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玉米褐色叶中脉突变体bm-like1的基因克隆与转录组分析
何田利1,2, 杨悦2, 张红伟2, 杨宗莹2, 卢雨晴2, 李润植1, 郑军2
0
(1.山西农业大学农学院;2.中国农业科学院作物科学研究所)
摘要:
本研究在田间发现了一个自然突变的玉米褐色叶中脉突变体,并命名为bm-like1。多代自交后发现bm-like1的褐色叶中脉性状能够稳定遗传。通过突变体bm-like1和自交系B73杂交构建了F2群体进行遗传分析发现,bm-like1的褐色叶中脉表型由隐性单基因控制,利用BSA-Seq和精细定位方法,对有褐色叶中脉表型的F2代植株进行定位,将目标区段定位在玉米第4号染色体32 Mb?36 Mb之间。通过玉米数据库检索发现,该定位区间内存在一个已报道的BM3基因。BM3基因编码咖啡酸-O-甲基转移酶,是玉米木质素生物合成途径中一个重要的酶。利用纯合突变体bm-like1与bm3突变体杂交进行等位测验,发现杂交后代表现出褐色叶中脉的表型。经序列分析发现突变体bm-like1中BM3基因在起始密码子ATG上游144 bp处有一个1439-bp的hAT转座子插入,而bm3突变体在BM3基因第2外显子上有一个5452-bp的序列插入。利用实时荧光定量PCR实验和转录组分析发现,突变体bm-like1中BM3基因的表达量显著低于自交系B73,突变体bm-like1与野生型之间的差异表达基因主要集中在次生代谢物的合成和代谢途径。
关键词:  玉米  褐色叶中脉  木质素  BM3  BSA-Seq
DOI:
投稿时间:2021-03-03修订日期:2021-03-24
基金项目:中国农业科学院创新工程
Gene Cloning and Transcriptome Analysis of Maize Brown Midrid Mutant bm-like1
Abstract:
In this study, bm-like1, a natural brown midrib mutant, was found to be steadily inherited after continual self-pollination. Phenotype analysis of the F2 population composed of homozygous bm-like1 mutant and B73 inbred lines indicated that the brown midrib phenotype of bm-like1 was controlled by a single recessive gene. Using BSA-seq and fine mapping, the target region was mapped to the interval of 32-36 Mb on maize chromosome 4, where BM3 was located. BM3 gene encodes a caffeic acid o-methyltransferase, which is an important enzyme in lignin biosynthesis. We crossed bm-like1 mutant with bm3 mutant for allelism testing and found that the F1 plants showed brown midrib phenotype, indicating that BM3 was the causal gene for bm-like1 mutant. Genomic sequence analysis revealed that a 1439-bp insertion at 144 bp upstream of transcription start site of BM3 might cause the brown midrib phenotype of bm-like1, which is different from bm3 mutation of 5452-bp insertion in the second exon of BM3 gene. Real-time quantitative PCR and transcriptomic analysis indicated that the expression of BM3 gene in bm-like1 mutants was significantly decreased compared with the wild type plants, the differentially expressed genes between bm-like1 and wild type plants were mainly enriched in the secondary metabolites biosynthesis and metabolic pathway.
Key words:  maize  brown midrib  lignin  BM3  BSA-Seq

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