野生大豆盐碱胁迫相关GsTIFY11b的克隆与功能分析

以耐盐碱野生大豆(Glycine soja L.G07256)为材料, 采用同源克隆方法和RT-PCR技术获得一个TIFY 类基因的全长cDNA(命名为GsTIFY11b)。进化树分析表明, 与其他物种相比, GsTIFY11b与拟南芥的AtTIFY11a基因相似性最高, 达到56%; 序列分析表明GsTIFY11b蛋白除具有 TIFY保守结构域外, 还具有一个N端保守结构域和一个C端保守的Jas结构域; 实时荧光定量PCR结果显示该基因受盐和碱胁迫诱导表达; 将GsTIFY11b转化拟南芥来验证其耐盐碱功能, 获得两个转基因纯合体株系, 盐碱胁迫分析结果表明, GsTIFY11b的超量表达没能提高拟南芥对盐碱胁迫的耐性, 并且与野生型相比, 转基因植株在种子萌发期和苗期表现出对盐胁迫更加敏感。盐胁迫信号通路相关marker基因在转基因拟南芥中的表达特性分析表明, GsTIFY11b可以调控RD29B、KIN1、DREB等基因的转录。在洋葱表皮细胞中瞬时表达GsTIFY11b-GFP融合蛋白的结果表明, GsTIFY11b定位于细胞核中。上述结果表明, 该基因在细胞核中起着转录调节子的作用, 可能是通过调控盐胁迫信号通路中关键基因的表达来改变植物对盐胁迫的耐受性。     

野生大豆盐碱胁迫相关GsTIFY11b的克隆与功能分析

以耐盐碱野生大豆(Glycine soja L.G07256)为材料,采用同源克隆方法和RT-PCR技术获得一个TIFY类基因的全长cDNA(命名为GsTIFY11b)。进化树分析表明,与其他物种相比,GsTIFY11b与拟南芥的AtTIFY11a基因相似性最高,达到56%;序列分析表明GsTIFY11b蛋白除具有TIFY保守结构域外,还具有一个N端保守结构域和一个C端保守的Jas结构域;实时荧光定量PCR结果显示该基因受盐和碱胁迫诱导表达;将GsTIFY11b转化拟南芥来验证其耐盐碱功能,获得两个转基因纯合体株系,盐碱胁迫分析结果表明,GsTIFY11b的超量表达没能提高拟南芥对盐碱胁迫的耐性,并且与野生型相比,转基因植株在种子萌发期和苗期表现出对盐胁迫更加敏感。盐胁迫信号通路相关marker基因在转基因拟南芥中的表达特性分析表明,GsTIFY11b可以调控RD29B、KIN1、DREB等基因的转录。在洋葱表皮细胞中瞬时表达GsTIFY11b-GFP融合蛋白的结果表明,GsTIFY11b定位于细胞核中。上述结果表明,该基因在细胞核中起着转录调节子的作用,可能是通过调控盐胁迫信号通路中关键基因的表达来改变植物对盐胁迫的耐受性。     

Overexpression of soybean ubiquitin-conjugating enzyme gene GmUBC2 confers enhanced drought and salt tolerance through modulating abiotic stress-responsive gene expression in Arabidopsis

Previous studies have shown that ubiquitination plays important roles in plant abiotic stress responses. In the present study, the ubiquitin-conjugating enzyme gene GmUBC2, a homologue of yeast RAD6, was cloned from soybean and functionally characterized. GmUBC2 was expressed in all tissues in soybean and was up-regulated by drought and salt stress. Arabidopsis plants overexpressing GmUBC2 were more tolerant to salinity and drought stresses compared with the control plants. Through expression analyses of putative downstream genes in the transgenic plants, we found that the expression levels of two ion antiporter genes AtNHX1 and AtCLCa, a key gene involved in the biosynthesis of proline, AtP5CS, and the copper chaperone for superoxide dismutase gene AtCCS, were all increased significantly in the transgenic plants. These results suggest that GmUBC2 is involved in the regulation of ion homeostasis, osmolyte synthesis, and oxidative stress responses. Our results also suggest that modulation of the ubiquitination pathway could be an effective means of improving salt and drought tolerance in plants through genetic engineering.     

Differential expression profiles of poplar MAP kinase kinases in response to abiotic stresses and plant hormones,and overexpression of PtMKK4 improves the drought tolerance of poplar

Mitogen-activated protein kinase (MAPK) cascades are universal signal transduction modules that play essential roles in plant growth, development and stress response. MAPK kinases (MAPKKs), which link MAPKs and MAPKK kinases (MAPKKKs), are integral in mediating various stress responses in plants. However, to date few data about the roles of poplar MAPKKs in stress signal transduction are available. In this study, we performed a systemic analysis of poplar MAPKK gene family expression profiles in response to several abiotic stresses and stress-associated hormones. Furthermore, Populus trichocarpa MAPKK4 (PtMKK4) was chosen for functional characterization. Transgenic analysis showed that overexpression of the PtMKK4 gene remarkably enhanced drought stress tolerance in the transgenic poplar plants. The PtMKK4-overexpressing plants also exhibited much lower levels of H₂O₂ and higher antioxidant enzyme activity after exposure to drought stress compared to the wide type lines. Besides, some drought marker genes including PtP5CS, PtSUS3, PtLTP3 and PtDREB8 exhibited higher expression levels in the transgenic lines than in the wide type under drought conditions. This study provided valuable information for understanding the putative functions of poplar MAPKKs involved in important signaling pathways under different stress conditions.     

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.     

Molecular evolution of plant P5CS gene involved in proline biosynthesis

The P5CS ({Delta} 1-Pyrroline–5-Carboxylate Synthetase) gene encodes for a bifunctional enzyme that catalyzes the rate limiting reaction in proline biosynthesis in living organisms. A wide range of multifunctional roles of proline have now been shown in stress defense. The proline biosynthetic genes, especially, P5CS is commonly used in metabolic engineering for proline overproduction conferring stress tolerance in plants. The gene is functionally well characterized at the molecular level, but there is more to learn about its evolutionary path in the plant kingdom, particularly the drive behind functional (osmoprotective and developmental) divergence of duplication of P5CS genes. In this review, we present the current understanding of the evolutionary trail of plant P5CS gene which plays a key role in stress tolerance.     

Ectopic Expression of a Glycine soja myo-Inositol Oxygenase Gene (GsMIOX1a) in Arabidopsis Enhances Tolerance to Alkaline Stress

Myo-inositol participates in various aspects of plant physiology, and myo-inositol oxygenase is the key enzyme of the myo-inositol oxygenation pathway. Previous studies indicated that myo-inositol oxygenase may play a role in plant responses to abiotic stresses. In this study, we focused on the functional characterization of GsMIOX1a, a remarkable alkaline stress-responsive gene of Glycine soja 07256, based on RNA-seq data. Using quantitative real-time PCR, we demonstrated that GsMIOX1a is rapidly induced by alkaline stress and expressed predominantly in flowers. We also elucidated the positive function of GsMIOX1a in the alkaline response in the wild type, atmiox1 mutant as well as GsMIOX1a-overexpressing Arabidopsis. We determined that atmiox1 mutant decreased Arabidopsis tolerance to alkaline stress, whereas GsMIOX1a overexpression increased tolerance. Moreover, the expression levels of some alkaline stress-responsive and inducible marker genes, including H⁺-Ppase, NADP-ME, KIN1 and RD29B, were also up-regulated in GsMIOX1a overexpression lines compared with the wild type and atmiox1 mutant. Together, these results suggest that the GsMIOX1a gene positively regulates plant tolerance to alkaline stress. This is the first report to demonstrate that ectopic expression of myo-inositol oxygenase improves alkaline tolerance in plants.     

Subcellular Localization and Functional Analysis of the Arabidopsis GTPase RabE

Membrane trafficking plays a fundamental role in eukaryotic cell biology. Of the numerous known or predicted protein components of the plant cell trafficking system, only a relatively small subset have been characterized with respect to their biological roles in plant growth, development, and response to stresses. In this study, we investigated the subcellular localization and function of an Arabidopsis (Arabidopsis thaliana) small GTPase belonging to the RabE family. RabE proteins are phylogenetically related to well-characterized regulators of polarized vesicle transport from the Golgi apparatus to the plasma membrane in animal and yeast cells. The RabE family of GTPases has also been proposed to be a putative host target of AvrPto, an effector protein produced by the plant pathogen Pseudomonas syringae, based on yeast two-hybrid analysis. We generated transgenic Arabidopsis plants that constitutively expressed one of the five RabE proteins (RabE1d) fused to green fluorescent protein (GFP). GFP-RabE1d and endogenous RabE proteins were found to be associated with the Golgi apparatus and the plasma membrane in Arabidopsis leaf cells. RabE down-regulation, due to cosuppression in transgenic plants, resulted in drastically altered leaf morphology and reduced plant size, providing experimental evidence for an important role of RabE GTPases in regulating plant growth. RabE down-regulation did not affect plant susceptibility to pathogenic P. syringae bacteria; conversely, expression of the constitutively active RabE1d-Q74L enhanced plant defenses, conferring resistance to P. syringae infection.