一份水稻OsWOX3B基因敲除突变体的鉴定

doi:10.16819/j.1001-7216.2021.0602

摘要/Abstract

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

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

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.

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.

图/表 8

表1 水稻OsWOX3B基因敲除T0代株系分子鉴定结果

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

图1 水稻OsWOX3B基因敲除株系的编辑位点方框表示外显子,线段表示内含子,箭头表示突变位点和结构域。

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

图2 突变体Oswox3b叶片和颖壳上表皮毛数目分析 A、B和E表示野生型R401和纯合突变株分蘖期叶片及成熟种子体式显微镜观察结果;C、D表示野生型R401和纯合突变株分蘖期叶片扫描电镜观察结果,箭头所指为宏毛。F-R401和oswox3b近轴面叶中部宏毛数目统计结果。柱形图上的数据点为平均值±标准差（n=3）。*表示突变体在P<0.05水平差异显著。

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.

图3 突变体Oswox3b的叶长和叶宽 A-抽穗期野生型R401与突变体Oswox3b的剑叶;B,C-A中红色区域;D-抽穗期野生型R401与突变体Oswox3b的叶宽;E-抽穗期野生型R401与突变体Oswox3b的叶长。柱形图上的数据点为平均值±标准差(n=10); *表示突变体在P<0.05水平上差异显著。

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.

图4 突变体Oswox3b叶片维管束分析 A、B-抽穗期野生型（A）与突变体（B）叶片大维管束之间小维管束扫描电镜观察结果,白色三角形代表小维管束;C-抽穗期大维管束数目统计分析;D-抽穗期剑叶大维管束之间小维管束数目;E-抽穗期剑叶小维管束总数;F-抽穗期剑叶小维管束间的距离。柱形图上的数据点为平均值±标准差（n=10）;*表示突变体在P<0.05水平差异显著。

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.

图5 突变体Oswox3b影响了维管束发育相关基因的表达柱形图上的数据点为平均值±标准差（n=3）,*表示突变体在P<0.05水平差异显著。

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.

图6 野生型R401与突变体Oswox3b不同生育期表型比较 A和B分别表示芽期和幼苗期野生型与突变体;C-芽长、根长和幼苗株高;D-抽穗期野生型与突变体植株的田间表型。柱形图上的数据点为平均值±标准差（n=10）,*表示突变体在P<0.05水平差异显著。

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.

图7 突变体Oswox3b的分蘖、穗型和千粒重 A-抽穗期野生型R401与突变体Oswox3b的植株表型;B-野生型R401与突变体Oswox3b穗形比较（比例尺为15 cm）;C-抽穗期野生型R401与突变体Oswox3b分蘖数目和株高,B中TT为总分蘖数,ET为有效分蘖数;D~G分别为野生型与突变体穗长,一次枝梗数,二次枝梗数和每穗总粒数的统计分析。柱形图上的数据点为平均值±标准差（n=10）,*表示突变体在P<0.05水平差异显著。

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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