Generation and Analysis of Expressed Sequence Tags (ESTs) from Halophyte Atriplex canescens to Explore Salt-Responsive Related Genes

Abstract

Little information is available on gene expression profiling of halophyte A. canescens. To elucidate the molecular mechanism for stress tolerance in A. canescens, a full-length complementary DNA library was generated from A. canescens exposed to 400 mM NaCl, and provided 343 high-quality ESTs. In an evaluation of 343 valid EST sequences in the cDNA library, 197 unigenes were assembled, among which 190 unigenes (83.1% ESTs) were identified according to their significant similarities with proteins of known functions. All the 343 EST sequences have been deposited in the dbEST GenBank under accession numbers JZ535802 to JZ536144. According to Arabidopsis MIPS functional category and GO classifications, we identified 193 unigenes of the 311 annotations EST, representing 72 non-redundant unigenes sharing similarities with genes related to the defense response. The sets of ESTs obtained provide a rich genetic resource and 17 up-regulated genes related to salt stress resistance were identified by qRT-PCR. Six of these genes may contribute crucially to earlier and later stage salt stress resistance. Additionally, among the 343 unigenes sequences, 22 simple sequence repeats (SSRs) were also identified contributing to the study of A. canescens resources.

Exploration for the Salinity Tolerance-Related Genes from Xero-Halophyte Atriplex canescens Exploiting Yeast Functional Screening System

“…Approximately, 1.76 × 10 6 colony-forming units (CFU) of E. coli transformants harboring A. canescens cDNA inserts by BP reaction were generated in pDONR222 with a recombinant rate of 91%. And the primary cDNA library was transferred to a yeast expressional destination vector pYES-DEST52 by LR reaction with a recombinant rate about 95% [ 20 ]. The size of cDNA inserts ranged from 0.6 to 2 kb and the average inserted fragment is sized above 1 kb ( Table 1 ).…”

“…In order to further explore molecular determinants of salt tolerance in A. canescens , a gateway cloning system compatible cDNA library was generated from A. canescens plants exposed to 400 mM NaCl [ 20 ]. 400 mM NaCl is high concentration for plants, but it does not affect A. canescens’ normal growth, which could significantly enhance A. canescens’ leaf osmotic potential compared to the control plants with no salt treatment in a recent report [ 26 ].…”

“…Xero-halophyte A. canescens was used in this study and the cDNA library generation was described in our previous report [ 20 ]. Briefly, surface-sterilized seeds were germinated and the seedlings were transferred to 1/2 Hoagland (pH 6.0) solution in controlled environmental facility.…”

“…After 50 days of growth, plants were then subjected to 400 mM NaCl treatment for 48 h, and the harvested samples (young leaves and stems) were immediately frozen with liquid nitrogen and kept at −80 °C for RNA preparation and cDNA library construction. The gateway system compatible vector pDONR222 was used for entry cloning and pYES-DEST52 [ 20 ] was used for functional expression in yeast. The E. coli strain DB3.1 was used to maintain the gateway compatible backbones pDONR222 and pYES-DEST52 with a ccdB cassette.…”

Proteomics Analysis of SsNsd1-Mediated Compound Appressoria Formation in Sclerotinia sclerotiorum

“…The FPKM values of histone H3 (SS1G_09608.3) and histone H2A (SS1G_02052) were used as endogenous control for quantitative comparison with SsNsd1 and other GATA-type protein genes. Hierarchical clustering was performed using the MeV program [ 62 ]. Clustering was based on the average of FPKM values.…”