玉米丁布葡萄糖苷-4-羟基甲基化酶基因bx12的克隆及其原核表达

Cloning and prokaryotic expression of the DIMBOA-Glc-4-OH methylase gene bx12 in maize

为阐明丁布葡萄糖苷-4-羟基甲基化酶在玉米抗蚜和抗螨中的作用,该研究以4个对蚜虫和叶螨抗性不同的玉米自交系为材料,克隆编码丁布葡萄糖苷-4-羟基甲基化酶的基因bx12,并进行了原核表达。结果表明:除自交系Mo17-476M外,其余3个自交系B73、ZaC546和Mo17-476W的基因组中都分离到含有由744个碱基所构成的bx12基因开放阅读框的目标DNA片段,都编码由247个氨基酸组成的同一种蛋白BX12P;该蛋白与参考基因编码的蛋白氨基酸序列的一致性为100%。生物信息学分析表明,BX12P是一种多功能酶,属于依赖于S-腺苷-L-蛋氨酸(SAM)的邻-甲基转移酶结构域超级家族,具有蛋白质二聚化功能、植物甲基转移酶功能和邻-甲基转移酶功能;该酶具有专一性地将丁布葡萄糖苷转化为4-甲氧基丁布葡萄糖苷的功能。利用克隆的bx12基因所构建的原核表达载体,经测序和通读结构鉴定,在大肠杆菌BL21(DE3)细胞中成功表达出了目的蛋白。

In order to elucidate the role of DIMBOA-Glc-4-OH methylase in the resistance of maize to aphid and mite,the gene bx12 encoding DIMBOA-Glc-4-OH methylase was cloned from four maize inbred lines with divergent resistance to aphids and tetranychids,and was prokaryotic expressed.The results showed that,except for inbred line Mo17-476M,the genomes of the other three inbred lines B73,ZaC546 and Mo17-476Wwere isolated to containthe target DNA fragment of the open reading frame of the bx12 gene composed of 744 bases,and they all encoded the same protein BX12P composed of 247 amino acids.The protein was 100%consistent with the amino acid sequence of the protein encoded by the reference gene.Bioinformatics analysis revealed that BX12P is a multifunctional enzyme,belonging to the S-adenosyl-L-methionine(SAM)-dependent o-methyltransferase domain superfamily,with protein dimerization,plant methyltransferase and O-methyltransferase functions.The enzyme has the function of converting DIMBOA-Glc to HDMBOA-Glcspecifically.The target protein was successfully expressed in Escherichia coli BL21(DE3)cells using the prokaryotic expression vector constructed from cloned bx12 geneafter sequencing and structural identification.