Screening And Identification Of Banana Fruit Softening Related Pectate Lyase Gene And Determination Of Its Upstream Regulatory Factors

Banana is a nutrient-rich fruit and a very important food crop in the world.It has important economic value.In recent years,more and more studies have shown that pectate lyase is closely related to fruit softening,and affects the storage and preservation of postharvest fruits.However,little is known about the functions and regulatory mechanisms of banana pectate lyase.In order to identify pectate lyase genes that related fruit maturity in banana and determine major upstream regulatory factors of key ripening-related pectate lyase genes.Firstly,the genetic structure,conserved domains,and phylogenetic relationship of the PL gene family in the banana A genome were systematically analyzed;Secondly,we identified the fruit ripening related PLs by quantitative PCR,and examined its subcellular localization;Third,we obtained two PLs mutated transgenic plants using CRISPR/Cas9 gene editing system;Finally,the yeast one-hybrid system was used to screen and identify the upstream regulatory factors of the two PL genes,and the interaction relationship was further clarified through point-to-point yeast one-hybrid and dual luciferase reporter assay.The results showed that:1)the members of the banana pectate lyase family were distributed on every chromosome in the banana genome,with 4 PLs on chromosome 6,3 PLs on chromosome3,and 4 PLs,2 PLs on chromosome 7 and 10 respectively,and 1 pectate lyase gene on other chromosomes.Analysis of the conserved domain of the PL gene showed that each PL protein contained predicted structures related to pectate lyase activity,such as the Pectate lyase domain,Amb＿all structure,and Parallel beta-helix repeats structure etc,indicating that it may have pectate lyase activity;2)Real-time quantitative PCR results showed that the expression of PL3.1 and PL11 were highly induced by ethylene,suggesting its ripening-related role in banana;3)Subcellular localization analysis reveals distribution of two pectate lyase enzyme proteins on nuclear and plasma membranes;4)Through yeast one-hybrid screening,3 WRKY transcription factor genes(Ma03＿t31070.1,Ma04＿t24790.1,and Ma06＿t34370.1)were determined and considered as upstream regulators of key PL genes;Point-to-point yeast one-hybrid hybrid and dual luciferase report experiments confirmed their interaction,and the expression of the three WRKY transcription factor genes showed an upward trend during fruit ripening5)Through Agrobacterium-mediated genetic transformation,we obtained 20 gene editing lines for both PL3.1 and PL11,and each lines have gene editing events vary with different pattern by common PCR amplification and regular sequencing analysis.Deep sequencing analysis for two of PL11-mutated lines shown that three alleles of one line are mutated,which means complete loss of function for PL11.In summary,all above results indicate that: 1)PL3.1 and PL11 might play the main role of pectate lyase in the softening of banana fruits,and its nuclear localization imply that they maybe have other functions except functioning on degradation of the cell wall;2)The PLs mutated lines are good research materials for further investigating the function of two PL genes and its ripening-related role in banana;3)The WRKY transcription factor might participate in banana fruit ripening process by regulating the expression of PL3.1 and PL11.These results are not only beneficial for deciphering molecular mechanism of PLs-mediated fruit ripening but also helpful for improving the molecular regulation network of banana fruit ripening,and lay the foundation for creating or breeding new banana germplasm that have high storage quality.