菠菜甜菜碱醛脱氢酶基因的克隆和序列分析

以耐盐的菠菜mRNA为模板,经反转录合成甜菜碱醛脱氢酶(BADH)基因第一链cDNA。在人工合成的两端引物引导下,通过多聚酶链式反应(PCR),扩增获得双链cDNA。把重组有BADH基因的pUC19转化至E.coli DH5α菌株,亚克隆后测定了基因的全序列。所得到的BADH基因全长序列为1491bp,编码497个氨基酸。与文献报道的相比较,核苷酸序列同源性99.8％,氨基酸序列同源性达99.6％。在此基础上,构建了BADH基因的高等植物表达载体。     

Compatibility of glycinebetaine in rice plants: evaluation using transgenic rice plants with a gene for peroxisomal betaine aldehyde dehydrogenase from barley

Glycinebetaine is synthesized in plants by the two‐step oxidation of choline, with betaine aldehyde as the intermediate. The reactions are catalyzed by choline mono‐oxygenase and betaine aldehyde dehydrogenase. Rice plants, which do not accumulate glycinebetaine, possess a gene encoding betaine aldehyde dehydrogenase, whose activity is detectable at low levels. To evaluate the compatibility in rice of glycinebetaine on growth and tolerance to salt, cold and heat, we produced transgenic rice plants by introduction of a cDNA for betaine aldehyde dehydrogenase of barley, which is localized in peroxisomes unlike the chloroplast‐specific localization of betaine aldehyde dehydrogenase in spinach and sugar beet. The transgenic rice plants converted high levels of exogenously applied betaine aldehyde (up to 10 mol m^–3) to glycinebetaine more efficiently than did wild‐type plants. The elevated level of glycinebetaine in transgenic plants conferred significant tolerance to salt, cold and heat stress. However, very high levels of glycinebetaine, resulting from conversion of applied betaine aldehyde to glycinebetaine or from exogenous application, inhibited increases in length of rice plants but not increases in dry weight. Our results suggested that the benefits of accumulation of glycinebetaine by rice plants might be considerable under high light conditions.     

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。     

甘菊BADH基因cDNA的克隆及在盐胁迫下的表达

利用PCR、RT—PCR和PCR—RACE技术,从菊科植物甘菊（Dendranthema lavandulifolium）中克隆到2个甜菜碱醛脱氢酶（betaine aldehyde dehydrogenase,BADH）基因的同源基因,分别命名为DlBADH1和DlBADH2,GenBank登录号分别为DQ011151和DQ011152。DlBADH1的cDNA全长1821bp,其开放阅读框编码503个氨基酸的蛋白质;DlBADH2全长1918bp,编码506个氨基酸的蛋白质。两个基因核苷酸序列的同源性为97％,推导的氨基酸序列的同源性为98％。与已发表的其它植物BADH基因氨基酸序列的同源性在64％以上。在推导的氨基酸序列中,均含有醛脱氢酶所具有的高度保守的十肽（VTLELGGKSP）以及与酶功能有关的半胱氨酸残基（C）。在推导的氨基酸序列的系统关系中,甘菊位于其它双子叶植物和单子叶植物之间,与其植物分类的系统关系相吻合。RT—PCR—Southern半定量表达分析表明,甘菊BADH基因家族中存在表达受盐诱导的成员。     

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.     

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。     

梭梭甜菜碱醛脱氢酶基因克隆及序列分析

采用RT-PCR、RACE等方法从超旱生、耐盐植物梭梭(Haloxylon ammodendron)中扩增出BADH基因的cDNA序列(命名为HaBADH),其开放阅读框为1 503 bp,推测的氨基酸序列全长为500个氨基酸残基,并含有醛脱氢酶所具有的高度保守的十肽(VTLELGGKSP)以及与酶功能有关的半胱氨酸残基(C).其核苷酸序列与藜科几种盐生植物如盐爪爪(Kalidium foliatum)、中亚滨藜(Atriplex centralasiatica)、三角叶滨藜(Atriplex triangularis)、菠菜(Spinacia oleracea)、山菠菜(Atriplex hortensis)和甜菜(Beta vulgaris)等的相似性均在85%以上,推导编码蛋白的氨基酸序列一致性均在87%以上,表明BADH基因在藜科植物中是一种比较保守的基因.研究结果为进一步从分子水平探明梭梭的抗旱、耐盐机制,挖掘并利用植物抗逆基因奠定基础.