甘蓝型油菜细胞质雄性不育材料1575A的分子鉴定

根据已报道的油菜育性相关基因orf224、orf138和orf222设计引物对1575A、陕3A、Ogura和nap等4份甘蓝型油菜的mtDNA进行PCR扩增.结果显示:引物对Syworf1382/ Xyworf1382在1575A和Ogura的mtDNA中均有1000 bp左右的扩增片段;测序结果表明,在1575A中的该扩增片段包含已报道基因orf138(登录号为AB055435)的全部编码序列;该引物对在陕3A和nap中都没有相应扩增片段.引物对Syworf222/Xyworf222在除Ogura外的其他3个材料的mtDNA中都有196 bp左右的扩增片段;测序结果显示,扩增产物与已报道基因orf222(登录号为DQ872162)同源性为100%.初步推测1575A属于Ogu CMS的改良类型.     

青花菜Ogu不育胞质分子鉴定和序列分析

雄性不育是农作物利用杂种优势、进行轮回选择和群体改良的重要手段,在农作物生产中具有巨大的利用价值。该研究为了鉴定青花菜细胞质雄性不育材料的不育胞质类型,以期今后为青花菜种质资源的收集、利用及分子标记辅助育种提供新的不育标记。根据Gen Bank中orf138基因保守序列设计特异引物,对20个青花菜种质资源基因组DNA进行PCR扩增。结果表明:特异引物P1/P2在12个青花菜雄性不育基因型中均扩增出392 bp的片段,在8个可育基因型中未扩增出条带,与田间育性鉴定结果相符。获得青花菜Ogu胞质雄性不育的特异基因orf138序列,Gen Bank中的登录号为HQ149728;用Blastn在Gen Bank中进行同源性比对分析,发现12个不育材料的特异片段与已报道的萝卜Ogu CMS所具有的Ogu orf138基因(Genbank登录号:Z18896.1)同源度高达100%。序列同源比对发现orf138基因存在变异位点。研究结果可为青花菜雄性不育细胞质的分子鉴定、进一步阐明胞质雄性不育败育机理,以及指导青花菜新型不育系的创建和杂种优势高效利用提供理论依据。     

花椰菜细胞质雄性不育基因特异PCR标记的筛选

基于同源序列的候选基因法(homology-based candidate gene method),通过检索NCBI核酸及蛋白数据库,获得细胞质雄性不育（cytoplasmic male sterility CMS）相关的基因或开放读码框。生物学软件分析,根据保守区设计5对特异引物,PCR扩增,其中引物P9/P10在花椰菜细胞质雄性不育系knxd612中特异扩增出313 bp的片段。单株检测,RT-PCR分析,斑点杂交鉴定,确定此片段为花椰菜细胞质雄性不育系knxd612所特有。序列分析表明该片段与Ogura型胞质不育萝卜,不育相关开放读码框orf138的同源性高达98％。初步结果显示实验所用不育花椰菜胞质亦可能为Ogura型。该结果为进一步从分子水平研究花椰菜细胞质雄性不育打下了坚实的基础。Abstract: The homology-based candidate gene method was used to identified the specific PCR markers linked to cytoplasmic male sterility (CMS) in cauliflower( Brassica oleracea var botrytis.).Searching the DNA and protein data-base of NCBI , correlative genes or open reading frames were indentified .Analysis of biosoft, based on the conservative regions ,five primers were designed . Among them, only primer P9/P10 produced a 313- bp specific fragment. Identified by individual plant testing , analysis of RT-PCR and dot blot ,this fragment was only existed in CMS cauliflower knxd612.Analysis of the sequence indicated it was high homologous(98%) with orf138 of Ogura CMS radish. Primary result suggested that the cytoplasmic type of CMS cauliflower knxd612 may belong to Ogura type. This research offered a good foundation to further investigate the CMS mechanism of cauliflower in molecular level.     

红树植物秋茄中受盐胁迫抑制的cyclophilin基因的鉴定与表达分析

利用代表性差异分析方法获得秋茄中两个编码亲环素(cyclophilin)蛋白的cDNA片段(称为SRGKC2和SRGKC3),该片段大小分别为282 bp和160 bp;序列分析表明:SRGKC2和SRGKC3是同一基因区域的不同长度片段,SRGKC3是SRGKC2片段的一部分。SRGKC2在84个氨基酸范围内与大戟属cyclophilin蛋白的氨基酸序列的一致性达到90%,SRGKC3在47个氨基酸范围内与蚕豆cyclophilin蛋白的一致性达到93%。Northem分析表明:盐分抑制SRGKC2片段的表达。依赖SRGKC2片段的序列资料,利用cDNA快速末端扩增(RACE)技术获取秋茄中cyclophilin基因的全长cDNA片段(命名为KCCYPl)(GenBank登录号:AY150052)。该cDNA全长约为0.9kb,含有一个516个核苷酸的完整开放阅读框,编码172个氨基酸,等电点为8.57,分子量18.2 KDa。42-49位氨基酸残基为推测的ATP/GTP结合位点A基序(P-loop),48-54位氨基酸残基是插入的7个氨基酸残基。文中还对SRGKC2在不同种中的表达状况进行了分析。     

红树植物秋茄中受盐胁迫抑制的cyclophilin基因的鉴定与表达分析

利用代表性差异分析方法获得秋茄中两个编码亲环素(cyclophilin)蛋白的cDNA片段(称为SRGKC2和SRGKC3),该片段大小分别为282bp和160bp;序列分析表明：SRGKC2和SRGKC3是同一基因区域的不同长度片段,SRGKC3是SRGKC2片段的一部分。SRGKC2在84个氨基酸范围内与大戟属cyclophilin蛋白的氨基酸序列的一致性达到90％,SRGKC3在47个氨基酸范围内与蚕豆cyclophilin蛋白的一致性达到93％。Northern分析表明：盐分抑制SRGKC2片段的表达。依赖SRGKC2片段的序列资料,利用cDNA快速末端扩增(RACE)技术获取秋茄中cyclophilin基因的全长cDNA片段(命名为KCCYP1)(GenBank登录号：AY150052)。该cDNA全长约为0．9kb,含有一个516个核苷酸的完整开放阅读框,编码172个氨基酸,等电点为8．57,分子量18．2KDa。42—49位氨基酸残基为推测的ATP／GTP结合位点A基序(P—loop),48—54位氨基酸残基是插入的7个氨基酸残基。文中还对SRGKC2在不同种中的表达状况进行了分析。     

西伯利亚蓼钙调蛋白基因的克隆及其在盐胁迫下的表达

已从西伯利亚蓼叶中cDNA文库中获得的钙调蛋白EST序列,采用cDNA末端快速扩增（RACE）技术克隆了具有完整编码区的钙调蛋白基因的cDNA序列（GenBank登录号GQ988382）,命名为PsCaM。该基因全长615bp,编码区为450bp,编码149个氨基酸,5＇非翻译区为63bp,3＇非翻译区为102bp。同源性分析表明,该蛋白与其他植物钙调蛋白高度保守,氨基酸同源性高达98%。用实时荧光定量PCR研究3%NaHCO3胁迫下西伯利亚蓼基因表达的结果显示,自然条件下,该基因在叶中表达量最高,地下茎次之,茎中最低;盐胁迫下CaM在西伯利亚蓼的地下茎、茎和叶中均有表达,表达模式不同。     

编码嗜麦芽黄单胞菌terminase-like蛋白基因的克隆与分析

根据Grover报道的XanthomonasmaltophiliaCG类受体的 342bp核酸序列设计的一特异引物P2和随机引物进行PCR扩增 ,将约 75 0bpPCR产物克隆到 pUCm T载体上 ,得到重组质粒 pUCm Ter。pUCm Ter上插入片段经M 13通用引物双向测序 ,其中ORF5 5 5核酸序列的 2 83～ 36 2bp部分与PseudomonasphageD3orf2基因的 1385～ 14 6 4bp部分有 86 %相同碱基。由ORF5 5 5编码 184aa蛋白序列的 1～ 16 5aa部分与PseudomonasphageD3orf2基因编码terminase的 2 2 9～ 393aa部分具有 6 6 %相同序列。因此 ,克隆到的ORF5 5 5核酸序列可能是编码嗜麦芽黄单胞菌terminase like蛋白的基因序列。     

绵羊CAST基因2型和4型转录本的克隆及特性分析

钙蛋白酶抑制蛋白(Calpastatin, CAST)是一种内源性的需要Ca²⁺激活的钙蛋白酶抑制剂, 在肌肉组织的蛋白质降解过程中起重要的调节作用。文章利用牛^CAST基因的mRNA序列, 通过逆转录RT-PCR首次克隆获得绵羊CAST基因2型转录本和4型转录本的部分cDNA序列, 并对序列进行生物信息学分析。CAST基因2型转录本的扩增片段为4 385 bp, 完整的开放阅读框为2 361 bp, 编码786个氨基酸; CAST基因4型转录本的扩增片段为1 467 bp, 完整的开放阅读框为1 317 bp, 编码438个氨基酸。CASTⅡ型蛋白序列存在4个保守结构域, CASTⅣ型蛋白序列存在3个保守结构域; 两者的二级结构均以螺旋为主, 富含疏水区域, 其氨基酸序列存在多个磷酸化位点以及蛋白激酶C(Protein kinase C, PKC)的磷酸化位点。通过RT-PCR分析CAST基因2型转录本和4型转录本的组织表达谱, 结果表明CAST基因2型转录本在所检测的10个组织中均表达, CAST基因4型转录本仅在睾丸组织中表达。     

桑泛素延伸蛋白基因片段的克隆及序列分析

目的:利用3’RACE技术克隆植物泛素基因,是进一步研究其功能的基础。方法:本研究从桑树(丰驰桑)(Morus bomby-cis)幼叶中提取总RNA,反转录成cDNA,根据已报道的泛素基因序列设计1条正向引物,利用3’RACE(Rapid Amplification of cDNAEnd)技术进行扩增。结果:扩增出1条690 bp的泛素基因片段。该片段5’端为编码156个氨基酸残基的阅读框,3’末端有219bp的非翻译区。结论:同源分析表明,此cDNA序列为泛素延伸蛋白基因(Genebank登录号为DQ839403)。用Genedoc软件对该片段编码的氨基酸序列进行同源性分析的结果表明:桑树泛素延伸蛋白与马铃薯、烟草、陆地棉、黄瓜的泛素延伸蛋白以及苜蓿的核糖体S27A蛋白的同源性都在96%以上。     

杨柳田头菇B交配型基因信息素受体片段的克隆与分析

利用简并PCR及DNA步移法,从杨柳田头菇Agrocybe salicacola YAASM0711菌株中扩增得到了一个4 231 bp的核酸片段.经过比对及序列预测,所获得序列中含有杨柳田头菇交配型编码基因中的信息素受体部分,其序列长度为1194 bp,包含4个内含子,5个外显子的长度分别为217 bp,113 bp,67 bp,138bp,449 bp.拼接后的ORF全长984 bp,编码327个氨基酸残基.该序列与灰盖鬼伞Coprinus cinerea、双色蜡蘑Laccaria bicolor信息素受体氨基酸序列较为相似,含有7个跨膜区.信息素受体遗传进化分析显示,其与多个物种信息素受体聚集在一起,可能与真菌信息素受体的多种起源有关.     

Identification and expression of the kosena radish (Raphanus sativus cv. Kosena) homologue of the ogura radish CMS-associated gene,orf138

A CMS-associated gene, orf125, present in the Japanese radish cultivar Kosena, has a sequence homologous to that of the ogura CMS-associated gene, orf138, except for two amino acid substitutions and a 39 bp deletion in the orf138 coding region. In Kosena radish, orf125 is linked with orfB, whereas the orf125 locus differs in a Brassica napus CMS cybrid derived from protoplast fusion between Kosena radish and B. napus. A novel mtDNA sequence is present in the 3-flanking region of orf125 in the B. napus kosena CMS cybrid. The orf125 is expressed both in the radish and the B. napus kosena CMS cybrid. Its accumulation is strongly associated with the CMS phenotype in B. napus. Fertility restoration was accompanied by a decrease in the amount of ORF125 in B. napus.     

鸡含锰超氧化物歧化酶cDNA克隆及序列分析

 为弄清鸡含锰超氧化物歧化酶 (manganese containingsuperoxidedismutase ,MnSOD)的cDNA序列 ,以开展动物锰营养学的深入研究 ,根据已知鸡MnSOD的N端氨基酸序列设计简并引物 ,应用 3′RACE(rapidamplificationofcDNAends)技术 ,扩增克隆了鸡心肌MnSOD 990bp的 3′cDNA片段 .再根据 3′RACE片段测序结果设计引物进行 5′RACE ,结果获取了一个与 3′RACE片段相互重叠的鸡心肌MnSOD 52 1bp的 5′RACE片段 ,并对其进行了克隆测序 .最后根据 3′RACE片段和 5′RACE片段序列信息进行拼接 ,从而获取鸡MnSODcDNA的全序列信息 .研究结果表明 :鸡MnSODcDNA全长为 110 8个核苷酸 ,其中 5′非翻译区 2 5个核苷酸 ,编码区 675个核苷酸 ,3′非翻译区 4 0 8个核苷酸 ,编码一个长 2 2 4个氨基酸残基的蛋白质前体 .其中信号肽长 2 6个氨基酸残基 ,成熟肽长 198个氨基酸残基 ,分子量为 2 2kD .与人、大鼠、线虫、果蝇等真核生物MnSOD氨基酸序列的同源性分别为82 4 %、84 .7%、62 .4 %、59.3% .     

Sequence analysis of a mitochondrial DNA fragment isolated from cultured cells of carrot cytoplasmic male-sterile strain.

2.0 kb Hind III fragment isolated from cytoplasmic male-sterile carrot mitochondria, designated PKT5, was hybridized to ORF13 which is the coding region of a unique polypeptide in maize CMS (Dewey et al., 1986). Sequence analysis indicated that PKT5 is consisted of 3 domains. Domain 1 was identical to the 5'-flanking region of atp6 in maize CMS-TURF2H3 sequence (Dewey et al., 1986). Domain 2 contained a novel ORF encoding 72 amino acids, which was extremely homologous to the amino-terminal 67 amino acids of the unique ORF13 in maize CMS. Domain 3 except an amino acid change (Ile87 = ATT for Asn87 = AAT), was identical to ORF25 polypeptide in maize CMS. Connective sequences of these 3 domains were also highly homologous to the maize CMS-TURF2H3 sequence. Out of 7 recombination points in maize CMS-TURF2H3 sequence, at least 4 points were conserved in PKT5 sequence.     

Molecular cloning and expression analysis of a new WD40 repeat protein gene in upland cotton

A new member of the WD repeat protein family, named GhWD40, was cloned from a near-isogenic line for glands in cotton. It has 2629 bp cDNA and a complete opening reading frame (ORF) of 1239 bp, containing the initial code (ATG) and terminal code (TAG); there is a 1061 bp non-coding sequence at the 5??-end, and a 329 bp non-coding sequence at the 3??-end, including the poly(A) sequence (accession number: JN714279). The predicted protein of the complete ORF comprised 412 amino acids with a calculated molecular mass of 47.1 kDa and an isoelectric point of 8.88. Protein domain scanning showed that the novel protein has five wd40 motifs and belongs to the WD40 family. From a search for GhWD40 cDNA and amino acid sequences in the database, it has 77% sequence identity and was 90% sequence positive with the WD-40 repeat protein from Trifolium pratense (accession number BAE71307.1), and 80% sequence identity and 89% sequence positivity with the ribosome biogenesis protein bop1 from Ricinus communis (accession number XP 002529002.1). We propose that GhWD40 may play the same role as bop1. In addition, expression of GhWD40 in near-isogenic lines 11 and 3 (with and without glands, respectively) was studied by quantitative RT-polymerase chain reaction, and the level in near-isogenic line 11 was higher than that in near-isogenic line 3, suggesting that GhWD40 may be related to gland formation.     

Cloning and characterization of Acmar1, a mariner-like element in the asiatic honey bee,Apis cerana japonica (Hymenoptera,Apocrita)

A mariner-like element was cloned from the genome of the Asiatic honey bee, Apis cerana japonica (Hymenoptera, Apocrita). The (composite) clone, named Acmar1, was 1,378 bp long, and encoded 336 amino acids corresponding to a transposase-like putative polypeptide in a single open reading frame. The D,D(34)D motif, the catalytic domain of the mariner transposase, was present, although there was a deletion of five amino acid residues within it as compared with the active transposase in Drosophila mauritiana. Nineteen-bp-long imperfect inverted terminal repeat-like sequences flanked by TA dinucleotides, the typical target site for mariner insertion, were observed. Southern blot analysis using a fragment covering two-thirds of the Acmar1 transposase coding sequence as a probe indicated the presence of multiple Acmar1-like elements in the genome. Maximum-parsimony phylogenetic analysis based on the transposase amino acid sequences of insect mariner-like elements revealed that Acmar1 is a member of the mellifera subfamily.     

A plant serpin gene. Structure, organization and expression of the gene encoding barley protein Z4

A 3133-bp nucleotide sequence of the gene Paz1 on chromosome 4 of barley, encoding endosperm protein Z4, has been determined. The sequence includes 1079 bp 5' upstream and 523 bp 3' downstream of the coding region. The 1079-bp 5' upstream region of the gene shows little similarity to 5' regions of other sequences genes expressed in the developing cereal endosperm. The coding sequence is interrupted by one 334-bp-long intron (bases 1497-1830). The deduced amino acid sequence, which was corroborated by peptide sequences, consists of 399 amino acids and has a molecular mass of 43,128 Da. This sequence confirms protein Z4 to be a member of the serpin superfamily of proteins. The similarity with other members of the family expressed as amino acids in identical positions is in the order of 25-30% and pronounced in the carboxy-terminal half of the molecule. Sequence residues assumed to form clusters stabilizing the tertiary structure are highly conserved. Protein Z4 is synthesized in the developing endosperm without a signal peptide and protein Z4 mRNA was evenly distributed among the free and membrane-bound polyribosomes of the endosperm cell. An internal hydrophobic region of 21 amino acids (residues 36-56) may serve as a signal for targeting the polypeptide into the lumen of the endoplasmic reticulum. The gene for protein Z4 could not be detected in the barley variety Maskin and some of its descendants. The 'high-lysine' allees, lys1 (Hiproly barley) and lys3a (Bomi mutant 1508) on chromosome 7, enhance and repress, respectively, the expression of the protein Z4 gene. Also, 1554 bp of another 8-kbp fragment of the barley genome Paz psi, similar to the protein-Z4-coding region, have been determined. Small insertions and deletions and the presence of an internal stop codon identify this fragment as part of a pseudogene related to the protein Z4 gene.     

Novel insertion sequence IS1380 from Acetobacter pasteurianus is involved in loss of ethanol-oxidizing ability.

Acetobacter pasteurianus NCI1380, a thermophilic strain isolated from the surface culture of acetic acid fermentation, showed genetic instability to produce at high frequency spontaneous mutants which were deficient in ethanol oxidation because of the loss of alcohol dehydrogenase activity. Southern hybridization experiments with the cloned alcohol dehydrogenase-cytochrome c gene cluster as the probe showed insertion of an unknown DNA fragment into a specific position in the cytochrome c gene in most of the mutant strains. Cloning and sequencing analyses revealed that the inserted sequence was 1,665 bp in length and had a terminal inverted repeat of 15 bp. In addition, this inserted sequence was found to generate a 4-bp duplication at the inserted site upon transposition. The target site specificity was not very strict, but a TCGA sequence appeared to be preferentially used. The inserted sequence contains two long open reading frames of 461 and 222 amino acids which are overlapped and encoded by different strands. Although these open reading frames showed no homology to any protein registered in the DNA data bases, the longer open reading frame contained many basic amino acids (87 of 461), as was observed with transposases of so-called insertion sequence (IS) elements. All of these characteristics are typical of IS elements, and the sequence was named IS1380. The copy number of IS1380 in a cell of A. pasteurianus NCI1380 was estimated to be about 100. Several strains of acetic acid bacteria also contained IS1380 at high copy numbers. These results suggest that IS1380 is associated with the genetic loss of ethanol-oxidizing ability as well as the genetic instability of acetic acid bacteria in general.     

Nucleotide sequence of IS26, a new prokaryotic mobile genetic element.

The DNA sequence of a new IS element, the IS26, is 820 bp long and carries 14 bp perfect terminal inverted repeats. Upon integration, IS26 generates an 8 bp duplication of its target sequence. A large open reading frame within IS26 could code for a protein of 234 amino acids. On its reverse strand, IS26 also carries one large open reading frame, 591 bp long, which contains no stop codon within IS26.