Cloning And Functional Analysis Of CDPK1 Genes In Two Different Habitats Plants

Ca²⁺ is an ubiquitous second messager in plant cell, which plays an vital role in plant growth and development, hormone regulation, immune defence, biotic stresses, abiotic stresses and so on. Calcium-dependent protein kinase（CDPK） is a major calcium receptor, that can be directly activated by calcium signal independent of calmodulin, specialized expression of CDPK genes will happen under many environmental factor, such as drought stress, salt stress and low temperature. Xinjiang as China’s major cotton production base, drought, soil salinization and coldness in the late spring, autumn frost and other adverse environmental factors often affecting cotton yield and quality, understanding the molecular mechanisms of stress tolerance in cotton will lay the foundation for improving the stress tolerance of cotton by gene manipulation. Saussurea involucrata as a unique plant, grow in adversity, which has extreme strong cold resistance and important value. It is significant to explore the resistance gene of stress resistant plants and study its molecular mechanism. So far, there were some reports, identification, characterization of CDPKs in cotton, but few studies of CDPKs in Saussurea involucrata have been carried out.In order to study the difference in function of CDPK1 gene in different habitats from the genetic level, this study successfully cloned GhCDPK1 and SikCDPK1 gene, detected the change of gene expression under different abiotic stresses by qRT-PCR. Then they were introduced into the model plant tobacco for verifing function. This study will provide a theoretical basis for the study of the resistance gene of the plant under extreme growth environment and breeding high efficiency and well resistance cotton varieties. The main contents of this study include the following aspects:1. The calcium-dependent protein kinase gene GhCDPK1 was obtained from cold-sensitive crops Gossypium hirsutum, and SikCDPK1 was firstly obtained from high-hardy plants Sasussured involucrata Kar.et Kir by RT-PCR. GhCDPK1 gene contains a 1764 bp complete open reading frame encoding 587 amino acids with a molecular weight 65.5 kDa theoretical isoelectric point 5.46. The GhCDPK1 was a unstable, hydrophilic protein, had no transmembrane region and signal peptide. There was a Ser/Thr kinase domain and four consevative EF-hands structural domain in GhCDPK1, and there have myristoylation site and palmitoylation site in N-terminal, presumably localized on the membrane. Blast on NCBI indicated that protein encoded by CDPK of cotton had more than 84% similarity with the Gossypium raimondii, Theobroma cacao, Populus trichocarpa, Citrus sinensis, Populus euphratica, Ricinus communis, Jatropha curcas, belongs to CDPKs family. SikCDPK1 contains a 1716 bp complete open reading frame encoding 571 amino acids with a molecular weight 63.5 kDa, theoretical isoelectric point 5.44. The SikCDPK1 was unstable and hydrophilic, had no transmembrane region and signal peptide. There was a Ser/Thr kinasedomain and two consevative EF-hands structural domain in SikCDPK1, also have N terminal myristoylation site and palmitoylation site. Blast on NCBI indicated that SikCDPK1 protein had more than 81% similarity with the Gossypium hirsutum, Ricinus communis, Populus trichocarpa, Nicotiana tomentosiformis, Sesamum indicum, Vitis amurensis.2. The cDNAs reversed transcripted from RNA of cotton and Sasussured involucrata leaves by treated such as PEG6000, NaCl and low temperature were used for real-time qRT-PCR analyses. The expression of GhCDPK1 and SikCDPK1 gene both can be induced by drought,NaCl and low temperature, This result indicated that CDPK1 may be play import role in plant stress signal process.3. Construction of subcellular localization vector pCAMBIA2300-35S-GhCDPK1-GFP-OCS. After infecting onion epidermal cells, GhCDPK1 was found to be distributed in cell membrane and cell wall in the laser scanning confocal microscopy.4. Construction of plant expression vectors pCAMBIA2300-35S-GhCDPK1-OCS and pCAMBIA2300-35S-SikCDPK1-OCS, transfer into tobacco with leaf disc method,tissue-cultured and get the seedlings, identified positive plants by RT-PCR. Transgenic lines and WT were treated with drought, high salt and low temperature stress, the plant phenotype was observed and the relative physiological indexes were determined. The results showed that the transgenic plants could reduce the damage to plants under stress to a certain extent, and enhance the plant stress resistance by accumulating more osmotic adjustment substances,enhancing the activity of the antioxidant system and maintaining the stability of the cell membrane.