Development of ten microsatellite loci for <Emphasis Type="Italic">Gentiana crassicaulis</Emphasis> (Gentianaceae)

Genetiana crassicaulis is one of famous Chinese medicinal plant. The over-collection for its root has caused its dramatic reduction. In order to devise adequate conservation and management strategies for this species, it is important to characterize its genetic diversity and understand its population structure. Here, 10 polymorphic microsatellite markers have been developed. AC/TG microsatellite was enriched by combining biotin capture method. Polymorphism of each locus was assessed in 30 individuals from six populations. The number of alleles ranged from 2 to 9 and the expected heterozygosity ranged from 0.32 to 0.78.     

滇龙胆GrWRKY5基因的克隆和植物表达载体构建

WRKY蛋白是目前被广泛研究的一类DNA特异结合转录因子,在植物次生代谢物生物合成、植物生长和发育及衰老等生理过程以及生物、非生物防御反应中起重要的调控作用。该研究从滇龙胆幼叶中克隆萜类合成的关键转录因子基因Gr WRKY5,并利用生物信息学方法对基因功能进行预测,构建植物过表达载体。结果表明:根据三年生滇龙胆转录组Gr WRKY5基因序列,设计特异性引物,通过RT-PCR扩增Gr WRKY5 ORF序列,并进行TA克隆、测序和序列分析;构建入门载体p ENTRTM2B-Gr WRKY5,经LR反应后构建植物过表达载体。Gr WRKY5 ORF全长591 bp,编码196个氨基酸,Gen Bank登录号为KF922375,其中第167与170之间的色氨酸(W)、精氨酸(R)、赖氨酸(K)、酪氨酸(Y)组成WRKY蛋白所特有的"WRKY"结构。序列分析表明Gr WRKY5是WRKY超家族的成员。经生物信息学在线软件分析发现,Gr WRKY5的等电点为6.29,脂肪族指数为61.37,不稳定指数为57.80。总平均疏水性为-0.708,为亲水蛋白;含有20种氨基酸,其中天冬氨酸(Asp)和脯氨酸(Pro)含量最高,为8.1%;半胱氨酸(Cys)含量最低,仅为1.0%。氨基酸序列系统发育分析表明,Gr WRKY5与拟南芥中WRKY家族中遗传距离最接近的是WRKY27,属于Ⅱe类成员;与Cr WRKY22和Vv WRKY22蛋白的亲缘关系较近;与Jc WRKY47和Tc WRKY27亲缘关系较远;BLASTp结果显示,Gr WRKY5与欧洲油菜Bn WRKY27-1的同源性最高(为69%);与拟南芥Aa WRKY22的一致性最低(仅为31%)。以Gateway入门载体p ENTR2B和目的载体p K2GW7为基础,成功构建了植物过表达载体p K2-35S-Gr WRKY5,该载体的成功构建为该基因在拟南芥、滇龙胆等植物中的遗传转化奠定了基础,同时为WRKY基因功能的深入研究提供依据。     

秦艽1-羟基-2-甲基-2-(E)-丁烯基-4-焦磷酸还原酶基因(GmHDR)的克隆和表达分析

龙胆苦苷(gentiopicroside)等裂环烯醚萜苷类化合物是中药秦艽中主要的有效成分,属于萜类化合物的衍生物,1-羟基-2-甲基-2-(E)-丁烯基-4-焦磷酸还原酶(1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate reductase,HDR)是植物萜类物质合成的关键酶之一。该研究在实验室高通量测序的基础上,采用RT-PCR方法克隆了秦艽HDR基因(即Gm HDR基因),利用生物信息学的方法分析了Gm HDR编码氨基酸序列的理化性质、信号肽、转运肽、亚细胞定位、保守结构域和高级结构等特征,并采用实时定量PCR分析了HDR的表达模式。结果表明:秦艽Gm HDR基因包含一个完整的长1 392 bp的ORF框,编码463个氨基酸;Gm HDR与萝芙木、艾菊等植物HDR蛋白具有很高的一致性(≥84%),无跨膜结构域,有叶绿体转运肽等结构,主要定位于叶绿体中。实时定量PCR结果显示,Gm HDR基因在秦艽的花中进行表达量高,在叶、茎和根等部位表达较低。Gm HDR在序列上与其他植物的HDR蛋白序列特征上存在很大的相似性;Gm HDR基因主要在秦艽花中表达,可能主要参与花中萜类物质的合成。该研究结果可为今后研究秦艽环烯醚萜类化合物的生物合成机制提供依据。     

坚龙胆环烯醚萜甙的HPLC分析

应用HPLC分析方法,建立了坚龙胆中5个主要环烯醚萜甙的含量测定方法。在ZORBAXSB—C18色谱柱,乙腈-0．2％磷酸水溶液为流动相,梯度洗脱,流速1．0mL/min,柱温为40℃,检测波长254nm的色谱条件下,化合物得到良好的分离,并具线性相关。本方法操作简便,重现性好,灵敏度高。对不同产地的样品进行比较分析的结果表明,产地对坚龙胆中环烯醚萜甙的组成和含量有显著的影响。     

栽培条叶龙胆根中龙胆苦苷含量差异的研究

采用高效液相色谱法,测定栽培条叶龙胆根中龙胆苦苷的含量,结果表明:花末期(9月3日)龙胆苦苷含量高于果期(9月18日)和枯萎期(10月10日);同一根系中二龄根的含量显著高于一龄根;有性后代个体之间龙胆苦苷含量差异显著,对条叶龙胆进行高含量育种研究是可行的。     

Studies on Formation of Flower Bud and Prerequisite of Flowering in Vitro in Gentiana manshurica Kitag

Large amounts of plantlets of Gentiana manshurica Kitag. from germinated seeds in vitro can be obtained by micropropagation. Flower buds differentiate on shoot tip and leaf axil when ptantlets are transferred into MS medium supplemented with 0.4 mg/l zeatin, 0.2 mg/l NAA, 0.4 mg/l kinetin and 2 mg/l succinic acid-2, 2-dimethyl hydrazide under the cultural conditions of 28±2℃, 1,300 lx and 8 hours per day. The anthesis of blue-purple flower can prolong to 4 weeks. The development of pollen and ovule is normal and is similar to that of Gentiaceae described in the monograph"Systematic embryology of the angiosperms", by Davis (1966). Flowers in vitro are utilized expectantly for breeding or for commerical use directly.     

HPLC Determination of Contents of Four Active Constituents in Tibetan Medicine Gentiana straminea ( Gentianaceae) During Different Growing Period

Contents of four iridoid glycosides viz. gentiopicroside, longanic acid, swertiamarin and sweroside of wild and cultivated Gentiana straminea roots gathered in different seasons were analyzed by means of HPLC determination. The results indicated that contents of the four iridoid glycosides varied greatly with different seasons between wild and cultivated Gentiana straminea. And according to the criterion of pharmacopoeia of China, after cultivation content of gentiopicroside in Gentiana str aminea root had accorded with the regulation and can preliminarily replaced the wild species to use as a plant medicine. The mechanism causing the content changes of the four iridoid glycosides may be due to the integrated effects of many factors, such as the ecological factors, the genetic factors.