CN110157729A - 一种获得高产黄酮的罗布麻毛状根诱导方法 - Google Patents

一种获得高产黄酮的罗布麻毛状根诱导方法 Download PDF

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CN110157729A
CN110157729A CN201910424172.4A CN201910424172A CN110157729A CN 110157729 A CN110157729 A CN 110157729A CN 201910424172 A CN201910424172 A CN 201910424172A CN 110157729 A CN110157729 A CN 110157729A
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范桂枝
姜黎
张李香
詹亚光
王海燕
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Abstract

本发明是一种利用Ar.1193菌株获得高产黄酮的罗布麻毛状根诱导方法，它属于生物工程领域。其特征在于：(1)Ar.1193菌株对罗布麻无菌苗茎段的遗传转化效率为69.17％；(2)在一个培养周期(23d)内，毛状根生长量增殖近9倍；(3)一个培养周期后收获毛状根中的总黄酮含量达63.28mg/g，是大田收获罗布麻植株根部含量的7倍。本发明提供一种遗传转化率高、生长速度快和黄酮含量高的罗布麻毛状根遗传转化体系建立方法。

Description

一种获得高产黄酮的罗布麻毛状根诱导方法
技术领域：
本发明是一种利用Ar.1193菌株获得生长速度快、黄酮含量高的罗布麻毛状根，它属于生物工程领域。
背景技术：
1952年，我国农业经济学家董正钧先生在新疆罗布泊首次发现罗布麻，并随发现地命名。一般认为我国的罗布麻植物分为1属2种，即罗布麻属(Apocynum)，罗布红麻(A.venetum)和罗布白麻(A.hendersonii)两个种(麻浩等，2017；徐宗昌等，2018)。罗布麻是一种名贵的中草药，入药历史可追溯到千年以前。据明朝李时珍《本草纲目》、《救荒本草》等药典记载：罗布麻具有平心悸、止眩晕、消痰止咳、强心利尿之功效。现代药理学研究发现，罗布麻还具有增强机体免疫力、降血压、强心、保肝、抗辐射、延缓衰老、抗癌和抗肿瘤等功效(杨博等，2016；Wen等，2016；Huang等，2017；Zhang等，2018)。目前有罗布麻茶、复方罗布麻片和罗布麻霜等产品在市场上销售。
在药理成分的研究方面发现，罗布麻的主要活性成分为黄酮类化合物，包括槲皮素、异槲皮甙和金丝桃甙等，同时发现黄酮类化合物的种类和含量存在组织部位、种类和分布区域的特异性(An等，2013；王慧竹等，2018；杨永涛等，2018)。李慕春(2018)等对33份不同生境来源的罗布麻和白麻叶片化学成分分析表明，从种植地域考虑，新疆阿勒泰、吉木乃和伊犁等地罗布麻品质最佳。另外，据不完全统计，我国生长罗布麻的土地约有133万公顷，产量达10万吨，其中，新疆约有53万公顷，产量约为5万吨。由此可见，新疆不仅是我国罗布麻分布最广的地区，也是罗布麻的最佳产地。
毛状根培养是20世纪80年代发展起来的基因工程和细胞工程相结合的一项技术，通过将发根农杆菌(Agrobacterium rhizogenes)中Ri质粒含有的T-DNA整合到植物的DNA上，诱导植物细胞产生毛状根(王成龙等，2015)。毛状根具备未转化的正常根的所有形态和生理特征，在生理上十分接近真正的根。由于毛状根生长迅速、周期短、生长条件简单、目的产物含量高且稳定，是规模化生物碱类、甙类和黄酮等次生代谢产物的途径之一(陈宇等，2016；Tsukagoshi等，2016；Mahmood等，2018)。
因此，利用发根农杆菌获得罗布麻毛状根，并对毛状根进行继代增殖培养，可规模化生产黄酮等次生代谢物，是罗布麻药用资源可持续发展的有效途径之一。
发明的内容：
本发明提供一种利用Ar.1193菌株获得高产黄酮的罗布麻毛状根诱导方法，其主要内容为：(1)Ar.1193菌株对罗布麻无菌苗茎段的遗传转化效率为69.17％；(2)一个培养周期(23d)收获毛状根中的总黄酮含量达63.28mg/g，是大田收获罗布麻植株根部含量的7倍。
本发明的目的是提供一种遗传转化率高，毛状根生长速度快且黄酮含量高的罗布麻毛状根遗传转化体系建立方法，为罗布麻有药用价值次生代谢产物生产源源不断地提供原料。
附图说明
附图1罗布麻无菌苗茎段获得毛状根的过程，(1)无菌苗茎段；(2)茎段诱导出的毛状根；(3)毛状根的增殖培养。
附图2转化毛状根的rolB基因的PCR检测电泳图，M，DL2000marker；1和2；阳性对照(质粒DNA)；3，空白对照(水)；4～5转化的不同毛状根无性系。
附图3一个培养周期内，3L小型生物反应器液体培养的罗布麻毛状根。
具体实施方案：
罗布麻无菌苗的获得，主要实施过程为：在超净工作台中，将清洗干净的罗布麻种子用75％的酒精杀菌30s,然后用无菌水洗2遍，再用的5％次氯酸钠消毒5min，无菌水清洗3遍，滤纸吸干种子表面水分，接种到WPM固体培养基上。培养条件为温度25℃，湿度40％～50％，光照强度1500～2000lux，光照16h/d。5d左右罗布麻种子发芽，15d左右可获得罗布麻的无菌苗。
利用Ar.1193菌株遗传转化罗布麻无菌苗茎段，主要实施过程为：采用培养至OD＝0.6～0.8的Ar.1193菌液分别侵染预培养0～3d的罗布麻茎段10～30min，然后共培养1～3d，对共培养后的茎段外植体利用400mg/L头孢噻肟钠进行除菌处理，最后接种于含量有头孢噻肟钠的WPM培养基上诱导毛状根。
罗布麻毛状根的鉴定，主要实施过程为：将外植体转入抑菌培养基后，大约一周左右茎段的切口部位将会有毛状根出现，诱导出的毛状根生长迅速，分枝多(图1)。取毛状根利用用植物基因组提取试剂盒提取发根农杆菌Ri质粒DNA和罗布麻毛状根基因组DNA，对所提取到的DNA样品进行PCR扩增和电泳检测，结果如图2所示，发根农杆菌所携带的目的基因已经整合进罗布麻植株中产生毛状根。同时研究发现，将OD＝0.6Ar.1193菌液分别侵染预培养2d的罗布麻茎段15min，然后共培养2d，其遗传转化效率最高，达69.17％。
罗布麻毛状根的液体继代增殖培养，主要实施过程为：将检测呈阳性、生长旺盛且分枝多的毛状根接种于WPM+20g/L蔗糖的液体培养基中增殖培养，罗布麻的毛状根生长速度快，一个月干物质增殖约9倍(图3)。
罗布麻毛状根中黄酮含量检测，主要实施过程为：采用紫外分光光度计法分析了培养一个生长周期(23d)、组培苗实生根和新疆大田收获期罗布麻成熟根中的总含量，发现黄酮含量毛状根(63.28mg/g)>大田根(9.35mg/g)>组培苗实生根(7.84mg/g)。

Claims (1)

1.一种获得罗布麻毛状根的诱导方法，其特征在于：利用Ar.1193菌株侵染罗布麻组培苗茎部，可获得生长速率快、黄酮含量高的毛状根。
CN201910424172.4A 2019-05-21 2019-05-21 一种获得高产黄酮的罗布麻毛状根诱导方法 Pending CN110157729A (zh)

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