Cloning Of Pectate Lyase Gene From Carica Papaya L.

Papaya, Carica papaya, is a member of the family Caricaceae. and one of the most important tropical and subtropical fruit trees. Because the papaya is very easy to soften or even rotten in a short period of time after mature, many scientists have been focusing on the study on how to improve the preserved quality of the papaya. To date, however, most of the researches were only concentrated on the ACC oxidase (ACO) and ACC synthesis (ACS), and no complete work has been done in relation to the enzymes which can play important roles in cell wall degradation and fruit softening. The Pectate lyase (PL) gene, that can resist softening, was cloned from the pulp of papaya, and its expression vectors of plant recombinant can be constructed for breeding new varieties which might have favorable preserving quality. The major results are summarized as follow:1. Development of a novel method for extraction and purification of total RNAAn improved TRIzol method was used for RNA extraction, by adding 10 % (V/V) absolute ethanol in the extraction buffer, extracting twice with chloroform, and precipitating RNA using LiCl. After comparied the methods of TRIzol with CTAB and SDS, a novel method for removing the impurities such as polyphenolics, polysaccharides and organic acids from Papaya pulps was developed, and the pure and intact RNA was obtained.2. Cloning of the consensus region of PL gene by RT-PCR.The 596 bp consensus sequences of PL gene which encodes a protein of 177 amino acid was cloned from cDNA reverse-transcripted from RNA. Blast showed that the Nucleotide sequence has high identities with that of PL gene of F.ananassa duch., Arabidopsis thaliana, Mangifera indica L and Vitis vinifera L.. The identities were 79ï¼…-82ï¼…3. Cloning of the sequence of 5' terminal in the PL gene by chromosome walking.The 1138 bp PL gene fragment, with a 721 bp ORF encoding 240 amino acid, was obtained by chromosome walking. Blast showed that it has high identities with that of...