Sugar Transporter SWEET In Narcissus Tazetta:cDNA Cloning And Functional Studies

Chinese narcissus(Narcissus tazetta L.Var.chinensis roem.),Lycoris perennial bulb plant,cultivated variants of multi-flower narcissus.It's one of the top ten traditional flowers in China,and also a characteristic flower in Fujian.It have an umbrella-shaped flower sequence and the scapes(style)with many small flowers,which contrast to the European narcissus.The small flowers on inflorescence opening in order and their life are about 3 to 4 days.In the process of flowering,the scapes are an important stock organ,which need to input sufficient carbohydrate(sucrose)and other nutrients from the source organs provide carbon skeleton and energy to complete the developmental process,such as flowering,fragrant composition synthesis,germ cell development and seed formation.Sucrose is not only an energy carrier,but also a signaling to regulate the formation,development and reproductive process of flowers.In order to understand the molecular mechanism and genetic of sugar transport in development of the Chinese narcissus,we had studied on SWEET gene family.The main research contents and results are as follows:1.The scales in different tissue parts of the bulb began flower bud differentiation after summer dormancy(July and September)and the tepal and corona at different development stages during flowering were collected to extract the RNA,and expression patterns were analyzed by fluorescent quantitative PCR.The results show that the expression of Nt SWEET14 and 15 gene were reduced with closer the flower bud,while Nt SWEET1a?1b?4b and 13 are gradually increases.However,these genes were a specific expression pattern in four stages of flowering.Nt SWEET1 a showed an increasing expression level in both the tepal and corona,and that was the highest about 12 times than other SWEET genes during the T5 period of the corona.The Nt SWEET4 a expressed in T3 period of the corona and Nt SWEET5 expressed in the T5 period were also highly expression.While Nt SWEET15 was the only express in the corona of T5,hardly express in other periods and tepal.2.Using the Gateway technology to construct a C-terminal with GFP tag in PK7FWG2 expression vector containing SWEET gene to infect tobacco leaves for instantaneously transformed express.The locate of cell fluorescence were observed under confocal laser confocal microscope.The results showed that Nt SWEET1 b,4a and 4b genes were located on the cell membranes of mesophyll cells,the Nt SWEET14 and 15 genes were also located on the cell membrane.But many small fluorescent spots can be seen in the cell,they were different from the fluorescence expressed separately.3.The Yeast expression vector p DRTXa-SWEETs were constructed by enzyme and digestion,p DRTXa-Nt SWEET1 b,4a,4b,5,15 and the control plasmid p DRTXa were respectively transformed into the yeast mutant EBYVW4000 that only grown on SD(-ura)medium with the substrate of maltose.Then,taking the yeast solution that containing different SWEET genes were cultured on SD(-ura)medium with different concentrations of sugar substrate.The experimental results showed that the p DRTXa-Nt SWEET1 b could grown on the medium with glucose,galactose and sucrose substrates,but not grown on the SD(-ura)medium with maltose and deoxy glucose as common substrates.However,PDRTXa-Nt SWEET4 a,4b,14 and 15 were only grown on the medium with maltose,deoxy glucose and maltose as common substrates.And could not grow on other mediums which containing glucose,mannose,galactose,fructose and sucrose.4.The Chinese narcissus ' jinzhan yintai' as the research material,using the scales at the bottom of the bulb and the not flowering of ovary were the explants to explore the optimal disinfection conditions,and the best medium for proliferation and differentiation of callus in vitro induced callus tissue regeneration,trying to establish the system of callus tissue culture.According to the experimental results showed that the optimal disinfection condition of bulb tablets and ovary were respectively disinfected 12 min,5 min by 1% Na Cl O.At the same time,the 6-BA and NAA were more suitable for callus induction.When the 6-BA and NAA hormones respectively were 9 mg/L,3 mg/L,the rate of callus induced was higher,which up to 73%.It was also found that 90% of the callus could differentiate into adventitious buds in the medium containing 3 mg/L 6-BA and 9 mg/L NAA plant hormones.The callus tissue samples from different development stages(90 days,130 days,150 days)in vitro culture were collected to analyse the expression pattern of SWEET genes and investigate the potential role of SWEET gene family in the process of callus tissues.The results showed that with the increase of the time of induced callus tissue differentiation of narcissus,the expression level of Nt SWEET1 a was also gradually increasing ecallus,while the expression of Nt SWEET15 gene decreased and then increased to 3 times in the later stage after 130 days.And that Nt SWEET14 gene was increasing and then decreasing the same as before.