野生大豆P5CS基因的克隆及对盐胁迫反应

逆境下植物大量积累脯氨酸是减轻胁迫伤害的一种自我保护机制。本研究应用同源克隆方法从NaCl处理的野生大豆中克隆获得一个脯氨酸合成酶(P5CS)基因,命名为GsP5CS。该基因核苷酸序列全长2.232 kb,含一个2148bp开放阅读框,编码715个氨基酸,包含有高等植物P5CS蛋白质的5个主要功能域,与菜豆PvP5CS1基因核苷酸序列相似性高达98.79%。Real Time PCR分析显示该基因受轻度盐胁迫诱导上调表达,根中表达高峰出现在200 mmol/L NaCl处理下,相对表达量为对照的5.83倍;叶片中表达高峰出现在300 mmol/L NaCl处理条件下,相对表达量为对照的12.78倍。并且该基因在根和叶片中的表达模式和脯氨酸含量的变化模式相同。上述结果说明,GsP5CS可能参与野生大豆脯氨酸合成。     

Salinity-induced expression of pyrrolline-5-carboxylate synthetase determine salinity tolerance in Brassica spp

The objective of the present study was to assess the role of salinity-induced expression of pyrrolline 5-carboxylate synthetase (P5CS), P5CS activity, and proline accumulation on salinity tolerance in Brassica genotypes. A pot culture experiment was conducted with four Brassica genotypes viz. CS 52, CS 54, Varuna, (B. juncea) and T 9 (B. campestris) under control and two salinity levels, i.e., 1.65, 4.50 and 6.76?dS?m^?1. Proline contents increased with increasing levels of salinity, and the highest content were recorded at post-flowering stage in CS 52 and CS 54. Activity of P5CS recorded at flowering stage was highest at higher level of salinity, with CS 52 and CS 54 recording highest activity. Gene expression of P5CS, which regulates the synthesis of proline, was higher in CS 52 and CS 54 under salt stress than Varuna and T 9. Comparison of partial nucleotide as well as amino acid sequence showed conserved domains, and inter and intra generic relatedness of these genes. The study suggests that salinity-induced expression of P5CS, pyrrolline-phosphate synthetase activity and proline accumulation may serve as one of the mechanism of salinity stress tolerance in Brassica genotypes.     

木薯及其他植物P5CS基因进化及表达分析

该研究采用生物信息学的方法,从木薯等31个已完成基因组测序的植物中,共鉴定出84个吡咯啉-5-羧酸合成酶(P5CS)基因,并对其进行系统发育分析。结果表明:(1)P5CS在内含子长度上差别较大,而在氨基酸长度、外显子数目、等电点和分子量上差别不大。(2)由于发生基因重复,在大多数单子叶和双子叶植物中都有2个P5CS,而且在单子叶和双子叶植物中均发现P5CS1基因聚类在一组,而P5CS2基因聚类在另一组,支持P5CS1和P5CS2基因是独立起源,且该重复事件发生在单子叶和双子叶植物分化之前。(3)在某些植物(如蒺藜苜蓿、大豆等)中存在3~7个P5CS基因,表明在单子叶和双子叶植物分化之后P5CS基因又发生了多次重复事件,并将它们归纳为4种进化模式。(4)木薯中有2个P5CS基因,表达分析显示,MeP5CS1和MeP5CS2在叶片、叶柄、茎、须根和储藏根中均可检测到,其中MeP5CS1在叶片中表达较高,而MeP5CS2在茎和储藏根中表达较高。(5)干旱胁迫下,MeP5CS1仅在第一片完全展开叶中被显著诱导,而MeP5CS2在第一片完全展开叶和老叶中均能被显著诱导;低温胁迫下,MeP5CS1和MeP5CS2在不同组织中均能被显著诱导,但具有不同的表达模式。研究表明,木薯的MeP5CS1和MeP5CS2基因在转录水平受到干旱、低温等非生物胁迫的调控。     

The evolution of pyrroline-5-carboxylate synthase in plants: a key enzyme in proline synthesis

Many plants synthesize and accumulate proline in response to osmotic stress conditions. A central enzyme in the proline biosynthesis is the bifunctional enzyme Δ¹-pyrroline-5-carboxylate synthase (P5CS) that includes two functional catalytic domains: the γ-glutamyl kinase and the glutamic-γ-semialdehyde dehydrogenase. This enzyme catalyzes the first two steps of the proline biosynthetic pathway and plays a central role in the regulation of this process in plants. To determine the evolutionary events that occurred in P5CS genes, partial sequences from four Neotropical trees were cloned and compared to those of other plant taxa. Molecular phylogenetic analysis indicated that P5CS duplication events have occurred several times following the emergence of flowering plants and at different frequencies throughout the evolution of monocots and dicots. Despite the high number of conserved residues in plant P5CS sequences, positive selection was observed at different regions of P5CS paralogous genes and also when dicots and monocots were contrasted.     

Isolation and characterization of a Δ1-pyrroline-5-carboxylate synthetase (NtP5CS) from Nitraria tangutorum Bobr. and functional comparison with its Arabidopsis homologue

Several functional and regulatory proteins play important roles in controlling plant stress tolerance. Proline (Pro) is one of the most accumulated osmolytes correlated with tolerance to stresses. Δ¹-Pyrroline-5-carboxylate synthetase (P5CS) is a rate-limiting enzyme in Pro biosynthesis. In the present study, we isolated the cDNA for a P5CS gene (NtP5CS) from the halophyte Nitraria tangutorum. Phylogenetic analysis and subcellular localization analysis of NtP5CS-GFP protein in onion cells showed that NtP5CS was a new P5CS gene and was involved in Pro synthesis in N. tangutorum. Expression of the NtP5CS gene was induced by salt stress, dehydration, and high and low temperatures. Escherichia coli overexpressing AtP5CS or NtP5CS exhibited better growth in all treatments, including high salinity, high alkalinity, dehydration, osmotic, heat and cold stresses. Additionally, NtP5CS recombinant E. coli cells grew better than did AtP5CS recombinant cells in response to abiotic stresses. Our data demonstrate that the P5CS from a halophytic species functions more efficiently than its homologue from a glycophytic species in improving the stress tolerance of E. coli.     

Reciprocal regulation of Δ 1-pyrroline-5-carboxylate synthetase and proline dehydrogenase genes controls proline levels during and after osmotic stress in plants

Plants generally accumulate free proline under osmotic stress conditions. Upon removal of the osmotic stress, the proline levels return to normal. In order to understand the mechanisms involved in regulating the levels of proline, we cloned and characterized a proline dehydrogenase (PDH) cDNA from Arabidopsis thaliana (AtPDH). The 1745?bp cDNA contains a major open reading frame encoding a peptide of 499 amino acids. The deduced amino acid sequence has high homology with both Saccharomyces cerevisiae and Drosophila melanogaster proline oxidases and contains a putative mitochondrial targeting sequence. When expressed in yeast, the AtPDH cDNA complemented a yeast put1 mutation and exhibited proline oxidase activity. We also determined the free proline contents and the Δ¹-pyrroline-5-carboxylate synthetase (P5CS) and PDH mRNA levels under different osmotic stress and recovery conditions. The results demonstrated that the removal of free proline during the recovery from salinity or dehydration stress involves an induction of the PDH gene while the activity of P5CS declines. The reciprocal regulation of P5CS and PDH genes appears to be a key mechanism in the control of the levels of proline during and after osmotic stress. The PDH gene was also significantly induced by exogenously applied proline. The induction of PDH by proline, however, was inhibited by salt stress.     

Three <Emphasis Type="Italic">P5CS</Emphasis> genes including a novel one from <Emphasis Type="Italic">Lilium regale</Emphasis> play distinct roles in osmotic,drought and salt stress tolerance

Proline accumulations in abiotically stressed plants is generally considered to benefit their stress tolerance. The Δ¹-Pyrroline-5-carboxylate synthetase (P5CS) gene family, which encodes the rate-limiting enzyme in proline biosynthesis pathway, usually contains two duplicated genes in most plants. However, three P5CS genes including LrP5CS1, LrP5CS2 as well as a third one, LrP5CS3, were isolated from Lilium regale. LrP5CS3 is highly identical to LrP5CS1 in amino acid sequences, indicating they could come from a paralogous duplication. The phylogenetic tree suggested that the duplication of LrP5CS occurred independently after the divergence of Liliales and commelinoids. The expression of LrP5CS1 was strongly induced in leaves and roots both under drought and salinity, while that of LrP5CS3 was upregulated more moderately. LrP5CS2 stayed almost constitutive under stress. LrP5CS1 exhibited the highest activity after expressed in E. coli. Overexpression of LrP5CS genes conferred enhanced osmotic, drought and salt tolerance on transgenic Arabidopsis without negative effects in unstressed condition. Under salt stress, lines LrP5CS2 accumulated fewer proline than others, and lines LrP5CS1 grew better in root elongation. The roots of lines LrP5CS3 grew better than all others under unstressed condition and osmotic stress. Our study suggests that the three LrP5CS genes play distinct roles respectively in proline accumulation and abiotic stress tolerance.     

Generation of transgenic wheat plants producing high levels of the osmoprotectant proline

Plasmid DNA (pBI-P5CS), containing the selectable neomycin phosphotransferase-II `npt II' gene for kanamycin resistance and the reporter -glucuronidase `gus' gene as well as the Vigna aconitifolia ¹-pyrroline-5-carboxylate synthetase `P5CS' cDNA that encodes enzymes required for the biosynthesis of proline, was delivered into wheat plants using Agrobacterium-mediated gene transfer via indirect pollen system. Southern, northern and western blot analysis demonstrated that the foreign gene had been transferred, expressed and integrated into wheat chromosomal DNA. Salinity test indicated that proline acts as an osmoprotectant and its overproduction in transgenic wheat plants results in the increased tolerance to salt.     

Structure and analysis of phospholipase D gene from Ricinus communis L

Phospholipase D (PLD; EC 3.1.4.4) has been proposed to play a pivotal role in various cellular processes, but molecular understanding of this enzyme is rather limited. This report describes the nucleotide sequence, structure, and genomic organization of a PLD gene from castor bean (Ricinus communis L. cv. Hale). The PLD gene was isolated from a castor bean genomic library using the PLD cDNA as a hybridization probe. Sequence comparison with the PLD cDNA revealed that the PLD gene consisted of four exons and three introns, one of which interrupts the 5-untranslated region. Southern blot analysis indicated that the cloned PLD gene was present as a single-copy gene, and yet there were other PLD or PLD-related sequences in the castor bean genome.     

Proline accumulation and the expression of Δ1-pyrroline-5-carboxylate synthetase in two safflower cultivars

Proline (Pro) accumulation under water stress was measured in safflower (Carthamus tinctorius L.) drought tolerant cv. A1 and sensitive cv. Nira. Activities of pyrroline-5-carboxylate reductase (P5C reductase) and pyrroline-5-carboxylate synthetase (P5C synthetase), two enzymes involved in the Pro biosynthetic pathway were also estimated. Water stress resulted in a reduction in the leaf dry mass and chlorophyll content along with a gradual accumulation of Pro. RT-PCR results show higher expression of Δ¹-pyrroline-5-carboxylate synthetase (p5cs) gene in correlation with up-regulated Pro accumulation in cv. A1. P5C reductase was found to be the Pro synthesis rate limiting whereas P5C synthetase did not show any specific response to the drought stress in both cultivars.     

Cloning and heterologous expression of nitrate reductase genes from <Emphasis Type="Italic">Dunaliella salina</Emphasis>

A cDNA corresponding to the nitrate reductase (NR) gene from Dunaliella salina was isolated by RT-PCR and (5′/3′)-RACE techniques. The full-length cDNA sequence of 3,694 bp contained an open reading frame of 2,703 bp encoding 900 amino acids, a 5′-untranslated region of 151 bp and a 3′-untranslated sequence of 840 bp with a poly (A) tail. The putative gene product exhibited 78%, 65%, 59% and 50% identity in amino acid sequence to the corresponding genes of Dunaliella tertiolecta, Volvox carteri, Chlamydomonas reinhardtii, and Chlorella vulgaris, respectively. Phylogenetic analysis showed that D. salina NR clusters together with known NR proteins of the green algae. The molecular mass of the encoded protein was predicted to be 99.5 kDa, with an isoelectric point of 8.31. This protein shares common structural features with NRs from higher plants and green algae. The full-length cDNA was heterologously expressed in Escherichia coli as a fusion protein, and accumulated to up to 21% of total bacteria protein. Recombinant NR protein was active in an enzyme assay, confirming that the cloned gene from D. salina is indeed NR.