Abstract:

【Objective】Excessive cadmium（Cd）in some rice grains seriously affect food safety in China. The aim of this study is to identify the novel gene that regulate accumulation of Cd in rice grains and provide the genetic resource for reducing Cd accumulation in rice grains. 【Method】OsLCT3, a member of the low- affinity cation transporter family in rice, was cloned by reverse transcription PCR and RACE techniques. Natural variations of OsLCT3 and physicochemical properties of its coding protein were analyzed by bioinformatics methods. Its expression profiles throughout the growth period and under Cd, Mn, Fe stress were analyzed by quantitative real-time PCR（RT-qPCR）. Its subcellular localization was explored by investigating localization of OsLCT3-GFP fusion protein in in rice protoplasts. The effect of OsLCT3 knockout on divalent cation transport in rice was analyzed by measuring cadmium and other divalent mineral metal elements in various parts of plants at seedling stage in cadmium-stressed hydroponics and at maturity stage in cadmium-polluted soil. In addition, the effect of OsLCT3 on the yeast tolerance to Cd was verified by heterologous functional complementarity in yeast cells. 【Result】OsLCT3 only existed in some indica and japonica rice varieties, with a total coding region of 1 263 bp. The amino acid sequences were classified into five haplotypes based on coding region variation, and the encoded proteins had 12 sub-transmembrane structural domains. OsLCT3 was similar to the LCT-like proteins in Triticum aestivum and Aegilops tauschii, whereas it shared only 52% sequence identity with OsLCT2, 49% and 47% sequence identity with OsLCT1 in indica and japonica rice subspecies, respectively. OsLCT3 was highly expressed in the roots at all stages of growth and development. Its expression levels in the roots were suppressed by Cd, excessive iron（Fe）and manganese（Mn）stress. OsLCT3 was localized to the plasma membrane. Compared with the wild type plants, the oslct3 knockout lines showed the reduced plant height, the lower concentrations of Cd, Fe, and Zn in the shoots, higher concentration of Fe in the roots, and the same concentrations of other bivalent mineral elements. Moreover, oslct3 knockout lines demonstrated the decreased root-to-shoot translocation rates of cadmium, iron and zinc. Under field conditions, Cd concentrations in straw and brown rice of oslct3 knockout lines at maturity were significantly lower than those of the wild type plants, and there were no significant differences in manganese, copper, iron and zinc concentrations in straw and brown rice compared with the wild type plants. Expression of OsLCT3 in yeast resulted in increased sensitivity of yeast to cadmium stress.【Conclusion】OsLCT3 is involved in the translocation of Cd, Fe, and Zn from roots to shoots, and positively regulated the accumulation of Cd in rice grains.

Key words: rice, OsLCT3, cadmium transport, iron transport, zinc transport, metal transporter