,1, 魏鑫,2Cloning and Functional Characterization of Sesame SiSAD Gene
ZHOU Rong1, LIU Pan1, LI DongHua1, ZHANG YanXin1, WANG LinHai1, ZHANG XiuRong,1, WEI Xin,2通讯作者:
编委: 李莉
收稿日期:2019-01-24接受日期:2019-03-11网络出版日期:2019-05-16
| 基金资助: |
Received:2019-01-24Accepted:2019-03-11Online:2019-05-16
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周瑢,E-mail：
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周瑢, 刘盼, 黎冬华, 张艳欣, 王林海, 张秀荣, 魏鑫. 芝麻硬脂酸脱饱和酶基因SiSAD的克隆及功能验证[J]. 中国农业科学, 2019, 52(10): 1678-1685 doi:10.3864/j.issn.0578-1752.2019.10.002
ZHOU Rong, LIU Pan, LI DongHua, ZHANG YanXin, WANG LinHai, ZHANG XiuRong, WEI Xin.
0 引言
【研究意义】芝麻（Sesamum indicum L.）属胡麻科,主要分布在亚洲和非洲,在中国种植已长达2 100余年。芝麻种子含油量高,平均可达55%,有“油料皇后”之美称,脂肪酸含量丰富,其中,油酸和亚油酸之和约占85%。油酸为具有一个双键的单不饱和脂肪酸,氧化稳定性高于其他多不饱和脂肪酸,因此,其货架期较长[1,2,3],另据报道,油酸可降低低密度胆固醇,减缓动脉粥样硬化,有效预防心血管疾病的发生[4,5,6,7],但人体自身合成的油酸远远不能满足身体需求,需要从食物中摄取,故食用油酸含量较高的食用油十分必要。主要植物油中油酸含量分别为橄榄油55%—83%、茶籽油74%—87%、花生油35%—67%,菜籽油61%—70%,而芝麻油中为34%—46%[8,9,10],相对较低。因此,发掘和利用芝麻中与油酸代谢相关的重要基因,为芝麻油酸的遗传改良提供重要的基因资源。【前人研究进展】前人研究表明,SAD编码的△9硬脂酰-ACP脱饱和酶（△9 stearoyl acyl-carrier-protein desaturase,SAD酶）是存在于细胞质体中的一种可溶性酶,该酶催化的反应是在硬脂酸（18:0）的第9、第10位间脱氢形成第一个双键[11],是硬脂酸向油酸转化的唯一催化酶,因此,SAD调控植物中饱和脂肪酸和不饱和脂肪酸比例方面起到关键作用[12,13,14,15]。目前,SAD已经在玉米、大豆、亚麻、蓖麻等多种植物中被克隆,并明确了其功能[16,17,18,19,20]。KLINKENBERG等[21]研究表明在干旱和缺氧逆境条件下,AtSAD6能够增加拟南芥体内的不饱和脂肪酸含量。WENDY等[22]发现通过转基因技术将土豆SAD转入到烟草中,烟草叶片和种子中的不饱和脂肪酸含量明显増加。KNUTZON等[23]通过反义抑制技术使SAD沉默,转基因油菜种子中硬脂酸含量、亚油酸含量均显著高于对照。【本研究切入点】目前,有关芝麻SAD的功能研究鲜见报道。国内应用的芝麻良种油酸含量偏低而饱和脂肪酸含量偏高,通过品种改良来提高芝麻品种的油酸含量是新时期重要育种目标。芝麻基因组测序已经完成,为解析芝麻油酸合成代谢等分子机制和发掘相关基因提供了重要的基础。【拟解决的关键问题】本研究从芝麻品种中芝13中克隆得到芝麻SiSAD的cDNA全长序列,通过进行多序列比对、系统进化树分析,构建表达载体转化拟南芥,研究SiSAD在芝麻油酸含量中的作用,为芝麻脂肪酸组分遗传改良提供基因资源。1 材料与方法
1.1 材料
中芝13、中芝33和中丰芝一号（由中国农业科学院油料作物研究所芝麻种质资源课题组育成和保存种子）种植于中国农业科学院油料作物研究所试验田,在开花授粉后,取发育25 d的种子,于液氮中迅速冷冻,-80℃保存待用。选用野生型拟南芥（Col-0）作为芝麻SiSAD的转化受体。野生型拟南芥及转基因后代均种植于植物培养间（22℃,120—150 μmol·m-2s-1光照强度,25%—75%相对湿度,光周期为16 h光照/8 h黑暗）,取叶片及幼嫩角果于-80℃保存备用。1.2 芝麻SiSAD的克隆
利用植物总RNA提取试剂盒（Aidlab,China）提取中芝13幼嫩叶片总RNA,用反转录试剂盒（BIO-RAD,USA）获得cDNA。根据Sinbase（http://ocri-genomics.org/Sinbase/index.html）[24,25]数据库中的SiSAD序列信息（序列号为SIN_1008977）,设计引物SiSAD-F/R（表1）扩增SiSAD的CDS序列,与pEASY-T1载体连接,得到pEASY-SiSAD,测序正确后备用。Table 1
表1
表1引物序列表
Table 1
| 引物名称 Primer | 引物序列（5′-3′） Primer sequence | 用途 |
|---|---|---|
| SiSAD-F | TCTAGAATGCAATCATCAGC | CDS克隆 CDS Cloning |
| SiSAD-R | GAGCTCCTACACAACTACTT | |
| SISAF | TGATGATTGAGAGGACAGT | 实时荧光定量PCR qRT-PCR |
| SISAR | CGTTCTTGGAATGAAGTGTA | |
| SIARactinF | CCCGCTATGTATGTCGCCA | |
| SIARactinR | AACCCTCGTAGATTGGCACAG | |
| PBISADF | ACTCTAGAGGATCCCCGGGATGCAATCATCAGCATTC | 植物表达载体构建 Constructs |
| PBISADR | ATCGGGGAAATTCGAGCTCCTACACAACTACTTCCC | |
| SiSAD-1F | CATTGCCAACTTACCAGA | 转基因鉴定引物 Transgenic line test |
| SiSAD-1R | TCGCCTACGGCTATCA |
新窗口打开|下载CSV
1.3 SiSAD及编码产物的生物信息学分析
用DNAMAN软件对测序结果进行分析,通过在线软件ProtParam（1.4 拟南芥表达载体的构建及转化
用限制性内切酶SmaⅠ和SacⅠ酶切植物表达载体pBI121,采用同源重组法获得重组质粒pBI121- SAD。利用农杆菌介导法将植物表达载体pBI121-SAD转化野生型拟南芥Col-0,使用Kana作为筛选标记。以转化pBI121空载体的拟南芥植株和野生型拟南芥为对照,获得T1种子,经50 μg·mL-1卡那霉素筛选后移栽至温室生长,自交得到T2种子,再经自交和筛选后得到纯系用于后续试验。1.5 目标基因的荧光定量PCR分析
根据SiSAD全长CDS序列设计荧光定量PCR引物（表1）。使用iScript cDNA Synthesis Kit（BIO-RAD,USA）试剂盒在Light Cycler 480 II（Roche,Germany）实时定量PCR仪上进行Real-time PCR反应。以芝麻β-actin为内参基因,采用3步法反应程序,进行融解曲线和荧光值变化曲线分析,用2-ΔΔCt法计算目的基因的相对表达量。1.6 转基因拟南芥中脂肪酸相对含量测定
分别收取3个转SiSAD拟南芥株系T3的种子,以转pBI121空载的转基因拟南芥T3种子为对照,每份样品种子量约为0.1 g,每个株系设置3个生物学重复,采用气相色谱法测定拟南芥种子中硬脂酸（C18:0）、油酸（C18:1）的相对含量,测定由农业部油料及制品质量监督检验测试中心完成。2 结果
2.1 芝麻SiSAD的克隆与氨基酸序列分析
以中芝13发育25 d的种子cDNA为模板,通过PCR扩增得到一条大于1 000 bp的条带（图1）,测序结果表明,目的片段的碱基序列与芝麻基因组数据库基因SiSAD（序列号SIN_1008977）编码区序列完全一致,起始密码子为ATG,终止密码子为TAG,全长1 152 bp,编码383个氨基酸,其分子量为43 kD,等电点为6.18。图1
新窗口打开|下载原图ZIP|生成PPT图1芝麻SiSAD的PCR扩增
Fig. 1PCR amplified product of SiSAD in sesame
利用NCBI在线分析工具分析,发现芝麻SiSAD蛋白序列中含有1个保守结构域,属于脂肪酸去饱和酶家族成员（图2）,位于氨基酸序列的第51—377位。利用ClustalX软件将SiSAD与橄榄（Olea europaea var. sylvestris）、牵牛花（Ipomoea nil）、蓖麻（Ricinus communis）、莴苣（Lactuca sativa）、葡萄（Vitis vinifera）、柑橘（Citrus sinensis）、拟南芥（Arabidopsis thaliana）等7个高等植物的SAD蛋白序列进行比对（图3）,结果表明,芝麻SiSAD蛋白和橄榄、牵牛花相似度较高。利用MAGA5.0软件构建系统发育树（图4）,结果表明,11个植物SAD氨基酸序列被聚为三大类,芝麻SAD蛋白与牵牛花、橄榄SAD蛋白亲缘关系最近,其次与葡萄、莴苣蛋SAD白亲缘关系较近,与蓖麻、拟南芥、柑橘等物种SAD蛋白的亲缘关系较远（图4）。另外,研究发现,植物SAD的氨基酸序列与酵母和藻类的没有同源性,说明高等植物的SAD是独立进化的[26]。
图2
新窗口打开|下载原图ZIP|生成PPT图2SiSAD蛋白的保守结构域
Fig. 2Putative conserved domain of SiSAD
图3
新窗口打开|下载原图ZIP|生成PPT图3芝麻SiSAD与其他物种SAD氨基酸序列比对分析
Fig. 3Multiple alignment of deduced amino acid sequences of SADs
图4
新窗口打开|下载原图ZIP|生成PPT图4芝麻SiSAD与其他植物蛋白的系统进化树
Fig. 4Phylogenetic tree analysis of SiSAD proteins from sesame and other plants
2.2 芝麻SiSAD组织特异性表达分析
通过对中33和中丰芝一号这2个芝麻品种的不同组织中SiSAD的表达分析（图5）,发现SiSAD在2个芝麻材料的根、茎、叶和蕾中表达量极低,而在种子中大量表达。图5
新窗口打开|下载原图ZIP|生成PPT图5芝麻SiSAD在中芝33和中丰芝一号不同组织器官中的表达分析图
Fig. 5Expression profiles of SiSAD in different organs of Zhongfengzhi1 and Zhongzhi33
2.3 SiSAD的功能验证
2.3.1 转基因拟南芥株系的SiSAD表达水平检测 利用农杆菌介导法获得纯系T2株系（SAD-1、SAD-2和SAD-3）,取这三个转基因纯系的幼嫩角果,以同一生长时期的野生型拟南芥（WT）为对照,对SiSAD的表达进行鉴定,结果表明,芝麻SiSAD在转基因拟南芥株系的角果中表达,表明SiSAD转化成功,并得到表达（图6）。图6
新窗口打开|下载原图ZIP|生成PPT图6转基因拟南芥SiSAD的表达分析
**：差异极显著（P<0.01）。下同
Fig. 6Expression profiles of SiSAD in transgenic Arabidopsis
**: Significant difference (P<0.01). The same as below
2.3.2 转基因拟南芥脂肪酸含量检测 用气相色谱法分析转基因拟南芥中硬脂酸（C18:0）和油酸（C18:1）2种脂肪酸的相对含量,与对照相比,转SiSAD拟南芥硬脂酸（C18:0）的含量分别降低了3.0%、4.8%和6.1%,而油酸（C18:1）分别升高了2.8%、4.3%和7.8%,平均升高4.97%。表明芝麻SiSAD的过量表达可以促进硬脂酸向油酸的转化（图7）。
图7
新窗口打开|下载原图ZIP|生成PPT图7转SiSAD拟南芥中脂肪酸相对含量的分析
*：差异显著（P<0.05）
Fig. 7Relative fatty acid content of transgenic Arabidopsis including SiSAD
*: Significant difference (P<0.05)
3 讨论
植物中的△9硬脂酰-ACP脱饱和酶（SAD）定位于质体上,催化硬脂酰-ACP脱饱和而在脂肪酸链的C9与C10间引入一个双键形成油酰-ACP的反应。本研究从芝麻品种中芝13中克隆SiSAD,其编码383个氨基酸。多重序列比对结果显示SiSAD属于脂肪酸去饱和酶家族成员,在C端具有相当高的同源性,含有脂肪酸去饱和酶的典型结构域。进化分析表明,SiSAD与牵牛花、橄榄等植物亲缘关系十分相近,属于同一个分支。表明这些特定结构在生物的进化汇中是稳定、保守的,是功能的基本单元。本研究表明SiSAD在种子中的表达量远高于其他组织器官。这与FOFANA等[27]发现亚麻SAD在种子子房中表达量最高这一结果相符。在拟南芥中进行了过表达研究,结果显示,转SiSAD拟南芥株系中油酸（C18:1）含量平均升高了4.97%。表明芝麻SiSAD的过量表达可以促进硬脂酸向油酸的转化。DU等[28]将ZmSAD1在玉米中进行超表达后,其成熟种子中的硬脂酸含量以及饱和脂肪酸与不饱和脂肪酸之比均低于对照。相反,通过RNAi干扰,转基因玉米种子中的硬脂酸含量、长链饱和脂肪酸含量及饱和脂肪酸与不饱和脂肪酸之比较对照均有增高,油酸含量显著低于对照。在拟南芥突变株ssi2/fab2中,SAD功能的缺失表现为突变体株中的硬脂酸（C18:0）含量升高,油酸（C18:1）含量降低[29]。LIU等[30]运用RNA干涉技术使棉花（Gossypium hirsutum）的SAD基因沉默,发现棉籽油中硬脂酸含量从20%上升到30%—40%,而其他3种主要脂肪酸棕榈酸、油酸和亚油酸的含量减少。
油酸含量是影响芝麻及其制品营养价值和理化稳定性的重要品质指标之一,近年来,油菜、花生等通过品种改良已经实现了高油酸化,而芝麻的高油酸品种改良尚未见成功案例报道。本研究获得了芝麻△9 硬脂酰-ACP脱饱和酶基因SiSAD,证明其在油酸合成过程中的作用,为进一步提高芝麻油酸含量提供了理论依据和基因资源,对高油酸芝麻育种具有实践意义。
4 结论
获得芝麻硬脂酰酰基载体蛋白脱饱和酶基因SiSAD的cDNA全长序列,全长1 152 bp,编码383个氨基酸,分子量为29.15 kD;SiSAD的过表达可以催化转基因拟南芥中硬脂酸向油酸转化,提高油酸的含量。（责任编辑 李莉）
参考文献 原文顺序
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目前食品科研与食品生产均注重营养与保健功能,因此,食用的油营养价值逐渐引起人们的重视.以花生、核桃、葵花籽为例,利用气相色谱法分析其脂肪酸的组成及含量,并着重探讨其中必需脂肪酸的含量.
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Off-flavor due to lipid degradation is an important factor in the shelf life of peanut products. The use of recently developed peanuts with high-oleic acid/linoleic acid (O/L) ratio has the potential to significantly extend the shelf life of roasted peanuts. To determine the full potential for shelf-life improvement of oil-roasted high-O/L peanuts, a study was conducted to examine the effects of roasting high-O/L peanuts (O/L=30) in high-O/L (O/L=23.2) or conventional (O/L=1.5) peanut oil. Peanuts were roasted at 177掳C to Hunter L values of 49卤1. Roasted peanuts were stored at 30掳C for 20 wk. Samples were taken at regular intervals to determine PV, oxidative stability index (OSI), moisture content, and water activity. The O/L ratio of high-O/L roasted peanuts was 27.9 vs. 13.6 for the conventional oil-roasted peanuts. After 20 wk of storage, PV of conventional oil-roasted peanuts was 10.8 compared to 5.3 for the high-O/L-roasted peanuts. OSI values were 88.5 and 52.4 immediately after roasting for the high-O/L-roasted vs. conventional oil-roasted peanuts. OSI for both decreased, but differences remained similar throughout the storage period. Shelf life of high-O/L peanuts decreased when roasted in conventional O/L-peanut oil vs. high-O/L peanut oil.
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<p>油菜是我国重要油料作物,菜籽油是我国城乡居民消费的主要食用油之一。菜籽油中的油酸营养价值很高,同时油酸也是一种重要工业原料。本文从国内外高油酸油菜育种现状、油菜籽油酸含量的诱变改良、遗传调控以及分子标记鉴定和QTL定位等几个方面介绍了油菜种子油酸含量遗传改良研究进展,旨在为今后进一步开展油菜高油酸育种研究提供参考。</p>
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<p>油菜是我国重要油料作物,菜籽油是我国城乡居民消费的主要食用油之一。菜籽油中的油酸营养价值很高,同时油酸也是一种重要工业原料。本文从国内外高油酸油菜育种现状、油菜籽油酸含量的诱变改良、遗传调控以及分子标记鉴定和QTL定位等几个方面介绍了油菜种子油酸含量遗传改良研究进展,旨在为今后进一步开展油菜高油酸育种研究提供参考。</p>
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Abstract : The epidemiological relation between increased levels of blood cholesterol and increased risk of future heart disease is clear, both within and between countries. These strong relationships have led to the adoption of consensus statements in most countries which recommend measures such as the reduction of dietary saturated fat/an increase in the polyunsaturated/saturated ratio and other dietary and sometimes drug methods to reduce serum cholesterol. There is controversy as to whether these measures should be targeted at individuals with high levels of cholesterol or whether there should be a public health approach to the whole population. The public and medical debate has become more heated since the data from intervention trials are conflicting. Taken overall the trials do appear to show reduction in risk of coronary which is stronger for non fatal, compared with fatal coronary events. Meta analysis suggests that increasing benefit accrues from larger reductions and also longer reductions in cholesterol by intervention. However, individual trials frequently show variable results and some, especially the recent 15 year follow up of a Finnish five year intervention (by diet, cholesterol lowering and blood pressure lowering drugs) was strikingly adverse鈥攁lthough the total number of events was not large. Total mortality is much harder to influence and the sum of the available trials is hopelessly inadequate in size to address these questions. As a result confusion abounds and is unlikely to be clarified by the present on going trials. The need for more data is clear. The pilot study for the Oxford Cholesterol Study will be presented as a prelude for a proposed main study in about 20 000 high risk individuals.
DOI:10.1136/bmj.306.6893.1648URLPMID:8518602 [本文引用: 1]
To investigate the level of risk of death from coronary heart disease above which cholesterol lowering treatment produces net benefits. Meta-analysis of results of randomised controlled trials of cholesterol lowering treatments. Published and unpublished data from all identified randomised controlled trials of cholesterol lowering treatments with six months or more follow up and with at least one death were included in the meta-analysis. The analyses were stratified by the rate of death from coronary heart disease in the control arms of the trials. Death from all causes, from coronary heart disease, and from causes other than coronary heart disease. In the pooled analysis, net benefit in terms of total mortality from cholesterol lowering was seen only for trials including patients at very high initial risk of coronary heart disease (odds ratio 0.74; 95% confidence interval 0.60 to 0.92). In a medium risk group no net effect was seen, and in the low risk group there were adverse treatment effects (1.22; 1.06 to 1.42). In a weighted regression analysis a significant (p < 0.001) trend of increasing benefit with increasing initial risk of coronary heart disease was shown. Raised mortality from causes other than coronary heart disease was seen in trials of drug treatment (1.21; 1.05 to 1.39) but not in the trials of non-drug treatments (1.02; 0.88 to 1.19). Cumulative meta-analysis showed that these results seem to have been stable as new trials appeared. Currently evaluated cholesterol lowering drugs seem to produce mortality benefits in only a small proportion of patients at very high risk of death from coronary heart disease. Population cholesterol screening could waste resources and even result in net harm in substantial groups of patients. Overall risk of coronary heart disease should be the main focus of clinical guidelines, and a cautious approach to the use of cholesterol lowering drugs should be advocated. Future trials should aim to clarify the level of risk above which treatment is of net benefit.
DOI:10.3969/j.issn.1672-979X.2005.10.007URL [本文引用: 1]
目的综述单不饱和脂肪酸与冠心病的关系.方法对国内外有代表性的文献加以归纳总结.结果与结论单不饱和脂肪酸通过对凝血功能、血脂和血压的影响,可降低冠心病的发病危险性.
DOI:10.3969/j.issn.1672-979X.2005.10.007URL [本文引用: 1]
目的综述单不饱和脂肪酸与冠心病的关系.方法对国内外有代表性的文献加以归纳总结.结果与结论单不饱和脂肪酸通过对凝血功能、血脂和血压的影响,可降低冠心病的发病危险性.
DOI:10.3969/j.issn.1001-0084.2010.03.011URL [本文引用: 1]
试验用气相色谱法测定常用的6种植物油中39种脂肪酸组成及含量。结果显示,植物油中脂肪酸组成以16碳和18碳脂肪酸为主,不饱和脂肪酸含量都在80%以上,6种植物油中葵花油、豆油和玉米油亚油酸含量较高。
DOI:10.3969/j.issn.1001-0084.2010.03.011URL [本文引用: 1]
试验用气相色谱法测定常用的6种植物油中39种脂肪酸组成及含量。结果显示,植物油中脂肪酸组成以16碳和18碳脂肪酸为主,不饱和脂肪酸含量都在80%以上,6种植物油中葵花油、豆油和玉米油亚油酸含量较高。
URL [本文引用: 1]
本文通过气相色谱法对市售油茶籽油、橄榄油、棕榈油、大豆油、菜籽油、葵花仁油、玉米油和花生油等植物油中的饱和脂肪酸如豆蔻酸、棕榈酸、十七烷酸、硬脂酸与不饱和脂肪酸如棕榈油酸、十七碳一烯酸、油酸、亚油酸组成进行分析,其中油茶籽油与橄榄油脂肪酸组成比例最为接近,油茶籽油中油酸含量高达80%,葵花仁油中亚油酸含量最高达63%,而棕榈油中饱和脂肪酸含量最高达42%,此法可通过主要脂肪酸的相对含量快速有效的区分不同种类的植物油。
URL [本文引用: 1]
本文通过气相色谱法对市售油茶籽油、橄榄油、棕榈油、大豆油、菜籽油、葵花仁油、玉米油和花生油等植物油中的饱和脂肪酸如豆蔻酸、棕榈酸、十七烷酸、硬脂酸与不饱和脂肪酸如棕榈油酸、十七碳一烯酸、油酸、亚油酸组成进行分析,其中油茶籽油与橄榄油脂肪酸组成比例最为接近,油茶籽油中油酸含量高达80%,葵花仁油中亚油酸含量最高达63%,而棕榈油中饱和脂肪酸含量最高达42%,此法可通过主要脂肪酸的相对含量快速有效的区分不同种类的植物油。
DOI:10.3969/j.issn.1006-9577.2013.03.015URL [本文引用: 1]
常用食用油有橄榄油、花生油、大豆油、茶油、芝麻油等。不同食用油的脂肪酸组成和含量不同，特别是一些重要的脂肪酸如油酸、亚油酸、亚麻酸等不饱和脂肪酸含量不近相同。橄榄油和茶油的油酸含量高达70％以上；花生油中油酸约占40％；大豆油的多不饱和脂肪酸含量较高，主要为亚麻酸；芝麻油中亚油酸含量占到40％以上。食用油除提供能量和必需脂肪酸外，还有脂溶性维生素和一些植物化合物，一些食用油对控制脂代谢异常具有一定的作用。
DOI:10.3969/j.issn.1006-9577.2013.03.015URL [本文引用: 1]
常用食用油有橄榄油、花生油、大豆油、茶油、芝麻油等。不同食用油的脂肪酸组成和含量不同，特别是一些重要的脂肪酸如油酸、亚油酸、亚麻酸等不饱和脂肪酸含量不近相同。橄榄油和茶油的油酸含量高达70％以上；花生油中油酸约占40％；大豆油的多不饱和脂肪酸含量较高，主要为亚麻酸；芝麻油中亚油酸含量占到40％以上。食用油除提供能量和必需脂肪酸外，还有脂溶性维生素和一些植物化合物，一些食用油对控制脂代谢异常具有一定的作用。
DOI:10.1021/bi981839iURL [本文引用: 1]
DOI:10.1146/annurev.pp.33.060182.000525URL [本文引用: 1]
Using a small substrate (thymidine 5'-(p-nitrophenyl phosphate) 3'-phosphate), the kinetics of staphylococcal nuclease insolubilized on CNBr-activated Sepharoses 4B and 6B are affected by internal diffusional limitations. Since we demonstrate that we are working under conditions in which external mass-transfer resistances do not influence the reaction rate, we propose a simple theoretical model that considers only the case of mixed enzymic reaction-internal diffusion kinetics. In the Eadie-Hofstee plots we find very good agreement between theory and experiment. The model accounts very well for the results obtained by changing support texture, reaction conditions, and/or enzyme concentration in the insoluble derivatives, variables that modify the diffusional restrictions of the system.
DOI:10.1073/pnas.0511079103URLPMID:16763049 [本文引用: 1]
We report the identification of bifunctional Δ12/ω3 desaturases from Fusarium moniliforme, Fusarium graminearum, and Magnaporthe grisea. The bifunctional activity of these desaturases distinguishes them from all known Δ12 or ω3 fatty acid desaturases. The ω3 desaturase activity of these enzymes also shows a broad ω6 fatty acid substrate specificity by their ability to convert linoleic acid (LA), γ-linolenic acid, di-homo-γ-linolenic acid, and arachidonic acid to the ω3 fatty acids, α-linolenic acid (ALA), stearidonic acid, eicosatetraenoic acid, and eicosapentaenoic acid (EPA), respectively. Phylogenetic analysis suggests that ω3 desaturases arose by independent gene duplication events from a Δ12 desaturase ancestor. Expression of F. moniliforme Δ12/ω3 desaturase resulted in high ALA content in both Yarrowia lipolytica, an oleaginous yeast naturally deficient in ω3 desaturation, and soybean. In soybean, seed-specific expression resulted in 70.9 weight percent of total fatty acid (%TFA) ALA in a transformed seed compared with 10.9%TFA in a null segregant seed and 53.2%TFA in the current best source of ALA, linseed oil. The ALA/LA ratio in transformed seed was 22.3, a 110- and 7-fold improvement over the null segregant seed and linseed oil, respectively. Thus, these desaturases have potential for producing nutritionally desirable ω3 longchain polyunsaturated fatty acids, such as EPA, with a significantly improved ratio of ω3/ω6 long-chain polyunsaturated fatty acids in both oilseeds and oleaginous microbes.
DOI:10.1007/s00122-013-2223-5URLPMID:24193357 [本文引用: 1]
Two new sources of elevated seed stearic acid were identified and the feasibility of an elevated stearic acid, high oleic acid germplasm was studied. Soybean [Glycine max (L.) Merr.] oil typically contains 2-4% stearic acid. Oil with at least 20% stearic acid is desirable because of its improved baking properties and health profile. This study identifies two new sources of high stearic acid and evaluates the interaction of high stearic and oleic acid alleles. TCHM08-1087 and TCHM08-755, high stearic acid 'Holladay' mutants, were crossed to FAM94-41-3, a line containing a point mutation in a seed-specific isoform of a 螖9-stearoyl-acyl carrier protein-desaturase (SACPD-C). F2-derived lines were evaluated for fatty acid content in four field environments. Sequencing of SACPDs in TCHM08-1087 and TCHM08-755 revealed distinct deletions of at least one megabase encompassing SACPD-C in both lines. After genotyping, the additive effect for stearic acid was estimated at +1.8% for the SACPD-C point mutation and +4.1% for the SACPD-C deletions. Average stearic acid in lines homozygous for the deletions was 12.2%. A FAM94-41-3-derived line and TCHM08-1087-11, a selection from TCHM08-1087, were crossed to S09-2902-145, a line containing missense mutations in two fatty acid desaturases (FAD2-1A and FAD2-1B). F1 plants were grown in a greenhouse and individual F2 seed were genotyped and phenotyped. No interaction was observed between either FAD2-1A or FAD2-1B and any of the SACPD-C mutant alleles. Seed homozygous mutant for SACPD-C/FAD2-1A/FAD2-1B contained 12.7% stearic acid and 65.5% oleic acid while seed homozygous for the SACPD-C deletion and mutant for FAD2-1A and FAD2-1B averaged 10.4% stearic acid and 75.9% oleic acid.
DOI:10.1016/j.gene.2014.03.047URLPMID:24680703 [本文引用: 1]
61Full length cDNA clone (SAD) was isolated from seeds of P. pinnata.61cDNA clone contained a single ORF, shares similarity with SAD from other plants.61The 3D structure of PpSAD is resembled to crystal structure of R. communis (SAD).61PpSAD shows distinct expression levels during various seed developmental stages.
DOI:10.2135/cropsci2005.06-0172URL [本文引用: 1]
ABSTRACT The delta9 stearoyl acyl-carrier protein desaturase (SACPD) gene of soybean [Glycine max (L.) Merrill] encodes a soluble enzyme that converts stearic to oleic acid. Understanding the regulation of SACPD expression and enzyme activity are thus important steps toward developing soybean lines with altered stearic or oleic acid content. Using primers designed to a G. max SACPD cDNA sequence, a 3648-bp product was cloned and sequenced from the genome of cultivar Dare. Comparison of the third SACPD exon protein sequence with other available Glycine SACPD sequences revealed unique amino acid variability at positions 310 and 313. Sequence-specific primers were designed for Real-time RT-PCR (reverse transcriptase-polymerase chain reaction) for this region of exon 3. Diagnostic and specific products were recovered with these primers using Dare cDNA template and Dare genomic DNA. Sequencing of a second genomic clone from Dare confirmed that there were two SACPD genes, designated A and B, in this cultivar. Survey of the genomes of 51 soybean lines and cultivars with PCR and the gene-specific primers indicated that all 51 had both A and B. Differences between SACPD-A and -B transcript abundance in soybean tissues, while quantifiable, were not dramatic. SACPD-A and -B transcript accumulation for three seed developmental stages between R5 and R6 was essentially equal. Biochemical analysis of the proteins encoded by these two SACPD genes may reveal whether the amino acid variability uncovered in this study has any relation to enzyme activity.
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DOI:10.1134/s1021443709050185URL [本文引用: 1]
As special maize with more than 6% oil concentration in the grain, high-oil maize has received increased interest recently. To date, little is known about the expressions of genes involved in fatty acid metabolism of high-oil maize. Stearoyl-acyl carrier protein desaturase (SAD) is a key enzyme that converts stearic acid to oleic acid. In this study, two-dimensional electrophoresis, gas chromatography, and real-time PCR were used to determine the expressions of SAD at three seed development stages in high-oil and normal maize inbred lines. SAD was significantly more abundantly expressed in high-oil maize than in normal maize, not only at the protein and mRNA levels, but also at the product level. These results suggested that a high expression of SAD may play an important role in increasing oil concentration in high-oil maize.
DOI:10.1007/s00438-016-1265-1URL [本文引用: 1]
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DOI:10.1007/s11248-008-9164-9URLPMID:18214708 [本文引用: 1]
The possibility of altering the unsaturation level of fatty acids in plant lipids by genetic transformation has implications for the stress tolerance of higher plants as well as for their nutritional value and industrial utilisation. While the integration and expression of transgenes in the plastome has several potential advantages over nuclear transformation, very few attempts have been made to manipulate fatty acid biosynthesis using plastid transformation. We produced transplastomic tobacco plants that express a 螖 desaturase gene from either the wild potato species desaturase protein in transplastomic plants was confirmed by northern and western blot analyses. In comparison with control plants, transplastomic plants showed altered fatty acid profiles and an increase in their unsaturation level both in leaves and seeds. The two transgenes produced comparable results. The results obtained demonstrate the feasibility of using plastid transformation to engineer lipid metabolic pathways in both vegetative and reproductive tissues and suggest an increase of cold tolerance in transplastomic plants showing altered leaf fatty acid profiles. This is the first example of transplastomic plants expressing an agronomically relevant gene produced with the 鈥渂inding-type鈥 vectors, which do not contain a heterologous marker gene. In fact, the transplastomic plants expressing the S. commersonii gene contain only plant-derived sequences, a clear attraction from a public acceptability perspective.
DOI:10.1073/pnas.89.7.2624PMID:1557366 [本文引用: 1]
Molecular gene transfer techniques have been used to engineer the fatty acid composition of Brassica rapa and Brassica napus (canola) oil. Stearoyl-acyl carrier protein (stearoyl-ACP) desaturase (EC 1.14.99.6) catalyzes the first desaturation step in seed oil biosynthesis, converting stearoyl-ACP to oleoyl-ACP. Seed-specific antisense gene constructs of B. rapa stearoyl-ACP desaturase were used to reduce the protein concentration and enzyme activity of stearoyl-ACP desaturase in developing rapeseed embryos during storage lipid biosynthesis. The resulting transgenic plants showed dramatically increased stearate levels in the seeds. A continuous distribution of stearate levels from 2% to 40% was observed in seeds of a transgenic B. napus plant, illustrating the potential to engineer specialized seed oil compositions
DOI:10.1186/gb-2014-15-1-r1URL [本文引用: 1]
DOI:10.1093/pcp/pcu175PMID:25480115 [本文引用: 1]
Abstract Sesame (Sesamum indicum L.) is an ancient and important oilseed crop grown widely in tropical and subtropical areas. It belongs to the gigantic order Lamiales, which includes many well-known or economically important species, such as olive (Olea europaea), leonurus (Leonurus japonicus) and lavender (Lavandula spica), many of which have important pharmacological properties. Despite their importance, genetic and genomic analyses on these species have been insufficient due to a lack of reference genome information. The now available S. indicum genome will provide an unprecedented opportunity for studying both S. indicum genetic traits and comparative genomics. To deliver S. indicum genomic information to the worldwide research community, we designed Sinbase, a web-based database with comprehensive sesame genomic, genetic and comparative genomic information. Sinbase includes sequences of assembled sesame pseudomolecular chromosomes, protein-coding genes (27,148), transposable elements (372,167) and non-coding RNAs (1,748). In particular, Sinbase provides unique and valuable information on colinear regions with various plant genomes, including Arabidopsis thaliana, Glycine max, Vitis vinifera and Solanum lycopersicum. Sinbase also provides a useful search function and data mining tools, including a keyword search and local BLAST service. Sinbase will be updated regularly with new features, improvements to genome annotation and new genomic sequences, and is freely accessible at http://ocri-genomics.org/Sinbase/. The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.
DOI:10.3969/j.issn.1000-1336.2006.02.014URL [本文引用: 1]
植物硬脂酰-酰基载体蛋白脱饱和酶(SAD)是植物脂肪酸合成代谢的酰-ACP脱饱和形成油酰-ACP的 反应.SAD决定着植物饱和脂肪酸和不饱和脂肪酸的比例,和植物响应温度变化,增强低温适应性有密切关系,并参与防御、膜形成等生理过程.SAD分子是同 源二聚体,具特异的二铁中心,其空间结构已被描述.对SAD进行基因工程操作具有广阔的前景.
DOI:10.3969/j.issn.1000-1336.2006.02.014URL [本文引用: 1]
植物硬脂酰-酰基载体蛋白脱饱和酶(SAD)是植物脂肪酸合成代谢的酰-ACP脱饱和形成油酰-ACP的 反应.SAD决定着植物饱和脂肪酸和不饱和脂肪酸的比例,和植物响应温度变化,增强低温适应性有密切关系,并参与防御、膜形成等生理过程.SAD分子是同 源二聚体,具特异的二铁中心,其空间结构已被描述.对SAD进行基因工程操作具有广阔的前景.
DOI:10.3923/ajps.2008.771.774URLPMID:17069354 [本文引用: 1]
Flax's recent popularity in human and animal foods is mostly due to its desirable FA composition. Flax is an excellent source of omega-3 FA, which have been shown to have many health benefits. To date, little is known about the genetic and environmental factors that control the FA composition of flax seeds. To elucidate some of the important genetic components, reverse transcriptase (RT)-PCR and real-time PCR were used to determine the expression profiles of two key FA biosynthetic genes during seed development. Plants of flax cultivar AC McDuff were grown under field conditions, and RNA was extracted from ovaries and developing bolls collected from 2 d after anthesis (DAA) to maturity. Desaturation enzymes stearoyl-ACP desaturase (SAD) and delta12 FA desaturase 2 (FAD2) were both expressed in ovaries, and their expression was differentially modulated throughout seed development. SAD was most highly expressed in ovaries. Its expression quickly decreased until 4 DAA; this was followed by a slight peak at 8 DAA, only to return to relatively low levels of expression in maturing bolls, ranging from 2.1% to 4.5% relative to the level observed in ovaries. FAD2 expression displayed a different temporal pattern. While expression of FAD2 did decrease in the early stages of seed development, expression increased starting at 8 DAA, peaking at 16 DAA, when it was 158% relative to the level observed in ovaries. FAD2, which desaturates oleic acid (18:1cisdelta9) into linoleic acid (18:2cisdelta9,12), is therefore controlled at the transcription level. To relate enzyme expression with FA profile, GC was performed on the same subsamples used for RT-PCR and real-time PCR, and proportions of palmitic, stearic, oleic, linoleic, and linolenic acids were determined for the same developmental stages. Although FAD2 expression increased from 8 to 16 DAA, relative changes in linoleic acid (18:2cis delta9,12) were not observed. However, linolenic acid (ALA; alpha-18:3; 18:3cisdelta9,12,15) l
DOI:10.1186/s12870-016-0827-zURLPMID:27297560 [本文引用: 1]
Stearoyl-acyl carrier protein desaturase (SAD) is a key enzyme that catalyses the conversion of stearoyl-acyl carrier protein (ACP) to oleoyl-ACP, a precursor for the biosynthesis of polyunsaturated fatty acids.ZmSAD1(GenBank: KU949326) is a major QTL for stearic acid content in maize seeds. To investigate the biological function and the application potential of maizeZmSAD1in oil biosynthesis, we isolated the full-lengthZmSAD1cDNA from maize B73 and overexpressed it inArabidopsisand maize. Under seed-specific overexpression ofZmSAD1inArabidopsis, the stearic acid content and the ratio of saturated to unsaturated fatty acids in the seeds were significantly decreased relative to those in the control. Conversely, in transgenicZmSAD1RNAiArabidopsisseeds, the contents of stearic acid and long-chain saturated acids and the ratio of saturated to unsaturated fatty acids were significantly increased; in addition, the oleic acid content was significantly decreased. More importantly, transgenicZmSAD1maize that expressed high levels ofZmSAD1in its mature seeds showed reduced stearic acid content (1.5702%) and a lower saturated to unsaturated fatty acid ratio (20.4002%) relative to those (1.6402% and 20.6102%, respectively) of the control. Conversely, down-regulation ofZmSAD1in maize resulted in increased levels of stearic acid (1.7802%), long-chain saturated acids (0.8502%) and the ratio of saturated to unsaturated fatty acids (21.5402%) relative to those (1.6402%, 0.7402%, and 20.6102%, respectively) of the control, whereas the oleic acid (32.0102%) level was significantly decreased relative to that (32.6802%) of the control. Our work demonstrates that the contents of stearic acid, oleic acid, and long-chain saturated acids, and the ratio of saturated to unsaturated fatty acids, are modified in maize seeds by seed-specific overexpression or down-regulation ofZmSAD1. Therefore, theZmSAD1gene is a useful tool for engineering the seed oil composition in maize and other crops. The online version of this article (doi:10.1186/s12870-016-0827-z) contains supplementary material, which is available to authorized users.
DOI:10.1007/s11103-006-9086-yURLPMID:17072561 [本文引用: 1]
In plants, changes in the levels of oleic acid (18:1), a major monounsaturated fatty acid (FA), results in the alteration of salicylic acid (SA)- and jasmonic acid (JA)-mediated defense responses. This is evident in the Arabidopsis ssi2/fab2 mutant, which encodes a defective stearoyl-acyl carrier protein-desaturase (S-ACP-DES) and consequently accumulates high levels of stearic acid (18:0) and low levels of 18:1. In addition to SSI2, the Arabidopsis genome encodes six S-ACP-DES-like enzymes, the native expression levels of which are unable to compensate for a loss-of-function mutation in ssi2 . The presence of low levels of 18:1 in the fab2 null mutant indicates that one or more S-ACP-DES isozymes contribute to the 18:1 pool. Biochemical assays show that in addition to SSI2, four other isozymes are capable of desaturating 18:0-ACP but with greatly reduced specific activities, which likely explains the inability of these SSI2 isozymes to substitute for a defective ssi2. Lines containing T-DNA insertions in S-ACP-DES1 and S-ACP-DES4 show that they are altered in their lipid profile but contain normal 18:1 levels. However, overexpression of the S-ACP-DES1 isoform in ssi2 plants results in restoration of 18:1 levels and thereby rescues all ssi2 -associated phenotypes. Thus, high expression of a low specific activity S-ACP-DES is required to compensate for a mutation in ssi2 . Transcript level of S-ACP-DES isoforms is reduced in high 18:1-containing plants. Enzyme activities of the desaturase isoforms in a 5-fold excess of 18:1-ACP show product inhibition of up to 73%. Together these data indicate that 18:1 levels are regulated at both transcriptional and post-translational levels.
DOI:10.1002/pssb.2221890110URLPMID:12177486 [本文引用: 1]
We have genetically modified the fatty acid composition of cottonseed oil using the recently developed technique of hairpin RNA-mediated gene silencing to down-regulate the seed expression of two key fatty acid desaturase genes, ghSAD-1-encoding stearoyl-acyl-carrier protein 9-desaturase and ghFAD2-1-encoding oleoyl-phosphatidylcholine 6-desaturase. Hairpin RNA-encoding gene constructs (HP) targeted against either ghSAD-1 or ghFAD2-1 were transformed into cotton (Gossypium hirsutum cv Coker 315). The resulting down-regulation of the ghSAD-1 gene substantially increased stearic acid from the normal levels of 2% to 3% up to as high as 40%, and silencing of the ghFAD2-1 gene resulted in greatly elevated oleic acid content, up to 77% compared with about 15% in seeds of untransformed plants. In addition, palmitic acid was significantly lowered in both high-stearic and high-oleic lines. Similar fatty acid composition phenotypes were also achieved by transformation with conventional antisense constructs targeted against the same genes, but at much lower frequencies than were achieved with the HP constructs. By intercrossing the high-stearic and high-oleic genotypes, it was possible to simultaneously down-regulate both ghSAD-1 and ghFAD2-1 to the same degree as observed in the individually silenced parental lines, demonstrating for the first time, to our knowledge, that duplex RNA-induced posttranslational gene silencing in independent genes can be stacked without any diminution in the degree of silencing. The silencing of ghSAD-1 and/or ghFAD2-1 to various degrees enables the development of cottonseed oils having novel combinations of palmitic, stearic, oleic, and linoleic contents that can be used in margarines and deep frying without hydrogenation and also potentially in high-value confectionery applications.
