Cloning And Functional Analysis Of Dehydrins In Prunus Mume

Mei （Prunus mume） belongs to the Rosaceae family. It is one of the most populous flowers in East Asia. Mei originated in the Yangtze River of China. Cold-hardiness breeding is important for mei utilization in the northern part of China. It has been demonstrated that dehydrins （also called group2late embryogenesis abundant proteins） play important roles in plant desiccation tolerance. In this study, we first did bioinformatics analysis to identify dehydrins in mei, and then cloned five dehydrin genes from P. mume ’Beijingyudie’ using RT-PCR method. At last, mei dehydrin genes were overexpressed in Escherichia coli and tobacco to analysis their function.1. The bioinformatics analysis of LEA gene family in P. mume:30LEA genes were identified from mei through genome-wide analysis. The PmLEA genes are distributed on all mei chromosomes except chromosome3. Twelve （40%） and four PmLEA genes are arranged in tandem and segmental duplications, respectively. The VmLEA genes could be divided into eight groups （LEA₁, LEA₂, LEA₃, LEA₄, LEA₅, PvLEA18, dehydrin and seed maturation protein）. Six PmLEA genes （PmLEA8, PmLEA9, PmLEA10, PmLEA19, PmLEA20, PmLEA29） were divided into dehydrin group. Ten gene conversion events were observed in four LEA groups, with most of them （70%） identified in dehydrin group.2. Cloning and expression analyses of dehydrin genes in P. mume:Five dehydrin genes （PmLEAs） were cloned from P. mume’Beijingyudie’using RT-PCR method. The entry vectors for PmLEAs were constructed using a TA method. The sequence of PmLEA8from P. mume’Beijingyudie’was longer than that from’Zangmei’genome database. The expressions of most genes were different among five organs （root, stem, leaf, flower, and fruit） except PmLEA29. The expression of most dehydrin genes was up-regulated under six abiotic stress treatments （cold, hot, drought, salt, ABA and SA treatment）, except PmLEA29under ABA treatment.3. Mei dehydrins confer the tolerance of E. coli:The E. coli expression vectors for dehydrin genes were established by LR recombination reaction. The mei dehydrins were expressed in E. coli BL21（DE3） after1mM IPTG induction. The results of a spot assay demonstrated that the expression of most mei dehydrins conferred the tolerance to the E. coli recombinant for cold and osmotic stresses.4. Transgenic analysis of dehydrin genes of P. mume:The dehydrin genes in entry vector was recombined into plant expression vector pEG203through LR reaction and then transferred into tobacco NC89through Agrobacterium mediated leaf disc transformation. Twelve PmLEA20transgenic plants were identified through Basta selection and PCR test. These T1lines were used for germination analysis under low temperature and the relative germination rates of transgenic lines were higher than that of wild-type. The amount of electrolyte leakage in wild-type tobacco seedlings was higher than that of transgenic lines.The cloning and functional analyses of P. mume dehydrin genes pave the way for their future utilization in plant resistance breeding.