生长素调控因子OsGRF4协同调控水稻粒形和稻瘟病抗性

doi:10.16819/j.1001-7216.2021.210206

摘要/Abstract

摘要：

【目的】水稻粒形为多基因控制的复杂数量性状,同时影响稻谷产量和稻米外观及碾磨品质,挖掘粒形相关基因并解析其遗传机制,对于水稻高产和优质品种选育具有重要意义。【方法】以前期利用大粒籼稻品种特大籼(TDX)为供体亲本、小粒粳稻品种日本晴(Nipponbare,NPB)为轮回亲本获得的一个大粒近等基因系,在水稻第2染色体上初定位粒形调控基因GS2.2(grain size 2.2)的基础上,对GS2.2基因进行了精细定位和候选功能基因的克隆鉴定。【结果】利用BC₄F₂群体中2887份极小粒单株及该群体中70份基因型杂合的大粒单株自交获得的BC₄F₃群体,将GS2.2基因精细定位在2M-7-1与2M-9之间的160.6 kb的区间内。该区间编码18个基因开放阅读框(ORF1-18)。测序发现ORF18基因在大粒近等基因系与小粒日本晴等位基因间存在5个SNP差异,ORF18编码生长素调控因子蛋白OsGRF4。采用CRISPR/Cas9基因编辑技术对大粒近等基因系中ORF18进行敲除,发现ORF18敲除株系粒长变短,证实ORF18是GS2.2的功能基因。进一步对ORF18大粒近等基因系和基因编辑敲除株系进行稻瘟菌室内离体和病圃接种鉴定,发现ORF18大粒近等基因系稻瘟病抗性增强,而基因编辑敲除株系稻瘟病抗性减弱,表明ORF18正调控水稻稻瘟病抗性。【结论】本研究发现的生长素调控因子OsGRF4协同调控水稻粒形和稻瘟病抗性,为协调水稻高产和抗性育种提供了重要的基因资源。

关键词: 水稻, 粒形性状, GS2.2, OsGRF4, 稻瘟病抗性

Abstract:

【Objective】Rice grain shape is a complex quantitative trait controlled by multiple genes, which affects rice yield, rice appearance and milling quality simultaneously. It is of great significance to explore the genes related to grain shape and elucidate its genetic basis. Previously, a gene, namely GS2.2, which controls rice grain size, was mapped on the chromosome 2, by using a set of near isogenic lines derived from the small-grain japonica rice variety Nipponbare (NPB) as the recurrent parent, and an indica rice variety Tedaxian(TDX) as the donor parent. 【Method】In this study, fine mapping of GS2.2 and identification of candidate functional genes were carried out. 【Result】 GS2.2 was fine mapped to the 160.6 kb interval between the makers of 2M-7-1 and 2M-9, by using a BC₄F₂ population with 2887 small grain size plants and a BC₄F₃population with 70 large grain size plants. Bioinformatics analysis showed that the 160.6 kb chromosome interval encodes 18 open reading frames (termed as ORF1-18, respectively). Sequence analysis showed that there were five SNP differences in ORF18 between the large grain near isogenic line and Nipponbare. ORF18 encoded an auxin regulatory factor protein OsGRF4. The grain length of ORF18 knockout lines, whose ORF18 was knocked out in large grain near isogenic lines by using CRISPR/CAS9 gene editing technology, became smaller. The above results confirmed that ORF18 was a functional gene of GS2.2. Further identification of ORF18 near isogenic lines (ORF18-NIL) and gene editing knockout lines by in vitro inoculation and disease nursery inoculation showed that the rice blast resistance of ORF18-NIL increased, while that of gene editing knockout lines decreased, indicating that ORF18 also positively regulates rice blast resistance. 【Conclusion】The auxin regulator OsGRF4 found in this study simultaneously regulates rice grain shape and blast resistance, which provides an important gene for breeding rice variety with high yield in coordination with enhanced resistance.

Key words: rice, grain shape, GS2.2, OsGRF4, rice blast disease resistance

曹煜东, 肖湘谊, 叶乃忠, 丁晓雯, 易晓璇, 刘金灵, 肖应辉. 生长素调控因子OsGRF4协同调控水稻粒形和稻瘟病抗性[J]. 中国水稻科学, 2021, 35(6): 629-638.

Yudong CAO, Xiangyi XIAO, Naizhong YE, Xiaowen DING, Xiaoxuan YI, Jinling LIU, Yinghui XIAO. Auxin Regulator OsGRF4 Simultaneously Regulates Rice Grain Shape and Blast Resistance[J]. Chinese Journal OF Rice Science, 2021, 35(6): 629-638.

图/表 11

表1 CRISPR/Cas9载体构建和基因敲除植株鉴定所用引物

Table 1 Primers for construction and identification of CRISPR/Cas9 vectors.

引物用途 Primer usage	引物名称 Primer name	引物序列 Primer sequence
CRISPR/Cas9载体构建 Constriction of CRISPR/Cas9 vector	CRS-GRF4-F	GCCGCAGCTCCTCGTACTGCGCCG
CRISPR/Cas9载体构建 Constriction of CRISPR/Cas9 vector	CRS-GRF4-R	AAACCGGCGCAGTACGAGGAGCTG
敲除转基因植株鉴定 Identification of knockout transgenic plants	GRF4-CRSJD-F	AACCCATTTTCTTGGCTC
敲除转基因植株鉴定 Identification of knockout transgenic plants	GRF4-CRSJD-R	CGCCTGATCGGAATAAAC

图1 GS2.2基因的精细定位与物理图谱构建 A-GS2.2精细定位遗传图谱;B-GS2.2位点BAC克隆重叠群和物理图谱;C-重组株系迭代法确定GS2.2物理区间,其中NIL为大粒近等基因系,NPB为日本晴,“L+数字”代表表型为大粒的重组株系,“S+数字”代表表型为小粒的重组株系,右侧为对应株系粒长和粒宽。

Fig. 1. Construction of genetic map and physical map of GS2.2. A, Genetic map of GS2.2; B, BAC clone contig and physical map of GS2.2 locus; C, The GS2.2 physical interval was determined by overlapping recombinant lines. NIL, Near-isogenic line with large grains; NPB, Nipponbare; “L+Number” represents a recombinant strain with large grains, and the “S+Number” represents a recombinant strain with small grains. The phenotype of grain length and width for NPB, NIL and recombinant lines was presented on the right.

表2 GS2.2候选基因预测分析

Table 2 Prediction and analysis of GS2.2 candidate genes.

候选基因 Candidate gene		蛋白编码结构 Protein coding structure
ORF1	Probable WRKY transcription factor 14
ORF2	Protein NRT1/ PTR FAMILY 8.3
ORF3	ADP-ribosylation factor
ORF4	Uncharacterized LOC4330423
ORF5	Putative surface protein SACOL0050
ORF6	60S ribosomal protein L12-1
ORF7	DNA topoisomerase 2
ORF8	Protein-coding
ORF9	Os02g0699800
ORF10	Transcription initiation factor TFIID subunit 8
ORF11	Uncharacterized protein At1g51745
ORF12	Cell division cycle 20.2, cofactor of APC complex
ORF13	Myb family transcription factor APL
ORF14	Ubiquinol oxidase 2, mitochondrial
ORF15	Polyamine transporter PUT1
ORF16	Cyclin-dependent kinase F-4
ORF17	Protein Brevis radix-like 2
ORF18	Growth-regulating factor 4

图2 NIL和日本晴等位基因的OsGRF4基因结构和自然变异

Fig. 2. OsGRF4 structure and natural variation in alleles from NIL and NPB.

图3 OsGRF4 CRISPR/Cas9基因编辑靶点序列和编辑株系基因型 A―OsGRF4基因编辑靶点序列;B―OsGRF4基因编辑株系敲除系靶点突变基因型。

Fig. 3. Sequence of target site for OsGRF4 CRISPR / Cas9 gene editing and genotype of OsGRF4 CRISPR / Cas9 gene editing lines in NIL background. A, Sequence of target site for OsGRF4 CRISPR / Cas9 gene editing; B, Genotype of OsGRF4 CRISPR / Cas9 gene editing lines in NIL.

表3 OsGRF4 CRISPR/Cas9基因编辑敲除突变类型

Table 3 OsGRF4 CRISPR / Cas9 gene editing knockout mutation types.

转基因株系 Transgenic lines	基因型 Genotype	基因编辑类型 Gene editing type
Reference	GCTGCCGCCGTTCACCGCGGCGCAGTACGAGGAGC
CR-NIL-15	GCTGCCGCCGTTCACCGCGGTCGCAGTACGAGGAGC	+1
CR-NIL-16	GCTGCCGCCGTTCACCGCGGACGCAGTACGAGGAG	+1
CR-NIL-16	GCTGCCGCCGTTCACCGCGGCCGCAGTACGAGGAG	+1
CR-NIL-17	AGGCTGCCGCCGTTCACCGC-(46 bp del)CTGGTGGCAGGCGTG	-46
CR-NIL-17	AGGCTGCCGCCGTTCACCGCGGGCGCAGTACGAGG	+1
CR-NIL-19	GGCTGCCGCCGTTCACCGCGGCGCAGTACGAGGAG (WT)	+0
CR-NIL-19	GGCTGCCGCCGTTCACCGCGTACGCAGTACGAGGA	+1
CR-NIL-20	GCTGCCGCCGTTCACCGCGGTCGCAGTACGAGGAG	+1
CR-NIL-23	GCTGCCGCCGTTCACCGCGGACGCAGTACGAGGAG	+1
CR-NIL-22	GCTGCCGCCGTTCACCGCGGGCGCAGTACGAGGAG	+1
CR-NIL-22	GCTGCCGCCGTTCACCGCGGTCGCAGTACGAGGAG	+1

图4 GS2.2-NIL背景下OsGRF4基因编辑转基因水稻的粒形表型 A和B―GS2.2-NIL背景下OsGRF4基因编辑转基因株系粒长和粒宽;C~F―GS2.2-NIL背景下OsGRF4基因编辑转基因株系粒长、粒宽、粒厚和千粒重统计。*P<0.05; **P<0.01(t测验)。误差线表示SD (n=30)。NIL―GS2.2-NIL; CR20-CR-NIL-20; CR23-CR-NIL-23.

Fig. 4. Grain shape of gene editing lines in GS2.2-NIL background. A and B, Phenotype of grain length and width. C-F, Statistic data of grain length, width, thickness and 1000-grain weight. *P<0.05; **P<0.01(t-test). Error bar indicated the SD value (n=30). NIL, GS2.2-NIL; CR20, CR-NIL-20; CR23, CR-NIL-23.

图5 NIL背景下OsGRF4基因编辑株系农艺性状表型 A―NIL背景下OsGRF4基因编辑株系株型;B~G, NIL背景下OsGRF4基因编辑株系株高、穗长、分蘖数、每穗总粒数、每穗实粒数和结实率。显著性分析采用t测验,图中误差值以SD表示（n=3）。NIL―GS2.2-NIL; CR20-CR-NIL-20; CR23-CR-NIL-23.

Fig. 5. Phenotype of agronomic traits of OsGRF4 gene editing lines in NIL background. A, Plant architecture for gene editing lines. B-G, Plant height, panicle length, tiller number, total grains per panicle, number of full grains per panicle and seed setting rate for gene editing lines. The t-test was used for significance analysis. Error bar indicated the SD value (n=3). **P<0.01.NIL, GS2.2-NIL; CR20, CR-NIL-20; CR23, CR-NIL-23.

图6 GS2.2-NIL和日本晴（NPB）在自然病圃发病症状 A―叶片病圃发病症状;B―叶片病圃发病级别统计。显著性分析采用t测验,图中误差值以SD表示(n=10)。**P<0.01(t测验)。

Fig. 6. Symptoms of GS2.2-NIL and Nipponbare(NPB) after infected by rice blast fungal in natural disease nurse. A, Symptoms of leaf after rice blast fungal infection in the natural disease nurse; B, Leaf blast rating after rice blast fungal infection in the natural disease nurse. The t-test was used for significance analysis. Error bar indicated the SD(n=10). **P<0.01。

图7 GS2.2-NIL和日本晴(NPB)稻瘟病离体接种发病症状 A―病斑症状;B―病斑相对菌丝量生物量;C―病斑相对面积。显著性分析采用t测验,图中误差值示SD(n=3)。**P<0.01(t测验)。

Fig. 7. Symptoms of GS2.2-NIL and Nipponbare(NPB) after in vitro inoculation with rice blast fungus. A, Leaf disease phenotype; B, Relative blast fungal biomass in the infected leaf; C, Area of leaf infected by blast fungal. The t-test was used for significance analysis, error bar indicated the SD value(n=3). **P<0.01(t-test).

图8 日本晴、GS2.2-NIL和GS2.2-NIL敲除株系稻瘟菌接种发病症状 A―病斑表型;B―病斑面积;C―病斑相对菌丝量生物量。显著性分析采用t测验, 图中误差线示SD（n=3）。*P<0.05; **P<0.01. NIL-15, CR-NIL-15.

Fig. 8. Symptoms of GS2.2-NIL, Nipponbare(NPB) and CRISPR/Cas9 editing lines after in vitro inoculation with rice blast fungus. A, Phenotype of infection; B, Infected leaf area; C, The relative fungal biomass. The t-test was used for significance analysis, error bar indicated the SD value (n=3). *P<0.05; **P<0.01(t-test). NIL-15, CR-NIL-15.

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