The Expression Pattern And Functional Analysis Of AtCPK30 Gene In Arabidopsis Thaliana

Calcium is a ubiquitous second messenger in eukaryotic signal transduction cascades. In plants, intracellular Ca2+ levels are modulated in response to various signals, including hormones, light, and abiotic stress. How response specificity is regulated during Ca2+-mediated signal transduction is an important biological issue. It appears that different stimuli elicit specific calcium signatures, different calcium sensors recognize specific calcium signatures and transduce them into downstream effects, including altered protein phosphorylation and gene expression patterns. CDPKs, calcium-dependent protein kinases, are wildly distributed in plants and algae, and also in some protistas. Currently, most of the known calcium-stimulated protein kinase activities in plants are associated with CDPKs. As calcium sensors, CDPKs mediate numerous responses including hormone signaling. Basing on previous studies, we made additional functional analysis of the gene AtCPK30 encoding a calcium-dependent protein kinase in Arabidopsis. The results of this study were listed as follows:In order to screen homozygous seedlings of T-DNA insertion, we had prepared a simple and feasible hydroponic device. This device is made up of two parts: some seed-holders and stainless steel net as support, a tank with fit cover. Results showed that T-DNA insertion of AtCPK30 grew well in this hydroponic system. Furthermore, this system has the advantage of its low cost of planting and easy maintenance. Owing to the support of steel net, Arabidopsis Plants can finish their whole life cycle successfully and can bear lots of normal seeds with characteristic of high germination rate. Moreover, multi-homozygous of T-DNA insertion was identified by PCR through double primer.Next, Expression pattern of AtCPK30 had been studied under different conditions. Results of semi-quantitative reverse transcription PCR (RT-PCR) analysis indicated that AtCPK30 was highly expressed in root and induced by ABA, IAA, 2,4-D, GA and BA treatment.Then cDNA of AtCPK30 was subcloned into the pEGAD binary vector with a GFP gene and a Bar gene under the cauliflower mosaic virus (CaMV) 35S promoter, and the recombinant was transformed into onion epidermis cells by gene gun. GFP-CPK30 fusion gene transient transformation of onion epidermis cells revealed the localization of CPK30 in cell wall and cell membrane.The physiological roles of AtCPK30 were studied using a gain-of-function approach. Seedlings of AtCPK30 transgenic lines had longer primary roots than those plants of wild-type at the early stages. Interestingly, when these plants grew on MS lack of Ca2+ including wild-type and transgenic lines, the roots of transgenic line were more sensitive to calcium, lack of Ca2+ had less effect on roots of transgenic lines than those of wild-type. Treated with several plant hormones, such as ABA, IAA, GA and BA, the roots of seedlings of transgenic line developed abnormally because they were more sensitive to hormones. Furthermore, NPA relatively less inhibited emergency of lateral roots of transgenic line than those of the wild-type. These results suggest that AtCPK30, as a positive regulator, involved in the hormone-signaling pathways.