| Luffa cylindrica is an a member of the Cucurbitaceae family,and it is native to tropical and subtropical regions.With a long history of evolution and selection,it has a strong adaptability.And it is now widely cultivated both in northern and southern China.WRKY transcription factors play a crucial role in a wide range of physiological processes as well as stress response mechanisms in plants.On the basis of whole genome sequencing data of Luffa cylindrica,the WRKY gene family was identified and analyzed,as well as LcWRKY34,which was involved in temperature stress responses.Using an Agrobacterium-mediated genetic transformation system,Arabidopsis thaliana LcWRKY34 was validated for function.The main results were as follows:1.57 members of LcWRKY family were identified in Luffa cylindrica.The LcWRKYs were divided into three groups(Group Ⅰ-Ⅲ),in which group II was further divided into five subgroups(Group Ⅱa-Ⅱe).It was evident that each LcWRKY protein had a highly conserved WRKYGQK and its characteristic zinc finger structure.In terms of gene structure and conserved motif,WRKY from the same group or subgroup showed some similarity.2.The LcWRKY gene expression was tissue-specific,with leaves and flowers showing high expression of LcWRKY40 and LcWRKY57.After low temperature treatment,14 LcWRKYs were up-regulated and one LcWRKY(LcWRKY39)was down-regulated,and LcWRKY7 and LcWRKY3 were more highly expressed.Under high temperature,expression of one LcWRKY(LcWRKY54)was up-regulated while expression of 17 LcWRKYs were down-regulated.3.In the LcWRKY34 sequence analysis: the full length of theopen reading frame of the LcWRKY34 was 867 bases,encoding 288 amino acids,with the protein molecular weight of31.08 k Da and an isoelectric point of 9.62.The N-terminus of LcWRKY34 contained a high conservation WRKY structural domain,while the C-terminus contains a zinc finger structure of the C2H2-type,indicating that LcWRKY34 was a member of group II.Based on blast comparisons,LcWRKY34 and cucumber,melon,and winter squash had over 90% amino acid sequence similarity.4.The p BWA(V)BS-LcWRKY34 overexpression vector was constructed using LcWRKY34 as the target gene,and infestation with Agrobacterium mediated transformation of Arabidopsis thaliana.A resistance screen was conducted on harvested seeds in order to obtain overexpressing LcWRKY34 plants.5.The relative conductivity of the overexpressing Arabidopsis strain was higher than that of wild-type plants,and the MDA and soluble protein contents were lower than that of wild-type plants,indicating that the overexpressing strain showed more severe membrane lipid peroxidation and cell membrane damage under high temperature stress treatment.The enzyme activities of the overexpression strain were significantly lower than those of wild-type plants,indicating that the overexpression strain had less antioxidant capacity under high temperature stress conditions.6.The relative conductivity of wild-type plants were higher than that of the overexpressing Arabidopsis strain,and the MDA and soluble protein contents were higher than that of wild-type plants,indicating that the overexpressing strain showed less membrane lipid peroxidation and cell membrane damage under low temperature stress treatment.The enzyme activities of CAT,POD and SOD in the overexpression strain were significantly higher than those of wild-type plants,indicating that the overexpression strain had a stronger antioxidant capacity under low temperature stress conditions. |
