Functional Comparison Of CAO Gene And CBN1 Gene In Chlamydomonas Reinhardtii

Photosynthesisis is one of important problems of plant physiology and plantcytobiology. Genetic control of photosynthetic pigment synthesis is one of thetopics, which is intensively studied nowadays. Chlorophyll b is one of primarypigments in photosynthesis. Chlorophyll a synthesis has been clarified, butchlorophyll b synthesis is not yet completely understood. Both the CAO and theCBN1gene are considered structural genes for chlorophyll b synthesis .CBN1 genehas been located in the linkage group â… in Chlamydomonas reihardtii, but theencoding product and sequence characteristic remain obscure; CAO gene has beencloned and the enzyme that it encodes has also been studied, but it has not beenmapped on Chlamydomonas chromosomes. It is not known if the biosynthesis ofchlorophyll b goes through single or many pathways. Comparing CAO gene andCBN1 gene and answering the question, whether they are allelic or not couldclarify the genetic control of chlorophyll b biosynthesize.Because CBN1 mutants lost both chlorophyll b and neoxanthin, neoxanthincontent of wild type CC-125, CBN1 gene mutant CC-1354 and five different CAOgene mutants has been analysed by HPLC. CAO gene mutants accumulatedifferent amounts of neoxanthin, which are much higher than amount ofneoxanthin accumulation CBN1 gene mutant. Neoxanthin amounts of CAO genemutants are close to wild type CC-125. Different amounts of neoxanthin in theCAO gene and CBN1 gene mutants suggest that CAO and CBN1 gene are notallelic.The plasmid Psp109-E8 was transformed into five CBN1 deficient strains byelectroporation. Transformants were screened by zeocin that the plasmid can resistand PCR, 30 transformants were obtained. According to fluorescence test andHPLC data, the transformants did not recover chlorophyll b, In other words, CAOgene cannot recovery chlorophyll b synthesis in CBN1 mutants. The experimentalresults confirm the hypothesis that CAO and CBN1 gene are not allelic. 5'end 220bp and 3'end 1067 bp DNA sequence of CAO gene were used forprobe1 and probe2 respectively, and they hybridized with plasmids of 21 BACclones carrying parts of chromosomeâ… around CBN1 gene position. The probe1(5'end 220bp) had no spot and the prob2 (3'end 1067 bp) showed a stronghybridization spot in 21st clone (33e2 in Chlamydomonas reinhardtii BAC). Theprobe 2 didn't have very good specificity. The sequence 33e2 is located betweenGBP1 and RB47 molecular markers, so CAO gene is not in this domain, Iconclude that CAO gene and CBN1gene do not locate in the same area in group â… of Chlamydomoans reihardtii. The research result further support the other twoexperimental conclusions and confirms that CAO gene and CBN1 gene are notallelic. The research confirms that CAO gene is not only structural gene inchlorophyll b synthesis .CAO gene and CBN1 gene may be two different gene.Their products could take part in the synthesis successively. The result paves aroad to make clear the genetic control of chlorophyll b.