Cloning And Preliminary Study Of Aegiceras Corniculatum AcTPC1 Gene

Ca²⁺ channels participate in and play a important role in signal transduction in high plant cells. When the study goes deeply, more and more Ca²⁺ channel types present themselves. Ishibashi et al discovered a new type of Ca²⁺ channel――TPC1(Two Pore Channel 1) in rat in 2000. After that, TPC1 genes were cloned in Arabidopsis, rice, tobacco and wheat too. In this article, we intend to clone and study the TPC1 gene in Aegiceras corniculatum etc..Genomic DNA was extracted from Aegiceras corniculatum etc., and degenerate primers were designed based on conserved domains of known TPC1s , and we successfully amplified TPC1 gene fragment from the genomic DNA with PCR.Then we isolated Aegiceras corniculatum RNA and reverse transcripted, the cDNA was used as PCR template, and a distinctive band of 643 bp was amplified by touchdown PCR. The fragment could encode 214 amino acids and shares high homology with known TPC1s. Translated to amino acids, the tentative peptide fragment contains 5 transmembrane segments (TMS) and 1 partial pore loop(P) segment. The 4th TMS segment harboring plenty of positively charged amino acids may serve as a voltage sensor. This structure is similar to S4 of voltage-dependent Na+/Ca²⁺ channels, indicating that TPC1 protein is probably voltage-dependent.Based on the TPC1 fragment, primers were designed to perform 3'and 5'RACE to get the unknown region. We achieved about 1.9 kb(in 3'RACE) and about 300 bp(in 5'RACE) DNA fragments. After sequencing and BLAST in GenBank, we confirmed that we got the unknown 3'and 5'regions.We devised new primers and successfully amplified the full length TPC1 gene in Aegiceras corniculatum. Its open reading frame(ORF) contains 2217 bp and encodes 738 amino acids, the protein has two conserved homologous domains, both of which contain 6 transmembrane segments and a pore loop between the S5 and S6 in each domain. In the center of AcTPC1, each domain has a EF binding arm. The gene's highly identities to known TPC1s demonstrate that we have successfully cloned the TPC1 gene in Aegiceras corniculatum. We nominate it as AcTPC1. We insert the AcTPC1 into the plasmid pET-22b, and the recombinant plasmid was transformed to BL21, and IPTG was used to induce the infusion protein to express. In SDS-PAGE, the 85 KD AcTPC1 appears at the right place, indicating the successful expression.The Ca²⁺ channel gene――CCH1 was directly disrupted with PCR products in Saccharomyces cerevisiae. Functional complementation assay was performed and the AcTPC1 can rescue mutant cch1 , indicating the AcTPC1 is a Ca²⁺ channel.From the above results, we confirm the successfully cloning of the AcTPC1 gene in Aegiceras corniculatum, a new member of TPC1 family. This work founds the groundwork for further investigating TPC1 on the origin, the evolution, the molecular structure and the mechanism of switch-on and switch-off; on the function mechanism in salt-tolerant; on the effects in signal transduction, cell metabolization, growth and development.