Comparative Analysis Of Mutant Gene Cbn1 With Mutant Gene Cao And Molecular Mapping Of CAO Gene In Chlamydomonas Reinhardtii.

When plasmid Psp109-E8 with CAO was transformed into Chlamydomonas cbn1 standart mutant strain CC-1354(cbnl-48 mt+)by electroporation, more than 20 transformants were cultured on the TAP culture contains zeocin, the covery expression of chlorophyll b was detected by PCR and HPLC, which verified primarily that cbn1 and cao without the ability of chlorophyll b systhesis both possess allelic characters. The mutant strain1641-1b(cbn1-43 mt+) and mutant strain CC-1354 ( cbn1-48 mt+) were taken to cross with the cao mutant strain CBS5-c1 (cao mt-) and 989 meiosis progeny were obtained from1641-1b×CBS5-c1 hybrid and 853 progeny was obtained from CC-1354×CBS5-c1 hybrid, total meiosis progeny number is1842.After that fluorescence detection was taken on these generations for whether they have Chlorophyll b, it showed that there was no wild type phenotype transformants that had Chlorophyll b, which exactly indicated that phenotypes of all segregants showed the lack of chlorophyll b. Statistics obtained from random analysis proved the close linkage between mutant gene cbn1 and mutant gene cao. And among the 50 segregants obtained by cell mating tetrad analysis no wild type segregants was detected, which further verified the allelic characters of mutant gene cbn1 and cao. According to the CAO DNA sequence in Chlamy DB and Genebank,we prepare two probes: probe1(220bp) and probe2 (1067bp).After that DNA dot blot analysis was performed with 21 BAC clones) between the GBP 1 and the RB 47 molecular marker on chlamydomonas linkagegroupⅠ. The result showed that positive control plasmid Psp 109-E 8 and two probe displayed cross with numbered 33e2 BAC clone and no else was found on other BAC clones. It verifies that this sequence and the DNA fragment, marked between GBP and RB47 on chlamydomonas linkagegroupⅠ, have the character of homology region, which means it is the position of CAO on 33e2 BAC clone. This result prooved that CAO exactly locate at cbn1 loci.RT-PCR was used to obtain the all cDNA sequence of CAO and cbn1, then the sequencing was performed, the result prove that CAO and CBN1 are one gene and cbn1 is a mutant of CAO gene.This research exactly proved that mutant gene cbn1 and mutant gene cao are allelic gene, CAO gene locate at almostly same loci of cbn1 on the between two molecular marker GBP and RB47 at the chlamydomonas linkagegroupⅠand also prove that CAO is the main gene of chlorophyllb biosynthesis and cbn1 gene is a mutant form of CAO gene.