Identification And Functional Analysis Of Larix Gmelinii Specific Calcium Binding Protein

Calcium ion(Ca²⁺)and calcium binding protein play an essential role in response to biotic and abiotic stresses.Plants originating from adverse environments are likely to have novel genes coding calcium binding proteins related to stress tolerance.Therefore,the plants with strong adaptability have great potential for identification of novel calcium binding protein.Dahurian larch,Larix gmelinii?Rupr.?,deciduous tree of Pinaceae,is the main species in Daxing'an Mountains of Inner Mongolia.Due to its strong adaptability to cold and humidity,it has been widely used for forestation project,paper industry and so on.In this study,the specific calcium binding proteins were identified using transcriptome database of L.gmelinii constructed in our previous study.Those proteins were further studied,and the major results are followed:?1?Transient expression of LgCaMBP12195 in Nicotiana benthamiana confirmed that the LgCaMBP12195 is a Golgi-localized protein.Phenotypic analysis of transgenic Arabidopsis expressing LgCaMBP12195 under the stress conditions of different concentrations of Ca²⁺,Fe²⁺,Zn²⁺,Mn²⁺ and Mg²⁺ showed that expression of LgCaMBP12195 enhanced tolerance of the transgenic plants to low-iron stress.Moreover,Inductively Coupled Plasma-Optical Emission Spectrometer?ICP-OES?experiments revealed that the contents of Fe in transgenic plants were higher than that in the wild type?WT?,while the contents of Ca and Zn had no significant difference under low-iron condition.These results indicated that LgCaMBP12195 obviously increased the accumulation of Fe and promoted growth of transgenic plants under low-iron stress condition.?2?Transient expression of LgCCHC-20045 in N.benthamiana showed that the LgCCHC-20045 localizes to the Golgi apparatus.Phenotypic analysis of transgenic Arabidopsis expressing LgCCHC-20045 under the stress conditions of five mineral nutrients showed that expression of LgCCHC-20045 enhanced tolerance of the transgenic plants to low-iron and high-zinc stress,respectively.The root length and fresh weight of transgenic plants were statistically different from those of the WT.Moreover,ICP-OES experiments revealed that the contents of Ca,Fe and Zn in transgenic plants were higher than that in the WT under low-iron condition,and the contents of Ca and Zn were higher than those in the WT under high-zinc condition.These results indicated that LgCCHC-20045 significantly increased the accumulation of Ca,Fe and Zn and promoted growth of transgenic plants under low-iron stress condition,and LgCCHC-20045 improved the accumulation of Ca and Zn to enhance growth of transgenic plants under high-zinc stress condition.?3?Subcellular localization analysis confirmed that the LgMog/PsbP15374 is a Golgi-localized protein.Phenotypic analysis of transgenic Arabidopsis expressing LgMog/PsbP15374 under the stress conditions of five mineral nutrients showed that expression of LgMog/PsbP15374 enhanced tolerance of the transgenic plants to low-iron and high-zinc stress,respectively.Furthermore,ICP-OES experiments revealed that the contents of Ca,Fe and Zn in transgenic plants were higher than that in the WT under low-iron condition,while the contents of Ca,Fe and Zn had no significant difference under high-zinc condition.These results indicated that LgMog/PsbP15374 markedly increased the accumulation of Ca,Fe and Zn and promoted growth of transgenic plants under low-iron stress condition,whereas LgMog/PsbP15374 did not enhance the accumulation of Ca,Fe and Zn in transgenic plants under high-zinc stress condition.In this study,nine of specific calcium binding proteins were identified in L.gmelinii.The LgCaMBP12195,LgCCHC-20045 and LgMog/PsbP15374,were confirmed to be localized in Golgi.The transgenic plants expressing the three proteins did not directly show Ca²⁺-specific affinity,whereas the proteins maybe play roles in regulating low-iron/ high-zinc tolerance by calcium signaling networks.These results lay a foundation for further research on calcium binding protein of L.gmelinii.