摘要
【目的】水稻叶片是光合作用的重要场所,也是理想株型的重要构成因素,通过对卷叶相关基因进行遗传分析和精细定位,为水稻卷叶基因分子标记辅助育种提供紧密连锁标记。【方法】从60Co-γ射线辐射诱变籼稻品种9311(wild-type,WT)所得突变体库中获得了一份卷叶突变体材料,暂时命名为rl16(t)(rolled leaf 16)。首先,对突变体rl16(t)进行连续多代套袋自交,确定突变表型的稳定性。在抽穗期,随机选取rl16(t)和WT各10株,分别测量剑叶卷曲度以及主要农艺性状。同时取rl16(t)和WT新鲜叶片的相同部位用FAA固定,乙醇系列脱水,石蜡包埋,用石蜡切片机切10μm薄片置于载玻片上,番红染色后置于显微镜下,拍照观察叶片泡状细胞显微结构,对泡状细胞个数和面积进行统计和测量。在分蘖期各取10株rl16(t)和WT剑叶,测定叶绿素含量。以rl16(t)为母本与WT杂交,观察F1和F2植株的叶片表型,进行χ2测验,分析突变体的遗传行为。将卷叶突变体rl16(t)与粳稻品种滇粳优杂交F2分离群体作为定位群体,同时利用SSR标记结合新发展的In Del分子标记用于定位目的基因。利用基因表达定量对定位区间内的3个已知结构域基因和已克隆的水稻卷叶相关基因进行定量表达分析。【结果】与WT相比,rl16(t)叶片出现显著内卷,株高降低,穗长变短,结实率降低等表型变化。rl16(t)自苗期(3叶1心)整株就出现叶片纵向内卷成近似筒状的表型。随着发育进程,植株叶片始终呈现卷曲表型,而WT叶片在发育进程中则始终呈平展状。剑叶石蜡切片观察发现,rl16(t)泡状细胞数量和面积与WT相比均减少。WT的泡状细胞数量为(385.1±43.6)个/mm2,rl16(t)泡状细胞数量为(1059.5±254.4)个/mm2。rl16(t)除泡状细胞发生变化外,叶片其他细胞结构与WT相比均无显著性变化。突变体rl16(t)的类胡萝卜素含量低于WT,而叶绿素a、叶绿素b、总叶绿素含量均显著高于WT。rl16(t)与WT杂交所得F1植株表现叶片平展,并且在包含423单株的F2中,分离出97株卷叶植株和326株平展叶植株,分离比符合3﹕1(χ2=0.86<χ20.05=3.84)。将Rl16(t)初步定位于第9染色体长臂SSR标记RM23769和RM23916之间,进一步扩大定位群体,最终将该卷叶基因定位在In Del标记DF70和SSR标记RM23818之间,该区段物理距离为51 kb。定位区间内有3个编码已知结构域的基因,分别是LOC_Os09g09320、LOC_Os09g09360和LOC_Os09g09370。rl16(t)与WT在基因LOC_Os09g09320与LOC_Os09g09370的表达量上无显著性差异。而rl16(t)中LOC_Os09g09360的表达量显著降低,只有WT的一半。对已克隆的8个卷叶基因进行表达定量分析,发现有7个基因(SLL1、ROC5、RL14、SRL1、ACL1、NRL1和NAL7)在rl16(t)中出现了不同程度的表达下调,只有OSZHD1表达上调。【结论】rl16(t)叶片发生内卷与泡状细胞数量变少,与面积变小相关。Rl16(t)是一个新的卷叶基因,LOC_Os09g09360有可能是目标基因。
【Objective】Rice leaf is not only a vital site for photosynthesis, but also an important factor for ideal plant architecture in rice. This study is useful in map-based cloning of target gene and marker-assisted transferring rolled gene in rice breeding programs. 【Method】A mutant rl16（t）（rolled leaf 16） with rolled leaves was derived from the Indica type rice 9311（wild-type WT） via the radiation of 60Co-γ. Then, it was planted for several generations to test the stability of mutant phenotype in the field. At heading stage, rl16（t） and WT were randomly selected 10 strains to measure the main agronomic traits including leaf rolling index, respectively. The same part of leaf of rl16（t） and WT was taken and fixed by FAA, then dehydrated in series ethanol and paraffin embedding. After that, the fixed leaf was incised to 10 microns slices and these slices were stained by sarranine. Finally, the bulliform cells were observed, the number of them was counted and their area was measured under the microscope. Chlorophyll content of rl16（t） and WT were tested at tilling stage. In addition, the leaf phenotype of the F1 plants and F2 population were investigated, and the mode of rolled gene inheritance was analyzed by chi-square test. To fine-map the Rl16（t）, an F2 population was generated by crossing between the rl16（t） mutant and a Japonica type rice Dianjingyou. Target gene was limited by simple sequence repeat（SSR） and In Del markers. Three known domain structure genes in the mapped region were analyzed by gene quantitative expression. 【Result】Compared with wild-type, the mutant had not only typically adaxially rolled leaves, but also decreased height and panicle length, as well as the seed setting percentage. The whole plant of rl16（t） appeared leaf longitudinally rolled into the approximate tubular phenotype at seedling stage（three leaves and one new）, then kept it at the rest of time. On the contrary, WT had flat leaves in the entire life. Paraffin section of flag leaf indicated that the number and area of bulliform cell of rl16（t） were lower than that of WT. The number of bulliform cell in WT was（385.1±43.6） cells/mm2, while that in the rl16（t） was（1059.5±254.4） cells/mm2. However, for the bulliform cell, there were no significant and obvious changes of rl16（t） and WT. The carotenoid content of rl16（t） was lower than that of WT, but the chlorophyll a, chlorophyll b and total chlorophyll content were significantly higher than that of WT. All of the F1 hybrid between rl16（t） and WT indicated flat leaves, meanwhile, in an F2 population, rolled and flat leaf plants segregated in a 3﹕1 ratio（97 rolled versus 326 flat leaves, chi-square = 0.86chi-square 0.05 = 3.84）. Cytological analysis indicated that the rolled leaf phenotype might be caused by the change of number and size of bulliform cells. Genetic analysis indicated that the rolled leaf characters were controlled by a recessive nuclear gene. Using SSR markers, Rl16（t） was initially mapped in the region between the RM23769 and RM23916 on the long arm of chromosome 9. Furthermore, with enlarged population and more developed In Del markers, Rl16（t） was finally delimited to a 51 kb region governed by the In Del marker DF70 and SSR marker RM23818. Three known structure coding genes（LOC_Os09g09320, LOC_Os09g09360, LOC_Os09g09370） were predicted in the limited interval. The expression of LOC_Os09g09320 and LOC_Os09g09370 in rl16（t） showed no obvious differences with that of WT, but the expression of LOC_Os09g09360 in rl16（t） was only the half of WT. As for quantitative expression analysis of the eight genes that contributed to the rolled leaf, seven of them（SLL1, ROC5, RL14, SRL1, ACL1, NRL1, NAL7） had a decline in the rl16（t） mutant, while OSZHD1 appeared a rise. 【Conclusion】The rolled leaf of rl16（t） was due to reduced number and area of bulliform cells. In addition, this gene would be a putative novel rolled leaf gene. As the result of quantitative gene expression, LOC_Os09g09360 may be the target genes.
出处
《中国农业科学》
CAS
CSCD
北大核心
2015年第13期2487-2496,共10页
Scientia Agricultura Sinica
基金
国家高技术研究发展计划(2014AA10A604)
