Analysis On The Function Of Myrosinase Genes TGG4 And TGG5 In Arabidopisis Thaliana

Thio-glucosidase/myrosinase defense system of cruciferous plants is unique.The enzymes and the substrates are stored separately in different cells,they are mixed upon infestation by pests or upon the mechanical damage,and thioglucosides are hydrolyzed into several toxic compounds to serve as defense weapons.The study of myrosinase has important significance.After searching the Arabidopsis gene database we found 6 myrosinase genes totally:TGG1,TGG2,TGG3,TGG4,TGG5 and TGG6.RT-PCR and Northern hybridization confirmed that the TGG1 and TGG2 genes were transcribed in leaves,cotyledons,stems and flowers with the myrosinase activity,but the two genes have the same function in pest-resistance;TGG3 and TGG6 are pseudogenes;TGG4 and TGG5 genes are exclusively transcribed in root tissue,while their functional roles are still unknown.In this study,reverse genetics was used to explore the TGG4,TGG5 genes'function and their possible interactive relationship:1.The TGG4 and TGG5 genes are located on the first chromosome with only 1.6 Mb apart.Their single gene T-DNA insertion mutants were crossed,and homozygous double mutant were obtained by PCR screening in the offsprings of the hybrid.2.The optimum assay condition of root myrosinase activity was determined:the concentration of Vc was 2 mmol/L,the reaction temperature was 37?,pH was 4.5.The root myrosinase activity of various genotypes was comparatively studied,results indicated that roots of the double mutant had the lowest myrosinase activity,the wild-type had the highest.3.Under the normal culture conditions(temperature 22?,relative air humidity 60%,illumination time 8 h/d,the light intensity 200 ?mol/(m2 · s),the aerial parts of TGG4,TGG5 double mutant is slightly better than the other types,the seed germination of double mutants is a little earlier than other genotypes,but reproductive growth was later than wild type and single mutants.4.In normal cultural conditions,analysis of the seed germination and root growth of the double mutants,single mutants and wild-type showed that TGG4 and TGG5 may delay the seed germination and inhibit the root growth.5.In the no-sugar culture condition,all the genotypes of Arabidopsis thaliana growed abnormally.In Sulfur-free culture condition,the double mutants grew better than the other genotypes,had the longest root length,the wild type were most sensitive to sulfur deficiency.In flooding situation,wild-type grew better than the single gene mutants,single mutants grew better than the double mutants,suggesting that TGG4,TGG5 help plant to tolerate waterlogging.