Selection of the Reference Gene for Expression Normalization in Salsola ferganica under Abiotic Stress.

Salsola ferganica is a natural desert herbaceous plant in the arid area of western and northwestern China. Because of its salt tolerance and drought resistance, it is of great significance in desert afforestation and sand-fixing capacity. There has been much research on the genes involved in plants under desert stresses in recent years. The application of the best internal reference genes for standardization was a critical procedure in analyzing the gene expression under different types. Even so, the reference gene has not been reported in the application of gene expression normalization of S. ferganica. In this study, nine reference genes (TUA-1726, TUA-1760, TUB, GAPDH, ACT, 50S, HSC70, APT, and U-box) in S. ferganica were adopted and analyzed under six different treatments (ABA, heat, cold, NaCl, methyl viologen (MV), and PEG). The applicability of candidate genes was evaluated by statistical software, including geNorm, NormFinder, BestKeeper, and RefFinder, based on their stability values in all the treatments. These results indicated that the simultaneous selection of two stable reference genes would fully standardize the optimization of the normalization research. To verify the feasibility of the above internal reference genes, the CT values of AP2/ERF transcription factor family genes were standardized using the most (ACT) and least (GAPDH) stable reference genes in S. ferganica seedlings under six abiotic stresses. The research showed that HSC70 and U-box were the most appropriate reference genes in ABA stressed samples, and ACT and U-box genes were the optimal references for heat-stressed samples. TUA-1726 and U-box showed the smallest value in gene expression levels of cold treatment. The internal reference groups of the best applicability for the other samples were U-box and ACT under NaCl treatment, ACT and TUA-1726 under MV stress, HSC70 and TUB under PEG treatment, and ACT in all samples. ACT and U-box showed higher stability than the other genes based on the comprehensive stability ranking of RefFinder, as determined by the geometric mean in this study. These results will contribute to later gene expression studies in other closely related species and provide an important foundation for gene expression analysis in S. ferganica.

A recombinant Sal k 1 isoform as an alternative to the polymorphic allergen from Salsola kali pollen for allergy diagnosis.

BACKGROUND: The incidence of Amaranthaceae pollen allergy has increased due to the desertification occurring in many countries. In some regions of Spain, Salsola kali is the main cause of pollinosis, at almost the same level as olive and grass pollen. Sal k 1 - the sensitization marker of S. kali pollinosis - is used in clinical diagnosis, but is purified at a low yield from pollen. We aimed to produce a recombinant (r)Sal k 1 able to span the structural and immunological properties of the natural isoforms from pollen, and validate its potential use for diagnosis. METHODS: Specific cDNA was amplified by PCR, cloned into the pET41b vector and used to transform BL21 (DE3) Escherichia coli cells. Immunoblotting, ELISA, basophil activation and skin-prick tests were used to validate the recombinant protein against Sal k 1 isolated from pollen. Sera and blood cells from S. kali pollen-sensitized patients and specific monoclonal and polyclonal antisera were used. RESULTS: rSal k 1 was produced in bacteria with a yield of 7.5 mg/l of cell culture. The protein was purified to homogeneity and structural and immunologically validated against the natural form. rSal k 1 exhibited a higher IgE cross-reactivity with plant-derived food extracts such as peanut, almond or tomato than with pollen sources such as Platanus acerifolia and Oleaceae members. CONCLUSIONS: rSal k 1 expressed in bacteria retains intact structural and immunological properties in comparison to the pollen-derived allergen. It spans the immunological properties of most of the isoforms found in pollen, and it might substitute natural Sal k 1 in clinical diagnosis.

Does an expressed sequence tag (EST) library of Salsola iberica (tumbleweed) help to understand plant responses to environmental stresses?

Weeds play an important role in agriculture and molecular techniques are useful to help understand traits that contribute to weediness and weeds' interactions with the environment. A total of 377 expressed sequence tags (ESTs) from a modest library were arranged into 227 unique fragments and 61 contigs, which consisted of two or more ESTs. From blastx results, we mapped and annotated unigenes using the gene ontology vocabulary according to biological process, cellular component and molecular function. These were then compared to a reference set of Arabidopsis thaliana sequences for statistically significant over- or underrepresented genes. The sequences were also compared against multiple protein databases for similarity of functional domains. Overall, the S. iberica sequences showed high similarity to response to stress, which included salt-induced proteins, betaine aldehydehyde dehydrogenase and calcium binding proteins. Only a modest number of transcripts were sequenced; however, the results presented here demonstrate the metabolic versatility of S. iberica in sub-optimal conditions that are likely to contribute to its cosmopolitan distribution. Here we propose that an EST library of an economically important weed species could be used to understand the weed's interactions with the environment.

A pectin methylesterase as an allergenic marker for the sensitization to Russian thistle (Salsola kali) pollen.

BACKGROUND: Chenopodiaceae pollen is considered the main cause of pollen allergy in desert countries and its incidence is world-wide increasing by the desertization of extensive zones. Although the correlation between the sensitization to Chenopodium album and Salsola kali pollens of patients suffering from allergy to Chenopodiaceae pollens is high, a significant number of patients exhibited IgE sensitivity exclusively towards S. kali. OBJECTIVE: To analyse this differential reactivity and to purify, clone and characterize the putative responsible allergen. METHODS: Immunoblotting was used to analyse the IgE binding to pollen extract for S. kali and C. album. The protein was isolated by two chromatographic steps and characterized by Edman degradation, mass spectrometry, finger print analysis and Concanavalin A lectin staining. Specific cDNA was amplified by polymerase chain reaction, cloned in Escherichia coli and sequenced. Immunologic characterization was performed by immunoblotting, enzyme-linked immunoassay detection and inhibition experiments using sera from 11 patients allergic to S. kali pollen. RESULTS: cDNA codifies for a mature protein of 339 amino acids plus a putative signal peptide of 23 residues and it belongs to the plant pectin methylesterase (PME) family. It is a mildly basic and polymorphic protein and was recognized by the IgE from all the patients allergic to S. kali included in the study, and was called Sal k 1. The protein was not recognized in the C. album pollen extract using the sera of these patients. CONCLUSION: Sal k 1 is a protein from the PME family with a high allergenic relevance. Considering this allergen as responsible for the different sensitization between S. kali and C. album pollen, it may be a useful marker to classify patients allergic to Chenopodiaceae allowing a safer and more specific immunotherapy.