天然产物厚朴酚存在水溶性较差、易氧化、结构不稳定等问题.通过对其进行化学结构修饰,再通过生物信息学分析初步探究厚朴酚抗中枢神经损伤的作用机制,以D-(+)-半乳糖、D-(+)-麦芽糖、D-(+)葡萄糖为糖基化的起始原料,对其上的活泼-OH进行一系列的修饰,然后与厚朴酚进行糖苷化反应,最后化合物进行脱保护处理,得到3个厚朴酚糖苷化衍生物,已通过1H-NMR、13C-NMR及MS确认结构.对3个厚朴酚糖苷衍生物进行生物信息学分析,分析讨论厚朴酚糖苷化衍生物对于中枢神经损伤疾病可能作用靶点及相关机制.结果显示,确定了厚朴酚糖苷衍生物治疗中枢神经损伤的主要潜在靶点6个,主要潜在生物过程4条,主要潜在信号通路4条,为接下来的药理学实验提供了道路指引.

目的:优化淫羊藿的最佳炮制工艺,研究淫羊霍炮制前后对DPPH和ABTS+的抗氧化清除能力.方法:选取炮制温度、炮制时间、加油量为考察因素,采用Box-Behnken试验设计对淫羊藿最佳工艺条件进行优化,并对其体外抗氧化活性进行测定.结果:淫羊藿5个成分总含量最佳炮制工艺为:炮制温度120℃,炮制时间4min,加油量9％.淫羊藿对DPPH及ABTS+皆有不同程度的清除能力,其中生品淫羊霍的IC50值分别为0.388mg/mL和0.158mg/mL,炙品淫羊藿的IC50值分别为0.404mg/mL和0.216mg/mL.结论:该研究建立的炮制工艺合理、操作简便易行,可为淫羊藿的炮制方法提供参考.通过测定淫羊藿炮制前后对DPPH及ABTS自由基的清除能力来验证抗氧化作用.

核因子Y(NF-Y)是真核生物中一种重要的转录因子.采用生物信息学方法从太子参转录组数据库中鉴定N F-Y蛋白家族成员,通过转录组数据分析各基因在不同组织中的表达水平,研究NF-Y基因对土壤水分及蔗糖信号的响应.通过对3个转录组数据库的检索,确定了太子参N F-Y蛋白家族的3个亚家族,包含9个PhNF-YA成员,10个PhNF-YB成员及5个PhNF-YC成员,发现PhNF-YA 4等9个成员在叶中有较高表达,PhNF-YA3在块根皮部及PhNF-YB3和PhNF-YB8在块根木质部中有较高表达.干旱胁迫可显著上调11个成员的表达,2个成员的表达量随土壤水分含量的上升表现出上调的趋势.大部分基因能够响应蔗糖浓度的变化.这些结果暗示了水分、蔗糖等因子可通过影响器官中NF-Y蛋白的表达,改变植物NF-Y蛋白间的相互作用关系,进而调控器官的形态建成及逆境响应.

近年来中药多糖的生物活性备受关注,研究也越来越深入.在现代研究中,太子参多糖具有抗糖尿病、免疫调节、保护心肌等多种生物活性,是太子参发挥功效及保健养身功能的最重要的物质基础成分.太子参多糖相关研究是解决太子参质量控制难题的关键,现已成为太子参研究领域的热门.本文综述了太子参多糖的提取分离、结构组成、生理功效等方面的研究内容,以期为太子参多糖的研究、开发与利用提供参考.

通过连接不同糖片段的三氯乙酰亚胺酯衍生物、甲基衍生物、对甲基苯硫酚衍生物等合成了4个常春藤皂苷元衍生物(4～7,其中6和7为新化合物),其结构经1H NMR和13C NMR表征.采用MTT法检测了化合物对人结肠癌细胞COLO205的抑制活性.结果表明:4和5对COLO205细胞有部分抑制作用,与浓度正相关;6对COLO205只在高浓度(1.00×10-4mmol/L)有较好抑制作用(98.36±0.43％);7只在中等浓度(1.00×10-5 mmol/L)对COLO205有微弱抑制作用(2.75±1.22％).

为提高齐墩果酸的水溶性和稳定性,以齐墩果酸苷元为起始原料,对其28-COOH进行甲基化修饰后制得齐墩果酸-28-羧甲酯(1).分别以D-半乳糖、D-葡萄糖、D-氨基葡萄糖为起始原料,通过对糖羟基的保护与去保护,得到一系列的二糖、四糖片段.通过三氯乙酰亚胺酸酯途径和对甲苯硫基途径,利用合成的糖片段对1的3-位羟基进行糖化学结构修饰,合成了4种新型的齐墩果酸糖苷化衍生物(2～5),其结构经1H NMR,13C NMR和MS(ESI)表征.采用MTT法测试了2～5对高表达人结肠癌细胞(HCT8)的体外抑制活性.结果表明:2～5对HCT8有一定的抑制作用,化合物5浓度为1×10-3 mmol/L时,抑制率达到(98.96±0.10)％.

针对熊果酸母核,经过甲基化修饰,再连接半乳糖、岩藻糖片段,合成了2个熊果酸糖苷衍生物(3、4),其中一个为新合成化合物(3),经1H NMR、13C NMR、MS表征结构.并采用MTT法,测定2种化合物对于人非小细胞肺腺癌细胞A549的生长抑制效果.实验结果表明,化合物3除最低浓度1×10-6 mmol/L对A549没有抑制率外,其余浓度(1×10-3 mmol/L、1×10-4mmol/L、1×10-5 mmol/L)均对肺癌细胞存在抑制作用,与浓度呈正相关;化合物4各浓度对A549均存在抑制作用,与药物浓度呈现正相关.化合物3在1×10-5 mmol/L时抑制率略高于对照组熊果酸,其余组抑制率均不如熊果酸组.化合物4的4组浓度,A549抑制率均不如对照组.

目的 研究太子参不同提取物及环肽HB对酪氨酸酶活性的抑制作用,为太子参用于化妆品中提供理论依据.方法 以L-酪氨酸作为底物,从马铃薯中提取酪氨酸酶,通过分光光度法测定太子参不同提取物及环肽HB对酪氨酸酶活性的抑制率,以生物美白剂熊果苷和Vc-PMG为对照.结果 熊果苷浓度在2mg/ml时对酪氨酸酶活性有较高抑制作用,抑制率为39％;Vc-PMG浓度1.67mg/ml时,酪氨酸酶活性抑制率随浓度增加不断上升,抑制率达55％.太子参甲醇提取物生药量在2.53～ 12.67mg/ml时,抑制率达42％;太子参水提液生药量在1.39mg/ml时抑制率达47％;太子参环肽HB浓度在4.98μg/ml时对酪氨酸酶抑制率达43％.结论 太子参不同提取物及环肽HB对酪氨酸酶抑制活性,可以考虑将其应用于化妆品中.

探明外源PBZ对IAA生物合成的相关基因YUCCA和GH3表达模式的影响,为太子参栽培提供理论参考.以太子参为实验材料,采用酶联免疫吸附测定法和实时荧光定量PCR技术对外源PBZ和GA3处理条件下太子参块根内源IAA的含量及其相关基因的转录表达水平的动态变化进行研究.结果显示,PBZ处理后太子参中内源IAA的含量在第1次处理后10、20和30 d均高于对照组,随后则低于对照组;经GA3处理后,除20 d外,其他时期内源IAA含量较对照组高.另外,内源IAA生物合成相关基因在外源PBZ和GA3处理后第50天时块根中的相应情况显示,GH3(Unigene37777)受外源PBZ的诱导而表达量上调,GH3(Unigene43146)和GH3(Unigene43412)的表达却受到抑制,但这3个基因均受外源GA3诱导;YUCCA(Unigene49937)对外源PBZ和GA3处理呈现相反的表现形式,受PBZ诱导,而受GA3抑制.PBZ和GA3对太子参发育过程中内源IAA的积累表现为诱导,同时对合成相关基因的表达也具有不同程度的响应作用.

Objective To discuss the influence of exogenous Gibberellin (GA3) and abscisic acid (ABA) on genetic expressions of key enzymes in ethylene synthesis in Taizishen (Pseudostillaria Root, Radix Pseudostellariae) , and provide reference for intelligent use of growth regulators during cultivation of Taizishen.Methods The genes of key enzymes in ethylene synthesis of Taizishen were screened and identified from transcriptome database by using local Blast method.Hai'ershen in full-bloom stage was treated with exogenous GA3 and ABA, and samples were collected at different time points (20 d, 40 d, 50 d and 60 d.The genetic expressions of key enzymes in ethylene synthesis of Taizishen were detected by using real-time fluorescence polymerase chain reaction (RT-PCR).Results There were totally 6 genes of key enzymes in ethylene synthesis in roots of Taizishen obtained, and they were named PhACO1, PhACO2, PhACO3, PhACS1, PhACS2 and PhSAMS.GA3 and ABA could regulated the expressions of above 6 genes, and expressions of PhACO1, PhACO2, PhACS1 and PhSAMS were up-regulated after treated with GA3, and expressions of PhACO3 and PhACS2 were up-regulated after 20 d to 40 d and down-regulated after 50 d to 60 d.The expressions of genes of key enzymes in ethylene synthesis in Taizishen showed down-regulation at first and then up-regulation after treated with ABA, but different genes had different demonstrations.The expression of PhACO1 was up-regulated after 20 d to 60 d, and down-regulated after 40 d to 50 d.The expressions of PhACO2 and PhACO3 were down-regulated after 20 d to 50 d, and up-regulated after 60 d.The expression of PhACS1 was down-regulated after 20, up-regulated after 40 d to 60 d.The expressions of PhACS2 and PhSAMS were down-regulated.Conclusion GA3 and ABA may control the ethylene signaling pathway to regulate plant growth through affecting the ethylene biosynthesis of Taizishen.It is speculated that Taizishen is treated with suitable concentration of GA3 in adventitious root growth period and treated with ABA treatment in root thickening period, the yield of Taizishen will be increased.

本研究采用同源克隆的方法分离得到太子参Ph CKX基因的cDNA序列,并对其序列进行生物信息学分析,分析表明,PhCKX基因的开放阅读框为1 611 bp,编码536个氨基酸,理论等电点为9.02,相对分子质量为61 kD,跨膜分析显示其为膜蛋白,含有CKX基因家族典型的功能位点FAD结合域和CTK结合域,并具有糖基位点。实时荧光定量PCR分析显示,太子参Ph CKX在块根发育初期表达量最高,随着块根的膨大,表达量逐渐降低。ABA激素处理后,Ph CKX的表达量呈现先上升后下降的趋势,生长中期达到最大;在GA3处理下,PhCKX的表达量则呈现先下降后上升的趋势。我们推测PhCKX的变化可能造成内源CTK含量的变化,ABA和GA3对内源CTK变化的调节作用说明ABA、GA3和CTK之间的激素平衡调节是太子参块根膨大的重要因素。本研究为细胞分裂素对太子参块根膨大及植物对环境刺激响应的分子机制奠定基础。

内源激素是植物生长发育的重要调节系统,对植物器官的形态建成具有重要意义.本研究采用外施赤霉素(GA3)或赤霉素抑制剂(PBZ)处理太子参种苗,探讨外源赤霉素对太子参内源赤霉素及块根形成的影响.结果表明,喷施外源赤霉素可显著增加株高、茎节数,但降低叶厚及地上茎粗,抑制太子参块根的形成.外施赤霉素早期迅速降低块根内源赤霉素含量,并在后期维持块根中内源赤霉素在较高水平.多效唑缓慢降低内源赤霉素水平,但在块根发育后期维持较高的赤霉素含量.外源赤霉素能显著降低GA20氧化酶、GA3氧化酶及受体蛋白基因GID1的表达水平;多效唑长期处理能显著提高降低GA20氧化酶、GA3氧化酶的表达水平,但降低受体蛋白基因GID1的表达水平.长期外施赤霉素与多效唑对植物内源赤霉素具有动态调节的作用,且内源赤霉素的生物合成具有显著的反馈调节机制.

目的:获取太子参醛氧化酶(abscisic acid aldehyde oxidase,AAO)基因.方法:基于AAO基因的同源性和太子参转录组数据库,以块根为材料,利用3’-RACE技术和PCR技术克隆获取AAO基因序列,通过qRT-PCR技术检测该基因在太子参茎、叶、须根,不同生长时期及经外源脱落酸及其抑制剂处理后的块根中表达情况.结果:克隆获得太子参PhAAO基因序列长6 773 bp,含完整开放阅读框(4 053 bp),由10个外显子和9个内含子组成,氨基酸结构预测其含有钼-黄素酶家族基因特有的结构域.qRT-PCR分析显示PhAAO基因在太子参叶、须根中显著表达,在块根形成的关键时期6月中旬表达量最高;脱落酸和氟啶酮处理后PhAAO基因表达上调,其中氟啶酮处理组较为明显.结论:获得太子参PhAAO基因序列,并进一步研究该基因的表达情况,为后续研究其功能特点,探讨太子参脱落酸生物合成分子机制奠定理论基础.

为了探讨ABA、GA3对太子参皂苷生物合成的影响,该研究通过根灌ABA及其抑制剂氟啶酮(Fluridone)、GA3及其抑制剂多效唑(PBZ),利用酶标仪分光光度法测定皂苷和内源茉莉酸甲酯(MeJA)的含量,采用实时荧光定量PCR技术检测三萜皂苷生物合成关键酶基因表达量的变化.结果表明,太子参总皂苷总含量与其生物合成关键酶基因的表达呈现一致的变化趋势,外源GA3长期处理后太子参中皂苷的含量明显增加,太子参SE1和IPPI基因的表达量明显上调;施加外源ABA则降低了太子参总皂苷的含量,太子参HMGS、MDD、SS1、SS2和β-A280各基因表达量均明显下调.因此,我们推测在太子参块根生长发育过程中GA3可能通过影响JA从而促进了皂苷的积累,ABA可能是通过抑制皂苷合成关键酶基因的表达进而降低皂苷的含量.此外,在块根发育的不同时期,ABA、GA对Me-JA的影响可能存在较大差异.

Objective To clone the gibberellin 2-oxidase (GA2ox) genes from Pseudostellaria heterophylla and perform the bioinformatic and expression mode analysis.Methods According to P heterophylla transcriptome annotation,two transcript codings of GA2ox were identified.The full-length cDNA PhGA2ox1 and PhGA2ox8 were determined using RT-PCR.Then the bioinformatic analysis of these genes and encoded proteins were performed.The coding sequences of PhGA2oxl and PhGA2ox8 were amplified by PCR.And then analyzed the bioinformationofthesesequences.The expression levels ofPhGA2oxl andPhGA2ox8 were analyzed using qPCR.Results Bioinformatic analysis showed that PhGA2oxl contained 981 bp and encoded a predicted protein of 326 amino acids with molecular weight of 36 871.3 and isoelectric point (PI) of 8.66;PhGA2ox8 contained 1 056 bp and encoded a predicted protein of 351 amino acids with molecular weight of 40 169.9 and PI of 6.91.Both PhGA2oxl and PhGA2ox8 contained GA2ox conserved domains DIOX_N and 20G-Fell_Oxy,without obvious hydrophobic region,transmembrane domain and signal peptide.Phylogenetic analysis showed that the GA2ox of plant could be divided into three classes,PhGA2oxl bolongs to Class Ⅰ and PhGA2ox8 belongs to Class Ⅲ.The qPCR.showed that the expression of PhGA2oxl in different tissues and organs ofP heterophylla was constant,but the expression of PhGA2ox8 in root xylem was significantly higher than that in other tissues and organs (P ＜ 0.05).Conclusion The PhGA2ox1 and PhGA2ox8 genes are cloned for the first time,and provide a foundation for they may play an important role in the growth and development process of P heterophylla.

According to the known fragment,specific primers ofPhA CT2 were designed.For a better understanding of bioinformatic characteristics,the full length of the gene was obtained from Pseudostellaria heterophylla by using rapid amplification of eDNA ends method (RACE).Sequence analysis indicated that the length of PhA CT2 with a 1 134 bp open reading frame (ORF) encoding 377 amino acid residues.The molecular weight of predicated protein was 41.79 kD and its theoretical isoelectric point was 5.30.Homologous analysis showed that it shared 88％ of nucleotide identities with Actin from Beta vulgaris and over 95％ of amino acid identities from other plants.Phylogenic analysis showed that PhA CT2 had close genetic relationship with AtACT7 with only 4 differences in amino acids.Real time PCR analysis revealed that PhA CT2 showed constantly expression in various organs of Pseudostellaria heterophylla and it can be considered as an internal reference gene.This work is beneficial to the functional analysis and utilization in molecular biology.

In our study,8 anthocyanins biosynthesis related MYB candidate genes were identified by using bioinformatics technologies and the expression profiles in different tissues and stages of fruits during the development and determined by transcriptome data and real time PCR.Phylogenetic analysis indicated that 4 MYB genes were closed to MYB genes from Vitis vinifera L.,Oryza sativa and Zea mays L.,while only 1 MYB gene was grouped into one cluster with those from Arabidopsisthaliana and Malusdomestica L..High level of transcripts of major genes in male flower was determined by transcriptome data.Contin-uous down-regulation expressions of MnMYBJ and MnMYB4 during the development of mulberry fruits were confirmed,while MnMYB330 had a continuous up-regulation expression.Therefore,MYB genes have positive regulation and negative regulation during the accumution of anthocyanins.

为了获取可靠内参基因,采用同源克隆技术和cDNA末端快速延伸技术,首次从太子参中克隆3-磷酸甘油醛脱氢酶基因(GAPDH)的部分cDNA序列,命名为PhGAPDH,序列长度为981 bp,包含699 bp的部分阅读框,推测其编码232个氨基酸,还含有282 bp的3'UTR.序列分析结果表明,太子参PhGAPDH基因编码的氨基酸序列与同科植物香石竹(Dianthus caryophyllus)氨基酸序列同源性高达99％.PCR分析结果表明,PhGAPDH基因在太子参不同种源块根、同一种源不同组织及不同生长时期的表达水平较稳定,可以作为太子参功能基因研究的内部参考基因.

In our study,LEA gene families have been identified and their expression profiles beenalso bioin-formaticaslly analyzed.The sequences of LEA genes in Arabidopsis thaliana,Rice,Glycine max have been downloaded from NCBI web site to search the LEA genes in poplar.Candidate gene have been deter-mined by means ofthe pfam program.Microarray Data and EST have been used to value the expression of LEA genes.According to motif analysis,a total of 87 LEA genes have been systematically identified from poplar classified into 8 subfamilies in our study.1 6 LEA genes have been highly expressed in flowers and 8 of them shared tissue-specific expression.PtLEA1.1 and PtLEA3.1 have high abundant expression in im-bibition seeds,as well as members of subfamilies LEAIV,LEAV,LEAVI and SMP.In germination seeds,1 2 LEA genes have beenhighly expressed and 4 of them shared tissue-specific expression.The ex-pression of 5 LEA genes has beeninvolved with light during the germination.3 LEA genes share down-reg-ulated expression in mature leaves.8 LEA genes havehigh expression in root and 3 LEA genes in xylem. These resultssuggest each subfamily of LEA have their function in different stages of seeds.High expres-sionof LEA genesare detected in formation and germination of seeds and these genes may play important roles in the development of seeds,not only formation but also germination.

目的 克隆太子参肌动蛋白(Actin)基因片段并进行序列分析.方法 利用植物Actin基因的保守序列设计简并引物,通过RT-PCR和抑制PCR扩增太子参Actin基因核心片段.利用半定量RT-PCR分析太子参Actin基因在不同种源、不同器官、不同生长发育时期的表达情况.结果 通过RT-PCR获得3条太子参Actin基因的核心片段,长度均为760 bp,依次命名为PhACT1、PhACT2、PhACT3.通过抑制PCR及序列拼接后3条核心片段依次延长至1 008、1 008、975 bp,分别编码336、336、325个氨基酸残基.半定量RT-PCR分析表明PhACT2和PhACT3基因在不同种源、不同器官、不同生长发育时期的表达量基本恒定,PhACT1基因存在一定的差异.结论 首次从太子参中克隆得到3条太子参Actin基因序列,并确定PhACT2基因适合作为太子参功能基因表达分析的内参基因.