The FunctionZmCBL1/9 Under Adversity Stress And Expression Pattern Analysis Of Six ZmERFs

CBLs(calcineurin B-like proteions),a unique kind of plant calcium sensor,and CIPK(CBL-interacting proteions kinase),its single down-stream target protein,play important role in plant response to biotic and abiotic stress.Previous studies have shown that,both Zm CBL1 and Zm CBL9 can interact with Zm CIPK8,which involved in drought stress.However,the functions of Zm CBL1 and Zm CBL9 in response to stress were not clear.The study showed that the sequence homology of Zm CBL1 and Zm CBL9 is as high as 90%,and can enhance the transgenic Arabidopsis response to stress,including drought,high salt,low temperatures etc.At the same time,6 Zm ERF genes were cloned and characterized.The expressions of these genes in maize roots and leaves of maize under ABA(abscisic acid),JA(jasmonic acid)and ET(ethylene)were analyzed.(1)In order to investigated the founctions of Zm CBL1 and Zm CBL9 in plant response to stress,different development periods of the over-expressed Arabidopsis phenotype were analyzed.In germination stage,under the mannitol,Na Cl,ABA,low potisum treatment,Zm CBL1 and Zm CBL9 over-expression seeds germinatin and seedling growth were promoted compared with WT.In the seedling stage,the transgenic plants showed prominent resistance to drought,and slightly resistance to ABA and Na Cl.Stressed in mature stage,over expressed Zm CBL1 and Zm CBL9 can improve plant salt resistance and over expressed Zm CBL1 also can improve the transgenic plants drought resistance but Zm CBL9 not.20 mg/L ET treatment seed in germination,the yellow WT seedling showed obviously "triple response",but the transgenic seeding have no the obviously phenomenon.The results showed that over expressed Zm CBL1 and Zm CBL9 can reduce the sensitivity to ET.These results suggested that over expressed Zm CBL1 and Zm CBL9 can enhance the transgenic Arabidosis response to abiotic stress;and the functions have differences in different plant development stages.(2)Using Maize g DNA as template,the p CAMBIA1931+pro-CBL1/9 fusion recombinant plasmid was constructed and transformed into Arabidopsis with floral-dip.GUS staining in early development stage showed that,there was no obvious staining in the early stage of germination,but can be dyed after the cotyledon greening.Under normal conditions of four leaf stage of seedling,Zm CBL1 mainly expressed in the hypocotyl top near leaf and cotyledon veins near leaf opex;Zm CBL9 mainly expressed in the hypocotyl top near leaf,cotyledon veins and leaves.After treatment by mannitol,Na Cl,ethephon and Ag NO3,it showed that Zm CBL1 and Zm CBL9 expression increased under drought stress but had no obvious change after high salt stress.Treated by the combined of 375 m M mannitol and 20?M Ag NO3,the expressions Zm CBL1 and Zm CBL9 were declined compared with seedling treated by 375 m M mannitol alone.The results indicated there is cross between the ethylene pathway and the CBL-CIPK signaling system.(3)Six maize ERF genes were isolated and identified by sequence analysis,named as Zm ERF4,26,28,64,89 and 202.Genetic structure analysis showed that Zm ERF was different from Arabidopsis thaliana.Phylogenetic analysis showed that the relationship between Zm ERFs and was closer,and the relationship between Os ERFs and At ERFs was a little farther.Expression analysis showed that,after ET treatment,the m RNAs levels of six Zm ERFs were accumulated in the leaves,but the four genes(Zm ERF4,28,64 and89)were negative regulation in maize roots.After ABA and JA treatment,the m RNAs levels of six Zm ERFs have changed more or less in leaves,but no significant changes in root of most genes,especially after JA treatment.These results suggest that Zm ERFs may play an important role in the hormone signaling pathway in maize roots and leaves.