水稻早衰突变体zs的鉴定与基因定位

Analysis and gene mapping of rice premature senescence mutant zs

[目的]本研究旨在对水稻叶片早衰突变体zs进行遗传分析及基因定位,探索水稻叶片早衰的分子机制。[方法]从甲基磺酸乙酯(EMS)诱变‘宁粳1号’突变体库中筛选鉴定了1个稳定遗传的早衰突变体zs,构建突变体zs与籼稻品种‘N22’的F2群体,对控制突变性状的基因进行定位,利用RT-q PCR对叶绿素合成以及质体发育相关基因的表达水平进行分析。[结果]突变体zs在28℃条件下生长14 d时,zs第2叶叶尖出现黄褐色斑点,并逐渐向叶基部蔓延。突变体zs叶绿素a和叶绿素b含量明显下降。zs叶肉细胞结构异常,叶绿体中类囊体片层结构排列松散。与野生型相比,zs的株高、分蘖数和千粒质量均显著降低。通过构建遗传分离群体,将突变基因定位在第12号染色体短臂上的600 kb的区间内。此区间内含有2个已报道的与叶片衰老相关的基因LOC_Os12g16410和LOC_Os12g16720,分别编码异黄酮还原酶和细胞色素单加氧酶(P450)。测序分析发现,仅在LOC_Os12g16720第2外显子上有1个单碱基突变,导致苏氨酸突变为甲硫氨酸。zs叶绿素合成相关基因和质体发育相关基因的表达量显著低于野生型。[结论]鉴定了1个早衰突变体zs,通过精细定位与序列分析,将LOC_Os12g16720作为候选基因,该基因可能参与调控叶绿素合成及质体的发育。

[Objectives]In this study,genetics analysis and gene mapping of zs,a mutant with early senescent leaf in rice,were conducted to explore the molecular mechanism or rice leaf premature senescence.[Methods]A premature senescence mutant zs was isolated from an ethylmethylsulfone-induced‘Ningjing 1’mutant library.The F 2 population from zs crossed to‘N22’(indica cultivar)was used to mapping the gene ZS.The expression level of the genes related to chlorophyll synthesis and plastid development was analyzed by RT-qPCR.[Results]Compared with the wild type,the second leaf tip of the mutant zs displayed yellow brown spots,spreading to the base of the leaf when it grew at 28℃for 14 days.The content of chlorophyll a and chlorophyll b decreased significantly in zs leaves.The structure of mesophyll cells in zs were abnormal,and the thylakoid lamellae of chloroplasts were loosely arranged.In addition,the plant height,tiller number and 1 000-grain weight of zs decreased significantly.The target gene was finally mapped on the short arm of chromosome 12 near to marker NL12-18 and ZS20 with the physical distance about 600 kb.There were two reported genes related to leaf senescence in this region,LOC_Os 12g16410 and LOC_Os12g16720,which encoded isoflavone reductase and cytochrome monooxygenase(P450),respectively.Sequencing analysis found that there was only a single base mutation on the LOC_Os 12g16720 gene exon,which caused the substitution of threonine to methionine.Compared with wild type,the expression level of chlorophyll synthesis and plastid development related genes decreased significantly in the mutant zs.[Conclusions]We identified a prematurely senescence mutant zs.The cytochrome monooxygenase gene(LOC_Os 12g16720)was the candidate gene by fine mapping and sequencing.ZS may be involved in the regulation of chlorophyll synthesis and plastid development.