Cloning And Functional Analysis Of A Ubiqutin Ligase E3 LrMCIP8-like Gene From Lycium Ruthenicum Murr

Lycium ruthenicum Murr. Belongs to the Solanaceae family and which is widely distributed in the salinized desert of Qinghai-Tibet Plateau. Its special physiological characteristics of drought-resistance and salt-resistance make it an ideal plant forpreventing soil desertification and alleviating the degree of soilsalinity-alkalinity.,which are very important for the ecosystem and agriculture in the remote area. A novel E3 ubiquitin ligase gene, LrMCIP8-like, was isolated from Lycium ruthenicum Murr in this study. We analyzed the structural characteristics of the sequence, heterologous overexpressed in Arabidopsis. The results are as follows:1. We got completed Lycium ruthenicum Murr E3 ubiquitin ligase gene LrMCIP8-like by using RACE technique amplify.The full-length cDNA sequence was 1271bp, encoding a predicted polypeptide of 363 amino acids.The deduced amino acids sequence of LrMCIP8-like had a calculated molecular weight of 40.094 kDa with a pI of 4.04. LrMCIP8-like gene and ubiquitin E3 ligase CIP8-like of solanaceae crops tomato are high homologous genes, their genetic similarity was 74%, and the full length cDNA amino acid sequence similarity was 79%.2.Sequence analysis showed that LrMCIP8-like protein was rich in hydrophilic amino acids, which had high hydrophilicity. Random coil and alpha helix structure built the major part of secondary structure. Software analysis showed that no transmembrane domain was detected in LrMCIP8-like protein. So we presumed LrMCIP8-like synthesized precursor protein in cytoplasm. No signal peptide guided it to other organelles. It just stays in cytoplasm, and modificates into the mature protein after mRNA translation, and works by combined with related metabolic substrate. Therefore it mainly synthesis precursor protein in cytoplasm.3. Heterologous overexpression of LrMCIP8-like improved drought and partial salt tolerance of transgenic Arabidopsis plants, as determined by physiological analyses of leaf water loss, electrolyte leakage and chlorophyll content, But, in plant germination period, the germination rate of over-expression of exogenous genes lower than the wild type. That means LrMCIP8-like is directly related to salt tolerance of plant.Confocal microscopic observation shows that LrMCIP8-like parts are mainly distributed in cell membranes.In summing up the appeal,LrMCIP8-like gene involved in the regulation of a variety of drought tolerant mechanisms. It not only improved the drought tolerant capability of transgenic Arabidopsis plants at seedling stage, but also improved the salt sensitivity of overexpressed Arabidopsis at germination and seedling stage. The study provided new gene resource and molecular tools for improving the salt tolerance of plant by providing a new genetic resources and molecular tools. It has shown widely application prospects. And it provided resources in genetic engineering of Lycium ruthenicum Murr.