Cloning And Expression Analysis Of Thaumatin-like Protein Gene From Litchi (Litchi Chenesis Sonn.)

Litchi (Litchi chinensis Sonn.) is one of the important tropical fruit trees, but the commercial value of litchi fruit is often reduced by the postharvest pericarp browning and diseases, which limits the development of of litchi industry. To explore the mechanism underline the change of postharvest fruit quality of different cultivars would provide basis for the control of postharvest losses. Thaumatin-like Protein (TLP), a protein belonging to the family of pathogenesis-related protein PR5, was proved to have closed relationship with plant disease resistance, drought tolerance and so on. However, there is no report on the TLP from litchi. Investigation on the relationship between the expression pattern of TLP gene in pericarp and the pericarp browning will contribute to the utility of the gene on litchi breeding. Storabilities of several litchi varieties in Hainan were studied. The results showed that for storage abilitity, Ziniangxi was the best and Wuhe was the weakest. Process of fruit senescence was affected by postharvest treatment. Low temperature and high moisture could delay the aging process of litchi.The full-length cDNA sequence of TLP gene (LTLP) and the DNA fragments corresponding the ORF had been isolated from the three cultivars of litchi. There was no intron in LTLP, since the nucleotide sequence of DNA fragment containing the ORF of LTLP was the same as that of the cDNA fragment. Three varieties of LTLP gene sequence containing single nucleotide polymorphisms (SNP). The TLP cDNA coded a polypeptide of 223 aa in length. The deduced polypeptide of LTLP had high identities with the amino acid sequence of TLP from Ricinus communis、Populus trichocarpa、Actinidia deliciosa and so on. Southern hybridization revealed that the gene was really from litchi and had two copies in litchi.Onion epidermal gene gun bombardment transient expression of GFP fusion gene showed that the gene expression in extracellular.A 2103 bp upstream promoter region of LTLP had been isolated. The transcription start site of LTLP promoter was within the 2003-2053 bp from the star codon. TATA box and CAAT boxes, which was the core promoter region in the region.,located in 21bp and 99bp upstream of the ATG, respectively. LTLP promoter contained ABRE, CGTCA-motif, HSE, TCT-motif and other components. The deleted plant expression vectors were constructed to identify the function of different promoter fragments. LTLP gene expressed in litchi pericarp, fruit, flower, stem, leaves and seed. LTLP expresses in pulp, stems, leaves, flowers and fruits. The expression level was the highest in flowers and lower in. pulp, stems and leaves.Along with the fruit development and senescence, LTLP gene expression was first increased and then decreased. Browning index of litchi pericarp increased at room temperature, LTLP gene expression was first increased and then decreased. Among the three cultivars, Ziniangxi had the highest expression level of LTLP, indicating that the differences of pericarp browning rate among different cultivars with different storage ability were negatively correlated with LTLP gene expressions. Expression of LTLP in preicarp was enhanced by fungal infection and reduced water loss in pericarp, however, was repressed by low temperature treatment. All the results indicated that the expression of LTLP was related with pericarp senescence.