Cloning And Functional Analysis Of OsCPK9Promoter In Rice (Oryza Sativa L.)

Calcium, as one of the important second messengers, plays an important role in plantgrowth and developmental processes and in response to environmental stresses. To date,several classes of calcium-sensing proteins have been characterized in plants. Amongthem, calcium-dependent protein kinases (CDPK) attracts special attention because it actsnot only as the Ca2+sensor, but also the effector, and is widely distributed in plants. Inrecent years, CDPKs have been shown to be involved in the plant development, hormoneresponses, metabolism, environmental stresses, defense responses and other processes. Itwas found OsCPK9transcript can be induced by drought, high salt stresses and rice blastinfection, indicating that OsCPK9may play important roles in response to rice bio-andabiotic stresses.Promoter, as cis-acting elements, is located upstream of the transcription initiationsite. It can be recognized and binding with RNA polymerase, there by initiating thedownstream gene expression. It determines temporal and spatial specificities of geneexpression in plants, and therefore is called "molecular switch. In order to investigateregulation mechanism of the OsCPK9gene, we cloned and sequenced about2kbupstream sequence of rice OsCPK9gene transcription initiation site. Cis-element analyseswere performed with PLACE database. POsCPK9::GUS expression vector wassuccessfully constructed for studying OsCPK9gene expression characteristics. The mainresults are as follows:(1) The2114-bp long upstream sequence of OsCPK9gene transcription initiation sitewas amplified by PCR. PLANTCARE online software analysis predicted that OsCPK9promoter POsCPK9contains a number of cis-acting elements including in plant growthand development, and in response to various biotic and abiotic stresses. The resultssuggested OsCPK9may play important roles in a variety of signaling pathways of plantgrowth and development, and in response to various biotic and abiotic stresses.(2) The plant expression vector POsCPK9::GUS was successfully constructed andwas transformed transiently into tobacco tissues using Agrobacterium-mediatedtransformation method. GUS histochemical staining results showed that gus gene driven by POsCPK9was expressed in leaves and stems at higher levels, expressed in pollens atmoderate levels, and expressed in the petals,calyx, root and stigma at lower levels. Resultsof GUS activities analyses were consistent with the results of GUS histochemical staining.These results show that POsCPK9has promoter function.(3) Gus gene driven by POsCPK9could be up-regulated by ABA and PEG-6000treatments in tobacco leaves transformed transiently with POsCPK9::GUS.(4) Plant expression vector POsCPK9::GUS was transformation into rice byAgrobacterium-mediated transformation method. Twenty transgenic plants wereregenerated, among then, four positive T0transgenic plants were further verified throughPCR identification. These transgenic plants will be utilized for subsequent experiments.In conclusion, the fndings of present study established the foundation for furtherOsCPK9gene functional analysis.