RNA secondary structure analysis for abiotic stress resistant and housekeeping genes in Arabidopsis thaliana and Oryza sativa

RNA is a single stranded nucleic acid type, produced by DNA via a process of transcription. RNA tend to form secondary structure by pairing the complementary base pairs within it. The RNA secondary structure is an essential parameter to understand its functions and expression into a protein. This research is based to understand the link between RNA secondary structure and abiotic stresses in two leading organisms viz; Arabidopsis thaliana and Oryza sativa. For this purpose the RNA secondary structures analyses for abiotic stress resistant and housekeeping genes were done in A. thaliana and O. sativa. Total 138 abiotic stress resistant and 2l housekeeping genes were selected through literature survey and subjected to RNA secondary structure tool. RNA secondary structure were analyzed and characterized in terms of minimum free energy, number of stems and loops. The 80% of abiotic stress and 67% of housekeeping genes show significant difference in the minimum free energy (MFE) while there were no significant variation in the number of stems and loops. The open reading frame analysis, as an additional parameter, of the selected genes were also done, that revealed the preference of +1 frame by most of the genes, i.e., 42% of abiotic stress and 43% of housekeeping genes. RNA secondary structure analysis help us to engineer stress resistant genes with stable RNA structures.


Introduction
The abiotic stresses such as cold, drought, osmotic, heat, salt and oxidative stress affects and reduce the growth and productivity yield of the plants.Plants also give response to these stress at the level of cell, molecule and at biochemical stage, that enable them to survive.At molecular level, stresses triggers the change in the expression of various genes in plants to combat and cope from it [1].A. thaliana because of its simple structure is modal plant in molecular genetics, therefore a wide study of stress tolerant genes is conducted [2].About 45% of all stress tolerant reported genes are from A. thaliana, while nearly 50% of all stress tolerated genes have been characterized using A. thaliana as the transgenic species [3].Seki  The current study is based on RNA secondary structure analyses of abiotic stress and housekeeping genes in A. thaliana and O. sativa.Main theme of this study is to find the relationship among the RNAs secondary structures of all abiotic stress and housekeeping genes.This would help us to better understand and manage the plant for abiotic stresses at molecular level.

Identification of abiotic stress resistant genes & housekeeping genes
Total 138 well annotated and characterized abiotic stress and 21 housekeeping genes were identified through literature survey in A. thaliana [2 & 3].These 159 genes were used as an initial data for downstream analyses.

Retrieval of sequences
The sequences of 159 abiotic stress and housekeeping genes for A. thaliana were retrieved from the Gene bank (NCBI).The FASTA format of these sequences was saved and used as query to find their homologue in O. sativa.For this purpose all the A. thaliana retrieved FASTA sequences were blast against O. sativa via BLASTn [7].The O. sativa sequences homologue to A. thaliana were also retrieved and saved.

RNA secondary structure prediction and analyses
The RNA secondary structures prediction and analyses were done for all 159 abiotic stress resistant and housekeeping genes transcripts of A. thaliana and O. stiva.For this purpose, the transcripts sequences in FASTA format were submitted to Zuker folding algorithm, MFOLD (version2.3)[8].publicly available at http://www.bioinfo.rpi.edu/applications/mfold/rna/form1.cgi.to produce the folded structure.The Mfold parameters were set similar as adopted earlier [9].All the secondary structures of the abiotic and housekeeping genes were selected on the basis of minimum free energy (MFE) and saved.These secondary structures were also subjected to manual analyses for number of loops and stems.

Open reading frame (ORF) analysis
All the selected 159 abiotic stress and housekeeping genes were also studied and characterized for the open reading frame (ORF).This would help us to better understand the link between transcripts and their translated proteins.For this purpose, the 159 abiotic stress resistant and housekeeping genes were analyzed through, available at NCBI (www.ncbi.nlm.nih.gov/gorf/gorf.html)[10].The longest frame produced by ORF finder were selected and subjected to physical analysis.

Results and discussion Characterization and analyses of RNA secondary structures
The RNA secondary structures of abiotic stress resistant and housekeeping genes in A. thaliana and O. sativa were analysed and characterized by Mfold algorithm.Some of these secondary structures are shown in Figure -1.The RNA secondary structures of 159 abiotic stress and housekeeping genes belonging to A. thaliana and O. sativa were characterized in terms of the number of loops, stems and minimum free energy (MFE).MFE is an important parameter to find the stability of the RNA secondary structure.The MFE of abiotic stress and housekeeping genes are evaluated as shown in (Table 1).The range of difference in MFE of abiotic stress resistant genes in A. thaliana and O. sativa are -0.7 KcalMole -1 to -287 KcalMole -1 while in case of housekeeping genes are -8.6 -1 to -128 KcalMole -1 .The average value of difference in MFE of abiotic stress resistant and housekeeping genes in A. thaliana and O. sativa are -144 KcalMole -1 and -68 KcalMole -1 respectively.The 110 abiotic stress resistant and 14 housekeeping genes showed the values greater than their average values respectively, while 28 and 6 genes showed values less than their respective average values correspondingly.All the 159 genes are classified into four different categories on the basis of their MFEs.These are category-I; -1 to -72, II; -73 to -144, III; -145 to -216 and IV; -217 to -288 KcalMole -1 .The maximum number of abiotic stress resistant and housekeeping genes are lies in the category-I, i.e. 60 and 15 respectively followed by category-II (50 and 6 respectively), category-III (16 and 0 respectively) and category-IV (04 and 0 respectively).Similar findings are also reported earlier for the characterization of secondary structures of viral biotic stress and housekeeping genes [5].

Figure 1. Secondary structures of few genes
et al. [2], reported 7000 gene responding to cold, drought and salinity in A. thaliana.Stress tolerant genes are also studied by Vandenbroucke [3], in A.

table 3 ,
[11]eas, no single A. thaliana gene is observed in -1, -2 and -3 frames.This indicates a little variation in the ORF preference between A. thaliana and O. sativa, and could serve a point to analyse in detail at individual gene level.The role of ORF in the expression of gene is reported in various organisms[11].Housekeeping genes also showed the remarkable results i.e., 9 genes out of 21 preferred +1 frame , 6 genes preferred +2 and +3 frame for each in A. thaliana.Five genes preferred +1 frame, 6 genes preferred +2 and +3 frame by each in O. sativa.Four genes in O. sativa also preferred -1 and -2 frame.ConclusionThis research is based on the relationship of secondary structure folding for abiotic stress resistant and housekeeping genes in A. thaliana and O. sativa.The concluded results showed that there are variation on the basis of RNA secondary structures and ORFs for abiotic stress resistant and housekeeping genes in A. thaliana and O. sativa.These findings would be helpful to understand and monocot plants under abiotic stresses.