Cloning And Analysis Of TaRab2 And TaFer Related To Drought Tolerance In Wheat (Triticum Aestivum L.)

Wheat(Triticum aestivum L.) is an important crop in the world. However, drought greatly affects the yield of wheat in many areas. Discovering and cloning genes related to drought tolerance are the foundation for improving the drought tolerance in crops with genetic modification, which also plays a crucial role in understanding the genetic mechanism of drought tolerance in crops.In present paper, the tested wheat genotype was Hanxuan 10, a commercial wheat cultivar with strong drought tolerance. Two suppression subtractive hybridization (SSH) cDNA libraries constructed from the 2-leaf seedlings of Hanxuan 10 treated with 24h and 48h water stress were used to screen ESTs candidate involved in drought tolerance, respectively. A total of 13 up-regulation ESTs and 3 down-regulation ESTs have been identified by Reverse Northern Blot. Of them, several were choosen as initial ESTs for cloning genes.Using No.731C8 clone from 48h cDNA library as a query probe, in silico cloning was used to extend the target sequence based on the sequence data of wheat dbEST. A full length cDNA with 824bp was isolated by RT-PCR, which was highly homologous to a small GTP-binding protein rab2 gene cloned from other plants and named as TaRab2 (accession number in Genebank: AY851657). Open reading frame of TaRab2 is 633bp and codes 210 ammo acids. Using the same approach, a 971bp full length cDNA of' No.730G14 EST clone that was highly identical with ferritin gene from other plants was obtained and named as TaFer. Its ORF is 768bp and codes a protein of 255 amino acids.Northern blot showed that both TaRab2 and TaFer were up-regulation expression genes induced by water stress, but the expression levels were different in the different time points. Their expression levels were the highest levels at 6h induced by water stress. The longer was duration, the lower was their expression level. The expression level of TaRab2 returned to original quantity at 48h.In order to understand the functions, TaRab2 was inserted into the plant expression vector, and the expression vector pCHF3-35Sp-TaRab2-NOSter was constructed. The recombined vector and empty vector were subsequently transferred into Agrobacterium tumefaciens, respectively. The TaRab2 transgenic Arabidopsis plants were generated through agro-bacterium-meditated transformation via infiltration. Up to now, twenty transgenic Tl lines including more than 700 T2 plants have been obtained. Phenotyping of drought tolerance in transgenic Arabidopsis plants is currently underway.