Cloning And Functional Analysis Of EnCIPK31 Gene From Elymus Nutans Griseb

Low temperature has become the biggest limiting factor for plant growth and the key factor affecting plant distribution in alpine regions.The special low temperature condition in Tibet restricts the production of plants.Therefore,it plays an important role in the development of animal husbandry in Tibet to accelerate the construction and restoration of artificial grassland by utilizing cold-resistant forage.Elymus nutans Griseb is the dominant grass species on the Tibet Plateau.It has high forage value and plays an important role in maintaining ecological balance in the alpine region.Meanwhile,Elymus verticosa is also a wild relative species of wheat crops and has valuable genes for rich genetic diversity of wheat crops and forage.Therefore,Elymus nutans Griseb is an ideal high quality forage to study low temperature adaptation.Based on the transcriptome response and q RT-PCR analysis of the low temperature response of Elymus nutans Griseb,it has been found that EnCIPK31-dependent calcium signaling pathway plays a positive role in the regulation of low temperature adaptation of Elymus nutans Griseb.However,how EnCIPK31 regulates the mechanism of low temperature adaptation of Elymus nutans Griseb populations remains unclear.Therefore,in this study,on the basis of the effect of Ca²⁺application on physiological mechanism to improve its cold resistance,the full-length CDS sequence of EnCIPK31 was cloned from the Elymus nutans Griseb and heterologously expressed into the model plant rice.The molecular mechanism of Ca²⁺signaling mediated by EnCIPK31regulating plant response to low temperature stress was further revealed.The main results of this study are as follows:（1）Under low temperature conditions,the physiological response mechanism of Elymus nutans Griseb was investigated by exogenous CaCl₂ spraying.The results showed that:Exogenous Ca²⁺regulation in the form of concentration dependent on male Elymus nutans Griseb with the low temperature adaptability of alkali grass including 15 mM CaCl₂treatment can significantly improve the cold resistance of hanging ear cover with alkali grass mainly for effectively alleviate cold Elymus nutans Griseb seedlings growth inhibition,sagging Elymus nutans Griseb low temperature stress in the ground/underground biomass increased significantly;Reduce the accumulation of malondialdehyde and reactive oxygen and increase the content of soluble sugar and free proline.At the same time,it can significantly increase the activity of peroxidase,superoxide dismutase,catalase and ascorbate peroxidase.Meanwhile,the expression levels of antioxidant enzymes related genes EnAPX,EnCAT2,EnGPX,EnGST4,EnMDAR,EnPER1,EnCu/ZnSOD,EnFe SOD and EnMn SOD were up-regulated.Moreover,exogenous Ca²⁺significantly up-regulated the expression of stress related genes EnCOR410,EnCS120,EnDHN5 and EnMYB4.（2）EnCIPK31 gene was successfully cloned from Elymus nutans Griseb by PCR technique.The full length CDS sequence of EnCIPK31 gene was 1350 bp.The CDS sequence of EnCIPK31 gene was consistent with the transcriptome data.The protein encoded by EnCIPK31 gene contained 449 amino acids,with a molecular weight of 50.9kD,an isoelectric point of 8.27,and no transmembrane structure.Subcellular localization prediction indicated that EnCIPK31 was localized in the nucleus and cytoplasm.Homology analysis showed that the amino acid sequence similarity of EnCIPK31 was 87.58%among Elymus nutans Griseb,Aegilops tauschii subsp.tauschii,Triticum aestivum,Hordeum vulgare subsp.Vulgare,Triticum dicoccoides Korn and Brachypodium distachyon（L.）Beauv.The amino acid sequence encoded by EnCIPK31 gene of Elymus nutans griseb is most closely related to Aegilops tauschii subsp.tauschii,Triticum aestivum and Hordeum vulgare subsp.Vulgare,followed by Triticum dicoccoides Korn and Brachypodium distachyon（L.）Beauv.（3）qRT-PCR was used to analyze the tissue expression characteristics of EnCIPK31and the transcriptional expression level of EnCIPK31 under drought,low temperature,NaCl and ABA stress.The results showed that EnCIPK31 was expressed in roots,stems and leaves,and the expression levels were different in different tissues.The highest expression was found in leaves and the lowest in stems.EnCIPK31 is involved in the response process of plants to drought,low temperature,NaCl and ABA stress,and its gene expression level is greatly affected by stress,and with the increase of stress treatment time,the gene expression level also shows an increasing trend.They also showed a sensitive response to low temperature stress.These results suggest that EnCIPK31 may be involved in the response to drought,low temperature and NaCl stress,and this regulation process may be related to ABA signaling.（4）The pCAMIBA1300:2×35S:EnCIPK31-EGFP expression vector was constructed,and the tobacco was transformed by Aturobacterium infection.The subcellular localization of EnCIPK31 in the epidermis and protoplasts of tobacco was observed by laser confocal microscopy,and the results showed that EnCIPK31 was mainly in the nucleus and cytoplasm.（5）In order to further clarify the regulatory role of EnCIPK31 gene in the low temperature response of plants,a complement plant（EnCIPK31Ox）was formed by Agrobacterium infection and the deletion of oscipk31 mutant was obtained by CRISPR/Cas9 editing technology.The phenotypic changes of wild type,mutant and complement plants were analyzed under normal temperature and low temperature stress,respectively.The results showed that the aboveground/underground biomass of rice seedlings showed that the wild type plant>complement plant>mutant plants at normal temperature.After low temperature treatment（4℃）,wild-type seedlings were less damaged by low temperature,followed by overexpressed seedlings,and mutant seedlings were more serious.（6）NMT non-invasive minimally invasive technique was used to analyze the changes of Ca²⁺flow in wild type（WT）,overexpression（OsCIPK31）and mutant（oscipk31）under low temperature treatment.The results showed that there was no Ca²⁺influx in wild type（WT）,overexpression（OsCIPK31）and mutant（oscipk31）at room temperature and low temperature,but they were all exogenous.The effervation rate of oscipk31 was significantly higher than that of WT and OsCIPK31,suggesting that EnCIPK31 may regulate the response to abiotic stress by preventing intracellular Ca²⁺effervation.In conclusion,under low temperature stress,EnCIPK31 may prevent intracellular Ca²⁺exogenous transport and maintain a relatively high intracellular Ca²⁺concentration,thus improving the cold resistance of plants by regulating EnCIPK31-dependent calcium signal transduction pathway,which provides a basic support for the breeding,cultivation and utilization of Elymus nutans Griseb.