Cloning And Functional Study Of WRKY Transcription Factor Gene From Narcissus Tazetta Var.'Yunxiang'

Chinese Narcissus(Narcissus tazetta var.chinensis)is one of the ten most famous traditional flowers in China.But the root of Chinese Narcissus is the fleshy tap root and requires a large amount of water during the growing period,and the good farmland that can be used for planting narcissus is greatly reduced.Therefore,it is of great significance to study Chinese narcissus in response process to stress of drought and salt stress,and to broaden the use of narcissus and enlarge its planting area.This study is mainly divided into three parts:the first part is the clone of the WRKY gene related to the abiotic stress of Narcissus tazetta var.'Yunxiang'.The second part mainly tests the tissue-specific expression,temporal and spatial expression,and the expression characteristics of exogenous plant hormone treatment and abiotic stress treatment of the WRKY gene in Narcissus tazetta var.'Yunxiang'.The third part is the preliminary analysis of the drought resistance and salt tolerant ability of the transgenic tobacco with the expression of the WRKY gene in Narcissus tazetta var.'Yunxiang'.This study provides a theoretical basis for the breeding of new varieties of narcissus with high resistance.During this study,we have achieved the following results:1.This study based on the early stage of the research on Narcissus tazetta var.'Yunxiang' different flowering petals and corona transcriptome database,using RT-PCR technology cloned NtWRKYY1(GenBank accession No.KX056495)and NtWRKYY2(GenBank accession No.KX056496)genes,carries on the preliminary analysis of the bioinformatics.The basic physicochemical properties of amino acids showed that NtWRKYY1 protein was an acidic and unstable hydrophobic protein,and NtWRKYY2 protein was an alkaline and unstable hydrophilic protein.These two proteins do not belong to secretory proteins and transmembrane proteins,which the phosphorylation of predominantly serine-modified,and supplemented by serine or tyrosine.Subcellular localization prediction indicates that these two proteins are more likely to be located in the nucleus.Sequence alignment and phylogenetic tree analysis showed that NtWRKYYl gene belongs to ?c sub-group of WRKY transcription factors,NtWRKYY2 gene belongs to II d sub-group of MWRKY transcription factors.2.Quantitative real-time PCR(RT-PCR)analysis demonstrated that NtWRKYY1 and NtWRKYY2 in Narcissus tazetta var.'Yunxiang' had a much significantly higher expression in flowers than in roots and leaves,with obvious tissue specificity.Temporal and spatial expression analysis indicates that NtWRKYY1 and NtWRKYY2 gene transcription level in petals and corona basic present gradually rising trend,suggesting that NtWRKYYl and NtWRKYY2 genes may be involved flower senescence process in Narcissus tazetta var.'Yunxiang'.The expression characteristics of exogenous plant hormone treatment of leave in Narcissus tazetta var.'Yunxiang' showed that the NtWRKYYl gene was inhibited by ABA induction and MeJA,while the NtWRKYY2 gene was induced by ABA and MeJA.The expression characteristics of abiotic stress treatment of leave in Narcissus tazetta var.' Yunxiang' showed that the NtWRKYYl and NtWRKYY2 gene may be induced by high temperature,drought and salt.3.We constructed that overexpressing vector pMDC140-NtWRKYY1 and pMDC140-NtWRKYY2 using In-Fusion cloning technique,and transformed into tobacco by the method of Agrobacterium though leaf disc transformation,the carrier of PCR and GUS staining results indicated the resistant plantlets were positive,respectively to obtain the overexpression of the NtWRKYYl gene T0 tobacco 3 strains,and the overexpression of the NtWRKYY2 gene T0 tobacco 8 strains.Harvest T0 generation seedling after seed sowing for T1 generation tobacco.The drought and salt stress treatment of T1 generation tobacco showed that overexpression of NtWRKYY1 and NtWRKYY2 transgenic tobacco showed less wilt and chlorosis than the wild type plants.Studies have shown that overexpression of the NtWRKYYl and NtWRKYY2 gene remarkably increased the drought and salt tolerance in transgenic tobacco.This study offers an alternative gene for drought and salt tolerant transgenic breeding of Narcissus.This study offers an alternative gene for drought and salt tolerant transgenic breeding of Narcissus.At the same time,for further screening of WRKY candidate genes into Narcissus,and establish work foundation to foster strong drought resistance and salt resistance and wide adaptability.