Identification and Gene Mapping of Outcurved Leaf Mutant ocl1 in Rice

doi:10.16819/j.1001-7216.2023.221004

Abstract

Abstract:

【Objective】 Moderate leaf rolling can enhance photosynthetic efficiency and improve rice yield. Identification of rice leaf rolling mutants will help elucidate the molecular mechanism behind rice leaf formation. 【Methods】 ⁶⁰Co-γ ray irradiation was used to mutagenize indica rice variety Yuzhenxiang and an outcurved leaf mutant was obtained, tentatively named ocl1 (outcurved leaf 1). The phenotypic and agronomic traits of the mutant were investigated. The F₂ population of a cross between ocl1 and 02428 was used for mapping of the OCL1 gene. Additionally, the expression of leaf-rolling related genes was analyzed by quantitative PCR. 【Result】 Compared with the wild-type, the leaves of the mutant was characterized by outcurved and drooping leaves from tillering to mature stage. The yield components including the seed setting rate, 1000-grain weight and yield per plant were obviously decreased in ocl1. Cross-section observation of leaves revealed that the bulliform cells between adjacent vascular bundles of ocl1 were larger than those of the wild-type. Genetic analysis implied that these phenotypes of the mutant were controlled by a pair of recessive nuclear gene. The OCL1 gene was fine-mapped to a 127 kb interval between markers RM19575 and ID02612 on the chromosome 6. Sequencing analysis indicated that one of the genes (LOC_Os06g10600) has a single-base mutation at the intron-exon junction, which leads to abnormal splicing and changes in amino acid sequence. The gene encodes a homologous domain-leucine zipper protein, which is an allele to the leaf rolling related gene ROC8 (URL1). Correspondingly, the expression of ROC5 and LAC17 genes related to bulliform cell development was down-regulated in ocl1, while that of XTH11 was up-regulated in ocl.【Conclusion】 The mutation of OCL1 gene results in outcurved leaf by affecting the development of bulliform cells, which in turn leads to a yield reduction.

Key words: rice, outcurved leaf, alternative splicing, bulliform cell

摘要：

【目的】 适度卷叶可增强叶片的光合效率，提高水稻的产量，筛选和鉴定水稻卷叶突变体有助于解析水稻叶片形成的分子机制。【方法】 利用280 GY钴⁶⁰ γ射线对籼稻品种玉针香进行诱变处理，筛选出一个外卷叶突变体，暂命名为ocl1(outcurved leaf 1)。考查了突变体的表型特征和农艺性状，并利用ocl1突变体与02428杂交的F₂群体定位目标基因，最后通过荧光定量PCR分析了卷叶相关基因的表达情况。【结果】 从分蘖期到成熟期，突变体的叶片表现外卷和披垂；在农艺性状方面，突变体的结实率、千粒重和单株产量显著降低；叶片的显微观察结果表明，突变体的外卷叶表型主要是由于相邻维管束之间的泡状细胞变大引起的；遗传分析表明，ocl1的突变表型受一对隐性核基因控制，将OCL1基因定位在第6染色体上SSR标记RM19575与InDel标记ID02612之间，物理距离约为127 kb；定位区间内的测序结果表明，其中一个基因（LOC_Os06g10600）的内含子-外显子连接处发生单碱基突变，导致异常剪接，进而引起氨基酸序列的改变。该基因编码同源异型域-亮氨酸拉链蛋白，与卷叶相关基因ROC8和URL1等位；荧光定量PCR结果显示，泡状细胞发育相关基因ROC5和LAC17在ocl1突变体中下调表达，而XTH11则在ocl1突变体中上调表达。【结论】 OCL1基因突变通过影响泡状细胞的发育导致叶片外卷，同时引起产量降低。

关键词: 水稻, 外卷叶, 选择性剪接, 泡状细胞

REN Zhiqi, XUE Kexin, DONG Zheng, LI Xiaoxiang, LI Yongzhao, GUO Yujing, LIU Wenqiang, GUO Liang, SHENG Xinnian, LIU Zhixi, PAN Xiaowu. Identification and Gene Mapping of Outcurved Leaf Mutant ocl1 in Rice[J]. Chinese Journal OF Rice Science, 2023, 37(4): 337-346.

任志奇, 薛可欣, 董铮, 李小湘, 黎用朝, 郭玉静, 刘文强, 郭梁, 盛新年, 刘之熙, 潘孝武. 水稻外卷叶突变体ocl1的鉴定及基因定位[J]. 中国水稻科学, 2023, 37(4): 337-346.

Figures/Tables 7

Table 1. Primers for gene mapping, sequencing and expression analysis.

引物名称 Primer	用途 Usage	正向引物(5'→3') Forward primer (5'→3')	反向引物(5'→3') Reverse primer (5'→3')
RM217	基因定位Gene mapping	ATCGCAGCAATGCCTCGT	GGGTGTGAACAAAGACAC
ID02356	基因定位Gene mapping	CTCACGTAGGTCTTGAGGAG	AGAAGAGGGCAGGAGGAG
RM19570	基因定位Gene mapping	CCCAGATATTCTGTGTGATCATGAGG	GAGTGAATGTGAGCCGTCTATTGG
RM19575	基因定位Gene mapping	TCATCACAAGCTCGTAATCAGG	CCAGAGAATAAGAGGACATGACG
ID02612	基因定位Gene mapping	GCAGTTAATTATTCCATGCG	TTTGAACTCTCCCATATTCG
ID03100	基因定位Gene mapping	CCATGGATGACTCTCTCTCT	ACACCTCCACTCCTCCAT
RM276	基因定位Gene mapping	CTCAACGTTGACACCTCGTG	TCCTCCATCGAGCAGTATCA
cDNA-S	测序Sequencing	AGAGAGGAAGAGGCAGAGGTAG	AGAAGATGCTGTCGTAGGTGTC
OsActin	内参Reference gene	CATTGGTGCTGAGCGTTTCC	AGAAACAAGCAGGAGGACGG
RL14	qRT-PCR	CTCTTTCAGGCATTCCATTGATG	CAACACCTTGTCAGCTTTCAAGC
ZHD1	qRT-PCR	CGAGAACGAATGCTCTCTCAG	CGGACCCCGGTATGGTAG
SLL1	qRT-PCR	GCCTCTGTGATTGCCATCTAAT	CAGGTGTCCAACCATGAGC
ROC5	qRT-PCR	CGCAAGAGGAAGAAGCGATAC	GCTCCAGTTGCGTCTTCATC
SRL1	qRT-PCR	TCTCCTGCCTCCTCTGTGTG	TAGGAGGGGTGGTGTTGAAG
NRL1	qRT-PCR	TCAGTAGTGTAGTGGTGTCGAGTTCA	GCACTCCTTCATGTGAGCTTCA
LAC17	qRT-PCR	CTGCAGATTTGGCACTCGAGAACGTC	CATGCTCTTGGTGTTGCACAG
EXPA2	qRT-PCR	GGGCACTCCTACTTCAACCT	TAGGAGTTGCTCTGCCAGTT
EXPA4	qRT-PCR	GGGCACTCCTACTTCAACCT	CTGGAAGGAGAGGCTCTGG
XTH11	qRT-PCR	ACCTTCTACTTGTCGTCGCA	TGCTGTGGGTTCCAGATGAT
CESA2	qRT-PCR	GGTATCCTTGAGATGAGGTGG	GCCTTTGAGGTGACAGTGAA
CESA3	qRT-PCR	AAGTTCTTCGGTGGGCTCT	TTTCCAGGATGCCAGTAGC

Fig. 1. Plant architecture of the wild type and ocl1. A, Plant architecture of WT and ocl1 at the seedling stage, bar=5 cm; B, Plant architecture of WT and ocl1 at the tillering stage, bar=15 cm; C, Leaf morphology of WT and ocl1 at the tillering stage, bar=1 cm; D, Leaf rolling index of WT and ocl1 at the tillering stage; E, Plant architecture of WT and ocl1 at the heading stage, bar=10 cm; F, Seed setting of WT and ocl1, bar=2 cm.

Fig. 2. Cross-sectional and leaf slice observation of WT and ocl1. A, Cross-sectional observation of WT and ocl1, bar=1 mm; B, Leaf slice observation of WT and ocl1, black lines point to bulliform cells of WT and ocl1, bar=100 μm; C, Number of bulliform cells of WT and ocl1; D, Area of bulliform cells of WT and ocl1; NS means there is no significant difference between the mutant and WT; ** Significant difference between the mutant and WT at 0.01 level. ad, Adaxial side; ab, Abaxial side.

Table 2. Agronomic traits of the WT and ocl1.

材料 Material	单株穗数	每穗总粒数	结实率		千粒重	单株产量
材料 Material	No. of panicles per plant	No. of spikelets per panicle	Seed-setting rate / %		1000-grain weight / g	Yield per plant / g
WT	11.9±0.4	94.2±1.3		74.4±0.8	29.6±0.1	24.7±1.1
ocl1	11.3±0.1	92.4±4.0		45.7±2.0^**	24.8±0.1^**	11.8±0.7^**

Fig. 3. Analysis of grain morphology between WT and ocl1. A, Grain length of WT and ocl1, bar=1 cm; B, Grain width of WT and ocl1, bar=5 mm; C, Grain plumpness of WT and ocl1, bar=1 cm; D, Statistical analysis of grain length, grain width and grain thickness of WT and ocl1; **Significant difference between the mutant and WT at 0.01 level.

Fig. 4. Map-based cloning of OCL1. A, Mapping of OCL1 gene. Black boxes, white boxes and black lines represent exons, UTR and introns, respectively. Black arrow indicates the mutation site; B, Sequence comparison of WT and ocl1 at the mutation site; C, Electrophoresis detection of cDNA amplification products; D, cDNA sequence alignment of the OCL1 gene between WT and ocl1; E, Alignment of the amino acid sequence between WT and ocl1.

Fig. 5. Relative expression level of genes associated with leaf rolling. **Significant difference between the mutant and WT at 0.01 level.

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