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作物学报 ›› 2025, Vol. 51 ›› Issue (2): 347-357.doi: 10.3724/SP.J.1006.2025.42030

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

水稻矮化多分蘖基因DT1的图位克隆与功能分析

李春梅(), 陈洁, 郎兴宣, 庄海民, 朱靖, 杜梓君, 冯浩天, 金涵, 朱国林, 刘凯()   

  1. 仲恺农业工程学院, 广东广州 510225
  • 收稿日期:2024-06-25 接受日期:2024-09-18 出版日期:2025-02-12 网络出版日期:2024-10-10
  • 通讯作者: 刘凯, E-mail: liukai5088@126.com
  • 作者简介:E-mail: lcm900306@hotmail.com
  • 基金资助:
    西南作物基因资源发掘与利用国家重点实验室开放基金课题(SKL-KF202315);省部共建农产品质量安全危害因子与风险防控国家重点实验室开放基金课题(2021DG700024-KF202408);广东省基础与应用基础研究基金项目(2024A1515013028);云浮市科技计划项目(2023020202);广东省普通高校特色创新类项目(2023KTSCX046);广州市科技计划项目(2024A04J4995)

Map-based cloning and functional analysis of Dwarf and Tillering 1 (DT1) gene in rice

LI Chun-Mei(), CHEN Jie, LANG Xing-Xuan, ZHUANG Hai-Min, ZHU Jing, DU Zi-Jun, FENG Hao-Tian, JIN Han, ZHU Guo-Lin, LIU Kai()   

  1. Zhongkai University of Agriculture and Engineering, Guangzhou 510225, Guangdong, China
  • Received:2024-06-25 Accepted:2024-09-18 Published:2025-02-12 Published online:2024-10-10
  • Contact: E-mail: liukai5088@126.com
  • Supported by:
    State Key Laboratory of Crop Gene Exploration and Utilization in Southwest China(SKL-KF202315);State Key Laboratory for Managing Biotic and Chemical Treats to the Quality and Safety of Agro-products(2021DG700024-KF202408);Basic and Applied Basic Research Foundation of Guangdong Province(2024A1515013028);Science and Technology Plan Project of Yunfu(2023020202);Guangdong Provincial Universities Characteristic Innovation Project(2023KTSCX046);Guangzhou Science and Technology Plan Project(2024A04J4995)

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摘要：

分蘖是影响水稻株型和产量的重要性状。本研究获得一个稳定遗传的矮化多分蘖自然突变体dt1 (dwarf and tillering 1)。此外, dt1突变体穗长、结实率、粒长、粒宽、千粒重、维管束鞘细胞数量及大小较野生型均显著降低。图位克隆证实dt1突变体是由编码独脚金内酯生物合成途径关键酶类胡萝卜素裂解双加氧酶(Carotenoid Cleavage Dioxygenase 7, CCD7)的D17/HTD1 (LOC_Os04g46470)第2个外显子上8 bp的插入导致的, 是一个D17/HTD1的新等位突变。此外, dt1突变体萌发率、根长、根直径均显著降低, 外施独脚金内酯类似物GR24能恢复dt1突变体的这些表型。转录组测序结果显示, dt1突变体有579个基因上调, 506个基因下调。GO分析显示上调基因显著富集在生长素响应、内源刺激响应和激素响应等通路, 下调基因显著富集在胞内碳水化合物代谢和组蛋白甲基化等通路。KEGG分析显示上调基因在植物激素信号转导等通路显著富集, 下调基因在氨基糖和核苷酸糖代谢及二萜生物合成等通路上显著富集。研究结果丰富和拓展了CCD7和独脚金内酯在水稻中的调控作用, 对水稻育种具有重要理论意义。

关键词: 水稻, 矮化多分蘖, 图位克隆, 生物学功能

Abstract:

Tillering is a crucial trait that influences plant architecture and yield in rice. In this study, we identified a natural mutant with dwarf stature and high tillering, which we designated as dwarf and tillering 1 (dt1). The dt1 mutant exhibited significant reductions in panicle length, seed setting rate, grain length, grain width, thousand-grain weight, and the number and size of vascular bundle sheath cells compared to the wild type. Map-based cloning revealed that the dt1 phenotype was caused by an 8 bp insertion in the second exon of D17/HTD1 (LOC_Os04g46470), which encodes Carotenoid Cleavage Dioxygenase 7 (CCD7), a key enzyme in strigolactone biosynthesis. Thus, dt1 represents a new allele of D17/HTD1. Additionally, the dt1 mutant showed significantly reduced germination rate, root length, and root diameter, all of which were restored by the exogenous application of the strigolactone analog GR24. Transcriptomic analysis identified 579 up-regulated and 506 down-regulated genes in the dt1 mutant. Gene Ontology (GO) analysis revealed that the up-regulated genes were significantly enriched in pathways related to auxin response, endogenous stimulus response, and hormone response, while the down-regulated genes were enriched in pathways involved in cellular carbohydrate metabolism and histone methylation. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated that the up-regulated genes were associated with plant hormone signal transduction, whereas the down-regulated genes were linked to amino sugar and nucleotide sugar metabolism, as well as diterpenoid biosynthesis. These findings enhance our understanding of the regulatory roles of CCD7 and strigolactones in rice and hold significant theoretical implications for rice breeding.

Key words: rice, dwarf and tillering, map-based cloning, biological function

表1

用于定位的InDel标记及引物序列"

名称Name 正向引物Forward primer (5′-3′) 反向引物Reverse primer (5′-3′)
C2783 ATGGCAAGCACAAGAAGTTGC CGGAGTGGGCATTGTTTTACA
C2782 AACCATTTCACCACAAACCAGC GAGCCTTCGATACTCACCGA
C2763 ACGAGTCCCATCCAAGTACG GAAACAGGAGTAGCTCCCCG
C2754 TTCTTCCGTACAGCAGGCAT AGGAAACTTGAAACGCGCCA

表2

用于PCR扩增和基因克隆的引物序列"

名称Name 正向引物Forward primer (5′-3′) 反向引物Reverse primer (5′-3′)
OsCCD7-E1 GGAGGCCAAGTCCAAAGATG ACCAGCGTCATATCAGTTCG
OsCCD7-E3 GTGAGTGACGCATGCAGGTT GTCCTTCATGCTCGCAGTGT
OsCCD7-E6 TGTCAAGCTGCTCCTACCAGT CCACAAGTGCATTCTCTGTCC
OsCCD7-E7 GCTCAGTCAGGAGGCTGTC TCCGAGACTGGATGTACAGTAG
OsCCD7-cds CTACAACCTCCTCTTCCGCT AGAAAGTGAAGTGGGATCGC

图1

野生型(WT)和dt1突变体的农艺性状 A~L: 分别为成熟期野生型和dt1突变体的植株表型、分蘖、株高、穗部表型、总粒数、实粒数、穗长、结实率、千粒重、粒型、粒长和粒宽。A中标尺为5 cm, D中标尺为2 cm, J中标尺为5 mm。"

图2

成熟期野生型和dt1突变体的叶片和茎秆组织学表型 A: 野生型和dt1突变体成熟期的叶片; B: 20×镜下野生型的叶片横截面; C: 20×镜下dt1突变体的叶片横截面; D: 野生型和dt1突变体叶片中脉的维管束鞘数量; E: 野生型和dt1突变体叶片中脉的维管束鞘面积; F: 野生型和dt1突变体成熟期的茎秆; G: 20×镜下野生型的茎秆横截面; H: 20×镜下dt1突变体的茎秆横截面; I: 野生型和dt1突变体茎秆中的维管束鞘数量; J: 野生型和dt1突变体茎秆中的维管束鞘面积。A和F中标尺为2 cm。"

图3

DT1基因的图位克隆 A: DT1的初定位; B~C: DT1的精细定位; D: 候选基因LOC_Os04g46470的基因结构(黑色方框代表外显子, 黑色线条代表内含子); E: 野生型(WT)及dt1突变体中LOC_Os04g46470的cds及编码氨基酸序列(红色表示突变后的核苷酸或氨基酸)。"

图4

GR24处理(GR24)和未处理(Mock)下野生型(WT)与dt1突变体种子的萌发 A: GR24未处理(Mock)下WT与dt1突变体种子萌发7 d后的表型; B: GR24处理下WT与dt1突变体种子萌发7 d后的表型; C: GR24处理和未处理7 d后WT与dt1种子的萌发率。"

图5

GR24处理(GR24)和未处理(Mock)下野生型与dt1突变体的根形态及参数的统计结果 A: GR24处理2周后野生型与dt1突变体的根形态; B: 野生型与dt1突变体的总根长; C: 野生型与dt1突变体的根直径; D: 野生型与dt1突变体的根表面积; E: 野生型和dt1突变体的根体积。"

图6

差异表达基因火山分布图 Padj表示多重假设检验矫正过的P值, Fold Change表示差异倍数。横虚线和竖虚线分别表示差异表达基因筛选的阈值线为-log10 Padj =1.301和log2 (Fold Change)=1或-1。"

图7

差异表达基因的GO和KEGG分析 A: 差异表达基因的GO富集分析; B: 差异表达基因的KEGG富集分析; C: ABA信号转导通路中的差异表达基因; D: GA生物合成中的差异表达基因。"

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