Determination And Cloning Of The Candidate Gene Involved In High Chlorophyll Content

Mutants are important resources for functional genomics. One dark green mutant of rice was found from breeding resources, Heredity research showed that the dark green mutant was controlled by a single dominant gene and. Three simple sequence repeats (SSR) markers (rm462, rm6340 and rm6464) that are linked to Gc (high chlorophyll content gene) were identified by amplification of DNA samples from near-isogenic lines using two hundred pairs of primers. The genetic distances on the short arm of rice chromosome 1 between Gc and rm6464, rm6340 and rm462 were 0, 0.588 and 1.18 cM, respectively.Study on the Gc was continued on the foundation.The main conclusions were followed.1. The target sequence of Gc was definited combined the rice genome.17 predicted genes was found on the targe sequence of Gc.2. candidate genes was definited after alignment and homologyy and function analysis to amino acid sequence of the 17 predicted genes. One encoded thylakoid lumenal protein, chloroplast precursor from Arabidopsis thaliana.The other one encoded chloroplast envelope membrane protein from chloroplast Pseudendoclonium akinetum.They were named Gccga and Gccgb respectively.3. PCR primers were designed according to sequence of Gccga to amplify the sequence contained complete candidate gene, probable promoter and Poly-A. Optimum conditions of PCR was get.The fragment was recoveried,then linked to pMD-18T vector,and transformed into E.coli JM109. It was determined that the candidate gene was successfully cloned after theidentification to transformed bacteria by PCR and restriction enzyme digestion. Positive clones were picked and then to be sequenced. The comparison analyses on the sequences of normal plant and mutant plant show that 73 nucleotides was inserted in the rejoin between the predicted promoter and the initiation codon of mutant plant.