Rna Interference On Atsus3 Arabidopsis Sus Family Expression Pattern And The Influence Of The Percentage Of Mature

Sucrose synthase (EC2.4.1.13, SuSy) was first identified in germ of wheat in1953. Since then, the enzyme has been widely studied and more than80genes from45plant species have been completely or partially cloned. SuSy is a key enzyme to sucrose matabolism, which catalyzes the reversible reaction of sucrose and UDP cleaved to UDP-glucose and fructose. SuSy exists in cell with two forms, particulate SuSy (P-SuSy) associated with the plasma membrane, and soluble SuSy (S-SuSy). The enzyme is ubiquitous in plants and has been found in nearly all plant organs, although its activity is predominantly expressed in sink organs. Previous studies have confirmed that SuSy has closed relationship with starch and cellulose biosynthesis, sink strength determination and sucrolysis regulation. Recently, some results of the studies revealed that SuSy also has important roles in stress response, seed development and nitrogen fixation.SuSy is encoded by multigene family; it occurs as isoforms and is encoded by at least two genes. A six-member multigene family has been found to encode different SuSy isoforms in Arabidopsis thaliana. There are evidences that loss of one gene expression increase other genes transcript levels in multigene family. AtSUSS is highly induced in seeds during the late maturation phase, and it is also induced in various organs under dehydration conditions including leaves deprived of water or plants suffered to kinds of osmotic stresses. It is still unclear whether other homologue genes can compensate for the absent expression of AtSUS3. So RNAi-SUS3vector was constructed and transferred into Arabidopsis using Agrobacterium tumefacien mediated vacuum infiltration. After harvesting pure T2transgenic lines, we observed phenotype alteration in both wild and transgenic plants. After that, we stained lignin of siliques via Wiesner reaction and observed endocarp cells using transmission electron microscopy. Meanwhile we analyzed enzyme activity, carbohydrate content, expression pattern of each member of AtSUS family and some sucrolysis genes in transcriptical level. These results offered evidence to further understanding the function of SUS family in Arabidopsis.The main results are listed as follows:1. In this research, we constructed RNAi-SUS3vector and transferred it into Arabidopsis thaliana. After harvesting pure transgenic lines, PCR analysis identified that transcriptional expression of AtSUS3is suppressed by RNA interference.2. Comparison to wild plant, no phenotype alteration was observed in transgenic plant, which implied the lack of AtSUS3expression may not influence the phenotype of Arabidopsis grown in normal conditions.3. In transgenic siliques, secondary wall of endocarp cells are thickening and lignification is enhancing. Meanwhile the valve is inclined to thicken in transgenic plants during fruit development and maturity.4. Siliques were harvested at5,10,15days after flowering (DAF) respectively, The activities of SuSy, INV and SPS are all increased first, and then decreased both in wild and transgenic plant. In transgenic siliques, SuSy activity is higher than wild type. In5DAF siliques, INV activity hasn’t significantly difference between wild and transgenic plant, but the sucrose content in transgenic siliques are lower than that in wild type, which has lower fructose content at the similar growth period. Both in10DAF and15DAF siliques, INV activities are higher in wild type compared with transgenic plant.5. Comparison to wild plant, transgenic Arabidopsis has earlier and finished early bolting. The productive and maturation rate of siliques are also higher in transgenic plant. These results indicate that transgenic siliques have an earlier development than wild siliques have. AtSUS3with RNA interference maybe facilitate siliques maturation.6. We analyzed AtSUS gene family expression pattern by RT-PCR, the expression of AtSUSl, AtSUS2and AtSUS4increased for compensation AtSUS3silence. The results of real-time PCR are consistent with RT-PCR analysis.7. We also analyzed some sucrolysis genes expression pattern. In5DAF transgenic siliques, the expression of AtCesA1, AtCesA7and AtCINV1are higher than that in wild siliques. In15DAF transgenic siliques, the expression of AtCesA1and AtCesA7are also higher than that in wild siliques, but the expression of AtCINV and AtCwINV are lower than that in wild siliques.