Identification Of WRKY Gene Family In Phyllostachys Edulis And Their Correlation With Leaf Senescence Regulation

Moso bamboo(Phyllostachys edulis)is a kind of single axis scattered bamboo plant of Gramineae and Phyllostachys,which is widely distributed in the Yangtze River Basin and southern provinces and regions of China.It is one of the best economic bamboo species with the largest area,the widest use and the most in-depth development and research in China.However,in the process of natural breeding and management,it is found that moso bamboo is susceptible to a variety of biotic and abiotic stress factors,leading to the early senescence phenomenon of plants and leaves,which results the reduce of the overall photosynthetic efficiency of moso bamboo forest,the amount of bioaccumulation,and then affects the quality of the timber,the expansion and extension of the underground rhizome and root system and the quality of the bamboo shoots,at last greatly influences the overall economic benefits of bamboo forest.WRKY transcription factor family is one of the most important transcription factor families in plants,which is widely involved in the response of plants to stress and the regulation of growth and development of plants.In this study,the WRKY gene family of moso bamboo was re-analyzed based on the second edition of the published genome of Phyllostachys edulis.On this basis,the relationship between WRKY gene family and leaf senescence regulation was discussed.Some WRKY genes that may regulate leaf senescence were found out and their functions were preliminarily identified.The results of this study are as follows:1.Based on the published second edition of the genomic data of moso bamboo,189 WRKY genes were screened.Compared with the first edition of the reported moso bamboo WRKY gene family,78 new WRKY genes were added,which greatly expands the number of WRKY gene family genes of Moso bamboo.2.The moso bamboo WRKY gene family was analyzed based on the protein sequence homology and the phylogenetic tree.According to the results,all the identified WRKY family proteins were divided into eight groups: In,Ic,IIa,IIb,IIc,IId,IIe and III.After analyzing the gene structure,conservative motif distribution and the 3D structure of the WRKYGQK protein domain of different groups of the WRKY gene family,it was found that the members of WRKY family in each group have different protein structure characteristics compared with other groups,which was consistent with the classification results of the phylogenetic tree.3.The promoter sequences of the WRKY family genes of Moso bamboo were analyzed.It was found that the promoter activities of these genes can be induced by many hormones(abscisic acid(ABA),methyl jasmonate(Me JA),salicylic acid(SA),auxin and gibberellins(GA))and exogenous abiotic stress factors(such as mechanical damage,drought,low temperature,etc.)related to plant senescence at the same time.A part of the WRKY family genes in Moso bamboo have different gene expression levels between leaves with different senescence degrees or different senescence degree regions of the same leaf,which indicates that there is a certain correlation between the WRKY transcription factor family and the senescence regulation of leaves in Moso bamboo.4.Referring to the previously identified MbLSAGs(Moso bamboo senescence related genes)in our laboratory,combined with the WRKY gene family obtained from this study,three WRKY genes MbLSAG28,MbLSAG77 and MbLSAG96 were selected for further research.After a series of studies,it was found that all the three genes were expressed at a certain level in bamboo leaves,their subcellular localization are in the nucleus and they are sensitive to the treatment with IAA,Me JA and Na Cl.GUS histochemical staining results showed that these three genes are mainly expressed in the main vein of leaves in transgenic Arabidopsis thaliana plants,and the expression level changed with leaf senescence.Meanwhile,by using yeast two hybrid experiment,the result showed these three WRKY proteins might have interactions among each other.5、The overexpression vectors of the three genes were respectively constructed and ectopicly transformed into Arabidopsis thaliana plants.The phenotype of MbLSAG77-OE transgenic plants was emphatically observed.The seedlings of transgenic plants are more sensitive to Na Cl stress,the leaves are more premature than the wild type,and the plants are also bolting in advance.The result indicates that MbLSAG77 has significant correlation with leaf senescence regulation..Based on the above results,from the perspective of bioinformatic analysis of WRKY family and functional identification of MbLSAG77 gene regulating leaf senescence,this study preliminarily analyzed the correlation between WRKY transcription factor gene family and the regulation of Moso bamboo leaf senescence,which laid a certain foundation for further research on the regulatory mechanism.At the same time,this study also provides theoretical reference and excellent gene resources for the subsequent selection of anti-senescence varieties and selection of anti-senescence genes for molecular breeding and variety improvement of Moso bamboo plants.