CN116334109B - 在青蒿中过表达AaMAPK6基因在提高青蒿素含量中的应用及方法 - Google Patents
在青蒿中过表达AaMAPK6基因在提高青蒿素含量中的应用及方法 Download PDFInfo
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Abstract
本发明公开了在青蒿中过表达AaMAPK6基因在提高青蒿素含量中的应用及方法,属于植物基因工程技术领域,该方法包含如下操作步骤:将AaMAPK6基因构建到植物双元表达载体上,然后在农杆菌介导下转化青蒿,经过荧光定量RCR检测、青蒿素含量测定后,筛选得到青蒿素含量提高的转基因植株,所述AaMAPK6基因的核苷酸序列如SEQ ID NO.3所示,过表达AaMAPK6基因的植株中,青蒿素含量升高明显,较野生型提高了1.26~1.56倍。
Description
技术领域
本发明涉及植物基因工程技术领域,具体涉及在青蒿中过表达AaMAPK6基因在提高青蒿素含量中的应用,还涉及提高青蒿中青蒿素的方法。
背景技术
青蒿素是一种含有过氧化桥结构的倍半萜内酯化合物,在治疗抗氯喹型疟疾方面发挥着重要作用。世界卫生组织推荐青蒿素联合疗法作为治疗疟疾的首选方法。青蒿植株中青蒿素含量太低,不能满足患者特别是发展中国家患者治疗需求。因此如何提高青蒿素产量成为青蒿素生产中的重大问题及国内外研究热点。丝裂原活化蛋白激酶(Mitogen-activated protein kinase ,MAPK)级联反应是真核生物中高度保守的信号通路,是由MAPKKK、MAPKK、MAPK三级蛋白激酶组成的磷酸化信号通路。参与调控植物生长发育、免疫应答,逆境胁迫等过程[Zhao et al. 2017]。青蒿中已报道多个转录因子受 JA 强烈诱导表达并参与调控青蒿素生物合成,如ORA、bHLH112、NAC1等。在青蒿中是否存在MAPK激酶会响应植物激素的诱导,并与调控青蒿素生物合成的转录因子发生相互作用,进而影响青蒿素的生物合成,因此,鉴定药用植物青蒿中的MAPK激酶基因成为一种试图提高青蒿素产量的方法。
发明内容
有鉴于此,本发明的目的之一在于提供在青蒿中过表达AaMAPK6基因在提高青蒿素含量中的应用;本发明的目的之二在于提供提高青蒿中青蒿素含量的方法。
为达到上述目的,本发明提供如下技术方案:
1、在青蒿中过表达AaMAPK6基因在提高青蒿素含量中的应用,所述AaMAPK6基因的核苷酸序列如SEQ ID NO.3所示。
2、一种提高青蒿中青蒿素含量的方法,包含如下步骤:通过在青蒿中过表达AaMAPK6基因,所述AaMAPK6基因的核苷酸序列如SEQ ID NO.3所示。
本发明优选的,所述在青蒿中过表达AaMAPK6基因的方法具体为:将AaMAPK6基因构建到植物双元表达载体上,然后在农杆菌介导下转化青蒿,筛选转基因植株,筛选到青蒿素含量提高的转基因植株。
本发明优选的,所述植物双元表达载体为以pHB载体为骨架载体。
本发明优选的,所述植物双元表达载体是将AaMAPK6基因通过BamHI 和XbaI位点构建到pHB载体上。
本发明优选的,所述农杆菌为EHA105。
本发明优选的,所述筛选转基因植株的方法是采用荧光定量RCR检测。
本发明优选的,所述荧光定量RCR的上游引物如SEQ ID NO.10所示,下游引物如SEQ ID NO.11所示。
本发明优选的,所述荧光定量RCR的反应条件为:94℃预变性3min;94℃变性10s,58℃退火20s,72℃延伸20s,40个扩增循环,每个循环的72℃延伸结束后采集荧光。
本发明的有益效果在于:本发明克隆了得到了青蒿AaMAPK6基因的编码序列,AaMAPK6基因的编码区核苷酸序列如SEQ ID NO.3所示,翻译后得到氨基酸序列如SEQ IDNO.4所示;将AaMAPK6基因通过BamHI和XbaI位点构建到植物双元表达载体pHB上,然后在农杆菌EHA105介导下转化青蒿,经过荧光定量RCR检测、青蒿素含量测定后,筛选得到青蒿素含量提高的转基因植株,过表达AaMAPK6基因的植株中,青蒿素含量升高明显,较野生型提高了1.26~1.56倍数,为培育青蒿素高含量的新品种和规模化生产具有重要意义。
附图说明
为了使本发明的目的、技术方案和有益效果更加清楚,本发明提供如下附图进行说明:
图1为AaMAPK6基因序列;
图2为pHB载体图谱;
图3为转基因植株的基因表达量检测图;
图4为AaMAPK6转基因中青蒿素含量图。
具体实施方式
下面结合附图和具体实施例对本发明作进一步说明,以使本领域的技术人员可以更好的理解本发明并能予以实施,但所举实施例不作为对本发明的限定。
实施例1、 AaMAPK6基因获得
提取青蒿RNA,反转为cDNA,根据青蒿基因组,设计AaMAPK6上、下游引物:
上游引物为:5’-atggatccatcaaatcaacaacc-3’(SEQ ID NO.1);
下游引物为:5’-cagattctggtactcgggat-3’(SEQ ID NO.2);
然后以青蒿cDNA为模板,以AaMAPK6上、下游引物进行PCR扩增,扩增条件为95℃预变性5分钟;95℃变性30秒,56℃退火30秒,72℃延伸105秒,共32个循环;72℃延伸2分钟,4℃保存,进行PCR扩增,扩增产物经回收后经测序获得AaMAPK6基因序列(图1),其编码区核苷酸序列如SEQ ID NO.3所示,翻译后得到的氨基酸序列如SEQ ID NO.4所示。
实施例2、构建AaMAPK6基因的植物表达载体
一、过表达载体pHB -AaMAPK6的构建
根据克隆到的AaMAPK6基因序列,分析其编码区的酶切位点和pHB载体(图2)上的多克隆位点,选择BamHI和SpeI作为载体构建的限制性酶切位点,在AaMED25全长的上游引入 BamHI 酶切位点,在AaMED25下游引入SpeI酶切位点,所用引物为:
AaMAPK6-F-BamH1:5’-tgcagcccgggggatccATGGATCCATCAAATCAACA-3’ (SEQ IDNO.5);
AaMAPK-R-SpeI :5’-cgataccgtcactagtCAGATTCTGGTACTCGGGAT-3’(SEQ IDNO.6);
用该对引物扩增AaMAPK6,然后将扩增的AaMAPK6序列和pHB同时用内切酶BamHI和XbaI酶切后进行同源重组连接。
二、干扰表达载体pBin19-AaMAPK6的构建
选取AaMAPK6基因组上300bp的特异片段(SEQ ID NO.7)作为干扰片段,设计一对引物,并在上下游引物 5′端分别引入HindIII和XbaI酶切位点:
imapk6-F:5’-gcggtaccaagcttTATCTAACATCACTGCACGATATCAG-3’(SEQ ID NO.8);
imapk6-R:5’-gcctcgagtctagaTATTTGGTTGCCATGACCTAACC-3’(SEQ ID NO.9);
将该片段正向克隆到干扰载体pHANNIBAL上;继续在上下游引物5’端再分别引入KpnI和XhoI酶切位点,将该片段反向克隆到引入了正向片段的干扰载体pHANNIBAL上,SacI和SpeI消化该载体,回收含有正、反向AaMAPK6片段的干扰表达框,通过T4连接酶将该片段连接到经SacI和XbaI消化的植物表达载体pBin19大骨架上,最终形成AaMAPK6的RNAi载体pBin19-AaMAPK6。
实施例3、转基因青蒿的获得
一、青蒿苗的准备:
(1)取适量青蒿种子于1.5 mL的Ep管中,加入1 mL体积分数为75%的酒精,浸泡30min,然后吸去酒精,用无菌水清洗3-5次;
(2)加入1 mL体积分数为10%的次氯酸钠溶液涡旋混匀,暗处消毒10 min,用无菌水洗3-5次;
(3)吸取1 mL无菌水,将种子一起吸入移液枪中,均匀涂布在MS培养基上,吸掉多余的水,吹干。4℃春化2d,放于光照培养室培养。(培养条件为:温度:25℃;光周期:16 h光/8 h暗;光照强度:80-220 μmo·m -2·s-1 ,湿度60%)培养一周左右,待幼苗长出第一对真叶,将其第一对真叶在叶柄基部剪下用于青蒿的遗传转化;
二、青蒿的遗传转化:
(1)将构建的过表达载体(pHB-AaMAPK6)和干扰表达载体pBin19-AaMAPK6分别转化农杆菌EHA105;
(2)在抗性板(Rif+Kan)上进行筛选,挑取单克隆菌斑进行 PCR 检测;
(3)扩大培养:在250 mL三角瓶中加入50 mL YEP(Rif+Kan)液,加入100 μL阳性菌液,28℃,200 rpm,过夜培养;
(4)待工程菌的菌液浓度培养至OD600为0.6左右时,4000 rpm,离心5 min,收集菌体。菌体沉淀用1/2MS液体培养基重悬至OD600为0.5左右,28℃,200 rpm 培养30 min;
(5)将培养好的菌液侵染青蒿叶片外植体20min,平铺于MS板(MS+NAA 0.1 mg/L+6-BA 1.0mg/L)上。25℃,暗培养2 d,2 d后将其转至分化板(MS+NAA+6-BA+Hygr)上。每隔10 天更换一次筛选培养基,一个月后,大量的丛生芽从分化培养基板上长起;
(6)将丛生芽剪下,依次转到壮苗培养基和生根培养基上,直到形成一颗完整的植株;
(7)将生根的青蒿植株移栽到土中生长;
三、阳性植株的基因表达量检测
荧光定量法检测阳性转基因植株的基因表达量,Actin作为内参基因,荧光定量检测的引物:
qAaMAPK6-F:5’-GAACATCATGCCCCAACC-3’(SEQ ID NO.10);
qAaMAPK6-R:5’-GGATTGTTTATATACAACAAACAGGAAG-3’(SEQ ID NO.11);
qActin-F:5’-CCAGGCTGTTCAGTCTCTGTAT-3’(SEQ ID NO.12);
qActin-R:5’-CGCTCGGTAAGGATCTTCATCA-3’(SEQ ID NO.13):
反应条件:94℃预变性3min,94℃变性10s,58℃退火20s,72℃延伸20s,40个扩增循环,每个循环的72℃延伸结束后采集荧光。
结果如图3所示,其中A图为AaMAPK6基因在干扰AaMAPK6 转基因青蒿(imapk6-1、imapk6-2、imapk6-3、imapk6-4)和野生型青蒿(CK)中的相对表达量,说明干扰AaMAPK6 转基因青蒿株系中AaMAPK6基因的表达量表现出不同程度的降低;B图为AaMAPK6 基因在过表达 AaMAPK6 转基因青蒿(OX-AaMAPK6-5、OX-AaMAPK6-7、OX-AaMAPK6-8、OX-AaMAPK6-11)和野生型青蒿(CK)中的相对表达量,说明过表达AaMAPK6 转基因青蒿株系中AaMAPK6基因的表达量表现出不同程度的升高。
实施例4、AaMAPK6过表达和干扰表达青蒿植株中青蒿素的含量测定
一、青蒿素的提取
(1)采集青蒿地上部分组织,于40℃烘箱中,烘烤至完全干燥,将其研磨成粉末;
(2)60℃恒温水浴锅预热石油醚;
(3)称取0.2 g粉末于50 mL Ep管中,加入25 mL石油醚,80 Hz超声40 min;
(3)用滤纸过滤,50 mL小烧杯收集滤液;再用25 mL石油醚清洗滤纸上残渣,同一烧杯再次收集滤液;
(4)将液体转移到100 mL的旋转蒸发瓶中,于50℃水浴中减压旋转蒸发,至石油醚挥发完全。
(5)向旋转蒸发瓶中加入1 mL色谱级甲醇,超声1 min;将重悬液体转移至1.5 mLEp管中,12000 rpm,离心10 min;上清液用0.22 μm的滤膜过滤,滤液用高效液相色谱联合蒸发光散射检测(HPLC-ELSD)测定青蒿素含量;
二、HPLC 测定青蒿素的含量
HPLC仪器为SPD20A***控制器,蒸发光散射检测器(EvaporativeLight-Scattering Detector,ELSD)。使用Waters C18色谱柱,流动相:乙腈和水,体积比 60%﹕40%,流速:1 mL/min;ELSD检测***为water alliance 2420,蒸发光散射检测器漂移管温度为40℃,载气压力5 bar;标准样品进样20 μL,每个样品进样20 μL,重复3次进样。青蒿素标准品的出峰时间为9.703 min,样品的保留时间为9.673 min,根据标准品的浓度和峰面积计算出样品中的青蒿素的含量,再除以青蒿粉末干重,从而计算出青蒿素占样品干重的含量。
转基因植株的青蒿素含量检测结果如图4所示,其中A图为干扰AaMAPK6转基因青蒿和野生型青蒿中的青蒿素含量对比,B图为过表达 AaMAPK6 转基因青蒿(imapk6-1、imapk6-2、imapk6-3、imapk6-4)和野生型青蒿(CK)中的青蒿素含量对比,说明干扰AaMAPK6基因的植株中青蒿素的含量没有明显升高,而过表达AaMAPK6基因的植株中,青蒿素含量升高明显,较野生型提高了1.26~1.56倍。以上所述实施例仅是为充分说明本发明而所举的较佳的实施例,本发明的保护范围不限于此。本技术领域的技术人员在本发明基础上所作的等同替代或变换,均在本发明的保护范围之内。本发明的保护范围以权利要求书为准。
Claims (9)
1.在青蒿中过表达AaMAPK6基因在提高青蒿素含量中的应用,其特征在于:所述AaMAPK6基因的核苷酸序列如SEQ ID NO.3所示。
2.一种提高青蒿中青蒿素含量的方法,其特征在于,包含如下步骤:通过在青蒿中过表达AaMAPK6基因,所述AaMAPK6基因的核苷酸序列如SEQ ID NO.3所示。
3.根据权利要求2所述提高青蒿中青蒿素含量的方法,其特征在于:所述在青蒿中过表达AaMAPK6基因的方法具体为:将AaMAPK6基因构建到植物双元表达载体上,然后在农杆菌介导下转化青蒿,筛选转基因植株,筛选到青蒿素含量提高的转基因植株。
4.根据权利要求3所述提高青蒿中青蒿素含量的方法,其特征在于:所述植物双元表达载体为以pHB载体为骨架载体。
5.根据权利要求3所述提高青蒿中青蒿素含量的方法,其特征在于:所述植物双元表达载体是将AaMAPK6基因通过BamHI和XbaI位点构建到pHB载体上。
6.根据权利要求3所述提高青蒿中青蒿素含量的方法,其特征在于:所述农杆菌为EHA105。
7.根据权利要求3所述提高青蒿中青蒿素含量的方法,其特征在于:所述筛选转基因植株的方法是采用荧光定量PCR检测。
8.根据权利要求7所述提高青蒿中青蒿素含量的方法,其特征在于:所述荧光定量PCR的上游引物如SEQ ID NO.10所示,下游引物如SEQ ID NO.11所示。
9.根据权利要求7所述提高青蒿中青蒿素含量的方法,其特征在于:所述荧光定量PCR的反应条件为:94℃预变性3min;94℃变性10s,58℃退火20s,72℃延伸20s,40个扩增循环,每个循环的72℃延伸结束后采集荧光。
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