Cloning And Subcellular Localization Of An Function Unknown Gene CkCAAD1from Caragana Korshinskii Kom

Caragana korshinskii is widely distributed in the northwest desert of China. It’s usually raised to protect environment, fix desertification and feed animals. C. korshinskii has a strong tolerance to abiotic stress, therefore, it’s extremely suitable for studying plant stress tolerance. Many investigations about C. korshinskii focus on its cultivation, little is about its physiology, biochemistry and the molecular mechanisms in stress tolerance. So far, only several genes responsive to drought in C. korshinskii was cloned and analyzed.CAAD domain was initially discovered in aminoacyl-tRNA synthetase (aaRS), its function still remains unknown. Previous data showed that CAAD domain also found in bacteria and eukaryon, it’s the unique domain in four kinds of aaRS, moreover, it’s essential for these aaRS to localize in thylakoid membrane.A gene from the suppression subtractive hybridization (SSH) library of C. korshinskii under drought treatment was cloned and analyzed. The full length CkCAAD1is1479bp, comprises5exons and4introns. It was predicated to encode a CAAD domain, and was named as CkCAAD1in this atudy. Coding sequence of CkCAAD1was cloned into the binary vector pCambia1302driven by the constitutive35S promoter and GFP encoding aequence was in-frame fused to the C terminus of CkCAAD1. The resulting construct was transformed into Arabidopsis leaf protoplast transiently. Our data indicated the fusion protein was localized in chloroplast. Expression analysis by real-time quantitative PCR showed that the transcript of CkCAADl increased slightly around lhr after drought treatment, and then dropped gradually. To further explore the function of this gene, the binary vector carrying CkCAADl,35S::CkCAAD1was constructed and transformed into wild type Arabidopsis thailiana(Columbia ecotype). Homozygous T3transgenic lines are under screening.