The Difference Analysis Of Gene Expression In Lonicera Confusa DC.under Calcium-rich Environment By GeneFishing

Ecological deterioration, soil erosion and desertification increase, have seriously restricted the development of local economy in Southwest karst recent years. The use of genetic engineering to repairing desertification is an important method in vegetation recovery of southwest karst. As one of typical species in southwest karst, the study of adaptation mechanisms of Lonicera confusa DC.to karst environment,especially to karst calcium-rich environment, can provide important theoretical basis for repairing desertification by genetic engineering. In this study, we simulated the growth environment of L. confusa and identified differentially expressed genes(DEGs) under different Ca2+ concentrations using GeneFishing technology. In order to verify the accuracy of the result,we also analyzed the expression changes of DEGs. In addition, the functions of these genes were preliminarily identified by using bioinformatics. The results as follows:(1) Established a suitable system for the technology of GeneFishing in L confusa. 2μl of total RNAs were used for the synthesis of first-strand cDNAs by reverse transcriptase using primer dT-ACP2. Reverse transcription was performed at least for 1 h at 42°C. First-strand cDNAs were diluted five timers for the GeneFishing PCR. In the PCR, 3μl diluted first-strand cDNAs and the PCR products were separated in 2% agarose gel stained with ethidium bromide for at least l h. So, the bands wil be clear.(2) Identified differentially expressed genes (DEGs) under different Ca2+ concentrations (0 and 125 mg/L) in L. confusa using GeneFishing technology. Twenty pairs of primers were used and twenty-four DEGs were found. Semi-quantitative analysis of DEGs in six different Ca2+ concentrations(0,25,50,75,100,125 mg/L) was used to verify the accuracy of the PCR results and analysis its expression changes.(3) Bioinformatics analysis the functions of the DEGs in adaption to calcium-rich environment in L. confuse. By the use of Blastn, we found that one DEG encoded channel protein, three DEGs were related with photosynthesis, three DEGs were related with protein sirting and secretion, five DEGs were related with redox reaction in plant, two DEGs were related with plant nutrition and material synthetic, one DEG encoded transcription factor. All of these DEGs reported before had close relationship with salt stress. Besides, there were also eight DEGs, the functions of which were not known, some may be new genes. The interactions between DEGs and Ca2+-target protein showed that they had close connections and were controlled by Ca2+.