6种川产姜黄属药用植物叶绿体trnK基因序列变异分析及其分子鉴定

目的建立6种川产姜黄属(Curcuma)药用植物快速简单的分子鉴定方法.方法采用叶绿体赖氨酸tRNA基因(trnK)测序与序列变异分析方法.结果 6种姜黄属药用植物(包括姜黄C. longa、莪术C. phaeocaulis、川郁金C. sichuanensis、川郁金C. chuanyujin、川黄姜C. chuanhuangjiang、川莪术C. chuanezhu)完整trnK基因长度在2699～2705 bp.序列可变区包括matK基因编码区和trnK外显子与matK内含子之间区域,共有6个单核苷酸多态性(SNPs)位点、1个9-bp的缺失重复序列和2个4-bp、14-bp插入重复序列.结论 trnK基因序列可变位点可以作为6种川产姜黄属药用植物快速简单的分子鉴定标记,并为它们之间种的归并提供了分子依据.     

中日产川芎的matK、ITS基因序列及其物种间的亲缘关系

目的分析中国产川芎Ligusticum chuanxiong Hort.及日本产川芎Cnidium officinale Makino的核基因组ITS和叶绿体基因组matK序列,为探讨中日产川芎物种间的亲缘关系提供分子依据。方法采用PCR直接测序技术测定川芎和日本川芎的ITS基因和matK基因核苷酸序列并作序列变异分析。结果川芎和日本川芎的matK序列长度均为1268 bp,编码422个氨基酸。ITS1-5.8S-ITS2序列长度均为699 bp,其中18S rRNA基因3′端序列54 bp,ITS1序列215 bp,5.8S rRNA基因序列162 bp,ITS2序列222 bp,26S rRNA基因5′端序列46 bp。根据排序比较,川芎原植物与其商品药材间的matK基因和ITS基因序列完全相同,而川芎与日本川芎间matK基因则仅有1个变异位点,即在上游959 nt处1个转换替代(T→C),反映在氨基酸序列则发生一个非同义取代V(GTG)→A(GCG);ITS基因也仅有1个变异位点,即在ITS1上游54 nt处1个转换替代(T→C)。结论通过进化速率较快的基因序列同源性分析,基本可以认为中日所产川芎基原一致,日本川芎学名似应改为Ligusticum chuanxiong Hort.。     

我国姜黄属植物的研究——Ⅱ.五种姜黄属药用植物根茎挥发油化学成分的比较

用GC,GC-MS和TLC分析了国产5种姜黄属植物(姜黄,温郁金,莪术,桂莪术和川郁金)根茎挥发油的化学成分。在OV-17柱上分离出30多个色谱峰,鉴定了24种成分,其中4种(柠檬烯,α-松油烯,芳樟醇和丁香烯)以前未见报道。不同姜黄属植物有许多相同成分,但也有很高含量的不同成分,例如川郁金中的桉叶素,莪术中的莪术酮,姜黄中的姜黄酮和芳姜黄酮。因此根据5种挥发油中主要成分的差别,列出了区分姜黄属品种的表。     

基于DNA条形码技术鉴定蒙药材刺柏叶及其混淆品

目的：基于DNA条形码技术鉴别蒙药材刺柏叶及其混淆品。方法：采用国际通用的条形码序列 ITS2、psbA-trnH、matK和rbcL,对刺柏叶及其混淆品圆柏叶共计11份样品进行DNA提取、扩增,采用CodonCode Aligner进行序列拼接,并用MEGA软件对拼接序列进行变异位点分析、邻接(NJ)聚类分析,并计算其平均种内、种间遗传距离。结果：4对引物的PCR扩增产物测序成功率分别为ITS2 100%、 psbA-trnH 100%、rbcL 100%、matK 0%;ITS2序列和psbA-trnH序列均可通过变异位点比较区分刺柏叶及其混淆品;NJ聚类分析的结果显示,psbA-trnH序列的NJ聚类树中刺柏叶与圆柏叶均能分别聚为一支,且psbA-trnH序列的平均种间遗传距离明显大于平均种内遗传距离。结论：psbA-trnH序列能有效区分圆柏叶与刺柏叶,可作为鉴别刺柏叶及其混淆品圆柏叶的条形码序列,为蒙药材刺柏叶及其混淆品的鉴别提供支持。     

射干及类似药用植物叶绿体rbcL基因序列分析

目的 对射干Belamcanda chinensis (L.) DC.,鸢尾Iris tectorum Maxim.,野鸢尾I.dichotoma Pall.,蝴蝶花I.japonica Thunb.和德国鸢尾I.germaica L.等5种药用植物进行叶绿体rbcL基因序列分析,并对其亲缘关系进行探讨。方法CTAB(Cetyl trimelhyl ammonium bromide,CTAB)法提取总DNA,用作者设计的引物对鸢尾科5种药用植物的叶绿体rbcL(ribulose 1,5-bisphosphate carboxylose Large Gene,rbcL)基因进行扩增,PCR扩增产物纯化后,用ABI310 DNA自动测序仪测序。结果获得射干和4种鸢尾属药用植物叶绿体rbcL基因部分序列(约750 bp),除德国鸢尾外, 其余4种药用植物的rbcL基因序列为首次获得;用clustal 8.0,MEGA 2.0等软件分析统计获得的目的基因片段,得到碱基突变点,遗传距离［碱基差异数(1.000～20.000)、颠换数为(0.000～9.000)、转换数为(0.000～14.000)］,根据rbcL基因部分序列数据建立分子系统树。结论根据叶绿体rbcL基因序列数据可以很好的鉴别5种鸢尾科植物。     

广藿香的基因序列与挥发油化学型的相关性分析

目的探讨“南药”广藿香Pogostemon cablin (Blanco) Benth.不同产地间的叶绿体和核基因组的基因型与挥发油化学型的关系，为广藿香道地性品质评价、规范化种植提供分子依据。方法用PCR直接测序技术对广藿香6个产地样本的叶绿体matK基因和核18S rRNA基因核苷酸序列进行测序分析研究。结果广藿香6个样本的matK基因序列长均为1 245 bp，编码415个氨基酸成熟酶。18S rRNA基因序列长为1 803～1 805 bp。根据排序比较，广藿香6个样本间的matK基因序列存在47个变异位点，18S rRNA基因存在17个变异位点，非加权组平均法构建的系统分支树表明广藿香基因序列分化与其产地、所含挥发油化学变异类型呈良好的相关性。结论结合挥发油分析数据，基因测序分析技术可作为广藿香道地性品质评价方法这一以及规范化种植过程关键技术“物种鉴定”的强有力工具。     

PP2C蛋白磷酸酶调控的细胞信号通路研究进展

2C类蛋白磷酸酶是蛋白磷酸酶家族的重要成员,可以对丝/苏氨酸残基特异性脱磷酸。近来研究表明,2C类蛋白磷酸酶控制着大量关键的细胞功能,如增殖、细胞周期阻滞、衰老和细胞程序性死亡、凋亡和自噬等,因而在介导机体免疫反应、衰老、神经发育及肿瘤发生发展中发挥重要的生物学作用。总结PP2C基因各亚型参与介导的重要细胞信号通路,如丝裂原活化蛋白激酶（MAPK）、磷脂酰肌醇3-激酶/丝苏氨酸蛋白激酶（PI3K/AKT）、转化生长因子-β（TGF-β）/Smads、核转录因子-κB（NF-κB）及DNA损伤应答通路,以期为阐明上述生理病理过程的分子基础和调控机制提供新的思路。     

纳米氧化铁颗粒tk及hprt基因突变试验比较研究

目的 使用L5178Y细胞分别开展tk基因突变试验和hprt基因突变试验评价纳米氧化铁颗粒（iron oxidenanoparticle, IONP）的潜在基因突变风险,比较两种方法的灵敏性。方法 不同质量浓度的PEG-IONP（31.25、62.5、125、250、500 μg/mL）分别与细胞作用3 h,于给药后第0、2、6天将细胞接种于96孔板继续培养9～10 d,用于计算平板接种效率。分别在给药后第2天或第6天加入三氟胸苷（TFT）和6-硫鸟嘌呤（6-TG）作为tk基因和hprt基因选择剂,继续培养14 d后分析基因突变频率。试验平行设置灭菌注射用水溶媒对照组和甲基甲烷磺酸酯（MMS,10 μg/mL）阳性对照组。结果 溶媒对照组和阳性对照组的hprt基因突变率均低于tk基因突变率,且统计学结果提示hprt基因突变试验数据获得显著性差异的起始浓度（250 μg/mL）高于tk基因突变试验（125 μg/mL）。但整体而言两种试验方法对PEG-IONP的评价结果一致。结论 PEG-IONP可引起小鼠淋巴瘤L5178Y细胞tk基因及hprt基因突变率显著性升高。该研究结果可为纳米材料基因突变风险评价的选择提供借鉴与参考。     

NUDT15c.415C>T和TPMT*3C基因多态性检测方法的比较与临床应用

目的 建立准确、快速、经济的方法,检测NUDT15 c.415C>T和TPMT*3C基因多态性,探讨临床应用价值。方法 收集2017年5月-2018年5月期间福建汉族患者服用硫唑嘌呤2周以上的血清样本,提取DNA或白细胞后分别采用PCR-RFLP法、PCR-Sanger测序法和荧光定量PCR法对NUDT15 c.415C>T和TPMT*3C进行基因多态性分型,比较这3种方法的准确性、简便性及经济性。根据白细胞值分组,结合临床资料,探讨基因多态性等因素与硫唑嘌呤致白细胞减少的相关性。结果 共纳入129例患者,其中硫唑嘌呤致白细胞减少15例（11.6%）。3种方法的基因多态性检测结果一致,TPMT*3C未发现突变纯合子。携带NUDT15c.415C>T突变等位基因者服用硫唑嘌呤致白细胞减少的风险高于携带野生等位基因者（OR=6.2,95%CI：2.5~15.4,P=0.000 054）,而携带TPMT*3C突变等位基因者与野生等位基因者出现白细胞减少比例并无显著性差异（P=0.393）。NUDT15c.415C>T基因多态性预测白细胞减少敏感度为53.3%,特异度为85.1%,ROC曲线AUC为0.69。结论 3种方法都可用于临床检测NUDT15 c.415C>T和TPMT*3C基因多态性。PCR-RFLP法不需要专用试剂盒,也不需要昂贵的仪器设备,成本较低,过程简单,易于操作,特别适合条件有限的单位开展工作。福建汉族患者在服用硫唑嘌呤前进行NUDT15c.415C>T基因多态性检测比TPMT*3C更具临床价值。     

福建产温郁金新鲜块茎中郁金、莪术、姜黄挥发油化学成分比较

目的研究比较泉州利鑫生物科技有限公司温郁金种植基地生产的温郁金新鲜块茎中郁金、莪术、姜黄挥发油化学成分及含量的差异。方法采用水蒸气蒸馏法提取同一植株不同部位（郁金、莪术、姜黄）的挥发油,用GC-MS法分析各挥发油的组分,对分离得化合物进行结构鉴定,应用色谱峰面积归一化法测定各成分得相对百分含量。结果郁金、莪术、姜黄的水蒸气蒸馏提取挥发油得油率在0．3％～3．3％之间,郁金鉴别出15种化学成分;莪术鉴别出4种化学成分;姜黄鉴别出26种化学成分。结论郁金、莪术、姜黄挥发油含量及其组分均存在差异,莪术挥发油成分以莪二酮、吉马酮为主;郁金、姜黄挥发油成分以按树脑为主。     

Molecular analysis of medicinally-used Chinese and Japanese Curcuma based on 18S rRNA gene and trnK gene sequences.

Curcuma drugs have been used discriminatingly for invigorating blood circulation, promoting digestion, and as a cholagogic in China. However, there is confusion about the drug's botanical origins and clinical uses because of morphological similarity of Curcuma plants and drugs. In order to develop an ultimate identification, molecular analysis based on 18S rRNA gene and trnK gene sequences were performed on 6 Curcuma species used medicinally in China and Japan. The 18S rRNA gene sequences were found to be of 1810 bps in length. In comparison with the common sequence of C. longa, C. phaeocaulis, C. wenyujin and C. aromatica, that of C. kwangsiensis had one base substitution, and the same base difference was observed between the Chinese and the Japanese populations of C. zedoaria. The trnK gene sequences were found to span 2698-2705 bps. There were base substitutions, small deletions or insertions at some sites between the trnK coding region and matK region among each species. Based on the base substitutions, C. zedoaria and C. kwangsiensis specimens were divided into two groups, respectively. An identical sequence was detected in C. phaeocaulis and in the Chinese population of C. zedoaria, as well as in the Japanese population of C. zedoaria and in one group of C. kwangsiensis with a purple-colored band in leaves. New taxonomic information to be used for authenticating Curcuma drugs was obtained.     

Phylogenetic relationship in the genus Panax: inferred from chloroplast trnK gene and nuclear 18S rRNA gene sequences

Chloroplast trnK gene and nuclear 18S rRNA gene sequences of 13 Panax taxa, collected mainly from Sino-Japanese floristic region, were investigated in order to construct phylogenetic relationship and to assist taxonomic delimitation within this genus. The length of trnK gene sequence varied from 2537 bp to 2573 bp according to the taxa, whereas matK gene sequences, embedded in the intron of trnK gene, were of 1512 bp in all taxa. Species-specific trnK/ matK sequence provided much insight into phylogeny and taxonomy of this genus. 18S rRNA gene sequences were of 1808 or 1809 bps in length, only 9 types of 18S rRNA sequences were observed among 13 taxa. Parsimony and neighbor-joining analyses of the combined data sets of trnK-18S rRNA gene sequences yielded a well-resolved phylogeny within genus Panax, where three main clades were indicated. P. pseudoginseng and P. stipuleanatus formed a sister group located at a basal position in the phylogenetic tree, which suggested the relatively primitive position of these two species. Monophyly of P. ginseng, P. japonicus (Japan) and P. quinquefolius, which are distributed in northern parts of Asia or America, was well supported (Northern Clade). The remaining taxa distributed in southern parts of Asia formed a relatively large clade (Southern Clade). The taxonomic debated taxa traditionally treated as subspecies or varieties of P. japonicus or P. pseudoginseng showed various nucleotide sequences, but all fell into one cluster. It might suggest these taxa are differentiated from a common ancestor and are in a period of high variation, which is revealed not only on morphological appearance, but also on molecular divergence. By comparing trnK and 18S rRNA gene sequences among 13 Panax taxa, a set of valuable molecular evidences for identification of Ginseng drugs was obtained.     

Authentication of Curcuma species (Zingiberaceae) based on nuclear 18S rDNA and plastid trnK sequences

Curcuma drugs have been used discriminatingly for invigorating blood circulation, promoting digestion, and as a cholagogic in China. However, there is confusion about the drug's botanical origins and clinical uses because of morphological similarity of Curcuma plants and drugs. Comparative sequencing of the 18S rRNA gene in nuclear ribosomal DNA (rDNA) and trnK gene in chloroplast DNA (cpDNA) was carried out in order to examine interspecies phylogeny and to identify ultimately Curcuma species. A total of a hundred of accessions of eighteen species were analyzed. This resulted in an aligned matrix of 1810 bp for 18S rDNA and 2 800 bp for trnK. 18S rDNA sequence divergence within the ingroup ranged from 0-0.05%, trnK ranged from 0-0.19%. One base transversion-substituted site (from cytosine to thymine) was observed from the upstream of 18S rDNA at nucleotide position 234 in C. kwangsiensis and Japanese population of C. zedoaria which have separated genetic distance to other Curcuma taxa. Two noncoding regions embedded in trnK intron showed higher variability, including nucleotide substitutions, repeat insertion and deletions. Based on consensus of relationship, eighteen major lineages within Curcuma are recognized at the species level. The results suggest that Curcuma is monophyletic with 100% bootstrap support and sister to the genera Hedychium and Zingiber. The trnK sequences showed considerable variations between Curcuma species and thus were revealed as a promising candidate for barcoding of Curcuma species, which provide valuable characters for inferring relationship within species but are insufficient to resolve relationships among closely related taxa.     

Sequence analysis of Chinese and Japanese Curcuma drugs on the 18S rRNA gene and trnK gene and the application of amplification-refractory mutation system analysis for their authentication

The botanical origins of Chinese and Japanese Curcuma drugs were determined to be Curcuma longa, C. phaeocaulis, the Japanese population of C. zedoaria, C. kwangsiensis, C. wenyujin, and C. aromatica based on a comparison of their 18S rRNA gene and trnK gene sequences with those of six Curcuma species reported previously. Moreover, to develop a more convenient identification method, amplification-refractory mutation system (ARMS) analysis of both gene regions was performed on plants. The ARMS method for the 18S rRNA gene was established using two types of forward primers designed based on the nucleotide difference at position 234. When DNAs of four Curcuma species were used as templates, PCR amplification with either of the two primers only generated a fragment of 912 base pairs (bp). However, when DNAs of the purple-cloud type of C. kwangsiensis and C. wenyujin were used, PCR amplifications with both primers unexpectedly generated the fragment, suggesting that these two were heterozygotes. The ARMS method for the trnK gene was also established using a mixture of four types of specific reverse primers designed on the basis of base substitutions and indels among six species, and common reverse and forward primers. C. phaeocaulis or the Chinese population of C. zedoaria, the Japanese population of C. zedoaria or the purple-cloud type of C. kwangsiensis, the pubescent type of C. kwangsiensis or C. wenyujin, and C. aromatica were found to show specific fragments of 730, 185, 527 or 528, and 641 or 642 bp, respectively. All species including C. longa also showed a common fragment of 897-904 bp. Using both ARMS methods, together with information on producing areas, the identification of Curcuma plants was achieved. Moreover, the ARMS method for the trnK gene was also useful for authentication of Curcuma drugs.     

Application of single-nucleotide polymorphism analysis of the trnK gene to the identification of Curcuma plants

We previously found that Curcuma plants and drugs derived from Curcuma longa, C. phaeocaulis, C. zedoaria, and C. aromatica could be identified by the nucleotide differences at two sites and the existence of a 4-base indel on trnK gene. In this paper, based on species-specific nucleotide sequences, the application of a new method, single-nucleotide polymorphism (SNP) analysis was investigated to identify Curcuma plants more conveniently. First, three types of reverse primer were synthesized in different lengths, 34 mer, 26 mer, and 30 mer, to anneal the template DNAs from each species at sites immediately upstream from substitution positions 177 and 645, and at the site including the 4-base insertion from 728 to 731, respectively. After single-base extension reaction of these primers using fluorescent-labeled ddNTPs and PCR products of the trnK gene region as template, the resulting products were detected using an ABI PRISM 310 Genetic Analyzer. The electrophoretogram showed three or two peaks at different positions depending on the 27 mer, 31 mer, and 35 mer product lengths. Each peak was derived from the incorporated fluorescent-labeled ddNMPs complementary to template nucleotides at positions 645, 724, and 177, respectively. C. phaeocaulis showed three peaks of ddCMP, ddAMP, and ddAMP. The other three species showed two peaks derived from 27 mer and 35 mer products: peaks of ddCMP and ddAMP in C. longa, those of ddCMP and ddTMP in C. zedoaria, and those of ddTMP and ddAMP in C. aromatica. Thus SNP analysis to identify four Curcuma plants was newly developed.     

Molecular identification of "Chuanxiong" by nucleotide sequence and multiplex single base extension analysis on chloroplast trnK gene

Chloroplast trnK gene sequences of Cnidium officinale and Ligusticum chuanxiong were determined to establish an effective method for identifying Japanese Senkyu and Chinese Chuanxiong, the two which have the same drug name in Chinese characters, similar external feature, but different botanical origins. Three sites of nucleotide differences were found between these 2 species at positions 767,924 and 964 from upstream in trnK gene sequence, allowing molecular identification of the two plants and crude drugs. Further, three kinds of specific primers of 14 mer, 23 mer and 30 mer long were designed to detect these 3 sites of marker nucleotides. By using multiplex single base extension (MSBE) analysis with the 3 specific primers, C. officinale and L. chuanxiong could be distinguished clearly by the electrophoretograms, where 3 peaks with different color of ddTMP, ddCMP and ddTMP were observed in case of C. officinale and those of ddGMP, ddAMP and ddGMP in L. chuanxiong. Moreover, trnK gene sequence of "Dongxiong," a kind of Chuanxiong cultivated in Northeast China, suggested that its botanical origin was C. officinale.     

Rapid identification of Curcuma longa and C. aromatica by LAMP

We have developed a novel method for the identification of Curcuma longa and C. aromatica called "loop-mediated isothermal amplification (LAMP)," based on trnK gene sequences. LAMP employs four primers that recognize six regions on the target DNA. Cycling elongation was initiated when the four primers were annealed to the target DNA. Amplifications were detected by measuring turbidity due to the formation of magnesium pyrophosphate. We designed allele-specific primer sets for C. longa and C. aromatica, respectively. LAMP using a primer set for C. longa and total DNA extracted from C. longa rhizome (0.5-10.0 ng) as template was detected up to 70 min. On the other hand, in the reaction using a primer set for C. longa and total DNA from C. aromatica as template, no amplifications were detected. The same tendency could be seen in the reactions using a set of primers for C. aromatica. LAMP enabled not only identification but also detection with high specificity. This rapid, specific, sensitive, and convenient method is expected to be applicable to the identification of the botanical origin of commercially available herbal products.