Cloning and characterization of a novel sulfate transporter gene from radish (Raphanus sativus L.).

A putative sulfate transporter gene, RSultr3.2A, was identified from radish (Raphanus sativus L.). The RSultr3.2A gene encodes a polypeptide of 651 amino acids with a calculated molecular mass of 71701 Da. The deduced amino acid sequence preserves the common motifs of the sulfate transporters and exhibits a high degree of homology to other plant sulfate transporters. RSultr3.2A was not induced by sulfur starvation and was expressed in roots, stems, leaves, flowers and young pods, suggesting a possible need for the nonregulated and constitutive sulfate transport system of the plant. The RSultr3.2A is a single copy gene in the radish genome. A genomic sequence information corresponding to RSultr3.2A cDNA was found by PCR and sequencing. Comparison of cDNA and genomic sequences revealed that the coding sequence of RSultr3.2A consisted of 12 exons. In addition, another cDNA (RSultr3.2B) from radish young pods was found. As compared with RSultr3.2A, RSultr3.2B encodes a 499 amino acid protein with modifications in the carboxy-terminus. Sequence analysis suggested an occasional donor splicing site mutation (GT to TT) in the RSultr3.2A gene from radish young pods leading to the truncated cDNA RSultr3.2B.     

Characterization of a cDNA encoding a proline-rich 14 kDa protein in developing cortical cells of the roots of bean (Phaseolus vulgaris) seedlings

A cDNA clone, corresponding to mRNAs preferentially expressed in the roots of bean (Phaseolus vulgaris L.) seedlings, was isolated. This clone contains a 381 bp open reading frame encoding a polypeptide of 13.5 kDa, designated PVR5 (Phaseolus vulgaris root 5). The amino acid sequence of this clone is rich in proline (13.5%) and leucine (12.7%) and shares significant amino acid sequence homology with root-specific and proline-rich proteins from monocots (maize and rice), and proline-rich proteins from dicots (carrot, oilseed rape, and Madagascar periwinkle). The precise biological roles of these polypeptides are unknown. PVR5 mRNA accumulation is developmentally regulated within the root, with high levels at the root apex and declining levels at distances further from the root tip. In situ hybridization shows that PVR5 mRNA specifically accumulates in the cortical ground meristem in which maximal cell division occurs. Southern blot analysis suggests that genomic DNA corresponding to PVR5 cDNA is encoded by a single gene or a small gene family.     

油菜烯醇酶基因的克隆与分析(英文)

烯醇酶(enolase)是糖酵解途径中的一个重要酶类,它能够催化磷酸甘油酸酯(2-PGA)生成磷酸烯醇丙酮酸酯(PEP)。我们通过RACE-PCR方法从油菜(Brassica napus L. )中克隆到了编码烯醇酶的全长基因。序列分析表明该基因全长cDNA为1624bp,拥有一个由444个氨基酸组成的开放读码框,所编码的蛋白质分子量为47.38kD,等电点为5.78。比较发现,油菜烯醇酶与已分离出的其他烯醇酶氨基酸序列有较高的同源性。Southern杂交结果显示烯醇酶以低拷贝形式在油菜基因组中存在。RT-PCR和Northern分析表明烯醇酶基因在100mmol/L盐浓度胁迫条件下表达量上升,而在低温诱导时表达量下降。该研究表明所克隆基因是植物烯醇酶基因家族的新成员。     

Molecular Cloning and Characterization of Enolase from Oilseed Rape （Brassica napus）

An enolase-encoding cDNA clone in oilseed rape (Brassica napus L.) was isolated. This gene (accession number: AY307449) had a total length of 1 624 bp with an open reading frame of 1 335 bp, and encoded a predicted polypeptide of 444 amino acids with a molecular weight of 47.38 kD. The deduced amino acid sequence shared identity with a number of enolases ranging from Bacillus subtilis to human beings and had much higher identity with other plant enolases than with enolases from Bacillus, yeast and human beings. Comparison of its primary structure with those of other enolases revealed the presence of an insertion of five amino acids in enolase of B. napus. Southern blotting analysis of genomic DNA indicated that enolase was likely to be a low-copy gene in the oilseed rape genome. Expression of the cloned enolase gene increased under salt stress, but decreased in response to low temperature. Our studies suggested that the cloned gene was a new member of plant enolase gene family, which contributed to the energy supply in stress-treated tissues.     

油菜烯醇酶基因的克隆与分析

烯醇酶(enolase)是糖酵解途径中的一个重要酶类,它能够催化磷酸甘油酸酯(2-PGA)生成磷酸烯醇丙酮酸酯(PEP).我们通过RACE-PCR方法从油菜(Brassica napus L.)中克隆到了编码烯醇酶的全长基因.序列分析表明该基因全长cDNA为1 624bp,拥有一个由444个氨基酸组成的开放读码框,所编码的蛋白质分子量为47.38 kD,等电点为5.78.比较发现,油菜烯醇酶与已分离出的其他烯醇酶氨基酸序列有较高的同源性.Southern杂交结果显示烯醇酶以低拷贝形式在油菜基因组中存在.RT-PCR和Northern分析表明烯醇酶基因在100 mmol/L盐浓度胁迫条件下表达量上升,而在低温诱导时表达量下降.该研究表明所克隆基因是植物烯醇酶基因家族的新成员.     

Primary Structure of Chlamydomonas reinhardtii Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase Activase and Evidence for a Single Polypeptide

Immunoblot analysis of ribulose 1,5-bisphosphate carboxylase/oxygenase (rubisco) activase from the green alga Chlamydomonas reinhardtii indicated the presence of a single polypeptide. This observation contrasts with the Spinacea oleracea (spinach) and Arabidopsis thaliana proteins, in which two polypeptide species are generated by alternative pre-mRNA splicing. A Chlamydomonas rubisco activase cDNA clone containing the entire coding region was isolated and sequenced. The open reading frame encoded a 408 amino acid, 45 kilodalton polypeptide that included a chloroplast transit peptide. The presumptive mature polypeptide possessed 62% and 65% amino acid sequence identity, respectively, with the spinach and Arabidopsis mature polypeptides. The Chlamydomonas rubisco activase transit peptide possessed almost no amino acid sequence identity with the higher plant transit peptides. The nucleotide sequence of Chlamydomonas rubisco activase cDNA provided no evidence for alternative mRNA splicing, consistent with the immunoblot evidence for only one polypeptide. Genomic DNA blot analysis indicated the presence of a single Chlamydomonas rubisco activase gene. In the presence of spinach rubisco activase, a lower extent and rate of activation were obtained in vitro with Chlamydomonas rubisco than with spinach rubisco. We conclude Chlamydomonas rubisco activase comprises a single polypeptide which differs considerably from the higher plant polypeptides with respect to primary structure.     

Bean abscission cellulase : characterization of a cDNA clone and regulation of gene expression by ethylene and auxin

The physiology and anatomy of abscission has been studied in considerable detail; however, information on the regulation of gene expression in abscission has been limited because of a lack of probes for specific genes. We have identified and sequenced a 595 nucleotide bean (Phaseolus vulgaris cv Red Kidney) abscission cellulase cDNA clone (pBACl). The bean cellulase cDNA has extensive nucleic and amino acid sequence identity with the avocado cellulase cDNA pAV363. The 2.0 kilobase bean mRNA complementary to pBACl codes for a polypeptide of approximately 51 kilodalton (shown by hybrid-selection followed by in vitro translation). Bean cellulase antiserum is shown to immunoprecipitate a 51 kilodalton polypeptide from the in vitro translation products of abscission zone poly(A)⁺ RNA. Ethylene initiates bean leaf abscission and tissue-specific expression of cellulase mRNA. If ethylene treatment of bean explants was discontinued after 31 h and then 2,5-norbornadiene given to inhibit responses resulting from endogenously synthesized ethylene, polysomal cellulase mRNA hybridizing to pBACl decreased. Thus, ethylene is required not only to initiate abscission and cellulase gene expression but also to maintain continued accumulation of cellulase mRNA. Explants treated with auxin 4 hours prior to a 48 hour treatment with ethylene showed no substantial accumulation of RNA hybridizing to pBACl or expression of cellulase activity.     

蚕豆豆类胰岛素基因的cDNA克隆及序列分析

为探明豆科植物中豆类胰岛素基因的结构特征与进化关系,在已获得大豆豆类胰岛素基因的基础上,以蚕豆种子胚根mRNA为材料,采用RT-PCR技术,克隆了蚕豆豆类胰岛素基因的cDNA序列,编码的前体多肽包括信号肽、成熟型豆类胰岛素及另一多肽的45个氨基酸残基。DNA序列分析表明,克隆片段与大豆和豌豆的同源性分别为62.5%和58.7%。在氨基酸水平上分别具有44.2%和43.6%的同源性,其中存在着高度保守的半胱氨酸位点,它们在维持豆类胰岛素的空间结构与生理功能方面,可能具有重要的作用。 Abstract:In order to elucidate the relationship between the structural features of leginsulin gene in legume plants and their phylogenetic significance,we have cloned the cDNA sequence of leginsulin gene from radicles of broad bean (Vicia faba) via RT-PCR techniques according to the leginsulin gene sequence we previously obtained from soybean (Glycine max).The cloned cDNA encoded for a precursor protein consisting of the signal peptide,mature leginsulin and an additional 45 amino acids of another polypeptide.A sequence search for homology comparison revealed the cloned leginsulin cDNA fragment shares 62.5% and 58.7% similarity to soybean and pea,respectively.The results also shown that leginsulin cDNA from broad bean presents 44.2% and 43.6% amino acid sequence homology with soybean and pea (Pisum sativum),respectively,and that there exists highly conserved cysteine sites among the leginsulin cDNAs,which may play a crucial role in maintaining the three-dimensional structure and the physiological functions of leginsulin.     

油菜蔗糖转化酶基因的电子克隆和生物信息学分析

运用电子克隆技术获得油菜中一个蔗糖转化酶基因eDNA序列,同时根据此段序列设计引物以油菜eDNA为模板进行扩增。经测序得到证实。采用生物信息学方法,对该基因编码蛋白从氨基酸组成、基本理化性质、跨膜结构域、信号肽导肽、疏水性／亲水性、二级结构、亚细胞定位等方面进行了预测和分析。结果表明：该基因eDNA序列长度为2150bp,包含一个1779bp开放阅读框,编码592个氨基酸;该编码蛋白含有蔗糖转化酶的多个典型的保守结构域。同源比对分析显示,该基因编码的氨基酸序列与拟南芥等植物的蔗糖转化酶基因具有高度的相似性,进一步确定该蛋白为蔗糖蛋白酶。研究结果为该基因进一步的实验克隆,表达分析,功能鉴定奠定基础。     

Dehiscence of Fruit in Oilseed Rape (Brassica napus L.): I. ANATOMY OF POD DEHISCENCE

The development sequence of anatomical changes taking placewithin the pericarp tissues of Brassica napus siliquae havebeen studied at a fine- and ultra-structural level. Tissue differentiationoccurred during the initial 20 d after anthesis (DAA) and allowedthe identification of dehiscence zone cells. This descrete tissuewas subsequently further delineated by extensive lignificationof adjacent valve edge and replar vascular cells. Concomitantwith the onset of pericarp lignification, cytoplasmic contentsof the thin-walled dehiscence zone cells exhibited progressivesenescence and degradation. Wall breakdown, initially evidentin pods by 60 DAA, exclusively affected cells within the dehiscencezone, and eventually extended throughout this tissue from theepidermal suture to the locule, thus precipitating valve detachment.Ultrastructural examination confirmed that this loss of cellularcohesion was primarily attributable to middle lamella degradationand, furthermore, the dissolution of wall material was apparentlydependent on rupture of the dehiscence zone protoplast. Thesignificance of dehiscence zone cell modifications in relationto autolytic cell wall breakdown, together with possible implicationsfor the regulation of pod shatter, are discussed. Key words: Oilseed rape, Brassica napus, pod shatter, dehiscence zone, cell wall breakdown