Isolation And Characterization Of MsCIPK8 From Alfalfa(Medicago Sativa L.)

In the process of growth and development,plants are faced with many abiotic stresses,such as low temperature,drought,salt and alkali.When plants are under abiotic stress,plants will adopt a series of complex and severe physiological and biochemical defense mechanisms.For example,in CBL-CIPK system,when the stress comes,the intracellular Ca2+concentration increases instantaneously.After Ca2+ combines with CBL,it promotes the formation of CBL-CIPK complex,so as to modify its downstream protein to cope with the stress.The MsCIPK8 gene cloned from alfalfa belongs to CIPK family.The transgenic plants were obtained through the genetic transformation of tobacco by MsCIPK8.The function of MsCIPK8 gene in plant salt and saline-alkali resistance was analyzed,and excellent gene resources were reserved for alfalfa genetic breeding.The main results are as follows:1.According to the analysis of transcriptome sequencing of Alfalfa under saline-alkali stress,the expression of MsCIPK8 was up-regulated by saline-alkali stress.According to the sequencing results,the primers were designed to clone MsCIPK8 gene.The gene CDs has a total length of 1341 bp,encoding a hydrophilic protein composed of 446 amino acid residues,which has no transmembrane domain.The secondary structure of MsCIPK8 protein was as follows:irregular curl accounted for 46.64%,?-helix accounted for 34.75%,and elongation chain accounted for 18.61%.Phylogenetic analysis showed that MsCIPK8 of alfalfa and MtCIPK8 of Medicago truncatula were closely related,and there were four heterotopia spots in the sequence alignment of two proteins,three of which were in the conservative domain.2.The expression of MsCIPK8 was different in different tissues of alfalfa,the lowest in seeds and the highest in roots.The expression of MsCIPK8 in alfalfa was induced by low temperature,drought,salt and alkcipksali stress,and changed with the time of stress treatment.Under low temperature stress,the expression of MsCIPK8 in roots and leaves reached the peak at 12 h and 3 h respectively;under salt stress,the expression of MsCIPK8 in roots and leaves was different;under saline-alkali stress,the expression of MsCIPK8 in roots and leaves was significantly up-regulated,reaching the peak at 24 h;under drought stress,the expression of MsCIPK8 in roots and leaves reached the peak at 12h.3.The MsCIPK8 gene was linked to the expression vector PCBM,which contained camv35s promoter and resistance screening gene glyphosate(PPT).Through resistance screening,PCR and qRT-PCR detection,16 positive strains were obtained.qRT-PCR results showed that the highest expression of target gene was found in strains 16,18 and 28.4.The phenotype of transgenic tobacco with mscipk8 gene was studied under the treatment of 300 mmol/L NaCl and 30 mmol/L NaHCO3.The results showed that the yellowing degree of leaves of transgenic tobacco was lower than that of wild type,and the chlorophyll content of transgenic tobacco was higher than that of wild type.After salt and alkali stress treatment,the MDA,O2-and H2O2 contents in the transgenic tobacco were significantly lower than those in the wild type,the soluble protein,soluble sugar and proline contents in the transgenic tobacco were significantly higher than those in the wild type,and the antioxidant enzyme activity was also significantly higher than that in the wild type.The above results showed that MsCIPK8 gene played an important role in salt and alkali resistance.