The Functions Of Ca²⁺/CaM And CDPK In The Enhancement Of Maize SHSPs-induced By Combined Drought And Heat Stress

sHSPs（small heat shock proteins）plays an important role in the plants.Our previous studies have shown that the expression of sHSP26,sHSP22,sHSP17.4,sHSP17.2 were induced significantly under heat or combine heat and drought stress.sHSP26 is localized in the chloroplast and interacts with 6 chloroplast proteins to protect the PSII activity.In addition,our study further demonstrats that exogenous H2O2 can increase the expression of sHSP26 and improve plant stress tolerance.However,it is not clear whether Ca²⁺/CaM,CDPK participate in the expression of sHSP26,sHSP22,sHSP17.4 under combine heat and drought stress.Therefore,by using zhengdan 958 as experimental material,and using these technologis such as RT-PCR,Western Blotting and so on,it was studied whether Ca²⁺/CaM,CDPK involved in the expression of sHSP26,sHSP22,sHSP17.4 under combine heat and drought stress,The main results were as follows:1.To determine the expression characteristics of sHSP26,sHSP22 and sHSP17.4 under heat stress,maize seedling were exposed to 42 and 44 ℃ ℃ for 0h（control）,2h,4h,6h,8h,10 h,respectively.RT-PCR and Western Blotting results indicated: the expression of sHSP26,sHSP22,sHSP17.4 was significantly upregulated under heat stress and was the most abundant at 2h.With treatment time lasting,the expression decreased gradually;the expression level of each gene at 44 ℃ was slightly higher than that at 42 ℃.Neverthless,the expression of sHSP26 was more abundant at 4h,with treatment time lasting,the expression level of each gene at 44 ℃ was slightly higher than that at 42 ℃.These results suggest that 44 ℃for 2h and 44℃ for 4h can better induce the expression of sHSP26,sHSP22,sHSP17.4 gene and protein.So,in subsequent experiments,44℃for 2h and 44 ℃ for 4h were used to study gene and protein expression of sHSPs in maize leaves.2.After pretreatment with 0mM,1mM,5mM,10 mM,15 mM,20 mM,30 mM CaCl₂ for 8 hours,maize seedlings were subjected to 44℃for 2h and 4h.The results indicated that the expression of sHSP17.4 and sHSP22 increased firstly,then decreased.Pretreatment with 10 mM CaCl₂ significantly upregulated the expression of sHSP17.4 and sHSP22.The expression of sHSP26 was significantly decreased after pretreatment with10 mM or 30 mM CaCl₂.Therefore,10 mM CaCl₂ was selected for subsequent experiments.3.After pretreatment with Ca²⁺ inducer CaCl₂,Ca²⁺ inhibitor EGTA and CaM analogist W7 or TFP,maize seedlings were subjected to 44℃ for 2h and 4h.The results showed that pretreatment with CaCl₂ leaded to the decrease of sHSP26 protein and gene expression,but the enhancement of sHSP17.4 and sHSP22.Pretreatment with EGTA,W7 and TFP reduced significantly the expression of sHSP26,sHSP17.4 and sHSP22 protein and gene,and the phosphorylation level of sHSP26.These results suggest CaM is involved in protein and gene expression of sHSP26,sHSP22 and sHSP17.4.4.As Ca²⁺ receptor,CDPK plays an important role in plant body.In order to explore the relationship between CDPK and sHSP26,sHSP22,sHSP17.4,we screened CDPK1（ID: 542762）,CDPK1（ID: 542526）,the CK3（ID: 101061019）,CDPK AK1 isoenzyme（ID: 100285166）,CDPK（ID: 542226）by homologous alignment techniques.We found that similarities between the expression characteristics of CDPK isozyme and sHSP26: CDPK isozyme and sHSP26 expression was induced under heat,combined drought and heat.The expression of CDPK isozyme and sHSP26 were decreased after pretreatment with CaCl₂.Therefor,in order to explore the function of CDPK isozyme,ds RNA transient silencing was applied.The results indicated that sHSP26,sHSP22,sHSP17.4 expression was significantly decreased after CDPK isozyme silence,suggesting that CDPK isozymes was involved in the expression of sHSP26,sHSP22,sHSP17.4.In summary,Ca²⁺/CaM and CDPK isozymes were involved in the expression of sHSP26,sHSP22,sHSP17.4.