CN113736784B - 银杏长链非编码RNA Lnc2L和Lnc2S及其载体和应用 - Google Patents
银杏长链非编码RNA Lnc2L和Lnc2S及其载体和应用 Download PDFInfo
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Abstract
本发明公开了银杏长链非编码RNA Lnc2L和Lnc2S及其载体和应用,所述银杏长链非编码RNA Lnc2L或者Lnc2S,其核苷酸序列分别如SEQ ID NO.1和SEQ ID NO.2所示。本发明提供的非编码RNA在银杏中过量表达后,可以用于调控银杏类黄酮合成,过量表达长链非编码RNA Lnc2L后类黄酮含量显著增加;而过量表达长链非编码RNA Lnc2S后类黄酮含量显著降低。这表明Lnc2L和Lnc2S是调控银杏类黄酮合成的关键lncRNA,通过Lnc2L和Lnc2S的过量表达或敲除来调控银杏类黄酮的含量,能够按照需求培育出类黄酮含量高的银杏,因此在银杏分子育种过程中具有重要的应用价值。
Description
技术领域
本发明属于植物基因工程技术领域,具体涉及两条新的银杏长链非编码RNALnc2L和Lnc2S及其载体和应用。
背景技术
银杏(Gingo biloba L.)是一种重要的经济树种﹐其叶﹑种仁和外种皮都含药用成分,被称为“全身都是宝的活化石”。银杏作为药用已有600多年的历史,其功效最早记载于《神农本草经》。银杏叶片中含有丰富的次生代谢产物,如类黄酮、萜内酯、聚戊烯醇等活性物质,被广泛大量用于药品、保健品和食品。银杏叶提取物(Ginkgo biloba Extract,GbE)是多种药物的原材料,对预防和治疗早期阿尔兹海默症、心血管疾病等有一定的效果。目前国际上银杏叶提取物相关药物已达30多种。类黄酮化合物是GbE的主要活性成分,目前已经从银杏中分离出40多种的类黄酮化合物,近年来被广泛研究的类黄酮化合物主要有黄酮醇、花青素和原花青素。类黄酮化合物种类较多,目前已知的类黄酮种类已经超过9000种,在植物的叶、根等器官中广泛存在,参与着植物的生长发育及抗逆反应调控。类黄酮化合物具有重要的药理学作用,在防治心血管硬化、抗氧化、抗衰老、抗肿瘤等方面起着重要作用,已经广泛应用在保健、医疗领域。
长链非编码RNA(Long non-coding RNAs,lncRNAs)是一种普遍存在的长度超过200nt的转录产物,这些非编码RNA具有很小的或没有蛋白编码能力,但有功能。lncRNA在转录、转录后和表观遗传水平上作为关键的调控分子参与各种基础生物学过程。目前lncRNA已被发现在植物的生长、发育和逆境耐受中起着重要的作用。
在植物中,一些重要的lncRNAs如OsPI、TPS11、IPS1、COLDAIR、LDMAR等在春化、雄性不育、结节形成、光形态建成和磷酸(Pi)摄取等各种基本过程中都发挥着重要的作用。ENOD40是第一个被预测在苜蓿、大豆的根瘤发生中发挥核糖调节作用的植物lncRNA。但是,相比草本植物,木本植物有关lncRNA的研究较少。
随着高通量测序技术的发展,很多lncRNA已经在植物中被发现。拟南芥中大约有40000个潜在的lncRNA,水稻中有27065个,玉米中有20163个。有关植物lncRNA研究报道较多,但大部分集中在拟南芥、水稻等模式植物,参与调控植物的生长、发育、信号转导、形态建成和胁迫应答等各个生命进程,而有关木本植物lncRNA,尤其裸子植物方面,研究报道相对较少,如银杏上鲜有报道,同时关于调控银杏类黄酮合成的lncRNA目前也没有相关报道。
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两条调控银杏类黄酮合成的关键lncRNA Lnc2L和Lnc2S,通过调控这两条lncRNA的表达能够控制杏类黄酮含量。
本发明还提供了所述的调控银杏类黄酮合成的关键lncRNA Lnc2L和Lnc2S表达的载体及其应用。
技术方案:为了实现上述目的,本发明所述两条新的银杏长链非编码RNA Lnc2L或者Lnc2S,其核苷酸序列分别如SEQ NO.1和SEQ NO.2所示。
本发明所述含有银杏长链非编码RNA Lnc2L或者Lnc2S的过量表达载体。
其中,所述过量表达载体在银杏长链非编码RNA Lnc2L或者Lnc2S的5’端组装组成型强表达启动子CAMV35S,它能使Lnc2L或者Lnc2S在银杏体内高效表达。
其中,所述过量表达载体在银杏长链非编码RNA Lnc2L或者Lnc2S的3’端组装了强终止子NOS-ter,其可有效终止Lnc2L和Lnc2S的转录。
其中,所述过量表达载体组装有NPTⅡ基因表达盒,作为转基因银杏的筛选标记,可以用卡那霉素进行转基因银杏的筛选。
其中,所述过量表达载体组装有LB和RB序列,促使组装于其间的基因表达框架和筛选标记基因NPTⅡ整合至银杏受体细胞染色体中。
本发明所述含有过量表达载体的宿主细胞。
其中,所述宿主细胞是以农杆菌为出发菌株。
本发明所述的银杏长链非编码RNA Lnc2L或者Lnc2S在调控类黄酮合成中的应用。
作为优选,所述应用为:将长链非编码RNA Lnc2L或Lnc2S转入银杏愈伤组织,过量表达长链非编码RNA Lnc2L的转基因银杏愈伤组织类黄酮含量显著增加,而过量表达长链非编码RNA Lnc2S的转基因银杏愈伤组织类黄酮含量显著降低。
本发明以银杏叶片为材料,克隆了两个新的lncRNA Lnc2L和Lnc2S。同时,通过酶切连接将该基因构建到过量表达载体pRI 101-AN(TaKaRa,日本),通过同源重组技术构建获得35S::Lnc2L和35S::Lnc2S载体。该基因位于启动子CaMV35S之后,在启动子CaMV35S的驱动下,Lnc2L和Lnc2S可在银杏愈伤组织中高效表达,从而调控类黄酮的合成。
本发明在银杏中首次克隆到两个新的基因lncRNA Lnc2L和Lnc2S,并且首次发现lncRNA Lnc2L和Lnc2S能够调控银杏类黄酮的合成基因lncRNA Lnc2L和Lnc2S是同一基因编码的两条银杏长链非编码RNA,两条全新的银杏长链非编码RNA Lnc2L和Lnc2S,它们相差98bp,其余序列完全一致。本发明提供的Lnc2L在银杏中过量表达后,可以用于调控银杏类黄酮合成,这表明Lnc2L和Lnc2S是调控银杏类黄酮合成的关键lncRNA,通过Lnc2L和Lnc2S的过量表达或敲除来调控银杏类黄酮的含量,能够按照需求培育出类黄酮含量高的银杏,因此在银杏分子育种过程中具有重要的应用价值。其研究结果将为采用基因调控技术改良提高银杏黄酮类物质合成与积累提供理论依据,并将为今后银杏产业生产中挑选优质种源和后期种植推广提供参考。
有益效果:与现有技术相比,本发明具有以下优点:
本发明首次从银杏中克隆到两个新的lncRNA——Lnc2L和Lnc2S基因,通过将Lnc2L和Lnc2S转入银杏愈伤组织,过量表达Lnc2L或Lnc2S的转基因银杏愈伤组织类黄酮含量明显增加或减少,说明Lnc2L和Lnc2S是调控银杏类黄酮合成的关键lncRNA,调控Lnc2L和Lnc2S的表达能够控制类黄酮的合成,因此调控Lnc2L和Lnc2S的表达在提高银杏叶片药用品质等方面具有重要的应用价值;同时本发明还构建了含Lnc2L或Lnc2S的过量表达载体和宿主细胞。通过Lnc2L和Lnc2S的表达量从而调控银杏类黄酮的含量,能够按照需求培育出类黄酮含量高的银杏,因此在银杏分子育种过程中具有重要的应用价值。
附图说明
图1是Lnc2L和Lnc2S的克隆电泳图;
图2是Lnc2L和Lnc2S的序列比对;
图3是构建好的植物表达载体35S::Lnc2L(a)和35S::Lnc2S(b)的结构示意图;
图4是Lnc2L和Lnc2S转基因银杏愈伤组织的表达量检测(**P<0.01,***P<0.001);
图5是Lnc2L和Lnc2S转基因银杏愈伤组织的类黄酮含量检测(***P<0.001)。
具体实施方式
以下结合附图和实施例对本发明作进一步说明。
实施例1
克隆Lnc2L和Lnc2S
(1)基于银杏lncRNA-seq数据,筛选得到了一个lncRNA,使用Primer Premier 5.0软件对该lncRNA进行人工设计引物。其中正向引物(F引物)为:5’-GTATTCGTTTCCTCATAAACCAGG-3’,反向引物(R引物)为:5’-TTTCAATTGGCAGGGATAATATA-3’。进行PCR扩增时出现了两个条带,可能是由于可变剪切造成的,将序列长的命名为Lnc2L,序列短的命名为Lnc2S(图1)。
(2)利用高保真酶PrimeSTAR Max(Takara,日本)PCR扩增,PCR体系如下:
将上述混合液轻柔混匀,瞬时低速离心后放置于普通PCR反应仪中,设置如下程序:
跑胶:取出PCR仪中的基因扩增产物,利用电泳仪将适量产物点在1%的琼脂糖凝胶上进行检测,25min左右后拿出应用成像系统进行观察,获得目的片段。
(3)纯化片段与克隆载体的连接反应
参照pEASY-Blunt Zero Cloning Kit(全式金,中国)操作说明书将胶回收产物连接到克隆载体上,具体体系如下:
在微型管中将体系中的溶液混合,室温反应5min。反应结束后放在冰上待用。
(4)大肠杆菌转化
参照Trans1-T1 Phage Resistant Chemically Competent Cell产品说明书(全式金,中国),将已连接的产物与感受态细胞混合,经过冰浴、热激、复苏后,取适量涂布于LB平板上,倒置平板,37℃过夜培养。
(5)阳性克隆筛选及测序分析
从筛选培养板上挑选单菌落接种于LB液体培养基中,37℃、250rmp摇菌过夜;直接以培养过夜的菌液为模板进行重组转化子的PCR检测。
反应体系:
反应程序:
菌液PCR检测为阳性的克隆送英骏生物技术公司(上海)测序鉴定,测得Lnc2L和Lnc2S的序列分别为676bp、578bp,序列如SEQ ID NO.1和SEQ ID NO.2所示,进行序列比对时发现Lnc2L比Lnc2S长了98bp,其他序列完全一致(图2)。
实施例2
Lnc2L和Lnc2S的植物表达载体构建
(1)本实验采用TaKaRa QuickCut限制酶(TaKaRa,日本)对pRI 101-AN载体(TaKaRa,日本,该载体上带有启动子CAMV35S,强终止子NOS-ter、NPTⅡ基因表达盒、LB和RB序列)和Lnc2L和Lnc2S的序列分别进行酶切反应实验,具体反应体系如下:
体系中各溶液混合后进行瞬时离心,在37℃水浴锅中保温30min后结束酶切反应,琼脂糖凝胶电泳观察酶切条带,随后分别将目的基因和载体片段切胶回收,用于后续的载体连接反应。
(2)参照TaKaRa T4 DNA Ligase(TaKaRa,日本)操作说明书,将双酶切反应后回收的表达载体与目的DNA片段产物相互连接,体系如下:
在微型管中将体系中的溶液混合,在金属浴中16℃反应5-6h。
通过PCR检测,确认Lnc2L和Lnc2S的过量表达载体构建成功,命名为35S::Lnc2L和35S::Lnc2S,如图3a、3b所示,所构建的表达载体在Lnc2L和Lnc2S的5’端组装了组成型强表达启动子CaMV35S,3’端组装了终止子NOS,表达载体上装NPTⅡ基因表达盒,作为转基因银杏的筛选标记,同时表达载体上组装LB和RB序列,促使组装于其间的基因表达框架和筛选标记基因NPTⅡ整合至银杏受体细胞染色体中。
(3)转化农杆菌
参照GV3101/EHA105 Chemically Competent Cell产品(全式金,中国)操作说明书,将步骤(2)构建的35S::Lnc2L和35S::Lnc2S表达载体质粒与EHA105农杆菌感受态细胞混合,依次经过静置5min、液氮5min、37℃水浴5min、冰浴5min后,加入培养基振荡培养。取适量涂布于LB平板上,28℃培养箱倒置培养。挑取平板上的单克隆,加入适量的LB液体培养基,培养48h,对菌液测序,分别获得含有35S::Lnc2L和35S::Lnc2S载体的农杆菌。
实施例3
Lnc2L和Lnc2S的遗传转化
1、银杏愈伤组织转化
(1)分别将实施例2获得含有35S::Lnc2L和Lnc2S载体的农杆菌涂在LB平板。经过培养后挑取LB平板上的农杆菌单克隆,将其接种到LB液体培养基中,28℃培养16h至OD600为0.5-0.6;
(2)菌液放入离心管中,18℃,3500rpm,15min离心后去掉上清液;
(3)向离心管加入重悬液(100mL MS液体培养基含100μM乙酰丁香酮)重悬底部菌体,并在室温放置2h;
(4)将大小一致的银杏愈伤组织小块放入农杆菌重悬液中,室温静置浸泡15min,然后用镊子轻轻夹出,用无菌滤纸吸掉表面的重悬液液体;
(5)将侵染过的愈伤组织放置在愈伤培养基(MS+4.0mg·L-1NAA+2.0mg·L-1KT+100μM乙酰丁香酮)上,25℃黑暗培养3d,取出放入液氮中速冻,保存在超低温冰箱中,应用于后续的类黄酮含量测定。
2、转基因材料的检测与类黄酮含量的测定
利用PrimeScriptTMReverse Transcriptase Reagent Kit(TaKaRa,日本)进行实时定量PCR,检测Lnc2L和Lnc2S在RNA水平的表达情况,步骤3得到的转基因银杏愈伤组织中Lnc2L和Lnc2S的表达量显著升高(图4),表明Lnc2L和Lnc2S已经成功转入银杏愈伤组织中。利用植物类黄酮提取试剂盒(苏州柯铭生物技术有限公司,中国)对未转基因(CK,未经过本发明含有35S::Lnc2L和35S::Lnc2S载体的农杆菌侵染,其他培养条件相同)和转基因(经含有35S::Lnc2L和35S::Lnc2S载体的农杆菌侵染)银杏愈伤组织的类黄酮含量进行测定,过量表达长链非编码RNA Lnc2L的转基因银杏愈伤组织类黄酮含量显著增加(增加了14.5%)(图5),而过量表达长链非编码RNA Lnc2S的转基因银杏愈伤组织类黄酮含量显著降低(降低了39.8%)(图5)。这些结果表明,Lnc2L有效促进类黄酮的合成,Lnc2S有效抑制了类黄酮的合成,二者配合可以有效调控类黄酮的合成。
序列表
<110> 扬州大学
<120> 银杏长链非编码RNA Lnc2L和Lnc2S及其载体和应用
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 676
<212> DNA
<213> 人工序列(Artificial Sequence)
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gtattcgttt cctcataaac caggtgagta tatggtatgg gaagggcgct ggtattcgtt 60
tcctcataaa ccagctgagt atatgttgtc tcataattta tgcacagctc ctttttattg 120
ggaagaagat gaattcgaat actcatttga acaagaaagt aacaagcaaa ggtattaatc 180
ccaagacgca tgctaacgac ttgtcttggg atagcagctc cttcaaatac gattttcatc 240
gaggaaggga ttggatgtac aatttgtctc tgaataacat aaaatgccgt gcaagaccgt 300
ctcaaatttt cgttagtcgg ggattgtttt aaatatacat tatttcagaa tttaaccatt 360
atagtgtcga tcacgaatag cacctatgta tctccctgtc tgacaaatct gtgaaggggt 420
ttgcgtcact ggaatcctta ttgggcattg tctacagaga aattatattc acaaattctg 480
aaatcacacg ccattatccg attttgatga tgattataat agtatatatg ccaagtaaag 540
tgttttggat gtgggtccat cttccctcac tatgctttaa ttgggaggta ctatggtgta 600
atatatatta tattatccct gccaatttaa ttgggaggta ctatggcgta atgtatatta 660
tccctgccaa ttgaaa 676
<210> 2
<211> 578
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
gtattcgttt cctcataaac caggaagaag atgaattcga atactcattt gaacaagaaa 60
gtaacaagca aaggtattaa tcccaagacg catgctaacg acttgtcttg ggatagcagc 120
tccttcaaat acgattttca tcgaggaagg gattggatgt acaatttgtc tctgaataac 180
ataaaatgcc gtgcaagacc gtctcaaatt ttcgttagtc ggggattgtt ttaaatatac 240
attatttcag aatttaacca ttatagtgtc gatcacgaat agcacctatg tatctccctg 300
tctgacaaat ctgtgaaggg gtttgcgtca ctggaatcct tattgggcat tgtctacaga 360
gaaattatat tcacaaattc tgaaatcaca cgccattatc cgattttgat gatgattata 420
atagtatata tgccaagtaa agtgttttgg atgtgggtcc atcttccctc actatgcttt 480
aattgggagg tactatggtg taatatatat tatattatcc ctgccaattt aattgggagg 540
tactatggcg taatgtatat tatccctgcc aattgaaa 578
Claims (9)
1.银杏长链非编码RNA Lnc2L或者Lnc2S,其核苷酸序列分别如SEQ NO.1和SEQ NO.2所示。
2.一种含有权利要求1所述的银杏长链非编码RNA Lnc2L或者Lnc2S的过量表达载体。
3.根据权利要求2所述的过量表达载体,其特征在于,所述过量表达载体在银杏长链非编码RNA Lnc2L或者Lnc2S的5’端组装组成型强表达启动子CAMV35S。
4.根据权利要求2所述的过量表达载体,其特征在于,所述过量表达载体在银杏长链非编码RNA Lnc2L或者Lnc2S的3’端组装了强终止子NOS-ter。
5.根据权利要求2所述的过量表达载体,其特征在于,所述过量表达载体组装有NPTⅡ基因表达盒,作为转基因银杏的筛选标记。
6.根据权利要求2所述的过量表达载体,其特征在于,所述过量表达载体组装有LB和RB序列,促使组装于其间的基因表达框架和筛选标记基因NPTⅡ整合至银杏受体细胞染色体中。
7.一种含有权利要求2所述过量表达载体的宿主细胞。
8.根据权利要求7所述的宿主细胞,其特征在于,所述宿主细胞是以农杆菌为出发菌株。
9.一种权利要求1所述的银杏长链非编码RNA Lnc2L或者Lnc2S在调控类黄酮合成中的应用,所述应用为:将长链非编码RNA Lnc2L或者Lnc2S转入银杏愈伤组织,过量表达长链非编码RNA Lnc2L的转基因银杏愈伤组织类黄酮含量显著增加,而过量表达长链非编码RNALnc2S的转基因银杏愈伤组织类黄酮含量显著降低。
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