成,表明通过转录因子调控花香合成有望成为提高特定花香成分生成量的有效手段,具有重大的应用价值。目前为止,对植物萜烯类花香相关转录因子的研究仅有5例,而在包括月季在内的观赏花卉中还未见报道。在拟南芥中,bHLH类转录因子AtMYC2通过结合到萜烯合成酶基因AtTPS11和AtTPS21启动子来提高倍半萜(E)-β-石竹烯的合成(Hong,G.,Xue,X.,Mao,Y.,et al.2012.Arabidopsis MYC2 interacts withDELLA proteins in egulating sesquiterpene synthase gene expression.The PlantCell 24(6):2635-2648)。APETALA2/ethylene-response factor(AP2/ERF)家族的一些转录因子成员,如黄花蒿(Artemisia annua)AaERF1和AaERF2可以直接激活青蒿素合成途径上的两个关键酶紫穗槐-4,11-二烯合酶基因ADS和P450单加氧酶基因CYP71AV1促进青蒿素(倍半萜)形成(Yu,Z.,Li,J.,Yang,C.,et al.2012.The jasmonate-responsive AP2/ERFtranscription factors AaERF1and AaERF2 positively regulate artemisininbiosynthesis in Artemisia annua L.Molecular Plant 5(2):353-365);在玉米(Zeamays)中,ZmEREB58直接结合到倍半萜合成酶TPS10基因启动子上促进两种倍半萜(E)-β-法呢烯和(E)-α-香柑油烯合成(Li,S.,Wang,H.,Li,F.,et al.2015.The maizetranscription factor EREB58 mediates the jasmonate-induced production ofsesquiterpene volatiles.The Plant Journal 84(2):296-308)等;在柑橘(Citrussinensis Osbeck)果实中,AP2/ERF转录因子CitERF71直接激活了香叶醇合成酶基因CitTPS16的表达,从而提高果实中的香叶醇产量(Li,X.,Xu,Y.,Shen,S.,etal.2017.Transcription factor CitERF71activates the terpene synthase geneCitTPS16 involved in the synthesis of E-geraniol in sweet orangefruit.Journal of experimental botany,68(17),4929-4938)。以上研究均未涉及观赏花卉中萜烯类的调控,同时,鉴定出的成员仅分布于bHLH和AP2/ERF两个家族。
发明内容
针对上述问题,本发明前期以香味显著超过亲本的月季品种‘甜蜜的梦’及其父本‘艾丽’、母本‘第一玫红’,以及另一个相比双亲花香变化不明显的同系子代‘情歌’为材料,使用权重共表达分析(weighted gene co-expression network analysis,WGCNA)、烟草瞬时过表达和月季花瓣VIGS等方法,鉴定到1个调控萜烯类花香合成的转录因子RhMYB2。本发明揭示了新型的MYB转录因子RhMYB2调控植物萜烯类花香合成,从而为月季花香品质改良提供基因储备,开辟一条新的花卉育种途径。
本发明首先提供了一个调控月季花香合成的转录因子基因RhMYB2,其核苷酸序列如SEQ No.1所示。
上述的转录因子基因RhMYB2在调控月季萜烯类花香合成中的应用。
进一步的,上述应用是通过基因RhMYB2与它的下游调控基因RhHDR(NCBI数据库,ID:112174324)互作来调控月季萜烯类花香合成的。
进一步的,上述基因RhMYB2在月季花香品质改良中的应用,其特征是,通过在月季中过表达RhMYB2基因来提高萜烯类花香物质合成量。
本发明的有益效果是:利用分子生物学和生物信息学等技术手段,筛选出一个月季花香具有调控作用的RhMYB2基因,并对RhMYB2的调控功能进行了进一步验证。通过在月季上的过表达分析及转录激活特性分析,验证RhMYB2调控萜烯类花香合成的功能;筛选出RhMYB2的下游调控基因RhHDR,验证了RhMYB2与其互作,并验证了RhHDR基因调控萜烯类花香合成的功能,最终证实了RhMYB2调控花香合成的功能。本发明揭示了新型的MYB转录因子RhMYB2调控植物萜烯类花香合成,从而为月季花香品质改良提供基因储备,开辟一条新的花卉育种途径。
附图说明
图1为月季品种‘甜蜜的梦’、‘艾丽’、‘第一玫红’和‘情歌’之间的花香释放量变化,其中,A图为萜烯类物质释放量,B图为单帖类物质释放量;
图2为月季品种‘甜蜜的梦’在四个开花阶段间的花香成分(萜烯类物质)的变化图;
图3为月季品种‘甜蜜的梦’相比双亲及另一子代‘情歌’,萜烯类合成途径相关基因发生显著变化图,其中颜色深浅,表示基因表达量的高低;
图4为月季品种‘甜蜜的梦’开花阶段间差异表达的转录因子基因进行重叠(A),初步筛选出可能调控萜烯类花香合成的278个转录因子,在转录因子家族中的分布图(B);
图5为使用WGCNA将月季品种‘甜蜜的梦’初开期、半开期、末花期及‘第一玫红’、‘艾丽’和‘情歌’的半开期共6个样本的代谢组和转录组数据,建立萜烯类花香成分与差异表达基因的关联图;结果,将6个样本间的29,241个差异表达基因聚类到了41个模块(A),并进一步生成了差异表达基因模块与19种花香成分之间的关联热图(B);
图6为RhMYB2基因调控香叶醇合成的功能验证图;结果显示:有RhMYB2基因过表达的烟草叶片中香叶醇指示物香叶醇异戊酸酯显著提高(A),RhMYB2基因沉默的月季花瓣相比对照香叶醇含量显著降低(B);
图7为RhMYB2基因的亲缘关系分析图;
图8为RhMYB2基因过表达的花瓣相比对照的基因表达量(A)及香叶醇释放量(B)的变化图;
图9为RhMYB2蛋白在拟南芥细胞中的亚细胞定位;DAPI染色和叶绿体标记分别显示细胞核(B)和叶绿体(C)的位置,携带GFP的RhMYB2-GFP融合蛋白显示位于细胞核位置(A),能够与细胞核位置(B)完整重叠(D),结果显示RhMYB2定位在细胞核内,具有转录因子的基本特性;
图10为RhMYB2蛋白的转录激活能力验证图;
图11为ChiP-seq结果中RhMYB2富集基因的peak分布图,由图可见,RhMYB2的靶序列在基因功能元件上的分布特征;
图12为RhMYB2基因沉默的花瓣相比对照基因表达量变化火山图;红色表示上调表达的1282个基因,绿色表示下调表达的1783个基因;
图13为qRT-PCR验证RhMYB2基因在其沉默的月季花瓣中相比对照基因表达量显著降低;qRT-PCR的基因相对表达量值RQ(relative quantity),与转录组中基因表达量FPKM值(Fragments Per Kilobase of transcript per Million mapped reads)变化一致;
图14为RhMYB2蛋白结合RhHDR基因启动子元件的酵母单杂交验证图;
图15为RhMYB2蛋白结合RhHDR基因启动子元件的EMSA验证图;
图16为RhMYB2下游靶基因RhHDR在其沉默花瓣及对照(TRV2::00)中的表达量(A)及萜烯类释放量在释放量变化(B)。
具体实施方式
以下结合实施例和附图来说明其效果。
一、调控花香合成的RhMYB2基因的鉴定
1、子代月季品种‘甜蜜的梦’相比父母本、另一子代‘情歌’萜烯类花香物质释放量发生显著的升高
使用动态顶空法联合GC-MS技术对月季品种‘甜蜜的梦’及其父本‘艾丽’、母本‘第一玫红’,以及另一个同系子代‘情歌’的花香挥发物进行了定量分析。四个品种的花香挥发物均以萜烯类物质为主,其中又都以单萜类的单萜醇及其衍生物为主(数量上均超过总数的80%,释放量上占总量的41.06%-78.27%)。四个品种间比较,结果如图1所示,从图中可以看出:‘甜蜜的梦’相比双亲及‘情歌’,萜烯类和单萜类花香成分合成量显著升高;‘甜蜜的梦’萜烯类(单萜类)的总量超过父母本2倍以上、超过‘情歌’25%以上。
2、月季品种‘甜蜜的梦’在开花进程中萜烯类花香物质释放量发生显著的变化
以月季品种‘甜蜜的梦’为试材,利用动态顶空法联合GC-MS技术检测了月季开花过程中4个开花阶段间的花香变化。结果如图2显示,萜烯类花香成分的总量在月季‘甜蜜的梦’花发育的不同阶段发生了显著的变化。具体成分看,绝大多数花香成分都在半开期达到最大的释放量,且在四个阶段间变化显著。
3、发现转录因子参与了月季品种‘甜蜜的梦’萜烯类物质合成变化的调控
对月季品种‘甜蜜的梦’初开期、半开期、末花期,及‘第一玫红’、‘艾丽’和‘情歌’半开期共计6个样本的花瓣材料,进行了Illumina测序。使用实验室生信分析平台,组装得到155,420条高质量的转录本,经过注释,总计90,788条得到注释。通过比对植物转录因子plantTFDB库,共鉴定出3,3491个转录因子,其中10,894个为差异表达的转录因子基因。
通过鉴定花香相关基因,发现萜烯类合成途径上发生了显著的基因表达变化(如图3所示)。其中,作为香叶醇合成的关键酶Nudix水解酶,相应的转录本在‘甜蜜的梦’中的表达量显著提高,分别比双亲的平均水平和‘情歌’高2-5倍。此外其它一些单萜或倍半萜合成酶基因,如β-罗勒烯合成酶、月桂烯酶、类胡萝卜素剪切双加氧酶(CCD)基因等,均在‘甜蜜的梦’中表达量最高,为双亲平均水平和‘情歌’的2倍以上。结果表明:萜烯类合成相关基因在‘甜蜜的梦’中受到了明显的调控。而对‘甜蜜的梦’相比亲本平均表达水平显著差异表达的基因(hybrid-MPV基因)进行的GO富集分析结果显示,转录因子参与了这种调控:富集的369个GO功能类别中,转录调控相关的功能类别,如‘transcription cofactoractivity’(GO:0003712)P值为5.89E-09、‘regulation of transcription,DNA-templated’(GO:0006355)P值为2.81E-07,显著性极高。
4、通过WGCNA工具筛选到潜在萜烯类相关转录因子基因
首先,通过对月季品种‘甜蜜的梦’开花进程的转录组分析,筛选差异表达基因(Differentially expressed gene,DEG)。如图4所示,根据花香合成从初开期经半开期、至末花期先上升后下降的变化趋势,通过重叠初开期经半开期、至末花期先上调后下调、或先下调后上调的差异表达的转录因子基因,获得了一个包含278个成员的集合,分布于bHLH、WOX、NAC等40个转录因子家族,可能与调控萜烯类花香合成有关。
然后,使用WGCNA工具建立差异表达基因与花香成分的关联。将月季品种‘甜蜜的梦’初开期、半开期、末花期及‘第一玫红’、‘艾丽’和‘情歌’的半开期共6个样本的代谢组和转录组数据,建立萜烯类花香成分与差异表达基因的关联图;结果将6个样本间的29,241个差异表达基因聚类到了41个模块,并进一步生成了差异表达基因模块与19种花香成分之间的关联热图(图5)。之后,根据关联系数,综合所有萜烯类成分,包括单萜类的香叶醇(geraniol)、橙花醇(nerol)、乙酸香叶酯(geranyl acetate)、乙酸橙花酯(nerylacetate)、乙酸香茅酯(citronellyl acetate)、柠檬醛(citral)、香茅醇(citronellol)、反式-β-紫罗兰酮(trans-β-ionone)、二氢-β-紫罗兰酮(dihydro-β-ionone)、4,6(E),8(Z)-大柱三烯(megastigma-4,6(E),8(Z)-triene)、二氢-β-紫罗兰醇(dihydro-β-ionol),以及倍半萜类的β-荜澄茄油烯(β-Cubebene)、(+)-香橙烯(Aromandendrene)和β-古巴烯(β-copaene)等,最终筛选出white、skyblue、royalblue、yellow、midnightblue、darkorange、darkred、magenta、violet、darkgreen、darkolivegreen、salmon、greenyellow和darkturquoise等14个模块作为萜烯类的关联模块。
最后,将上一步中筛选出的278个转录因子基因与以上14个模块中基因进行比照,发现142个转录因子基因为两者共有,为萜烯类合成相关的核心候选转录因子,分布于bHLH、MYB related、NAC、ERF等31个转录因子家族。
5、鉴定1个萜烯类合成调控相关的转录因子基因RhMYB2
从筛选出的142个转录因子中,依据在样本间差异表达的程度以及所在模块与萜烯类成分的相关程度,选取7个转录因子基因进行了全长克隆并构建到了pSol2092载体的35S启动子后面,进行了烟草瞬时过表达实验。GC-MS的结果如图6A所示,相比空载体pSol2092,有1个基因的过表达引起了烟草叶片中香叶醇指示物香叶醇异戊酸酯的提高,为RhMYB2,表明其可能调控了萜烯类物质的合成。
RhMYB2基因的扩增引物如下:
RhMYB2-WF:GCCCTTTTAAGCCATTGCAAGA;
RhMYB2-WR:ACACTTTGCGGGCTAATGGA。
RhMYB2基因全长918bp,编码305个氨基酸,序列(SEQ No.1)如下:ATGCAAACTCTGTACCCACCTTCATATATGGCTGATACTAATTGGTTTGTCCAAGAGAGCCAGAGCTCAGATTGGACTAGAGAAGAGAACAAGCAATTTGAGAGTGCTTTGGCTATC TACGATGAGAAAACGCCGGACCGGTGGGGAAAGATTGCGATGATGATTCCGGGGAAGACGGCTGTCGATGTGTTCAAGCAGTACAAGGAGCTGGAAGATGATGTTAGTGACATAGAAGCAGGGCTGGTCGAAATTCCAGGCTACCAACAGCCGTCTTCTTTCACATTGGAGTTGGTGGATGACCGAAATTTCGATGCTAACCGAAAGAGGTCTGCAGCAACTAGAGGTTCTGATCAGGAGAGGAAGAAGGGGATTCCTTGGACAGAAGATGAGCATCTGCGGTTTCTGAAGGGACTTCTAAAATATGGGAAAGGGGATTGGAGAAATATCTCCAGGAACTTTGTTATTTCTAAAACTCCAACTCAAGTTGCTAGCCATGCTCAGAAATACTTTATGAGGCAACACTCAGGAGGAAAAGATAAAAGGAGACCTAGCATCCATGATATAACAACTGTCAACCTCACGAGCACTACCACTACTAGTCCATCACAGAATAATAATAGGCCTCCTTTAGACCAGTCTCCACCACCAGAGCACAATAGCAAGTCCACTGAATCACCAAGAGTCGTACCCGACTGGAATATGCCTAATGATGGATTAGCAGGAGCTATGATCTTTGACGCAACACATGGCGATCTTTTTGAGTCATCGTCTCCATTTGATGTTGGATCTCCAGACGATCTTAAGCGACATTGGCAAAAAATTTACGCCAGTGCCACTCATTATGCTGCTCATGCTAGATCCCCGAGTTCGATGTATCTAATGCAGACCTCAAGACATCAGATACATGGATGCCGCTGA。
月季“嫁接VIGS”验证中,克隆RhMYB2基因约200bp的片段,通过Gateway BP反应克隆到pDONR207(Invitrogen,Carlsbad,California)中构建入门载体pDONR207::RhMYB2,随后通过Gateway LR反应将目的基因转移到TRV2载体上构建沉默载体TRV2::RhMYB2(以未连接基因RhMYB2的TRV2载体作为对照)。
克隆RhMYB2基因片段时使用的引物如下:
RhMYB2-SF:CTCCAGGGCTGGTCGAAATTCCAG;
RhMYB2-SR:CGGCAAGACAGAATAACTCACAGA。
在此基础上,使用“嫁接VIGS”方法对其中RhMYB2基因在月季上进行了功能验证。结果如图6B所示,沉默植株相比对照香叶醇含量显著降低,进一步证实了RhMYB2对香叶醇合成具有正调控作用。
二、RhMYB2基因功能进一步验证
在月季中过表达RhMYB2基因,GC-MS分析过表达植株中花香成分变化,进一步验证其调控萜烯类花香合成的功能。
1、RhMYB2基因的进化、亲缘关系分析
RhMYB2基因序列与已知其它植物同一家族进行比对,分析其在植物中的进化过程。相关工作主要使用Vector NTI、ClustalW2、MEGA等软件。
RhMYB2基因的亲缘关系分析图如图7所示,经过对比拟南芥、绿薄荷、长春花、矮牵牛等MYB家族成员,RhMYB2不属于现有MYB家族分类,具有较大的创新性。
2、RhMYB2基因过表达载体构建
月季花瓣过表达分析中,使用酶切法构建载体。首先使用分别添加HindⅢ和KpnⅠ酶切位点的RhMYB2基因上、下游引物,扩增基因全长,将获得的目的基因全长通过酶切连接到super1300表达载体上,构建super1300::RhMYB2-GFP载体(以未连接基因RhMYB2的super1300::GFP载体作为空载体对照),并转化农杆菌GV3101。
使用引物如下:
RhMYB2-WF:CAAGCTTGGCCCTTTTAAGCCATTGCAAGA;
RhMYB2-WR:GGGTACCCACACTTTGCGGGCTAATGGA。
3、月季花瓣过表达分析
将构建好的空载体对照和super1300:RhMYB2-GFP载体的农杆菌菌株分别在LB固体培养基(含Kan和Rif)上重新活化,取单菌落用5ml LB液体培养基(50μg/ml Kan,50μg/mlRif)28℃培养过夜,再用100ml的LB培养基(50μg/ml Rif,50μg/ml Kan和20mM乙酰丁香酮)28℃进行二次摇菌过夜,5,000rpm常温离心10min收集菌体。菌体沉淀用侵染缓冲液(10mMMgCl2,10mM MES和200mM乙酰丁香酮)悬浮,调节OD600值接近2.0,室温暗处静置3-6h。
以月季花瓣作为实验材料。用真空抽吸法对月季花瓣进行农杆菌侵染(抽真空至0.5-0.7atm后缓慢放气),用去离子水清洗花瓣和disc上多余的菌液。将抽真空后的圆盘放于清水中暗平衡1天,再将花瓣置于清水中光平衡2天,即可取样。
使用qRT-PCR技术鉴定转基因植株,利用GC-MS技术检测过表达转基因植株的花香成分,进一步明确RhMYB2基因对萜烯类花香成分合成代谢的影响。
qRT-PCR所用引物如下:
RhGAPDH-F:ATCCATTCATCACCACCGACTACA;
RhGAPDH-R:GCATCCTTACTTGGGGCAGAGA;
RhMYB2-F:AAGACGGCTGTCGATGTGTT;
RhMYB2-R:AGAAGACGGCTGTTGGTAGC。
结果如图8所示:相比对照super1300::GFP,RhMYB2基因过表达的花瓣中其表达量升高,萜烯类物质合成量显著提高,进一步证实了其调控花香合成的功能。
三、RhMYB2的生化功能分析
1、亚细胞定位
使用构建的super1300::RhMYB2-GFP瞬时表达载体,转染拟南芥原生质体。23℃弱光培养过夜,离心后用枪头吸取弃上清,在激光共聚焦显微镜下进行观察,确定RhMYB2蛋白的亚细胞定位。瞬时表达结果发现(如图9所示):RhMYB2定位在细胞核内,具有转录因子的基本特性。
扩增RhMYB2基因全长时使用引物如下:
pGBKT7-WF:ATATGGCCATGGAGGCCAGTGAATTCATGCAAACTCTGTACCCACCTT CA;
pGBKT7-WR:ATCTGCAGCTCGAGCTCGATGGATCCTCAGCGGCATCCATGTATCTGA T。
2、转录激活能力验证
将RhMYB2基因的全长构建到酵母表达载体pGBKT7上,融合到蛋白DNA结合域的编码序列末端,获得目的基因载体pGBKT7-RhMYB2,以pGADT7-T载体为诱饵载体,共转化酵母菌株AH109。pGBKT7-P53载体(包含DNA结合域和转录激活域的编码序列)+pGADT7-T组合和pGBKT7载体(仅包含DNA结合域的编码序列)+pGADT7-T组合分别为阳性、阴性对照。结果如图10所示:在SD/-Trp培养基上生长全部正常,证明载体及转化操作无误,而阴性对照不能在SD/-His培养基上生长,阳性对照pGBKT7-P53+pGADT7-T及实验组pGBKT7-RhMYB2+pGADT7-T可以在SD/-His培养基上正常生长,表明RhMYB2基因可以激活His元件。结果表明RhMYB2基因有转录激活活性。
四、RhMYB2下游调控基因的筛选
1、ChIP-seq
使用ChIP-seq(染色质免疫共沉淀结合二代测序技术;ChromatinImmunoprecipitation,ChIP)结合qRT-PCR技术筛选RhMYB2的下游调控基因。对RhMYB2的ChIP-Seq数据进行统计分析,确认Input和RhMYB2-IP的raw reads分别为7.92G和7.86G,然后使用软件fastqc(version:0.11.5)对raw resds进行质控分析,再使用软件Trimmomatic(version:0.36)对其进行数据过滤,去除接头和低质量的reads后得到clean reads分别为7.77G和7.64G。将得到的clean reads进一步比对到番茄参考基因组上,其中Input和SlAREB1-IP的比对比率分别为43.24%和40.85%。
对于得到的比对到基因组的有效reads,利用MACS分析软件(version:2.1.1.20160309)在基因组范围内分析peak信息,筛选显著性的peak的阈值设为q-value<0.05。RhMYB2-IP共得到3227个peak,片段平均长度为196bp。统计peak在各基因功能元件分布个数,使用ChIPseeker R包绘制peak在基因功能元件上分布。RhMYB2的靶序列在基因功能元件上的分布特征(如图11所示)为:51.11%在基因间,38.64%在启动子区域,1.94%在外显子区域,3.73%在内含子区域,0.49%在3’-UTR端,4.1%在5’-UTR端。
2、RhMYB2靶序列GO和KEGG分析
为了进一步探讨RhMYB2的结合位点特征,理解RhMYB2对基因调控的机制,从peaksummit位置(若无summit位点则取中点)找到最近的转录起始位点(transcription startsite,TSS)对应的基因进行GO注释,并对GO注释对应的基因数目按照分子功能(MolecularFunction)、细胞组分(Cellular Component)和生物学过程(Biological Process)进行绘图,结果:RhMYB2的靶序列参与生物学过程和分子功能较多,没有参与细胞组分功能。在生物学过程中,其主要与代谢过程(organic cyclic compound metabolic process)、DNA代谢(DNA metabolic process)、酶结合(coenzyme binding)和催化活性(catalyticactivity)有关。
KEGG通路分析反映了RhMYB2的靶序列代谢通路及具体分布情况。对于得到的KEGG富集结果,将p.adjust<0.05筛选出显著性富集结果,共有10个显著富集的KEGG terms,发现其主要分布在次级代谢物合成(Biosynthesis of secondary metabolites)和倍半萜和三萜生物合成(Sesquiterpenoid and triterpenoid biosynthesis)通路,与其调控花香物质合成的功能较为吻合。
3、RhMYB2基因沉默花瓣的转录组分析
将RhMYB2基因沉默与TRV2空载体转化的月季花瓣,进行了转录组分析。采半开期的花瓣,分别构建cDNA文库之后,使用Illumina Hiseq 2500测序平台进行双末端PE150测序。测序完成后,经过严格的质量控制步骤,6个样本一共产生了255M Reads,共38.21Gbpdata,每个样本(包含重复)的数据量均大于6.36Gbp,且Q30范围从90.08%~93.28%,满足了转录组分析对数据的质量要求。
使用HISAT2软件将Clean Reads与参考基因组进行快速精确的比对,获取Reads在参考基因组上的定位信息,6个重复的比对比率都达到了69%以上。根据基因比对在参考基因组上的位置信息,统计每个基因(包括新预测基因)从起始到终止范围内覆盖的reads数。分别过滤掉比对质量值低于10的reads,非成对比对上的reads,比对到基因组多个区域的reads。差异基因的筛选标准是|log2(FoldChange)|>=1&padj<=0.05。结果如图12所示,相比对照,RhMYB2基因沉默的花瓣中,共有3065个差异表达基因,表达量上调的是1282个,下调的是1783个。
4、RhMYB2的下游候选基因
将得到的3227个peak基因,结合其GO和KEGG富集结果,以及RhMYB2基因沉默花瓣中基因差异表达情况,我们筛选出36个基因可能是其靶基因,根据与花香合成调控的相关信息,初步确定基因RhHDR为RhMYB2的下游靶基因。
将RhMYB2基因沉默与TRV2空载体转化的月季花瓣,进行了qRT-PCR验证,结果如图13所示:RhHDR基因在沉默花瓣中表达量减少60%左右,结果进一步佐证了ChiP-seq的结果。
五、RhMYB2与下游调控基因互作关系的验证
1、载体构建
酵母单杂实验中,使用酶切法构建载体。首先使用分别添加EcoRⅠ和BamHⅠ酶切位点的RhMYB2基因上、下游引物,扩增基因全长,将获得的目的基因全长通过酶切连接到酵母表达载体pGADT7上,构建猎物载体pGADT7::RhMYB2。
在构建诱饵载体时,为了避免单杂过程中的自激活现象,月季‘荔枝’RhHDR基因启动子被短截为700bp左右的3条片段序列,分别构建成Pabai-ProRhHDR1,Pabai-ProRhHDR2和Pabai-ProRhHDR3三个载体。载体构建时,片段两侧分别添加HindⅢ和KpnⅠ酶切位点。
构建好的猎物载体同诱饵载体共转化酵母菌株Y1H。以pGADT7-53载体(包含P基因DNA结合域和转录激活域的编码序列)+Pabai-P53(包含P基因启动子序列)组合为阳性对照,以pGADT7载体(只包含转录激活域的编码序列)+Pabai-P53(包含P基因启动子序列)组合为阴性对照。
使用引物如下所示:
pGADT7-MF:
ATATGGCCATGGAGGCCAGTGAATTCATGCAAACTCTGTACCCACCTTCA;
pGADT7-MR:
ATCTGCAGCTCGAGCTCGATGGATCCTCAGCGGCATCCATGTATCTGAT;
Pabai-MF1:CAAGCTTGAATGAACGGTCTTGACAGG;
Pabai-MR1:GGGTACCCCGAAACTGGAGCTTGGGAT;
Pabai-MF2:CAAGCTTGGAAAAGTACTGTAAGCAGC;
Pabai-MR2:GGGTACCCTTCACTAATAATACAAAGCTGC;
Pabai-MF3:CAAGCTTGGCCTAGTCGAGTTTGGTTGTC;
Pabai-MR3:GGGTACCCCGGAGAGGCGTGTGAGAGAG。
结果如图14所示,所有载体在SD/-Leu培养基上都生长正常,证明载体及转化操作无误;然而,仅有阳性对照和Pabai-ProRhHDR1+pGADT7-RhMYB2组合在SD/-Leu/ABA(500ng/ml)培养基上生长正常,表明RhMYB2蛋白可以结合RhHDR基因启动子片段1,与启动子元件分析中片段1包含MYB转录因子结合位点的结果吻合,表明RhMYB2对RhHDR基因有转录激活活性。
2、凝胶迁移实验(EMSA)
凝胶迁移实验(EMSA)包括RhMYB2蛋白的原核表达和纯化、融合蛋白与探针的结合两部分。
EMSA操作中,利用PCR扩增得到RhHDR基因(NCBI数据库,ID:112174324)ATG前2111bp的启动子序列,用-1631~-1596bp之间35bp作为探针,这个探针含有一个6bp的MYB转录因子结合的顺式作用原件CAACTG(图15A)。经生物素标记的探针,标为Biotin-P,未经标记的探针用Cold-P表示(图15B)。将RhMYB2全长构建到原核表达载体pGEX-2T上,与载体上的GST标签融合,构建蛋白表达载体,转化大肠杆菌BL21菌株,在28℃下诱导融合蛋白,并利用GST标签纯化GST-RhMYB2蛋白。将纯化的GST-RhMYB2融合蛋白和探针共孵育然后在Native-PAGE上跑胶,结果如图15B所示,未加入GST-RhMYB2融合蛋白时,无特异条带(泳道1),仅加入GST蛋白以后,也未出现特异条带(泳道2);当加入GST-RhMYB2融合蛋白后,出现特异条带(泳道3),但随着竞争探针(Cold-P)的加入和浓度的增加结合条带逐渐变弱(泳道4,5)。结果表明,GST-RhMYB2能特异结合RhHDR启动子序列。
六、RhMYB2下游靶基因的功能验证
1、载体构建
对于VIGS实验,使用二元TRV载体pTRV1和pTRV2。为了克隆RhHDR基因片段,将RhHDR的392bp编码序列进行PCR扩增,并通过Gateway BP反应克隆到pDONR207(Invitrogen,Carlsbad,California)中构建入门载体pDONR207::RhHDR,随后通过LR反应,将RhHDR片段转移到Gateway兼容的目的载体pTRV2-Gateway上得到TRV2::RhHDR。
扩增RhHDR的392bp编码序列所使用的序列和引物如下:
HDR-SF:ctctagaaggcctccatggggatccCGGCCATAAGGAAGAGACCCTCG;
HDR-SR:
cctcgagacgcgtgagctcggtaccGCCTTCATCGAATCGAAAATGAAGAATTTATAACGTACC。
产物(SEQ No.2):
CGGCCATAAGGAAGAGACCCTCGAGCTCATGAACCGCGAATACACCAGTAAGCTACTAG
CTAGCTATGAATAATTTCAGTCATTAATCGAAGTATTGAAGAAGAATTATTCGGTAAATAA
TTGAAGGATTGAACGATTTGAATTTGAAAATTTTAGGTGATGTTATTAAGAAGCTGAAGG
AGAACGGGTTCGAGTACACCTGGGGAAACGTGACCGTGAAGCTCGCCGAGGCGCACG
GGTTCTGTTGGGGCGTTGAGAGAGCCGTGCAGATTGCTTACGAGGCCAGAAAGCAATTT
CCAGTTGAGAGGATTTGGATTACCAACGAGATCATTCACAATCCGACCGTCAATAAGGTA CGTTATAAATTCTTCAATTTTCGATTCGATGAAGGC。
2、月季花瓣沉默
将构建好的空载体对照(TRV2::00)和TRV2::RhHDR载体的农杆菌菌株分别侵染月季花瓣圆盘(用约8mm打孔器取下的月季花瓣圆片),然后进行qRT-PCR和月季花香检测。
RhMYB2下游靶基因RhHDR基因在其沉默花瓣及对照中的表达量及萜烯类释放量在释放量变化如图16所示。在qRT-pCR检测RhHDR基因表达量降低65%左右的花瓣圆盘中,相比TRV2空载体侵染的圆盘,萜烯类物质含量显著降低,证明了RhHDR基因对萜烯类物质合成具有正调控作用,揭示了RhMYB2基因通过调控RhHDR基因调控花香物质合成的分子机理。
Claims (4)
1.一个调控月季花香合成的转录因子基因RhMYB2,其核苷酸序列如SEQ No.1所示。
2.权利要求1所述的转录因子基因RhMYB2在调控月季萜烯类花香合成中的应用。
3.如权利要求2所述的应用,其特征是,通过转录因子基因RhMYB2与它的下游调控基因RhHDR互作来调控月季萜烯类花香合成。
4.权利要求1所述的转录因子基因RhMYB2在月季花香品质改良中的应用,其特征是,通过在月季中过表达RhMYB2基因来提高萜烯类花香物质合成量。
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