Study On Lignification Of Pleurotus Eryngii Stored At Low Temperature And Cloning And Expression Analysis Of Key Enzyme Genes Involved In Lignin Biosynthesis

The king oyster mushroom(Pleurotus eryngii) has unique delicious taste and high nutritional value. In the past decade, the commercial production of Pleurotus eryngii has grown rapidly. Under refrigerated conditions, Pleurotus eryngii will occur several texture deterioration phenomenons, such as the increase of hardness, soft and hard to chew, resulting in the loss of the unique crisp taste of the mushroom, and causing the decline of nutrient content, which will seriously affect the storage quality of mushroom. In this research, we studied the texture variation of Pleurotus eryngii stored at low temperature to explore the possible inducor of Pleurotus eryngii texture deterioration, and cloned key enzyme genes involved in lignin biosynthesis to investigate their expression level in Pleurotus eryngii lignification process. The aim of this research is to explore mechanism of Pleurotus eryngii postharvest lignification at the molecular level. The results are as follows:(1) Characteristics of texture change of Pleurotus eryngii stored at low temperatureFresh king oyster mushrooms, packed in low-density polyethylene bags, were stored at different low temperatures for 18 days to measure textural attribute. It was found that toughening occurred twice during the entire storage time. Highly associated change profiles were observed for the lignin content and activities of three important enzymes involved in lignin synthesis. The chitin and cellulose contents exhibited low correlation with toughening. Our results suggested that toughening may be mainly caused by lignification and can affect the postharvest quality of the tested mushrooms.(2) Cloning of key enzyme genes involved in lignin biosynthesisUsing RT-PCR and RACE techniques, we successfully cloned the full-length c DNA sequence of a pal gene(named Pe Pal)and partial sequences(include 3' terminal sequences) of four 4CL genes(named Pe4cl1, Pe4cl2, Pe4cl3 and Pe4cl4, respectively), and one POD gene(named Pe Pod)from Pleurotus eryngii. The sequence of Pe Pal consists of 2441 bp with an intact open reading frame of 2229 bp, encoding a polypeptide of 743 amino acids. The length of Pe4cl1?Pe4cl2?Pe4cl3?Pe4cl4 and Pe Pod sequences that cloned is 1898 bp?1620 bp?1406 bp?1012 bp and 754 bp,respectively.(3) The expression pattern of key enzyme genes at different stages in Pleurotus eryngii stored at low temperatureThe expression patterns of these cloned key enzyme genes was detected at different stages in Pleurotus eryngii stored at low temperature using quantitative Real-Time PCR(q RT-PCR). The results showed that Pe Pal?Pe4cl1 and Pe4cl3 genes had similar expression pattern in storage time(up- down- up- down); and the expression levels of Pe Pal?Pe4cl1?Pe4cl3 and Pe Pod genes showed similar trends with POD and PAL enzyme activity, toughness and lignin content curve in the first 12 days of storage at different storage temperatures. Therefore, we hypothesized that Pe Pal,Pe4cl1, Pe4cl3 and Pe Pod gene expression may be induced by low temperature and other environmental factors, and they played an important role in Pleurotus eryngii lignification process under low temperature storage.