Preliminary Exploration On Mechanism Of Lignin Metabolism Related Genes Of Pomelo During Fruit Development Stages

Lignin is an important product of phenylpropane metabolism and an important component of plant cell wall.It plays an important role in maintaining the normal development of plants,enhancing the overall mechanical strength of plants,and increasing the ability of plants to resist stress.At the same time,juice cell granulation usually occurs at pomelo fruit development,the content of lignin is also an important index to measure the pomelo fruit development and juice granulation.Phenylalanine ammonia lyase(PAL),4-coumaric acid Co A ligase(4CL)and Cinnamyl alcohol dehydrogenase(CAD)play key roles in the synthesis of lignin.In order to explore the role of PAL,4CL and CAD in lignin metabolism of pomelo fruit.In this study,we measured the content of lignin and the changes of enzyme activities in juice cells in different part of ‘Sanhongmiyou' and other varieties of pomelo during different stages of growth and development;target genes were cloned by reverse transcription PCR,proteins encoded by these genes were analyzed by series of bioinformatic methods;meanwhile we used real-time quantitative PCR to analyzed the expression patterns of target genes in ‘Sanhongmiyou' and other varieties pomolo during theirs growth and development stages;The potential function of relevant gene members in the lignin synthesis pathway of pomelo was preliminarily explored.The main results are as follows:1.We measured the content of lignin in three varieties of pomelo and different juice sacs of 'Sanhongmiyou' pomelo during fruit development satges.Results showed that there were significant differences in lignin contents among three varieties of pomelo.At the early stage of fruit development,there was no significant difference in lignin content between two kinds of juice sac,the contents of lignin increased significantly in the late stage of fruit development,The increase of lignin content in juice sac near the column which prone to granulation was larger than that in juice sac sway the column.2.By measuring the changes of enzyme activities in three varieties of pomelo and different juice sacs of 'Sanhongmiyou' pomelo during fruit development stages.Result showed that the enzyme activities of PAL,4CL and CAD in three pomelo varieties were different,which indicated that the degree of lignification in different varieties of pomelo were different either;there were two peaks of PAL activity in the fruit development of ‘Sanhongmiyou' pomelo,and the trends of PAL activity in juice sac near the column and away the column were different;the change trends of 4CL enzyme activity in juice sac near the column and away the column were different,the enzyme activity of 4CL peaked at 180 days after flowering;the changes of CAD enzyme activity in the two parts of juice were similar,and the peak appeared 180 days after flowering;after the peak activity,the activity of three enzymes were decreased,however,the activity of three enzymes increased in the late stage of fruit development,the enzyme activities of three enzymes in juice sac near the column were higher than those in away the column,it is suggested that the fruit juice sac near the column may be more likely to accumulate lignin and lignify.3.According to the genome and transcriptom data,through Blast N,gene annotation,NCBI website conservative domain comparison tool and FPKM value heat map analysis we screened three Cm PAL,five Cm4 CL and three Cm CAD gene members,proteins encoded by these genes were analyzed by series of bioinformatic methods,The results showed that the promoter regions of Cm PAL,Cm4 CL and Cm CAD genes contain elements that respond to light,hormone and stress,the proteins encoded by Cm PAL,Cm4 CL and Cm CAD gene members have typical conserved domains of their gene family,respectively.They are relatively conservative in structure and also have transmembrane helix structure and phosphorylation sites.The results of phylogenetic tree show that three Cm PAL members belong to dicotyledonous plant group,which closest to Cs PAL;Different species 4CL and CAD divided into several groups,Cm4CLl4 and Cm CAD4 were closely related and belong to the same group of key members involved in the regulation of lignin synthesis,respectively.It indicates that they may have similar functions.4.We used real time quantitative PCR to study the relative expression of the related gene members in the fruit development stages of pomelo,the results showed that the expression of Cm PAL,Cm4 CL and Cm CAD gene members were different in three varieties of pomelo during fruit development stages;the relative expression level of Cm PAL gene members increased gradually in ‘Sanhongmiyou' pemelo at fruit development stages,expression levels in juice sac near the column were significantly higher than that in away the column at the middle and late stage of fruit development,there was significant correlation between the expression of three Cm PAL genes and lignin content;the expression patterns and abundances of Cm4 CL and Cm CAD gene members were different;the expression levels of Cm4CL4 and Cm CAD4 increased significantly in the late stage of fruit development and both of them were significantly correlated with lignin content.It is speculated that the specific Cm PAL gene members are widely involved in the lignin synthesis of pomelo,and Cm4CL4 and Cm CAD4 may play a more important role in the lignin biosynthesis of pomelo fruit.