Map-based Cloning and Functional Analysis of a New Allele of D1, a Gene Controlling Plant Height in Rice (Oryza sativa L.)

doi:10.16819/j.1001-7216.2024.230502

Abstract

Abstract:

【Objective】Plant height is an important agronomic trait in rice breeding. Identification of genes controlling plant height and their functional characterization can provide useful genetic resources for high-yield breeding.【Methods】A dwarf mutant, d1-11, was screened from the rice variety Nipponbare by EMS mutagenesis. Phenotypic and cytological observations, gene expression, hormone content and drought resistance were analyzed. The d1-11 mutant gene was identified through map-based cloning.【Results】The d1-11 mutant exhibits dwarfism, with more shortened and widened leaves, and more rounded grains compared to the wild type. The d1-11 mutant leaf has a smaller midvein, reduced number and area of large and small veins, resulting in abnormal leaf morphology in the d1-11 mutant. The d1-11 gene is genetically mapped between two molecular markers on rice chromosome 5. Map-based cloning reveals that a single base substitution at the junction of the ninth exon-intron in the D1 gene results in the loss-of-function mutation of d1-11. The D1 gene has a higher expression level in various tissues at the seedling stage, but the expression levels decrease from the tillering stage. Exogenous abscisic acid (ABA) treatment for 24 hours induces D1 gene expression, exogenous gibberellin (GA) treatment inhibits D1 gene expression, and salt stress treatment for 24 hours can strongly induce D1 gene expression. The contents of several hormones such as GA, brassinosteroid (BR), and indole-3-acetic acid (IAA) were increased in the d1-11 mutant. The d1-11 mutant shows a significant increase in relative water content (RWC) and a reduced rate of water loss in leaves. Furthermore, d1-11 mutant plants exhibit stronger resistance to drought stress.【Conclusion】The d1-11, a novel allele in D1 locus was identified in the present study. It was showed that the d1-11 mutant had increased levels of various endogenous hormones, increased leaf water content, and enhanced resistance to drought stress. This study will further enrich the genetic resources related to dwarfism and reveals some new biological roles of the D1 gene in rice.

Key words: plant height, dwarfism, D1 gene, d1-11 mutant, plant hormones, drought stress

摘要：

【目的】株高是作物重要的农艺性状，挖掘株高控制基因并解析其分子功能，可为作物高产育种提供更多有用的基因资源。【方法】利用EMS诱变水稻日本晴获得的矮化突变体d1-11为材料，进行表型和细胞学观察，通过图位克隆的方法鉴定d1-11基因并对基因表达、激素含量和抗旱性进行了分析。【结果】d1-11突变体表现出植株矮化、叶片变短变宽和籽粒形态变圆表型；d1-11突变体叶片中脉萎缩，大脉和小脉数量和面积减少，导致叶片形态变异。d1-11基因被定位到水稻5号染色体R5M15.2和R5M15.8两个分子标记之间；图位克隆结果表明d1-11突变体中D1基因第11外显子和内含子交界处单碱基替换导致基因功能缺失。D1基因在苗期各组织中表达量较高，从分蘖期开始表达量降低；外源脱落酸(ABA)处理24 h后诱导D1基因表达，外源赤霉素(GA)处理抑制D1基因表达，盐胁迫处理24 h诱导D1基因剧烈上调表达。d1-11突变体植株GA、油菜素内酯(BR)和生长素(IAA)等激素含量均上升，叶片相对含水量上升、叶片失水速率降低，植株对干旱胁迫抗性显著增强。【结论】鉴定到水稻D1基因新等位突变d1-11，发现d1-11突变体多种内源激素水平上升、叶片含水量增加、植株对干旱胁迫抗性增强。本研究进一步丰富了水稻矮化基因资源并揭示D1基因新的生物学功能。

关键词: 株高, 矮化, D1基因, d1-11突变体, 植物激素, 干旱胁迫

GAO Junru, QUAN Hongyu, YUAN Liuzhen, LI Qinying, QIAO Lei, LI Wenqiang. Map-based Cloning and Functional Analysis of a New Allele of D1, a Gene Controlling Plant Height in Rice (Oryza sativa L.)[J]. Chinese Journal OF Rice Science, 2024, 38(2): 140-149.

高郡茹, 权弘羽, 袁刘珍, 李钦颖, 乔磊, 李文强. 水稻D1基因新等位突变体的鉴定与功能分析[J]. 中国水稻科学, 2024, 38(2): 140-149.

Figures/Tables 8

Table 1. Primer sequences used in this study.

标记名称 Marker name	正向引物序列 Forward primer sequence(5′-3′)	反向引物序列 Reverse primer sequence(5′-3′)	用途 Use
R5M8	CGAGATGGAGGGATTTGAC	CCCGATGACTAGGGATTAGAG	基因定位Gene mapping
R5M10	TACAAAATATGCCCGTGTCG	GATCGTTCCGCAAACAGAG	基因定位Gene mapping
R5M12	GGTAGGAGGTGGAAGAAGG	TTTAGAGGGTCGGGCGATT	基因定位Gene mapping
R5M14	CGTCAACAGGCTTGAAAGA	TTGCCAGTCCCAAAGAGTA	基因定位Gene mapping
R5M14.5	AACGCAGTTGTGCTAATCAG	AGATCTTGTTGCCAAAAAAT	基因定位Gene mapping
R5M15	GACATTTCGTGTGAATTGTT	TTCACAACTTCACTCGAGAC	基因定位Gene mapping
R5M15.2	ATTAAGGTAGGGGCATGAAT	ATCACAAAGCAGTTTCCAGA	基因定位Gene mapping
R5M15.6	ACAAAAATATGATGTGGCAA	CAACCCAGAAACCATCTAGT	基因定位Gene mapping
R5M15.8	CTGAGAATGACTGCTCCGAA	CTTAGCGCATGAGTGGTTTT	基因定位Gene mapping
R5M16	CCCACTGTATTGGATTCTGC	CCACCAGGTCCCACGTTAT	基因定位Gene mapping
R5M18	AATGCCCTTCTCATCCGTGTG	GTCTATGCGTGCTGTGGGCTA	基因定位Gene mapping
D1-1	AAGAGAGAGGCTCAGGCATG	GGAACTCAAAAGCTCAAGGT	突变位点检测Mutation site detection
D1-2	GGTGCAAAACTGCTGTGAAT	GTGGGCTATGCAAATTAACA	突变位点检测Mutation site detection
CDS-9F/13R	AAACAAAAGAGGTGGAGAGG	TCTCATGCTCTCATCAATCA	cDNA测序 cDNA sequencing
CDS-10F/12R	GAATGATGGAGACCAAGGAA	CTGTTTCCCAGGTGCAATAG	cDNA测序 cDNA sequencing
UBQ-qPCR	GGACTGGTTAAATCAATCGTCA	CCATATACCACGACCGTCAAAA	qRT-PCR
D1-qPCR	CAGGAGGTTGAACATGCATATG	CCTGTGTGACTTACAACCTAGT	qRT-PCR

Fig. 1. Phenotypic characterization of WT and d1-11 mutant A, Seedlings of 15-day-old WT and d1-11; B, Morphology of WT and d1-11 at the heading stage; C, Phenotypes of WT and d1-11 leaves at the heading stage. From left to right, three leaves represent flag leaf, the second leaf and the third leaf; D and E, Seed morphology; F-L, Agronomic comparison between WT and mutant. Bars represent mean ± SD (n=20). **P<0.01（the Student’s t-test）.

Fig. 2. Cross section of leaves of the wild type and the mutant A-C, Leaf midrib, large vein and small vein in WT; D-F, Leaf midrib, large vein and small vein in mutant; G-I, Area of leaf midrib, large vein and small vein in WT and mutant; J-K, Number of large and small veins per leaf in WT and mutant. LV, large vein; SV, small vein; As, Air space. Bars represent mean ± SD (n=9). **P<0.01（the Student’s t-test）

Fig. 3. Comparison of hormone levels in the wild type and the d1-11 mutant Bars represent mean ± SD (n=9). *P<0.05; **P<0.01(t-test).

Fig. 4. Genetic mapping and map-based cloning of the d1-11 gene A and B, Genetic mapping of d1-11; C, Gene structure of D1; D, Sequence comparison between WT and d1-11; E, RT-PCR analysis of D1 gene in WT and the d1-11 mutant. M, DNA marker; F, Alignments of D1 gene cDNA sequence in WT and d1-11.

Fig. 5. Temporal and spatial expression pattern of the D1 gene in wild type plants

Fig. 6. Expression analyses of D1 gene under applications of plant hormones and abiotic stresses conditions

Fig. 7. Analysis of drought resistance in the d1-11 mutant A, Phenotypes of WT and mutant under drought stress; B, Survival rate of WT and the mutant after recovery for 5 days; C and D, Relative water content (RWC) and rate of water loss (RWL) in WT and the mutant under non-stressed conditions. Bars represent mean ± SD（n=9）. * and ** indicate significant difference between WT and the d1-11 mutant by the Student’s t-test (P<0.05, P<0.01), respectively.

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