Analysis Of MiRNA Expression Profiling Related Autotoxicity And Mechanism Of Some Autotoxins In Cucumis Melo L.

Continuous cropping obstacles is very serious in melon cultivation and autotoxicity is one of the main reasons.Root exudates are the main source of autotoxins in melon.The continuous accumulation of related components in soil is an important factor leading to autointoxication and continuous cropping obstacles.However,due to its complex composition,research of the molecular mechanism of root exudates in melon toxicity is very difficult.The regulatory role of miRNA in plant response to different stresses has been widely recognized.There is no report about the role of miRNAs related to melon toxicity.Salicylic acid(SA)and epicatechin(EC)are two important components of melon autotoxins and belong to phenolic acids and flavonoids respectively.In this study,the differentially expressed miRNAs related to melon toxicity were analyzed,melon varieties ’Xinyinhui’ treated by root exudates was used as the research materials.In order to further analyze partial important miRNAs in physiological and molecular biology ways,the content of flavonoids,total phenolics and anthocyanins and the activity of PAL in melon were measured after treated with 100μM salicylic acid and epicatechin respectively.The expression of key genes in the phenylpropane metabolic pathway was also measured in melon after treated with 100μM salicylic acid.The expression of some differentially expressed miRNAs after salicylic acid and epicatechin treatment was also measured at the same time.These results will explore the possible regulatory mechanisms of these important miRNAs against melon’s autotoxicity.The main conclusions are as follows.1、Differential expression analysis of miRNA related to melon autotoxicity mediated by root exudates.A total of 148 miRNAs were identified,among which 114 weredifferentially expressed.Eight miRNAs closely related to autotoxicity were selected for subsequent studies by bioinformatics analysis such as COG and GO enrichment.2、Effects of SA and EC treatment on the content of total phenolics,flavonoids,anthocyanins and the PAL activitiy.After SA and EC treatment,the contents of total flavonoids decreased.The content of flavonoids decreased significantly at 1 day after SA treatment,and then decreased and maintained at one similar low level.After EC treatment,the content of flavonoids was lower than that of control,but there was no obvious change.The content of anthocyanin decreased slightly after treatment with SA,and then decreased rapidly and maintained at a low level.After the EC treatment,the anthocyanin content decreased significantly except 1 day,and in other detected time showed an increasing trend,and the content was higher than control.The change of PAL activity was not obvious at 1d,and reached its peak at 2d,then decreased rapidly to normal.After the EC treatment,the change trend of PAL activity was similar to SA treatment,the peak appeared on the third day.3、Effects of SA treatment on expression of key genes closely related to autotoxicity in the phenylpropane metabolic pathway.After SA treatment,the relative expression of PAL increased continuously and reached the peak at 3d.The expression of C4H decreased at 1 day after treatment and significantly increased at 4 days.There was no obvious change in the relative expression of 4CL after treatment.The relative expression of CHS was rapidly increased at 1 day after treatment,and then slightly lower than the control,no significant difference was observed.CHI and F3H decreased significantly at the beginning of treatment,and then maintained a low level of expression.The relative expression of R2R3-MYB and WD40 increased slightly at the beginning of treatment,and then some fluctuation were observed,but both below control.In general,the expression of WD40 and CHI was significantly inhibited after SA treatment.There was a high correlation between WD40 and CHI.The expression of F3H,CHS and R2R3-MYB was significantly decreased with the prolongation of treatment time,and there was a high correlation.Especially,the relative expression pattern of R2R3-MYB and CHS are nearly the same.4、The relative expression pattern of several important miRNAs related to melon autotoxicity after SA and EC treatment.The relative expression pattern of different miRNAs after SA treatment was complicated.MiR171＿1 and miR396 were significantly increased at 1d after treatment,and decreased at 2d and 3d,but the former increased again at 4d and higher than that of control.The miR171＿1 and miRukn continued to decrease;miR172 had no obvious rule;miR398 could not be detected.MiR408 was lower in the first 3 days after treatment,and the increase rate increased rapidly at 4 days.The trend of different expression of miRNAs after EC treatment was relatively simple.MiR177,miR171,miR172 and miRukn decreased to a low level in the early stage of treatment,and 3d began to increase significantly.MiR396 continued to decrease after treatment and miR408 and miR398 had no obvious change.The above-mentioned miRNAs have positive or negative effects on the growth and development of plant roots,stress response and resistance.The relative expression of these miRNA can partially explain the phenomenon that slow growth and development of plant roots,weak resistance to stress and continuous cropping obstacles under the allelopathy stress.