【作者】 杨阳；

【导师】 蔡一林；

【作者基本信息】 西南大学 ， 作物遗传育种， 2021， 硕士

【摘要】 玉米是世界上最广泛种植的粮食作物之一,在我国的农业生产中具有至关重要的地位,在黑色作物中,黑玉米果皮中的花色苷含量要远远高于其他黑色作物,花色苷作为一种天然色素普遍存在于自然界中,为植物提供丰富多彩的植物外在颜色,鲜艳的颜色能吸引鸟类昆虫等为植物授粉传播种子,同时花色苷也具有强大的药用价值,例如抗氧化、抗癌、抗突变、预防心血管疾病等,因此研究植物花色苷的合成调控机制对作物高花色苷育种具有重要的理论指导意义。前人已经利用QTL定位到了控制玉米籽粒果皮花色苷合成的候选基因pl1,本研究对黑玉米籽粒果皮发育过程中花色苷的累积进行量化,从玉米自交系MOSB中克隆获得pl1基因,对其进行序列分析,蛋白功能定位,组织特异性分析,转录激活特性分析,蛋白互作分析,对pl1的过表达株系进行表型分析和转录组测序,结果表明在黑玉米籽粒果皮发育的过程中,pl1具有至关重要的作用。本研究的主要结果如下:(1)从亲本MOSB中克隆获得pl1的全长序列,pl1基因全长由3个外显子和2个内含子构成,外显子共816bp编码272个氨基酸。将不含终止密码子的pl1序列插入过表达载体Ubi:Myc得到融合蛋白表达过表达载体Ubi:pl1-Myc,利用农杆菌介导的玉米幼胚遗传转化法,以草甘膦作为筛选压力,利用跨基因区和载体启动子区的引物检测转基因株系,得到pl1的过表达株系OE-4、OE-7、OE-8。(2)表达模式分析结果表明,pl1在种皮中表达量最高,在花药中表达量最低。(3)构建了35S:pl1-GFP融合蛋白表达载体,利用PEG介导的玉米原生质体瞬时转化法将核Marker与35S:pl1-GFP共同转化玉米原生质体,利用激光共聚焦显微镜观察到在玉米原生质体中核Marker信号与GFP信号共定位于细胞核,表明pl1是一个在细胞核发挥功能的核蛋白。(4)pl1的过表达造成了转基因玉米籽粒果皮中总类黄酮的积累量增加,相比于受体材料的果皮表现出更高的总类黄酮含量,利用q RT-PCR对超表达玉米株系中花色苷合成通路下F3H、F3′H、LC、DFR、CHS、PAL、CHI、4CL等的结构基因的表达量进行检测,发现在pl1超表达的玉米背景下,这些结构基因相较于野生型表现出更高的表达量,证明pl1参与到了花色苷的合成通路中,且对花色苷合成通路下各结构基因具有调控作用。(5)对pl1的过表达株系和野生型株系的同一时期果皮进行转录组测序和分析,结果表明pl1的高表达引起了玉米果皮内如IAA、CTK、GA、ABA等植株激素信号转导的抑制,由于pl1能够被植物激素所诱导表达,所以推测过高的pl1表达量对植物激素信号转导的抑制现象可能是一种反馈调节机制影响整个花色苷的合成过程。(6)在酵母双杂交实验中,单独转化了pl1的酵母菌株能够在缺陷培养基SD-Trp-His-Ade中生长,证明pl1是一个具有转录激活活性的基因,通过分步构建pl1的截短载体转化酵母感受态细胞,将pl1的转录激活结构域定位于蛋白C端20个氨基端的区间内,利用同样的截短策略构建了双荧光素酶报告基因系统,利用REN作为内参,同时以pl1的各截短蛋白激活LUC的表达,以LUC和REN的比值评估截短各pl1蛋白的转录激活能力,结果表明pl1 3′端60bp序列所编码的蛋白对LUC的激活能力要远远高于其他截短蛋白,进一步证明了pl1的转录激活结构域位于蛋白C端20个氨基酸的区间内。(7)在黑玉米果皮发育的6个时期中发现有两个bHLH型转录因子GRMZM5G822829、GRMZM2G378653的表达量始终高于黄玉米,将这两个基因构建到PGAD-T7载体中,将切除了转录激活结构域的pl1构建到PGBK-T7载体中,将两者共同转化酵母感受态细胞,转化的酵母细胞能够在缺陷培养基SD-Trp-Leu-His-Ade中生长,表明pl1与GRMZM5G822829、GRMZM2G378653均存在互作关系。更多还原

【Abstract】 Maize is one of the most widely planted food crops globally and plays a vital role in our country’s agricultural production.In black crops,the anthocyanins in black maize pericarp are much higher than other black crops.As a natural pigment,anthocyanins are commonly found in nature,providing plants with colorful external.The bright colors attract birds and insects to pollinate and spread seeds for plants.At the same time,anthocyanins also have substantial medicinal value,for instance,anti-oxidation,anti-cancer,anti-mutation,prevention of cardiovascular disease,etc.Therefore,studying the synthesis and regulation mechanism of plant anthocyanins biosynthesis has important theoretical guiding significance for crop high-anthocyanin breeding.Predecessors have used QTL to locate the candidate gene pl1 that controls anthocyanin synthesis in maize kernels.This study quantified the accumulation of anthocyanins in the different developments of black maize kernels.The pl1 gene was cloned from the maize inbred line MOSB,its sequence analysis,subcellular localization,tissue-specific analysis,transcription activation characteristic analysis,protein interaction analysis,phenotype analysis,and transcriptome sequencing of pl1 overexpression lines were performed.The results showed that in the development of the black maize pericarp,pl1 plays a vital role.The main results of this study was as follows:(1)The full-length sequence of pl1 was cloned from the parental MOSB.The pl1 gene is composed of three exons and two introns.The total exons are 816 bp,encoding272 amino acids.Insert the pl1 sequence without the stop codon into the overexpression vector Ubi:Myc to obtain the fusion protein overexpression vector Ubi:pl1-Myc,using Agrobacterium-mediated genetic transformation of immature maize embryos to obtained pl1 overexpression lines OE-4,OE-7,and OE-8.The transgenic lines were detected by primers across the gene region and the promoter region of the vector.(2)The results of expression pattern analysis showed that pl1 had the highest expression in pericarp and the lowest in anthers.(3)The 35S:pl1-GFP fusion protein expression vector was constructed,and the nuclear Marker and 35S:pl1-GFP were co-transformed into maize protoplasts with the PEG-mediated transient transformation of maize protoplasts.The nuclear Marker signal and GFP signal co-localized in the nucleus in protoplasts,indicating that pl1 is a nuclear function protein.(4)The overexpression of pl1 caused an increase in the accumulation of total flavonoids in the pericarp of transgenic maize kernels,which showed a higher total flavonoid content than the recipient material.The expression levels of F3 H,F3’H,LC,DFR,CHS,PAL,CHI,4CL,and other structural genes in the anthocyanin synthesis pathway in the overexpression maize lines were detected by q RT-PCR,it was found that these structural genes showed higher expression levels than the wild-type,which proved that pl1 participates in the anthocyanin synthesis pathway and has a regulatory effect on the structural genes in the anthocyanin synthesis pathway.(5)Transcriptome sequencing analysis showed that the overexpression of pl1 caused inhibition of plant hormone signals transduction,pl1 can be induced to be expressed by plant hormones,it is speculated that the inhibition of plant hormone signal transduction by excessive pl1 expression may be a feedback regulation mechanism that affects the entire anthocyanin synthesis process.(6)In the yeast two-hybrid experiment,the yeast strain transformed with pl1 can grow in the defective medium SD-Trp-His-Ade,which proves that pl1 is a transcriptionally activating gene.The truncation of pl1 is constructed step by step.The short vector was used to transform competent yeast cells,and the transcription activation domain of pl1 was located in the 20-amino acid interval of the C terminal of pl1 protein.The same truncation strategy was used to construct a dual-luciferase reporter gene system.The REN was used as an internal reference,and the pl1 truncated protein activates the expression of LUC,and the ratio of LUC to REN is used to evaluate the transcriptional activation ability of each truncated pl1 protein.The results showed that the activation capacity of the protein encoded by the 60 bp sequence of 3’terminal of pl1 was much higher than that of other trunked proteins,which further proved that the transcriptional activation domain of pl1 was located in the 20-amino acid interval of C terminal of pl1 protein(7)The expression levels of two bHLH-type transcription factors GRMZM5G822829 and GRMZM2G378653 were found to be higher than those of yellow maize during the six stages of black maize pericarp developments.These two genes were constructed into the PGAD-T7 vector.The pl1,which removed the transcriptional activation domain,was constructed into the PGBK-T7 vector,and these two vectors were co-transformed into competent yeast cells.The transformed yeast cells can grow in the defective medium SD-Trp-Leu-His-Ade,indicating that pl1 interacts with GRMZM5G822829 and GRMZM2G378653.更多还原