Cloning And Analysis Of Calmodulin Gene From Phaseolus Radiatus L. And Triticum Aestivum L.

Calmodulin(CaM) is an important regulative protein that exists in signal transferring and highly conserves in eukaryotes. CaM is combined with Ca²⁺ and takes part in modulating target enzymes, so that it can modulate the cell's growth and take action to irritation outside the cell. Calmodulin joins in many aspects: gene expression, cell division and metabolism, cell membrane transportation and reproductive development etc. By far, lots of calmodulin genes have been cloned from many species: arabidopsis thaliana, barely, rice, hot pepper, beet, salvia miltiorrhiza and cowpea etc.Phaseolus radiatus L. is an annual herbaceous plant as both grain crops and offcinals. Its seeds and seed capsules take important effects in clearing summer-heat and detoxifcating. Triticum aestivum L. is one of important grain crops in the world. It accounts for 17% of the size of white crops, which is the main food of 35% of population of the world.In this paper, Calmodulin genes from Phaseolus radiatus L. and Triticum aestivum L. have been cloned, and the structure of Calmodulin gene have been analyzed. Total RNA was isolated from leaves of wheat and mung bean and reverse transcripted to cDNA. By PCR, the Calmodulin gene from Triticum aestivum L. and Phaseolus radiatus L. was cloned. The Calmodulin cDNA from Phaseolus radiatus L. and Triticum aestivum L. has 447bp, including a full Open Reading Frame(ORF) and coding 148 amino acids. By comparing with two Calmodulin genes, we find that the similarity of nucleotide is 84.6% and the similarity of amino acid is 95.6%. There are high similarities in Calmodulin genes among many other different plants. The similarity of nucleotide is over 80% and the similarity of amino acid is over 90%. The Calmodulin gene of Phaseolus radiatus L. was registered in Genebank, DQ778070; that of Triticum aestivum L. was registered as EF577270.The Calmodulin gene was inserted into pCambial304 plant expression vector positively and reversely. By PCR and enzyme cutting, the Calmodulin gene from Phaseolus radiatus L. had been inserted into the plant expression vectors. The antisense had been transferred into Salvia miltiorrhiza Bunge. This will greatly help us to understand the calmodulin influence in plants.Using bioinformatics softwares such as TMHMM, ProtScale, SOPMA and Pfam, we analyzed hydrophilicity and hydrophobicity, transmembrane structure, curl-helix structure, function region of Calmodulin from Phaseolus radiatus L., identified the sub-cell position and predicted secondary structure. The Calmodulin from Phaseolus radiatus L. is a hydrophilicity protein, and has no signal peptide. It exists, is produced and stays in the cytoplasm. The secondary structure is mainlya-helix, including four EFhands that can combine with Ca²⁺ to transfer signals.Heat Shock Protein is a kind of protein that expresses highly under high temperatures or other environment irritations. There are some main Hsps existing in cells such as Hsp60, Hsp70, Hsp90 and sHsps. Hsps can eliminate the effect of heat shock to protect cells and organisms. Many organisms including plants can produce Hsps at high temperatures. This article cloned Heat Shock Protein sequence from Brassica campestris L. and analyzed the gene sequence. By PCR, we obtained the fragment of 834bp, coding 278 amino acids. By comparing in NCBI, a high similarity of Heat Shock Protein cDNA is found between Brassica campestris L. and Arabidopsis. The similarity of nucleotide is 88.1% and the similarity of amino acid is over 90%. Using DNAstar and DNAman bioinformatics softwares, we analyzed nucleotide sequence, amino acid sequence, hydrophilicity and hydrophobicity of an ORF and curl-helix structure. At last, secondary structure of Heat Shock Protein was predicted.