CN116769823A - 一种提高青蒿中青蒿素含量的方法 - Google Patents
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Abstract
本发明公开了一种提高青蒿中青蒿素含量的方法。所述方法包括:将青蒿遗传改造为缺乏功能性AaMYBTL3蛋白。本发明利用hairpinRNA介导的RNA干扰或Cas9介导的基因敲除方法抑制AaMYBTL3的表达,获得了青蒿素含量显著提高的转基因青蒿植株,青蒿素的含量可提高1.9倍,为青蒿素的规模化生产提供高产、稳定新药源具有重要意义。
Description
技术领域
本发明属于生物技术技术领域,涉及一种提高青蒿中青蒿素含量的方法。
背景技术
青蒿素(artemisinin)作为一种有效的抗疟药物已经得到全世界的认同。目前,青蒿素的主要来源是从青蒿植株的地上部分提取。Artemisia annua L.(青蒿)又名黄花蒿,是一年生草本药用植物。由于青蒿中青蒿素的含量非常低(占干重的0.01%-1%),使得这种药物的大规模商业化生产受到了限制。虽然现在已经能够通过化学合成或生物合成的方式生产青蒿素,但由于难度大,产量低,成本高,不具备商业化生产的可行性。
目前,利用组织培养及细胞工程来生产青蒿素是一种很有吸引力的方法。然而青蒿素在愈伤组织中含量低于干重的0.1%,在芽中最高也只有干重的0.16%,而大多数研究在根中没有检测到青蒿素。因此利用组织培养及细胞工程来生产青蒿素的可行性不高。
利用基因工程技术获得稳定的青蒿素含量较高的青蒿新品种是一种比较可行的方法,包括采用过量表达青蒿合成途径中的关键酶法尼基焦磷酸合酶(farnesyldiphosphate synthase,FPS)的方法等,如CN106755060A公开共转FPS和DBR2基因提高青蒿素含量的方法及制备的青蒿,将法尼基焦磷酸合酶FPS基因和青蒿醛Δ11(13)双键还原酶DBR2基因共转化青蒿。通过高效液相色谱-蒸发光散射检测器测定青蒿中青蒿素含量,筛选获得青蒿素含量提高的转基因青蒿植株。本发明获得的转基因青蒿中青蒿素的含量显著提高,最高达到非转化对照植株的3.36倍。
综上所述,开发提高青蒿素产量的方法,扩展高效生产青蒿素的手段,对于青蒿素的推广应用具有重要意义。
发明内容
针对现有技术的不足和实际需求,本发明提供一种提高青蒿中青蒿素含量的方法,本发明首次采用抑制AaMYBTL3基因表达的策略提高青蒿中青蒿素的含量,为高效生产青蒿素提供新手段和思路。
为达上述目的,本发明采用以下技术方案:
第一方面,本发明提供一种提高青蒿中青蒿素含量的方法,所述方法包括:
将青蒿遗传改造为缺乏功能性AaMYBTL3蛋白。
本发明中,发现采用如RNA干扰或基因敲除等手段抑制青蒿中AaMYBTL3基因表达水平,能够提高青蒿素含量,为提高青蒿中青蒿素的含量提供了一条可行的方法,为利用转基因青蒿大规模生产青蒿素奠定坚实的基础。
优选地,所述遗传改造为缺乏功能性AaMYBTL3蛋白包括敲除AaMYBTL3基因或弱化AaMYBTL3基因表达量。
优选地,所述敲除AaMYBTL3基因的方法包括:
从青蒿中克隆AaMYBTL3基因片段,构建含AaMYBTL3基因靶点的把AaMYBTL3基因部分序列构建于Cas9载体上,得到含AaMYBTL3基因靶点的Cas9-AaMYBTL3植物表达载体Cas9-AaMYBTL3,将Cas9-AaMYBTL3载导入青蒿中获得再生植株。
优选地,所述敲除AaMYBTL3基因的方法具体包括:
从青蒿中克隆AaMYBTL3基因片段,设计sgRNA,并把AaMYBTL3基因部分序列构建于Cas9载体(市场有公开出售的生物材料)上,得到含AaMYBTL3基因靶点的植物表达载体Cas9-AaMYBTL3,将Cas9-AaMYBTL3载导入青蒿中获得再生植株。
优选地,所述Cas9包括Cas9-1300载体。
优选地,所述弱化AaMYBTL3基因表达量的方法包括:
从青蒿中克隆AaMYBTL3基因片段,构建含AaMYBTL3 hairpin的植物表达载体pHellsgate-AaMYBTL3,将pHellsga4te-AaMYBTL3导入青蒿中获得再生植株。
优选地,所述弱化AaMYBTL3基因表达量的方法具体包括:
从青蒿中克隆AaMYBTL3基因片段,把AaMYBTL3基因片段构建成hairpin结构后可操作性地连于表达调控序列,重组到phellsgate12载体(为市场有公开出售的生物材料,可以从多家公司如澳大利亚CAMBIA公司购得)上得到含AaMYBTL3 hairpin基因的植物表达载体pHellsgate-AaMYBTL3,将pHellsgate-AaMYBTL3导入青蒿中获得再生植株。
优选地,所述提高青蒿中青蒿素含量的方法包括:
从青蒿中克隆AaMYBTL3基因片段,构建含AaMYBTL3 hairpin的植物表达载体pHellsgate-AaMYBTL3和/或含AaMYBTL3基因靶点的Cas9-AaMYBTL3载体,用根癌农杆菌介导,将pHellsgate-AaMYBTL3和/或Cas9-AaMYBTL3载体转入青蒿中获得再生植株,检测目的基因AaMYBTL3的整合和表达情况,测定青蒿素的含量,筛选获得青蒿素含量提高的转基因青蒿植株。
优选地,所述检测目的基因AaMYBTL3的整合和表达情况的方法包括PCR和qRT-PCR。
优选地,所述PCR的方法包括:
设计合成AaMYBTL3 hairpin基因的引物,进行转化植株进行DNA扩增,紫外线下观察到目的条带的阳性植株即为转基因青蒿植株;设计合成Cas9-AaMYBTL3基因的引物,对转化植株进行DNA扩增,紫外线下观察到目的条带的阳性植株即为转基因青蒿植株。
优选地,所述qRT-PCR的方法包括:
分别设计合成AaMYBTL3基因和看家基因actin的引物,进行qRT-PCR扩增,利用相对定量法分析基因相对表达量,包括青蒿素合成途径关键酶ADS,CYP71AV1,DBR2和ALDH1基因的表达等。
优选地,所述测定青蒿素的含量的方法包括高效液相色谱法,进一步优选地,使用ZORBAX SB-C183.5μm,2.1×100mm,SN861753-902,SNUSRY002682色谱柱,流动相为乙腈-0.1%甲酸水溶液(65:35),柱温为30℃,进样量为5μL,流速为0.3mL/min,单针进样时间为5min。
作为优选的技术方案,所述的提高青蒿中青蒿素含量的方法包括以下步骤:
(1)从青蒿中克隆AaMYBTL3基因片段;
(2)设计sgRNA,并把AaMYBTL3基因部分序列构建于Cas9载体上,得到含AaMYBTL3基因靶点的植物表达载体Cas9-AaMYBTL3,和/或,
把AaMYBTL3基因片段构建成hairpin结构后可操作性地连于表达调控序列,得到含AaMYBTL3 hairpin基因的植物表达载体pHellsgate-AaMYBTL3;
(3)将Cas9-AaMYBTL3和/或pHellsgate-AaMYBTL3转化根癌农杆菌,获得用于转化青蒿的根癌农杆菌菌株;
(4)利用所构建的根癌农杆菌菌株转化青蒿,利用PCR和qRT-PCR检测AaMYBTL3的整合和表达情况,利用荧光显微镜统计青蒿腺毛密度;
(5)检测转基因青蒿植株中青蒿素含量,获得青蒿素含量提高的转基因青蒿植株。
与现有技术相比,本发明具有以下有益效果:
本发明利用hairpinRNA介导的RNA干扰或Cas9介导的基因敲除方法抑制AaMYBTL3的表达,实现了提高青蒿中青蒿素含量,获得了青蒿素含量显著提高的转基因青蒿植株,pHellsgate-AaMYBTL3转基因青蒿植株中青蒿素的含量最高可达到16mg/g DW(即干重的1.6%),是非转化普通青蒿(9mg/g DW,即干重的0.9%)的1.8倍,Cas9-AaMYBTL3转基因青蒿植株中青蒿素的含量最高可达到17.1mg/g DW(即干重的1.71%),是非转化普通青蒿(9mg/g DW,即干重的0.9%)的1.9倍,为青蒿素的规模化生产提供高产、稳定新药源具有重要意义。
附图说明
图1为AaMYBTL3表达水平图;
图2为青蒿素合成途径中基因表达水平图;
图3为荧光显微镜观察青蒿腺毛图;
图4为青蒿腺毛密度图;
图5为青蒿素含量图。
具体实施方式
为进一步阐述本发明所采取的技术手段及其效果,以下结合实施例和附图对本发明作进一步地说明。可以理解的是,此处所描述的具体实施方式仅仅用于解释本发明,而非对本发明的限定。
实施例中未注明具体技术或条件者,按照本领域内的文献所描述的技术或条件,或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者,均为可通过正规渠道购买获得的常规产品。
本发明具体实施例中未注明具体条件的实验方法,通常按照常规条件,例如Sambrook等分子克隆:实验室手册(NewYork:Cold Spring Harbor Laboratory Press,1989)中所述的条件,或按照制造厂商所建议的条件。
实施例1
本实施例进行青蒿AaMYBTL3基因片段的克隆。
1.青蒿基因组总RNA的提取
取少量青蒿(采用产于重庆酉阳青蒿素含量较高的青蒿品种)幼嫩叶片,用液氮速冻后,迅速用研钵研碎,加入盛有1mL TRIzol(TRIzol Reagents,GIBCO BRL,USA)的1.5mLEppendorf管中,充分振荡后,于25℃下放置5min,加200μL氯仿,用力振荡15sec,25℃放置2min后,于4℃、12,000g离心15min;将上清液(约600μL)吸入干净的1.5mL Eppendorf管中,加入等体积的异丙醇,颠倒混匀,25℃下放置10min后,于4℃、12,000g离心10min;弃上清,加1mL 75%乙醇清洗,振荡后,于4℃、7,500g离心5min;25℃干燥15-20min后溶于适量(30-50μL)RNAase-free水中;用甲醛变性胶电泳鉴定总RNA质量,然后在分光光度计上测定RNA含量。
2.青蒿AaMYBTL3基因片段的克隆
将所获的青蒿基因组总RNA通过反转录酶XL(AMV)反转录获得第一链cDNA,根据所述青蒿AaMYBTL3基因的编码序列,设计扩增出含有部分AaMYBTL3编码序列(序列1的1bp到621bp)的上下游引物,并在上游和下游引物上分别引入接头序列(这可视选用的载体而定),以便构建表达载体。以所述的第一链cDNA为模板,经PCR扩增后进行测序。DNA序列测定由苏州金唯智技术服务有限公司采用3730自动测序仪完成。测序结果表明,所克隆的部分序列与GenBank中所报道的青蒿AaMYBTL3基因的编码序列(序列1的1bp到621bp)一致。
序列1:
atgaggaatgtgttaccggcctctgttaagaaagggccatggacacatgaagaagatgaaTtgttgtcgagttacatcgctagagaaggagaggggcagtggcaggccctacccaagaagGcgggacttctccgttgtgggaagagctgcagactccgttggatgaactaccttcgtccttctgtcaagcgaggccacatctccgccgatgaagaagacctcatcatccggctccaccgccttcttggtaataggtggtcattaatagcgggaaggattcctgggcgtacagataacgagattaagaactactggaatactcaccttagcaagaagttaacaagccaagggattgatccaaagactcacaaaccattatcatcaccatcttctctaaacccaaatcacaatctaaaccattcatcttcttcaccggatcaaaccaaaactatttcaacaaattcccaagaaaacatctcaaacctcaactccatcaacccgcacagtggtcctcgatctgatgataatcttagccactcgcctgatgatgggttctcttcgttcttggattcactgatcaacgacgaaatgtgcagcgaacgcaccccaactgctctatag。
本实施例采用基因克隆方法从青蒿中获得序列正确的青蒿中青蒿腺毛负调控基因AaMYBTL3的编码序列,为通过RNA干扰抑制、Cas9敲除AaMYBTL3的表达从而提高青蒿素含量提供了基因片段。
实施例2
本实施例进行含AaMYBTL3 hairpin基因的植物双元表达载体的构建。
1.中间载体pENTR-AaMYBTL3 hairpin的构建
将实施例1中AaMYBTL3基因片段通过基因引物PF1:CACCCTCCGCC GATGAAGAAGACC,PR1:TCCGGTGAAGAAGATGAATGG扩增出来,通过GATAWAY的入门克隆的方法连接到pENTR上形成PENTR-AaMYBTL3。具体操作如下:用高保真酶KOD plus(日本东洋纺)同引物F1R1扩增出AaMYBTL3片段。通过和pENTR载体(赛默飞公司商品化载体)的同源重组30min后(反应体系如表1所示),将重组产物转化大肠杆菌DH5α,挑取单克隆,提取质粒做PCR检测,获得中间载体pENTR-AaMYBTL3。
表1
pENTR反应体系 | 体积 |
pENTR载体(20ng/μL) | 0.25μL |
盐缓冲液 | 0.5μL |
PCR产物(2.5mmol/L) | 0.5μL |
ddH2O | 1.75μL |
总体积 | 3μL |
2.植物双元表达载体pHellsgate::AaMYBTL3 hairpin的构建
以所述的pENTR-AaMYBTL3载体为基础,同载体pHellsgate12进行LR反应,连接到pHellsgate上形成pHellsgate-AaMYBTL3。具体操作如下:抽提pENTR-AaMYBTL3的质粒,通过和pHellsgate载体(赛默飞公司商品化载体)同源重组30min后(反应体系如表2所示),将重组产物转化大肠杆菌DH5α,连接转化,挑取单克隆,提取质粒做PCR检测,获得植物双元表达载体pHellsgate-AaMYBTL3。
表2
本实施例将青蒿腺毛发育负调控基因AaMYBTL3的部分编码序列构建成hairpin结构后可操作性地连于表达调控序列,形成含AaMYBTL3 hairpin基因的植物表达载体pHellsgate-AaMYBTL3,该表达载体可用于通过代谢工程策略来提高青蒿中青蒿素含量。
实施例3
本实施例进行含AaMYBTL3基因Cas9敲除载体的构建
按照CHOPCHOP网站(http://chopchop.cbu.uib.no/)的描述,输入实施例1中AaMYBTL3编码区序列(序列1)作为Target,选择拟南芥基因组为参照,以CRISPR/Cas9的方法进行knock-out,点击“FindTarget Sites”,搜索该段序列的靶位点。根据基因组位置、正反义链、GC含量、预测敲除效率等,选择最优的sgRNA链。
根据酶切位点设计引物,PF1:GATTAACTCCATCAACCCGCACAG,PR1:AAACCTGTGCGGGTTGATGGAGTT,合成后按照1:1的比例加入PCR管中,PCR反应程序为:94℃保持1min,按照5℃/min从90℃梯度降火至15℃,10℃5min,保存于4℃即可。
按照表3中的体系进行载体连接,25℃连接30min,将重组产物转化大肠杆菌DH5α,连接转化,挑取单克隆,提取质粒做PCR检测,获得植物敲除表达载体Cas9-AaMYBTL3.
表3
组分 | 添加量 |
PCR产物 | 6.5μL |
10×T4 DNA缓冲液 | 1μL |
Cas9-1300载体 | 2μL |
T4DNA连接酶 | 0.5μL |
本实施例将青蒿腺毛发育负调控基因AaMYBTL3的靶点序列构建形成含AaMYBTL3部分基因的植物表达载体Cas9-AaMYBTL3,该表达载体可用于通过代谢工程策略来提高青蒿中青蒿素含量。
实施例4
本实施例利用根癌农杆菌介导AaMYBTL3 hairpin、Cas9-AaMYBTL3基因遗传转化青蒿获得转基因青蒿植株。
1.含AaMYBTL3 hairpin基因双元植物表达载体pHellsgate-AaMYBTL3、含AaMYBTL3基因靶点的Cas9-AaMYBTL3根癌农杆菌工程菌的获得
将实施例2中含AaMYBTL3 hairpin基因的植物双元表达载体pHellsgate-AaMYBTL3、实施例3中Cas9-AaMYBTL3载体转入根癌农杆菌(如EHA105,为市场有公开出售的生物材料,可以从澳大利亚CAMBIA公司购得,菌株编号为Gambar 1),并进行PCR验证。结果表明,含AaMYBTL3 hairpin基因植物双元表达载体pHellsgate-AaMYBTL3、含AaMYBTL3基因靶点的Cas9-AaMYBTL3已成功构建到根癌农杆菌菌株。
2.根癌农杆菌介导AaMYBTL3 hairpin、Cas9-AaMYBTL3的基因转化青蒿
2.1.外植体的预培养
青蒿种子用75%乙醇浸泡1min,再用20%NaClO浸泡20min,无菌水冲洗4次,用无菌吸水纸吸干表面水分,接种于无激素的MS(Murashige and Skoog,1962)固体培养基中,25℃、16h/8h(光/暗)光照培养,即可获得青蒿无菌苗。待苗长至5cm左右后,剪取无菌苗叶片外植体用于转化。
2.2.农杆菌与外植体的共培养
将所述的叶片外植体,转到共培养培养基(1/2MS+AS 100μmol/L)中,分别滴加含活化好的所述含AaMYBTL3 hairpin基因植物双元表达载体、含AaMYBTL3基因靶点的Cas9-AaMYBTL3的根癌农杆菌工程菌的1/2MS悬液,使外植体与菌液充分接触,28℃暗培养3天(d)。以滴加在不带有目的基因的根癌农杆菌的1/2MS液体培养基悬液的叶片外植体为对照。
2.3.抗性再生植株的筛选
将所述的共培养3d的青蒿外植体转入到发芽筛选培养基(MS+6-BA 0.5mg/L+NAA0.05mg/L+Kan 50mg/L+Cb 500mg/L)上于25℃、16h/8h光照培养,每两周继代培养一次,经过3次继代后即可获得Kan抗性丛生芽。将生长良好的抗性丛生芽剪下转入生根培养基(1/2MS+Cb 125mg/L)上培养至生根,从而获得Kan抗性再生青蒿植株。
3.转基因青蒿植株的PCR检测
根据目的基因所在载体序列AaMYBTL3 hairpin、Cas9-AaMYBTL3分别设计正向引物设计和反向引物对转基因青蒿植株中目的基因进行检测。结果表明,利用所设计的PCR特异引物(表4),pHellsgate-AaMYBTL3转化植株能扩增出约400bp的特异DNA片段,而以非转化青蒿基因组DNA为模板时,没有扩增出任何片段;Cas9-AaMYBTL3转化植株能扩增出约200bp的特异DNA片段,而以非转化青蒿基因组DNA为模板时,没有扩增出任何片段,且转基因植株AaMYBTL3序列上存在编辑位点。
表4
pHellsgate-AaMYBTL3-PF1 | GAGCTACACATGCTCAGG |
pHellsgate-AaMYBTL3-PF2 | GGGATGACGCACAATCC |
pHellsgate-AaMYBTL3-PR | TCCGGTGAAGAAGATGAATGG |
Cas9-AaMYBTL3-PF | GTAAAACGACGGCCAGT |
Cas9-AaMYBTL3-PR | AAACCTGTGCGGGTTGATGGAGTT |
AaMYBTL3-PF | ATGAGGAATGTGTTACCGGC |
AaMYBTL3-PR | TAGAGCAGTTGGGGTGCGTT |
本实施例将所述的植物表达载体转化根癌农杆菌,获得用于转化青蒿的含AaMYBTL3 hairpin、含AaMYBTL3基因靶点的Cas9-AaMYBTL3基因植物表达载体的根癌农杆菌菌株,利用所构建的根癌农杆菌菌株转化青蒿,获得经PCR检测的转基因青蒿植株。
实施例5
本实施例进行QRT-PCR检测转基因青蒿植株中AaMYBTL3基因的表达。
1.引物的设计和合成
根据青蒿AaMYBTL3基因和看家基因actin全序列设计引物。引物扩增基因片段长度分别为100bp、300bp。所用引物由苏州金唯智合成。引物序列如下:
q-AaMYBTL3-PF:CGCACAGTGGTCCTCGATCTGA;
q-AaMYBTL3-PR:TCGCTGCACATTTCGTCGTTGA;
q-Aaactin-PF:CCAGGCTGTTCAGTCTCTGTAT;
q-Aaactin-PR:CGCTCGGTAAGGATCTTCATCA。
2.RNA的提取和反转录
参照实施例1中所述方法,从青蒿植株中提取RNA,用DNaseI除去RNA中基因组DNA,用反转录酶XL(AMV)进行第一链cDNA的合成。
3.RT-PCR检测转基因青蒿中AaMYBTL3基因和看家基因actin的表达
为调整各样品间在RNA抽提和逆转录过程中反应效率的差异,检测目的基因AaMYBTL3表达量的同时需检测看家基因actin基因的表达。检测actin和目的基因的PCR的反应体系如表5所示。
表5
反应体系 | 体积 |
GSP1(10μmol/L) | 0.5μL |
GSP2(10μmol/L) | 0.5μL |
dNTP(2.5mmol/L) | 1.5μL |
10×PCR缓冲液 | 2.5μL |
MgCl2(25mmol/L) | 1.5μL |
模板cDNA(100ng/μL) | 1μL |
Taq酶(5U/μL) | 0.3μL |
ddH2O | 17.2μL |
总体积 | 25μL |
PCR反应条件:热启动和变性94℃5min,1个循环;94℃30s,54℃30s,72℃10s,35个循环;72℃8min,1个循环;4℃保存。电泳检测结果。
本实施例采用qRT-PCR技术测定转基因青蒿中腺毛发育负调控因子AaMYBTL3基因表达量的高低,如图1所示,结果表明RNA干扰抑制(RNAi)AaMYBTL3基因表达量降低至对照的80%,Cas9敲除AaMYBTL3基因表达量降低至对照的45%-62%;同时检测青蒿素合成途径基因的表达量的高低,如图2所示,与未转基因处理的对照组相比,RNA干扰抑制(RNAi)AaMYBTL3基因表达和Cas9敲除AaMYBTL3基因的转基因青蒿中青蒿素合成途径基因的表达量均升高,可以初步筛选出可能的青蒿素高产的青蒿植株。
实施例6
利用荧光显微镜测定青蒿腺毛密度。由于青蒿腺毛会自发荧光,通过奥林巴斯荧光显微镜BX43系统,在激发光波长为430nm下观察荧光,计算面积统计腺毛密度,结果如图3和图4所示,与未转基因处理的对照组相比,RNA干扰抑制(RNAi)AaMYBTL3基因表达和Cas9敲除AaMYBTL3基因的转基因青蒿中青蒿腺毛密度显著提高。
实施例7
本实施例利用HPLC-MS/MS测定转基因青蒿中青蒿素含量。
1.HPLC-MS/MS条件及系统适用性以及标准溶液的配制
HPLC:使用ZORBAX SB-C183.5μm,2.1×100mm,SN861753-902,SNUSRY002682色谱柱,流动相为乙腈-0.1%甲酸水溶液(65:35),柱温为30℃,进样量为5μL,流速为0.3mL/min,单针进样时间为5min。
精密称取青蒿素标准品(Sigma公司)2.0mg用1mL甲醇完全溶解,得到2mg/mL青蒿素标准品溶液,保存于-20℃备用。
2.标准曲线的制作
将所述对照品溶液在相应色谱条件下分别进样2μL,4μL,6μL,8μL,10μL记录图谱及色谱参数,分别以峰面积(Y)对标准品含量(X,μg)进行回归分析。通过研究,本发明中青蒿素在4-20μg范围内呈现良好的log-log线性关系。青蒿素对照品的log-log线性回归方程为:
Y=1.28e+000X+4.71e+000;R=0.979546。
3.样品的制备和青蒿素含量的测定
青蒿素的提取过程基于Van Nieuwerburgh et al.(Quantitationofartemisinin and its biosynthetic precursors in Artemisia annua L.by highperformance liquid chromatography-electrospray quadrupole time-of-flighttandem mass spectrometry.JChromatogrA.2006,1118(2):180-187)中报道的方法:取少量新鲜的青蒿叶片(2g鲜重),于50mL试管中将其浸没在10mL氯仿中摇荡1min,将浸出液倒入新的试管中使氯仿挥发完全,取3mL无水乙醇充分溶解提取物,用于HPLC检测。同时,氯仿提取后的叶片收集放入60℃烘箱进行烘干,称重(计算青蒿叶片的干重);
采用HPLC-MS/MS测定青蒿素含量,样品进样体积为20μL,根据峰面积代入线形回归方程计算出样品中的青蒿素含量(mg),再除以样品的青蒿叶干重(g),从而计算出青蒿植株中青蒿素的含量。
结果如图5所示,AaMYBTL3 hairpin和Cas9-AaMYBTL3转化植株青蒿素含量显著提高,pHellsgate-AaMYBTL3转基因青蒿植株中青蒿素的含量最高可达到16mg/g DW(即干重的1.6%),是非转化普通青蒿(9mg/g DW,即干重的0.9%)的1.8倍,Cas9-AaMYBTL3转基因青蒿植株中青蒿素的含量最高可达到17.1mg/g DW(即干重的1.71%),是非转化普通青蒿(9mg/g DW,即干重的0.9%)的1.9倍。
综上所述,本发明利用hairpinRNA介导的RNA干扰或Cas9介导的基因敲除方法抑制AaMYBTL3的表达,实现了提高青蒿中青蒿素含量,获得了青蒿素含量显著提高的转基因青蒿植株,为规模化生产青蒿素提供了一种理想方法。
申请人声明,本发明通过上述实施例来说明本发明的详细方法,但本发明并不局限于上述详细方法,即不意味着本发明必须依赖上述详细方法才能实施。所属技术领域的技术人员应该明了,对本发明的任何改进,对本发明产品各原料的等效替换及辅助成分的添加、具体方式的选择等,均落在本发明的保护范围和公开范围之内。
Claims (10)
1.一种提高青蒿中青蒿素含量的方法,其特征在于,所述方法包括:
将青蒿遗传改造为缺乏功能性AaMYBTL3蛋白。
2.根据权利要求2所述的提高青蒿中青蒿素含量的方法,其特征在于,所述遗传改造为缺乏功能性AaMYBTL3蛋白包括敲除AaMYBTL3基因或弱化AaMYBTL3基因表达量。
3.根据权利要求2所述的提高青蒿中青蒿素含量的方法,其特征在于,所述敲除AaMYBTL3基因的方法包括:
从青蒿中克隆AaMYBTL3基因片段,构建含AaMYBTL3基因靶点的把AaMYBTL3基因部分序列构建于Cas9载体上,得到含AaMYBTL3基因靶点的Cas9-AaMYBTL3植物表达载体Cas9-AaMYBTL3,将Cas9-AaMYBTL3载导入青蒿中获得再生植株。
4.根据权利要求3所述的提高青蒿中青蒿素含量的方法,其特征在于,所述敲除AaMYBTL3基因的方法具体包括:
从青蒿中克隆AaMYBTL3基因片段,设计sgRNA,并把AaMYBTL3基因部分序列构建于Cas9载体上,得到含AaMYBTL3基因靶点的植物表达载体Cas9-AaMYBTL3,将Cas9-AaMYBTL3载导入青蒿中获得再生植株;
优选地,所述Cas9包括Cas9-1300载体。
5.根据权利要求2-4任一项所述的提高青蒿中青蒿素含量的方法,其特征在于,所述弱化AaMYBTL3基因表达量的方法包括:
从青蒿中克隆AaMYBTL3基因片段,构建含AaMYBTL3hairpin的植物表达载体pHellsgate-AaMYBTL3,将pHellsga4te-AaMYBTL3导入青蒿中获得再生植株。
6.根据权利要求5所述的提高青蒿中青蒿素含量的方法,其特征在于,所述弱化AaMYBTL3基因表达量的方法具体包括:
从青蒿中克隆AaMYBTL3基因片段,把AaMYBTL3基因片段构建成hairpin结构后可操作性地连于表达调控序列,得到含AaMYBTL3hairpin基因的植物表达载体pHellsgate-AaMYBTL3,将pHellsga4te-AaMYBTL3导入青蒿中获得再生植株。
7.根据权利要求1-6任一项所述的提高青蒿中青蒿素含量的方法,其特征在于,所述方法包括:
从青蒿中克隆AaMYBTL3基因片段,构建含AaMYBTL3hairpin的植物表达载体pHellsgate-AaMYBTL3和/或含AaMYBTL3基因靶点的Cas9-AaMYBTL3载体,用根癌农杆菌介导,将pHellsgate-AaMYBTL3和/或Cas9-AaMYBTL3载体转入青蒿中获得再生植株,检测目的基因AaMYBTL3的整合和表达情况,测定青蒿素的含量,筛选获得青蒿素含量提高的转基因青蒿植株。
8.根据权利要求7所述的提高青蒿中青蒿素含量的方法,其特征在于,所述检测目的基因AaMYBTL3的整合和表达情况的方法包括PCR和qRT-PCR;
优选地,所述PCR的方法包括:
设计合成AaMYBTL3hairpin基因的引物,进行转化植株进行DNA扩增,紫外线下观察到目的条带的阳性植株即为转基因青蒿植株;
设计合成Cas9-AaMYBTL3基因的引物,对转化植株进行DNA扩增,紫外线下观察到目的条带的阳性植株即为转基因青蒿植株;
优选地,所述qRT-PCR的方法包括:
分别设计合成AaMYBTL3基因和看家基因actin的引物,进行qRT-PCR扩增,利用相对定量法分析基因相对表达量。
9.根据权利要求7或所述的提高青蒿中青蒿素含量的方法,其特征在于,所述测定青蒿素的含量的方法包括高效液相色谱法。
10.根据权利要求1-9任一项所述的提高青蒿中青蒿素含量的方法,其特征在于,所述方法包括以下步骤:
(1)从青蒿中克隆AaMYBTL3基因片段;
(2)设计sgRNA,并把AaMYBTL3基因部分序列构建于Cas9载体上,得到含AaMYBTL3基因靶点的植物表达载体Cas9-AaMYBTL3,和/或,
把AaMYBTL3基因片段构建成hairpin结构后可操作性地连于表达调控序列,得到含AaMYBTL3hairpin基因的植物表达载体pHellsgate-AaMYBTL3;
(3)将Cas9-AaMYBTL3和/或pHellsgate-AaMYBTL3转化根癌农杆菌,获得用于转化青蒿的根癌农杆菌菌株;
(4)利用所构建的根癌农杆菌菌株转化青蒿,利用PCR和qRT-PCR检测AaMYBTL3的整合和表达情况,利用荧光显微镜统计青蒿腺毛密度;
(5)检测转基因青蒿植株中青蒿素含量,获得青蒿素含量提高的转基因青蒿植株。
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