【作者】 张雪梅；

【导师】 乐毅； 杨前进；

【作者基本信息】 安徽农业大学 ， 农业硕士（专业学位）， 2024， 硕士

【摘要】 干旱是全球农业面临的主要挑战之一,对作物的产量和质量造成严重影响。与主要粮食作物相比,杂粮作物在干旱适应性方面具有独特优势,但是对其干旱适应机制的研究还相对有限。深入挖掘杂粮作物在干旱适应方面的关键基因和调控网络,对于改善作物的耐旱性和提高作物产量具有重要意义。本文以大麦、高粱、玉米、绿豆、赤小豆和藜麦作为研究对象,甄选干旱处理组和对照组的转录组原始测序数据,通过多物种转录组学分析方法、WGCNA和同源比对,挖掘杂粮作物抗旱关键基因。具体研究内容和结果如下:(1)对30组转录组测序数据进行RNA-Seq数据分析,得到六种作物全基因组表达矩阵。通过DESeq2差异表达分析,筛选出干旱胁迫差异基因表达矩阵,并对差异基因进行富集分析。结果发现差异基因主要富集在对非生物刺激的反应、氧化还原酶活性和次生代谢物的生物合成等方面,其中苯丙烷代谢通路在植物对非生物胁迫(如干旱)响应中发挥重要作用。(2)基于WGCNA挖掘关键基因模块。对差异基因表达矩阵采用动态树切割和层次聚类方法划分模块。通过数据融合将模块和样本分组进行关联分析,依据相关系数和显著性p值,筛选出赤小豆3个、绿豆4个、大麦2个、玉米4个、高粱2个和藜麦5个关键基因模块。并对禾本科大麦、豆科绿豆和藜科藜麦关键模块内的基因构建互作网络,筛选出可能抗旱hub基因8～9个,进行蛋白功能预测。预测结果显示:HORVU.MOREX.r3.7HG0644400和Vradi03g04780可能是抗旱关键基因。(3)同源比对推测物种间的抗旱关键基因。利用Ortho Venn3对六种作物差异基因蛋白序列进行同源分析,得到663个直系同源蛋白簇和58个单拷贝基因簇。构建系统发育树和蛋白质互作网络,筛选出3～6个核心蛋白,展开生理生化分析和亚细胞定位。结果发现:MAD2、A0A816WU08、C5YAX4＿SORBI与已经验证的水稻Q0JC85均是含HORMA结构域蛋白。LOC106757504、A0A0L9U8L9和大豆研究中发现的K7MZ95(Gm NAC家族)具有类似的生物学功能。据此推测蛋白质对应的基因很可能是抗旱关键基因。另外LOC106757504与Vradi03g04780基因翻译的蛋白功能一致,也在一定程度上验证了预测结果的准确性。(4)针对上述研究,设计作物耐旱基因查询系统。主要包括浏览、查询、提交、下载和BLAST五个功能模块。旨在为农学爱好者提供一个便捷友好地学习平台,为育种工作者提供数据参考。更多还原

【Abstract】 Drought is one of the major challenges facing global agriculture,causing serious impacts on crop yields and quality.Compared with major grain crops,miscellaneous crops have unique advantages in drought adaptation,but research on their drought adaptation mechanisms is still relatively limited.In-depth exploration of key genes and regulatory networks of miscellaneous crops in drought adaptation is of great significance for improving crop drought tolerance and increasing crop yield.This thesis used barley,sorghum,maize,mung beans,adzuki beans,and quinoa as the research subjects,and selected the raw transcriptome sequencing data of the drought-treated groups and the control groups,and used multi-species transcriptomics analysis methods,WGCNA,and homology alignment to identity key drought-resistant genes in miscellaneous crops.The specific research content and results are as follows:(1)RNA-Seq data analysis was performed on 30 transcriptome sequencing datasets,and obtained whole-genome expression matrices for six crop species.Using DESeq2 for differential gene expression analysis,differentially expressed gene matrices under drought stress were identified,and enrichment analysis was conducted on these genes.The results revealed that the differentially expressed genes were mainly enriched in response to abiotic stimuli,oxidoreductase activity,and biosynthesis of secondary metabolites.Among which,the phenylpropanoid metabolic pathway plays a crucial role in the response of plants to abiotic stresses,such as drought.(2)Mining key gene modules based on WGCNA.The differentially expressed gene matrices were divided into modules by dynamic tree cutting and hierarchical clustering.Through data integration,the modules and samples were grouped for association analysis,according to the correlation coefficient and significance p-value,three key gene modules of adzuki bean,four of mung bean,two of barley,four of maize,two of sorghum,and five of quinoa were screened out.In addition,constructing interactome networks of genes in the key modules of gramineous barley,leguminous mung bean and chenopodiaceous quinoa,and filtering 8-9 hub genes with potential drought-resistant to predict the protein functions.The predicted results indicated that HORVU.MOREX.r3.7HG0644400 and Vradi03g04780 might be drought-resistant key genes.(3)Homologous alignment to infer drought-resistant key genes among species.Ortho Venn3 was employed to perform homology analysis on differentially expressed genes protein sequences from six crop species,resulting in 663 orthologous protein clusters and 58single-copy gene clusters.Phylogenetic trees and protein-protein interaction networks were constructed,and 3～6 core proteins were slected for further physiological and biochemical analysis as well as subcellular localization.The results revealed that MAD2,A0A816WU08,C5YAX4＿SORBI,and the previously validated rice protein Q0JC85 all belong to the HORMA domain-containing protein family.LOC106757504,A0A0L9U8L9,and the soybean protein K7MZ95(Gm NAC family)discovered in soybean research exhibited similar biological functions.Based on this,it can be inferred that the corresponding genes of these proteins are likely to be drought-resistant key genes.Furthermore,the functional consistency between LOC106757504 and the protein translated by the Vradi03g04780 gene partially validates the accuracy of the predicted results.(4)Designing a crop drought tolerance gene query system based on the above research.It primarily consists of five functional modules: Browse,Search,Submit,Download and BLAST.It aims to provide a convenient and user-friendly learning platform for agronomy enthusiasts and offer data references for breeders.更多还原