کلونینگ و ارزیابی بیوانفورماتیکی ژن مونودهیدروآسکوربات ردوکتاز (MDHAR) در گیاه آلوروپوس لیتورالیس و بررسی بیش‌بیان آن در گیاه توتون (Nicotina tobacum)

Salinity as one of the most important stresses disturbs favorable growth conditions of the plants leading to several metabolic disorders such as reactive oxygen species (ROS). Monodehydroascorbate reductase (MDHAR) with FAD (Flavin adenine dinucleotide) co-factor is one of the key enzymes in Glutathione-ascorbate pathway which reduces monodehydroascorbate (MDHA) radicals. In this study MDHAR gene was isolated from Aeluropus littolaris, a halophyte plant with, characterized and was transferred to tobacco plants after cloning. Gene specific primers were designed using similar sequences in NCBI and the desired sequence was amplified via RT-PCR, cloned in pTZ57R/T vector and sequenced.  Primary bioinformatics assessments revealed that MDHAR gene consisted of 1436 bp lacking intron sequences. For tobacco transformation, cDNA fragment was amplified with Pfu DNA polymerase, inserted into pGreen0029 containing 35s promoter and terminator sequences. The recombinant vector was then introduced to LBA4404 agrobacterium cells, which were used for tobacco transformation via leaf disc method PCR analysis of the putative transgenic plants, indicated successful gene insertion into plant cells and gene transcription was confirmed by RT-PCR. Comparing which MDHAR sequences form other plants in NCBI, A. littoralis gene exhibited a high similarity with genes isolated from gramineae family. The highest similarity (89%) was observed for MDHARs from sorghum (Sorghum bicolor). Protein also analysis has proven that this gene is outside cell, pyr-redoxin and NAD-binding motif area. Secondary structure showed 78.3% alpha helix, 41.1% beta sheet and structure 13.6%  B-turn structure or reverse turn.