Abstract:Soluble acid invertase (SAI) is a key enzyme in sucrose metabolism which takes a pivotal role in sugar sensing and plant development. It is necessary to find a simple method of cloning soluble acid invertase gene for genotyping a large quantity of breeding materials or germplasm. Based on known full-length sequence of SAI gene obtained by 5 segments PCRs and assembled in our lab, in order to further find a simpler one, we tried several strategies, i.e. one time cloning for full-length sequence, nested PCR, two even fragments PCR, and two uneven fragments PCR. And only was success achieved in two uneven fragments PCRs with a set of special PCR parameters. Sequence analysis showed bases of GC is strongly unevenly distributed in SAI-1 gene sequence, with a high GC content (69.6%) in 635bp PCR product of the first fragment, with highest over 80% in some regions of this segment. Except for high GC content, steep change of GC content may be another factor that affects PCR result. The suitable strategy for PCR cloning SAI-1 gene is through an approach of two uneven segments PCR with an annealing temperature difference.