木质素生物合成中C3H/HCT的研究进展

木质素是由三种不同的木质素单体聚合而成的,其含量和单体组成与植物体的综合利用密切相关。木质素单体的生物合成途径涉及到许多酶的参与,C3H/HCT是发现较晚的两个酶,在从对-香豆酰辅酶A（p-coumaroyl CoA）到咖啡酰辅酶A（caffeoyl CoA）的羟基化过程中起作用,是控制木质素H-单体与G/S-单体相互转化的关键酶。该文主要对C3H/HCT的研究进展及在木质素生物合成中的作用进行了阐述。     

白桦CESA7基因启动子的克隆与表达分析

克隆得到了一个白桦纤维素合成酶基因（CESA7）GenBanK登录号（EU591531）启动子序列,通过序列分析发现该启动子含有多个不同功能的顺式作用元件,包括光响应元件、激素响应元件、叶片形态发育元件等,推测该启动子在白桦生长发育过程中具有关键作用。将BpCESA7启动子克隆至带有GUS报告基因的植物表达载体,命名为proBpCESA7-121-GUS,并利用农杆菌介导方法侵染白桦和拟南芥,然后通过GUS组织化学染色观察BpCESA7基因启动子的组织表达特性。结果在白桦的根、茎、叶和拟南芥的根,叶,萼片、雌蕊中检测到了GUS活性,说明BpCESA7基因启动子具有启动子活性,并且在白桦的根和叶中染色最深,表明BpCESA7基因在白桦根和叶中表达量较高,并且其存在组织表达特异性。     

拟南芥AHAl基因启动子的表达特性分析

从拟南芥中分离到编码质膜H^＋-ATPase的AHA1基因启动子序列813bp,并构建了此种启动子与GUS嵌合的重组载体,通过农杆菌介导转化拟南芥,得到转基因拟南芥。用组织化学方法分析AHA1基因启动子驱动GUS转基因拟南芥的结果表明,GUS基因在转基因的拟南芥根、茎、叶、花和荚的维管组织中均有表达,且不同发育时间内GUS基因表达也不同。以上研究表明拟南芥AHA1基因可能参与植物的生长发育与抗逆胁迫反应。     

拟南芥非特异性磷脂酶C4基因表达模式的研究

拟南芥非特异性磷脂酶C4（AtNPC4）具有降解磷脂酰胆碱（PC）,产生二酰甘油（DAG）和磷酸胆碱的活性。本研究从拟南芥基因组中分离了NPC4基因起始密码子上游1 379bp的启动子序列,与GUS报告基因融合后转化拟南芥,获得转基因植株。GUS组织化学染色表明,AtNPC4基因主要在处于衰老过程中的叶片中高水平表达,在根、茎、种荚和花中也有一定程度的表达,这种表达模式与RT-PCR结果相一致。另外,通过RT-PCR发现,AtNPC4基因在转录水平上受脱落酸的诱导,但不受水杨酸和茉莉素诱导。     

玉米脱氧麦根酸分泌通道蛋白基因YS3启动子的克隆与启动活性分析

缺铁是世界范围内农业生产面临的严重问题,玉米通过分泌脱氧麦根酸(2’-deoxymugineic acid, DMA)吸收利用土壤中的难溶性铁。为探明玉米DMA分泌通道蛋白基因YS3的表达和调控机制,本文通过克隆获得长为2813 bp的YS3基因启动子,该序列含有大量TATA-box、CAAT-box等启动子基本元件,以及光响应、激素调控等多个顺式调控元件;构建YS3启动子驱动GUS基因的植物表达重组载体pCAMBIA-YS3GUS,利用农杆菌介导转化拟南芥,获得pYS3::GUS转基因植株,对转基因植株进行GUS组织化学染色,并通过石蜡切片技术对转基因植株进行组织观察,分析pYS3::GUS转基因植株中YS3基因启动子的活性。结果表明,YS3启动子主要驱动GUS基因在拟南芥根部表达,且主要集中在根部表皮细胞,机械损伤可激发YS3启动子活性,驱动GUS基因在损伤临近部位表达。本研究对于理解玉米DMA分泌的分子调控机理方法od3 gmaigensuan有重要意义。     

拟南芥γ-生育酚甲基转移酶(γ-TMT)启动子的分离及表达特性分析

维生素E是一类人体所必需的脂溶性的维生素,具有重要的生理功能。γ-生育酚甲基转移酶(γ-TMT)是维生素E生物合成途径中的关键酶之一,催化γ、δ-生育酚甲基化,生成α、β-生育酚。从拟南芥中分离了γ-生育酚甲基转移酶基因1552bp的启动子序列,构建了含有该启动子和GUS报告基因的植物表达载体,通过农杆菌介导转化拟南芥,获得了转基因植株。GUS组织化学染色结果表明,在γ-TMT启动子的驱动下,报告基因GUS在拟南芥的叶、茎以及花均有表达,且在茎尖、雄蕊和幼叶中表达最强,而在根、种子和种荚中则没有检测到GUS基因的表达,表明γ-TMT基因可能仅在拟南芥某些组织中特异性高表达。     

拟南芥ats1A基因启动子的克隆和功能分析

通过PCR扩增,从拟南芥中克隆出ats1A基因启动子(包括叶绿体转运肽),将此启动子与GUS基因相连构建植物瞬时表达载体,用基因枪法将之导入烟草进行瞬时表达。GUS基因检测分析表明,ats1A基因启动子能特异的启动GUS基因在烟草叶片中高效表达。     

拟南芥钙调素结合蛋白基因启动子的克隆及表达

采用PCR技术从拟南芥中克隆了SCBP60g基因的启动子,并与GUS报告基因融合构建重组表达载体,转化野生型拟南芥,对获得的转基因株系进行GUS组织染色,从基因调控水平上探讨其在功能方面的差异。结果显示:SCBP60g基因的启动子能指导GUS报告基因在拟南芥的根、茎、叶和花中表达,并且在这些部位的维管束表达较强。这种表达方式与LCBP60g基因的启动子指导的GUS基因组织化学染色有差异,表明这个启动子的表达调控具有一定的特异性。     

拟南芥2-甲基-6-叶绿基-1,4-苯醌甲基转移酶启动子的分离及表达特性分析

维生素E是一类人体必需的脂溶性抗氧化剂, 具有重要的生理功能。2-甲基-6-叶绿基-1,4-苯醌甲基转移酶(MPBQ MT)是天然维生素E合成途径中的关键酶之一, 催化MPBQ甲基化, 生成DMPBQ。从拟南芥分离了MPBQ MT基因1018bp的启动子序列, 构建了含该启动子和GUS报告基因的植物表达载体, 通过农杆菌介导转化拟南芥, 获得了转基因植株。GUS组织化学染色结果表明, 在MPBQ MT启动子驱动下, 报告基因GUS在拟南芥的茎、叶、花萼、雄蕊、种荚均有表达, 且在茎、叶、种荚中表达量较高, 而在根、花瓣和种子中则没有观察到GUS基因的表达, 表明MPBQ MT基因可能仅在拟南芥幼嫩茎、叶、种荚等绿色组织中特异性高表达。     

白桦APETALA2基因启动子的克隆及其表达分析

APETALA2（AP2）转录因子亚家族普遍存在于植物中,参与植株的生长发育、胁迫应答和多种生理生化反应的信号传导。本研究从白桦（Betula platyphylla Suk.）基因组中克隆了AP2基因2 308 bp的启动子序列,生物信息学分析发现,该序列除具有TATA-box和CAAT box等高等植物普遍具有的保守元件外,还具有大量光响应元件和激素响应元件,如响应赤霉素、脱落酸、茉莉酸甲酯等的元件。将白桦AP2基因启动子克隆至pBI121-35S：：GUS植物表达载体中,命名为pBI121-proAP2：：GUS,用农杆菌介导法侵染白桦和拟南芥,并进行GUS染色分析,结果表明AP2基因启动子驱动下的GUS报告基因在整个拟南芥中都表达,在白桦的营养器官和雌花种翅及花柄中也有表达,说明其具有启动活性,可能参与该器官的发育。     

白桦BpSPL2基因启动子的克隆及表达分析

SPL(SQUAMOSA promoter-binding protein-like)是植物特有的转录因子,研究表明其在参与发育阶段转变、花和果实发育等方面起着重要作用。利用PCR技术从白桦基因组DNA中扩增获得BpSPL2基因上游1 960 bp启动子序列,使用PLACE和Plant CARE在线软件分析序列,发现BpSPL2基因启动子序列中含有与开花、非生物胁迫及激素响应等相关的顺式作用元件,暗示其在植物的生长发育和胁迫应答中起重要作用。进而构建了BpSPL2基因启动子驱动GUS报告基因的植物表达载体,并利用农杆菌介导将其瞬时转化至白桦和拟南芥,通过GUS组织化学染色检测BpSPL2基因启动子的组织表达特性,结果表明BpSPL2基因启动子具有启动子活性,能够驱动GUS基因在白桦和拟南芥中表达;而其表达活性在白桦的叶片、芽及根部中较强,在拟南芥的花药、雌蕊和叶片较强,为进一步研究白桦BpSPL2基因的表达调控及其功能分析提供参考。     

Oxidation of 1,4-alkanediols into γ-lactones via γ-lactols using Rhodococcus erythropolis as biocatalyst

Whole cells of Rhodococcus erythropolis DSM 44534 grown on ethanol, (R)- and (S)-1,2-propanediol were used for biotransformation of racemic 1,4-alkanediols into γ-lactones. The cells oxidized 1,4-decanediol (1a) and 1,4-nonanediol (2a) into the corresponding γ-lactones 5-hexyl-dihydro-2(3H)-furanone (γ-decalactone, 1c) and 5-pentyl-dihydro-2(3H)-furanone (γ-nonalactone, 2c), respectively, with an EE(R) of 40–75%. The transient formation of the γ-lactols 5-hexyl-tetrahydro-2-furanol (γ-decalactol, 1b) and 5-pentyl-tetrahydro-2-furanol (γ-nonalactol, 2b) as intermediates was observed by GC–MS. 1,4-Pentanediol (3a) was transformed into 5-methyl-dihydro-2(3H)-furanone (γ-valerolactone, 3c) whereas (R)- and (S)-2-methyl-1,4-butanediol (4a) was converted to the methyl-substituted γ-butyrolactones 4-methyl-dihydro-2(3H)-furanone (4c₁) and 3-methyl-dihydro-2(3H)-furanone (4c₂) in a ratio of 80:20 with a yield of 55%. Also cis-2-buten-1,4-diol (5a) was transformed resulting in the formation of 2(5H)-furanone (γ-crotonolactone, 5c). At the higher pH values of 8.8 the yield of lactone formed was improved; however, the enatiomeric excesses were slightly higher at the lower pH of 5.2.     

双管基因枪介导的基因瞬时表达技术在拟南芥中的应用

基因瞬时表达是植物中研究目标基因功能的常用手段。在模式植物拟南芥(Arabidopsis thaliana)中, 相比原生质体和农杆菌介导的基因异源表达技术, 利用粒子轰击进行基因瞬时表达一直鲜有报道。其主要原因是拟南芥叶型相对较小、基因枪操作相对烦琐以及基因表达效率差异较大。该研究通过优化双管基因枪系统, 在营养生长旺盛的拟南芥莲座叶中实现GFP和GUS基因高效表达。同时, 通过GUS报告基因明确了坏死诱导因子BAX、Avh238和ATR13/Rpp13激发拟南芥细胞坏死的表型。但在本氏烟(Nicotiana benthamiana)中明显诱导细胞坏死的Avrblb1/RB基因对, 在拟南芥中却丧失了诱导细胞坏死的活性。由于双管基因枪系统每次轰击时设置平行对照, 可有效降低转化实验中的样本变异度, 为拟南芥及其突变体研究中准确评价基因功能和高通量筛选目标基因提供新的技术参考。     

Xylem-specific expression of a GRP1.8 promoter::4CL gene construct in transgenic tobacco

Lignin is a complex aromatic polymer of vascular plants that provides mechanical strength to the stem and protects cellulose fibres from chemical and biological degradation. 4-Coumarate:CoA ligases (EC 6.2.1.12) are key enzymes for the biosynthetic pathway of monolignols which is an important complex aromatic polymer for lignin biosynthesis and tree growth. Recently, 4-coumarate:CoA ligase has been used as exogenous gene in transgenic plants to genetically modify the lignin biosynthesis pathway. Since most lignin is produced in the vascular cells, a tissue-specific-expressed promoter in the vascular cell would be important and useful to change and modify the content of lignin. Here we report the existence of a promoter of GRP1.8 (the glycine-rich protein 1.8) in Sopho japonica L. (GenBank accession number AF250149) and studies on its function in transgenic tobacco. The promoter activity was analyzed in transgenic tobacco plants by histochemical staining of GUS gene expression driven by a 613-bp sjGRP1.8p promoter sequence. In sjGRP1.8p-GUS transgenic plants, intense GUS staining was detected in the xylem of the stem. To further investigate the regulation of the tissue-specific expression of the 4CL1 gene, we analyzed the activity of the 4CL1 gene which is sense orientated with the sjGRP1.8p promoter in transgenic tobacco. The Pto4CL1 gene was expressed in the stem of transgenic tobacco. The activity of the 4CL1 enzyme was increased 1–2-fold in the stem but not increased in the leaves of transgenic tobacco. In comparison with the control plants, the content of lignin was increased 25% in the stem but there was no increase in the leaves of transgenic tobacco.     

白桦BpPIN3基因启动子序列及应答特性分析

PIN蛋白家族作为植物中重要的生长素外排载体家族,在植物生长和发育过程中表现出广泛的生理效应。为了进一步了解BpPIN3的功能,探究其在白桦（Betula platyphylla）发育过程中及其对不同激素信号和非生物胁迫的响应,采用生物信息学方法分析白桦BpPIN3启动子序列。以1年生和2年生白桦无性系苗木的根、茎、叶和顶芽为材料进行组织部位表达模式分析。以白桦幼苗为材料,用100 μmol·L^-1生长素（IAA）、100 μmol·L^-1赤霉素（GA₃）、200 μmol·L^-1脱落酸（ABA）和长光照条件下分别进行激素诱导和光胁迫处理,并取激素处理后0、2、4、8、16、24、48 h以及光胁迫后0、1、3、6,12、24、48、72 h时的白桦叶片和根提取RNA,利用qRT-PCR技术分析BpPIN3基因的表达情况。结果显示：BpPIN3启动子序列包含赤霉素、脱落酸、茉莉酸甲酯等不同类型的生长素响应元件,以及多个与逆境相关的顺式调控元件。BpPIN3在不同生长年份白桦的多个组织部位都有表达,尤其在叶片中表达量较高,并且所有组织部位中BpPIN3第二年的表达量均高于第一年。BpPIN3基因在不同处理条件下,不同部位间的相对表达量的变化存在一定差异,IAA及GA能够诱导白桦叶片组织细胞中的BpPIN3上调表达;而在ABA处理下除16、48 h外,BpPIN3基因表现出与IAA处理下相反的表达模式。在根组织中,IAA、GA₃及ABA均能诱导BpPIN3的表达。在叶片组织中,遮光胁迫诱导了BpPIN3基因的表达;在根组织中,随着处理时间的推移,12 h开始BpPIN3基因的相对表达量均显著高于对照（0 h）。根据试验结果,推测BpPIN3基因在白桦生长发育过程,以及IAA、GA₃和ABA信号转导途径和植物光响应过程中发挥重要调控作用。     

Opioid-binding cell adhesion molecule (OBCAM)-related clones from a rat brain cDNA library.

The AKin10 gene from Arabidopsis thaliana encoding a putative Ser/Thr protein kinase (PK) has been isolated and characterized. The AKin10-encoding gene is located on a genomic 5.4-kb BamHI fragment and contains ten introns, one being located in the 5' untranslated region. The deduced amino acid sequence of AKin10 is 65% identical over the catalytic domain to the yeast PK (SNF1). SNF1 is essential for the derepression of many glucose-repressible genes, including Suc2 which encodes invertase. Southern blot hybridization experiments suggested the presence of one copy of the gene per haploid genome of A. thaliana. Northern hybridization experiments indicated that this gene is expressed in roots, shoots and leaves. AKin10 may play an important role in a signal transduction cascade regulating gene expression and carbohydrate metabolism in higher plants.     

Characterization of a cinnamoyl-CoA reductase that is associated with stem development in wheat

Cinnamoyl-CoA reductase (CCR) is responsible for the CoA ester to aldehyde conversion in monolignol biosynthesis, which diverts phenylpropanoid-derived metabolites into the biosynthesis of lignin. To gain a better understanding of lignin biosynthesis and its biological function, a cDNA encoding CCR was identified from wheat (Triticum aestivum L.), and designated as Ta-CCR1. Phylogenetic analysis indicated that Ta-CCR1 grouped together with other monocot CCR sequences while it diverged from Ta-CCR2. DNA gel-blot and mapping analyses demonstrated that Ta-CCR1 is present as a single copy gene in the wheat genome. Recombinant Ta-CCR1 protein converted feruloyl CoA, 5-OH-feruloyl CoA, sinapoyl CoA, and caffeoyl CoA, but feruloyl-CoA was the best substrate, suggesting the preferential biosynthesis of G-type lignin. RNA gel-blot analysis indicated that Ta-CCR1 was highly expressed in stem, with lower expression in leaves, and undetectable expression in roots. CCR enzyme activity was increased progressively along with the lignin biosynthesis and stem maturity. During stem development, Ta-CCR1 mRNA levels remained high at elongation, heading, and milky stages in the wheat H4564 cultivar, while they declined dramatically at the heading and milky stages in stems of the C6001 cultivar. Ta-CCR1 mRNA expression paralleled extractable CCR enzyme activity in these two cultivars. Furthermore, high Ta-CCR1 mRNA levels and high CCR enzyme activity in wheat stem were correlated with a higher Klason lignin content and greater stem mechanical strength in the H4564 cultivar. This suggests that Ta-CCR1 and its related CCR enzyme may be involved in the regulation of lignin biosynthesis during stem maturity and then contributes to stem strength support in wheat.