木薯MeMLO12基因克隆及其CRISPR-Cas9表达载体的构建OA北大核心CSTPCD

MLO gene is an unique negative regulatory factor for disease resistance in plants,and the mutation in the gene can lead to broad-spectrum disease resistance in plants.In this study,a DNA and cDNA sequence of cassava MLO12 gene were cloned from the entire cassava genome and named MeMLO12.This gene has a total length of 3743 nt and a coding region(ORF)of 1728 nt,with a complete open reading frame containing 15 exons and 14 introns,encoding 586 amino acids.The protein has a molecular weight of 67.2 kDa and an isoelectric point of 8.85.MeMLO12 protein is lo-cated on the endoplasmic reticulum membrane,without signal peptides,and forms seven transmembrane domains at 23-45,74-96,161-183,285-307,312-334,371-393,413-435 aa.Quantitative analysis by qRT PCR revealed signifi-cant differences in the expression of MeMLO12 gene in resistant and susceptible cassava germplasms after infection with Xanthomonas axonopodis,indicating a negative regulatory effect in the interaction between cassava andXam.Se-lecting the 11th exon of the gene for Snap Gene Viewer analysis,10 455 seed sequences of sgRNA were obtained.Three target sequences of approximately23nt were selected,with a G terminus at the 3'end of the base composition,and they were constructed onto the CRISPR-Cas9 vector.After verification,it was confirmed that the three target sequences of MeMLO12 have been successfully constructed onto the gene editing vector,named pSGR-Cas9-AT-MeMLO12 vector.