菜薹蛋白精氨酸甲基转移酶基因BrcuPRMT5的克隆及表达分析

以‘油青49’和‘油青甜菜薹80’菜薹茎尖为材料,采用RT-PCR和RACE技术克隆获得组蛋白甲基转移酶基因Brcu PRMT5的全长cDNA和gDNA序列。BrcuPRMT5 cDNA序列全长为2 117 bp,其中完整开放阅读框为1 929 bp,编码642个氨基酸,相对分子量为71.55 kD,理论等电点(p I)为5.87;多序列比对结果表明,Brcu PRMT5编码的氨基酸序列含有高等植物PRMT5基因1个高度保守的结构域;系统发育分析结果显示与大白菜、油菜及甘蓝的亲缘关系最近;亚细胞定位软件分析得知,Brcu PRMT5蛋白无跨膜区域,可能定位于线粒体中;对应的gDNA全长为4 151 bp,含有23个外显子和22个内含子,最长的外显子长度为140 bp,内含子的长度范围为50~150 bp。利用半定量RT-PCR和实时荧光定量PCR技术分析基因的表达,BrcuPRMT5在菜薹不同组织中均有表达,其中在花中表达量最高,叶次之,根中最低;BrcuPRMT5从苗期至完全抽薹开花期的表达量呈现上升趋势。BrcuPRMT5在菜薹花发育过程中可能起一定的调控作用。     

天然富含可可碱茶树的研究进展

天然富含可可碱茶树是一类以可可碱为代表性生物碱的稀有茶树种质资源,具有独特的保健功效与挖掘利用价值。从生化成分组成特征、生物代谢途径和可可碱在茶树体内富含的分子机理,对可可茶、红芽茶、秃房茶和厚轴茶4份新近发现的天然富含可可碱茶树进行比较研究与综述。结果发现,可可碱占茶叶干重的质量分数高达2%～6%,咖啡碱则低于0.5%;优势儿茶素单体为没食子酸儿茶素没食子酸酯,而表没食子酸儿茶素没食子酸酯含量较低;咖啡碱合成酶活性位点精氨酸突变为组氨酸使N-甲基转移酶的底物特异性发生变化,从而导致咖啡碱合成酶终止合成咖啡碱并使得可可碱在茶树体内富集。天然富含可可碱茶树资源丰富了茶种的多样性,但开发利用度尚有限,结合当前茶树育种技术的发展状态,对天然富含可可碱茶树种质的繁育与资源挖掘利用进行了展望。     

合成茶树咖啡碱相关的N-甲基转移酶基因家族的克隆及序列分析

咖啡碱是茶叶中重要的功能成分,它以黄苷为底物,以S-腺苷甲硫氨酸（SAM）为甲基供体,通过N-甲基转移酶（NMT）类催化的一系列甲基化反应合成的产物。根据NMT基因高度相似的特性,利用长片段PCR法和侧翼序列克隆技术分离了白叶一号6种NMTs的基因组DNA全长,其中有2种为已报道的茶树咖啡碱合成酶基因TCS1、TCS2,1种为假基因,另3种基因分别被命名为TCS3、TCS4、TCS5,基因结构分析发现这6种基因均由4个外显子和3个内含子组成。山茶属植物的NMTs可聚为5类,其中TCS4和TCS5为与其他基因的相似性相对较低的一类。这些结果为今后更好地从基因组水平上剖析茶树咖啡碱的遗传机制提供有用参考。     

A real-time polymerase chain reaction (PCR) method for the identification of Nicotiana tabacum in tobacco products

A simple and specific real-time PCR assay based on TaqMan^® technology has been developed for the identification of cultured tobacco (Nicotiana tabacum) in various commodities such as cigars, cigarettes and reconstituted tobacco. The TaqMan^® assay targets a sequence of the putrescine N-methyltransferase gene family encoding an enzyme that plays a crucial role in the biosynthesis of nicotine. To reduce the possibility of false negatives, universal plant chloroplast primers were also used in a separate real-time PCR reaction to give indication if DNA is amplifiable in the matrix. The TaqMan^® assay successfully identified tobacco in over 40 commercial tobacco products, while negative results were obtained from the assay for DNA extracted from a variety of other botanical products. In our study, two commercial DNA isolation kits were used, namely, the Qiagen DNeasy^® Plant Mini kit and the Qiagen Gentra^® Puregene^® kit. They produced good quality DNAs in sufficient quantities for real-time PCR analysis. In a few cases, an additional purification step with the Promega DNA IQ™ system had to be implemented to obtain amplifiable DNA.     

Ontogeny of hepatic methionine catabolic enzyme activities (Transmethylation and Transsulphuration) and associated physiological amino acids in E10-21 chick embryos and D1-49 broilers

Developmental changes in hepatic methionine adenosyltransferase, cystathionine β-synthase, cystathionase, and glycine N-methyltransferase were determined in broiler chick embryos and hatched chicks by using radiometric and spectrometric methods. Hepatic free methionine, S-adenosylmethionine, S-adenosylhomocysteine, homocysteine, cystathionine, and cysteine levels were also investigated. Results showed an increase in hepatic MAT activity from E10 to E21 during embryogenesis, suggesting greater transmethylation rates throughout the rapid embryonic growth and development period. A strong positive correlation between embryo BW and MAT activity also supports this idea. The MAT specific activity continued to increase after hatching, but there was a negative correlation between chick BW and MAT activities from D1 to D49. This may indicate different MAT isozymes exist for chick embryo hepatic tissue compared to hepatic tissue of hatched chick and growing broilers. The developmental pattern of MAT isozymes could be critical for methionine metabolism to cope with the demand imposed on the embryo, chicks, and growing broilers. Additionally, the specific activity of hepatic CBS in chick embryos was determined to be lower compared to that observed in older broilers (35 and 49 days). Since liver CBS specific activity is at the lowest point from D1-7 in young chicks, the ability to convert adequate homocysteine to cysteine through transsulphuration may be limiting for cysteine synthesis at this time. Steady-state hepatic homocysteine levels in chick embryos and chicks may be a function of the rates of homocysteine formation, remethylation, and catabolism via the transsulphuration pathway. The present study indicates young chicks from D1 to D7 may have a limited ability for adequate transsulphuration; therefore, dietary cystine may be needed for optimum performance.     

茶树N-甲基转移酶基因启动子克隆及功能分析

咖啡碱是茶叶中重要的功能物质,N-甲基转移酶（NMT）是其生物合成的关键酶。本研究以英红9号茶树新梢为材料,利用hiTAIL-PCR克隆NMT1基因启动子,并采用Plant CARE等在线软件分析其顺式作用元件。根据其元件组成,设计5'递减的启动子与GUS基因一起组建了融合载体转入烟草,利用GUS染色和定量PCR方法,分析了克隆的启动子功能及其对环境因子的响应。结果表明,克隆的NMT1基因启动子长767βbp,含有TATA-box、CAAT-box等真核生物启动子基本元件及多个与植物非生物胁迫相关的响应元件。以5'递减的NMT1启动子替换pBI121中的CaMV35S启动子,构建出与GUS融合的pA、pB、pC、pD载体。在烟草叶片中进行瞬时表达,不同长度的NMT1启动子均具驱动GUS表达功能,且长度越长驱动能力越强。以含全长NMT1启动子载体pA对烟草转基因,转基因烟草各组织均检测到GUS基因的表达且表达量叶>茎>根,叶片中的表达量为根部的3倍。对转基因烟草进行不同程度光照、温度、模拟干旱和脱落酸处理后,除40℃温度条件外,其他处理的叶片中GUS基因表达在处理前后均存在显著变化,表明NMT1启动子功能受外界环境因子的胁迫影响。     

植物中的咖啡碱：从合成途径研究到转基因作物

 在植物细胞中咖啡碱的核心合成途径为：黄嘌呤核苷→7–甲基黄嘌呤核苷→7–甲基黄嘌呤→可可碱→咖啡碱，即3次顺序甲基化反应和1次核糖水解过程。目前已经从茶树等植物中分离出催化上述甲基化反应的酶，并克隆得到编码这些酶的基因。在此基础上，利用RNAi干扰和农杆菌介导法等技术，已成功培育出低咖啡碱饮料作物茶树和咖啡以及能合成咖啡碱的烟草和菊花植株。     

咖啡碱合成N-甲基转移酶研究进展

咖啡碱是茶叶、咖啡等饮料的主要品质成分及功能成分之一。在植物体内咖啡碱生物合成的核心途径为：黄嘌呤核苷→7-甲基黄嘌呤核苷→7-甲基黄嘌呤→可可碱→咖啡碱,其中包括3步由N-甲基转移酶催化的转甲基化反应和1步由核糖核苷水解酶催化的脱核苷反应。N-甲基转移酶是参与咖啡碱生物合成的关键酶类。介绍了植物中咖啡碱的基本情况及其生物合成途径,重点综述了咖啡碱合成N-甲基转移酶的酶学特性、NMTs的克隆、基因结构与功能的关系以及基因表达调控研究等方面国内外的研究进展,并对未来该领域的研究重点进行了探讨和展望。     

颠茄PMT·TR-I和H6H基因植物高效表达载体的构建

[目的]构建1,4-丁二胺-氮-甲基转移酶（Putrescine N-methyltransferase,PMT）基因、托品酮还原酶-I（Tropinone reductase-I,TR-I）基因和莨菪碱6-β-羟化酶（Hyoscyamine 6β-Hydroxylase,H6H）基因的植物高效表达载体。[方法]采用RT-PCR方法从颠茄中克隆到PMT、TR-I和H6H基因编码区序列,并连接到植物表达载体pCAMBIA1304^＋（p1304^＋）中。[结果]成功构建了植物高效表达载体p1304^＋-PMT、p1304^＋-TR-I、p1304^＋-H6H、p1304^＋-PMT-H6H、p1304^＋-TR-I-PMT和p1304^＋-TR-I-H6H,并转化农杆菌,获得可直接用于遗传改良颠茄的工程菌。[结论]该研究为利用植物基因工程技术提高东莨菪碱的产量奠定了基础。