| As a Ca2+-binding protein, Calmodulin (CaM) is one of most important signal transduction molecule now. Ca2+/CaM complex has been studied in a most in-depth and extensive way in the plant cell signal system field. In plants, multiple CaM genes were found, which encode the same or similar proteins. These proteins have EF-hand chiral Ca2+-binding domain, which can combine with Ca2+, therefore the same or similar proteins known as CaM isoforms. Different subtypes of plant CaM genes are have tissue, organ and developmental specificity of different periods, different various subtypes of CaM gene responses to external stimuli differently, therefore plants can better resist to the touch, hot and cold, light, pathogens and hormones (growth hormone) and other kinds of external stimuli. In addition, many studies find that not only transient increase of Ca2+ is early signals triggered by of the plant defense responses, but also involved in the Ca2+ the plant defense responses signaling pathway which Ca2+ involved in is often mediated by CaM.Using wheat-leaf rust interaction system as investigated subject, the studies of the blocking or activating the Ca2+ signaling, Ca2+ subcellular localization, rising of cytoplast Ca2+ in different wheat varieties in elicitor stimulate protoplasts of mesophyll cell show that Ca2+ is involved in the signal transduction of wheat leaf rust infection. On this basis, Real-time quantitative RT-PCR and western blotting were performed. These works show that the expression of wheat calmodulin has increased after the stimulation of the compatible leaf rust race. We have initially proved that Ca2+/CaM involved in the resistance of wheat leaf rust infection-induced hypersensitive reaction (HR). To this end, in this article wheat (Triticum aestivum L.) cultivars L10 and leaf rust (Puccinia triticina) race 260 constituted the incompatible combination, and the L10-165 was considered as compatible combination, in order to study the distribution of CaM in subcellular level after infection by leaf rust. The results showed that the calmodulin distribution of incompatible combination is obviously higher (increase) than compatible combination and mock inoculated after inoculation 4 h, 8 h in the intercellular spaces, but on the other organelles (e.g.mitochondria, chloroplasts, nucleus, etc.) distribution among the three treatment is consistent, it is provides experimental evidence in ultrastructural level for further argued CaM function in wheat-leaf rust interaction. For the wheat CaM exists in three subtypes, in order to further explore the role of CaM, preparation of the specific CaM isoform antibody is necessary. It is the most effective and specific reagents in localization, quantitation and physiological functions research of CaM.This experiment according to known sequences, using wheat cultivars L10 leaf as template, amplified calmodulin gene TaCaM2-3, 4-1. Then the recombinant expression vectors pET28a-TaCaM2-3 and pET28a-TaCaM4-1 were constructed and transformed into E. coli strain BL21. Using E.coli expression system, TaCaM2-3,4-1 fusion protein were inducted by IPTG. The optimal expression conditions of pET28a-TaCaM2-3 and pET28a-TaCaM4-1: when the cell OD600 reachs 0.5-0.8 in LB medium at 37℃, adding IPTG to a final concentration of 0.8mmol/L, induced 6 h at 37℃. Further using Ni-NTA column, the protein were extracted and purified TaCaM2-3, and SDS-PAGE showed to remove the most complex protein.Protein concentration was about 1.5mg/mL, consistent with the requirements of the antibody preparation. Purified TaCaM2-3 were used as antigen immuned rabbit, acquire the anti-wheat CaM-specific rabbit antiserum. The study made a foundation of further exploring CaM in wheat-leaf rust interaction of spatial and temporal distribution and function.
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