Analysis On The Function Of Myrosinase Gene TGG6 In Arabidoposos Thaliana

Cruciferous plants are very important in regard of economic value and theoretical research. These plants include many oil crops and vegetables, such as brassica, radish,many ornamental plants, and nectariferous plant. Cruciferous plants contain a kind of specific secondary metabolites—glucosinolates. Upon mechanical damage and insect attack or pathogens infection, myrosinase is released and mixed with its substrates,glucosinolates were then hydrolyzed to toxic compounds, and serve as defense reagents.In Arabidopsis, three myrosinase genes denoted as TGG1, TGG2 and TGG3 have been found previously. Recently,a new myrosinase subfamily were found including called TGG4, TGG5, and TGG6 by database mining. These genes have high similarity to the typical myroinase genes, and TGG4 and TGG5 have been confirmed to have myrosinase activity.In previous studies, TGG6 has long been regarded as a "pseudogene" due to multiple frameshift mutations in its coding region . In this study,We cloned a TGG6 gene in Arabidopsis ecotype Ty-0. All the frameshift mutations in the previously reported TGG6 genes did not exist in this gene. Therefore this TGG6 is considered as a functional allele to the previously reported TGG6. GUS expression analysis indicated that the functional allele was expressed in the same pattern as the non-functional allele, in which they were specifically expressed in pollens, except that the functional allele expressed a little earlier and at a higher level compared to the non-functional allele. The full-length functional gene including promoter, coding region and its terminator was cloned and constructed in the expression vector pCAMBIA - 1300-221, by replacing the CaMV35S promoter. The functional allele was then transformed into Arabidopsis ecotype Col-0 (with non-functional allele), via Agrobacterium infiltration method. RT-PCR results showed that the functional allele was transcribed in the same pattern as in the original ecotype Ty-0. Phenotypic analysis of the transgenic plants showed that the transgenic plants had larger leaves,taller plants, and more siliques compared to the wild type Col-0. The transgenic plants also yielded more seeds compared to wild type Col-0 plants. Statistic analysis supported these difference to be significant. These results suggested that functional TGG6 allele helps the plants to grow bigger and thus improve seed yield. The cDNA of the functional TGG6 was cloned and constructed in the yeast expression vector pPic9k, and introduced into Pichia strain GS115. TGG6 protein was obtained. This experiment will help in subsequent enzymatic anaylsis of TGG6.