Functional Identification Of OsCPK2, OsCPK15 And OsCPK29 In Rice (Oryza Sativa L.ssp. Japonica)

Cytoplasmic Ca²⁺ is an important second messenger.Phosphorylation signal cascades are generated through Ca²⁺ binding proteins,regulating the expression of downstream genes.Calcium-dependent/calmodulin-independent protein kinases(CDPKs) are a Ser/Thr protein kinase family which exist only in the plant and parts of protista,and play a very important role in Ca²⁺ signal transduction.More and more evidences indicate that CDPKs participate widely in singnal transduction processes of physiological responses,including plant development,pathogen defense and abiotic stress response etc. Thirty one CDPK genes have been found in rice so far,however only few of them have been well characterized.The objective of this research is to reveal the functions of OsCPK2,OsCPK15 and OsCPK29 through over-expression and RNA interference technology,which can provide new gene resources and theoretical guidances for rice breeding using genetic engineering.Main research results of this study are as follows:1.The expression profiles of OsCPK2,OsCPK15 and OsCPK29 in the root at tillering stage,the mature leaf,the distance from panicle tip to flag leaf pulvinus is 0 cm, 0-1cm,1-3 cm,3-5 cm,5-8 cm,8-12cm,12-16 cm,16-20 cm,20-25cm,25-30 cm,the panicle at filling stage(named P1 to P12 stages) of Oryza sativa cv.Nipponbare have been analysised by RT-PCR.The results show that OsCPK2 and OsCPK29 have high expression level in the panicle at P2 to P11 stages,and weak expression level in the mature leaf and the panicle at P12 stage,while no expression in the root at tillering stage and the young panicle.OsCPK15 has high expression level in all detected tissue stages. These results indicate that the expression of OsCPK2 and OsCPK29 is tissue-specific and temporal-spatial,while OsCPK15 expresses constitutively.2.The OsCPK2-overexpressing vector(p1300S-CPK2) was constructed and transformed by Agrobacterium tumefaciens-mediated transformation using Nipponbare as the recipient material.Fourty two regenerated plants have generated through transformation.Southern blot and Northern blot analysis indicate that 16 among them are single-copy inserted and 4 are over-expression plants within 16 single-copy inserted transgenic plants.3.The OsCPK15-overexpressing vector(p1300S-CPK15) was constructed and transformed by Agrobacterium tumefaciens-mediated transformation using Nipponbare. Fourty three regenerated plants have generated through transformation.Southern blot and Northern blot analysis indicate that 11 among them are single-copy inserted and 2 are over-expression plants within 11 single-copy inserted transgenic plants.4.The OsCPK29-overexpressing vector(p1300S-CPK29) was constructed and transformed by Agrobacterium tumefaciens-mediated transformation using Nipponbare. Seventy four regenerated plants have generated through transformation.The positive rate of transgenic plants is 92%by PCR analysis.Southern blot and Northern blot analysis indicate that 11 among them are single-copy inserted and 4 are over-expression plants within 11 single-copy inserted transgenic plants.5.The pollen fertility was observed in T₂ families of OsCPK29-overexpressing plants.The results show that there is no difference on pollen fertility between transgenic plants and wild type.6.T₂ families of OsCPK29-overexpressing plants were treated under three different abiotic stresses(drought,salt and cold).The results showed that there is no significant difference on drought,salt and cold tolerence between transgenic plants and the control based on phenotypic observation.Mannitol and ABA treatments were also conducted on T₂ families of OsCPK29-overexpressing plants and the transgenic plants did not show significant differences from the control.7.Leaf senescence process of T₂ families of OsCPK29-overexpressing plants were also detected,and the results showed that the leaf senescence process of OsCPK29-overexpressing plants delayed compared with the control.Meanwhile, chlorophyll content of the flag leaf were determined in vitro before and after treatments, and the result showed that the chlorophyll content of transgenic plants is significantly higher than that of the control after the treatment but no difference before that.8.The OsCPK2,OsCPK15 and OsCPK29 RNAi vectors(pDS1301-CPK2, pDS1301-CPK15 and pDS1301-CPK29) were constructed and transformed by Agrobacterium tumefaciens-mediated method.Thirty nine,thirty eight and fourty three regenerated plants were generated respectively.The positive rate of transgenic plants is more than 90%by PCR analysis.9.The expression level of OsCPK2,OsCPK15 and OsCPK29 in the corresponding RNAi transgenetic plants have been detected by RT-PCR,and the results showed that all of three target genes were not suppressed.10.The OsCPK2,OsCPK15 and OsCPK29 subcellular location vectors (p1391aGFP-CPK2,p1391aGFP-CPK15 and p1391bGFP-CPK29) were constructed,and coated on gold particles respectively,and then bombarded into onion epidermal cells.All of the fusion proteins are localized in the cytoplasm by laser scanning confocal microscope.