| Hami melon is an important cash crop in Xinjiang, however the rot during storage and transport caused a lot of economic losses in cantaloupe industry. One of the main reasons caused rot is fungi infection, so it is an important way to solve this problem by controling fungal pathogens. When faced with germs stress, Hami melon would start resistance related proteins to constitute the first line of defense against infection. Chitinase is an important pathogenesis-related(PR) protein. A variety of studies have showned that the chitinase has a positive effect in plant response to abiotic stress.Object: In this study, two chitinase genes in Hami melon associated with defense were cloned and various software were used to analysis its bioinformatics.To study changes of this two chitinase genes expression of Hami melon infected with Penicillium, and changes of enzyme activity, speculating the relationship between gene expression and enzyme activity. All of this provided not only a basis for the study of the molecular mechanisms of cantaloupe response to abiotic stress, but also a theoretical basis for the biological preservation of cantaloupe.Methods: According the previous transcriptome library of Hami melon, we selected chitinase genes for this study. Two chitinase gene fragments were cloned by RT-PCR. A variety of software were used to analyze the open reading frame of these two genes, and to deduced amino acids sequence, to predicte the physicochemical properties of the encoded protein, speculate the signal peptide, the secondary structure, subcellular localization, etc. Using RT-q PCR to analyze the expression levels of these two genes after Hami melon infected with Penicillium. And chitinase activity was determined by reducing sugar assay.Results and Conclusion:(1) These two sequences were named as Hm CHI and Hm CHT-2, accession numbers KX083345, KX083346 were obtained with submited to NCBI Genbank database. Length of Hm CHI was 1153 bp, and it had the largest open reading frame of 445 nucleotides, encoding 145 amino acids, which was a Class III chitinase gene belonging to familie 18. The molecular weight of th protein was 15.04 ku, theoretical isoelectric point of 8.82, a hydrophobic protein. There was a signal peptide, and it was non-transmembrane protein, located in extracellular. The length of Hm CHT-2 was 1011 bp, and its largest open reading frame was 936 nucleotides, encoding 312 amino acids, which was a Class Ia chitinase gene belonging to familie 19 after analyzed by CDs. Molecular weight of encoded protein was 33.81, the theoretical isoelectric point of 8.42, hydrophilic protein with a signal peptide and non-transmembrane domain, located in the vacuole.(2) The results of fluorescence quantitative showed that expression of Hm CHI continued to rise after Penicillium infection, until reached the maximum at the 60 hours, and the expression level was significantly higher than the control, high expression level last longer. Fluorescence quantitative results showed that expression of Hm CHT-2 after infection overall upward.In the first 48 hours, the expression rose slowly, at the 60 hour, the expression level increased rapidly, and reached maximum at 72 hour. The expression peak of the experimental group appeared later and the increase rate is much greater than control.The expression of two genes in the control group reached the maximum at the 24 hour, then declined, which indicated that Hm CHI and Hm CHT-2 both played an important role in the immune systerm response to Penicillium infection in Hami melon, and Hm CHI precede Hm CHT-2, but the increase rate of Hm CHT-2 was greater than Hm CHI.(3) Activity assay results indicated that chitinase activity increased rapidly after inoculation, and reached the maximum at 48 hour. Howevere, the control group continued to rise slowly, and was always lower than the experimental group. It showed that Penicillium infection could induce chitinase activity’s changes. Penicillium infection induced up-regulation of chitinase genes in Hami melon, and enzymatic activity increased. The enzymatic activity was similar to the trend of expression of chitinase genes. |
