CN101353661A - 具有△6脂肪酸脱氢酶功能的基因及其应用 - Google Patents
具有△6脂肪酸脱氢酶功能的基因及其应用 Download PDFInfo
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Abstract
本发明利用来源于黑茶藨子(Ribes nigrum L.)DNA中的两个具有完整阅读框架的基因RnD6C和RnD6D,将其在酵母表达体系中进行了表达。通过GC-MS分析表明,在酵母中这两个基因所编码的蛋白可以分别催化外加底物亚油酸(LA)和α-亚麻酸(ALA)分别生成γ-亚麻酸(GLA)和十八碳四烯酸(SDA),具有Δ6脂肪酸脱氢酶功能。本发明可应用于采用基因工程技术以低等真核细胞表达体系和植物表达体系生产GLA和SDA。
Description
技术领域
本发明涉及植物编码基因及其应用。具体地,涉及两个具有完整阅读框架的基因RnD6C和RnD6D,其分别来源于黑茶藨子(Ribes nigrum L.)的两段DNA序列,以及以此为基础克隆的两个具有完整阅读框架的基因序列。本发明还涉及该基因所编码具有Δ6脂肪酸脱氢酶功能的多肽,以及含有该DNA序列的低等真核细胞表达载体和植物表达载体、宿主细胞以及利用该基因分别转化低等真核生物和植物生产GLA和SDA的应用。
背景技术
多不饱和脂肪酸(Polyunsaturated fatty acids,PUFAs)是指含有两个或两个以上双键且碳链长为18~22个碳原子的直链脂肪酸,主要包括亚油酸(linoleic acid,LA,18:2Δ9,12)、γ-亚麻酸(γ-linolenic acid,GLA,18:3,Δ6,9,12)、花生四烯酸(Arachidonic Acid,AA,20:4Δ5,8,11,14)、二十碳五烯酸(Eicosapentaenoic Acid,EPA,20:5Δ5,8,11,14,17)、二十二碳六烯酸(Decosahexaenoic Acid,DHA,20:5Δ4,7,10,13,16,19)等。其中,亚油酸及亚麻酸被公认为人体必需脂肪酸(Essential fatty acids,EFA),可进一步衍化成具有不同功能的高度不饱和脂肪酸,如AA、EPA、DHA等。
PUFAs具有非常重要的生理功能。第一,PUFAs在心血管运动、大脑、视网膜和神经组织发育等生理功能中发挥重要作用。第二,DHA和EPA具有较好的抗癌作用,如可以预防(尤其是绝经后妇女)乳腺癌的发生。第三,花生四烯酸、EPA和DHA等多不饱和脂肪酸在免疫调节中发挥了重要的作用。第四,多不饱和脂肪酸还能具有防止皮肤老化、延缓衰老、促进毛发生长等作用。
在脂肪酸代谢过程中,Δ6脂肪酸脱氢酶(Δ6-fatty acid desaturase,D6D)分别催化底物亚油酸(LA)和α-亚麻酸(ALA)的第6位碳原子脱氢形成γ-亚麻酸(GLA)[1]和十八碳四烯酸(Stearidonic acid,SDA,18:4Δ6,9,12,15)。
γ-亚麻酸又可通过碳链延长和脱氢作用进一步形成AA、前列腺素和白三烯类生理活性物质,这些产物在人的大脑发育、视觉、过敏反应及心血管运动等一系列生理功能中产生重要影响。作为人体的一种必需的不饱和脂肪酸,γ-亚麻酸具有降血脂、抗脂质过氧化、减肥、抑制溃疡、增强胰岛素、抗血栓性心血管疾病等一系列生物学功能[1,2],目前国际上已作为一种新的维生素-维生素F被研究利用[3]。SDA是EPA和DHA的代谢前体,在人体内很容易转化为EPA和DHA,利用效率是ALA的4倍,可以有效缓解与EPA和DHA缺乏有关的生理疾病。当然通过直接补充EPA或DHA也可以获得同样效果。
然而,PUFAs的现行商业来源主要是特定的种子植物、海洋鱼类和一些动物组织。除了LA以外,现有的来源在总产量和质量两个方面都不能满足日益增长的PUFAs的市场需求。这主要是基于下面的一些原因:
(1)PUFAs的植物资源受到季节和地域限制,PUFAs产量和质量的不稳定;而且植物资源大都产量低、不适合大面积种植。例如:含有γ-亚麻酸的月见草、琉璃苣和黑茶藨子等植物产量仅300~600kg/ha,远远低于常规油料作物如油菜的3t/ha的产量[4]。
(2)有限的天然海洋渔业资源由于过度捕捞造成渔业资源缺乏,而且鱼油固有的鱼腥味和氧化不稳定性,限制了PUFAs的进一步利用;
(3)由于需对低品质的油进行提炼,大大增加了PUFAs的生产成本。由此造成了纯品PUFAs的高昂市场价格,阻碍了其许多潜在的用途。
鉴于PUFAs的供应量很快将不能满足其需求量及其应用于生物药品所需纯品PUFAs供应量的不足,因此有必要寻求商业化生产的可替代性来源。
所以,作为GLA和SDA生产中的关键酶-Δ6脂肪酸脱氢酶已被越来越多的研究和利用。迄今为止,Δ6脂肪酸脱氢酶基因已从动物[5]、植物[4]、真菌[6]和线虫[7]等不同生物中克隆获得,并在酿酒酵母[7,8]、番茄[9]、烟草[4,10]、油菜[6,8]和大豆[11]中成功获得了表达。
在植物中,GLA和SDA仅存在于月见草(Oenothera sp.)、琉璃苣(Borago officinalis L.)、黑茶藨子等少数几种植物中[12],而这些植物的种子油生产亦成为目前世界上GLA和SDA的主要商业来源。利用产油真菌发酵也可提取获得GLA和SDA。但通过现有的这些生产方式获得的GLA和SDA产量都很低,远远不能满足日益增长的市场需求[13]。因此,利用酿酒酵母或转基因油料作物植株表达外源Δ6脂肪酸脱氢酶来生产GLA和SDA具有重要的研究意义和经济价值。此外,虽然琉璃苣[5]具有Δ6脂肪酸脱氢酶基因在国外已有研究报道,但本申请的两个基因是来源于黑茶藨子的Δ6脂肪酸脱氢酶基因,迄今在国内外未见报道。
发明内容
本发明提供了两个基因RnD6C、RnD6D,其核苷酸酸序列分别如SEQID NO:1和SEQ ID NO:2所示,这两个基因分别来源于黑茶藨子(Ribesnigrum L.)的DNA片段,所编码的多肽分别如SEQ ID NO:3,SEQ ID NO:4所示。
本发明的另一个目的是提供含基因RnD6C、RnD6D的低等真核细胞表达载体如酵母表达载体,以及利用该表达载体转化的酵母细胞。在转化的酵母细胞中所表达的多肽具有Δ6脂肪酸脱氢酶功能,可以分别催化外加底物亚油酸和α-亚麻酸分别生成γ-亚麻酸(GLA)和十八碳四烯酸(SDA)。
由于这类基因所编码的是一个蛋白,可采用酿酒酵母表达体系来进行体外表达,但由于该蛋白为膜蛋白,在体外表达体系中即使表达出来,亦较难分离纯化。故一般采用考察所表达的蛋白对外加底物的催化活性的方法来进行功能鉴定。
在所采用的酿酒酵母表达体系中,受体菌是尿嘧啶缺陷型INV Sc I,本身不含LA(亚油酸)、ALA(α-亚麻酸)、GLA(γ-亚麻酸)和SDA,因此,在添加底物亚油酸的条件下,如若在酵母工程菌中检测到催化产物GLA或SDA,就可以确定外源基因所编码蛋白在酿酒酵母表达体系中获得了表达,对外加底物产生了催化作用。所以酿酒酵母已经作为研究外源的Δ6、Δ12-脂肪酸脱氢酶基因功能性鉴定的最常用、最有效的表达体系,且已有许多成功的研究报道[4,6,7]。脂肪酸的检测主要采用GC-MS(气相色谱-质谱联用技术)来完成。其原理是通过GC(气相色谱)将不同的脂肪酸组分分离,然后通过MS(质谱)检测器分别对每种组分进行质谱定性分析,从而确定每一种组分的成分及含量的比较成熟的联用分析技术。
本发明的又一个目的是提供一种含基因RnD6C、RnD6D的植物表达载体。可使用任何一种可以引导外源基因在植物中表达的表达载体。这些植物表达载体包括但不限于,双元农杆菌载体,例如pBIN19、pBI121、pB221,pCambia 1300,pGreen等的植物表达载体。
本发明的载体也可含有适当的启动子。在本发明中可使用任何一种强启动子。这些启动子包括但不限于花椰菜花叶病毒(CaMV 35S)、Ubiqutin、Actin启动子。它可单独使用或与其它的植物启动子结合使用。
本发明的表达载体可通过使用Ti质粒,Ri质粒,植物病毒载体,直接的DNA转化,微注射,电穿孔等方式导入植物细胞。
可使用本发明的方法转化的低等真核宿主选自由酵母、藻类等低等真核生物组成的组。
可使用本发明的方法转化的植物宿主包括烟草、油菜、向日葵、大豆、番茄、蓖麻、芝麻和花生等植物。
本发明还涉及基因RnD6C、RnD6D在生产GLA和SDA中的应用。
附图说明
图1.RnD6C片段核苷酸序列,SEQ ID NO:1。
图2.RnD6D片段核苷酸序列,SEQ ID NO:2。
图3.基因RnD6C所编码的多肽序列,SEQ ID NO:3,其中划线部分分别为Cytb5功能域和三个His Box功能域。
图4.基因RnD6D所编码的多肽序列,SEQ ID NO:4,其中划线部分分别为Cytb5功能域和三个His Box功能域。
图5.基因RnD6C和RnD6D的酵母表达载体图。
图6.基因RnD6C和RnD6D的植物表达载体图。a)以潮霉素(Hyg)作为筛选标记的RnD6C/D的植物表达载体图;b)去除筛选标记的RnD6C/D的植物表达载体图。
图7.基因RnD6C和RnD6D在酿酒酵母表达中表达的总脂肪酸GC-MS分析
a)INV Sc I酵母菌株脂肪酸组成的GC-MS;
b)INV Sc I酵母菌株脂肪酸组成和加有LA(亚油酸)、GLA(γ-亚麻酸)标准样品的GC-MS;
c)含空载pYES2表达质粒酵母的总脂肪酸GC-MS;
d)含空载pYES2表达质粒酵母在添加LA底物诱导后的总脂肪酸GC-MS;
e)表达RnD6C基因的酵母在添加LA底物诱导后的总脂肪酸GC-MS,红色箭头所指为产物峰;
f)表达RnD6D基因的酵母在添加LA底物诱导后的总脂肪酸GC-MS,红色箭头所指为产物峰;
g)含空载pYES2表达质粒酵母在添加ALA底物诱导后的总脂肪酸GC-MS;
h)表达RnD6C基因的酵母在添加ALA底物诱导后的总脂肪酸GC-MS,红色箭头所指为产物峰;
i)表达RnD6D基因的酵母在添加ALA底物诱导后的总脂肪酸GC-MS,红色箭头所指为产物峰;
j)由napin启动子启动RnD6D基因表达的转基因油菜H165种子的总脂肪酸GC-MS,红色箭头所指为产物峰;
k)对照-未转化的油菜H165种子的总脂肪酸GC-MS。
图8.酿酒酵母中RnD6C、RnD6D基因表达催化产物GC-MS分析中MS图谱,a:RnD6C、RnD6D的酵母表达催化产物GLA的MS图;b:γ-亚麻酸标准品的MS图。
具体实施方式
下面参考实施例和附图详细描述本发明。本领域的普通技术人员可以理解的是,下述实施例是举例说明的目的,其不应以任何方式解释为对本发明的限制。本发明的保护范围由后附的权利要求所限定。
实施例一、RnD6C、RnD6D基因的获得
1.黑茶藨子基因组DNA的制备
以北京植物园中种植的黑茶藨子(Ribes nigrum L.)活体植株为材料,取其幼嫩叶片(约100mg),于7ml Ep管中加入钢珠(直径5mm),置于液氮中冷冻20-30min,于漩涡器上高速破碎,反复操作2-3次,至材料完全破碎。加入1-2ml预热的CTAB抽提液(参见“精编分子生物学实验指南”2001,科学出版社,颜子颖,王海林译),混匀。65℃水浴30min。加入等体积氯仿,轻柔抽提约5min。于12000rpm室温离心10min。取上清,加入1/2体积异丙醇混匀,室温放置10min沉淀DNA。用Tip头将沉淀出的DNA挑出,用70%乙醇中洗涤两次,于70%乙醇中室温放置30min。除去70%乙醇,空气中吹干。溶于适量灭菌ddH2O,-20℃保存。
2.RnD6C、RnD6D DNA片段的克隆
设计引物分别从前面所提取的DNA中克隆RnD6C、RnD6D的DNA片段。所用反应体系如下:
PCR反应体系(50μl):
PCR产物电泳(1%琼脂糖凝胶浓度)后切胶回收目的片段(RnD6C、RnD6D,约1.3kb),连入pGEM-T载体(购自Promega公司),测序验证。
所用引物分别如下:
1).RnD6C DNA片段的克隆所用引物
正向引物
Kpn I
5′-CGCGGGTACCATGGCTAATGCAATCAAG-3′(SEQ ID NO:5)
反向引物SacI
5′-CGCCGAGCTCTCAGCCATAGGTGTTGAC-3′(SEQ ID NO:6)
2).RnD6D DNA片段的克隆所用引物
正向引物Kpn I
5’CGCGGGTACCATGGGTGAAAATGGAAGG-3’(SEQ ID NO:7)
反向引物Sac I
5’-CGCCGAGCTCTCAACCATAGGTGTTGAC-3’(SEQ ID NO:8)
实施例二、RnD6C、RnD6D基因酵母载体的构建
从含有基因RnD6C和RnD6D的pGEM-T载体中,利用KpnI、Sac I酶切位点,双切后获得RnD6C和RnD6D基因,定向克隆于酵母表达载体pYES2(购自Ivitrogen公司),获得酵母表达质粒pYRnD6C和pYRnD6D,转化大肠杆菌DH-5α(由本实验室保存)保存。其载体图见图5。
实施例三、RnD6C、RnD6D基因在酵母中的表达
1.酵母的转化
参照Invitrogen公司pYES2Kit(Cat#V285-20)所述方法,将上述嵌合基因的酵母表达质粒pYRnD6C和pYRnD6D,采用醋酸锂介导转化酿酒酵母营养缺陷型菌株INV Sc I(购于Invitrogen公司),以空载pYES2质粒为对照,获得含有各表达质粒的酵母细胞。
2.转化酵母细胞的诱导表达
取含目的基因酵母表达质粒转化的酵母单菌落,接种于50ml SC-U培养液(参照Invitrogen公司pYES2Kit所述配方)含2%绵子糖的SC-U培养液中,250rpm,28℃,培养过夜;加入NP-40(购自BBI公司)(终浓度1%)、外源亚油酸和α-亚麻酸底物(购自Sigma公司)(终浓度为0.003%),以及酵母表达诱导物D-半乳糖(购自Amresco公司)(终浓度为2%)250rpm,22℃培养48h诱导表达。
实施例四、RnD6C、RnD6D基因酵母表达物对Δ6脂肪酸脱氢酶底物催化产物的GC-MS分析
1.脂肪酸的提取与甲酯化。
离心收集取诱导后的酵母菌体,去离子水洗涤,50℃烘干。取40mg酵母粉(在实例3.2中所诱导获得的酵母经50℃烘干即得)充分研磨,加入5ml 5%KOH-CH3OH,70℃水浴5h后加入HCl酸化至其pH值达2.0。再加入4ml14%BF3-CH3OH(购自Aldtich公司)溶液,70℃水浴1.5h。加入2ml 0.9%NaCl溶液,混匀后静止片刻。加入2ml氯仿∶正己烷(V/V1∶4)抽提,吸取抽提液,N2吹干。最后溶于100μl乙酸乙酯。
2.终产物GC-MS检测分析实验。
所用GC-/MS仪为TurboMass(PerkinElmer公司),柱子:BPX-70,30m ×0.25mm×0.25vm,柱温120℃,气化室温度230℃。取1μl终产物上样,分流比10∶1。
3.GC-MS结果分析。
对比图7-1a、7-1b和7-1c,表明在不添加亚油酸底物条件下,所用酵母工程植株以及含经诱导的空载pYES2表达质粒酵母中没有新的产物峰出现,说明所用酵母工程菌株及空载pYES2表达质粒酵母的总脂肪酸中不含外加底物亚油酸(LA)、α-亚麻酸(ALA)和催化产物γ-亚麻酸(GLA)、十八碳四烯酸(SDA)。
当添加亚油酸底物,并进行诱导表达时,对照(空载pYES2)亦没有产物峰γ-亚麻酸(GLA)的出现(参见图7-1d)。而pYRnD6C/D的酵母表达菌被诱导后,外加底物亚油酸(LA)可以被催化生成γ-亚麻酸(GLA)(参见图7-1e和7-1f)。
对比图7-1d、7-1e和7-1f,可以发现在保留时间为7.25min左右时,有一个新的物质生成。其出峰时间和质谱图与标准品γ-亚麻酸(购自Sigma公司)的峰图及质谱图一致(参见图8a和图b),确定为γ-亚麻酸。
当添加α-亚麻酸底物,并进行诱导表达时,对照(空载pYES2)亦没有产物峰SDA的出现(参见图7-2g)。而pYRnD6C/D的酵母表达菌被诱导后,外加底物α-亚麻酸可以被催化生成SDA(参见图7-2h和7-2i)。
对比图7-2g、7-2h和7-2i,可以发现在保留时间为7.75min左右时,有一个新的物质生成。根据其出峰时间和质谱图确定为SDA。
以上结果证明来源于黑茶藨子的两个基因RnD6C、RnD6D在酿酒酵母中成功表达,所表达的蛋白可以分别催化底物亚油酸(LA)和α-亚麻酸(ALA)生成γ-亚麻酸和SDA。
实施例五、RnD6C、RnD6D基因植物表达载体的构建
1.napin启动子napin-T中间载体的构建
提取(芥菜型)油菜总DNA,(方法参见实施例1.1)。napin启动子片段的克隆所用引物:
正向引物:Hind III
5′-CGCGAAGCTTACTACAATGTCGGAGAGACAAGG-3′(SEQID NO:9)
反向引物:BamH I
5′-CGCGGGATCCTTGTGTATGTTCTGTAGTGATGAGTTTTG-3′(SEQ ID NO:10)
用Pyrobest DNA Polymerase PCR扩增(反应体系参见实施例1.2),PCR产物电泳(1%琼脂糖凝胶浓度)后切胶回收目的片段(约1.8kb),连入pGEM-T载体(购自Promega公司),测序验证。
2.RnD6C、RnD6D基因的RnFD6C/D-T中间载体的构建
根据RnFD6C/D的基因序列,分别设计含酶切位点为BamH I和Sac I的上下游特异引物,用Pyrobest DNA Polymerase扩增目的片段RnFD6C和RnFD6D,回收目的片段定向克隆到pGEM-T载体上,挑取单菌落,经PCR、酶切和测序验证,得到RnFD6C/D-T中间载体。
3.RnD6C、RnD6D基因的植物表达载体的构建
1)pC1300-napin的构建
用Hind III和BamH I分别双酶切Napin-T和pCambia 1300,并回收napin启动子片段和pCambia 1300载体片段,连接转化大肠杆菌,随机挑取单克隆,经PCR,酶切验证得到替换35S启动子为napin启动子的pC1300-napin植物表达载体。
2)pC1300-nRnD6C/D植物表达载体的构建
用BamH I和Sac I分别双酶切RnD6C/D-T和pC1300-napin,并回收RnD6C/D目的片段和pC1300-napin载体片段,连接转化大肠杆菌,随机挑取单克隆,经PCR,酶切验证得到pC1300-n RnD6C/D的植物表达载体。其载体图见图6a。
3)去除潮霉素(Hyg)筛选标记的pC1300-nRnD6C/D的构建
在构建好之后,用Xho I单酶切,然后回收大片断,连接转化后,随机挑取单克隆进行PCR、酶切验证获得去除潮霉素(Hyg)筛选标记的pC1300-nRnD6C/D植物表达载体。其载体图见图6b。
实施例六、RnD6C、RnD6D基因及在植物中的转化
1.植物表达载体转入农杆菌
挑取农杆菌单菌落接种在5ml YEP液体培养基中,28℃,200rpm,振荡过夜培养。将2ml过夜培养的菌液加到含有50ml YEP培养基中,28℃,220rpm,振荡培养至OD600在0.5-1.0之间。5000rpm离心菌液,弃上清,沉淀悬浮于10ml 0.5M NaCl,4℃,5000rpm离心5min,弃上清,用1ml预冷的20mM CaCl2重悬细胞。取0.2ml加入约0.5-1μg质粒DNA,轻轻混匀,液氮速冻1min,37℃热激5min,加入1ml YEP溶液,28℃振荡培养2-4h。5000rpm离心菌液,弃上清,将细胞重悬于0.2ml YEP培养基中,均匀涂布在含Kan的YEP平板上,28℃培养48h。随机挑取单菌落进行PCR和酶切验证。
2.Floral-dip法转化油菜(H165)
选择初花期的甘蓝型油菜植株处理,前1d田间浇透水。去除主花序和每个分枝花序的顶端花蕾,同时除去已开放的花朵,只保留快要开放的花蕾用于转化。
用10ml新鲜的YEP+kan的培养基28℃过夜培养含有目标基因植物表达载体的农杆菌至次日10:00,再取5ml转入100ml新鲜的YEP+kan的培养基,过夜培养22h。2500rpm离心农杆菌菌液,弃上清后,用100ml转化Bufer(MS基本培养基+藨糖5%+Silwet-77,pH5.8)重悬至OD600=1.0后浸渍油菜花序。10d内间隔1d连续处理5次。浸渍完后套上纸袋。浸渍处理24h后除去纸袋直至收获种子。
实施例七、T1代种子的筛选
对浸渍处理后获得的油菜T1代种子经75%酒精表面消毒30s,0.1%HgCl2消毒10min,铺在预先倒好的筛选培养基上,经7~15d筛选培养,将正常生长的绿色苗进行第2次抗性筛选(7~15d),将第2次筛选后的抗性植株进行第3次抗性筛选(7~15d),第2次,第3次筛选培养基为:Ms+Kan(150mg/L)。将第三次筛选后的绿苗转入花盆,温室培养至种子收获。
对于用含有去除筛选标记的pC1300-n RnD6C/D的农杆菌处理后获得的T1代油菜种子直接播种于田间,提取叶片DNA,通过PCR筛选阳性植株。
实施例八、RnD6C、RnD6D基因植物表达物对Δ6脂肪酸脱氢酶底物催化产物的GC-MS分析
1.脂肪酸的提取与甲酯化。
取50-100mg成熟对照或转基因油菜种子,液氮中充分研磨,加入5ml 5%KOH-CH3OH,70℃水浴5h后加入HCl酸化至其pH值达2.0。再加入4ml14%BF3-CH3OH(购自Aldtich公司)溶液,70℃水浴1.5h。加入2ml 0.9%NaCl溶液,混匀后静止片刻。加入2ml氯仿∶正己烷(V/V 1∶4)抽提,吸取抽提液,N2吹干。最后溶于100μl乙酸乙酯。
2.终产物GC-MS检测分析实验。
所用GC/MS仪为TurboMass(PerkinElmer公司),柱子:BPX-70,30m×0.25mm×0.25vm,柱温120℃,气化室温度230℃。取1μl终产物上样,分流比10∶1。
3.GC-MS结果分析。
对比图7-2j和7-2k,与未进行转化的油菜种子对照相比,转化株油菜出现两个新的产物峰,与标准品和酵母GC-MS结果比较,可以发现在保留时间为7.25min左右时,出现GLA的产物峰;在保留时间为7.75min左右时,出现SDA的产物峰。以上结果证明来源于黑茶藨子的基因RnD6D在油菜种子中成功表达,所表达的蛋白可以分别催化底物亚油酸(LA)和α-亚麻酸(ALA)生成GLA和SDA。这也就表明,将RnD6C/D转入油菜,可以用转基因油菜作为生物反应器生产GLA和SDA。
参考文献:
1.Gunstone FD(1992)Gamma linolenic acid-occurrence and physical andchemical properties.Prog Lipid Res 31:145-161.
2.Horrobin DF(1992)Nutritional and medical importance of gamma-linolenicacid.Prog Lipid Res 31:163-194.
3.Huang YS and Milles DE(1996)Gamma-Linolenic Acid:Metabolism andIts Roles in Nutrition and Medicine.AOCS Press,Champaign,IL.
4.Sayanova O,Smith MA,Lapinskas P,Stobart AK,Dobson G,Christie WW,Shewry PR and Napier JA(1997)Expression of a borage desaturase cDNAcontaining an N-terminal cytochrome b5 domain results in the accumulationof high levels of Δ6-desaturated fatty acids in transgenic tobacco.Proc NatlAcad Sci USA 94:4211-4216.
5.Hyekyung PC,Manabu TN and Steven DC(1999)Cloning,Expression,andNutritional Regulation of the Mammalian Δ6-Desaturase.J Biol Chem274(1):471-477.
6.Hong HP,Datla N,Reed DW.,Covello PS,MacKenzie SL and Qiu X(2002)High-Level Production of γ-Linolenic Acid in Brassica juncea Using a Δ6Desaturase from Pythium irregulare.Plant Physiol 129:354-362.
7.Napier JA,Hey SJ,Lacey DJ and Shewry PR (1998)Identification of aCaenorhabditis elegans Δ6-fatty-acid-desaturase by heterologousexpression in Saccharomyces cerevisiae.Biochem J 330:611-614.
8.Qiu X,Hong HP,Datla N,MacKenzie SL,Tayler CD and Thomas LT(2002)Expression of borage Δ6-desaturase in Saccharomyces cerevisiaeand oilseed crops.Can J Bot 80:42-49.
9.Cook D,Grierson D,Jones C,Wallace A,West G and Tucker G(2002)Modification of fatty acid composition in tomato(Lycopersicon esculentum)by expression of a borageΔ6-desaturase.Mol Biotechnol.21(2):123-128.
10.Sayanova O,Smith MA,Lapinskas P,Stobart AK,Dobson G,ChristieWW,Shewry PR and Napier JA(1997)Expression of a borage desaturasecDNA containing an N-terminal cytochrome b5 domain results in theaccumulation of high levels of Δ6-desaturated fatty acids in transgenictobacco.Proc Natl Acad Sci USA 94:4211-4216.
11.Sato S,Xing AQ,Ye XG,Schweiger B,Kinney A,Graef G and Clemente T(2004)Production of γ-Linolenic Acid and Stearidonic Acid in Seeds ofMarker-Free Transgenic Soybean.Crop Sci 44:646-652.
12.Ucciani E(1995)Nouveau Dictionnaire des Huiles Végétales-Composition en Acides Gras.Lavoisier,Paris pp 3-596.
13.Gyves EM,Sparks CA,Sayanova O,Lazzeri P,Napier JA and Jones HD(2004)Genetic manipulation of γ-linolenic acid(GLA)synthesis in acommercial variety of evening primrose(Oenothera sp.)Plant Biotech2(4):351-357.
序列表
<110>中国科学院遗传与发育生物学研究所
<120>具有Δ6脂肪酸脱氢酶功能的基因及其应用
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Claims (14)
1.基因RnD6C,其核苷酸酸序列如SEQ ID NO:1所示。
2.基因RnD6D,其核苷酸酸序列如SEQ ID NO:2所示。
3.权利要求1的基因RnD6C所编码的多肽,其氨基酸序列如SEQ ID NO:3所示。
4.权利要求2的基因RnD6D所编码的多肽,其氨基酸序列如SEQ IDNO:4所示。
5.一种表达载体,其特征在于包含权利要求1和/或2所述的基因。
6.权利要求5的表达载体,其为低等真核细胞表达载体或植物表达载体。
7.权利要求6的表达载体,其中低等真核细胞表达载体选自pYES2等用于低等真核细胞表达的载体。
8.权利要求6的表达载体,其中所述植物表达载体选自pBIN19、pBI121、pB221、pCambia 1300,pGreen等的植物表达载体。
9.权利要求5的表达载体,其特征在于还包含启动子,所述启动子选自花椰菜花叶病毒CaMV35S、Ubiqutin、Actin、或植物组织部位特异表达启动子,所述启动子单独或与其它植物启动子结合使用。
10.一种宿主细胞,其特征在于包含权利要求5的表达载体。
11.权利要求10的宿主细胞,其中所述表达载体是通过Ti质粒、Ri质粒、植物病毒表达载体、直接的DNA转化、微注射或电穿孔的方式导入。
12.权利要求10的宿主细胞,其为低等真核细胞或植物细胞。
13.权利要求12的宿主细胞,其中所述低等真核细胞为酵母、藻类等的细胞,所述植物细胞包括烟草、油菜、向日葵、大豆、番茄、蓖麻、芝麻和花生等细胞。
14.权利要求1的基因RnD6C或权利要求2的基因RnD6D在生产GLA和SDA中的应用。
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Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
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CN101889630A (zh) * | 2010-06-28 | 2010-11-24 | 浙江大学 | 一种富含高度不饱和脂肪酸的开口饵料制备方法 |
CN102399758A (zh) * | 2010-09-17 | 2012-04-04 | 中国科学院遗传与发育生物学研究所 | 具有△6脂肪酸脱氢酶功能的突变基因及其应用 |
CN102424827A (zh) * | 2010-09-10 | 2012-04-25 | 兰州大学 | 微孔草Δ-6脂肪酸脱氢酶基因(Ms-Δ6-FAD)及用途 |
CN107236679A (zh) * | 2017-04-27 | 2017-10-10 | 广州弘宝元生物科技有限公司 | 一种高产不饱和脂肪酸重组工程菌株及其构建方法 |
CN110982797A (zh) * | 2019-12-25 | 2020-04-10 | 湖南农业大学 | 一种检测油菜脂肪酸脱氢酶的多克隆抗体的制备方法及其产品与应用 |
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WO2000021557A1 (en) * | 1998-10-09 | 2000-04-20 | Merck & Co., Inc. | Delta 6 fatty acid desaturase |
CN1195060C (zh) * | 2003-06-06 | 2005-03-30 | 南开大学 | 少根根霉△6-脂肪酸脱氢酶的核苷酸序列及其应用 |
CN100413969C (zh) * | 2005-04-21 | 2008-08-27 | 中国科学院遗传与发育生物学研究所 | 具有δ6脂肪酸脱氢酶功能的嵌合基因及其应用 |
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Cited By (8)
Publication number | Priority date | Publication date | Assignee | Title |
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CN101889630A (zh) * | 2010-06-28 | 2010-11-24 | 浙江大学 | 一种富含高度不饱和脂肪酸的开口饵料制备方法 |
CN101889630B (zh) * | 2010-06-28 | 2013-06-05 | 浙江大学 | 一种富含高度不饱和脂肪酸的开口饵料制备方法 |
CN102424827A (zh) * | 2010-09-10 | 2012-04-25 | 兰州大学 | 微孔草Δ-6脂肪酸脱氢酶基因(Ms-Δ6-FAD)及用途 |
CN102424827B (zh) * | 2010-09-10 | 2015-06-03 | 兰州大学 | 微孔草Δ-6脂肪酸脱氢酶基因(Ms-Δ6-FAD)及用途 |
CN102399758A (zh) * | 2010-09-17 | 2012-04-04 | 中国科学院遗传与发育生物学研究所 | 具有△6脂肪酸脱氢酶功能的突变基因及其应用 |
CN102399758B (zh) * | 2010-09-17 | 2012-12-12 | 中国科学院遗传与发育生物学研究所 | 具有△6脂肪酸脱氢酶功能的突变基因及其应用 |
CN107236679A (zh) * | 2017-04-27 | 2017-10-10 | 广州弘宝元生物科技有限公司 | 一种高产不饱和脂肪酸重组工程菌株及其构建方法 |
CN110982797A (zh) * | 2019-12-25 | 2020-04-10 | 湖南农业大学 | 一种检测油菜脂肪酸脱氢酶的多克隆抗体的制备方法及其产品与应用 |
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