CN118127043A - 一种玫瑰黄酮类化合物调控基因Rr4CL3及其应用 - Google Patents
一种玫瑰黄酮类化合物调控基因Rr4CL3及其应用 Download PDFInfo
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Abstract
本发明公开了一种玫瑰黄酮类化合物调控基因Rr4CL3及其应用,Rr4CL3基因的核苷酸序列如SEQ ID NO.1所示,其mRNA序列如SEQ ID NO.2所示,蛋白氨基酸序列如SEQ ID NO.3所示。本发明鉴定并克隆玫瑰中可能调控黄酮类化合物合成的基因Rr4CL3,通过沉默Rr4CL3基因后玫瑰花瓣中黄酮类物质含量变化,证明Rr4CL3能够调控玫瑰中黄酮类代谢物质的积累,在增强黄酮类代谢物质合成、改良玫瑰综合品质方面有重要应用价值。
Description
技术领域
本发明属于植物基因工程技术领域,具体涉及一种玫瑰黄酮类化合物调控基因Rr4CL3及其应用。
背景技术
玫瑰(Rosarugosa Thunb.)属蔷薇科蔷薇属多年生灌木,花色鲜艳,香气浓郁,是一种药食同源花卉,其药用和营养益处主要源于其次生代谢产物,如挥发油、类黄酮和花青素等。黄酮类化合物是广泛存在于植物体内的次生代谢产物,种类众多,根据环C3的氧化程度和取代模式,黄酮类化合物可分为多个亚群,包括查尔酮、黄烷醇、二氢黄酮、二氢黄酮醇、黄酮、黄酮醇、花青素和异黄酮等,但其在植物中含量低且不易提取。玫瑰花中的黄酮类物质含量较高,主要包括槲皮素、二氢黄酮、山奈酚、花色苷等成分,不仅赋予花朵色素沉着,而且具有抗氧化、抗肿瘤、抗炎抑菌、降血糖、降血脂等多种生理活性,在新药研制、食品贮藏、化妆品工业等领域均有重要应用。因此,研究玫瑰黄酮类化合物的合成基因及合成路径具有重要意义。
目前,黄酮类化合物的生物合成途径已经在许多植物物种中得到了广泛的研究,前三个步骤称为一般苯丙烷途径,几乎在所有双子叶植物中都是保守的,涉及到苯丙氨酸解氨酶(phenylalanine ammonia-lyase,PAL)、肉桂酸4-羟化酶(cinnamic acid-4-hydroxylase,C4H)和香豆酰-CoA连接酶(4-coumaryl-CoA ligase,4CL)。4CL作为植物苯丙烷代谢途径中类黄酮生物合成的第一步,在黄酮类化合物的合成中起关键调节作用。有研究表明,拟南芥(Arabidopsis thaliana)中的4CL基因主要分为两个亚家族,其中,第一亚家族成员4CL1和4CL2参与木质素合成,第二亚家族成员4CL3参与黄酮类物质的生物合成。目前,玫瑰中未明确鉴定到参与合成黄酮类化合物的4CL基因,因此,鉴定并克隆玫瑰黄酮类化合物的合成基因,有助于阐明玫瑰黄酮类化合物的合成路径,为高黄酮类物质含量玫瑰的基因工程育种提供分子工具,具有提高玫瑰营养品质潜在的经济价值。
发明内容
本发明的目的在于针对现有技术中存在的不足,提供一种玫瑰黄酮类化合物调控基因Rr4CL3,以满足使用需求。
本发明的另一目的还在于提供上述玫瑰黄酮类化合物调控基因Rr4CL3的应用。
为了实现上述发目的,本发明采用的技术方案如下:
一种玫瑰黄酮类化合物调控基因Rr4CL3,其核苷酸序列如SEQ ID NO.1所示,所述玫瑰黄酮类化合物调控基因Rr4CL3的表达蛋白的氨基酸序列如SEQ ID NO.3所示。
所述玫瑰黄酮类化合物调控基因Rr4CL3在调控玫瑰黄酮类代谢物质合成中的应用。
含有所述玫瑰黄酮类化合物调控基因Rr4CL3的植物沉默载体。
进一步,所述载体包括辅助载体以及重组载体,所述辅助载体为烟草脆裂病毒pTRV1,重组载体为含玫瑰黄酮类化合物调控基因Rr4CL3片段的烟草脆裂病毒pTR V2-Rr4CL3;所述重组载体表达盒5'端组装有启动子CaMV 35S,可干扰Rr4CL3基因在植株中的表达。
本发明的有益效果:
本发明鉴定并克隆了‘丰花’玫瑰中可能调控黄酮类化合物合成的且与拟南芥At4CL3高同源性的氨基酸序列(ID:evm.model.Chr3.5119),命名为Rr4CL3,其核苷酸序列如SEQ ID NO.1所示;发明人通过构建植物沉默载体pTRV-Rr4CL3同源转化‘墨红’玫瑰,并通过实时荧光定量检测技术和生理表型测定,证明Rr4CL3能够正向调控玫瑰黄酮类代谢物质,在改良玫瑰品质方面具有重要应用价值。
附图说明
图1是植物沉默载体pTRV2-Rr4CL3质粒图谱;
图2是沉默Rr4CL3后的‘墨红’玫瑰花瓣和野生型‘墨红’玫瑰花瓣表型;
图3为Rr4CL3基因在pTRV-Rr4CL3处理组和Mock对照组花瓣中的相对表达量测试结果;
图4为pTRV-Rr4CL3处理组和Mock对照组花瓣中黄酮含量测试结果。
具体实施方式
下面结合附图和具体实施例对本发明的技术方案作清楚、完整地描述。
需要说明的是,下述实施例中,采用的农杆菌感受态购于国药集团;大肠杆菌感受态购于擎科生物;限制性内切酶购于NEB公司;抗生素与激素购于索莱宝公司;RNA提取试剂盒购于天根生化科技有限公司;反转录试剂盒、高保真酶购于TAKA RA生物;产物纯化试剂盒、菌液PCR Taq酶、质粒提取试剂盒、同源重组酶、荧光反转录试剂、荧光定量PCR试剂购于南京诺唯赞生物公司。
实施例1
通过转录本同源比对,鉴定并克隆了Rr4CL3基因:
基于拟南芥中已报道能调控黄酮类化合物的At4CL3基因,同源比对玫瑰转录本,鉴定到相似性为73.84%的玫瑰4CL转录本,包含完整的开放阅读框(ORF)。将其氨基酸序列与拟南芥的原始序列进行蛋白序列比对,发现均含有保守区BOXⅠ和BOXⅡ,初步认定找到的基因是4CL基因,并命名为Rr4CL3。以玫瑰花器官总RNA为模板,克隆Rr4CL3的ORF序列(cDNA序列),转入克隆载体,对插入片段进行阳性筛选后进行测序,测序结果比对正确后确认其为完整的Rr4CL3基因。
I.引物设计
Rr4CL3 ORF引物对为ORF-F:5’-ATGATATCCATTGCGTCTAACAACAAC-3’和为ORF-R:5’-TTAAACGTTCGGAGTGGCTAGC-3’;
II.ORF序列克隆
(1)反转录;
反转录反应体系为:1μg Total RNA,1μL dNTP Mix,1μL Oligo dT Primer,4μL10XRT Buffer,0.5μL RNase Inhibitor,1μL M-MLV Reverse Transcriptase,Nuclease-free Water补至20μL。反应程序为:42℃反应60min,70℃反应15min,得到cDNA模板。
(2)PCR扩增;
PCR反应体系为:KeyPo Master Mix(2×)25μL,ORF-F primer 2μL,ORF-R primer2μL,cDNA 1μL,Nuclease-free ddH2O 20μL;反应程序为:98℃10sec,60℃5sec,72℃9sec,35循环;得到PCR产物。
(3)连接克隆载体;
与5×TA/Blunt-Zero Cloning Mix克隆载体进行连接,连接反应体系(5μL)为:1μL PCR纯化产物,1μL 5×TA/Blunt-Zero Cloning Mix,3μL ddH2O。反应条件为:37℃,5min,得到连接产物。
(4)大肠杆菌转化;
取5μL连接产物,加入到50μL大肠杆菌感受态细胞中,轻弹混匀,冰上静置30min;42℃水浴45~60sec,迅速置于冰上2min;加入700μL无抗LB液体培养基,37℃,100rmp复苏1h;4000rmp离心3min,吸掉上层550μL培养基,混匀剩余菌液,均匀涂抹于含有Kan的LB筛选培养板上,37℃倒置培养过夜。
(5)阳性克隆筛选及测序;
从过夜的培养板上挑选单菌落接种于含有Kan的LB液体培养基中,37℃,200rmp过夜培养,得到菌液,以菌液为模板进行PCR检测,PCR反应体系为:ddH2O8.5μL,Rapid TaqMaster Mix(2×)12.5μL,M13F primer 1μL,M13Rprimer 1μL,菌液2μL;反应程序为:95℃3min(预变性);95℃15sec,60℃15sec,72℃25s,25cycles;72℃5min(彻底延伸),电泳检测。检测为阳性的菌液送生工生物公司(上海)测序。
分析测序结果发现,Rr4CL3的DNA长1767bp,其序列如SEQ ID NO.1所示;Rr4CL3的ORF长1767bp,其序列如SEQ ID NO.2所示;对应的Rr4CL3蛋白序列为588个氨基酸残基,其序列如SEQ ID NO.3所示。
实施例2
Rr4CL3沉默载体构建和‘墨红’玫瑰花瓣盘侵染:
I.载体构建
(1)设计引物,扩增Rr4CL3基因片段,上游引物5’-TTCAAGTGGTCACCAT CGAC-3’和下游引物5’-ACAACAAGACACTGTTCAAGGAGT-3’,PCR扩增、克隆载体连接、大肠杆菌转化以及阳性克隆筛选和测序具体步骤同实施例1;
(2)pTRV2载体线性化,双酶切位点为BamH1和Sma1;
(3)将扩增片段插入pTRV2反义载体上,获取重组质粒pTRV2-Rr4CL3,将重组载体通过液氮冻融法转化农杆菌GV101感受态。所述的液氮冻融法具体方法为:取农杆菌GV3101感受态细胞,冰上融化,加入1μg待转质粒,冰浴5min,液氮速冻5min,37℃水浴锅水浴5min,再次冰浴5min,加入700μL无抗LB液体培养基,28℃,200rpm震荡培养2-3h,涂布于固体LB培养基上(含Kan(卡拉霉素)和Ri f(利福平)),28℃倒置避光培养3天长出单克隆。挑取单克隆于含有Kan和Rif抗生素的液体LB培养基中,28℃,200rpm震荡培养24h,经菌液PCR鉴定阳性后,得到含有质粒pTRV1、pTRV2和pTRV-Rr4CL3农杆菌GV3101阳性菌株,保菌备用。
II.花瓣盘的准备
采集‘墨红’玫瑰盛花期花瓣,用打孔机从花瓣对称位置分离出两个直径为1cm的花瓣盘。随后将花瓣盘置于去离子水中,以达到组织培养增殖后的平衡,备用。
III.侵染液的准备
(1)将含有pTRV1、pTRV2、pTRV-Rr4CL3质粒的农杆菌GV3101菌液,在LB平板培养基上培养(含Kan和Rif),长出单克隆;
(2)分别挑取单克隆放于LB液体培养基中培养(含Kan和Rif),实现单克隆的增殖;
(3)将得到的pTRV1、pTRV2、pTRV-Rr4CL3的单克隆培养液置于锥形瓶中扩大培养,待菌液OD600至1.0左右,室温离心收集菌体。
(4)向步骤(3)所得的菌体中加入重悬液(重悬液中含10mM氯化镁、10mM 2-(N-吗啡啉)乙磺酸(MES)缓冲液和200mM的乙酰丁香酮(AS)),将菌体重悬至终浓度为OD600=1.0。分别将pTRV1与pTRV-Rr4CL3、pTRV1与pTRV2空载的重悬液按体积比1:1混合均匀配置成侵染液,放置于超净工作台中备用。
Ⅳ.玫瑰花瓣盘的侵染
用镊子将玫瑰花瓣从去离子水中小心取出并擦净,等量放入100mL离心管中,记为离心管1和2,离心管1中加入50mL含pTRV1与pTRV2的转化菌液(记为Mock对照组),离心管2中加入50mL含pTRV1与pTRV-Rr4CL3的转化菌液(记为pTRV-Rr4CL3处理组),使用无菌棉球将花瓣浸没在转化菌液中,保鲜膜封口并戳出4-5个孔后置于真空泵中。使用负压装置抽真空(负压装置需将旋钮与吸口垂直,按动开关即可启动真空泵),抽真空至侵染液不再冒泡,缓慢放气,真空渗透2-3次。取出花瓣可观察到颜色变为半透明状即可,后将侵染过的玫瑰花瓣用无菌水短暂洗涤并吸干多余水分,转接到覆于湿润滤纸的培养皿上并封口,置于8℃暗培养。
Ⅴ.总黄酮含量的测定
称取液氮研磨后的玫瑰花瓣0.2g,使用磁力搅拌器将样品与5mL 60%的无水乙醇于120rpm下混合15min,后5000rpm,4℃收集上清液并重复相同的程序两次。将收集到的上清液装于10mL离心管中进行后续的测定。
芦丁标准曲线的制作:精确称取芦丁标准品0.020g,用60%乙醇溶液定容至100mL,配成200μg/mL的溶液,备用。取7个比色管,分别加入0、0.5、1.0、1.5、2.0、2.5、3.0mL芦丁标准溶液,用60%乙醇定容至5mL,涡旋混匀,各加入0.3mL NaNO2(5%),混合均匀后静置5分钟,再加入0.3mL Al(NO3)3(10%),混匀后静置6分钟,加入4mL NaOH(1M),再加入0.4mL 60%乙醇使总样体积为10mL,混匀后静置10分钟,在波长510nm处测定吸光值,以芦丁标准品浓度为横坐标,吸光度为纵坐标,制作标准曲线。标准曲线为:y=0.0011x+0.0008(相关性系数R2=0.9997)。
样品总黄酮含量的测定:取提取液1mL,按以上标准曲线绘制方法进行测定,每组各3个平行。根据上述标准曲线计算相应的总黄酮含量,结果以每克鲜样中含有的芦丁等效物表示,计算公式如下:
W(mg/g)=(X×D×V)/m
其中,X:标准曲线计算的总黄酮含量,mg;
D:稀释倍数;
V:反应混合物中玫瑰花瓣提取液体积,mL;
m:反应体系中含有的玫瑰花瓣提取物干物质质量,g。
图2是沉默Rr4CL3后的‘墨红’玫瑰花瓣和野生型‘墨红’玫瑰花瓣表型,可以看出,Rr4CL3基因沉默后的花瓣盘于第5天开始变色,颜色较对照组变浅。
培养10天后,通过荧光定量PCR技术检测Rr4CL3的沉默效果,于Rr4CL3基因的沉默片段外设计荧光定量正向引物5’-GTCGCTCCCTCGGCTACAAC-3’和反向引物5’-CGTGGCCTCATCGTCGTTCA-3’,基于内参基因设计的正向引物5’-CGGCAACGGATATCTCGG-3’和反向引物5’-TGTGACGCCCAGGCAGACG-3’。利用TaKaRa Premix Taq(Takara公司)和荧光定量PCR仪CFX96TM(Bi o-RAD公司),参照生产商说明书的PCR体系和程序,获取荧光定量PCR获取达到荧光阈值的循环数,计算出Rr4CL3基因在不同处理组花瓣中相对表达量。
Rr4CL3基因在pTRV-Rr4CL3处理组和Mock对照组花瓣中的相对表达量测试结果见图3,结果显示,第十天的Rr4CL3在pTRV-Rr4CL3处理组花瓣中的表达量明显低于对照组。
图4为pTRV-Rr4CL3处理组和Mock对照组花瓣中总黄酮含量测试结果,可以看出,相较于对照组,pTRV-Rr4CL3处理组花瓣的类黄酮含量也明显减少,下降约44.33%,结果与花瓣盘表型相一致。
以上结果表明,Rr4CL3对黄酮类化合物的合成过程起着重要的调节作用,玫瑰的Rr4CL3基因对增强黄酮类物质合成、改良玫瑰综合品质具有重要应用价值。
Claims (4)
1.一种玫瑰黄酮类化合物调控基因Rr4CL3,其核苷酸序列如SEQ ID NO.1所示,所述玫瑰黄酮类化合物调控基因Rr4CL3的表达蛋白的氨基酸序列如SEQ ID NO.3所示。
2.权利要求1所述玫瑰黄酮类化合物调控基因Rr4CL3在调控玫瑰黄酮类代谢物质合成中的应用。
3.含有权利要求1所述玫瑰黄酮类化合物调控基因Rr4CL3的植物沉默载体。
4.如权利要求3所述的植物沉默载体,其特征在于,所述载体包括辅助载体以及重组载体,所述辅助载体为烟草脆裂病毒pTRV1,重组载体为含玫瑰黄酮类化合物调控基因Rr4CL3片段的烟草脆裂病毒pTRV2-Rr4CL3;所述重组载体表达盒5'端组装有启动子CaMV 35S,可干扰Rr4CL3基因在植株中的表达。
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