盐胁迫下甘菊叶片中BADH基因表达及甜菜碱含量的变化

利用实时荧光定量PCR(real-time PCR)和雷氏盐紫外分光光度计法分别测定了400mmol/L NaCl胁迫处理0,0.5h,2h,12h,1d,2d,4d,6d,8d,10d和12d,甘菊叶片中BADH基因表达和甜菜碱含量的变化,并讨论了二者间的相互作用关系。试验结果表明,在高盐胁迫下甘菊叶片中BADH基因和甜菜碱含量均呈现先上升后下降的趋势。在处理初期(0.5h和2h)BADH基因的表达量与对照相比略有下降,此后随处理时间的增加BADH基因表达持续增大,在胁迫处理6d时BADH基因表达量最大为对照的4.6倍,6d之后BADH基因表达量逐渐降低。甜菜碱含量在NaCl处理0.5h突然增大以应对胁迫反应,此后其含量出现了小幅的震荡上升,在胁迫处理4d时达到了最大值,此后随胁迫处理时间的增加甜菜碱含量逐渐降低。二者之间的变化并不是同步的,而是存在滞后性,分析认为甘菊叶片中BADH基因表达与甜菜碱积累间存在相互抑制的作用。     

钾离子对盐诱导菠菜甜菜碱累积的影响

无钾条件下盐胁迫处理菠菜幼苗所诱导的甜菜碱积累量及甜菜碱醛脱氢酶（BADH）活性均比含钾条件下盐处理时低。无K 培养离体叶片可引起组织甜菜碱含量下降;用无K 培养或K 通道抑制剂TEA处理菠菜幼苗,BADH活性随处理时间延长出现下降。表明K 可能参与对甜菜碱合成积累及BADH酶活性的调节。     

外源甜菜碱对低温胁迫下香蕉内源甜菜碱合成的影响

以巴西香蕉(MusaAAA Giant Cavendish cv.Brazil)幼苗为试验材料,用不同浓度外源甜菜碱(BT)预处理香蕉幼苗后,置于人工气候箱中模拟低温(7℃)胁迫,分别测定香蕉叶片和根系内源甜菜碱的含量和甜菜碱合成关键酶甜菜碱醛脱氢酶(BADH)活性,以探讨外源甜菜碱对香蕉叶片和根系内源甜菜碱合成的影响.结果显示:7℃低温胁迫16 h后,10 mg/L外源甜菜碱即可极显著提高香蕉幼苗叶片BADH活性,叶片内源BT含量也同步极显著增加,低温胁迫24h后根系内源甜菜碱的含量虽显著高于常温对照,其BADH活性却无显著提升.同时,香蕉幼苗叶片内源BT含量的积累与叶片BADH活性的提高具有显著正相关关系,与根系内源BT含量的增加呈极显著正相关关系,与外源BT浓度无显著相关性.研究表明,外源甜菜碱可促进低温胁迫下香蕉内源甜菜碱的合成和积累,叶片和根系均具有合成内源BT的能力.     

菠菜甜菜碱醛脱氢酶基因在烟草中的表达

质粒pLS9含有1．5kb的编码菠菜甜菜碱醛脱氢酶(BADH)基因。经限制酶切后克隆到植物表达载体的35S启动子和PolyA终止子之间。经农杆菌介导转化烟草,获得90多株抗卡那霉素再生植株。经PCR检测证明60％以上再生植株含有BADH基因。转基因植株经Western blot,BADH酶活性测定,BADH酶活性特异性染色法检查和耐盐性分析,证明菠菜BADH基因在烟草正常表达。在叶绿体和胞液中均有BADH酶存在。转基因植株能耐较高浓度盐。     

盐胁迫下三色苋甜菜碱及有关酶含量的变化

三色苋(Amaranthus tricolor)不同器官中的甜菜碱(GB)含量显著不同.除子叶外,根、茎和叶的GB含量和茎、叶中的胆碱单加氧酶(CMO)含量都因300 mmol/L的NaCl处理而增加.甜菜碱醛脱氢酶(BADH)的表达无论盐处理与否在所有器官中都能检测到,其含量变化不大.当种子发芽时,具备合成GB的能力,CMO含量增加;在此之前未能检测到CMO,也不能合成GB.研究结果表明三色苋响应盐胁迫而合成GB的关键酶是CMO.     

盐生植物中亚滨藜甜菜碱醛脱氢酶(AcBADH)基因的表达及其启动子的分离

克隆了盐生植物中亚滨藜 (Atriplexcentralasiat icaIljin)甜菜碱醛脱氢酶 (AcBADH)cDNA ,推测的氨基酸序列与其它物种的BADH有较高的同源性。Northern杂交结果表明 ,ABA 1 0 0 μmol L和NaCl40 0mmol L处理 48h后 ,BADH的表达增加 1 0倍左右。通过筛选中亚滨藜基因组文库 ,得到了AcBADH的基因组序列。它全长 7.7kb ,包含 1 5个外显子和 1 .2kb启动子区。除了TATA box和CAAT box以外 ,在AcBADH启动子区还发现了一些与胁迫有关的其它元件 ,如GC motif、EIRE、MRE、WUN motif、ABRE和HSE。     

植物甜菜碱合成酶的分子生物学和基因工程

甜菜碱是一种非毒性的渗透调节剂,多种高等植物在盐碱或缺水的环境下在细胞中积累甜菜碱,以维持细胞的正常膨压,甜菜碱的积累使得许多代谢中的重要酶类在渗透胁迫下能保持活性,在植物中甜菜碱由胆碱经两步氧化得到,催化第一步反应的酶是胆碱单加氧酶（CMO）,催化第二步反应的酶是甜菜碱醛脱氢酶（BADH）。本文综述了这两种酶的分子生物学及基因工程研究的最新进展,讨论了基因工程研究的意义。     

植物甜菜碱合成酶的分子生物学和基因工程

甜菜碱是一种非毒性的渗透调节剂。多种高等植物在盐碱或缺水的环境下在细胞中积累甜菜碱 ,以维持细胞的正常膨压。甜菜碱的积累使得许多代谢中的重要酶类在渗透胁迫下能保持活性。在植物中甜菜碱由胆碱经两步氧化得到 ,催化第一步反应的酶是胆碱单加氧酶 (CMO) ,催化第二步反应的酶是甜菜碱醛脱氢酶 (BADH)。本文综述了这两种酶的分子生物学及基因工程研究的最新进展 ,讨论了其基因工程研究的意义。     

甜菜碱对NaCl胁迫下小麦细胞保护酶活性的影响

本文以小麦为材料,研究甜菜碱对NaCl胁迫下小麦幼苗细胞保护酶系统的影响。结果表明,外源甜菜碱能够提高NaCl胁迫下小麦幼苗超氧化物歧化酶、过氧化物酶等细胞保护酶的活性,抑制过氧化作用产物丙二醛的积累,降低叶片质膜透性和盐害对细胞膜的伤害。所有这些变化都有利于提高NaCl胁迫下小麦幼苗细胞膜的稳定性、完整性和对NaCl胁迫的适应性。     

中亚滨藜甜菜碱醛脱氢酶基因的表达特性

根据已发表的几种植物的甜菜碱醛脱氢酶（BADH）基因的同源保守区设计了一对兼并引物,通过RT-PCR方法从中亚滨藜中扩增出BADH基因的近5′端序列,共395bp,与菠菜、山菠菜、甜菜、千穗谷、大麦的BADHcDNA相应片段的同源性较高,以此片段为探针,对中亚滨藜的基因组进行Southern杂交分析,证明该基因可能是单拷贝的。Northern印迹杂交结果表明：NaCl250mmol/L处理的植株的BADHmRNA水平比对照植株约高2倍。说明中亚滨藜中BADH基因的表达受盐诱导。     

Betaine lipids

Diacylglyceryltrimethylhomoserine (DGTS) and diacylglyceryl hydroxymethyltrimethyl-β-alanine (DGTA) belong to a new type of glycerolipids called betaine lipids, which are composed of diacylglycerol andN-permethylated hydroxyamino acids, that are linked by an ether linkage. Betaine lipids are widely distributed in lower plants and algae as well as in some non-photosynthetic microorganisms. They are no longer regarded as “unusual” lipids but are important constituents of membranes of lower organisms. The present state of knowledge of the phylogenetic distribution, the fatty acid composition, the thermal properties, and the biosynthesis of the bataine lipids is briefly summarized in this review in a perspective of a future search for the biological roles and activities of betaine lipids. Recipient of the Botanical Society Award of Young Scientists, 1991.     

Oxidation of Betaine Aldehyde by Betaine Dehydrogenase from Spinach Leaves

Betaine content in leaves of fifteen plant species was determined. The results showed higher betaine levels in those salt-, drought-, and chilling-resistant species. Betaine aldehyde dehydrogenase (BADH, EC 1.2.1.8 ) was isolated and partially purified from spinach leaves. Some properties of this enzyme were studied. BADH was precipitated by 60% saturation of (NH4)2SO4. Its activity was not detected in 70% saturation of (NH4)2SO4. BADH has two isoenzymes. The activity of BADH was quite stable below –80℃. It was inhibited by 0.125–1.0 mol/L NaG1 or KC1 but not by Mn2+ and Mo6+, and slightly increased by Mg2+.     

Betaine distribution in Angiosperms

Over 140 Angiosperm species, included in 45 of the 62 orders listed by Engler, have been investigated for the presence of betaines, which were detected in 86% of the species examined and in 43 of the orders. Moreover, betaines were reported earlier in a further seven of the orders. Thus, it can be concluded that betaines are very widely distributed in Angiosperms. The most commonly detected betaines in the study were glycinebetaine and trigonelline, although others, such as prolinebetaine, trans-4-hydroxyprolinebetaine and pipecolatebetaine were found, although with a very restricted distribution. In the large majority of species tested, betaine levels were low (below 0.1%, dry weight).     

甜菜碱增强长片段PCR的扩增

聚合酶链式反应(PCR)作为一项非常成熟的技术可以用于基因组序列的扩增。普通的PCR技术只适合于短片段DNA的扩增,一般在6kb以下。对于6kb至十几kb甚至几十kb以上的DNA片段的扩增就非常困难。通过添加不同化学物质,发现甜菜碱对长片段PCR的扩增有非常有效的增强作用。通过对玉米总DNA以及质粒DNA的扩增,发现1mol／L到2．5mol／L甜菜碱对改进PCR扩增效果明显。通过添加甜菜碱,可以从玉米基因组中扩增出9kb以上的单拷贝片段,从质粒中扩增出16kb以上片段。经过试验,发现不同GC含量的引物需要使用不同浓度的甜菜碱。甜菜碱可以减少甚至消除长片段PCR中的非特异性扩增。同时,我们发现其它的添加物,如DMSO,甘油,甲酰胺对长片段PCR的作用不明显。     

Betaine lipids in chlorarachniophytes

Evolving from the endosymbiosis of a green algal cell by a filose amoeba or amoeboflagellate, the chimearic chlorarachniophytes combine unique features retained from both of their ancestral units. They have preserved from the endosymbiont only the nucleomorph and chloroplast. Four strains from three genera of this algal class were studied to identify a set of non‐phosphorous‐containing polar lipids and their associated fatty acids using the techniques of positive‐ion electrospray ionization/mass spectrometry (ESI/MS) and electrospray ionization/mass spectrometry/mass spectrometry (ESI/MS/MS). Fourteen non‐phosphorous‐containing polar lipids, classified as betaine lipids were primarily identified as forms of diacylglyceryl‐N,N,N‐trimethylhomoserine (DGTS) and its structural isomer diaclyglycerylhydroxymethyl‐N,N,N‐trimethyl‐β‐alanine (DGTA). Though the number of forms of DGTA and DGTA were roughly equal, DGTS composed more of the polar lipid portion present in three of the strains tested, while the fourth, Lotharella globosa, was dominated by forms of DGTA. In addition, a lipid tentatively identified as diacylglycerylcarboxyhydroxymethylcholine (DGCC) was observed twice in minor amounts. The polar lipid‐associated fatty acids of the aforementioned algal strains generally included dodecanoic acid (12:0), tetradecanoic acid (14:0), hexadecanoic acid (16:0), octadecanoic acid (18:0), octadecenoic acid (18:1), and eicosapentaenoic acid [20:5(n‐3)]. The differences in betaine lipid content among the species studied may allow for further conclusions to be drawn regarding the taxonomy of chlorarachniophytes.     

Betaine improved restriction digestion

Here we report that supplementation of a common compound betaine (1-carboxy-N,N,N-trimethylmethanaminium inner salt) enhances restriction digestion of DNA molecules being resistant to digestion despite the existence of recognition sites. A previous study reported total isostabilization of DNA was achieved in the presence of 5.2M of betaine, however, we have observed the enhancement of restriction kinetics at 0.3M of betaine, therefore, it likely provided some catalytic proficiency to restriction enzymes rather than the induction of DNA conformational changes. Betaine also enhances catalytic efficiency of PCR, and our result of restriction digestion, taken together, suggests potential application of betaine in other enzymatic reactions in an aqueous solution.     

外源甜菜碱与植物抗逆性

甜菜碱是一种季铵型生物碱,广泛存在于动物、植物和微生物体内.甜菜碱在高等植物体内是一种重要的非毒性渗透调节物质,具有稳定生物大分子的结构和功能以及降低逆境条件下渗透失水对细胞膜、酶及蛋白质结构与功能的伤害,从而提高植物对各种胁迫因子的抗性.该文对外源甜菜碱以不同的作用方式(灌根、浸种、喷施叶片)作用于逆境(如干旱、盐碱等)条件下的作物并提高作物抗逆性能进行了综述,经过全面系统的讨论,阐明外源甜菜碱提高逆境下作物抗逆的机理,为甜菜碱的农业利用提供理论依据.     

Levels of Betaine and Betaine Aldehyde Dehydrogenase Activity in the Green Leaves, and Etiolated Leaves and Roots of Barley

The accumulation of betaine and the induction of betaine aldehydedehydrogenase, which catalyzes the last step in the synthesisof betaine, were analyzed in salt-stressed barley leaves. Whenhydroponically grown barley plants were transferred to a mediumthat contained 200 mM NaCl, the levels of both betaine and thetotal extractable betaine aldehyde dehydrogenase activity inthe leaves increased approximately 7-fold and 3-fold when calculatedon the basis of total leaf protein, respectively, over the courseof 7 days. Betaine aldehyde dehydrogenase activity was alsodetected in either etiolated leaves or roots of barley plantsgrown under aseptic conditions. Betaine was detected in bothetiolated leaves and roots at levels that were about 20% ofthat in green leaves when calculated on a fresh weight basis. ¹ This research was supported financially by a research grantfrom the Ministry of Education, Science and Culture (63560080) (Received March 9, 1990; Accepted May 29, 1990)     

Betaine distribution in the Bromeliaceae

Aerial parts of 38 taxa, distributed in 21 genera and all three subfamilies of the Bromeliaceae have been examined for the presence of betaines. Glycinebetaine and trigonelline were isolated from all the plants studied, although the yields of both compounds were low. For glycinebetaine, values (based upon dry weight) varied from 0.0004% for Pitcairnia corallina to 0.087% for Tillandsia usneoides. Trigonelline levels varied from 0.0003 to 0.022%; the highest yields were recorded for Billbergia rubicunda (0.022%) and Vriesia splendens (0.020%).     

Betaine accumulation in salt-stressed sorghum

Two analytical methods for measuring betaine were compared in a study of betaine accumulation in salt-stressed sorghum. Spectrophotometric determination of betaine as the p -bromophenacyl ester is highly sensitive and specific. However, a periodide assay was found to be more convenient for screening numerous plant samples without undue sacrifice in accuracy.
The accumulation of betaine in grain sorghum [ Sorghum bicolor (L.) Moench] cvs NK 265 and Double TX was measured in salt-stressed plants grown hydroponically and in the field and in drought-stressed potted plants. Neither drought nor mild salinity (-0.2 MPa) stress was effective in stimulating betaine accumulation. However, when the osmotic potential of the culture solution was lowered to -0.8 MPa, betaine levels in the shoots rose rapidly for 12 days after initiation of salination, and then declined, apparently because of dilution by plant growth. In young leaf blades, betaine was strongly accumulated up to 70–75 μmol (g dry weight)⁻¹; the concentration in leaf sheaths was less than 6 μmol (g dry weight)⁻¹.
In the field, betaine levels in salt-stressed sorghum increased 6- to 7-fold over the basal level of the control plants. In a comparable study of two wheat species ( Triticum aestivum L. cv. Probred and T. durum Desf. cv. 1000-D), betaine increased only 3- to 4-fold over unstressed plants.