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The Study Of Glycinergic-Fipronil Uptake In Cotyledon Of Ricinus Communis Mediated By ATF And APC Family Transporters

Posted on:2017-03-25Degree:MasterType:Thesis
Country:ChinaCandidate:J L ZhaoFull Text:PDF
GTID:2323330509961520Subject:Plant pathology
Abstract/Summary:PDF Full Text Request
Guided pesticides is the theory that parent compounds possess better targeting transport ability by adding endogenous amino acid or hexose derivatives to them. In order to fill the gap between transport ability and the transporters, GlyF, a new conjugate possessing a glycine core was synthesized and its uptake mechanism in cotyledon of Ricinus was studied in plant physiology and molecular biology level in this paper.A fluorescent indicator dye, Di BAC4(3), was used in cytometric analysis for plasma membrane potential measurement of Ricinus cotyledons. The results indicated GlyF was not phytotoxic to protoplast of Ricinus in the short-term experiments and can be used for subsequent experiments.The Ricinus seedlings were utilized to test the phloem uptake of GlyF. Within the five times phloem sap collection with interval of one hour, GlyF concentration was gradually higher. This result showed that adding glycine to fipronil could confer phloem mobility to fipronil.Kleier model is a classic theory, which is usually used to predict phloem mobility of chemical compounds. The model showed GlyF is not mobile in phloem, which was not agreeable with the phloem mobility test. This result indicated that there could be more than one kind of uptake mechanism of GlyF uptake.Foliar disk uptake of GlyF was conducted. The results showed four kinds of amino acid(Glu, Gly, His, Phe), CCCP and p CMBS can significantly inhibit the uptake of GlyF. The uptake of GlyF was concentration-dependent and could reach the maximum uptake on 100?mol/L. And the uptake process was also sensitive to p H and temperature.Total of 62 amino acid transporters in Ricinus were identified. These 62 transporters possess two families, ATF and APC family. The ATF family consists of at least fivesubfamilies, including RcAAP, RcLHT, RcAUX, RcANT and RcProT, within 10, 9, 4, 15, 7members, respectively. APC family includes RcCAT and RcBAT within 15 and 2 members,respectively. All 62 transporters showed different expression profile against GlyF. And 17 high expression transporters have been screened out by RT-q PCR. Combining results of semi RT-PCR and RT-q PCR, four candidates, Rc LHT6, Rc ANT15, Rc Pro T2 and Rc CAT2 were picked out for the further study on details.Generally, four candidate transporters showed different expression profile when treated at three time points under 100 ?mol/L GlyF. At 1h, all four candidates displayed up-regulation over 1-fold, especially 3.5-fold for Rc CAT2. At 3h and 6h, except up-regulation for Rc LHT6, the rest three transporters showed down-regulation(Rc ANT15 showed no significant change at 3h).Each of the GlyF induced proteins was infusioned to green florescence protein(GFP)and expressed in BY-2 tobacco protoplast transiently. Result showed the four proteins were distributed both in cytomembrane and cytoplasm. Taken together, the four transporters are involved in uptake of GlyF in cotyledons of Ricinus and served as good candidates for further exploring their function in GlyF uptake.
Keywords/Search Tags:guided pesticides, amino acid transporter, Ricinus communis, RT-qPCR
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