Identification of a Knockout Mutant of OsWOX3B Gene in Rice (Oryza sativa L.)

doi:10.16819/j.1001-7216.2021.0602

Abstract

Abstract:

【Objective】The OsWOX3B gene regulates leaf morphology and trichome development in rice. This study aims to further understand the role of OsWOX3B gene in regulating rice development. 【Method】In this study, OsWOX3B of indica rice variety R401 was knocked out by CRISPR/Cas9 gene editing technique. The mutation sites and phenotype of the obtained materials were analyzed as well as the expression levels of related genes. 【Result】The 341^st base of the coding region of OsWOX3B of the obtained plant was changed from T to C, and the 395th-397th bases were absent, and the leaves and glumes of the mutant were glabrous, the same as the phenotype of dep/nuda/glr1. Therefore, the plant was Oswox3b mutant. In addition to the reported loss-of-function phenotype of OsWOX3B, new phenotypes of Oswox3b were found in this study. Compared with wild-type R401, mutant Oswox3b showed prolonged growth duration, decreased tiller number, wider and shorter leaves, longer panicle and more grains per panicle. At the same time, the mutant Oswox3b had increased number of vascular bundles in the blade, increased distance between the small vascular bundles. The changes in the expression levels of the two genes related to the development of lateral organs in the mutant Oswox3b were also consistent with the phenotypic changes. 【Conclusion】A new mutant of rice OsWOX3B gene was identified that affects lateral organ development in rice.

Key words: rice (Oryza sativa L.), lateral organs, vascular bundle, growth duration

摘要：

【目的】水稻OsWOX3B基因调控叶片形态和表皮毛发育,根据表型被命名为LSY1、DEP、NUDA和GLR1等。深入了解OsWOX3B基因对水稻发育调控的功能具有重要意义。【方法】利用CRISPR/Cas9基因编辑技术对籼稻品种R401的OsWOX3B进行基因敲除。对所获材料进行突变位点分析和表型分析,同时进行相关基因的表达分析。【结果】所获材料的OsWOX3B基因的编码区第341位碱基由T变为C,第395–397位碱基缺失,其叶片和颖壳光滑,与突变体dep、nuda、glr1的表型相同,可确认其为Oswox3b突变体。除了与已报道的OsWOX3B的功能缺失突变表型相关外,Oswox3b突变体也有新表型出现。与野生型R401相比,突变体Oswox3b表现为生育期延长、分蘖数减少、叶片变宽、稻穗变长和每穗粒数增多。同时,突变体Oswox3b的剑叶维管束增多,小维管束间距增大,2个水稻侧生器官发育相关基因在突变体Oswox3b中的表达变化也与其表型变化一致。【结论】鉴定了一个新的水稻OsWOX3B基因突变体,其影响水稻侧生器官发育。

关键词: 水稻, 侧生器官, 维管束, 生育期

Yali ZHENG, Linchuang YU, Xiaoxiao AN, Xinle CHENG, Lijun REN, Zilong SU, Xiaoya ZHENG, Tao LAN. Identification of a Knockout Mutant of OsWOX3B Gene in Rice (Oryza sativa L.)[J]. Chinese Journal OF Rice Science, 2021, 35(2): 112-120.

郑亚莉, 余林闯, 安肖肖, 程心乐, 任丽君, 苏子龙, 郑小雅, 兰涛. 一份水稻OsWOX3B基因敲除突变体的鉴定[J]. 中国水稻科学, 2021, 35(2): 112-120.

Figures/Tables 8

Table 1 Molecular identification results of OsWOX3B knockout T0 generation lines.

再生株系	阳性株系	阳性率	编辑株系	编辑率	纯合株系	杂合株系
Regenerated lines	Positive lines	Positive rate/%	Edited lines	Editing rate/%	Homozygous lines	Heterozygous lines
18	18	100	12	66.7	2	10

Fig. 1. Editing sites of OsWOX3B knockout line. Box represents exon, line segment represents intron and arrow represents mutation site and domain.

Fig. 2. Analysis of the number of epidermal hairs on the leaf and seed glume of Oswox3b mutant. A, B and E show the microscopic observation results of leaf and mature seed of wild-type R401 and homozygous mutants in tillering stage; C and D show the tillering stage leaf observation results in wild-type R401 and homozygous mutants by SEM (the arrow: macro-hair). F shows the macro-hair number in the middle adaxial surface of wild type R401 and oswox3b. The data points on the chart are mean ± standard deviation (n=3). *shows the difference is significant at P < 0.05 level.

Fig. 3. Leaf length and leaf width of mutant Oswox3b. A, Wild type R401 and mutant Oswox3b at heading stage; B, C, The red area in A under a body microscope; D, Leaf width of wild type R401 and mutant Oswox3b at heading stage; E, Leaf length of wild type R401 and mutant Oswox3b at heading stage. The data points on the column chart are mean value ± standard deviation (n=10); *shows the difference is significant at P < 0.05 level.

Fig. 4. Analysis of vascular bundles in leaves of mutant Oswox3b. A and B, Small vascular bundle between the wild type (A) and the mutant (B) leaf at heading stage by SEM. White triangle represents small vascular bundle; C, Large vascular bundle quantity at heading stage; D, Small vascular bundle quantity between the large vascular bundles of the flag leaf at heading stage; E, Total small vascular bundle quantity of the flag leaf at heading stage; F, Spacing between small vascular bundles of the flag leaf at heading stage. The data points on the column chart are mean value ± standard deviation (n=10); *shows the difference was significant at P < 0.05 level.

Fig. 5. The mutant Oswox3b affected the expression of genes related to vascular bundle development. The data points on the column chart are mean value ± standard deviation (n=3); *shows the difference was significant at P < 0.05 level.

Fig. 6. Comparison of phenotypes between wild type R401 and mutant Oswox3b at various growth stages. A, B, Phenotypes of wild type and mutant in bud stage and seedling stage, respectively; C, Bud length, root length and plant height in A and B; D, Field phenotype of wild type and mutant plants in heading stage. The data points on the column chart are mean value ± standard deviation (n=10); *shows the difference was significant at P < 0.05 level.

Fig. 7. Tiller, panicle and 1000-grain weight of mutant Oswox3b. A, Plant phenotype of wild type and mutant at heading stage; B, Comparison of panicle shape between wild type R401 and mutant Oswox3b (scale: 15 cm); C, Statistical analysis of tiller number and plant height of wild type R401 and mutant Oswox3b; D–G, Statistical analysis of panicle length, primary rachis branch number, secondary rachis branch number and total grain number of wild type and mutant, respectively. The data points on the column chart are mean value ± standard deviation (n=10); *Means the difference was significant at P < 0.05 level.

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