硫腺苷甲硫氨酸合成酶基因的克隆及其在盐胁迫条件下的不同表达

硫腺苷甲硫氨酸作为甲基供体在转甲基反应中起到重要作用.为了解硫腺苷甲硫氨酸在盐地碱蓬(Suaedasalsa (L.)Pall)耐盐中的作用,我们对可能编码硫腺苷甲硫氨酸合成酶的基因(SsSAMS2)进行了分析.该基因在经400 mmol/L NaCl处理的盐地碱蓬地上部分的λ-Zap cDNA文库中克隆到,其插入片段全长1 531 bp,包含一个395个氨基酸的开放阅读框架,该基因推断的分子量约为43 kD.SsSAMS2与长春花(Catharanthus roseus)的SAMS2在氨基酸水平上的一致性为93%.Southern杂交显示,SsSAMS2在盐地碱蓬基因组中可能是两个拷贝.Northern分析显示硫腺苷甲硫氨酸合成酶基因受NaCl等胁迫的正调控.酶活性检测表明,NaCl胁迫条件下该酶活性增强.     

盐地碱蓬GST基因的克隆、序列分析及其表达特征

从盐地碱蓬 (Suaedasalsa)幼苗的cDNA文库中克隆到一个 0 .9kb的全长cDNA ,同源性分析表明该全长cDNA与已报告的大豆 (Glycinemax)GST基因相应序列的同源性达 5 5 % ,可能编码由 2 35个氨基酸组成的谷胱甘肽转移酶 (glutathioneS transferase ,GST)。Southern杂交结果证明GST基因在碱蓬基因组中可能有至少两个以上的拷贝 ;Northern杂交结果表明 ,4 0 0mmol/L的NaCl处理 4 8h ,幼叶中GSTmRNA的表达量是对照的 2～ 3倍 ,说明碱蓬中GST基因受盐诱导     

简并PCR结合RACE技术克隆申克孢子丝菌未知过氧化氢酶基因

目的 克隆孢子丝菌未知过氧化氢酶基因,命名为Sscat基因.方法 根据生物信息库中7种已知真菌过氧化氢酶氨基酸序列的高度保守区域设计简并引物,PCR扩增获得部分Sscat基因cDNA片段,随后应用RACE技术分别扩增其3’端和5’端未知序列.结果 Sscat基因cDNA序列全长1746 bp,其中包括5’端121 b...     

盐地碱蓬(Suaeda salsa)中SsINPS基因的分子克隆及表达特性分析

肌醇 1 磷酸 (I 1 P)合成酶 (EC5 .5 .1 .4,INPS)是肌醇生物合成中的关键酶 ,催化葡萄糖 6 磷酸 (G 6 P)到I 1 P的反应。从该实验室已构建的NaCl40 0mmol/L处理的盐地碱蓬 (Suaedasal sa)cDNA文库中克隆了肌醇 1 磷酸合成酶的全长cDNA (S .salsamyo inositol 1 phosphatesynthase,SsINPS) ,基因注册号为AF43 3 879。SsINPS全长约 1 986bp ,含有开放式阅读框架 1 5 3 0bp ,3′和 5′的非翻译区分别为 1 3 9bp和 3 1 7bp ;推导的氨基酸序列全长 5 1 0个氨基酸残基 ,分子量约为 5 6 .7kD ,pI值为 5 .3 5。BLAST同源性分析表明 ,该cDNA与已报告的冰叶日中花 (Mesembryanthemumcrys tallinum)的INPS基因同源性最高 ,其中 ,核苷酸水平的同源性为 91 % ,氨基酸水平上的同源性为84%。以SsINPS全长cDNA为探针进行的South ern杂交结果表明 ,SsINPS基因在盐地碱蓬基因组中只有一个拷贝 ;Northern结果表明 ,在盐处理(40 0mmol/L的NaCl)下 ,SsINPS在叶中的表达量有显著的增加。从而说明SsINPS在盐胁迫下是上升调节的     

盐地碱蓬延伸因子(SsEF-1α)的克隆与表达分析

从盐胁迫下的盐地碱蓬(Suaeda salsa)的cDNA文库中获得了一个延伸因子(SsEF-1α)的cDNA片段。以此片段为探针,对其在不同胁迫处理下的盐地碱蓬中表达利用Northern杂交进行分析,结果表明,不同盐浓度处理、同一盐浓度不同时间处理下及双氧水刺激条件下延伸因子在碱蓬叶中的表达量先呈现减少然后增加的趋势;聚乙二醇(PEG)及低温诱导下均呈现上升趋势。由此可见该基因在盐、氧化、高渗和低温胁迫条件下产生一定的反应。     

Expressed sequence tags from a NaCl-treated Suaeda salsa cDNA library

Past efforts to improve plant tolerance to osmotic stress have had limited success owing to the genetic complexity of stress responses. The first step towards cataloging and categorizing genetically complex abotic stress responses is the rapid discovery of genes by the large-scale partial sequencing of randomly selected cDNA clones or expressed sequence tags (ESTs). Suaeda salsa, which can survive seawater-level salinity, is a favorite halophytic model for salt tolerant research. We constructed a NaCl-treated cDNA library of Suaeda salsa and sequenced 1048 randomly selected clones, out of which 1016 clones produced readable sequences (773 showed homology to previously identified genes, 227 matched unknown protein coding regions, 16 anomalous sequences or sequences of bacterial origin were excluded from further analysis). By sequence analysis we identified 492 unique clones: 315 showed homology to previously identified genes, 177 matched unknown protein coding regions (101 of which have been found before in other organisms and 76 are completely novel). All our EST data are available on the Internet. We believe that our dbEST and the associated DNA materials will be a useful source to scientists engaging in stress-tolerance study.     

Construction and characterization of a Schistosoma mansoni bacterial artificial chromosome library

A bacterial artificial chromosome (BAC) library has been established from genomic DNA isolated from the trematode parasite of human, Schistosoma mansoni. This library consists of more than 21,000 recombinant clones carrying inserts in the pBeloBAC11 vector. The mean insert size was 100 kb, representing an approximate 7.95-fold genome coverage. Library screening with eight chromosome-specific or single-copy gene probes yielded between 1 and 9 positive clones, and none of those tested was absent from the library. End sequences were obtained for 93 randomly selected clones, and 37 showed sequence identity to S. mansoni sequences (ESTs, genes, or repetitive sequences). A preliminary analysis by fluorescence in situ hybridization localized 8 clones on schistosome chromosomes 1 (2 clones), 2, 3, 5, Z, and W (3 clones). This library provides a new resource for the physical mapping and sequencing of the genome of this important human pathogen.     

费氏中华根瘤菌042BS结瘤调节基因的克隆及功能检测

费氏中华根瘤菌 (Sinorhizobiumfredii) 0 4 2BS可以在大豆和苜蓿上结瘤。用费氏中华根瘤菌USDA2 5 7的nodD1和nodD2基因分别作为探针 ,与 0 4 2BS总DNA进行Southern杂交 ,发现其DNA经EcoRI酶切后分别在 3 0kb和 6 0kb处各有一条阳性带。回收这两条阳性带附近的DNA片段 ,建立部分基因文库 ,克隆到带有nodD1基因的 3 0kb片段 ,以及带有nodD2基因的 6 0kb片段。对nodD1和nodD2进行序列分析 ,结果表明 0 4 2BS的nodD1与费氏中华根瘤菌根瘤菌USDA2 5 7和USDA1 91的同源性高达 99% ,而nodD2与USDA2 5 7的同源性为1 0 0 %。再将nodD1的片段克隆到pBBRIMCS 5载体上 ,导入豌豆根瘤菌蚕豆生物变种 (Rhi zobiumleguminosarumbv.viciae)LPR5 0 5 4中进行功能检测 ,显示 0 4 2BS的nodD1均可被大豆分泌的类黄酮物质染料木黄酮以及苜蓿分泌的类黄酮物质毛地黄黄酮所诱导     

cDNA clones encoding bovine interphotoreceptor retinoid binding protein

We have isolated a cDNA clone (lambda IRBP-1) for bovine interphotoreceptor retinoid-binding protein (IRBP) by immunological screening of a bovine retinal lambda gt11 cDNA expression library. This clone contained a cDNA insert 325 bp in length. A 250 bp fragment of this cDNA was used to screen a bovine retina lambda gt10 cDNA library, resulting in the isolation of two larger cDNA clones containing inserts of 2.5 kb (lambda IRBP-2) and 1.5 kb (lambda IRBP-3). Restriction endonuclease mapping revealed all three clones to have an EcoR I restriction site. The 250 bp fragment of lambda IRBP-1 and the 2000 bp fragment of lambda IRBP-2 both hybridized to a single bovine retinal mRNA species approximately 8 kb in length; there was no hybridization with either chicken lens or liver RNA. The amino acid sequence of a tryptic peptide from authentic IRBP has been obtained. The deduced amino acid sequence from the cDNA nucleotide sequence is the same as this authentic peptide. This definitively establishes the identity of the cDNA clones as encoding bovine IRBP.     

Nuclear-encoded chloroplast ribosomal protein L12 of Nicotiana tabacum: characterization of mature protein and isolation and sequence analysis of cDNA clones encoding its cytoplasmic precursor.

Poly(A)+ mRNA isolated from Nicotiana tabacum (cv. Petite Havana) leaves was used to prepare a cDNA library in the expression vector lambda gt11. Recombinant phage containing cDNAs coding for chloroplast ribosomal protein L12 were identified and sequenced. Mature tobacco L12 protein has 44% amino acid identity with ribosomal protein L7/L12 of Escherichia coli. The longest L12 cDNA (733 nucleotides) codes for a 13,823 molecular weight polypeptide with a transit peptide of 53 amino acids and a mature protein of 133 amino acids. The transit peptide and mature protein share 43% and 79% amino acid identity, respectively, with corresponding regions of spinach chloroplast ribosomal protein L12. The predicted amino terminus of the mature protein was confirmed by partial sequence analysis of HPLC-purified tobacco chloroplast ribosomal protein L12. A single L12 mRNA of about 0.8 kb was detected by hybridization of L12 cDNA to poly(A)+ and total leaf RNA. Hybridization patterns of restriction fragments of tobacco genomic DNA probed with the L12 cDNA suggested the existence of more than one gene for ribosomal protein L12. Characterization of a second cDNA with an identical L12 coding sequence but a different 3'-noncoding sequence provided evidence that at least two L12 genes are expressed in tobacco.