The Screening Of A Novel Mutant Gfoc In Arabidopsis And The Study On The Genetic And Phenotype

Starting with screening of T-DNA insertion Arabidopsis thaliana stocks,a TDZ insensitive mutant,gfc1,which grows faster than the wild type on MS medium containing TDZ,was isolated.The clone of the mutant gene and the analysis of flanking sequence by inverse-PCR revealed that the mutant gene encodes a acyltransferase belong to BAHD family.The members of this family play an important role in the biosynthesis of a variety of secondary metabolites and catalyse the first committed reaction of phytoalexin biosynthesis in many other plants.There are approximately 70 BAHD gene family members in the model organism Arabidopsis,although to date,the substrates and products of the enzymes encoded by these genes have not been determined.The homozygote gfc2,allelic mutant of gfc1,has the same phenotype when cultured on the MS medium containing TDZ.0.1mg/l as gfc1.While the probability of spoontaneous mutation at the same site in two different mutant is almost zero even if they have the same phenotype.According to the linkage relationship between gene and phenotype,we confirmed that the mutant was induced by the novel gene discovered.By choosing the homozygous gfc1 as a male parent crossed to wild type,F1_BC1 progeny showed Kam resistance and confirmed to be heterozygous by PCR.Incubated on MS medium containing TDZ 0.1mg/l,the mutant phenotype was eliminated and F1_BC1 exhibited the wild-type phenotype,which demonstrates that the mutation is recessive.Incubated on MS basal medium for 10 days,the roots of gfc1 were longer than wild type,and the difference of hypocotyls between mutant and the wild type was slight.After transferred into soil,little difference of them was found during the vegetative growth.Incubated on MS medium containing cytokinin,gfc1 developed much longer root than that of wild type,but shorter hypocotyl.It implies that gfc1 might be insensitive to cytokinin by interrupting the cytokinin signal transduction.The difference of phenotype was insignificant between mutant and wild type grown on MS medium containing IAA.It indicates that the mutation doesn't affect the metabolism of endogenous IAA.The detection of endogenous IAA concentration by high performance liquid chromatography(HPLC)revealed that IAA concentration in wild type and mutant after treated with TDZ for 6 hours were decreased,while the quantity of IAA in wild type dropped much faster than in gfc1.It might results from inhibition of the decrease of endogenous IAA by exogenous cytokinin in mutant.RNA expression of GFC1 in inflorescence revealed by semi-quantity RT-PCR is much higher than in other organs and followed the order silique > juvenile leaf > inflorescence stem > adult leaf > roots > senescent leaf.The result was consistent with the data from the gene chips published on genevestigator website.The semithin sections of Arabidopsis leaves incubated on MS medium containing TDZ were observed under light microscope,and then analyzed by means of computer image analysis system.Compared with the mutant,mesophyll cells from wild type arranged more densely and the size was smaller.The arrangement of mesophyll cells in gfc1 was looser.The sequence of promoter has been cloned in this experiment.Nevertheless,three vectors(cDNA_gfc1-pBI121,full length_gfc1-pCAMBIA1300,P_gfc1v-pBI121)need to be constructed and further verification will be made,such as complementation test,Gus stain and so on.In addition,as the crucial enzyme during the biosynthesis pathway of phytoalexin in other plants,the study on biochemical activity of benzoyltransferase has important significance for plant defense response.