姚兴兰^,, 杨文竹, 罗彦忠, 陈茹梅, 王磊^,, 张兰^,中国农业科学院生物技术研究所,北京 100081

Acquisition and Characteristic Analysis of Transgenic Maize with phyA2, ZmTMT, and Bar

YAO XingLan^,, YANG WenZhu, LUO YanZhong, CHEN RuMei, WANG Lei^,, ZHANG Lan^,Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081

通讯作者: 张兰,E-mail： zhanglan01@caas.cn; 王磊,E-mail： wanglei01@caas.cn

责任编辑: 李莉
收稿日期:2020-03-7接受日期:2020-04-20网络出版日期:2020-12-16

基金资助:

转基因生物新品种培育科技重大专项.2016ZX08003-002

Received:2020-03-7Accepted:2020-04-20Online:2020-12-16
作者简介 About authors
姚兴兰,E-mail： 15751004149@163.com。








摘要
【目的】玉米是家禽和单胃动物饲料的主要原料,但玉米籽粒中高活性的α-生育酚含量较低,且籽粒中的磷主要是以植酸磷的形式存在,动物不能有效吸收利用,因此,饲料中需要添加化学合成的维生素E和无机磷或微生物来源的植酸酶以满足动物生长发育的需要,增加了饲料成本,同时又容易造成磷污染。通过转基因获得α-生育酚含量和植酸酶活性大量提高的玉米,为饲用玉米育种提供材料基础。【方法】构建含有3个基因表达盒的载体,采用农杆菌侵染的方法转化玉米,草铵膦作为筛选压力;通过喷施草铵膦和PCR鉴定的方法筛选转基因阳性植株,RT-PCR分析目的基因在转录水平的表达情况;Western blot分析目的基因在翻译水平上的表达情况,采用分光光度计测定转基因玉米籽粒中的植酸酶活性,利用HPLC测定籽粒中的维生素E含量,同时比较转基因株系与野生型在其他营养成分和农艺性状上的差异。【结果】构建了胚乳特异性启动子123387驱动的植酸酶基因phyA2表达盒、胚特异性启动子13387驱动的玉米γ-生育酚甲基转移酶基因ZmTMT表达盒以及组成型启动子CaMV 35S驱动的草铵膦抗性基因Bar表达盒串联的植物表达载体;转化玉米获得转基因植株;喷施草铵膦和PCR鉴定得到阳性植株;经过多个世代回交转育,获得目标性状均较好的2个转基因纯合玉米株系TPB1和TPB2;RT-PCR和Western blot分析结果表明phyA2、ZmTMT和Bar在转基因玉米中显著高表达。植酸酶活性测定结果表明,转基因玉米籽粒中植酸酶活性达到10 000—13 000 U·kg^-1。维生素E含量测定结果表明,转基因玉米籽粒中90%以上的γ-生育酚转化为α-生育酚,α-生育酚的含量达到50—70 mg·kg^-1,α-三烯生育酚的含量也有明显增加。转基因玉米中的植酸酶活性和α-生育酚含量完全能够满足动物饲料的需要。转基因株系的农艺性状与野生型无显著差异,TPB1的营养成分与野生型总体无显著差异,TPB2稍高于野生型但是未产生不利影响;且均具有草铵膦抗性。【结论】获得的富含维生素E和植酸酶、且具有除草剂抗性的玉米新材料可以用于玉米杂交种的开发应用,降低饲料成本,提高磷的利用率,减少环境污染。
关键词： 玉米;植酸酶;维生素E;α-生育酚;草铵膦抗性

Abstract
【Objective】Corn seeds are the main feed ingredient for poultry and monogastric animals, but the content of α-tocopherol with high-activity in corn seeds is low, and maize is rich in phosphorus mainly existed in the form of phytate, which cannot be effectively utilized by animals. Therefore, synthesized DL-α-tocopheryl acetate and phosphorus or microbial phytase are supplemented in the feed for optimal animal growth. These methods not only greatly increase the feed cost, but also cause the phosphorus pollution due to the undigested phytate. The aim of this study is to acquire maize seeds rich in α-tocopherol and high in phytase activity, and provide resources for forage maize breeding.【Method】Construct an expression vector with three expression cassette, and introduced into maize through agrobacterium infection method, glufosinate was used for screening; The positive transgenic plants were identified by spraying glufosinate and PCR analysis; RT-PCR analysis of target gene expression at the transcription level; Western blot analysis of target gene expression at the translation level; Phytase activity was measured by spectrophotometric method; vitamin E content was determined by HPLC. At the same time, the differences of other nutrients and agronomic traits between transgenic lines and wild types were compared.【Result】Constructed an expression vector with three expression cassette, phyA2 driven by endosperm-specific promoter 123387, ZmTMT driven by embryo-specific promoter 13387, Bar driven by constitutive promoter CaMV35S, and introduced into maize; Transgenic plants were identified by spraying glufosinate and PCR; Two transgenic homozygous maize lines TPB1 and TPB2 with good target traits were obtained after multiple generations of backcross transfer. RT-PCR and Western blot analysis showed that phyA2, ZmTMT and Bar were all highly expressed in transgenic lines. Phytase activity determination showed phytase activity reached to 10 000-13 000 U·kg ^-1, which is able to meet the needs of animal growth and development, vitamin E content measurement showed more than 90% of γ-tocopherol was transformed to α-tocopherol, and α-tocopherol content increased to 50-70 mg·kg ^-1. In addition, there were no significant differences on agronomic traits between transgenic lines and wild type, and there was no significant difference on nutrient component between TPB1 and wild type, slight difference but without adverse effect between TPB2 and wild type; and the transgenic plants were resistant to BASTA.【Conclusion】These transgenic maize, which are rich in α-tocopherol and phytase, and also with herbicide resistance, can be applied for maize breeding to reduce feed cost, improve the utilization rate of phosphorus and reduce environmental pollution.
Keywords：maize;phytase;vitamin E;α-tocopherol;glufosinate resistance


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本文引用格式
姚兴兰, 杨文竹, 罗彦忠, 陈茹梅, 王磊, 张兰. 转phyA2、ZmTMT和Bar玉米的获得及其特性分析[J]. 中国农业科学, 2020, 53(24): 4982-4991 doi:10.3864/j.issn.0578-1752.2020.24.002
YAO XingLan, YANG WenZhu, LUO YanZhong, CHEN RuMei, WANG Lei, ZHANG Lan. Acquisition and Characteristic Analysis of Transgenic Maize with phyA2, ZmTMT, and Bar[J]. Scientia Acricultura Sinica, 2020, 53(24): 4982-4991 doi:10.3864/j.issn.0578-1752.2020.24.002


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0 引言

【研究意义】玉米（Zea mays）是动物和家禽重要的饲料作物之一,全球用于饲料的玉米占总量的65%,相当于4.5×10⁸亿t^[1]。磷和维生素E是动物生长发育所需的重要元素,磷在谷物、豆类等籽实作物中主要以植酸磷的形式存在,占植物种子磷含量的52%—80%^[2,3]。而单胃动物（例如猪、鸡等）消化道中不能分泌植酸酶,导致植酸磷不能得到有效利用^[3,4,5]。此外,植酸是抗营养因子,能与动物体内的重要矿质元素（如Fe³⁺、Zn²⁺、Ca²⁺、Mg²⁺和K⁺）和蛋白结合形成共沉淀,从而导致动物不能有效吸收矿物质^[6,7,8]。因此,饲料中需外源添加无机磷或者微生物来源的植酸酶才能满足动物生长的需要。维生素E是一类脂溶性抗氧化剂,是人体和动物营养所必须、但又不能自身合成的重要维生素。维生素E只能在光合组织合成（植物和光合细菌）,具有维持生物膜完整性、防止脂质氧化等重要的生物学功能^[9,10]。根据天然维生素E植基侧链的饱和程度分为生育酚和三烯生育酚,根据色满醇环上甲基数量和位置的不同分为α-、β-、γ-、δ-生育酚/三烯生育酚^[10],其中,以α-生育酚的活性最高^[11]。玉米作为动物饲料的主要原料,籽粒中低活性的γ-生育酚含量最高,而高活性的α-生育酚含量较低。为了满足动物生长发育需要,饲料中需要外源添加合成的DL-α-生育酚醋酸酯。DL-α-生育酚醋酸酯的生物活性仅为天然α-生育酚的三分之二,其生物利用率也仅为天然α-生育酚的五分之一左右^[12,13]。因此,改善玉米籽粒中植酸酶的表达量和维生素E的组成,提高玉米中植酸酶活性和高活性α-生育酚的含量,则饲料中可以不再外源添加植酸酶或磷和合成的维生素E,大大降低饲料成本。【前人研究进展】植酸酶可以催化植酸水解产生肌醇衍生物和无机磷^[14],目前,已从植物和微生物中分离了植酸酶基因,其中来源于微生物、尤其是曲霉属的植酸酶报道比较多^{[15,16,17,18]}。曲霉来源的植酸酶phyA因其在酸性环境酶活性高、耐热性好而备受关注^[18]。酵母中表达的phyA2具有较高的酶活性（13 000—16 000 U·mL^-1）和良好的热稳定性^[14],目前已被用作饲料添加剂^{[15,16,17,18]}。玉米中胚特异性表达phyA2,籽粒中植酸酶活性达到2 200 U·kg^{-1 [19]},胚乳特异性表达phyA2,籽粒中植酸酶活性达到125 000 U·kg^-1[20],此胚乳特异表达phyA2的玉米已获得安全生产证书（BVLA430101）^[21],可应用于饲料生产。而在维生素E的合成途径中,γ-生育酚甲基转移酶（γ-TMT）催化低活性的γ-生育酚转化为高活性的α-生育酚。因此,提高γ-TMT的表达来增加高活性的α-生育酚含量是进行维生素E基因工程的首选。1998年,SHINTANI等^[22]率先过表达拟南芥的γ-TMT（AtTMT）,使得拟南芥种子中α-生育酚含量提高约80倍;水稻中过表达AtTMT,转基因水稻胚乳中α-三烯生育酚含量提高1.7倍^[23];莴苣中过表达AtTMT,T₂代转基因株系中α-生育酚含量提高约38倍^[24];过表达拟南芥AtTMT和白苏的PfTMT,转基因大豆种子中α-生育酚含量分别提高4倍和10.4倍^[25,26]。ZHANG等^[23,27]分别研究了大豆的GmTMT2a和玉米的ZmTMT,转GmTMT2a和ZmTMT玉米株系α-生育酚含量分别提高3—4.5倍和5—6.5倍。【本研究切入点】前期分别研究了phyA2和ZmTMT在玉米籽粒中的表达,转phyA2玉米中,植酸酶活性得到大幅度提高^[19,20];转ZmTMT玉米中,α-生育酚含量与野生型相比提高5—6.5倍^[27]。但是通过同时表达phyA2和ZmTMT聚合高α-生育酚和植酸酶活性2种性状的相关研究却未见报道,因此,利用phyA2和ZmTMT共表达获得多种目标性状聚合的转基因玉米,将为饲料加工提供便利,还可以大大降低成本;同时,研究多性状聚合是否会影响受体材料的农艺性状以及营养成分,为今后聚合更多性状饲用玉米育种提供参考。【拟解决的关键问题】本研究分别利用胚、胚乳特异表达的启动子驱动目的基因的表达,获得多性状聚合的转基因玉米,为饲料加工提供便利,大大降低成本;同时为饲料玉米育种提供资源。

1 材料与方法

1.1 试验材料

双元载体pCAMBIA3301（含有Bar表达盒,具有草铵膦抗性）、质粒pPHP20754-phyA2（含植酸酶基因phyA2表达盒、LEG1启动子、LEG1终止子）^[20]、pSP-ZmTMT（含有ZmTMT表达盒,Glb1的启动子和终止子）^[27]、pEU13387G3（含有13387启动子）^[28]、pUM3G-123387（含有123387启动子）、农杆菌菌株EHA105、含有30a-ZmTMT质粒的大肠杆菌菌株^[27]均为王磊研究员实验室保存。

转化受体材料Hi-II^[29]和回交转育材料（自交系郑58）为王磊研究员实验室保存。玉米大田种植于试验基地;转基因幼苗在25℃的生长箱中以16 h光照/8 h黑暗周期生长约2周,再在16 h光照/8 h黑暗周期中移至28℃的温室中。

1.2 载体构建和转基因植株的获得

利用引物ZT3301 FW：5′-ATCGCAAATTTGGTC ATGGCTCACGCGGCGCTGCT-3′,ZT3301 RV：5′- CGATCGGGGAAATTCGAGCTGGGTCACCTCTTTTATGAATAATAATAA-3′,以质粒pSP-ZmTMT为模板,PCR扩增获得ZmTMT片段（含有ZmTMT序列和Glb1终止子序列）。利用引物13387 FW0：5′-CTATGACCATGATTACGGTTTAAACAAGGAACATCTTAGGAAGTG-3′（加入PmeⅠ酶切位点）,13387 RV1：5′-GACCAAATTTGCGAT GTGTCGTCG TCCGCCACCCGA-3′,以质粒pEU13387G3为模板,PCR扩增得到13387启动子序列。质粒载体pCAMBIA3301做EcoRⅠ和BstEⅡ酶切处理。利用试剂盒（Vazyme,C112-01）,将酶切后的载体pCAMBIA3301、13387启动子序列以及ZmTMT片段进行重组反应,得到3301-13387-ZT质粒载体。

利用引物CPAO3301 FW：5′-TGGTCACAAATT TCGATGGCCCCACGCCGCCTGCT-3′,CPAO3301 RV1：5′-CTAAGATGTTCCTTGTTTAAACCAGTAT AACTATGCCGAGGT-3′, 以质粒pPHP20754-phyA2为模板,PCR扩增得到phyA2片段（含有信号肽SP,靶向液泡序列VTS, phyA2序列以及LEG1终止子）。利用引物123387 FW：5′-CTATGACCATGATTACG GTTTATTTAAATATGCTTCGACCAAAACACCC-3′, 123387 RV：5′-CGAAATTTGTGACCAGCATGCAG TCAACAATGGCCA-3′（加入SwaⅠ酶切位点）, 以质粒pUM3G-123387为模板,PCR扩增得到123387启动子序列。质粒载体3301-13387-ZT做PmeⅠ酶切处理。将酶切后的3301-13387-ZT、123387启动子序列以及phyA2片段进行重组反应, 得到3301-phyA2- ZT质粒载体。

将3301-phyA2-ZT载体转化农杆菌菌株EHA105,侵染玉米Hi-II幼胚,参照HYUN等^[30]方法进行,草铵膦BASTA作为筛选压力,获得转基因植株。转基因植株与自交系郑58杂交获得F₁植株,然后再与郑58回交5代,自交2代获得BC₅F₃。最后,获得转基因纯合株系TPB1和TPB2。每个世代的转基因玉米植株均通过叶片涂抹草铵膦或者喷施草铵膦鉴定阳性,再经过PCR验证。

1.3 phyA2、ZmTMT和Bar的RT-PCR分析

利用RNA提取试剂盒（Transgene,ER501-01）提取转基因株系玉米授粉15 d的籽粒总RNA,cDNA第一链的合成按照MonScript? RTIII All-in-One Mix（Monad,MR05001S）的操作说明进行。

采用RT-PCR的方法分析phyA2、ZmTMT、Bar的RNA水平表达。以ZmActin作为内参^[31],引物：Actin-F：5′-ATGTTTCCTGGGATTGCCGAT-3′,Actin-R：5′-CCAGTTTCGTCATACTCTCCCTTG-3′;phyA2引物：AO1229F1：5′-TTGTCTGGCGTGACTCT CAC-3′,AO1229R1：5′-TCCTGCTCAGACTGGCAT TG-3′;ZmTMT引物：ZT1229F1：5′-ATGTCGGATGTG GCATTGG-3′,ZT1229R1：5′-CCCTGGATCATTAG CGGC-3′;Bar引物：bar1225F：5′-GGAAGTTGACCG TGCTTGTC-3′,bar1225R：5′-CGAGTCAACCGTGT ACGTCT-3′。

1.4 phyA2、ZmTMT和Bar蛋白的Western blot分析

转基因株系成熟籽粒研磨成粉末,称取100 mg提取总蛋白（蛋白提取液：50 mmol·L^-1 Tris-HCl（pH 8.0）、150 mmol·L^-1 NaCl、1 mmol·L^-1 EDTA、2% NP-40（v/v）和10 mmol·L^-1 PMSF）进行Western blot分析,参考CHEN等^[19]方法进行。SDS-PAGE使用12%浓度胶,使用NC膜（Merk,Germany）转膜。phyA2杂交一抗为phyA2蛋白的兔抗血清^[19],二抗为辣根过氧化物酶（horseradish peroxidase,HRP）标记的羊抗兔IgG（CW Bio,CW0156S）;ZmTMT和Bar杂交的一抗分别为2个蛋白的鼠抗单克隆抗体,二抗为辣根过氧化物酶标记的羊抗鼠IgG（CW Bio,CW0110S）,杂交时一抗/二抗稀释5 000—10 000倍进行杂交。

1.5 植酸酶活性以及植酸磷和磷含量测定

参考MARKUS等^[32]和CHEN等^[19]方法测定植酸酶活性、植酸磷和磷含量。

植酸酶活性测定：称取上述成熟籽粒粉末50 mg加入1 mL提取液,振荡孵育1 h,10 000 r/min,离心15 min,上清液即为植酸酶提取液。酶活性测定：取20 μL植酸酶提取液,加入80 μL植酸钠,37℃孵育30 min,100 μL 15%TCA终止反应;以孵育前加入TCA的反应体系作为对照。取20 μL反应液加入80 μL水和100 μL显色液,37℃显色20 min,酶标仪检测酶活性。

磷含量测定：称取上述成熟籽粒粉末50 mg加入1 mL 0.4 mol·L^-1 HCl和15% TCA的混合液;室温置于摇床振荡3 h;5 000 r/min,离心15 min;取200 μL上清加入100 μL H₂O,混匀,4 000 r/min,离心10 min;取50 μL上清加入96孔板中;每孔加入100 μL显色液,37℃反应30 min后读数。

植酸磷测定：取50 μL上述磷含量测定时的提取液上清,加入550 μL 36.3 mmol·L^-1 NaOH和200 μL显色液,混匀,5 000 r/min,离心10 min;取200 μL混合液加入96孔板中进行读数。

1.6 维生素E含量测定以及其他营养成分分析

参考KONDA等^[33]方法测定维生素E含量,在中国农业科学院作物科学研究所重大工程中心完成。称取上述成熟籽粒粉末50 mg加入1 mL维生素E提取液（0.01% BHT,甲醇﹕二氯甲烷=9﹕1（v/v）,标准品1.5 ng·μL^-1）,混匀静置20 min,10 000 r/min,离心10 min,上清经0.22 μm滤器（津腾,LQ0002）过滤,HPLC上样检测。标准品为Rac-5,7-dimethyltocol（Abcam,ab143879）,分离柱为安捷伦XDB-C18柱（258×4.6×5）,用荧光检测器（Ex 292 nm,Em 330 nm）进行测定。

根据饲料中所需检测的主要营养成分^[34],检测转基因玉米材料籽粒中钙、18种氨基酸、干物质、粗脂肪以及粗蛋白的含量,委托中国农业大学农业农村部饲料效价与安全监督检验测试中心完成。

1.7 农艺性状分析

对成熟期的玉米进行农艺性状分析,各取40个单株,对其株高、穗位,雌穗的穗行数、行粒数、穗长以及轴粗等数据进行统计分析。

2 结果

2.1 转基因植株的获得

为了获得籽粒中α-生育酚和植酸酶含量提高的材料,分别选用胚乳特异表达的启动子和胚特异表达的启动子驱动phyA2和ZmTMT的表达,与载体自带的组成型启动子驱动的Bar表达盒串联,如图1所示,采用农杆菌侵染的方法转化玉米,经过多个世代回交转育,获得了目标性状均较好的2个转基因纯合玉米株系TPB1和TPB2,对其进行比较深入的研究。

图1

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图1载体构建示意图

phyA2表达盒由胚乳特异表达的启动子123387驱动;ZmTMT表达盒由胚特异表达的启动子13387驱动;Bar表达盒由组成型启动子CaMV 35S驱动
Fig. 1Schematic diagram of vector construction

phyA2 cassette is driven by endosperm-specific promoter 123387; ZmTMT cassette is driven by embryo-specific promoter 13387; Bar cassette is driven by the constitutive promoter CaMV 35S

2.2 phyA2、ZmTMT和Bar的转录水平分析

为了明确转基因株系籽粒中phyA2、ZmTMT和Bar的RNA水平表达,提取授粉后15 d籽粒的RNA进行RT-PCR分析（图2）。phyA2、ZmTMT和Bar在转基因株系TPB1和TPB2的籽粒中均有高表达,但由于phyA2和Bar均是玉米外源基因,二者在WT（郑58）中无表达;而ZmTMT是玉米内源基因,在WT中存在一定水平的表达。

图2

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图2授粉后15 d的籽粒中phyA2、ZmTMT和Bar的RT-PCR分析

WT：野生型郑58;TPB1、TPB2：转基因纯合株系
Fig. 2RT-PCR analysis of phyA2, ZmTMT and Bar genes in maize seeds 15 days after pollination

WT: Wild type Zheng58; TPB1, TPB2: Transgenic homozygous lines

2.3 phyA2、ZmTMT和Bar的翻译水平分析

为了确定转基因株系籽粒中phyA2、ZmTMT和Bar蛋白的表达情况,提取成熟籽粒的总蛋白进行Western blot分析（图3）。phyA2、ZmTMT和Bar蛋白在转基因株系TPB1和TPB2的籽粒中均有表达。ZmTMT蛋白为玉米内源蛋白,不但在转基因株系有表达,在WT中也有表达,但在WT中的表达与在转基因株系中的表达差异并不明显,推测可能的原因是,ZmTMT是维生素E合成途径中的关键酶,ZmTMT蛋白可能部分在发挥功能之后被降解;另外,用于阳性对照的ZmTMT蛋白是原核表达的融合蛋白,大小约38 kD,而ZmTMT蛋白本身含有叶绿体定位信号肽,在植物体内信号肽被去除之后其蛋白大小约为33 kD,这也是ZmTMT的蛋白杂交条带与阳性对照有差异的原因（图3-A）。Bar和phyA2蛋白均为外源蛋白,因此,2个蛋白只在转基因株系TPB1和TPB2中有表达,而在WT中无表达（图3-B和图3-C）。

图3

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图3成熟籽粒中phyA2、ZmTMT和Bar蛋白的Western blot分析

A：ZmTMT蛋白的表达分析;B：Bar蛋白的表达分析;C：phyA2蛋白的表达分析。WT：野生型郑58;TPB1、TPB2：转基因纯合株系;Marker：蛋白分子量标准;ZmTMT CK：原核表达的融合ZmTMT蛋白阳性对照;Bar CK：Bar蛋白阳性对照;phyA2 CK：已报道的转基因玉米表达的phyA2蛋白阳性对照^[19]
Fig. 3Western blot analysis of phyA2, ZmTMT and Bar in mature maize seeds

A, B, and C show the protein accumulation of ZmTMT, Bar and phyA2, respectively; WT: Wild type Zheng58; TPB1, TPB2: Transgenic homozygous lines; ZmTMT CK: Positive control of ZmTMT; Bar CK: Positive control of Bar; phyA2 CK: Positive control of phyA2

2.4 植酸酶活性以及植酸磷和磷含量测定

植酸酶phyA2蛋白已在转基因玉米成熟籽粒得到高表达,接下来对其酶活性进行了测定（图4）。转基因株系TPB1和TPB2中phyA2的酶活性均超过10 000 U·kg^-1,TPB1中phyA2的酶活性是10 884 U·kg^-1,TPB2中phyA2的酶活性达到12 864 U·kg^-1。已有报道饲料中对植酸酶的需求量是750—1 000 U·kg^{-1 [35]},TPB1和TPB2中植酸酶活性已到达需求量的10倍以上,完全能够满足饲料的需要。

图4

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图4籽粒中植酸酶活性分析

WT：野生型郑58;TPB1、TPB2：转基因纯合株系
Fig. 4Analysis of phytase activity in maize seeds

WT: Wild type Zheng58; TPB1, TPB2: Transgenic homozygous lines


植酸酶能够分解植酸磷生成肌醇衍生物和无机磷,进一步测定了植酸磷和磷的含量,结果显示,转基因株系TPB1和TPB2中植酸磷和磷的含量与WT相比并无差异（表1）,这是由于植酸磷主要存在于胚中,而TPB1和TPB2中的植酸酶主要在胚乳中特异高表达,酶和底物存在部位的不同导致植酸酶不能直接分解植酸磷。而植酸酶在胚中特异高表达时则可以有效分解植酸磷^[19]。

Table 1
表1
表1籽粒中植酸磷和磷含量分析
Table 1Analysis of the contents in phytic acid and phosphorus of maize seeds

株系 Lines	植酸磷 Phytic acid (mg·g-1)	磷 Phosphorus (mg·g-1)
WT	2.77±0.41	0.22±0.02
TPB1	2.60±0.22	0.22±0.06
TPB2	2.65±0.19	0.20±0.02

WT：野生型郑58;TPB1、TPB2：转基因纯合株系
WT: Wild type Zheng58; TPB1, TPB2: Transgenic homozygous lines

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2.5 维生素E含量以及其他营养成分分析

γ-生育酚转化为高活性的α-生育酚,为了确定ZmTMT蛋白的高表达对维生素E组分的影响,采用内标法,利用HPLC检测了转基因株系TPB1和TPB2中维生素E的含量（图5）。转基因株系TPB1和TPB2中,α-生育酚的含量显著提高,TPB1中达到51.58 mg·kg^-1,TPB2中达到66.18 mg·kg^-1,分别是WT中α-生育酚含量的4.9倍和6.3倍;同时,γ-生育酚的水平则均显著下降（P<0.01）,表明ZmTMT充分发挥了其功能,将90%以上的γ-生育酚转化为了α-生育酚。另外,ZmTMT蛋白的高表达,也将部分γ-三烯生育酚转化为了α-三烯生育酚（图5-B）。

图5

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图5籽粒中维生素E含量分析

A：维生素E测定色谱图。α-、γ-、δ-T/T3表示α-、γ-、δ-生育酚/生育三烯酚;IS：标准品;Rac-5,7-dimethyltocol;B：籽粒中维生素E含量分析。WT：野生型郑58;TPB1、TPB2：转基因纯合株系
Fig. 5Analysis of vitamin E content in maize seeds

A: Chromatogram of vitamin E determination. α-, γ-, δ-T/T3: α-, γ-, δ-tocopherol/tocotrienol, IS Internal standard: Rac-5,7-dimethyltocol; B: Analysis of Vitamin E content in maize seeds. WT: Wild type Zheng58; TPB1, TPB2: Transgenic homozygous lines


为了确定转基因玉米中α-生育酚和植酸酶的积累是否影响玉米籽粒其他营养成分的含量,对转基因株系籽粒中的营养成分进行了测定（表2）。结果显示,TPB1的营养成分总体与WT无显著差异,TPB2总体稍高于WT,推测原因为：不同转基因株系之间由于插入位点的不同可能会导致性状之间有一定的差异;另外,转基因材料最初受体是玉米Hi-II,之后转育到郑58中,转基因株系中存留了少量的Hi-II遗传信息,这也可能是导致差异的原因之一;除此之外,并未发现任何不利影响。

Table 2
表2
表2籽粒中其他营养成分分析
Table 2Analysis of other nutrient components in maize seeds (%)

营养成分Nutrient component	WT	TPB1	TPB2
天门冬氨酸 Aspartic acid	0.55±0.001	0.54±0.004	0.58±0.005
苏氨酸 Threonine	0.33±0.001	0.32±0.001	0.36±0.004
丝氨酸Serine	0.41±0.003	0.40±0.002	0.46±0.003*
谷氨酸 Glutamic acid	1.46±0.008	1.42±0.006	1.72±0.001**
脯氨酸 Proline	0.88±0.009	0.8±0.007*	0.97±0.015**
甘氨酸 Glycine	0.32±0.005	0.31±0.002	0.33±0.002
丙氨酸 Alanine	0.59±0.001	0.57±0.002	0.68±0.005**
胱氨酸 Cystine	0.18±0.001	0.19±0.003	0.21±0.007
缬氨酸 Valine	0.42±0.010	0.41±0.002	0.47±0.002*
蛋氨酸 Methionine	0.31±0001	0.44±0.005**	0.38±0.005*
异亮氨酸 Isoleucine	0.28±0.001	0.27±0.001	0.32±0.001*
亮氨酸 Leucine	1.02±0.075	0.99±0.004	1.22±0.007**
酪氨酸 Tyrosine	0.30±0.001	0.24±0.008*	0.25±0.005*
苯丙氨酸 Phenylalanine	0.43±0.003	0.40±0.010	0.47±0.010*
组氨酸 Histidine	0.23±0.001	0.23±0.007	0.26±0.008*
赖氨酸 Lysine	0.26±0.001	0.26±0.005	0.28±0.008
精氨酸 Arginine	0.37±0.002	0.36±0.010	0.38±0.002
色氨酸 Tryptophan	0.07±0.001	0.07±0.001	0.07±0.001
钙 Calcium	0.03±0.001	0.03±0.001	0.03±0.001
粗蛋白 Crude protein	8.47±0.063	8.35±0.008	9.89±0.050**
粗脂肪 Crude fat	3.10±0.015	2.50±0.001*	2.80±0.010
干物质 Dry matter	91.10±1.330	91.80±1.410	90.80±1.270

WT：野生型郑58;TPB1、TPB2：转基因纯合株系;结果以平均值±标准差（SD）表示;*表示P≤0.05,**表示P≤0.001
WT: Wild type Zheng 58; TPB1, TPB2: Transgenic homozygous lines; Results are expressed as mean ± standard deviation (SD); *P≤0.05, **P≤0.001

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2.6 农艺性状分析

为了检测转基因玉米中α-生育酚和植酸酶的积累是否影响玉米本身的农艺性状,对转基因株系TPB1和TPB2的株高、穗位,雌穗的穗长、穗行数、行粒数和轴粗等进行了统计分析。结果显示,转基因植株的株高、穗位以及雌穗的性状与WT相比均无显著差异（图6）,表明转基因玉米籽粒中维生素E组分的改变以及植酸酶含量的提高并未对其农艺性状产生影响。

图6

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图6农艺性状分析

WT：野生型郑58;TPB1、TPB2：转基因纯合株系
Fig. 6Analysis of agronomic traits

WT: Wild type Zheng58; TPB1, TPB2: Transgenic homozygous lines

3 讨论

玉米是最重要的畜禽饲料原料之一,约占饲料成分的二分之一。玉米中低植酸酶活性和维生素E含量使得饲料中必须外源添加大量微生物来源的植酸酶和合成的维生素E。中国饲料工业年鉴2006/2007数据显示,中国饲料工业一年需要合成的维生素E达17 500 t,按市场价150元/kg计算,则需要花费26亿元;而且近些年的数据还在不断增加^[27]。而目前,国际市场上植酸酶占饲料酶市场总量的40%,高达3×10⁵ t以上,对于标准化为5 000 FTU·g^-1的产品（工业植酸酶制剂的浓度范围通常为5 000—100 000 FTU·g^-1）,市场价格7—35元/kg不等,花费在21亿—25亿元,大大增加了饲料的成本^[36]。转phyA2玉米与微生物来源的植酸酶分别同时添加到饲料中饲喂肉鸡发现,玉米表达的phyA2对肉鸡的营养作用与微生物来源的植酸酶在减少饲料无机磷添加和动物粪便中磷酸盐的排泄方面相似^[20],表明表达phyA2的玉米可以直接应用于饲料生产。转GmTMT2a玉米与合成的DL-α-生育酚醋酸酯分别同时添加到饲料中饲喂肉鸡发现,尽管玉米表达的维生素E与合成的维生素E对肉鸡生长性能无显著差异,但富含维生素E的转基因玉米能够提高肉仔鸡血清中α-生育酚含量,改善血清和肝脏的抗氧化能力^[37],表明α-生育酚含量高的玉米不但可以用于饲料,而且要优于合成的维生素E。本研究将phyA2、ZmTMT和Bar表达盒串联转化玉米,获得了具有植酸酶活性>10 000 U·kg^-1、α-生育酚含量达50—70 mg·kg^-1以及草铵膦抗性的性状聚合的玉米新材料,该玉米用于饲料,则饲料中不再需要外源添加维生素E和植酸酶就完全能够满足动物生长发育的需求,可以降低饲料成本,增加生产性能,减少磷污染。另外,此转基因玉米在同时表达3个基因、聚合3种性状时,其植株农艺性状以及籽粒的营养成分（除目标性状外）等与WT相比无显著差异;表明获得的转基因材料既保持了该品种自身的所有性状,同时又增加了除草剂抗性、改良了品质性状。这也为多性状聚合育种提供了参考。

本研究中,转基因玉米中不仅α-生育酚含量升高,α-三烯生育酚的含量也得到了提高。α-三烯生育酚的生物活性约相当于合成的DL-α-生育酚醋酸酯生物活性的45%^[38],即本研究获得的转基因材料中活性维生素E成分有额外的改善。另外,多项研究表明,三烯生育酚也同样具有清除自由基、抗氧化等作用。CHE等^[39]通过同时表达HGGT和PSY,使得高粱中生育酚和三烯生育酚含量以及β-胡萝卜素含量都得到了提高,并且大量积累的维生素E对β-胡萝卜素储存过程中的稳定性起到了非常关键的作用。KONDA等^[33]同时表达HGGT和γ-TMT,使得大豆中维生素E含量提高;此转基因大豆与高SDA的大豆材料杂交后,改善了PUFA的氧化稳定性。在医疗方面,三烯生育酚还具有降低胆固醇、抑制癌细胞生长等重要作用^[40,41]。因此,从生物强化的角度来看,维生素E仍将是未来具有潜力的研究方向,通过同时转化多个基因的手段,不但能够获得多性状聚合的植物材料,而且还可以具有营养丰富、耐储存等优良性状。

4 结论

同时表达生育酚合成相关基因ZmTMT、植酸酶基因phyA2和草铵膦抗性基因Bar,获得了富含α-生育酚和植酸酶、且具有除草剂抗性的转基因玉米,可应用于饲料生产。

参考文献 View Option 原文顺序
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[12] CHENG K, NIU Y, ZHENG X C, ZHANG H, CHEN Y P, ZHANG M, HUANG X X, ZHANG L L, ZHOU Y M, WANG T. A comparison of natural (d-alpha-tocopherol) and synthetic (dl-alpha-tocopherol acetate) vitamin e supplementation on the growth performance, meat quality and oxidative status of broilers
Asian-Australasian Journal of Animal Sciences, 2016,29(5):681-688.
DOI:10.5713/ajas.15.0819URLPMID:26954216 [本文引用: 1]
The present study was conducted to compare the supplementation of natural (D-alpha-tocopherol) and synthetic (DL-alpha-tocopherol acetate) vitamin E on the growth performance, meat quality, muscular antioxidant capacity and genes expression related to oxidative status of broilers. A total of 144 1 day-old Arbor Acres broiler chicks were randomly allocated into 3 groups with 6 replicates of 8 birds each. Birds were given a basal diet (control group), and basal diet supplemented with either 20 IU D-alpha-tocopherol or DL-alpha-tocopherol acetate for 42 days, respectively. The results indicated that treatments did not alter growth performance of broilers (p>0.05). Compared with the control group, concentration of alpha-tocopherol in the breast muscle was increased by the supplementation of vitamin E (p<0.05). In the thigh, alpha-tocopherol content was also enhanced by vitamin E inclusion, and this effect was more pronounced in the natural vitamin E group (p<0.05). Vitamin E supplementation increased the redness of breast (p<0.05). In the contrast, the inclusion of synthetic vitamin E decreased lightness of thigh (p<0.05). Dietary vitamin E inclusion reduced drip loss at 24 h of thigh muscle (p<0.05), and this effect was maintained for drip loss at 48 h in the natural vitamin E group (p<0.05). Broilers given diet supplemented with vitamin E showed decreased malondialdehyde (MDA) content in the breast (p<0.05). Additionally, natural rather than synthetic vitamin E reduced MDA accumulation in the thigh (p<0.05). Neither natural nor synthetic vitamin E supplementation altered muscular mRNA abundance of genes related to oxidative stress (p>0.05). It was concluded that vitamin E supplementation, especially the natural vitamin E, can enhance the retention of muscular alpha-tocopherol, improve meat quality and muscular antioxidant capacity of broilers.

[13] MARUSICH W L, ACKERMAN G, BAUERNFEIND J C. Biological efficacy of D-and DL-α-tocopheryl acetate in chickens
Poultry Science, 1967,46(3):541-548.
DOI:10.3382/ps.0460541URL [本文引用: 1]

[14] YAO B, ZHANG C Y, WANG J H, FAN Y. Recombinant Pichia pastoris overexpressing bioactive phytase
Life Sciences, 1998,41:330-336.
DOI:10.1007/BF02895110URLPMID:18425641 [本文引用: 2]
Phytase genephyA2, whose signal peptide encoding sequence and intron sequence had been removed, was modified. The Arg-encoding codons CGG and CAG inphyA2 were mutated into synonymous codon AGA. The modifiedphyA2 was fused behind a-factor signal sequence under the control ofAOX1 promoter in plasmid pPIC9, then introduced into the hostPichia pastoris by electroporation. The results of Southern blotting analysis and Northem blotting analysis demonstrated that thephyA2 gene had integrated into the genome ofP. pastoris and transcribed. The result of SDS-PAGE of the phytase expressed by P.pastoris showed that the modifiedphyA2 had been overexpressed and secreted. The concentration of the phytase expressed by P.pastoris with modifiedphyA2 exceeded 15 000 U/mL, which had a 3 000-fold increase over that of originAspergillus niger 963 and was 37 times higher than that of recombinantP. pastoris with non-modifiedphyA2.

[15] THEO C V, PETER A P, ALBERT J J, JAN W M, ANDRé H, PEN J. Stable accumulation of Aspergillus niger phytase in transgenic tobacco leaves
Plant Physiology, 1995,109:1199-1205.
DOI:10.1104/pp.109.4.1199URLPMID:8539288 [本文引用: 2]
Phytase from Aspergillus niger increases the availability of phosphorus from feed for monogastric animals by releasing phosphate from the substrate phytic acid. A phytase cDNA was constitutively expressed in transgenic tobacco (Nicotiana tabacum) plants. Secretion of the protein to the extracellular fluid was established by use of the signal sequence from the tobacco pathogen-related protein S. The specific phytase activity in isolated extracellular fluid was found to be approximately 90-fold higher than in total leaf extract, showing that the enzyme was secreted. This was confirmed by use of immunolocalization. Despite differences in glycosylation, specific activities of tobacco and Aspergillus phytase were identical. Phytase was found to be biologically active and to accumulate in leaves up to 14.4% of total soluble protein during plant maturation. Comparison of phytase accumulation and relative mRNA levels showed that phytase stably accumulated in transgenic leaves during plant growth.

[16] HENRIK B P, LISBETH D S, HOLM P B. Engineering crop plants: Getting a handle on phosphate
Trends in Plant Science, 2002,7(3):118-125.
DOI:10.1016/s1360-1385(01)02222-1URLPMID:11906835 [本文引用: 2]
In plant seeds, most of the phosphate is in the form of phytic acid. Phytic acid is largely indigestible by monogastric animals and is the single most important factor hindering the uptake of a range of minerals. Engineering crop plants to produce a heterologous phytase improves phosphate bioavailability and reduces phytic acid excretion. This reduces the phosphate load on agricultural ecosystems and thereby alleviates eutrophication of the aquatic environment. Improved phosphate availability also reduces the need to add inorganic phosphate, a non-renewable resource. Iron and zinc uptake might be improved, which is significant for human nutrition in developing countries.

[17] ALAN E R, PAUL A H, HAYES J E. Extracellular secretion of Aspergillus phytase from Arabidopsis roots enables plants to obtain phosphorus from phytate
The Plant Journal, 2001,25(6):641-649.
DOI:10.1046/j.1365-313x.2001.00998.xURLPMID:11319031 [本文引用: 2]
Phosphorus (P) deficiency in soil is a major constraint for agricultural production worldwide. Despite this, most soils contain significant amounts of total soil P that occurs in inorganic and organic fractions and accumulates with phosphorus fertilization. A major component of soil organic phosphorus occurs as phytate. We show that when grown in agar under sterile conditions, Arabidopsis thaliana plants are able to obtain phosphorus from a range of organic phosphorus substrates that would be expected to occur in soil, but have only limited ability to obtain phosphorus directly from phytate. In wild-type plants, phytase constituted less than 0.8% of the total acid phosphomonoesterase activity of root extracts and was not detectable as an extracellular enzyme. By comparison, the growth and phosphorus nutrition of Arabidopsis plants supplied with phytate was improved significantly when the phytase gene (phyA) from Aspergillus niger was introduced. The Aspergillus phytase was only effective when secreted as an extracellular enzyme by inclusion of the signal peptide sequence from the carrot extensin (ex) gene. A 20-fold increase in total root phytase activity in transgenic lines expressing ex::phyA resulted in improved phosphorus nutrition, such that the growth and phosphorus content of the plants was equivalent to control plants supplied with inorganic phosphate. These results show that extracellular phytase activity of plant roots is a significant factor in the utilization of phosphorus from phytate and indicate that opportunity exists for using gene technology to improve the ability of plants to utilize accumulated forms of soil organic phosphorus.

[18] 范云六. 植酸酶的分子生物学与基因工程
生物工程学报, 2000,16(1):1-5.
URLPMID:10883265 [本文引用: 3]
Phytase is a new-style enzyme used in monogastric animal feed. The addition of phytase to feed can increase phosphorus availability, decrease environmental phosphorus pollution and improve the performance of animal. This paper reviews the research progress and trend in recent studies related to molecular biology and gene engineering of phytase.
FAN Y L. Molecular biology and genetic engineering of phytase
Chinese Journal of Biotechnology, 2000,16(1):1-5. (in Chinese)
URLPMID:10883265 [本文引用: 3]
Phytase is a new-style enzyme used in monogastric animal feed. The addition of phytase to feed can increase phosphorus availability, decrease environmental phosphorus pollution and improve the performance of animal. This paper reviews the research progress and trend in recent studies related to molecular biology and gene engineering of phytase.

[19] CHEN R, XUE G, CHEN P, YAO B, YANG W, MA Q, FAN Y, ZHAO Z, TARCZYNSKI M C, SHI J. Transgenic maize plants expressing a fungal phytase gene
Transgenic Research, 2008,17(4):633-643.
DOI:10.1007/s11248-007-9138-3URL [本文引用: 7]
Maize seeds are the major ingredient of commercial pig and poultry feed. Phosphorus in maize seeds exists predominately in the form of phytate. Phytate phosphorus is not available to monogastric animals and phosphate supplementation is required for optimal animal growth. Undigested phytate in animal manure is considered a major source of phosphorus pollution to the environment from agricultural production. Microbial phytase produced by fermentation as a feed additive is widely used to manage the nutritional and environmental problems caused by phytate, but the approach is associated with production costs for the enzyme and requirement of special cares in feed processing and diet formulation. An alternative approach would be to produce plant seeds that contain high phytase activities. We have over-expressed Aspergillus niger phyA2 gene in maize seeds using a construct driven by the maize embryo-specific globulin-1 promoter. Low-copy-number transgenic lines with simple integration patterns were identified. Western-blot analysis showed that the maize-expressed phytase protein was smaller than that expressed in yeast, apparently due to different glycosylation. Phytase activity in transgenic maize seeds reached approximately 2,200units per kg seed, about a 50-fold increase compared to non-transgenic maize seeds. The phytase expression was stable across four generations. The transgenic seeds germinated normally. Our results show that the phytase expression lines can be used for development of new maize hybrids to improve phosphorus availability and reduce the impact of animal production on the environment.

[20] CHEN R, ZHANG C, YAO B, XUE G, YANG W, ZHOU X, ZHANG J, SUN C, CHEN P, FAN Y. Corn seeds as bioreactors for the production of phytase in the feed industry
Journal of Biotechnology, 2013,165(2):120-126.
DOI:10.1016/j.jbiotec.2013.01.030URL [本文引用: 4]
Corn seed is a major ingredient of animal feed worldwide. However, it contains phytate, a major phosphate storage form that is unavailable to monogastric animals like pigs and poultry. We report a transgenic corn with bioavailable phosphate, achieved by seed-specific overexpression of Aspergillus niger phytase, an enzyme catalyzing the release of phosphate from phytate. We obtained maximal phytase activity of 125 FTU/g kernels, 1000-fold above that of the wild type, with 1000g of kernels containing up to 67 times the feed industry requirement. Enzymatic characterization of Zea mays recombinant phytase (ZmrPhy) showed it to be equivalent to yeast (Pichia pastoris) recombinant phytase (PprPhy), a commercially available phytase product. An animal feeding trial demonstrated that ZmrPhy had similar nutritional effects on broiler chickens to PprPhy in terms of reducing inorganic phosphorus addition to feed and phosphate excretion in animal manure. These results suggest that transgenic phytase corn can be used directly in the feed industry. Experiments were conducted to assess the food safety of the corn; the results demonstrated no difference versus regular corn. This is the first genetically modified corn officially issued with a biosafety certificate in China and has great potential in the animal feed industry. (C) 2013 Elsevier B.V.

[21] 曹茸. 我国首度批准发放转基因粮食作物安全证书
农业知识, 2010,6:49-50.
[本文引用: 1]

CAO R. China's first approval to issue safety certificate of genetically modified food crops
Agricultural Knowledge, 2010,6:49-50. (in Chinese)
[本文引用: 1]

[22] SHINTANI D, DELLAPENNA D. Elevating the vitamin E content of plants through metabolic engineering
Science, 1998,282:2098-2100.
DOI:10.1126/science.282.5396.2098URLPMID:9851934 [本文引用: 1]
alpha-Tocopherol (vitamin E) is a lipid-soluble antioxidant synthesized only by photosynthetic organisms. alpha-Tocopherol is an essential component of mammalian diets, and intakes in excess of the U.S. recommended daily allowance are correlated with decreased incidence of a number of degenerative human diseases. Plant oils, the main dietary source of tocopherols, typically contain alpha-tocopherol as a minor component and high levels of its biosynthetic precursor, gamma-tocopherol. A genomics-based approach was used to clone the final enzyme in alpha-tocopherol synthesis, gamma-tocopherol methyltransferase. Overexpression of gamma-tocopherol methyltransferase in Arabidopsis seeds shifted oil compositions in favor of alpha-tocopherol. Similar increases in agricultural oil crops would increase vitamin E levels in the average U.S. diet.

[23] ZHANG L, LUO Y, ZHU Y, ZHANG L, ZHANG W, CHEN R, XU M, FAN Y, WANG L. GmTMT2a from soybean elevates the alpha- tocopherol content in corn and Arabidopsis
Transgenic Research, 2013,22(5):1021-1028.
DOI:10.1007/s11248-013-9713-8URL [本文引用: 2]

Tocochromanol, or vitamin E, plays a crucial role in human and animal nutrition and is synthesized only by photosynthetic organisms. γ-Tocopherol methyltransferase (γ-TMT), one of the key enzymes in the tocopherol biosynthetic pathway in plants, converts γ, δ-tocopherols into α-, β-tocopherols. Tocopherol content was investigated in 15 soybean cultivars and GmTMT2 was isolated from five varieties based on tocopherol content. GmTMT2a was expressed in E. coli and the purified protein effectively converted γ-tocopherol into α-tocopherol in vitro. Overexpression of GmTMT2a enhanced α-tocopherol content 4–6-fold in transgenic Arabidopsis, and α-tocopherol content increased 3–4.5-fold in transgenic maize seed, which correlated with the accumulation of GmTMT2a. Transgenic corn that is α-tocopherol-rich may be beneficial for animal health and growth.



[24] CHO E, LEE C, KIM Y S, BAEK S H, REYES B, YUN S. Expression of c-tocopherol methyltransferase transgene improves tocopherol composition in lettuce (Latuca sativa L.)
Molecules and Cells, 2005,19:16-22.
URLPMID:15750335 [本文引用: 1]
A cDNA encoding gamma-tocopherol methyltransferase (gamma-TMT) from Arabidopsis thaliana was overexpressed in lettuce (Latuca sativa L.) to improve the tocopherol composition. Seven lines of lettuce (T0) containing the gamma-TMT transgene were produced by Agrobacterium-mediated transformation. The inheritance and expression of the transgene were confirmed by DNA and RNA gel blot analyses as well as quantification of tocopherols and gamma-TMT activities. The ratio of alpha-/gamma-tocopherol content (TR) varied from 0.6 to 1.2 in non-transformed plants, while the T0 plants had ratios of 0.8 to 320. The ratio ranged from 0.4 to 544 in 41 T1 progenies of the T0 transgenic line gTM3, and the phenotypic segregation indicated monogenic inheritance of the transgene (i.e., 3:1 = dominant:wild-type classes). There was a tight relationship between the TR phenotype and gamma-TMT activity, and enzyme activities were affected by the copy number and transcript levels of the transgene. The TR phenotype was stably expressed in T2 progenies of T1 plants. The results from this study indicated that a stable inheritance and expression of Arabidopsis gamma-TMT transgene in lettuce results in a higher enzyme activity and the conversion of the gamma-tocopherol pool to alpha-tocopherol in transgenic lettuce.

[25] TAVVA V S, KIM Y H, KAGAN I A, DINKINS R D, KIM K H, COLLINS G B. Increased alpha-tocopherol content in soybean seed overexpressing the Perilla frutescens gamma-tocopherol methyltransferase gene
Plant Cell Reports, 2007,26(1):61-70.
DOI:10.1007/s00299-006-0218-2URL [本文引用: 1]
Tocopherols, with antioxidant properties, are synthesized by photosynthetic organisms and play important roles in human and animal nutrition. In soybean, γ-tocopherol, the biosynthetic precursor to α-tocopherol, is the predominant form found in the seed, whereas α-tocopherol is the most bioactive component. This suggests that the final step of the α-tocopherol biosynthetic pathway catalyzed by γ-tocopherol methyltransferase (γ-TMT) is limiting in soybean seed. Soybean oil is the major edible vegetable oil consumed, so manipulating the tocopherol biosynthetic pathway in soybean seed to convert tocopherols into more active α-tocopherol form could have significant health benefits. In order to increase the soybean seed α-tocopherol content, the γ-TMT gene isolated from Perilla frutescens was overexpressed in soybean using a seed-specific promoter. One transgenic plant was recovered and the progeny was analyzed for two generations. Our results demonstrated that the seed-specific expression of the P. frutescens γ-TMT gene resulted in a 10.4-fold increase in the α-tocopherol content and a 14.9-fold increase in the β-tocopherol content in T2 seed. Given the relative contributions of different tocopherols to vitamin E activity, the activity in T2 seed was calculated to be 4.8-fold higher than in wild-type seed. In addition, the data obtained on lipid peroxidation indicates that α-tocopherol may have a role in preventing oxidative damage to lipid components during seed storage and seed germination. The increase in the α-tocopherol content in the soybean seed could have a potential to significantly increase the dietary intake of vitamin E.

[26] LEE B K, KIM S L, KIM K H, YU S H, LEE S C, ZHANG Z, KIM M S, PARK H M, LEE J Y. Seed specific expression of perilla γ-tocopherol methyltransferase gene increases α-tocopherol content in transgenic perilla (Perilla frutescens)
Plant Cell, Tissue and Organ Culture, 2007,92(1):47-54.
[本文引用: 1]

[27] ZHANG L, LUO Y, LIU B, ZHANG L, ZHANG W, CHEN R, WANG L. Overexpression of the maize gamma-tocopherol methyltransferase gene (ZmTMT) increases alpha-tocopherol content in transgenic Arabidopsis and maize seeds
Transgenic Research, 2020,29(1):95-104.
DOI:10.1007/s11248-019-00180-zURLPMID:31673914 [本文引用: 5]
The vitamin E family includes tocopherols and tocotrienols, which are essential lipid-soluble antioxidants necessary for human and livestock health. The seeds of many plant species, including maize, have high gamma (gamma)-tocopherol but low alpha (alpha)-tocopherol contents; however, alpha-tocopherol is the most effective antioxidant. Therefore, it is necessary to optimize the tocopherol composition in plants. alpha-Tocopherol is synthesized from gamma-tocopherol by gamma-tocopherol methyltransferase (gamma-TMT, VTE4) in the final step of the tocopherol biosynthetic pathway. In the present study, the full-length coding sequence (CDS) of gamma-TMT was isolated from Zea mays, named ZmTMT. The ZmTMT CDS was 1059 bp in size, encoding 352 amino acids. Recombinant ZmTMT was expressed in Escherichia coli and the purified protein effectively converted gamma-tocopherol into alpha-tocopherol in vitro. A comparison of enzyme activities showed that the activity of ZmTMT was higher than that of GmTMT2a (Glycine max) and AtTMT (Arabidopsis thaliana). Overexpression of ZmTMT increased the alpha-tocopherol content 4-5-fold in transgenic Arabidopsis and around 6.5-fold in transgenic maize kernels, and increased the alpha-/gamma-tocopherol ratio to approximately 15 and 17, respectively. These results show that it is feasible to overexpress ZmTMT to optimize the tocopherol composition in maize; such a corn product might be useful in the feed industry in the near future.

[28] LIU X, TIAN J, ZHOU X, CHEN R, WANG L, ZHANG C, ZHAO J, FAN Y. Identification and characterization of promoters specifically and strongly expressed in maize embryos
Plant Biotechnology Journal, 2014,12(9):1286-1296.
DOI:10.1111/pbi.12227URL [本文引用: 1]
The use of maize seeds as bioreactors has several advantages for the production of recombinant proteins in plant biotechnology, but available embryo-specific and strong promoters are limited. Here, we describe a genome-scale microarray-based approach to identify embryo-specifically and strongly expressed genes and their promoters in maize. We identified 28 embryo-preferred and abundantly expressed genes based on our microarray data. These embryo-preferred genes were further analysed using the UniGene database and by quantitative reverse transcriptase-PCR leading to the identification of seven genes (Zm.2098, Zm.13387, Zm.66589, Zm.85502, Zm.68129, Zm.3896 and Zm.2941) as embryo-specific genes with higher expression levels relative to maize globulin-1. The putative promoters of five embryo-specific genes (Zm.13387, Zm.66589, Zm.85502, Zm.3896 and Zm.2941) were isolated and all exhibited strong promoter activities when transiently expressed in maize embryos of 20 DAP. The embryo specificity and expression levels of the promoters of four genes (Zm.13387, Zm.85502, Zm.3896 and Zm.2941) were further examined in transgenic maize plants, revealing that they are strong promoters in embryos of all four developmental stages tested compared with reference globulin-1 promoter. Moreover, Zm.2941 and Zm.3896 promoters are stringently embryo-specific promoters, while Zm.85502 promoter is basically embryo specific yet wounding inducible in non-seed tissues, and Zm.13387 promoter is developmentally expressed in both embryo and aleurone with wounding-induced activity in non-seed tissues. Our study provides novel embryo-specific and strong promoters that are suitable for production of high-level recombinant proteins in maize embryos.

[29] ARMSTRONG C L, GREEN C E, PHILLIPS R L. Development and availability of germplasm with High Type-II culture formation response
Maize Genetic Coop News Letter, 1991,65:92-93.
[本文引用: 1]

[30] HYUN A K, SETYO D U, SUK Y K, SUNG R M, JIN S K, HAN S Y, CHOI P S. The development of herbicide-resistant maize: Stable Agrobacterium-mediated transformation of maize using explants of type II embryogenic calli
Plant Biotechnology Reports, 2009,3:277-283.
DOI:10.1007/s11816-009-0099-2URL [本文引用: 1]
One of the limitations to conducting maize Agrobacterium-mediated transformation using explants of immature zygotic embryos routinely is the availability of the explants. To produce immature embryos routinely and continuously requires a well-equipped greenhouse and laborious artificial pollination. To overcome this limitation, an Agrobacterium-mediated transformation system using explants of type II embryogenic calli was developed. Once the type II embryogenic calli are produced, they can be subcultured and/or proliferated conveniently. The objectives of this study were to demonstrate a stable Agrobacterium-mediated transformation of maize using explants of type II embryonic calli and to evaluate the efficiency of the protocol in order to develop herbicide-resistant maize. The type II embryogenic calli were inoculated with Agrobacterium tumefaciens strain C58C1 carrying binary vector pTF102, and then were subsequently cultured on the following media: co-cultivation medium for 1day, delay medium for 7days, selection medium for 4×14days, regeneration medium, and finally on germination medium. The T-DNA of the vector carried two cassettes (Ubi promoter-EPSPs ORF-nos and 35S promoter–bar ORF-nos). The EPSPs conferred resistance to glyphosate and bar conferred resistance to phosphinothricin. The confirmation of stable transformation and the efficiency of transformation was based on the resistance to phosphinothricin indicated by the growth of putative transgenic calli on selection medium amended with 4mgl⁻¹ phosphinothricin, northern blot analysis of bar gene, and leaf painting assay for detection of bar gene-based herbicide resistance. Northern blot analysis and leaf painting assay confirmed the expression of bar transgenes in the R₁ generation. The average transformation efficiency was 0.60%. Based on northern blot analysis and leaf painting assay, line 31 was selected as an elite line of maize resistant to herbicide.

[31] LI Y, LIU X, LI J, LI S, CHEN G, ZHOU X, YANG W, CHEN R. Isolation of a maize ZmCI-1B promoter and characterization of its activity in transgenic maize and tobacco
Plant Cell Reports, 2015,34(8):1443-1457.
DOI:10.1007/s00299-015-1799-4URLPMID:25941157 [本文引用: 1]
KEY MESSAGE: The 2-kb ZmCI - 1B promoter is active in the root and embryo and induced by wounding in maize and the 220-bp 5'-deleted segment maybe the minimal promoter. The subtilisin-chymotrypsin inhibitor gene, CI-1B of Zea mays (ZmCI-1B), has been suggested to induce the maize defense system to resist insect attack. Real-time RT-PCR showed that ZmCI-1B gene exhibited especially high expression in roots and embryos. The 2-kb full-length promoter of ZmCI-1B gene was isolated from the maize genome and used to drive expression of a beta-glucuronidase (GUS) reporter gene for transient expression and stable expression analysis in maize. The results of GUS histochemical staining in transgenic maize plants revealed that the ZmCI-1B promoter induced GUS expression preferentially in roots and embryos and in response to wounding. A series of 5'-deleted segments of the ZmCI-1B promoter were cloned individually to drive GUS expression for further analysis. Deletion analysis combined with the histochemical staining of transgenic tobacco plants revealed 220-bp segment could drive GUS in a tissue-specific and wounding-induced expression in tobacco; thus, it maybe the minimally active promoter of ZmCI-1B gene. Furthermore, it revealed that the ZmCI-1B promoter contained tissue-specific and wounding-induced elements.

[32] MARKUS W, LUIS P, ARNO F, ROLAND R, MICHEL T, ALEXANDRA K, ANKE M, MARTIN L, LINE S, R?THLISBERGER U. Biochemical characterization of fungal phytases (myo-Inositol Hexakisphosphate Phosphohydrolases): catalytic properties
Applied and Environmental Microbiology, 1999,65(2):359-366.
DOI:10.1128/AEM.65.2.359-366.1999URLPMID:9925554 [本文引用: 1]
Phytases (myo-inositol hexakisphosphate phosphohydrolases) are found naturally in plants and microorganisms, particularly fungi. Interest in these enzymes has been stimulated by the fact that phytase supplements increase the availability of phosphorus in pig and poultry feed and thereby reduce environmental pollution due to excess phosphate excretion in areas where there is intensive livestock production. The wild-type phytases from six different fungi, Aspergillus niger, Aspergillus terreus, Aspergillus fumigatus, Emericella nidulans, Myceliophthora thermophila, and Talaromyces thermophilus, were overexpressed in either filamentous fungi or yeasts and purified, and their biophysical properties were compared with those of a phytase from Escherichia coli. All of the phytases examined are monomeric proteins. While E. coli phytase is a nonglycosylated enzyme, the glycosylation patterns of the fungal phytases proved to be highly variable, differing for individual phytases, for a given phytase produced in different expression systems, and for individual batches of a given phytase produced in a particular expression system. Whereas the extents of glycosylation were moderate when the fungal phytases were expressed in filamentous fungi, they were excessive when the phytases were expressed in yeasts. However, the different extents of glycosylation had no effect on the specific activity, the thermostability, or the refolding properties of individual phytases. When expressed in A. niger, several fungal phytases were susceptible to limited proteolysis by proteases present in the culture supernatant. N-terminal sequencing of the fragments revealed that cleavage invariably occurred at exposed loops on the surface of the molecule. Site-directed mutagenesis of A. fumigatus and E. nidulans phytases at the cleavage sites yielded mutants that were considerably more resistant to proteolytic attack. Therefore, engineering of exposed surface loops may be a strategy for improving phytase stability during feed processing and in the digestive tract.

[33] KONDA A R, NAZARENUS T J, NGUYEN H, YANG J, GELLI M, SWENSON S, SHIPP J M, SCHMIDT M A, CAHOON R E, CIFTCI O N. Metabolic engineering of soybean seeds for enhanced vitamin E tocochromanol content and effects on oil antioxidant properties in polyunsaturated fatty acid-rich germplasm
Metabolic Engineering, 2019,57:63-73.
DOI:10.1016/j.ymben.2019.10.005URLPMID:31654815 [本文引用: 2]
Soybean seeds produce oil enriched in oxidatively unstable polyunsaturated fatty acids (PUFAs) and are also a potential biotechnological platform for synthesis of oils with nutritional omega-3 PUFAs. In this study, we engineered soybeans for seed-specific expression of a barley homogentisate geranylgeranyl transferase (HGGT) transgene alone and with a soybean gamma-tocopherol methyltransferase (gamma-TMT) transgene. Seeds for HGGT-expressing lines had 8- to 10-fold increases in total vitamin E tocochromanols, principally as tocotrienols, with little effect on seed oil or protein concentrations. Tocochromanols were primarily in delta- and gamma-forms, which were shifted largely to alpha- and beta-tocochromanols with gamma-TMT co-expression. We tested whether oxidative stability of conventional or PUFA-enhanced soybean oil could be improved by metabolic engineering for increased vitamin E antioxidants. Selected lines were crossed with a stearidonic acid (SDA, 18:4(Delta6,9,12,15))-producing line, resulting in progeny with oil enriched in SDA and alpha- or gamma-linoleic acid (ALA, 18:3(Delta9,12,15) or GLA, 18:3(Delta6,9,12)), from transgene segregation. Oil extracted from HGGT-expressing lines had >/=6-fold increase in free radical scavenging activity compared to controls. However, the oxidative stability index of oil from vitamin E-enhanced lines was ~15% lower than that of oil from non-engineered seeds and nearly the same or modestly increased in oil from the GLA, ALA and SDA backgrounds relative to controls. These findings show that soybean is an effective platform for producing high levels of free-radical scavenging vitamin E antioxidants, but this trait may have negative effects on oxidative stability of conventional oil or only modest improvement of the oxidative stability of PUFA-enhanced oil.

[34] 王薇薇, 宋歌, 王丽, 周航, 董正林, 刘宽博, 王永伟, 宋丹, 李爱科. 黑龙江粮库库存玉米的营养成分、 脂肪酸值和霉菌毒素含量分析
粮油食品科技, 2019,27(5):69-74.
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WANG W W, SONG G, WANG L, ZHOU H, DONG Z L, LIU K B, WANG Y W, SONG D, LI A K. Analysis of nutritional content, fatty acid value and mycotoxins content of corn in Heilongjiang Grain Depot
Science and Technology of Cereals Oils and Foods, 2019,27(5):69-74. (in Chinese)
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[35] HUMER E, SCHWARZ C, SCHEDLE K. Phytate in pig and poultry nutrition
Journal of Animal Physiology and Animal Nutrition(Berl), 2015,99(4):605-625.
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[36] GUERRAND D. Chapter 26 - Economics of food and feed enzymes: Status and prospectives. In: Enzymes in Human and Animal Nutrition
Edited by Nunes C S, Kumar V: Academic Press; 2018: 487-514.
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[37] 杨欢, 汤超华, 张凯. 富维生素E转基因玉米对0~3周龄肉仔鸡生产性能和抗氧化能力的影响
甘肃农业大学学报, 2016,194(2):28-34.
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YANG H, TANG C H, ZHANG K. Effects of vitamin E-rich transgenic corn on production performance and antioxidant capacity of broilers at 0-3 weeks of age
Journal of Gansu Agricultural University, 2016,194(2):28-34. (in Chinese)
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[38] 李海娜, 高仁喜, 田胜军, 刘艳. 天然维生素,生产技术现状
湿法冶金, 2003,22(3):113-117.
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LI H N, GAO R N, TIAN S J, LIU Y. Natural vitamins, current production technology
Hydrometallurgy of China, 2003,22(3):113-117. (in Chinese)
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[39] CHE P, ZHAO Z Y, GLASSMAN K, DOLDE D, HU T X, JONES T J, GRUIS D F, OBUKOSIA S, WAMBUGU F, ALBERTSEN M C. Elevated vitamin E content improves all-trans β-carotene accumulation and stability in biofortified sorghum
Proceedings of the National Academy of Sciences of the USA, 2016,113(39):11040-11045.
DOI:10.1073/pnas.1605689113URLPMID:27621466 [本文引用: 1]
Micronutrient deficiencies are common in locales where people must rely upon sorghum as their staple diet. Sorghum grain is seriously deficient in provitamin A (beta-carotene) and in the bioavailability of iron and zinc. Biofortification is a process to improve crops for one or more micronutrient deficiencies. We have developed sorghum with increased beta-carotene accumulation that will alleviate vitamin A deficiency among people who rely on sorghum as their dietary staple. However, subsequent beta-carotene instability during storage negatively affects the full utilization of this essential micronutrient. We determined that oxidation is the main factor causing beta-carotene degradation under ambient conditions. We further demonstrated that coexpression of homogentisate geranylgeranyl transferase (HGGT), stacked with carotenoid biosynthesis genes, can mitigate beta-carotene oxidative degradation, resulting in increased beta-carotene accumulation and stability. A kinetic study of beta-carotene degradation showed that the half-life of beta-carotene is extended from less than 4 wk to 10 wk on average with HGGT coexpression.

[40] WANG L, NEWMAN R K, NEWMAN C W, JACKSON L L. Tocotrienol and fatty acid composition of barley oil and their effects on lipid metabolism
Plant Foods for Human Nutrition, 1993,43:9-17.
DOI:10.1007/BF01088091URLPMID:8464850 [本文引用: 1]
Barley oil was extracted with hexane from the grain of a high oil waxy hull-less barley. Twelve male broiler chicks were fed corn-based diets with either 10% barley oil, 10% corn oil or 10% margarine ad libitum for ten days. Total plasma cholesterol concentration of the chicks fed barley oil was 34% lower (p < 0.05) than that of the chicks fed margarine. Plasma low density lipoprotein cholesterol concentration of chicks fed barley oil was 53% and 59% lower (p < 0.05) than those of chicks fed corn oil and margarine, respectively. Plasma high density lipoprotein cholesterol and triglyceride concentration of the barley oil group were similar to those of the margarine but higher (p < 0.05) than those of the corn oil group. Chicks fed the barley oil gained more (p < 0.05) body weight than those fed the corn oil and margarine. Barley oil had an effect in suppression of TC and LDLC in chicks compared to margarine. Barley oil suppressed LDLC but not HDLC in chicks compared to corn oil. A greater weight gain of the chicks fed barley oil suggested that these chicks had normally functioning digestion and absorption. alpha-Tocotrienol and gamma-tocotrienol content of the barley oil were 24 and 17 times greater, respectively, than those observed in the corn oil, while the same fractions were not detectable in the margarine. Polyunsaturated fatty acid content of the barley oil was more than threefold that of margarine. These data suggest that alpha-tocotrienol and polyunsaturated fatty acids are hypocholesterolemic components in barley oil.

[41] PARKER R, PEARCE B, CLARK R W, GORDON D A, WRIGHT J. Tocotrienols regulate cholesterol production in mammalian cells by post-transcriptional suppression of 3-hydroxy-3-methylglutaryl-coenzyme A reductase
The Journal of Biological Chemistry, 1993,268:11230-11238.
URLPMID:8388388 [本文引用: 1]
Tocotrienols are natural farnesylated analogues of tocopherols which decrease hepatic cholesterol production and reduce plasma cholesterol levels in animals. For several cultured cell types, incubation with gamma-tocotrienol inhibited the rate of [14C]acetate but not [3H] mevalonate incorporation into cholesterol in a concentration- and time-dependent manner, with 50% inhibition at approximately 2 microM and maximum approximately 80% inhibition observed within 6 h in HepG2 cells. 3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase total activity and protein levels assayed by Western blot were reduced concomitantly with the decrease in cholesterol synthesis. In HepG2 cells, gamma-tocotrienol suppressed reductase despite strong blockade by inhibitors at several steps in the pathway, suggesting that isoprenoid flux is not required for the regulatory effect. HMG-CoA reductase protein synthesis rate was moderately diminished (57% of control), while the degradation rate was increased 2.4-fold versus control (t1/2 declined from 3.73 to 1.59 h) as judged by [35S]methionine pulse-chase/immunoprecipitation analysis of HepG2 cells treated with 10 microM gamma-tocotrienol. Under these conditions, the decrease in reductase protein levels greatly exceeded the minor decrease in mRNA (23 versus 76% of control, respectively), and the low density lipoprotein receptor protein was augmented. In contrast, 25-hydroxycholesterol strongly cosuppressed HMG-CoA reductase protein and mRNA levels and the low density lipoprotein receptor protein. Thus, tocotrienols influence the mevalonate pathway in mammalian cells by post-transcriptional suppression of HMG-CoA reductase, and appear to specifically modulate the intracellular mechanism for controlled degradation of the reductase protein, an activity that mirrors the actions of the putative non-sterol isoprenoid regulators derived from mevalonate.