Cloning And Expression Of Expansin Gene In Papaya Fruit

Papaya is a kind of typical respiration climacteric fruit, which has important theoretical and practical meanings to regulate its ripening and to study the mechanism of ripening. Expansin is a group of cell-wall-localized proteins which inducing extension of cell wall. Expansins have been shown to play an important role in softening of fruits. In recent, expansin have been studied in many fruit, such as banana, mango and litchi, but it has no report about expansin in papaya.PCR degenerate primers were used to amplify cDNA fragments in papaya fruit by RT-PCR, which were designed with reference to the conserved amino acid sequences of known expansins'amino acid sequences, a cDNA fragment of 531 bp was cloned, named as Cp-EXP1. The Cp-EXP1 cDNA encodes a polypeptide of 177 amino acids, has the conserved structure of expansin amino acid sequence. The result of blast suggested that Cp-EXP1 has 85.66% sequence simility with both Pc-Exp1 and Pc-Exp2, 84.67% with Md-Exp1 from apple and 79.92% with Cs-EXP1 from cumcuber.The primers were designed according to the conserved sequence of Cp-EXP1, then the 3'end fragment of Cp-EXP1 by 3'RACE was amplified. A cDNA fragment of 673 bp was cloned. The result of Genscan indicated that the 3'end of Cp-EXP1 included an integrated ORF and there were 206 amino acids.RNAs of the root,stem,leaf,flower and fruit were extracted, and reverse transcripted to cDNA as the templates for expression. The result of RT-PCR showed that Cp-EXP1 expressed in all of the five organs.In order to further analyse the expression in the different stages during the fruit ripening, RNA was extracted from papaya fruit in different mature period, and quantitative analysis of Cp-EXP1 gene expression was carried by using Real Time PCR method. It was suggested that expression of Cp-EXP1 was regulated by ethylene.It was the first time to clone the expansin gene in the papaya fruit and then analyzed the expression at different organs,which had important theoretical meaning to the relationships between expansin gene and the produce of ethylene and the mechanism of fruit ripening, and build a great base of papaya growth research.