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活性均比含钾条件下盐处理时低。无Ｋ＾＋２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。     

干旱和NaCl胁迫下梭梭幼苗中甜菜碱含量和甜菜碱醛脱氢酶活性的变化（简报）

梭梭幼苗的甜菜碱含量和甜菜碱醛脱氢酶（BADH）活性随外界NaCl浓度的增加和干旱胁迫时间的延长而增加。干旱和NaCl促进旱生植物梭梭体内甜菜碱积累与BADH活性有关。     

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.     

盐生植物盐爪爪甜菜碱醛脱氢酶基因的克隆及在盐胁迫下的BADH 基因的表达

根据已发表的几种藜科植物甜菜碱醛脱氢酶(BADH) 基因的同源保守区设计了一对引物, 采用RT-PCR 方法从盐生植物盐爪爪( Kalidium foliatum) 中扩增出BADH 基因的1 个开放阅读框架, 其核苷酸序列长1 503 bp , 推测的氨基酸序列全长为500 个氨基酸残基。核苷酸序列与藜科几种盐生植物如滨藜、碱蓬、菠菜、山菠菜和甜菜等的同源性为81% , 与甜土植物水稻的同源性为69%。氨基酸序列与以上两类植物(盐生植物和甜土植物) 的同源性比对为80% 和71% , 说明BADH 基因在藜科盐生植物中是一种较高保守的基因。BADH 基因编码的多肽在高等植物中行使重要的功能。用不同浓度的NaCl 胁迫处理盐爪爪植株, BADH mRNA 的表达水平比对照植株高, 说明盐爪爪BADH 基因的表达受盐诱导, 间接说明甜菜碱醛脱氢酶催化合成的甜菜碱作为渗透调节的小分子物质, 它的积累与盐胁迫存在紧密关联, 本研究为进一步从生理和分子水平阐明盐爪爪的耐盐机制提供一定的参考。     

盐生植物盐爪爪甜菜碱醛脱氢酶基因的克隆及在盐胁迫下的BADH基因的表达

根据已发表的几种藜科植物甜菜碱醛脱氢酶(BADH)基因的同源保守区设计了一对引物,采用RT-PCR方法从盐生植物盐爪爪(Kalidium foliatum)中扩增出BADH基因的1个开放阅读框架,其核苷酸序列长1503bp,推测的氨基酸序列全长为500个氨基酸残基。核苷酸序列与藜科几种盐生植物如滨藜、碱蓬、菠菜、山菠菜和甜菜等的同源性为81%,与甜土植物水稻的同源性为69%。氨基酸序列与以上两类植物(盐生植物和甜土植物)的同源性比对为80%和71%,说明BADH基因在藜科盐生植物中是一种较高保守的基因。BADH基因编码的多肽在高等植物中行使重要的功能。用不同浓度的NaCl胁迫处理盐爪爪植株,BADHmRNA的表达水平比对照植株高,说明盐爪爪BADH基因的表达受盐诱导,间接说明甜菜碱醛脱氢酶催化合成的甜菜碱作为渗透调节的小分子物质,它的积累与盐胁迫存在紧密关联,本研究为进一步从生理和分子水平阐明盐爪爪的耐盐机制提供一定的参考。     

ABA对低温胁迫下小桐子幼苗耐冷性及甜菜碱积累的影响

旨在探讨外源脱落酸(ABA)对低温胁迫下小桐子幼苗耐冷性及甜菜碱积累的影响。用150μmol/L ABA处理低温胁迫下的小桐子幼苗,研究其对小桐子幼苗存活率,根系活力,电解质渗漏率,MDA含量,甜菜碱含量及其代谢关键酶BADH活性和Jc BADH基因表达水平的影响。结果表明,外源150μmol/L ABA处理可显著提高低温胁迫下小桐子幼苗的存活率和根系活力,降低电解质渗漏率和MDA含量;ABA也提高了低温胁迫下小桐子幼苗的甜菜碱含量,上调了BADH的活性和Jc BADH的表达水平。因此,ABA可提高低温胁迫下小桐子幼苗的耐冷性,并且在ABA诱发的小桐子幼苗耐冷性的提高过程中,甜菜碱发挥着重要作用。     

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。     

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。     

外源甜菜碱提高小麦幼苗抗盐性的研究

以小麦品系山农215953(SN215953)为材料,采用水培的方法,于幼苗期(两叶一心)根灌15mmol·L-1甜菜碱,研究了外源甜菜碱对盐胁迫下小麦幼苗水分状况、脯氨酸和可溶性糖含量及抗氧化酶活性的影响。结果表明:(1)外源甜菜碱可缓解盐渍造成的小麦幼苗叶片的水分损失,改善小麦幼苗的水分状况,并发现甜菜碱的这种作用主要是通过促进脯氨酸和可溶性糖的积累进而提高小麦叶片的渗透调节能力来实现。(2)外源甜菜碱可以维持盐胁迫下小麦幼苗叶片的抗氧化酶(SOD、APX、POD)活性,缓解盐渍造成的盐胁迫伤害,对盐胁迫下小麦幼苗生物膜的稳定性和完整性起到保护作用。     

甜菜碱醛脱氢酶基因在水稻中的表达及转基因植株的耐盐性研究

甜菜碱醛脱氢酶(BADH)是渗透调节剂甜菜碱生物合成中涉及的第2个酶,我们将含盐生植物山菠菜BADH基因的植物双元表达载体经基因枪法导入水稻,经盐胁迫筛选得到转化植株,经RAPD检测全部阳性,在随机选择的10株转化植株中全部测出BADH活性,而对照未见.Northern杂交表明,其中7株为阳性.在含0.5%氯化钠的盐池中大多数转基因植株生长基本正常,结实率约为10%,而对照受盐害现象显著,几乎全部枯萎.     

Overproduction of spinach betaine aldehyde dehydrogenase in Escherichia coli. Structural and functional properties of wild-type, mutants and E. coli enzymes.

Betaine aldehyde dehydrogenase (BADH) catalyzes the last step in the synthesis of the osmoprotectant glycine betaine from choline. Although betaine aldehyde has been thought to be a specific substrate for BADH, recent studies have shown that human and sugar beet BADHs also catalyze the oxidation of omega-aminoaldehydes. To characterize the kinetic and stability properties of spinach BADH, five kinds of expression vectors encoding full length, mature, E103Q, E103K, and chimera BADHs were constructed. These enzymes together with Escherichia coli BADH were expressed in E. coli and purified. The affinities for betaine aldehyde were similar in the spinach and E. coli BADHs, whereas those for omega-aminoaldehydes were higher in spinach BADH than in E. coli BADH. A chimera BADH in which part of the Rossmann type fold in the spinach BADH was replaced with that of E. coli BADH, showed properties which resembled spinach BADH more than E. coli BADH. The spinach E103K mutant was almost inactive, whereas the E103Q mutant showed a similar activity for the oxidation of betaine aldehyde to that of wild type BADH, but a lower affinity for omega-aminoaldehydes. All spinach BADHs were dimers whereas E. coli BADH was a tetramer. E. coli BADH was more stable at high temperature than spinach BADHs. The E103Q mutant was most labile to high temperature. These properties are discussed in relation to the structure of spinach BADH.     

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。     

Genetic engineering of the biosynthesis of glycinebetaine enhances photosynthesis against high temperature stress in transgenic tobacco plants

Genetically engineered tobacco (Nicotiana tabacum) with the ability to synthesis glycinebetaine was established by introducing the BADH gene for betaine aldehyde dehydrogenase from spinach (Spinacia oleracea). The genetic engineering enabled the plants to accumulate glycinebetaine mainly in chloroplasts and resulted in enhanced tolerance to high temperature stress during growth of young seedlings. Moreover, CO2 assimilation of transgenic plants was significantly more tolerant to high temperatures than that of wild-type plants. The analyses of chlorophyll fluorescence and the activation of Rubisco indicated that the enhancement of photosynthesis to high temperatures was not related to the function of photosystem II but to the Rubisco activase-mediated activation of Rubisco. Western-blotting analyses showed that high temperature stress led to the association of Rubisco activase with the thylakoid membranes from the stroma fractions. However, such an association was much more pronounced in wild-type plants than in transgenic plants. The results in this study suggest that under high temperature stress, glycinebetaine maintains the activation of Rubisco by preventing the sequestration of Rubisco activase to the thylakoid membranes from the soluble stroma fractions and thus enhances the tolerance of CO2 assimilation to high temperature stress. The results seem to suggest that engineering of the biosynthesis of glycinebetaine by transformation with the BADH gene might be an effective method for enhancing high temperature tolerance of plants.     

Salt-inducible betaine aldehyde dehydrogenase from sugar beet: cDNA cloning and expression

Members of the Chenopodiaceae, such as sugar beet and spinach, accumulate glycine betaine in response to salinity or drought stress. The last enzyme in the glycine betaine biosynthetic pathway is betaine aldehyde dehydrogenase (BADH). In sugar beet the activity of BADH was found to increase two- to four-fold in both leaves and roots as the NaCl level in the irrigation solution was raised from 0 to 500 mM. This increase in BADH activity was paralleled by an increase in level of translatable BADH mRNA. Several cDNAs encoding BADH were cloned from a gt10 libary representing poly(A)⁺ RNA from salinized leaves of sugar beet plants, by hybridization with a spinach BADH cDNA. Three nearly full-length cDNA clones were confirmed to encode BADH by their nucleotide and deduced amino acid sequence identity to spinach BADH; these clones showed minor nucleotide sequence differences consistent with their being of two different BADH alleles. The clones averaged 1.7 kb and contained an open reading frame predicting a polypeptide of 500 amino acids with 83% identity to spinach BADH. RNA gel blot analysis of total RNA showed that salinization to 500 mM NaCl increased BADH mRNA levels four-fold in leaves and three-fold in the taproot. DNA gel blot analyses indicated the presence of at least two copies of BADH in the haploid sugar beet genome.     

Isolation of cDNA and enzymatic properties of betaine aldehyde dehydrogenase from Zoysia tenuifolia

We isolated cDNAs encoding betaine aldehyde dehydrogenase (BADH, EC 1.2.1.8) from the salt-tolerant Poaceae, Zoysia tenuifolia by polymerase chain reactions. Zoysia betaine aldehyde dehydrogenase 1 (ZBD1) is 1892bp long and codes for 507 amino acids. The deduced amino acid sequence of ZBD1 is 88% similar to the sequence of rice BADH. Ten cDNA clones were isolated from a cDNA Library of salt-treated Z. tenuifolia by using the ZBD1 fragment as a probe. The proteins coded in some clones were more homologous to BBD2, the cytosolic BADH of barley, than to ZBD1. To investigate their enzymatic properties, ZBD1 and spinach BADH were expressed in Escherichia coli and purified. The optimal pH of ZBD1 was 9.5, which was more alkaline than that of spinach BADH. ZBD1 was less tolerant to NaCl than spinach BADH. ZBD1 showed not only BADH activity but also aminoaldehyde dehydrogenase activity. The Km values of ZBD1 for betaine aldehyde, 4-aminobutyraldehyde (AB-ald), and 3-aminopropionaldehyde (AP-ald) were 291, 49, and 4.0 microM, respectively. ZBD1 showed higher specific activities for AB-ald and AP-ald than did spinach BADH.     

根施甜菜碱对水分胁迫下烟草幼苗光合机构的保护

以烟草品种大黄金5210（抗旱性强）和中烟100（抗旱性弱）为材料,研究了水分胁迫对烟草叶片光合机构的影响,并通过根部施用甜菜碱的方法,探讨了甜菜碱对烟草光合机构的保护作用。结果表明：水分胁迫导致烟草幼苗光合机构损伤,表现在叶绿素含量、PSII光化学效率、希尔反应活力以及类囊体膜ATPase活性下降,且对抗旱性弱的中烟100损伤更加严重。外源甜菜碱处理减轻了水分胁迫对以上指标的降低程度,特别是对干旱敏感型烟草品种中烟100的效果更加明显。甜菜碱的这种保护作用可能与它能够维持叶片中各种抗氧化酶活性、减轻活性氧的积累、保护类囊体膜上各种色素蛋白的功能以及缓解水分胁迫对膜的破坏作用有关。     

Effect of salinity and abscisic acid on accumulation of glycinebetaine and betaine aldehyde dehydrogenase mRNA in Sorghum leaves (Sorghum bicolor)

The effects of salt stress and abscisic acid (ABA) on the expression of betaine aldehyde dehydrogenase (BADH) were determined in sorghum (Sorghum bicolor L.) plants. BADH mRNA expression was induced by salinity, and the timing coincided with the observed glycinebetaine (betaine) accumulation. The leaf water potential in the leaves of the sorghum plants was significantly affected by salinity. In response to salinity, betaine, ABA, Na and Cl accumulations increased 6-, 16-, 90-, and 3-fold, respectively. In the leaf disks from unsalinized plants incubated on NaCl, or ABA solution, the BADH mRNA level was lower than in the ABA-treated disks. Exogenous application of the ABA biosynthetic inhibitor fluridone to the NaCl-treated disks reduced the ABA accumulation and BADH mRNA levels compared with NaCl-treated leaves. The results indicate that the salt-induced accumulation of betaine and BADH mRNA coincides with the presence of ABA.     

Possible Involvement of Greening in Cell Growth of Suaeda japonica under Salt Stress

Suaeda japonica, a member of the family Chenopodiaceae, is ahalophyte that grows on the shores of the Ariake Sea in Japan.Using yellowish and selected green callus tissues of S. japonica,we examined the correlation between cell growth and glycinebetainecontent, as well as the activity of betaine aldehyde dehydrogenase(BADH), under salt stress. Although the growth of yellowishcallus tissues was markedly inhibited by 0.1 M NaCl, that ofgreen callus tissues was not. However, in 0.3 M NaCl, the freshweight of green callus tissues was decreased by 15%. Both theendogenous level of glycinebetaine and the activity of BADHin yellowish callus tissues were very low, but green callustissues contained very high levels of glycinebetaine and highBADH activity. The activity in green callus increased significantlywith increases in the concentration of NaCl. The increase inlevels of Chl was also observed, but the increase occurred earlierthan that in the level of glycinebetaine. Functional developmentof chloroplasts (an increase in the level of Chl) seems to beinvolved in the increase in the activity of BADH, and the resultantincrease in levels of glycinebetaine seems to facilitate callusgrowth under salt stress. (Received October 2, 1996; Accepted November 18, 1996)     

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+.