Allelic variation in the coumarate 3-hydroxylase gene associated with wood properties of Catalpa fargesii Bur.

Coumarate 3-hydroxylase(C3h)genes participate in the synthesis of lignin and may affect the properties of wood that are important for its commercial value.A better understanding of the natural variation in C3h genes and their associations to wood properties is required to effectively improve wood quality.We used a candidate gene-based association mapping approach to identify CfC3h allelic variants associated with traits that affect the wood properties of Catalpa fargesii.We first isolated the full-length CfC3h cDNA(1825 bp),which was expressed at relatively high levels in xylem according to real time-polymerase chain reaction.In totally,17 common single-nucleotide polymorphisms(minor allele frequency>5%)were identified through cloning and sequencing the CfC3h locus from a mapping population(including 88 unrelated natural C.fargesii individuals collected from main distribution area).Nucleotide diversity and linkage disequilibrium(LD)in CfC3h indicate that CfC3h has low nucleotide diversity(π_(t)=0.0031 andθ_(w)=0.0103)and relatively low LD(within 1800 bp;r^(2)≥0.1).An association analysis identified eight common single-nucleotide polymorphisms(SNPs)(false discovery rate,Q<0.10)and ten haplotypes(Q<0.10)associated with wood properties,explaining 4.92-12.09%of the phenotypic variance in an association population consisted of 125 unrelated natural individuals(The 88 individuals from the mapping population were comprised in the association population).Our study would provide new insight into C3h gene affecting wood quality,and the SNP markers identified would have potential applications in marker-assisted breeding in the future.

Functional analysis of flavanone 3-hydroxylase(F3H)from Dendrobium officinale,which confers abiotic stress tolerance

Flavonoids are important bioactive components in Dendrobium officinale,a medicinal orchid.They are involved in many biological activities,including protecting plants against biotic and abiotic stresses.Research on the key genes related to flavonoid biosynthesis in D.officinale is limited.In this study,one of the key flavonoid biosynthesis genes,flavanone 3-hydroxylase(F3H),was characterized from D.officinale.The open reading frame of DoF3H was 1134 bp long and it encoded a 377-amino acid protein.The DoF3H protein showed considerably high homology with F3H proteins from other plant species and shared a common evolutionary ancestor with other F3Hs.DoF3H transcripts were detected in different organs of adult plants and mainly accumulated in flowers,followed by roots,stems and leaves,a pattern that was similar to the content of flavonoids.Recombinant DoF3H protein,which was localized in the cytosol,could convert naringenin to dihydrokaempferol.The mRNA levels of DoF3H were significantly induced by salt and cold stresses.Furthermore,the heterologous expression of DoF3H in Escherichia coli conferred it higher tolerance to salt and cold stresses.These results provide insight into the molecular function of DoF3H in the biosynthesis of flavonoids,and provide a new application for improvement of abiotic tolerance in D.officinale.

金银花香豆酸3-羟化酶基因LjC3H2的生物信息学分析

香豆酸3-羟化酶广泛存在于植物中,属于细胞色素P450家族的CYP98A亚家族。该酶能在植物苯丙素代谢中催化苯环C3位置的羟基化反应,是绿原酸生物合成的关键酶之一。本文以金银花香豆酸3-羟化酶基因LjC3H2为研究对象,采用生物信息学方法进行分析。结果表明,LjC3H2无信号肽,无跨膜结构域,偏亲水,定位于内质网上;二级结构以α螺旋为主,无规则卷曲较多。LjC3H2与其他植物同源蛋白氨基酸序列比对和系统发育分析表明,LjC3H2与蓟、桔梗的C3H亲缘关系较近,与同属于金银花的LjC3H亲缘关系较远;该蛋白的三级结构与细胞色素P450三级结构相似。

Correlation between expression of 1 α-hydroxylase and hypocalcaemia in rats with severe pancreatitis

Objective:To investigate the essential biochemical indices like 1-hydroxylase and hypocalcaemia in the rats with severe acute pancreatitis and explore the correlation between them.Methods:A total of 120 SPF grade Wistar male rats which were in similar physiological status were selected and randomly divided into two groups:sham group(SO group) and severe acute pancreatitis group(SAP group).Then they were divided into 1 h,3 h,6 h,and 12 h subgroups according to the killing lime.The severe acute pancreatitis model was established by retrograde injection of 5%sodium taurocholate.Serum calcium,serum creatinine,serum urea nitrogen and serum amylase were measured at different time.Serum 1,25 dihydroxy vitamin D3 level was determined by enzyme linked immunosorbentassay.The expression of 1-hydroxylase protein in the kidney tissue was determined with Western blotting and immunohistochemistry to observe its location.The pathologic features of the kidney tissue section was observed under light microscope and submicroscopic structure of the proximal convoluted tubule epithelial cell was observed under transmission electron microscope.Results:Compared with the SO group,rats in the SAP group showed continuous pathological injury as time went by.There was significant increase in serum creatinine,serum urea nitrogen and serum amylase in SAP group compared with the SO group 1,3,6,12 hours after the operation(P<0.05).There was significant decrease in serum calcium and 1,25 dihydroxy vitamin D3 3.6,12 hours after the operation(P<0.05).It also showed that the expression of the 1-hydroxylase protein in kidney tissues was upregulated at 1 h.3 h and decreased at 6h,12 h compared with the SO group.The serum calcium,1,25 dihydroxy vitamin D3 and the expression of the 1-hydroxylase protein in kidney tissues of the SAP group showed sustaining decrease.Western blotting showed positive correlation between the 1-hydroxylase expression and serum calcium at 3 h.6 h and 12 h(r=0.976,P<0.001;r=0.948.P<0.001;r=0.742,P=0.001) and also positive correlation between the 1-hydroxylase expression and serum1,25 dihydroxy vitamin D3 at 1 h,3 h,6 h and 12 h(r=0.935,P<0.001;r=0.952,P<0.001;r=0.917.P<0.001:r=0.874,P<0.001).Conclusions:At the early stage of the kidney injury,the expression of 1-hydroxylase in the kidney tissue is reduced with the progress of the disease and the decrease in its activity has a correlation with the hypocalcaemia.

毛竹香豆酸-3-羟基化酶基因的克隆与表达研究

香豆酸-3羟-基化酶(C3H)是木质素单体合成中的一个关键酶。采用文库筛选的方法从毛竹萌发种子全长cDNA文库中克隆了1个C3H基因,命名为PheC3H。PheC3H基因全长1842bp,包括1个完整的开放阅读框,编码1个由513个氨基酸残基组成的蛋白(PheC3H),C端具有1个细胞色素P450蛋白(CYP450)特异的保守结构域。相似性比对显示,PheC3H与其他植物的C3H有着较高的相似性,其中与小麦的CYP98A蛋白相似性最高,达91.2%;聚类分析表明,PheC3H属于CYP98A亚家族蛋白;实时定量PCR结果表明,PheC3H在一年生实生毛竹叶鞘中表达量最高,其次是在根中,在叶片和茎中表达量相对较低。

杨树木质素合成酶c3h基因的克隆及其序列分析

根据CYP98A3(GenBank登录号为AY064170)cDNA序列保守区设计引物,从正在分化的2年生‘欧美杨107’次生木质部提取的总RNA经RT-PCR扩增出一基因片段,然后与pMD20-T载体连接。重组质粒经限制性内切酶酶切、特异引物PCR扩增和测序鉴定。结果表明,扩增片段长度为994bp,其中包含一个长为495bp的开放阅读框,编码的氨基酸序列(登录号为CAP47423)与NCBI中AY064170的CYP98A3编码氨基酸序列的相似性为91%,初步断定其为CYP98A3家族中的一员,其cNDA序列GenBank登录号为AM920690。参照国内外已发表的部分植物的c3h基因序列,构建了c3h的遗传进化树,分析了不同植物c3h的遗传进化关系。

苎麻C3H基因的克隆、生物信息学分析及表达分析

旨在为后期验证C3H基因编码蛋白功能并最终实现低木质素优良苎麻品种的基因工程育种奠定基础。基于苎麻高通量测序信息,利用RACE技术克隆C3H基因全长序列,对其进行生物信息学分析;并利用荧光定量技术对苎麻C3H的表达模式进行研究。获得了全长为1940 bp的苎麻C3H基因c DNA序列Bn C3H-1(Gen Bank登录号为KY078743)。Bn C3H-1基因编码蛋白理化性质和结构的分析表明,该基因编码一个含511个氨基酸,分子量为57.80 k D的亲水性蛋白,它可能定位在内质网上。氨基酸序列和结构分析显示Bn C3H-1有一个保守区域,即P450结构域。系统进化分析表明,该基因与巨桉和苦荞C3H基因具有很高的同源性。

苎麻香豆酸–3–羟化酶基因的原核表达

以湘苎3号为材料,采用RT–PCR克隆,获得苎麻香豆酸–3–羟化酶(Bn C3H)基因的开放阅读框(ORF)序列,测序正确后将该基因片段连接到原核表达载体p ET–22b中,将构建成功的重组子p ET–22b–C3H转入BL21并诱导表达。结果显示:Bn C3H基因ORF区大小为1 536 bp,编码511个氨基酸,推测蛋白相对分子质量为57 800,理论等电点为8.61,编码的蛋白质为亲水性蛋白质;重组子在IPTG浓度为1 mmol/L时,融合蛋白的表达量在诱导2 h后达到最高,经IPTG诱导和SDS–PAGE检测,获得的融合蛋白与预期蛋白相符。

反相HPLC同时测定白花蛇舌草口服液中4种组分含量

目的：建立反相HPLC同时测定白花蛇舌草口服液中3，4-二羟基苯甲酸甲酯（Ⅰ）、对香豆酸（Ⅱ）、阿魏酸（Ⅲ）和反式6-0-对香豆酰鸡屎藤苷甲酯（Ⅳ）的含量。方法：采用Diamonsil^TM C18柱（4．6mm×250mm，5μm）；流动相A乙腈，B甲醇-水-冰醋酸（5：95：0.25），梯度洗脱：0～20min，1％～16％A；20～42min，16％A；42～46min，16％～20％A；46～65min，20％A。检测波长265nm；流速1．0mL·min^-1。结果：Ⅰ，Ⅱ，Ⅲ与Ⅳ分别在2．1～105（r=0．9998），3．5～175（r=0．9998），1．72～86（r=0．9999），4．0～200mg·L^-1（r=1．0000）线性关系良好，方法平均回收率分别为99．9％，97．9％，98．6％，98．1％。结论：方法简便快速、准确度高，可用于白花蛇舌草口服液中4种组分的同时测定。

Changes of chlorogenic acid content and its synthesis-associated genes expression in Xuehua pear fruit during development

According to synthetic pathway of plant chlorogenic acid （CGA）, the expression patterns of genes encoding enzymes that are associated with CGA synthesis were studied in normally developed Xuehua pear fruit. The study demonstrated that CGA content in peel and flesh of Xuehua pear decreased as fruit development progressed, with a higher level in peel. The expression levels of PbPAL 1, PbPAL2, PbC3H, PbC4H, Pb4CL 1, Pb4CL2, Pb4CL6, PbHC T1 and PbHC T3 genes decreased in fruit, which was consistent with the pattern of variation in CGA content. That indicated that these genes might be key genes for influencing fruit CGA synthesis in Xuehua pear. However, Pb4CL7 gene expression profile is not consistent with variation of CGA content, hence, it may not be a key gene involved in CGA synthesis.

当归香豆酸-3-羟化酶基因ASC3H的克隆及表达模式与阿魏酸含量相关性分析

目的:对当归中香豆酸-3-羟化酶基因(C3H)全长进行克隆,进行生物信息学与基因表达模式分析,结合当归根不同组织部位阿魏酸的含量,推测ASC3H基因功能。方法:基于前期转录组测序结果,通过实时荧光定量聚合酶链式反应(Real-time PCR)克隆全长cDNA序列,运用生物信息学方法分析该序列特征,并利用Real-time PCR和高效液相色谱法(HPLC)分别测定当归不同组织中ASC3H基因表达量及阿魏酸含量。结果:克隆获得当归ASC3H全长基因(登录号MN2550298),开放阅读框(ORF)长度为1530 bp,编码509个氨基酸,理论相对分子质量为57.86 kDa,等电点8.36,为不含信号肽的亲水性不稳定蛋白,定位于叶绿体中,有跨膜区,具有多个磷酸化位点,含有细胞色素P450的保守结构域CGYDWPKGYGPIINVW_P450(383~399 aa);多重氨基酸序列比对分析结果显示ASC3H与其他植物的C3H基因具有较高相似性,并与同科植物大阿米芹的同源性最高;Real-time PCR结果显示ASC3H基因在当归不同组织的表达量不同;HPLC结果表明阿魏酸成分在当归根皮层组织含量最高,周皮、中柱次之。结论:成功获得当归C3H基因序列,分析了其蛋白结构与特异性表达特征,推测ASC3H可能参与当归阿魏酸的合成,为进一步研究C3H基因在当归阿魏酸生物途径中的功能提供了参考依据,也为遗传改良当归品质奠定了研究基础。

The Pathogen and Wound Induces Expression of Genes Related to Proanthocyanidins (PAs) Synthesis in Cotton Leaves

Responses to biotic and abiotic stress have been extensively studied in plants. In the current proteomic study, the cotton (Gossypium hirsutum L.) seedlings were infected with Verticillium dahliae by root-dip inoculation using suspension of fungal conidia. The different proteins were analyzed by two-dimensional gel elactrophoresis (2-DE), and flavanone 3-hydroxylase (F3H) showed a significantly up-regulation in cotton leaf after V. dahliae infection. Further research revealed F3H and the downstream genes of F3H in proanthocyanidins (PAs) biosynthesis were also significantly induced and showed coordinate expression patterns during wounding. The results indicate that PAs in cotton act an important role in response to infection V. dahliae and wounding.

甘薯香豆酸3'-羟化酶基因的克隆和表达分析

香豆酸3'-羟化酶(coumarate-3-hydroxylase,C3H)是绿原酸代谢途径中的关键酶之一。为了揭示甘薯绿原酸生物合成途径和挖掘绿原酸合成相关基因,本研究从甘薯中克隆一个C3H的同源基因IbC3H。该基因全长1656 bp,包含一个1530 bp的完整开放阅读框,编码509个氨基酸。IbC3H蛋白具有典型P450家族蛋白结构特征,与牵牛花(Ipomoea nil)、马铃薯(Solanum tuberosum)、浆果状椒(Capsicum baccatum)和烟草(Nicotiana attenuate)中相应蛋白的同源性分别达到97.2%、85.4%、84.8%和84.8%。IbC3H启动子序列中含有脱落酸、赤霉素、乙烯和干旱等多个激素和胁迫响应元件,以及髓细胞组织增生病毒癌基因同源物(MYB)、碱性螺旋-环-螺旋蛋白(bHLH)、和WRKY蛋白等转录因子结合元件。荧光定量PCR结果显示,IbC3H在甘薯叶片中表达量最高,在茎中次之,而在块根中的表达量最低,与甘薯不同组织绿原酸含量正相关。本研究结果为进一步揭示IbC3H在绿原酸生物合成中的功能提供了依据。

丹参香豆酸-3-羟化酶基因(SmC3H)的生物信息学及其组织表达模式分析

通过分析丹参转录组数据库(SRX021907)得到一条香豆酸-3-羟化酶(C3H)基因,blast比对发现该基因为丹参C3H(EU358957.1),该基因c DNA全长1 704 bp,包括1个1 503 bp的开放阅读框,编码508个氨基酸。生物信息学分析表明,其编码蛋白的分子量为57.21 k D,等电点为8.53,含琢-螺旋,延伸链和无规则卷曲,具有1个细胞色素P450蛋白(CYP450)特异的保守结构域。聚类分析表明其与芝麻的亲缘关系最近。组织特异性表达分析结果表明,该基因在茎中的表达量最丰富,其次是叶和花,根中最低。

Vitamin D_3 increased intestinal Na/Pi-Ⅱb and CYP27B1 mRNA level in rats fed low-phosphorus diets

The objective of the study was to determine the role of vitamin D3(VD3) in regulating adaptation and mechanism of rats to low-phosphorus(P) diets. Rats were assigned to 4 diets containing 0.2%, 0.4%, 0.6%,or 0.8% P consisting of 5 replicate cages with 6 rats per replicate cage and fed for 7 days. Four rats from each replicate cage were treated with ethane-1-hydroxy-1,1-diphosphonicacid, tetrasodium salt(EHDP)and 2 rats remained untreated. Twelve hours prior to preparation on d 7, two of the EHDP-treated rats received an intraperitoneal injection of VD3 [1,25-(OH)_2 D_3] at 600 ng per kg body weight, while two rats did not receive the injection. Rats that did not receive VD_3 injection had decreased(P < 0.001) P absorption, but injection of VD3 resulted in increased(P < 0.001) absorption. The effect of VD3 injection was greater(P < 0.001) for rats fed 0.2% P diet than rats fed 0.8% P diet in ileum. Sodium dependent phosphate cotransporter type IIb(Na/Pi-IIb) and 25-hydroxyvitamin D 1-α hydroxylase(CYP27 B1) mRNA level showed the same trend with P absorption. Serum concentration of VD3 and la-hydroxylase activity in rats fed 0.2% P diet were lower than those fed 0.8% P diet. The injection of VD3 increased(P < 0.001)serum concentration of VD3 and la-hydroxylase activity. Thus, VD3 increased Na/Pi-Ⅱb and CYP27 B1 mRNA level and improved serum concentration of VD3 and la-hydroxylase activity in rats fed low-P diets.

Ce(NO_3)_3与4-香豆酸对黑曲霉合成黄酮类化合物的影响

培养基中添加Ce(NO_3)_3和4-香豆酸,研究黑曲霉合成黄酮类化合物的变化。超声波法提取黑曲霉胞内黄酮类化合物,分光光度法表征黄酮总量(芦丁计),高效液相色谱(HPLC)法分析Ce(NO_3)_3对黑曲霉黄酮类化合物合成的影响。Ce(NO_3)_3和4-香豆酸都可以提高黑曲霉胞内黄酮产量,与对照相比差异均显著(p<0.05);Ce(NO_3)_3浓度分别为20,25和30 mg·L^(-1)时,黑曲霉黄酮类产量与添加4-香豆酸相比差异均显著(p<0.05),表明此浓度下Ce(NO_3)_3促进黑曲霉合成黄酮类化合物能力比前体物更强;HPLC结果表明与对照相比,Ce(NO_3)_3(25 mg·L^(-1))可以提高黑曲霉芦丁(2.3倍)、大豆苷元(2.5倍)、槲皮素(2.7倍)的产量,降低了山奈酚的产量(1/2)。Ce(NO_3)_3和4-香豆酸都可以提高黑曲霉合成黄酮类化合物总量,可以考虑在发酵产黄酮类工业应用中添加Ce(NO_3)_3做刺激因子,提高黄酮类化合物产量。

大花益母草的化学成分研究

目的研究大花益母草Leonurus macranthus的化学成分。方法采用硅胶、Sephadex LH-20、ODS柱色谱和半制备HPLC等色谱技术进行分离纯化,并根据理化性质和波谱数据鉴定化合物的结构。结果从大花益母草地上部分70%丙酮提取物的二氯甲烷萃取部位分离鉴定了19个化合物,分别鉴定为(+)-丁香脂素(1)、(+)-1-羟基丁香脂素(2)、rayalinol(3)、erythro-guaiacylglycerol-β-O-4′-coniferyl ether(4)、(7R,7′R,7″S,8S,8′S,8″S)-3′,4″-dihydroxy-3,5,4′,5″-tetramethoxy-7,9′:7′,9-diepoxy-4,8″-oxy-8,8′-sesquineo-lignan-7″,9″-diol(5)、(7R,7′R,7″S,8S,8′S,8″S)-4′,5″-dihydroxy-3,5,3′,4″-tetramethoxy-7,9′:7′,9-diepoxy-4,8″-oxy-8,8′-sesquineo-lignan-7″,9″-diol(6)、芫花素(7)、3′-羟基芫花素(8)、圣草酚(9)、异莨菪亭(10)、对香豆酸(11)、咖啡酸甲酯(12)、反式-阿魏酸(13)、丁香醛(14)、香草酸(15)、oct-1-en-3-ylβ-glucopyranoside(16)、5-羟基-2-吡咯烷酮(17)、pterolactam(18)和烟酰胺(19)。结论化合物1~6和9~19为首次从益母草属植物中分离得到,化合物7和8为首次从大花益母草中分离得到。

竹根化学成分的研究(Ⅱ)

目的研究竹根的化学成分。方法采用色谱方法分离化学成分,根据波谱数据及理化常数分析进行结构鉴定。结果分离鉴定了9个化合物,分别为3-(p-羟基苯)-(2,3)环氧-1-丙酸(Ⅰ)、2,3-二羟基-1-(4-羟基-3,5-二甲氧基苯基)-1-丙酮(Ⅱ)、(+)-鹅掌揪树脂醇B(Ⅲ)、原儿茶醛(Ⅳ)、原儿茶酸(Ⅴ)、荭草苷(Ⅵ)、牡荆苷(Ⅶ)、异荭草苷(Ⅷ)、异牡荆苷(Ⅸ)。结论化合物Ⅰ为新化合物,命名为环氧香豆酸(epoxy coumaric acid)。

丹红注射液多元指纹图谱及多成分定量分析研究

目的采用HPLC-UV-MS法建立丹红注射液多元指纹图谱，并对其中9种主要药效成分同时定量分析。方法采用AtiantisT3色谱梓（250mm×4．6mm，5μm），流动相为0．05％甲酸水溶液-50％乙蜻水溶液，梯度洗脱；质谱检测采瑚负离子选择离子（SIM）模式。结果该多元指纹图谱较全面地体现了制剂中2味处方药材片参与红化的化学信息，11批制剂指纹蚓讲相似度均在0．988以上。5-羟甲基糠醛、丹参素钠、原几茶醛、香豆酸、丹酚酸D、迷迭香酸、丹酚酸B、丹酚酸A、山奈酚-3-O-苷香糖9种成分在各自质量浓度范围内呈良好线性关系，r≥0．9990：9种化合物的平均加样回收半分别为（99．0±1．4）％、（102．0±1．7）％、（99．3±1．6）％、（97．6±1．6）％、（100．0±1．8）％、（97．9±1．6）％、（100．5±4．4）％、（100．6±2．0）％、（106．0±4．7）％（n=9）；方法重复性的RSD均小于1．45％；测定的11批制剂中9种成分总量在2．61～3．06mg／mL。结论该方法简单、准确、重复性好，多元指纹图谱结合定量测定能更全面地反映丹红注射液的质量，可用于制剂的质量控制。

大苞鞘石斛化学成分及细胞毒活性研究

目的研究大苞鞘石斛Dendrobium wardianum的主要化学成分及部分化合物的肿瘤细胞毒活性。方法应用多种色谱分离技术,结合半制备液相色谱对化合物进行分离纯化,通过核磁共振谱、质谱、薄层色谱对照等鉴定化合物结构;MTS法检测部分化合物的肿瘤细胞毒活性。结果从大苞鞘石斛中分离得到16个化合物,分别鉴定为愉悦石斛素(1)、钩状石斛素(2)、反式-阿魏酸二十烷基酯(3)、反式-阿魏酸二十二烷基酯(4)、对香豆素酸二十四烷基酯(5)、4-羟基-3-甲氧基肉桂醛(6)、二氢松柏醇(7)、3,7-二甲氧基-5-羟基-1,4-菲醌(8)、3-乙氧基-5-羟基-7-甲氧基-1,4-菲醌(9)、4-甲氧基-9,10-二氢菲-2,7-二醇(10)、5,7,4′-三羟基二氢黄酮(11)、5,7,4′-三羟基-3′,5′-二甲氧基二氢黄酮(12)、苜蓿素(13)、3,5,4′-三甲氧基-4-羟基联苄(14)、3,5,3′-三甲氧基-4′-羟基联苄(15)和3,3′-二甲氧基-4,5′-二羟基联苄(16)。对所得化合物3、6、8、9、11、14、15进行了肿瘤细胞毒活性筛选,其中化合物6、8有一定的体外肿瘤生长抑制活性。结论化合物3、5、6为首次从石斛属植物中分离得到,化合物10、11、14、15为首次从大苞鞘石斛中分离得到;大苞鞘石斛活性成分有待挖掘。