Cloning And Functional Analysis Of Genes Involved In Flesh Linification Of Loquat Fruit

Loquat (Eriobotrya japonica) is a typical fruit in China. Red-fleshed loquat tend to exhibit lignification phenomenon during postharvest storage which seriously influences fruit eating quality. Here,’Luoyangqing’loquat was chosen as test material and heat shock (HT), low temperature conditioning (LTC) and 1-Methylcyclopropene (1-MCP) treatment were used to examine the changes of related indexes during lignification, including lignin content, enzyme activity and gene expression. EjLIMwas also isolated and its participation in loquat fruit lignification was preliminarily proved. The main results are as follows:1. HT, LTC and 1-MCP treatment were effective for loquat fruit lignification. The activity of up-stream enzymes of the phenylpropanoid pathway, including L-phenylalanine ammonia-lyase (PAL), Cinnamate 4-hydroxylase (C4H) and 4-coumarate:coenzyme A ligase (4CL) were investigated. It’s found that the activity of these enzymes were positively correlated with loquat fruit lignification and suppression of these increases by HT, LTC and 1-MCP treatment can significantly alleviate loquat fruit lignification, which suggesting the important roles of PAL, C4H and 4CL in lignification.2. Gene cloning and expression analysis of related coding genes. Based on information from an RNA-seq database, eight new genes were subsequently isolated and were designated as EjC4H1-2 and Ej4CL6-11, respectively. EjPAL1, EjPAL2, Ej4CL1, Ej4CL6 and Ej4CL8 were proposed as key candidates for loquat fruit lignification while EjC4H1-2 showed no correlations with lignification. Expression of Ej4CL1 was found to be the most responsive to low temperature and HT, LTC treatment can significantly inhibit its expression in chilling-injured fruit. Furthermore, Ej4CL1 expression in senescent fruit can also be significantly suppressed by 1-MCP treatment. The function of Ej4CL1 was analyzed by transient over-expression in tobacco leaves, where it stimulated lignin accumulation.3. Identification and analysis of lignification-related transcription factor EjLIM. EjLIM can participate in lignification by activating the promoter of AtPALl and At4CL1 in Arabidopsis. Similarly, it can also enhance the promoter activity of loquat EjPAL1 and Ej4CLl gene. However, transient over-expression of EjLIM in tobacco leaf didn’t result in significant accumulation of lignin content, indicating that the mechanism of EjLIM participating in lignification of loquat may need further exploration.