Abstract: 【Objective】 The objective of this study is to clone and characterize GhPPO1 from cotton (Gossypium hirsutum), then to study the dynamic changes of GhPPO1 mRNA expression level and PPO activity induced by Helicoverpa armigera in order to clarify the function of this gene in defensing response in cotton. 【Method】 A fragment of PPO gene which has an obvious response to insect feeding from cotton SSH library was got. Specific primers were designed to conduct the 5′RACE reaction. After sequencing and assembly, homologous retrieval was carried out in the NCBI cotton dbEST database and an EST (GenBank accession number: DR461072.1) which have a 410 bp overlap with targeted PPO gene were found. After assembly and electronic extension by DNAstar software, the complete sequences of cotton PPO gene, named GhPPO1, was got. To verify the sequence authenticity of GhPPO1, primers were designed at the two ends of the gene. Using genomic DNA as template, PCR was done to check whether there exist introns in GhPPO1. After verification, the tool of BLASTX was used to analyze the sequence homologous and ClustalW software was used to do multiple sequence alignment and MEGA 4.0 was used to construct a phylogenetic tree and other softwares including ANTHEPRO5.0, ExPASy and InterProscan were used to predict the existence of functional sites and physicochemical properties in the speculated coding regions. The GhPPO1 mRNA expression level and PPO activity in cotton leaves were detected by real-time quantitative PCR and spectrophotometric method, respectively. 【Result】 The complete cDNA sequence of GhPPO1 is 2 022 bp, with a 1 797 bp open reading frame which encodes 598 amino acids (M= 67.18 kD and pI= 6.11). The 5′ and 3′ untranslated regions were 102 and 123 bp, respectively. There were no introns in this gene. In the amino acid coding sequence, there were CuA and CuB binding sites, 3 N-glycosylation sites, 8 N-nutmeg acylation sites, 6 protein kinase C phosphorylation sites, 8 casein kinase Ⅱ phosphorylation sites, 1 cAMP and cGMP dependent protein kinase phosphorylation site and 1 amidation site. In the CuA and CuB binding regions, there existed the histidine and cysteine which play a key role in PPO protein. The similarity of PPO protein between cotton and other plants were more than 50%, however, the relationship of GhPPO1 protein was relatively far away with other kinds of PPO proteins. After mechanical damage, cotton leaf GhPPO1 expression showed a trend of rise and fall. The expression level was the highest at 6 h and the maximum fold change was 3.29 times compared with un-treated leaves. GhPPO1 mRNA level showed a variable trend which was increased firstly and then decreased and then increased at last after 1st late instar cotton bollworm larvae feeding. Twelve hours after feeding, the expression reached the highest level which was 6.04 times higher than the un-treated leaves. However, the mRNA expression level was lower in cotton bollworm feeding leaves than in mechanically injured ones at both 3 h and 6 h. PPO activity showed an increasing trend after both insect feeding and mechanical wounding treatment. The enzyme activity was lower in former treatment. Compared with the untreated cotton leaves, the mRNA expression level and enzyme activity significantly up-regulated after mechanical injury and water jointly treated leaves. However, there were no significant difference between the oral secretion treated and untreated cotton leaves in both GhPPO1 mRNA expression level and PPO activity. It indicated that oral secretion from cotton bollworm larvae might have a suppression role in both GhPPO1 mRNA expression and enzyme activity.【Conclusion】The full-length cDNA sequence of GhPPO1 which plays an important role in defensing cotton bollworm was cloned from cotton. It is speculated that some inhibitors may exist in the oral secretion of cotton bollworm larvae, which may cope with the defense system induced by insects.

Key words: Gossypium hirsutum , polyphenol oxidase , gene cloning , mRNA expression level , PPO activity