The RAPD Markers Linked To The Red Leaf Traits Of Prunus And CDNA Cloning Of Chalcone Synthase Gene

Chalcone Synthase (CHS) is a key enzyme in the biosynthesis of all classes of flavonoids. Increase or decrease its expression in plant would change leaf color. In this study, five kinds of Prunus plant as the experiment materials, a molecular marker linked to the red leaf traits was detected with RAPD technology, which related with the formation of anthocyanins chalcone synthase gene cDNA sequence, specially expressed in Prunus, was cloned by homology cloning method. The main results were submited as follows:1. P. salicina×P. atropurupurea and Prunus salicina cv. 'Angenuo' was the hybrid combination, after reciprocal cross, 56 F1 individuals were obtained. red / green leaf gene pools were constructed respectively by BSA (Bulked Segregate Analysis) method. Using RAPD markers, two gene pools, the DNA of male / female parents and other F1 individuals were amplified by PCR method with random primers. The results showed that: a marker linked to the red leaf traits was revealed from 350 RAPD primers, which was appeared in red leaf gene pool but not appeared in green leaf gene pool. The size of this marker was about 2 300 bp, and was designated as S450-2 300. After repeating tests and checking up individuals of population, this marker could only be amplified in the red leaf individuals (except recombinants), and the linkage distance was 11.2 cM.2. At different times, collected leaves of five kinds of Prunus plant, and extracted RNA by RNA OUT kit method. The first-strand of cDNA was obtained using reverse transcriptase, in which RNA as templates and oligo (dT) as reverse primer. PCR was performed with specific primers CHS-F and CHS-R. Agarose gel electrophoresis illustrated a 1 200 bp DNA band, which is in accordance with expectation. PCR products were recovered using DNA Gel Extraction Kit and cloned to pGEM-T Vector. The results of sequence indicated that: CHS gene cDNA sequence of five kinds of Prunus plant were 1 170 bp~1 176 bp and encoded 389~391 amino acid. Among which the homology was more than 95%, and that of Prunus×bliriana'Meirenmei'and Prunus cerasifera var. atropurpurea is 99.3%. When CHS gene sequence of Prunus plant was compared with the sequences of other species in NCBI database using Blastn searching program, the identity is more than 80%.