Functional Analysis Of CdCIPK5 And OsCIPK5

Cynodon spp.is a turf grass with high tolerance to drought and salt stress.ABA participates in the regulation of drought,salt and cold stress.The differentially expressed gene CdCIPK5 was selected from the gene expression profile of triploid bermudagrass treated by exogenous ABA.And overexpression of CdCIPK5 increased the salt tolerance of rice.This paper preliminarily revealed the mechanism of CdCIPK5 regulating salt tolerance,and studied the function of CdCIPK5 homologous gene OsCIPK5 in rice.The main results are as follows:1.CdCBL4-CdCIPK5 complex regulates plant salt toleranceFive CdCBL genes were cloned by homologous cloning according the gene sequences of Setaria italica.The interaction between CdCIPK5 and CdCBL4 was verified by yeast two-hybrid and BiFC.Three CdCBL4 overexpression transgenic rice lines were selected for salt tolerance identification.It was found that one week after 125 mM NaCl treatment,the transgenic lines had higher RWC and Fv/Fm,indicating that overexpressing of CdCBL4 improved salt tolerance in rice.The results of sodium and potassium ion content determination showed that the overexpressing CdCBL4 and CdCIPK5 transgenic lines had lower sodium ion accumulation and higher K+/Na+ ratio under salt stress,which maintained the balance of sodium and potassium ions in plants.Results of qRT-PCR showed that the expression levels of OsNHX3,OsHKT1;1,OsHKT1;4,OsHKT1;5 in overexpressing CdCIPK5 lines were higher than those in WT.So it was speculated that CdCBL4-CdCIPK5 complex could regulate the absorption,transportation and excretion of Na+ under salt stress by regulating the expression level of Na+transporter genes,thus improving plant salt tolerance.2.Genetic transformation and identification of bermudagrassA large number of transgenic lines were obtained through callus induction,suspension subculture,Agrobacterium infection,co-culture,selecting,differentiation,rooting and transplanting,but all were negative after PCR identification.In the later stage,due to the lack of Dicamba(Sigma)and the failure to find alternatives,the whole regeneration system was affected,so we tried to rebuild the regeneration system of bermudagrass.Finally,the callus was successfully induced,and a suspension culture system independent of Dicamba was established.However,it was still unsuccessful to get the regeneration plant.At the same time,suspension culture with Dicamba from other companies was also tried,but it still failed to differentiate and regenerate into plants.It is speculated that Dicamba(Sigma)can not only promote callus growth,but also maintain its callus regeneration ability.3.Characteristics of OsCIPK5 gene and proteinThe OsCIPK5 gene has only one exon,and the CDS has a full length of 1386 bp,encoding 461 amino acids.OsCIPK5 has a typical kinase domain,Activation loop and a NAF domain that mediates the interaction between CBL and CIPK,with the highest homology with AtCIPK20 and AtCIPK6 in Arabidopsis.OsCIPK5 located in cell membrane,cytoplasm and nuclear OsCIPK5 is expressed in roots,leaves and flowers,and its expression is induced by ABA,cold,dehydration and salt stress.It indicates that OsCIPK5 may be involved in a variety of regulatory pathways.4.Functional study of rice OsCIPK5A rice OsCIPK5 overexpression vector and a CRISPR-Cas9 knockout vector were constructed,and the overexpressing transgenic lines and gene knockout mutant lines were obtained through Agrobacterium-mediated genetic transformation.Homozygous T-DNA insertion mutants were obtained by PCR identification,and we have generated homozygous RNAi interference lines before.After evaluation of abiotic resistance,it is found that OsCIPK5 positively regulate salt stress and negatively regulate cold stress.After 10 days of seed germination,the plant height of the gene knockout lines and T-DNA insertion mutant was significantly shorter than that of the wild type while there were no significant difference among overexpressing lines,RNAi lines and wild type.When the growth reached the maturity stage on the 85th day,the plant height of the overexpressing lines remained the same as that of the wild type,and the RNAi lines,knockout lines and the T-DNA insertion mutant had lower plant height than the wild type,indicating that OsCIPK5 is involved in the regulation of rice growth(plant height).There were no differences in spike length,grain number per spike and seed setting rate between the overexpression lines and the wild type,but the spike length,grain number per spike and seed setting rate of the RNAi lines,knockout lines and T-DNA insertion mutant were significantly lower than those of the wild type,indicating that OsCIPK5 is involved in the regulation of rice reproductive development.5.Preliminary analysis of the regulation mechanism of rice OsCIPK5The interaction between OsCIPK5 and OsCBLl/4/7/8 was verified by yeast two-hybrid.Homozygous OsCBLl and OsCBL8 knockout mutants were obtained by CRISPR-Cas9 technique.OsCBLl mutant showed more sensitivity to salt stress and more tolerant to cold stress than wild type.The plant height,panicle length,grain number per panicle and seed setting rate of two homozygous OsCBL8 mutant lines were significantly lower than those of wild type,and showed the same growth and development phenotype as OsCIPK5 mutant.After yeast library selection and verification,OsCIPK5 interacted with OsSOS1,OsACN1,OsK4,OsDIP1,OsDjA6 and OsDjA7.It has been shown that OsSOS1 is involved in salt stress regulation,OsDIP1 regulates osmotic stress,and OsK4 regulates photoperiod flowering.Therefore,it is speculated that OsCBL1/4 senses calcium signal and then transmits it to OsCIPK5.OsCIPK5 phosphorylates OsSOSl and OsDIP1,regulates its activity and regulates salt stress.OsCBL1-OsCIPK5 negatively regulates rice cold resistance.The OsCBL8-OsCIPK5 complex may regulate growth and development by regulating the activity of OsK4.Due to the high homology between OsCIPK5 and AtCIPK6,it is speculated that there is a pathway similar to AtCBL4-AtCIPK6-AtAKT2 in rice,OsCBL4-OsCIPK5-OsAKT2,which regulates rice growth and development.OsDjA6 and OsDj A7 may be involved in the regulation of OsCIPK5 activity as molecular chaperones.