超长链多不饱和脂肪酸在棉花中的异源合成

引用本文

刘江, 马燕斌, 孙全喜, 吴霞, 李雪滢, 孙美红, 李燕娥, 李新征, 亓宝秀. 超长链多不饱和脂肪酸在棉花中的异源合成. 作物学报, 2014, 40(1): 86-92
LIU Jiang, MA Yan-Bin, SUN Quan-Xi, WU Xia, LI Xue-Ying, SUN Mei-Hong, LI Yan-E, LI Xin-Zheng, QI Bao-Xiu. Production of Very Long Chain Polyunsaturated Fatty Acids in Cotton. Acta Agronomica Sinica, 2014, 40(1): 86-92 复制到剪切板

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超长链多不饱和脂肪酸在棉花中的异源合成

刘江^1,^**, 马燕斌^2,^**, 孙全喜^1,³, 吴霞², 李雪滢¹, 孙美红¹, 李燕娥², 李新征^1,^*, 亓宝秀^1,^*

¹作物生物学国家重点实验室 / 山东农业大学生命科学学院, 山东泰安271018

²山西省农业科学院棉花研究所, 山西运城044000

³山东省花生研究所, 山东青岛266100

^* 通讯作者(Corresponding authors): 亓宝秀, E-mail:qbx126@sdau.edu.cn, Tel: 0538-8246205; 李新征, E-mail:lxz@sdau.edu.cn, Tel: 0538-8246205

第一作者联系方式: E-mail: liujiang2582006@126.com

^** 对本文有相同贡献.

收稿日期:2013-09-16

基金:本研究由国家转基因生物新品种培育科技重大专项(2009ZX08005-024B), 国家自然科学基金项目(30970222)和山东省现代农业产业技术体系建设专项资金资助。

摘要

从球等鞭金藻、眼虫、高山被孢霉和拟南芥中分别克隆到Δ9链延长酶、Δ8去饱和酶、Δ5去饱和酶和Δ15去饱和酶基因, 利用我们的多基因聚合方法, 将这4个基因聚合到植物表达载体pCambia2300上, 其中每个基因都含有独立的CaMV35S启动子和Tnos终止子。利用农杆菌介导法将该表达载体转入棉花, 通过卡纳霉素和PCR筛选获得转基因阳性植株, 提取转基因阳性植株叶片总脂肪酸, 用气相色谱分析法检测到花生四烯酸(AA, 20:4Δ^5,8,11,14)和二十碳五烯酸(EPA, 20:5Δ^5,8,11,14,17)含量分别达1.0%和5.0%。表明通过基因代谢工程在棉花中异源合成EPA是可行的, 为进一步在棉籽中生产VLCPUFAs奠定了基础。

关键词: 去饱和酶; 链延长酶; 转基因; EPA; 棉花

Production of Very Long Chain Polyunsaturated Fatty Acids in Cotton

LIU Jiang^1,^**, MA Yan-Bin^2,^**, SUN Quan-Xi^1,³, WU Xia², LI Xue-Ying¹, SUN Mei-Hong¹, LI Yan-E², LI Xin-Zheng^1,^*, QI Bao-Xiu^1,^*

¹ State Key Laboratory of Crop Biology, College of Life Sciences, Shandong Agricultural University, Tai’an 27101, China

² Cotton Research Institute, Shanxi Academy of Agricultural Sciences, Yuncheng 044000, China

³Shandong Peanut Research Institute, Qingdao 266100, China

^** Contributed equally to this work

Abstract

We have isolated four genes encoding a Δ9 elongase, a Δ8 desaturase, a Δ5 desaturase, and a Δ15 desaturase fromIsochrysis galbana,Euglena gracilis,Mortierella alpina, andArabidopsis thaliana respectively. Using a multigene transfer technology that we developed, these genes were stacked together in the plant expression vector pCambia2300. Each gene contained its own CaMV35S promoter and Tnos terminator. This plant expression vector was then transferred into cotton byAgrobacterium-mediated transformation method. Transgenic cotton seedlings were first identified by screening them based on kanamycin-containing media and followed by PCR with gene-specific primers of the four transgenes. Finally, these transgenic plants were subjected to gas liquid chromatography analysis for their fatty acid composition and the results showed that the contents of arachidonic acid (ARA, 20:4Δ^5,8,11,14) and eicosapentaenoic acid (EPA, 20:5Δ^5,8,11,14,17) were 1.0% and 5.0% respectively in the leaves of the transgenic plants, indicating that the four genes were expressed in cotton. Therefore, our data clearly demonstrated the feasibility for the heterologous production of EPA in cotton and this will lay a foundation for the production of VLCPUFAs, including EPA and DHA in cotton seed through transgenic technology in the future.

Keyword: Desaturase; Elongase; Transgenes; Eicosapentaenoic acid; Cotton

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超长链多不饱和脂肪酸(very long chain poly unsaturated fatty acids, VLCPUFAs)是指含有4~6个双键且碳链长度大于等于20个碳原子的直链脂肪酸^{[ 1]}。VLCPUFAs根据双键的位置通常分为ω3脂肪酸和ω6脂肪酸。深海鱼油是公认的保健品, 其有效成分为EPA (20:5, eicosapentaenoic acid)和DHA (22:6, docosahexaenoic acid)等ω3脂肪酸。EPA是哺乳动物细胞膜的组分, 也是前列腺素、白三烯和血栓素等激素的前体, 对调节血压、炎症反应以及信号转导有重要作用, 从而有效防止多种心血管疾病和炎症的发生^{[ 2, 3]}。DHA对胎儿神经系统的形成至关重要^{[ 4]}, 但人体内合成EPA/DHA的效率比较低, 因此在日常饮食中补充足够的EPA/DHA对维持身体健康极为重要。

VLCPUFAs的合成主要包括两条途径(图1), 第一条为经典的Δ6途径, 亚油酸(LA, 18:2Δ^9,12)和α-亚麻酸(ALA, 18:3Δ^9,12,15)在Δ6去饱和酶作用下生成γ-亚麻酸(GLA, 18:3Δ^6,9,12)和十八碳四烯酸(SDA, 18:4Δ^6,9,12,15), 然后经过Δ6链延长生成二高-γ-亚麻酸(DGLA, 20:3Δ^8,11,14)和二十碳四烯酸(ETA, 20:4Δ^8,11,14,17), 而后再经过Δ5去饱和生成AA和EPA。EPA又可经Δ5链延长和Δ4去饱和生成DHA, 并且ω6脂肪酸可在Δ15和Δ17去饱和酶作用下生成ω3脂肪酸。在某些微藻如球等鞭金藻中, VLCPUFAs的合成还存在另一条“替代途径”, LA和ALA先经Δ9链延长生成二十碳二烯酸(EDA, 20:2Δ^11,14)和二十碳三烯酸(ETrA, 20:3Δ^11,14,17), 然后经Δ8去饱和以及Δ5去饱和生成花生四烯酸(AA, 20:4Δ^{5, 8,11,14})和二十碳五烯酸(EPA, 20:4Δ^5,8,11,14,17)^{[ 1, 5]}。

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	图 1 超长链多不饱和脂肪酸合成途径依据Qi等^{[ 5]}和Abbadi等^{[ 1]}。Fig . 1 VLCPUFA biosynthetic pathwaysAccording to Qi et al.^{[ 5]} and Abbadi et al.^{[ 1]}.

高等植物由于缺乏VLCPUFAs合成途径中的去饱和酶以及链延长酶, 一般只能合成亚油酸和亚麻酸, 不能合成VLCPUFAs, 通过转基因技术将VLCPUFAs合成相关酶转入高等植物, 来生成VLCPUFAs已成为研究热点^{[ 5, 6, 7, 8, 9]}。2004年, Qi等^{[ 5]}将球等鞭金藻中的Δ9链延长酶、眼虫的Δ8去饱和酶以及高山被孢霉的Δ5去饱和酶转入拟南芥中, 在转基因拟南芥叶片中生成3.0%的EPA, 首次证明了高等植物通过基因代谢工程合成VLCPUFAs是可行的。

棉花是重要的纤维作物, 其副产品棉籽是食用植物油脂的重要来源之一。精炼的棉籽油是一种优质的食用植物油, 含有大量人体必需的脂肪酸, 如亚油酸和亚麻酸, 其中亚油酸的含量可达50%^{[ 10]}。与菜籽油和大豆油相比, 棉籽油虽然产量高, 但价值相对较低, 在不影响纤维产量和质量的条件下, 可以通过改良棉籽中的脂肪酸成分来提高其营养和经济价值。本研究旨在探索棉花中合成EPA的可能性, 为此构建了含有Δ9链延长酶^{[ 11]}、Δ8去饱和酶^{[ 12]}、Δ5去饱和酶^{[ 13]}和Δ15去饱和酶^{[ 14]}4个基因的植物表达载体, 以农杆菌介导法转化棉花, 通过气相色谱检测转基因棉花叶片中的脂肪酸成分, 研究在棉花中异源合成EPA的可行性, 为进一步在棉籽中生产VLCPUFAs奠定基础。

1 材料与方法

1.1 试验材料及试剂

大肠杆菌XL10 (Stratagene)、植物表达载体pCambia2300、辅助载体pAUX2为实验室保存; Taq酶、限制性内切酶、T4DNA连接酶和琼脂糖凝胶回收试剂盒等均购自TAKARA公司; 脂肪酸底物等购自Nuchek公司; PCR引物由上海生工生物工程技术服务有限公司合成。

1.2 引物设计

根据NCBI上已经提交的Δ9链延长酶基因(AF390174)、Δ8去饱和酶基因(AF139720)、Δ5去饱和酶基因(AF054824)、Δ15去饱和酶基因(AT3G 11170)设计D9L ( Xho I)-D9R ( Xho I)、D8L ( Xho I)-D8R ( Xho I)、MortD5L ( Xho I)-MortD5R ( Xho I)和AtD15L-AtD15R ( Xho I) 4对引物(两端分别引入适宜的酶切位点)(表1), 分别扩增4个基因。

表1 基因扩增与检测引物 Tabl e 1 Primer for genes amplification detection

1.3 多基因表达载体的构建

我们分别克隆了4个基因(Δ9链延长酶基因、Δ8去饱和酶基因、Δ5去饱和酶基因、Δ15去饱和酶基因)的cDNA片段, 并亚克隆到辅助载体pAUX2中。首先以D9L ( Xho I)、D9R ( Xho I)为引物, 以含有球等鞭金藻Δ9链延长酶基因的质粒pCR2.1-Δ9Elo为模板, PCR扩增出约0.8 kb的条带, 获得Δ9链延长酶基因; 以D8L ( Xho I)、D8R ( Xho I)为引物, 以含有眼虫( Euglena gracilis) Δ8去饱和酶基因的pESC-TrpΔ8质粒为模板, PCR扩增出大约1.3 kb的条带, 获得Δ8去饱和酶基因; 以转基因拟南芥的基因组DNA为模板, 用高山被孢霉( Mortierella alpine) Δ5去饱和酶的引物MortD5L ( Xho I)和MortD5R ( Xho I)进行PCR扩增, 得到大约1.4 kb的条带, 获得Δ5去饱和酶基因; 以反转录的野生型拟南芥第一链cDNA为模板, 用拟南芥Δ15去饱和酶基因引物AtD15L和AtD15R ( Xho I)进行PCR扩增, 得到大约1.4 kb的条带, 获得Δ15去饱和酶基因。利用多基因载体构建的方法^{[ 15]}, 将5个基因表达盒串联起来; 用 XbaI将4个基因表达盒从pAUX2-4EC上切下, 酶切产物进行琼脂糖凝胶电泳纯化回收, 用T4 DNA连接酶将线性化载体pCambia2300与4个基因表达盒片段按摩尔比1∶3~1∶9连接, 得到pCambia2300-4EC后转化大肠杆菌XL10, 提取质粒, 经酶切及测序验证后, 转化农杆菌GV3101。

1.4 农杆菌转化及抗性植株再生

将质粒pCambia2300-4EC转入农杆菌菌株GV3101, 挑取重组农杆菌GV3101单菌落于YEP (含50 mg L^-1利福平和 50 mg L^-1卡那霉素)液体培养基中, 28℃、180转 min^-1振荡培养过夜; 取过夜活化后的菌液5 mL加至100 mL新鲜的 YEP液体培养基中, 28℃、180转min^-1培养过夜, 至菌液OD₆₀₀为0.6~0.8, 于4℃、5000转 min^-1离心 5 min 收集菌体, 用 MS 液体培养基洗涤一次后悬浮于MS液体培养基, 调节OD₆₀₀至0.4~0.6。用农杆菌侵染无菌的棉花子叶, 将侵染后的子叶置于共培养基(MS+2,4-D 0.1 mg L^-1+KT 0.1 mg L^-1)中培养3 d, 然后将其转移到诱导培养基中(MS+2,4-D 0.1 mg L^-1+KT 0.1 mg L^-1+Km 100 mg L^-1+Cef 500 mg L^-1), 在黑暗条件下培养2个月后, 将诱导出的愈伤组织放入增殖培养基(MS+MgCl₂ 0.9 g L^-1+Celrit 2.0 g L^-1+葡萄糖30 g L^-1, pH 5.8)分化, 愈伤组织继代3~5次后转入分化培养基中(MS+谷氨酰氨1.0 g L^-1+ MgCl₂ 0.9~1.5 g L^-1+ Celrit 2.0~3.0 g L^-1+葡萄糖20~30 g L^-1, pH 5.8), 分化成胚状体, 进一步长成小苗, 将小苗嫁接到新的植株。

1.5 转基因棉花的筛选与鉴定

用SDS法提取转基因棉花基因组, 利用基因特异性引物99JC-L和99JC-R、8JC-L和8JC-R、MortD5JC-L和MortD5JC-R、AtD15L和AtD15R ( Xho I)进行PCR检测(表1)。

1.6 脂肪酸提取及气相色谱分析

取转基因棉花叶片0.5 g, 烘干并加液氮研磨, 置于3 mL的玻璃瓶中, 添加1 mL甲基化溶液(含甲醇0.85 mL, 2,2-二甲氧丙烷 0.05 mL, 盐酸0.1 mL), 85℃甲基化反应1 h; 冷却后, 添加1%氯化钠1 mL和正己烷0.5 mL, 漩涡振荡后于室温放置10 min或更长时间; 离心取上层液相, 进行气相色谱分析^{[ 5]}。

2 结果与分析

2.1 植物表达载体pCambia2300-4EC的构建

将高等植物中丰富的油酸LA或亚麻酸ALA转化成EPA, 需要至少3个酶催化, 即Δ9链延长酶、Δ8去饱和酶和Δ5去饱和酶, 为了增加目的产物EPA的最终产量, 还可以增加ω3去饱和酶基因, 即可将油酸转化成亚麻酸的Δ15去饱和酶。我们将这4个基因分别亚克隆到辅助载体pAUX2中, 经酶切鉴定, 获得1.9、2.3、2.5和2.6 kb的目的片段(图2), 分别含有35s启动子、目的基因和终止子。

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图2 Δ9、Δ8、Δ5、Δ15基因辅助载体单酶切鉴定M: DNA分子量; 1~4: pAUX2-Δ9、pAUX2-Δ8、pAUX2-Δ5和pAUX2-Δ15载体 XbaI单酶切; a~e: 线性化的pAUX2片段, E9表达盒, D8表达盒, D5表达盒, D15表达盒。Fig.2 Verification of vectors containing individual transgenes of Δ9, Δ8, Δ5, Δ15 with XbaI digestionM: DNA marker; 1-4: pAUX2-Δ9, pAUX2-Δ8, pAUX2-Δ5 and pAUX2-Δ15 digested with Xba I, respectively; a-e: fragment of pAUX2, fragment of E9 expression cassette, fragment of D8 expression cassette, fragment of D5 expression cassette, and fragment of D15 expression cassette.

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	图3 辅助载体pAUX2-Δ9Δ8Δ5Δ15的构建Fig. 3 Construction of the plant expression vector pAUX2-Δ9Δ8Δ5Δ15

利用本实验室多基因表达载体构建专利技术^{[ 15]}, 构建由Δ8去饱和途径中Δ9链延长酶、Δ8去饱和酶、Δ5去饱和酶及Δ15去饱和酶4个基因组成的辅助表达载体pAUX2-Δ9Δ8Δ5Δ15 (图3), 首先将pAUX2- Δ8中的Δ8基因的表达盒(35S/Δ8/Tnos) XbaI切下, 连接到经限制性内切酶 AvrII线性化的质粒pAUX2-Δ9中, 鉴定方向后, 获得质粒pAUX2- Δ9Δ8。然后, 用 XbaI将Δ5基因的表达盒在质粒pAUX2-Δ5上切下来, 连接到用 AvrII线性化的质粒pAUX2-Δ9Δ8中, 鉴定方向后, 获得pAUX2- Δ9Δ8Δ5。接着, 将pAUX2-Δ9Δ8Δ5用 AvrII线性化, 来接受用 XbaI在pAUX2-Δ15上切下的Δ15基因的表达盒, 获得带有4个基因表达盒的过渡质粒pAUX2-Δ9Δ8Δ5Δ15。对各个中间质粒用 XbaI酶切验证, 每个过渡载体除能切出3.9 kb的辅助载体的骨架片段外, 还能分别切出带有1~4个基因表达盒的片段(图4), 其中每个基因都含有独立的CaMV35S启动子和Tnos终止子, 最后将4个基因的表达盒酶切连入植物表达载体pCambia2300, 以抗 kan基因作为选择标记。

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图4 重组载体pAUX2-Δ9、pAUX2-Δ9Δ8、pAUX2-Δ9Δ8Δ5和pAUX2-Δ9Δ8Δ5Δ15的 XbaI酶切检测所有的质粒都用 XbaI酶切, 并在0.8%的琼脂糖电泳上分离。a: pAUX3-E9D8D5载体的结构示意图; b: 中间载体的酶切检测, 红色箭头所指是载体主干(3.9 kb); c: 各个独立表达盒的大小。Fig. 4 Recombinant plasmids pAUX2-Δ9, pAUX2-Δ9Δ8, pAUX2-Δ9Δ8Δ5, and pAUX2-Δ9Δ8Δ5Δ15 digested by XbaIAll the intermediate constructs were digested by XbaI and separated on a 0.8% agarose gel. a: diagram of pAUX2-Δ9Δ8Δ5Δ15, showing the arrangement and linkage of individual cassette; b: restriction digestion of all intermediate vectors. Red arrow indicates the position of the vector backbone (3.9 kb); c: sizes of individual inserts.

2.2 多基因载体的转化以及转基因棉花的筛选与鉴定

利用农杆菌介导法, 转化棉花珂201子叶(图5), 获得46个再生克隆, 嫁接367棵再生苗, 以PCR鉴定筛选到阳性植株37棵, 其中12株收获棉籽。SDS法提取T₂代转基因株系叶片基因组DNA, 利用基因特异性引物进行PCR检测, 在琼脂糖凝胶电泳图上有目的条带(图6), 在大部分植株中能检测到目的基因, 初步确定这些株转基因株系为阳性株系。

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	图5 棉花的再生过程A: 叶片诱导出初级愈伤组织; B: 分化中的愈伤组织; C: 愈伤分化形成的小苗; D: 小苗嫁接到新的植株。Fig. 5 Regenerative process of transgenic cottonA: primary calli from the leaf; B: differentiating calli; C: regenerated plantlets calli; D: new plant grafted.

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	图6 转基因棉花的PCR检测M: marker; 1~12: 转基因植株。M: marker; 1-12: transgenic lines.Fig. 6 Detection of transgenic lines by PCR

2.3 转基因棉花叶片脂肪酸检测

分别提取上述12株转基因棉花以及非转基因棉花叶片的脂肪酸, 并利用气相色谱进行脂肪酸含量分析(图7)。其中6号和10号株转基因棉花叶片在18 min左右产生了2个明显的峰, 与标样对比, 鉴定这2个峰分别为AA和EPA, 平均含量分别为1.0%和5.0% (表2)。通过与非转基因棉花叶片比较可以看出, 转基因棉花叶片中其他脂肪酸成分及含量也发生了变化, 其LA、ALA的含量明显下降, 其他脂肪酸变化不大。表明转化的EPA合成途径中的这4个基因已经整合到棉花基因组中并成功表达, 其编码产物即4种酶在棉花中成功组建了EPA的合成代谢途径, 并以棉花内源LA和ALA为底物经过多步连续酶促反应生成了AA和EPA。

表2 转基因棉花叶片脂肪酸组成分析 Tabl e 2 Total fatty acid composition of leaves from transgenic cotton

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	图 7 6号转基因棉花叶片脂肪酸气相色谱分析Fig . 7 GC profiles of the sixth transgenic cotton leaf fatty acid methyl esters

3 讨论

棉花是我国重要的农作物, 精炼的棉籽油是我国重要的食用油来源之一, 其含有丰富的必需脂肪酸, 为了进一步改良棉籽油, 可以通过转基因技术改变棉籽油的成分, 培育出优质的棉花新品种, 理论上所有外源基因都可以转入棉花^{[ 16]}。目前, 向棉花中转入的基因主要涉及抗虫、抗除草剂和耐盐等基因。

在棉花中生产EPA, 至少需要转化3个酶基因才能实现, 目前常用的就是分次转化法^{[ 17]}, 每次转化都要一个新的筛选标记基因来筛选转基因植株, 同时需要筛选纯合体, 来进行下一个基因的转化。由于高等植物生长周期长、转化困难, 因此较难运用于实践, 同时分次转化获得的植株后代容易发生基因分离, 导致性状不稳定, 因此需要寻找更简单快捷的转化方法。

本研究中, 为在高等植物中重组EPA合成代谢途径, 我们分别从球等鞭金藻、眼虫、高山被孢霉和拟南芥中克隆到Δ9链延长酶、Δ8去饱和酶、Δ5去饱和酶和Δ15去饱和酶基因, 利用我们自己的多基因表达载体构建专利技术, 将EPA合成途径中的4个基因串联在一起, 一次性转化棉花, 通过卡那霉素筛选, 获得12株转基因株系。提取转基因植株叶片基因组, 利用基因特异性引物检测, 确定转基因株系为阳性株系, 从图2可以看出, 在12株转基因株系中, 均能扩增出Δ8和Δ5去饱和酶基因, 而Δ9链延长酶基因和Δ15去饱和酶基因却只在部分植株中扩增出来, 部分原因可能是这些基因都具有相同的启动子与终止子, 且方向相同, 在基因整合到染色体过程中发生重组而丢失掉了。提取12株转基因棉花叶片脂肪酸进行气相色谱检测, 在转基因棉花叶片中, 可以明显看出亚油酸和亚麻酸的含量比野生型含量低, 其中有2株生成了AA和EPA, 平均含量达到1.0%和5.0%。在转基因棉花叶片中并没有检测到中间产物的生成, 这可能是本研究将EPA合成途径的4个基因表达盒串联在一个基因片段上, 转入棉花中, 由于基因之间紧密连锁, 相同的启动子使其表达量也相同, 而且由于在EPA合成中, 催化第一步反应的酶一般都是限速酶, 其他酶活性相对较高, 因此不会造成中间产物积累, 所以在转基因棉花叶片中检测不到中间产物是正常的; 这与一些富含EPA的微藻和真菌中情况类似, 如马铃薯治病疫霉, 含有丰富的EPA, 占总脂肪酸的15.2%, 但EPA合成途径的中间产物含量却极低, 有的甚至检测不到^{[ 18]}。

转基因植物合成VLCPUFAs后, 需要将其转化为三酰甘油(TAG)才能被利用, 种子是TAG储存的主要部位, 因此选择强的种子特异性启动子来实现外源基因在种子中的超表达, 是提高VLCPUFAs在植物中含量的有效方法之一。2010年, Cheng等^{[ 19]}以含有Napin启动子的多基因载体转化埃塞俄比亚芥( Brassica carinata), 在转基因种子中检测到EPA, 含量达到总脂肪酸的20%, 是迄今为止, 在转基因植物中合成的EPA含量最高记录, 这与埃塞俄比亚芥自身脂类代谢及Napin启动子的高效表达相关。为了进一步提高棉花中EPA含量, 我们将克隆棉花种子特异性启动子及高活性的酶, 从而促进EPA的积累。EPA可以通过Δ5链延长和Δ4去饱和生成DHA, 因此在棉花中生产DHA还需转入Δ5链延长酶基因和Δ4去饱和酶基因, 我们将进一步克隆高催化活性的Δ5链延长酶基因和Δ4去饱和酶基因以及调控脂肪酸合成的转录因子 WRI1和 LEC1等^{[ 20]}, 一起转入可以高效合成EPA的转基因棉花中, 来促进外源基因在棉花种子中的表达以及脂肪酸的合成, 从而提高VLCPUFAs的产量。当棉籽油中AA、EPA和DHA的含量各占总脂肪酸的5%, 10 g这样的油脂就能满足1个人1天对VLCPUFAs的需求量^{[ 21]}; 转基因植物就有可能作为鱼油的良好替代资源应用于生产, 为更多的人提供廉价且高品质的食用油, 从而大大增进人类健康。

4 结论

通过转基因技术, 将Δ9链延长酶、Δ8去饱和酶、Δ5去饱和酶和Δ15去饱和酶4个基因转化棉花, 在转基因棉花叶片中检测到1.0%和5.0%的AA和EPA, 没有检测到其他非特异性脂肪酸, 证明在棉花中异源合成EPA是可行的, 为进一步在棉籽中生产VLCPUFAs奠定基础。

The authors have declared that no competing interests exist.

作者已声明无竞争性利益关系。

参考文献

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2004

8.987

0.0

... 超长链多不饱和脂肪酸(very long chain poly unsaturated fatty acids, VLCPUFAs)是指含有4~6个双键且碳链长度大于等于20个碳原子的直链脂肪酸^[1] ...

... 在某些微藻如球等鞭金藻中, VLCPUFAs的合成还存在另一条“替代途径”, LA和ALA先经Δ9链延长生成二十碳二烯酸(EDA, 20:2Δ^11,14)和二十碳三烯酸(ETrA, 20:3Δ^11,14,17), 然后经Δ8去饱和以及Δ5去饱和生成花生四烯酸(AA, 20:4Δ^{5, 8,11,14})和二十碳五烯酸(EPA, 20:4Δ^5,8,11,14,17)^[1,5] ...

2001

0.0

Science. 2001, 294(5548):1871-1875

Prostaglandins and Leukotrienes: Advances in Eicosanoid Biology

Colin D. Funk

Center for Experimental Therapeutics and Department of Pharmacology, University of Pennsylvania, Philadelphia, PA 19104, USA.E-mail:colin{at}spirit.gcrc.upenn.edu

... EPA是哺乳动物细胞膜的组分, 也是前列腺素、白三烯和血栓素等激素的前体, 对调节血压、炎症反应以及信号转导有重要作用, 从而有效防止多种心血管疾病和炎症的发生^[2,3] ...

2002

2.29

0.0

2001

2.129

0.0

Lipids. 2001, 36(9):885 - 895 DOI:10.1007/s11745-001-0798-1

Essential fatty acids in visual and brain development

Ricardo Uauy(12) Dennis R. Hoffman(2) Patricio Peirano(1) David G. Birch(2) Eileen E. Birch(2)

1.University of Chile Institute of Nutrition and Food Technology (INTA) Macul 5540, Casilla 138-11 Santiago Chile
2.Retina Foundation of the Southwest Dallas Texas

Essential fatty acids are structural components of all tissues and are indispensable for cell membrane synthesis; the brain, retina and other neural tissues are particularly rich in long-chain polyunsaturated fatty acids (LC-PUFA). These fatty acids serve as specific precursors for eicosanoids, which regulate numerous cell and organ functions. Recent human studies support the essential nature of n-3 fatty acids in addition to the well-established role of n−6 essential fatty acids in humans, particularly in early life. The main findings are that light sensitivity of retinal rod photoreceptors is significantly reduced in newborns with n−3 fatty acid deficiency, and that docosahexaenoic acid (DHA) significantly enhances visual acuity maturation and cognitive functions. DHA is a conditionally essential nutrient for adequate neurodevelopment in humans. Comprehensive clinical studies have shown that dietary supplementation with marine oil or single-cell oil sources of LC-PUFA results in increased blood levels of DHA and arachidonic acid, as well as an associated improvement in visual function in formula-fed infants matching that of human breast-fed infants. The effect is mediated not only by the known effects on membrane biophysical properties, neurotransmitter content, and the corresponding electrophysiological correlates but also by a modulating gene expression of the developing retina and brain. Intracellular fatty acids or their metabolites regulate transcriptional activation of gene expression during adipocyte differentiation and retinal and nervous system development. Regulation of gene expression by LC-PUFA occurs at the transcriptional level and may be mediated by nuclear transcription factors activated by fatty acids. These nuclear receptors are part of the family of steroid hormone receptors. DHA also has significant effects on photoreceptor membranes and neurotransmitters involved in the signal transduction process; rhodopsin activation, rod and cone development, neuronal dendritic connectivity, and functional maturation of the central nervous system.

... DHA对胎儿神经系统的形成至关重要^[4], 但人体内合成EPA/DHA的效率比较低, 因此在日常饮食中补充足够的EPA/DHA对维持身体健康极为重要 ...

2004

23.268

0.0

. 2004, 22(6):739-745

Production of very long chain polyunsaturated omega-3 and omega-6 fatty acids in plants

Kunyan Liu¹ ,Yufeng Jing¹ ,Chuanrang Zhu¹ ,Xinghua Wei³ ,Bin Han^1,2 ,Marco A Oropeza-Rosas¹ ,Namiko Satoh Nagasawa^1,2 ,Edward S Buckler^2,3,5,7 ,Qi Feng¹ ,Randall J Wisser⁴ ,Qian Qian³ ,Araby R Belcher⁵ ,Doreen Ware³ ,Stephen Kresovich³ ,Yan Zhao¹ ,Meng Li^5,6 ,Ahong Wang¹ ,Canyang Li¹ ,Zhiwu Zhang⁵ ,Tao Huang¹ ,Tingting Lu¹ ,Wei Li¹ ,Peter J Bradbury^2,3 ,Peter J Balint-Kurti^5,7 ,Tao Sang⁴ ,Taoying Zhou¹ ,John C Zwonitzer⁵ ,Qi-Fa Zhang⁸ ,Liuwei Deng¹ ,Wenjun Li¹ ,James B Holland^1,7 ,David Jackson¹ ,Qiang Zhao^1,2 ,Jiayang Li⁹ ,Lu Wang¹ ,Xuehui Huang^1,2 ,Michael D McMullen⁶ ,Yiqi Lu¹ ,Yunli Guo¹ ,Kristen L Kump¹ ,Peter Bommert¹ ,Qijun Weng¹ ,Danlin Fan¹ ,Zhang Lin¹

¹ Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, USA.
² Present address: Laboratory of Plant Genetics and Breeding, Akita Prefectural University, Akita, Japan.
³ State Key Laboratory of Rice Biology, China National Rice Research Institute, Chinese Academy of Agricultural Sciences, Hangzhou, China.
⁴ Department of Plant Biology, Michigan State University, East Lansing, Michigan, USA.
⁵ Institute for Genomic Diversity, Cornell University, Ithaca, New York, USA.
⁶ National Center for Soybean Improvement, State Key Laboratory of Crop Genetics and Germplasm Enhancement, College of Agriculture, Nanjing Agricultural University, Nanjing, China.
⁷ US Department of Agriculture–Agricultural Research Service, Ithaca, New York, USA.
⁸ National Key Laboratory of Crop Genetic Improvement, National Center for Plant Gene Research (Wuhan), Huazhong Agricultural University, Wuhan, China.
⁹ National Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China.

We report the production of two very long chain polyunsaturated fatty acids, arachidonic acid (AA) and eicosapentaenoic acid (EPA), in substantial quantities in a higher plant. This was achieved using genes encoding enzymes participating in theω3/6 Δ⁸-desaturation biosynthetic pathways for the formation of C20 polyunsaturated fatty acids.Arabidopsis thaliana was transformed sequentially with genes encoding a Δ⁹-specific elongating activity fromIsochrysis galbana, a Δ⁸-desaturase fromEuglena gracilis and a Δ⁵-desaturase fromMortierella alpina. Instrumental in the successful reconstitution of these C20 polyunsaturated fatty acid biosynthetic pathways was theI. galbana C18-Δ⁹-elongating activity, which may bypass rate-limiting steps present in the conventional Δ⁶-desaturase/elongase pathways. The accumulation of EPA and AA in transgenic plants is a breakthrough in the search for alternative sustainable sources of fish oils.

... 高等植物由于缺乏VLCPUFAs合成途径中的去饱和酶以及链延长酶, 一般只能合成亚油酸和亚麻酸, 不能合成VLCPUFAs, 通过转基因技术将VLCPUFAs合成相关酶转入高等植物, 来生成VLCPUFAs已成为研究热点^[5,6,7,8,9] ...

... 2004年, Qi等^[5]将球等鞭金藻中的Δ9链延长酶、眼虫的Δ8去饱和酶以及高山被孢霉的Δ5去饱和酶转入拟南芥中, 在转基因拟南芥叶片中生成3 ...

... 离心取上层液相, 进行气相色谱分析^[5] ...

2011

9.272

0.0

2010

10.667

0.0

. 2010, 49(2):108â119-null

2005

23.268

0.0

. 2005, 23(8):1013-1017

Stepwise engineering to produce high yields of very long-chain polyunsaturated fatty acids in plants

Very long chain polyunsaturated fatty acids (VLCPUFAs) such as arachidonic acid (AA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are valuable commodities that provide important human health benefits^1,^2,^3,^4,⁵. We report the transgenic production of significant amounts of AA and EPA inBrassica juncea seeds via a stepwise metabolic engineering strategy. Using a series of transformations with increasing numbers of transgenes, we demonstrate the incremental production of VLCPUFAs, achieving AA levels of up to 25% and EPA levels of up to 15% of total seed fatty acids. Both fatty acids were almost exclusively found in triacylglycerols, with AA located preferentially atsn-2 andsn-3 positions and EPA distributed almost equally at all three positions. Moreover, we reconstituted the DHA biosynthetic pathway in plant seeds, demonstrating the practical feasibility of large-scale production of this importantω-3 fatty acid in oilseed crops.

2008

4.773

0.0

2009

0.0

0.5439

... 精炼的棉籽油是一种优质的食用植物油, 含有大量人体必需的脂肪酸, 如亚油酸和亚麻酸, 其中亚油酸的含量可达50%^[10] ...

2002

3.538

0.0

. , :null-null

... 本研究旨在探索棉花中合成EPA的可能性, 为此构建了含有Δ9链延长酶^[11]、Δ8去饱和酶^[12]、Δ5去饱和酶^[13]和Δ15去饱和酶^[14]4个基因的植物表达载体, 以农杆菌介导法转化棉花, 通过气相色谱检测转基因棉花叶片中的脂肪酸成分, 研究在棉花中异源合成EPA的可行性, 为进一步在棉籽中生产VLCPUFAs奠定基础 ...

1999

2.935

0.0

. 1999, 365(2):307â316-null

1998

4.773

0.0

1993

6.535

0.0

2013

4.15

0.0

... 利用多基因载体构建的方法^[15], 将5个基因表达盒串联起来 ...

... 利用本实验室多基因表达载体构建专利技术^[15], 构建由Δ8去饱和途径中Δ9链延长酶、Δ8去饱和酶、Δ5去饱和酶及Δ15去饱和酶4个基因组成的辅助表达载体pAUX2-Δ9Δ8Δ5Δ15 (图3), 首先将pAUX2- Δ8中的Δ8基因的表达盒(35S/Δ8/Tnos) Xba I切下, 连接到经限制性内切酶Avr II线性化的质粒pAUX2-Δ9中, 鉴定方向后, 获得质粒pAUX2- Δ9Δ8 ...

2007

0.0

1.4495

... 3 讨论棉花是我国重要的农作物, 精炼的棉籽油是我国重要的食用油来源之一, 其含有丰富的必需脂肪酸, 为了进一步改良棉籽油, 可以通过转基因技术改变棉籽油的成分, 培育出优质的棉花新品种, 理论上所有外源基因都可以转入棉花^[16] ...

2005

0.0

. 2005, 3(2):141-155

Gene stacking in transgenic plants – the challenge for 21st century plant biotechnology

Claire Halpin^*

Plant Research Unit, School of Life Sciences, University of Dundee at SCRI, Invergowrie, Dundee DD2 5DA, UK
^*Correspondence (fax 44 (0)1382 568505; e-mailc.halpin@dundee.ac.uk)

One of the major technical hurdles impeding the advance of plant genetic engineering and biotechnology is the fact that the expression or manipulation of multiple genes in plants is still difficult to achieve. Although a small proportion of commercial genetically modified (GM) crops present ‘stacked’ or ‘pyramided’ traits, only a handful of products have been developed by introducing three or more novel genes. On the research front, a variety of conventional and more novel methods have been employed to introduce multiple genes into plants, but all techniques suffer from certain drawbacks. In this review, the potential and problems of these various techniques and strategies are discussed, and the prospects for improving these technologies in the future are presented.

... 在棉花中生产EPA, 至少需要转化3个酶基因才能实现, 目前常用的就是分次转化法^[17], 每次转化都要一个新的筛选标记基因来筛选转基因植株, 同时需要筛选纯合体, 来进行下一个基因的转化 ...

2012

3.425

0.0

... 2%, 但EPA合成途径的中间产物含量却极低, 有的甚至检测不到^[18] ...

2010

2.754

0.0

Transgenic Res. 2010, 19(2):221 - 229 DOI:10.1007/s11248-009-9302-z

Towards the production of high levels of eicosapentaenoic acid in transgenic plants: the effects of different host species, genes and promoters

Bifang Cheng(12) Guohai Wu(1) Patricia Vrinten(1) Kevin Falk(2) Joerg Bauer(3) Xiao Qiu(4)

1.Bioriginal Food and Science Corporation 110 Gymnasium Place Saskatoon SK S7N 0W9 Canada
2.Saskatoon Research Centre Agriculture and Agri-Food Canada 107 Science Place Saskatoon SK S7N 0X2 Canada
3.BASF Plant Science GmbH 67117 Limburgerhof Germany
4.University of Saskatchewan Department of Food and Bioproduct Sciences Saskatoon SK S7N 5A8 Canada

Eicosapentaenoic acid (EPA, 20:5n-3) plays an important role in many aspects of human health. In our efforts towards producing high levels of EPA in plants, we investigated the effects of different host species, genes and promoters on EPA biosynthesis. Zero-erucic acidBrassica carinata appeared to be an outstanding host species for EPA production, with EPA levels in transgenic seed of this line reaching up to 25%. Two novel genes, an 18-carbon ω3 desaturase (CpDesX) fromClaviceps purpurea and a 20-carbon ω3 desaturase (Pir-ω3) fromPythium irregulare, proved to be very effective in increasing EPA levels in high-erucic acidB. carinata. The conlinin1 promoter from flax functioned reasonably well inB. carinata, and can serve as an alternative to the napin promoter fromB. napus. In summary, the judicious selection of host species and promoters, together with the inclusion of genes that enhance the basic very long chain polyunsaturated fatty acid biosynthetic pathway, can greatly influence the production of EPA in plants.

... 2010年, Cheng等^[19]以含有Napin启动子的多基因载体转化埃塞俄比亚芥(Brassica carinata), 在转基因种子中检测到EPA, 含量达到总脂肪酸的20%, 是迄今为止, 在转基因植物中合成的EPA含量最高记录, 这与埃塞俄比亚芥自身脂类代谢及Napin启动子的高效表达相关 ...

2011

9.272

0.0

... EPA可以通过Δ5链延长和Δ4去饱和生成DHA, 因此在棉花中生产DHA还需转入Δ5链延长酶基因和Δ4去饱和酶基因, 我们将进一步克隆高催化活性的Δ5链延长酶基因和Δ4去饱和酶基因以及调控脂肪酸合成的转录因子WRI1和LEC1等^[20], 一起转入可以高效合成EPA的转基因棉花中, 来促进外源基因在棉花种子中的表达以及脂肪酸的合成, 从而提高VLCPUFAs的产量 ...

2005

11.047

0.0

. , :null-null

... 当棉籽油中AA、EPA和DHA的含量各占总脂肪酸的5%, 10 g这样的油脂就能满足1个人1天对VLCPUFAs的需求量^[21] ...