The Preliminary Study Of The Expression And Functions Of The HECT-Type Ubiquitin-protein Ligases (UPL) Gene Family In Arabidopsis

As a major type of E3s, homologous to E6-AP C-terminus ubiquitin ligases (HECT E3s) are present in all eukaryotes. It was characterized according to C-terminal HECT domain which contains approximately 350 amino acids and is essential for substrate recognition. HECT E3s are able to specifically recognize and ubiquitinate the substrate protein and make them degraded by 26S proteasome, leading to regulate many biological processes. To date, in animal the functions of HECT E3s have been well studied, they have been proved to play critical roles in disease-associated process. Recently some of plant HECT E3s members have been discovered to play roles in regulation of senescence, trichome development and abiotic stress responses, but the specific mechanism is still unclear. In Arabidopsis, there are seven HECT E3s like genes, but except UPL5 and UPL3, there are no more expression patterns and function information of the other five UPL genes. Therefore, in this study, the promoters of six Arabidopsis HECT E3s genes UPL1, UPL2, UPL3, UPL4, UPL6 and UPL7 were amplified from genome DNA and subcloned into the promoter analysis plasmid pCAMBIA1381Xb to drive the GUS reporter gene expression. The transgenic lines were obtained by Col-0 type Arabidopsis transformed with Agrobacterium tumefaciens carrying the UPL::GUS constructs by vacuum infiltration. Materials with different organs were obtained at different developmental stage to do GUS histochemical staining, thereby, the expression patterns of Arabidopsis HECT E3s will be more systematically learnt and understood. Moreover, the T-DNA insertion mutant of Arabidopsis HECT E3s purchased from ARBC were screened and identified, the phenotype and the function of the single UPL gene knockout lines were analyzed. The results are showed detailed as follow:(1) At the early germination stage of transgenic Arabidopsis seeds, only promoter of UPL6 has activity at cotyledon on 2DAG, the promoter of other family members have activity at cotyledon, hypocotyls and pericycle of maturation zone of root tip on 2DAG and 5DAG, The promoters of UPL2 and UPL4 have activity at meristematic zone and roothair of root tip, promoter of UPL4 has high activity in pericycle of root elongation zone, UPL3’s promoter has a specific activity in euphylla forming region on 5DAG. At the later germination stage after 7DAG, the whole family members’promoters have activity in new born tussue like euphylla and trichome.(2) Systematically obtain all rosette leaves and then do GUS staining at different reproductive growth period of ProUPLs-GUS promoter transgenic Arabidopsis plants. From the GUS staining results we find that promoter activity of UPL1 and UPL4 gene in old rosette leaves are higher than young leaves; UPL2 and UPL3 have much higher promoter activity in middle leaf age leaves than in old and young rosette leaves; UPL6’s promoter has much stronger activity in old and young rosette leaves than in middle leaf age leaves; UPL7 has no significant difference of promoter activity in different leaf age rosette leaves.(3) From the GUS staining of tissue and organ of inflorescence on reproductive phase, we can see that the six family members’promoters all have activity at stem, peduncle, receptacle and sepal; Promoters of UPL2, UPL3, UPL4, UPL6, UPL7 also have activity at filament and stigma; and UPL2’s promoter has activity at anther.(4) At last we choose the silique to do GUS staining, the results show that all six promoters have activity at silique carpopodium, the promoters of UPL2, UPL3, UPL4, UPL6 and UPL7 also have activity at silique pod and pedicel. UPL3, UPL4, UPL6’s promoters have activity at the top of the silique pod at early development of silique as well, but only UPL2’s promoter has activity at seeds.(5) The T-DNA insertion mutant of Arabidopsis HECT E3s were used three-primer method to identify and screen the T-DNA insertion homozygote lines, then to identify transcriptional level of candidate homozygote lines by qRT-PCR. The identified upl2, upl3, upl4 and upl6 homozygote lines are used to subsequently analyze their phenotype including chlorophyll content, photosynthesis efficiency of the 5th and 7th leaf, the proportion of yellow leaves and green leaves statistic from 15 independent plants and some related gene expression. The results show that in the obserbation period from 25DAG to 67DAG of knock out mutant plants, KO:UPL2, KO:UPL3, KO:UPL4, KO:UPL6 all have a slightly delay senescence phenotype on 60DAG. Using semi qRT-PCR and qRT-PCR to screen possible downstream gene of each mutant on 53DAG, at last we screen the senescence negatively regulating related gene named AtNEET which has up-regulated expression. We can draw a conclution that UPL2, UPL3, UPL4 and UPL6 may have a correlation with senescence.