热带作物学报2024，Vol.45Issue(8)：1528-1537,10.DOI:10.3969/j.issn.1000-2561.2024.08.002
木薯MeMLO12基因克隆及其CRISPR-Cas9表达载体的构建
Cloning of Cassava MeMLO12 Gene and Construction of Its CRISPR-Cas9 Expression Vector
摘要
Abstract
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.关键词
木薯/MeMLO12基因/克隆/表达分析/CRISPR-Cas9载体/构建Key words
cassava/MeMlo12 gene/cloning/expression analysis/CRISPR-Cas9 vector/construction分类
农业科技引用本文复制引用
蔡吉苗,李博勋,黄贵修,李超萍,时涛,王国芬..木薯MeMLO12基因克隆及其CRISPR-Cas9表达载体的构建[J].热带作物学报,2024,45(8):1528-1537,10.基金项目
国家木薯产业技术体系项目(No.CARS-11). （No.CARS-11）
