Data_Sheet_1_Allelic Variation in Zmfatb Gene Defines Variability for Fatty Acids Composition Among Diverse Maize Genotypes.docx

Edible oil with lower saturated fatty acids is desired for perceived quality and health benefits to humans and livestock. fatb gene encoding acyl-ACP thioesterase is a key player in the conversion of palmitic acid to oleic acid, thereby modifying the ratio of saturated to unsaturated fatty acids in maize kernels. The present investigation characterised the full-length sequence of the Zmfatb gene (4.63 kb) in two mutants (Zmfatb) and eight wild-types (ZmfatB) inbreds to study allelic variation, gene-based diversity, phylogenetic-relationship, protein-modelling, and molecular-docking to identify novel candidates for modification of fatty acid profile. Sequence alignment revealed wide genomic variability for Zmfatb among the inbreds; identified five novel SNPs and two InDels that clearly differentiated the wild-type and mutant genotypes. Gene-based diversity using 11-InDel markers categorised 48-diverse maize-inbreds into two-clusters. The majority of mutant and wild-type inbreds were grouped in separate clusters and led to the generation of 41 haplotypes. Genetic relationship of maize fatb gene with orthologues among 40 accessions of 12 oilseed-crops using both nucleotide and protein sequence clustered maize, soybean, sunflower, opium-poppy, Citrulus lanata, quinoa, and prunus species into one cluster; and brassica, camelina, and arabidopsis into the different cluster. The clustering pattern revealed that the plant oil with higher unsaturated fatty acids, particularly oleic, linoleic, and linolenic acids grouped together in one cluster and higher proportions of other fractions like arachidic, eicosenoic, and erucic acids grouped in another cluster. Physico-chemical properties highlighted more similarity between maize and 29 orthologue proteins, but orthologues were found to have better thermostability. Homology models have been developed for maize mutant and wild-type inbreds using Umbellularia californica (PDB ID: 5x04) as a template. Predicted protein models possessed optimum confidence-score and RMSD values and validated stability via., Ramachandran plots. Molecular docking indicated most of the interactions of protein-ligand were having similar binding-affinity due to the broader specificity of fatty acyl-ACP thioesterases and the presence of conserved-domains across crops. This is the first report on the comprehensive molecular characterisation of the fatb gene in maize and various orthologues. The information generated here provided new insights into the genetic diversity of fatb gene which can be utilised for the enhanced nutritive value of oil in the breeding programme.

饱和脂肪酸含量更低的食用油，因其优异的感官品质以及对人类与畜禽的健康益处，成为市场与育种领域的热门目标。编码酰基-ACP硫酯酶（acyl-ACP thioesterase）的fatb基因，是棕榈酸转化为油酸的关键调控因子，可通过该过程改变玉米籽粒中饱和脂肪酸与不饱和脂肪酸的比例。本研究针对2个Zmfatb突变体自交系与8个ZmfatB野生型自交系的Zmfatb基因全长序列（4.63 kb）进行了系统表征，旨在通过分析等位基因变异、基因多样性、系统发育关系、蛋白质建模与分子对接，筛选可用于改造玉米脂肪酸谱的新型候选材料。序列比对结果显示，不同玉米自交系间的Zmfatb基因存在广泛的基因组变异；本研究共鉴定出5个全新的单核苷酸多态性（Single Nucleotide Polymorphisms, SNPs）位点与2个插入缺失（Insertion-Deletion, InDels）标记，可有效区分野生型与突变体基因型。利用11个InDel标记开展的基因多样性分析，将48个遗传多样性丰富的玉米自交系划分为2个类群。大部分突变体与野生型自交系分别聚类于不同类群，最终共产生41个单倍型。基于核苷酸与蛋白质序列，对12种油料作物共40份材料的Zmfatb基因直向同源基因进行系统发育分析，结果将玉米、大豆、向日葵、鸦片罂粟、西瓜（Citrulus lanata）、藜麦以及李属（Prunus）物种聚类为一类；而芸苔属、亚麻荠与拟南芥则单独聚类为另一类。聚类模式显示，不饱和脂肪酸（尤其是油酸、亚油酸与亚麻酸）含量更高的植物油聚为一类，而花生酸、二十碳烯酸与芥酸等其他脂肪酸组分占比更高的植物油则聚为另一类。理化性质分析表明，玉米Zmfatb蛋白与29个直向同源蛋白的相似度更高，但直向同源蛋白的热稳定性更优。本研究以加州月桂（Umbellularia californica，PDB编号：5x04）为模板，分别构建了玉米突变体与野生型自交系的蛋白质同源建模模型。预测得到的蛋白质模型具备最优的置信度评分与均方根偏差（Root Mean Square Deviation, RMSD）值，并通过拉马钱德兰图（Ramachandran plots）验证了结构稳定性。分子对接结果显示，由于脂肪酸酰基-ACP硫酯酶具有较广的底物特异性，且不同作物间存在保守结构域，大部分蛋白质-配体相互作用的结合亲和力相近。本研究首次对玉米fatb基因及其各类直向同源基因开展了全面的分子表征。本研究获得的结果为fatb基因的遗传多样性研究提供了全新视角，可应用于提升食用油营养品质的玉米育种计划中。