Expression Analysis Of Eustoma Grandiflorum Relative Salt-Tolerance Genes

Eustoma grandiflorum is a kind of Gentianaceae herbaceous plant. It is very popular as a fresh cut flower and has salt tolerance. Eustoma grandiflorum can grow normally in the saline- alkaline grasslands (soil pH at 9.0). The purpose of this study was to get salt induced genes and genes related to salt tolerance from Eustoma grandiflorum using the technique of suppression subtractive hybridization (SSH) and the technique of cDNA microarray. The library used Eustoma grandiflorum's mRNA treated with NaCl for 1, 3 and 6 h, where equally commix materials were regarded as tester, and the mRNA of non-stress materials as driver.The secondary PCR amplification products of SSH library were inserted in pGEM-T vector, and then transferred into competent JM109 cells. After screening by blue/white spot test and subsequent cell culture, the plasmid DNA was extracted. Sequencing PCR reaction of the plasmid DNA was performed using Dynamic ET Dye Terminator Cycle Sequencing Kit (Amersham). Purified PCR product sequences were read by MegaBACE 500 DNA Analysis Systems. These genes were clustered by SeqClust 2.1 software. As a result, 1153 high quality sequences longer than 200bp were isolated with the average length of 375bp, removing the vector and redundancy sequencing and we got 658 non-repeat sequences. The library redundancy was 42.9%.These 658 sequences were classified by gene ontology. They were sorted to 14 classes in Biological Process classification, including mitotic spindle checkpoint, response to oxidative stress, protein sumoylation, transport, electron transport etc., 21 classes in Molecular Function classification, including catalase activity, catalytic activity, epoxide hydrolase activity, monooxygenase activity, glutathione dehydrogenase (ascorbate) activity etc., and 10 classes in Cell Component classification, including mitochondrion, chloroplast, peroxisome, glyoxysome, nucleus etc.Inserts were amplified from 658 sequences of Eustoma grandiflorum SSH library clones, using "primer I" and "primer 2R". All above sequences were used for making cDNA microarray. Salt stress and non-stress Eustoma grandiflorum's mRNA was labeled with Cy3- or Cy5- fluorescence dye respectively during reverser transcription and then mixed as targets. Following the direction of user manual of 3DNA 900, we accomplished microarray hybridization. The array was scanned by ScanArray 4000 microarray scanner, and the results was analized by limma package. We got 14 up-regulated genes, 16 down-regulated genes and 5 of them with known functions, including ubiquitin-like protein SUMO, peptide transport protein, cytochrome P450, thioredoxin, and dehydroascorbate reductase.