CN108070603B - 一种提高油用牡丹籽含油率的转基因方法 - Google Patents
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Abstract
一种提高油用牡丹籽含油率的转基因方法，属于生物工程领域，该方法首先构建多基因植物表达载体，然后通过根癌农杆菌将其转入到牡丹植株组织中表达并遗传，其中，所述多基因植物表达载体的构建是在pBI1211‑GUS上35S启动子的3’末端引入一个多克隆位点，构建载体pBI121‑35S MCS，再将外源基因WRI1、LEC2和FAD2依次插入到pBI121‑35S MCS中，构建形成多基因植物表达载体pBI121‑WRI1‑LEC2‑FAD2。该方法已构建的多基因植物表达载体为基础，使其能够在牡丹植株中有效表达，收获的牡丹籽能够稳定遗传且含油率高。
Description
技术领域
本发明属于生物工程领域，具体涉及一种提高油用牡丹籽含油率的转基因方法。
背景技术
牡丹是我国特有的传统名花，已有2000多年的栽培历史，栽培品种多达800个以上，牡丹极具观赏、药用、保健等方面价值，尤其是油用牡丹，以其牡丹籽油较高的经济价值和食用、药用价值受到广泛关注，一般油用牡丹籽含油率在27%—33%之间，实践中通常采用传统育种或改变栽培条件来提高牡丹籽的含油率，但这些方法随机性强，且增幅较小，要想较大幅度提高含油量仅依靠传统育种方法通过表型选择十分困难。尤其是面对当前牡丹产业的飞速发展，依靠传统育种技术已远远不能满足规模化种植和生产需要、更难以满足产业化深加工需求。
目前，牡丹在基因功能研究和资源保护、牡丹籽油育种开发等方面的研究基础仍较为薄弱，而含油量又是一种个非常复杂的对基因控制的数量性状，采用基因工程技术来改变牡丹籽油的含油率，必须建立对油分形成和积累的基因调控机制有全面和深入了解的基础，进而利用现代分子生物学技术手段与常规技术相结合才能得以实现，而在此过程中，如何合理且有效的利用已知油料基因，并使其能在牡丹植株中有效表达和稳定遗传是目前面临的一大难题。
WRI1是在拟南芥中发现的一个与种子含油量相关的基因；在植物的胚胎发育过程中，有许多转录因子参与，其中在胚胎发育的中晚期起主要作用的主要有ABI3和LEC类转录因子，LEC2是LEC类转录因子中的其中一种。FAD2基因是油酸去饱和酶的编码基因，是控制油酸向亚油酸转化的关键基因，该基因编码的酶定位于内质网，在亚油酸合成过程中，相位于内质网的单不饱和脂肪酸引入第二个双键，是多不饱和脂肪酸的关键酶。
发明内容
针对上述在提高牡丹籽含油率方面所面临的问题，本发明提供一种提高油用牡丹籽含油率的转基因方法，该方法运用基因工程技术手段，扩增出已知油料基因，构建多基因表达载体并使其能够在牡丹植株中有效表达，收获的牡丹籽能够稳定遗传且含油率高。
本发明是通过以下技术方案实现的：
一种提高油用牡丹籽含油率的转基因方法，具体包括以下步骤：
步骤一：设计引物，分别扩增3’和5’端均带有酶切位点的WRI1和FAD2基因；在pBI1211载体上引入一个多克隆位点；通过多克隆位点将WRI1和FAD2基因插入到pBI1211载体上，构建多基因植物表达载体pBI121-WRI1 -FAD2；
步骤二、制备感受态根癌农杆菌GV3101，将构建的多基因植物表达载体进行根癌农杆菌的转化，验证正确后获取阳性根癌农杆菌；无菌条件下将阳性根癌农杆菌培养至对数期，形成浸染液，备用；
步骤三、取生长状况良好的牡丹新生幼嫩叶片，在无菌条件下消毒并置于1/2MS培养基上，20-22 ℃于暗处预培养2-3天，用浸染液浸染后在黑暗条件下共培养2-3天，然后转入到诱导培养基上进行愈伤组织的诱导；愈伤组织形成后，将愈伤组织从基部切下，转至MS培养基上培养，获取无根苗，再将无根苗转入生根培养基上培养，获取牡丹苗，驯化移栽于温室内，之后按照常规方法进行管理。
进一步的，步骤三所述愈伤组织诱导培养中，控制培养光照为20h/d，光照强度为15-18μmol/(m2.s)。
进一步的，所述诱导培养基为1/2 MS + 0.1-0.5mg/L GA3 + 0.2-1.0mg/L 激动素（KT）+0.1-0.3mg/L谷胱甘肽+0.2-0.4mg/L硫酸亚铁。
进一步的，所述生根培养基为1/2 MS + 0.2mg/L NAA + 1.0mg/L IBA + 25g/L蔗糖 + 0.5g/L聚乙烯比咯烷酮（PVP）+ 1.0-1.3g/L维生素C+3.0g/L活性炭（AC）。
有益效果;
1、本发明所述方法以构建的多基因植物表达载体为基础，利用表达载体本身的转录单元在牡丹营养器官中保证了外源基因的有效表达，同时借助组织培养的方法，以幼嫩叶片为外植体，充分发挥植物器官的全能性，并随着植株的生长发育，使外源基因牡丹的生殖器官中也能高效表达，使牡丹种子的含油率较常规种子的含油率提高15%，且不饱和和饱和脂肪酸的含量均有所增高，大大增加了牡丹的经济价值和营养价值。
2、本发明所述方法选用的两种外源基因，其中的WRI1对植物油脂的合成起重要作用，FAD2可编码形成脂肪酸去饱和酶，影响种子种储存脂中多不饱和脂肪酸的含量和比例，以后两种基因能在子叶原基和子叶中表达，通过对上述基因的综合考虑，选择合适的表达载体，根据基因大小和ORF分析，对表达载体进行改造，使其能够较好地容纳和兼容两种外源基因，将多克隆位点引入到35S启动子的末端，使外源基因能够有效表达，同时不影响内源基因的正常表达，最终从油脂合成的整个过程中增强代谢进程，提高种子中油脂的合成和积累，改善脂肪酸的含量和比例，尤其在亚油酸、油酸、亚麻酸和硬脂酸在内的18-20个碳原子的不饱和脂肪酸，其含量可增加一倍以上。
附图说明
图1是pBI121-35S MCS的质谱图。
具体实施方式
下面通过具体的实施方式对本发明做进一步的解释说明。
实施例1
一、拟南芥总DNA的提取
采用改良CTAB法提取总DNA，具体步骤如下：称取300mg新鲜幼叶于液氮中迅速形成粉末，转入1.5ml离心管中，加入700微升 65℃预热的DNA提取液（1.4mol/L NaCl；100mmol/L Tris-HCl，pH 值8.0；20mmol/L EDTA，pH 值8.0；2% CTAB），同时加入15微升β-巯基乙醇，混匀，65℃水浴60min；取出离心管，加入等体积酚/氯仿（1:1）混合液，充分混匀，10000rpm/min离心10min，取上清液，转入另一离心管中；加入等体积氯仿，充分混匀，8000rpm/min离心5min；取上清液，加入2倍体积-20℃预冷的无水乙醇，颠倒混匀，静置5min；将上清液缓慢倒出，用浓度75%乙醇反复冲洗，振荡并离心，倒去上清液，自然干燥；加入等体积的氯仿再抽提一次，轻轻混匀，8000rpm/min离心5min，将上清液转入1.5ml离心管，加入2倍体积预冷的无水乙醇，混匀，8000rpm/min离心5min，收集沉淀；用浓度70%乙醇冲洗2-3次后在超净工作台吹干至无酒精味，根据沉淀的多少溶于适量1 倍TE缓冲液，置于-20℃，备用。
二、基因扩增
以上述步骤获得的DNA为模板，设计引物，分别PCR扩增WRI1和FAD2基因，然后经琼脂糖凝胶电泳和测序进行验证，验证正确。其中，WRI1的核苷酸序列SEQ ID NO：1所示；FAD2的核苷酸序列SEQ ID NO：2所示；另外，基因WRI1的上下游引物中分别引入BamHＩ和XmaＩ酶切位点，基因FAD2的上下游引物中分别引入XhoI和SacI酶切位点。相关引物序列如下表1所示。
表1：引物序列表
三、植物表达载体的构建
以质粒pBI121为模板，用引物YW5和YW6进行PCR扩增，获得35S promoter。然后以35S promoter为模板，分别用2对引物YW5和YW7，YW5和YW8依次对其纯化回收产物进行PCR扩增，最终获得目的基因35S-MCS。其中多克隆位点包括BamHI，XmaI，XhoI和SacI。通过酶切位点HindIII和SacI将目的基因35S-MCS克隆到载体pBI121上，从而获得新的双元载体pBI121-35S MCS。相关引物序列如下表1所示。
表2：引物序列表
将基因WRI1、 LEC2和FAD2依次载入到双元载体pBI121-35S MCS中，首先用BamHI和XmaI酶切双元载体，回收大片段，将步骤二扩增的WRI1基因片段连接到经酶切的载体上；再用XhoI和SacI酶切载体，回收大片段，将FAD2基因片段连接到载体上，验证正确后，得到pBI121- WRI1 -FAD2表达载体。
四、农杆菌浸染液的制备
制备感受态根癌农杆菌GV3101，将构建的多基因表达载体pBI121- WRI1- -FAD2进行根癌农杆菌的转化，验证正确后获取阳性根癌农杆菌；将阳性根癌农杆菌培养至对数期，形成浸染液，备用；
五、转基因牡丹苗的获取
取生长状况良好的牡丹新生幼嫩叶片，在组织培养室的无菌条件下消毒并置于1/2MS培养基上，20-22 ℃于暗处预培养2-3天，用浸染液浸染后在黑暗条件下共培养2-3天，转入到诱导培养基上进行愈伤组织的诱导；愈伤组织形成后，将其从基部切下，转至MS培养基上培养50d，获取无根苗，再将无根苗转入生根培养基上培养15d，获取牡丹苗，驯化移栽于温室内，之后按照常规方法进行管理。其中，组织培养室内光照为20h/d，光照强度为15-18μmol/(m2.s)，温度为20-22℃，湿度为60%-70%。
牡丹苗移栽后，按照常规方法进行管理，直至开花结籽，牡丹籽的一部分留作种子于次年进行播种，另一部分进行含油率和脂肪酸分析。
其中，牡丹籽含油率测定是将去皮牡丹籽适量研磨成粉末，放入索氏提取器，以体积比7:1的石油醚-乙酸乙酯回流提取6h，得提取液；提取液减压浓缩回收石油醚-乙酸乙酯，得淡黄色油状液体，敞口放置2d后称重，计算含油率。试验设置五组平行。最后测定本发明所得牡丹籽的含油率高达41.6%，而用同样方法测定的常规牡丹的含油率为27%。
种子脂肪酸的提取和分析，具体过程如下：
1、样品准备：将成熟的种子于自然光下通风干燥48h，磨样前于干燥箱中45℃恒温干燥24h，在研钵中研磨成粉末，称取500mg于玻璃试管中备用，取10株单独植株上的种子，每种取5次重复；
2、总脂的提取：在试管中加入2mL氯仿：异丙醇（2:1），加入500微升内标，旋紧盖子，涡旋震荡后与避光条件下静置2h，期间每半小时震荡一次，最后2500rpm离心5min。
3、酯化：吸取500微升上清液至另一干净试管中，加入2ml 1%的MeOH/H2SO4，旋紧盖子后震荡混匀，90℃金属浴1h。
4、终止反应：金属浴结束后取出冷却至室温，加入2ml 1%NaCl和1ml 正己烷，剧烈震荡混匀，2500rpm离心2min，吸取上清液至另一干净玻璃管中，在分别用1ml正己烷提取2次，合并上清液备用，准备上样。
5、气相色谱操作条件：柱温：采取程序升温方式。程序开始160℃保持1min，然后以4℃/min的速率上升至240℃，保持16min；氢火焰离子检测器温度：250℃；进样口温度：250℃；进样量1微升。
结果表明，本发明所得牡丹籽不饱和和饱和脂肪酸的含量均有所增高，相对于常规牡丹籽，不饱和脂肪酸含量提高5%左右，饱和脂肪酸含量提高2%左右；其中，包括亚油酸、油酸、亚麻酸和硬脂酸在内的18-20个碳原子的不饱和脂肪酸增幅较大，相对于常规牡丹籽，其含量增加一倍以上。
最后说明的是，以上实施例仅用以说明本发明的技术方案而非限制本发明的保护范围，在不偏离所附权利要求书所限定的精神和范围的前提下，在形式和细节上对本发明所做的任何改变，均属于本发明的保护范围。
SEQUENCE LISTING
<110> 河南健特生物科技集团有限公司
<120> 一种提高油用牡丹籽含油率的转基因方法
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gtggt 425
Claims (1)
1.一种提高油用牡丹籽含油率的转基因方法，其特征在于：包括以下步骤：
步骤一：设计引物，分别扩增3’和5’端均带有酶切位点的WRI1和FAD2基因；在pBI1211载体上引入一个多克隆位点；通过多克隆位点将WRI1和FAD2基因插入到pBI1211载体上，构建多基因植物表达载体pBI121-WRI1 -FAD2；所述WRI1的核苷酸序列如SEQ ID NO：1所示；FAD2的核苷酸序列如SEQ ID NO：2所示；
步骤二、制备感受态根癌农杆菌GV3101，将构建的多基因植物表达载体进行根癌农杆菌的转化，验证正确后获取阳性根癌农杆菌；无菌条件下将阳性根癌农杆菌培养至对数期，形成浸染液，备用；
步骤三、取生长状况良好的牡丹新生幼嫩叶片，在无菌条件下消毒并置于1/2MS培养基上，20-22 ℃于暗处预培养2-3天，用浸染液浸染后在黑暗条件下共培养2-3天，然后转入到诱导培养基上进行愈伤组织的诱导；愈伤组织形成后，将愈伤组织从基部切下，转至MS培养基上培养，获取无根苗，再将无根苗转入生根培养基上培养，获取牡丹苗，驯化移栽于温室内，之后按照常规方法进行管理；
步骤三所述诱导培养基为1/2 MS + 0.1-0.5mg/L GA3 + 0.2-1.0mg/L 激动素+0.1-0.3mg/L谷胱甘肽+0.2-0.4mg/L硫酸亚铁；
步骤三所述生根培养基为1/2 MS + 0.2mg/L NAA + 1.0mg/L IBA + 25g/L蔗糖 +0.5g/L聚乙烯比咯烷酮+ 1.0-1.3g/L维生素C+3.0g/L活性炭；
步骤三所述愈伤组织诱导培养中，控制培养光照为20h/d，光照强度为15-18μmol/(m2.s)。
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