| Pear is one of the major temperate fruit crops in the world with a production over 23.7million tons in 2018.In our country,the planting area and yield of pear are second only to citru and apple,making it the third largest fruit tree.For a long time,pear lacks a stable genetic transformation system,which seriously hinders the research on functional genomics and molecular breeding of pear.By inducing pear calli,some genes function can be verified quickly and reliably.This work aims to establish the CRISPR/Cas9 gene editing system and ChIP-Seq system using European pear calli,and provide a technical platform for functional genomics study and molecular breeding of pear.At the same time,we hope to use ChIP-Seq technology to discover the downstream target genes of DAM genes(PpDAM2,PpDAM3,PpDAM4),so as to further reveal their functions.The specific work and main results are as follows:1.Establishment of dual-cut CRISPR/Cas9 gene editing system in pear calli:We edited the GUS gene in pro DAM3:GUS transgenic pear calli using a dual-cut strategy.Based on the sequence characteristics of the GUS gene,two sg RNAs were designed to target the second exon of the GUS gene.The corresponding GUS-p YLCRISPR/Cas9 plasmids were constructed and transformed into the pro DAM3:GUS transgenic calli by Agrobacteriummediated transformation.The results from sequencing data revealed that 4 out of 6 independent transgenic lines carried mutations at the target sites,with mutation rates of 66.7%.We further analyzed the mutation types of the 4 GUS editing lines,and found that #1,#2,#3 lines showed deletion and insertion of nucleotides,while #5 lines showed deletion of large fragments between two target sites,whereas no nucleotide substitution was observed in any edited line.GUS staining showed that the control calli was blue,while the 4 gene editing lines were completely white,indicating that the CRISPR-Cas9 system was a powerful and precise method to induce targeted mutagenesis in pear calli.2.High throughput screening of DAM target genes in pear calli:The ChIP-Seq system was eatablished in pear calli to discover the potential target genes of DAM genes(PpDAM2,PpDAM3,PpDAM4).The results showewd that PpDAM2 had peaks in the promoter regions of Pc PEPKR1,Pc RS/Pc SIP,Pc AHL,Pc HSFA,Pc SAE2,Pc SYP7.PpDAM3 had peaks in the promoter regions of Pc PEPKR1,Pc RS/Pc SIP,Pc CHS,Pc RABA1 G,Pc DUF567.PpDAM4 had peaks in the promoter regions of Pc PEPKR1,Pc CHS,Pc SYP7.The above-mentioned genes may be potential target genes of PpDAM2,PpDAM3 and PpDAM4.In this work,the CRISPR/Cas9 dual-cut gene editing system and ChIP-Seq system were successfully established,and provide a technical platform for functional genomics study and molecular breeding of pear.At the same time,we successfully discovered the downstream target genes of DAM genes by using ChIP-Seq technology,which laid a foundation for further elucidating the molecular mechanism of DAM genes regulating dormancy process. |
