Saline-alkali Stress Research Articles

Virus-induced gene silencing in two novel functional plants, Lycium barbarum L. and Lycium ruthenicum Murr.

Two species of Goji, Lycium barbarum L. and Lycium ruthenicum Murr., are novel functional vegetables and functional fruits widely used in China and other Asian countries. Both species possess complex secondary metabolic pathways and show high tolerance and adaptability to saline-alkali stress, making them novel targets for functional genetic analysis of the biochemical pathways involved. Although stable transgenic Goji lines have been produced, the process is very labor-intensive and time-consuming. Virus-induced gene silencing (VIGS) presents an effective and rapid alternative for creating targeted gene knock-outs to study gene function in plants. In this study, the first application of VIGS in Lycium species is presented, using the Tobacco rattle virus (TRV) vectors. A number of vector delivery methods were trialed, including leaf syringe-infiltration, agrodrench, seedling vacuum-infiltration and sprout vacuum-infiltration (SVI). Vacuum-infiltration was the most effective method and was used to successfully silence two reporter genes, phytoene desaturase (PDS) and Mg-chelatase H subunit (Chl H), concomitant with photobleaching and yellow leaf phenotypes, respectively. The proven application of VIGS to these Lycium sp. will expedite the functional characterization of novel genes involved in the biosynthesis of functional components both in leaves and fruits, as well as the abiotic stress tolerances.

Expression Analysis of <i>Puccinellia tenuifolra</i> Gene <i>PutSTE24</i> and Response to Aluminium Stresses

Alkaline grass ( Puccinellia tenuiflora ) can serve as a model of salt tolerance in monocotyledon crops. However, little is known about its mechanism of aluminum stress. In this work, we screened PutSTE24 gene under aluminum stress to predict its subcellular localization of proteins by using bioinformatics analysis software, and the result showed that this gene was in the endoplasmic reticulum, which was the same with expressing PutSTE24 gene in yeast INVsI. Under aluminum and saline-alkali stress conditions, the relative mRNA expression levels of the PutSTE24 of alkaline grass were measured by real-time quantitative PCR, and the results demonstrated that its expression was induced up regulation. We also constructed a plant expression vector of PutSTE24 gene, and then transferred into Arabidopsis. For further determine the correlation between PutSTE24 gene and aluminum stress, we choose transgenic seeds for resistance experiment, which provided a wealth of theoretical and experimental basis for further research on interaction of related genes and regulatory mechanism of aluminum stress.

Alfalfa seeding root characteristics under complex saline-alkali stress

为研究紫花苜蓿根系对混合盐碱胁迫的响应, 探索紫花苜蓿对混合盐碱的适应特点, 为紫花苜蓿在盐碱地的栽培实践提供理论依据。本试验以"甘农三号"紫花苜蓿为试验材料, 采用两种中性盐NaCl和Na₂SO₄及两种碱性盐NaHCO₃和Na₂CO₃, 按不同比例混合成为A(NaCl∶Na₂SO₄=1∶1)、B(NaCl∶NaHCO3=1∶1)、C(NaCl∶Na₂SO₄∶NaHCO₃∶Na₂CO₃=1∶1∶1∶1)、D(Na₂SO₄∶Na₂CO₃=1∶1)、E(NaHCO₃∶Na₂CO₃=1∶1)5种混合盐, 分别以25 mmol·L^-1、50 mmol·L^-1、100 mmol·L^-1、150 mmol·L^-1 4种盐浓度, 模拟出20种盐度和碱度各不相同的混合盐碱条件对紫花苜蓿幼苗进行处理, 测定了根系总长度、根系平均直径、根表面积、根尖数等指标, 以了解紫花苜蓿根系在混合盐碱胁迫下的生长特性。结果表明, 盐浓度为影响紫花苜蓿根系生长的主要因素。混合盐碱对根尖数的影响表现出明显的负效应, 对根系平均直径的影响并不明显。随着混合盐浓度的升高, 根系总长度、根表面积呈现先增大后减小的趋势。A、B处理的根长分别在盐浓度为25 mmol·L^-1和50 mmol·L ^-1时达到最大值, 分别比对照(0 mmol·L^-1)增加50.7%和37.9%, 而且差异均达到显著水平(<i>P</i><0.05); 在盐浓度达到150 mmol·L^-1时, A、B、C、D、E的根系总长度分别比对照减少26.6%、37.7%、51.6%、37.0%和55.7%, 差异显著。A、C、D组处理下仅在25 mmol·L ^-1时, 根表面积显著增加, 分别比对照增加21.1%、43.4%和12.7%; 在浓度为150 mmol·L^-1时, E处理下根表面积显著减小, 比对照减少49.6%。表明低浓度混合盐碱对紫花苜蓿根系的生长影响不显著或具有一定的促进作用, 高浓度胁迫下抑制根系的生长; 在高盐浓度下随着碱性盐比例的增加, 对紫花苜蓿根系生长的抑制作用越明显。

Coexpression of ScNHX1 and ScVP in Transgenic Hybrids Improves Salt and Saline-Alkali Tolerance in Alfalfa (Medicago sativa L.)

Salt and saline-alkali are major environmental factors limiting the growth and productivity of alfalfa, the most economically important forage legume worldwide. In this study, alfalfa plants transgenic for both ScNHX1 (encoding vacuolar membrane Na+/H+ antiporter from Suaedacorniculata) and ScVP (encoding vacuolar H+-PPase from S.corniculata) were produced by cross-pollination. Transgenic alfalfa plants coexpressing ScVP/ScNHX1 showed enhanced salt and saline-alkali tolerance to 300 or 200 mM NaCl with 100 mM NaHCO3 treatments, compared with wild-type plants. In addition, ScVP/ScNHX1-coexpressing alfalfa plants accumulated more Na+ in leaves and roots than wild-type plants and showed increased tolerance to higher salt and saline-alkali stress. Using the fluorescent carboxy-SNARF probe, the intracellular pH was visualized in the transgenic and wild-type plants under salt and saline-alkali stress. The results showed that the overnight treatment caused a massive change in pH in ScVP/ScNHX1-coexpressing alfalfa plants and they showed that there was significantly higher vacuolar alkalization under salt stress compared with wild-type plants. However, saline-alkali treatment enhanced vacuolar acidification more in the wild-type plants than in transgenic plants. Taken together, our results indicate that coexpression of multiple, effective genes in transgenic plants can enhance resistance to salt and saline-alkali stress.

Agronomic Traits of Transgenic Soybean Overexpressing TaDREB3

Dongnong50 was overexpressing TaDREB3, a stress-tolerance gene, and transgenic progenies with TaDREB3 showing stable heredity characters were obtained in this study. The seeds of T4 generation were sowed on saline land of which salt concentration was 0.19% and pH was 8.43, and agronomic traits including growth period, plant height, numbers of main nodes, branching numbers, pods per plant, numbers of seeds per plant, and weight of 100-seed were collected and comparing to control plants Dongnong50 were made. The impacts of TaDREB3 gene on agronomic traits of soybeans under high concentration of salt were discussed. The results showed that the growth period was not altered in transgenic line with TaDREB3 under high concentration of saline and alkali, the extremely significant or significant increase in the numbers of main nodes, pods per plant and in branching numbers and other agronomic traits in transgenic lines in comparing with non-transgenic lines was an indicator of their resistance to saline-alkali stress.

Expression profiling of the genes induced by Na 2CO 3 and NaCl stresses in leaves and roots of Leymus chinensis

Alkaline soil is more challenging factor to grow plants than saline soil based on the size of its affected area. To reveal genetic expression changes by alkaline and saline soil, Leymus chinensis (Trin.) Tzvel. was examined response to Na 2CO 3 and NaCl stresses by using microarray chips comprising 1642 cDNA clones, previously reported by us. A total of 536 genes were responsive to Na 2CO 3 and NaCl stresses by up-regulation or down-regulation. We observed transcriptional changes arose more to Na 2CO 3 stress, or leaves than to NaCl stress or roots, respectively, in L. chinensis genome. Overall, 99 and 59 genes were up-regulated, while 365 and 176 genes were down-regulated in leaves and roots, respectively; demonstrating down-regulation occurs more as the response to the Na 2CO 3 and NaCl stress. The majority of the down-regulated genes (30.1%) were classified to photosynthesis related proteins while that of the up-regulated genes were categorized into metabolism proteins or noble proteins. Only 37 genes in the stressed leaves and 6 genes in the stressed roots were showed the same expression changing patterns between Na 2CO 3 and NaCl stresses during three time points of detections. Among those genes, 70% were constantly up-regulated or down-regulated during our detection presenting difference in the gene regulation systems against two difference stress, the saline-alkali stress (Na 2CO 3) and saline stress (NaCl). In addition, a total of 87 genes detected in this study were characterized to the unclassified proteins, whereas 72 had no similarity to the current GenBank databases which were considered as novel proteins detected in the alkali and saline stressed L. chinensis genome. The four important genes to abiotic stress tolerance, ACC, GDP, hsp70, and elF1 were detected constantly for all of three time points of stressed which are the grand candidates for establishing stress tolerance plants by developing transgenic.

Osmotic Regulation of Betaine Content in Leymus chinensis Under Saline-alkali Stress and Cloning and Expression of Betaine Aldehyde Dehydrogenase(BADH) Gene

Abstract The potted Leymus chinensis seedlings were treated with saline-alkali solution of six different(from I to VI) concentrations. The results demonstrate that the betaine content and Betaine-aldehyde dehydrogenase( BADH: EC 1.2.1.8) activities have a direct relation with increased stressing time in the same treatment; both exhibit a single peak with increasing the concentration of saline-alkali solution, and number V shows the highest value. The BADH gene of Leymus chinensis was cloned by RT-PCR and RACE technology and was designated as LcBADH . The cDNA sequence of LcBADH was 1774bp including the open reading frame(ORF) of 1521bp(coding 506 amino acids). The vector of prokaryotic expression was constructed by inserting the LcBADH gene fragment into pET30a(+) and transformed into E. coli BL21 (DE3). The result of SDS-PAGE shows that the idio-protein with a molecular mass of 56.78 kDa was effectively expressed in the recombinant bacteria induced by isopropyl β-D -thiogalactoside(IPTG).

Expression profiling of salinity-alkali stress responses by large-scale expressed sequence tag analysis in Tamarix hispid

Tamarix hispida, a woody halophyte, thrives in saline and saline-alkali soil. To better understand the gene expression profiles that manifest in response to saline-alkali stress, three cDNA libraries were constructed from leaf tissue of T. hispida plants that were well watered and exposed to NaHCO3 for 24 and 52 h. A total of 9,447 high quality expressed sequence tags (ESTs) were obtained from the three libraries. These ESTs represent 3,945 unigenes, including 986 contigs and 2,959 singlets. The numbers of unigenes obtained from the three libraries were 1,752, 1,558 and 1,675, respectively. The EST analysis was performed to compare gene expression in the three cDNA libraries; the transcripts responsive to NaHCO3 were identified. The differentially expressed transcripts were identified. The up-regulation genes were involved in a variety function areas, such as stress-related proteins, hormone signaling transduction, antioxidative response, transcriptional regulators, protein synthesis and destination, ion homeostasis, photosynthesis and metabolism. The results indicated that the response to NaHCO3 in T. hispida is a complex one, involving multiple physiological and metabolic pathways. Nine gene expression patterns were compared in response to NaHCO3 and NaCl using real time reverse transcription-polymerase chain reaction (RT-PCR). Gene expression trends were similar after a 24-h exposure to either NaCl or NaHCO3, however, great variability was found after a 52-h exposure, indicating that short-term responses to either salt may not be obviously different.

Development of a cDNA microarray to identify gene expression of Puccinellia tenuiflora under saline–alkali stress

Puccinellia tenuiflora is the main grass species growing in the saline–alkali soil of the Songnen plain in northeastern China, suggesting it has a high tolerance to saline stress. In this study, cDNA microarrays containing 1067 clones of P. tenuiflora were constructed to investigate gene expression patterns resulting from saline–alkali (NaHCO 3) stress. RNA was extracted from P. tenuiflora treated with 400 mmol L −1 NaHCO 3 for 6, 12, 24 and 48 h. Untreated (no saline–alkali stress) samples were used as control. A total of 95 transcripts were differentially regulated under the conditions studied, and 38, 35, 25 and 49 genes were differentially expressed with NaHCO 3 stress for 6, 12, 24 and 48 h, respectively. Among these, approximately 40% were putative novel or functionally unknown genes, and the remainder function in photosynthesis, cell rescue, defense, transport, metabolism, transcription regulation and protein destination, etc. Analysis of the P. tenuiflora genes demonstrated the complexity of, and differences in, gene expression patterns resulting from different NaHCO 3 stress times. The genetic relationship between P. tenuiflora and other plants was investigated by BlastN analysis. The results showed nearly 20% of the expressed sequence tags from P. tenuiflora shared significant similarities with rice Oryza sativa, an important food crop. The close genetic relationship between these two species suggests that P. tenuiflora may be a good plant model for studying saline–alkali tolerance mechanisms in O. sativa.