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

MLO基因是植物中特有的一类抗病性负调控因子,该基因突变导致植物产生广谱抗病性。本研究从木薯全基因组中克隆获得木薯MLO12基因的DNA和cDNA序列,并将其命名为MeMLO12。该基因全长3743 nt、编码区(ORF)全长1728 nt,具有一个完整的开放阅读框,含有15个外显子和14个内含子,编码586个氨基酸,蛋白的分子质量为67.2 kDa,等电点为8.85。该基因编码的蛋白定位在内质网膜上,无信号肽,在23~45、74~96、161~183、285~307、312~334、371~393、413~435 aa处形成7次跨膜结构域。qRT-PCR定量分析发现,受木薯黄单胞病菌侵染后,MeMLO12基因在木薯抗、感品种中的表达量存在明显的差异,参与木薯与黄单胞菌之间的互作,表现出负调控作用。选择该基因第11个外显子进行Snap Gene Viewer分析,获得了10 455条sgRNA的种子序列,从中选取3条靶序列约23 nt,碱基组成上3'末端含G结尾,将其构建到CRISPR-Cas9载体上,经验证,确认MeMLO12的3条靶序列已经成功构建到基因编辑载体上,将其命名为pSGR-Cas9-AT-MeMLO12载体。

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 and Xam.Se-lecting the 11th exon of the gene for Snap Gene Viewer analysis,10455 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.