Cloning PhyA Gene CDNA Fragment From Alfalfa And Construction Of Its RNAi Corresponding Vector

Fall dormancy of alfalfa may be a photoperiodic response. We speculate that phytochrome A (phytochrome, phyA) plays an important role in the fall dormancy, as it is a most important light receptors in plants. According to the principle of RNA interference (RNAi) technology, a cDNA fragment of phyA was cloned from Alfalfa to construct the corresponding RNAi vector, which laid a theoretical foundation for abtaining transgenic plants which miss phyA. On this basis, we can study the function of phyA, especially in the fall dormancy. In this study, fresh young leaves were chosed to extract RNA, then the positive and negative target fragments of phyA was cloned by the method of RT-PCR on the basis of the specific primers with double enzyme sites according to the principle of hpRNA (hairpin RNA)in plants. The length of the fragment is 604bp. Then insert both the positive and negative target fragments into the corresponding sites of the intermediate vector pKANNIBAL. Next using NotI restriction, a fragment containing promoter, positive fragments, intron, negative fragments and terminator was got, which can form a hairpin structure. Finally the fragment was linked with expression vector pART27, the RNAi vector would be finished. From this study the following conclusions were obtained:(1)Both positive and negative fragments were obtained;(2)The appropriate ratio of linking fragment and vector was obtained; (3)Detected by restriction enzyme digestion, we got the fragment which can form a hairpin structure;(4)We got the RNAi vector of phyA successfully;(5)The recombinant plasmid was transformed into Agrobacterium EHA105. This will provide an effective tool for the further study of GLDH gene function.