【作者】 代敏；

【导师】 胡育峰；

【作者基本信息】 四川农业大学 ， 作物遗传育种， 2025， 硕士

【摘要】 玉米(Zea mays L.)是分布于世界各地的主要粮食作物之一,其产量及品质的主要影响因素是籽粒灌浆,而籽粒灌浆受到淀粉合成、细胞增殖与发育、光合作用、非生物胁迫及环境条件等内外因素的共同调控,因此完善玉米籽粒灌浆的分子调控网络对提高玉米产量及品质具有重要意义。课题组前期对包含262份玉米自交系的自然群体进行灌浆速率及百粒重等性状的全基因组关联分析(GWAS),发现一个多环境共定位转录因子ZmABI3,其可能在调控玉米籽粒灌浆速率中起到关键作用。故本论文对转录因子ZmABI3调控籽粒灌浆的分子机制进行解析,主要研究内容包括转录因子ZmABI3基本特性研究、zmabi3敲除材料农艺性状和生理性状考察分析、下游靶基因筛选验证。主要结果如下:1.转录因子ZmABI3不具转录激活活性、在核内行使功能。蛋白结构分析结合进化树分析显示转录因子ZmABI3位于AFL亚家族,且无同源基因;实时荧光定量PCR表明ZmABI3基因在玉米的根、茎及花丝中不表达,在18 DAP(days after pollination)的胚、叶、胚乳、籽粒中表达,且在胚乳中高表达;酵母自激活研究发现ZmABI3蛋白不具有转录激活活性;亚细胞定位分析表明ZmABI3蛋白是在细胞核内行使功能的蛋白。2.转录因子ZmABI3参与调控玉米株高及籽粒大小。通过构建转录因子ZmABI3敲除载体,创制其敲除材料,通过对回交后的敲除材料进行基因型鉴定,从多个敲除株系中筛选出分离比符合1:2:1的zmabi3-1/zmabi3-2两个敲除株系;对敲除材料与野生型材料进行性状考察,与野生型相比敲除材料的株高均略有增加、成熟期百粒重均显著性下降、粒长分别呈现显著和极显著降低、粒宽无显著性差异、粒厚呈现显著降低、粒形整体变小、扫描电镜显示成熟籽粒胚变小,胚重和除胚之外的籽粒重显著性低于野生型。3.转录因子ZmABI3通过调控总淀粉和脂肪的生物合成过程,影响籽粒灌浆速率。对敲除材料及野生型材料的生理性状进行测定分析,成熟期敲除材料与野生型材料的醇溶蛋白与非醇溶蛋白含量及蛋白条带宽度无明显区别、胚中粗脂肪含量显著下降;24年夏温江和24年冬海南敲除材料的灌浆速率均低于野生型,敲除材料籽粒百粒重与野生型相比在25 DAP后呈现显著性下降,敲除材料的总淀粉含量自15 DAP后均低于野生型,且成熟期敲除材料淀粉颗粒变小,籽粒中AGPase、SSs和GBSS酶活均低于野生型,且AGPase和GBSS酶活呈现显著下降。4.转录因子ZmABI3可能通过调控基因Miniature1(Mn1)、Trpp6、Acc1和Acc2的表达量,促进总淀粉和脂肪的生物合成。RNA-seq共筛选到447个差异表达基因(DEGs),包括319个差异表达上调基因,128个差异表达下调基因。其中与淀粉和蔗糖代谢相关的基因有34个,与脂肪合成相关的基因有16个。我们对其中已报道的影响淀粉、油分合成的关键基因细胞壁转化酶INCW2的编码基因Miniature1(Mn1)、6-磷酸海藻糖磷酸酶及脂肪合成的主要限速酶乙酰辅酶A羧化酶的编码基因Acc1和Acc2的表达量进行q RT-PCR验证,发现敲除材料中四个关键基因的表达量均显著或极显著低于野生型,与RNA-seq结果一致。综上所述,转录因子ZmABI3可能通过调控基因Mn1、Trpp6、Acc1和Acc2的表达量,促进淀粉和脂肪的合成,影响籽粒灌浆和玉米产量。明确了ZmABI3在玉米胚乳籽粒灌浆过程中的功能,为玉米高产和优质育种提供优异基因资源。更多还原

【Abstract】 Maize(Zea mays L.)is one of the major food crops distributed all over the world.The main influencing factors of its yield and quality are grain filling,which is jointly regulated by internal and external factors such as starch synthesis,cell proliferation and development,photosynthesis,abiotic stress and environmental conditions.Therefore,improving the molecular regulatory network of corn grain filling is of great significance for increasing the yield and quality of corn.The research group previously conducted a genome-wide association study(GWAS)on traits such as grain filling rate and 100-grain weight in a natural population containing 262 maize inbred lines,and discovered a multi-environment co-localization transcription factor,ZmABI3,which may play a key role in regulating the grain filling rate of maize.Therefore,this thesis analyzes the molecular mechanism by which the transcription factor ZmABI3 regulates grain filling.The main research contents include the study of the basic characteristics of the transcription factor ZmABI3,the investigation and analysis of the agronomic and physiological traits of zmabi3 knockout materials,and the screening and verification of downstream target genes.The main results are as follows:1.The transcription factor ZmABI3 does not have transcriptional activation activity and functions in the nucleus.Protein structure analysis combined with phylogenetic tree analysis revealed that the transcription factor ZmABI3 was located in the AFL subfamily and had no homologous genes.Real-time fluorescence quantitative PCR indicated that the ZmABI3 gene was not expressed in the roots,stems and filaments of maize,but was expressed in the embryos,leaves,endosperm and grains of 18 DAP(days after pollination),and was highly expressed in the endosperm.Yeast self-activation studies have found that the ZmABI3 protein does not have transcriptional activation activity.Subcellular localization analysis indicated that the ZmABI3protein is a protein that functions within the cell nucleus.2.The transcription factor ZmABI3 is involved in regulating the plant height and grain size of corn.By constructing the transcription factor zmabi3 knockout vector and creating its knockout material,and through genotype identification of the backcrossed knockout material,two knockout lines zmabi3-1/zmabi3-2 with a segregation ratio of 1:2:1 were screened out from multiple knockout lines.The properties of the knockout materials and the wild-type materials were investigated.Compared with the wild-type materials,the plant height of the knockout materials slightly increased,the weight per 100 grains at maturity significantly decreased,the grain length showed significant and extremely significant decreases respectively,there was no significant difference in grain width,the grain thickness showed significant decrease,the overall grain shape became smaller,and the embryo of mature grains became smaller as shown by scanning electron microscopy.The embryo weight and grain weight other than the embryo were significantly lower than those of the wild type.3.The transcription factor ZmABI3 affects the grain filling rate by regulating the biosynthesis process of total starch and fat.The physiological properties of the knockout material and the wild-type material were determined and analyzed.There was no significant difference in the contents of alcohol-soluble protein and non-alcohol-soluble protein and the width of protein bands between the knockout material and the wild-type material at the mature stage,and the crude fat content in the embryo decreased significantly.The filling rates of the knockout materials in Wenjiang in the summer of 24 and Hainan in the winter of 24 were both lower than those of the wild type.The grain weight per 100 grains of the knockout materials showed a significant decrease after 25 DAP compared with the wild type.The total starch content of the knockout materials was lower than that of the wild type after 15 DAP,and the starch particles of the knockout materials became smaller at the mature stage.The enzyme activities of AGPase,SSs and GBSS in the grains were all lower than those in the wild type,and the enzyme activities of AGPase and GBSS showed a significant decrease.4.The transcription factor ZmABI3 may promote the biosynthesis of total starch and fat by regulating the expression levels of genes Miniature1(Mn1),Trpp6,Acc1 and Acc2.A total of447 differentially expressed genes(DEGs)were screened by RNA-seq,including 319differentially expressed up-regulated genes and 128 differentially expressed down-regulated genes.Among them,there are 34 genes related to starch and sucrose metabolism,and 16 genes related to fat synthesis.We conducted q RT-PCR verification on the expression levels of Miniature1(Mn1),the coding genes of the key genes that have been reported affecting the synthesis of starch and oil,namely the cell wall converting enzyme INCW2,6-phosphotrehalose phosphatase,and Acc1 and Acc2,the coding genes of the main rate-limiting enzyme acetyl-Co A carboxylase in fat synthesis.It was found that the expression levels of the four key genes in the knockout material were significantly or extremely significantly lower than those in the wild type,which was consistent with the RNA-seq results.To sum up,the transcription factor ZmABI3 may promote the synthesis of starch and fat by regulating the expression levels of genes Mn1,Trpp6,Acc1 and Acc2,and affect grain filling and corn yield.The function of ZmABI3 in the grain filling process of corn endosperm was clarified,providing excellent gene resources for high-yield and high-quality corn breeding.更多还原