Isolation And Functional Analysis Of Malus Xiaojinensis WRKY Transcription Factors WRKY55 And WRKY64

In this study,the Mx WRKY55 and Mx WRKY64 genes were cloned from Malus xiaojinensis Cheng et Jiang.By using the sequence analysis,subcellular localization analysis,tissue-specific expression analysis and transgenic Arabidopsis resistance identification,we preliminarily investigated the functions of Mx WRKY55 and Mx WRKY64 genes.The main results were as follows:We designed the specific primers according to the known partial sequences of ‘Golden Delicious’ apple,and obtained the full lengths of Mx WRKY55 and Mx WRKY64 by PCR amplification using c DNA of Malus xiaojinensis.The open reading frames of Mx WRKY55 and Mx WRKY64 were 984 bp and 1116 bp,respectively.Subcellular localization of Mx WRKY55 and Mx WRKY64 proteins revealed that they were localized in the nucleus.Semi-quantitative PCR results showed that the expression of Mx WRKY55 and Mx WRKY64 genes was organ-specific: In the normal Hoagland nutrient solution culture(0h),Mx WRKY55 and Mx WRKY64 genes were expressed in all organs tested.The expression level of Mx WRKY55 was higher in new leaves and stem,the expression level of Mx WRKY64 only in new leaves was higher.Under salt stress(200 m M Na Cl),low temperature stress(2°C),high iron stress(160 μM Fe-EDTA)and low iron stress(4 μM Fe-EDTA),the expression levels of Mx WRKY55 and Mx WRKY64 went up in early times,as time went on,they declined.The results of real-time PCR showed that under the salt stress,low temperature stress,high iron stress and low iron stress,the expression levels of Mx WRKY55 and Mx WRKY64 went up in early times and as time went on,they declined: the highest expression level of Mx WRKY55 and Mx WRKY64 in new roots was appeared on 6h/9h,9h/6h,9h/9h,6h/9h,and the peak time of Mx WRKY55 and Mx WRKY64 expression level in new leaves was 9h/12 h,6h/3h,12h/12 h,12h/12 h.We constructed Mx WRKY55 and Mx WRKY64 overexpression vectors respectively,and transferred then in Arabidopsis thaliana by Agrobacterium-mediated method to obtain Mx WRKY55 and Mx WRKY64 transgenic plants.The wild-type and transgenic Arabidopsis thaliana were treated with low iron stress,high iron stress and normal iron concentration for 2 weeks.It was found that under low iron stress,the etiolation of wild-type plants were serious,the main root length and fresh weight of plants decreased significantly,while only few leaves of transgenic lines turned yellow,and all physiological indexes of transgenic lines were significantly better than wild type.Under high-iron stress,the leaves of wild-type Arabidopsis thaliana were yellow or even purple,the growth of roots was severely hindered,and the wild-type plants tended to die.However,there were only some old leaves’ color changed in Mx WRKY55 and Mx WRKY64 overexpressing plants,and most of new leaves were still green.The physiological indicators of transgenic Arabidopsis thaliana were obviously surpassed wild-type.Under salt stress,the leaves of wild-type Arabidopsis thaliana were yellow and wilting,while the leaves of Mx WRKY55 and Mx WRKY64 overexpressing plants were still green,and physiological indexes of them were significantly better than wild-type Arabidopsis thaliana.After 7 days of salt treatment,Mx WRKY55 and Mx WRKY64 transgenic plants’ survival rates were obviously surpassed that of wild-type plants.In conclusion,overexpression of Mx WRKY55 and Mx WRKY64 genes enhanced the tolerance of Arabidopsis thaliana to high iron stress,low iron stress and salt stress.