水稻早衰突变体es5的鉴定及其突变基因的精细定位

doi:10.3864/j.issn.0578-1752.2018.04.002

摘要/Abstract

摘要： 【目的】对水稻叶片早衰突变体es5进行遗传分析及精细定位，探索水稻叶片衰老的分子机制。【方法】es5是嘉禾212经EMS诱变获得的一个叶片早衰突变体。利用es5与籼稻中恢8015杂交获得F1，F1自交获得分离群体F2。在杭州和海南观察F1和F2的表型，并分析该突变表型的遗传行为。取F2中隐性单株，利用图位克隆方法精细定位该基因。抽穗期时对突变体和野生型叶片进行SOD活性、MDA含量、可溶性蛋白含量、叶绿素含量、净光合速率测定、组织化学分析、透射电镜观察以及衰老相关基因的表达量分析。成熟后调查es5和嘉禾212的几个重要农艺性状。【结果】四叶期前es5表型与嘉禾212相比无明显差异，四叶期时下部叶片叶尖开始黄化衰老，随着植株的生长发育，叶片的衰老面积逐渐变大，至拔节期，中下部叶片黄化衰老严重。与野生型相比，es5的叶绿素a、叶绿素b、类胡萝卜素含量和净光合速率均显著下降，es5的株高、有效穗数、每穗总粒数、千粒重、结实率等农艺性状都极显著下降。伊文思蓝染色和二氨基联苯胺染色分析结果表明，es5叶片中含有更多的死亡细胞和过氧化氢积累。酶活性和衰老相关参数的测量结果显示抽穗7 d和抽穗21d，es5叶片中的SOD活性均显著高于嘉禾212中SOD活性；抽穗21 d时，es5中MDA含量显著高于嘉禾212中MDA含量；抽穗7 d和抽穗21des5叶片中可溶性蛋白含量均极显著低于嘉禾212中可溶性蛋白含量。透射电镜观察结果表明es5衰老部位细胞质溶解，叶绿体结构异常，叶绿体膜溶解，基粒模糊，基质片层疏松，类囊体发育异常，淀粉体和嗜饿颗粒增多。qRT-PCR结果显示es5中促进衰老的基因Osh36、Osh69、OsI85、RCCR1表达量极显著上调。2个衰老的标志基因Osh69和OsI85表达量分别上调12.7和36.6倍。同时叶绿素降解相关基因SGR也显著上调。遗传分析结果表明该性状是由单隐性核基因控制。采用图位克隆的方法将该基因精细定位在第5染色体上的BF-10和RM3664两标记之间，物理距离约52.7 kb，该区间包含8个开放阅读框。【结论】es5由于叶片的过早衰老造成与产量相关的几个重要农艺性状显著下降。将该基因定位在第5染色体介于标记BF-10和RM3664之间52.7 kb的物理区间内。

关键词: 水稻, 早衰, es5, 图位克隆, 精细定位

Abstract: 【Objective】In this study genetic analysis and gene mapping of es5, a mutant with early senescent leaf in rice, were conducted to explore the molecular mechanism of rice leaf senescence.【Method】 es5 was isolated from the progeny of japonica rice cv. Jiahe212 treated by ethyl methane sulfonate (EMS). F2 population was derived from the cross between es5 and Zhonghui8015. The phenotypes of the F1 and the segregation ratio of the F2 were investigated in Hangzhou and Hainan separately. The recessive individuals in F2 were used to locate the gene by the map cloning method. The SOD activity, MDA content, soluble protein content, chlorophyll content, the net photosynthetic rate, the histochemical analysis and the expression of senescence-related genes were analyzed for es5 and Jiahe212 leaves at heading stage. Several main agronomic traits of es5 and Jiahe212 were analyzed under field conditions at maturation stage in Hangzhou.【Result】There was no difference between Jiahe212 and es5 before 4-leaf-stage. Leaf senescence in es5 occurred at 4-leaf-stage. The etiolated area of the rice leaf increased gradually with the growth and development. At elongation stage, the middle and lower leaves showed serious aging. Compared with the Jiahe212, Chl a, Chl b, carotenoid content and photosynthetic capacity of es5 significantly decreased and the plant height, the number of productive panicles per plant, the number of spikelets per panicle, seed setting rate and 1 000-grain weight also significantly reduced. The results of Evans blue and DAB staining assay indicated that there were more dead cells and H2O2 in leaves of es5. At 7 days to 21 days and heading, the SOD activities increased significantly in es5, MDA content increased significantly in 21days after heading, while the soluble protein content significantly decreased at 7 days and 21 days after heading. Dissolved cytoplasm, abnormal chloroplast structure, dissolved chloroplast membrane, blurred base, loose lamellar lamellae, abnormal thylakoid development and more starches grains and osmiophilic granules were observed in the senescent leaf region of es5 by Transmission Electronic Microscope (TEM). qRT-PCR analysis showed that senescence-associated genes Osh36, Osh69, OsI85, RCCR1 were up regulated significantly in es5. Especially, the expression of Osh69 and OsI85, two marker genes about aging, were up regulated 12.7 and 36.6 times respectively. SGR, a gene about degradation of chlorophyll, was up regulated significantly at the same time. Genetic analysis suggested that the phenotype of es5 was controlled by single recessive nuclear gene, and ES5 was fine mapped in a 52.7 kb interval including 8 open reading frames between BF-10 and RM3664 on chromosome 5 by map-based cloning strategy.【Conclusion】In this study several agricultural traits significantly in es5 for the leaf senescence ES5 was located in a 52.7 kb range between BF-10 and RM3664 by map-based cloning strategy.

Key words: Oryza sativa L., early senescence, es5, map-based cloning, fine mapping

王备芳，陈玉宇，张迎信，刘群恩，孙滨，向小娇，曹永润，程式华，曹立勇. 水稻早衰突变体es5的鉴定及其突变基因的精细定位[J]. 中国农业科学, 2018, 51(4): 613-625.

WANG BeiFang, CHEN YuYu, ZHANG YingXin, LIU QunEn, SUN Bin, XIANG XiaoJiao, CAO YongRun, CHENG ShiHua, CAO LiYong. Identification and Fine Mapping of an Early Senescent Leaf Mutant es5 in Oryza sativa L.[J]. Scientia Agricultura Sinica, 2018, 51(4): 613-625.

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