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作物学报 ›› 2023, Vol. 49 ›› Issue (8): 2288-2295.doi: 10.3724/SP.J.1006.2023.22056

• 研究简报 • 上一篇    下一篇

水稻种子快速萌发突变体rgs1的鉴定及调控基因克隆

贾璐绮(), 孙悠, 田然, 张学菲, 代永东, 崔志波, 李杨羊, 冯新宇, 桑贤春(), 王晓雯()   

  1. 西南大学农学与生物科技学院 / 转基因植物与安全控制重庆市重点实验室, 重庆 400716
  • 收稿日期:2022-09-27 接受日期:2022-11-25 出版日期:2023-08-12 网络出版日期:2022-12-02
  • 通讯作者: 桑贤春,王晓雯
  • 作者简介:E-mail: 632063809@qq.com
  • 基金资助:
    重庆市自然科学基金项目(cstc2020jcyj-msxm0539);国家重点研发计划项目(2022YFD1201600);国家级大学生创新创业训练计划项目(202210635039);国家自然科学基金项目(32171964)

Identification of the rgs1 mutant with rapid germination of seed and isolation of the regulated gene in rice

JIA Lu-Qi(), SUN You, TIAN Ran, ZHANG Xue-Fei, DAI Yong-Dong, CUI Zhi-Bo, LI Yang-Yang, FENG Xin-Yu, SANG Xian-Chun(), and WANG Xiao-Wen()   

  1. College of Agronomy and Biotechnology, Southwest University / Chongqing Key Laboratory of Application and Safety Control of Genetically Modified Crops, Chongqing 400716, China
  • Received:2022-09-27 Accepted:2022-11-25 Published:2023-08-12 Published online:2022-12-02
  • Contact: SANG Xian-Chun,and WANG Xiao-Wen
  • Supported by:
    Natural Science Foundation of Chongqing, China(cstc2020jcyj-msxm0539);National Key Research and Development Program of China(2022YFD1201600);College Students’ Innovation and Entrepreneurship Training Scientific Research Project(202210635039);National Natural Science Foundation of China(32171964)

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摘要：

水稻种子快速萌发, 可减短吸水膨胀至出土成苗时间, 减少有害生物危害, 进而有效提高种子成苗率, 有利于培养强壮秧苗。为此, 本研究对缙恢10号EMS (甲基磺酸乙酯)诱变体库进行了筛选, 从中鉴定到一个种子萌发快且矮化多蘖的突变体rgs1, 进一步分析发现矮化是由于各节间均缩短造成的, 而多蘖则是分蘖芽发育较快引起的。遗传分析表明rgs1的突变性状受单隐性核基因调控, 定位区间内叶绿体ζ-胡萝卜素异构酶编码基因LOC_Os12g21710的第3个外显子发生了G至A的碱基替换, 导致蛋白翻译提前终止, 从而确定为目的基因。qPCR分析发现, 与野生型相比, 突变体中独角金内酯(SL)合成基因HTD1、D27、D10及信号传导相关基因D14、TB1的表达均极显著降低, SL信号途径抑制因子D53的表达则极显著升高, 表明rgs1矮化多蘖性状与SL缺陷有关。在rgs1中, 脱落酸(ABA)合成代谢关键酶基因OsNCED1几乎无表达, MOC3和FON1的表达极显著升高, 表明rgs1种子的快速萌发与ABA合成缺陷有关。RGS1可能是水稻体内精准调控SL和ABA协同作用的一个关键基因, 从而调控水稻种子萌发、株高、分蘖等的发育。

关键词: 水稻(Oryza sativa L.), 矮化多蘖, 种子萌发, 叶绿体ζ-胡萝卜素异构酶(T20/MIT1), 脱落酸(ABA)

Abstract:

Rice seed absorbed water and germinated rapidly in the paddy field, which could reduce the probability of damage from the pest and germs by shortening the stay time in soil, and thus enhancing seed surviving rate and facilitating the formation of strong seedlings. To exploer the molecular mechanism of seed germination, a mutant rgs1 (exhibiting rapid seed germination, dwarfism, and multi-tillers etc) was identified by screening the EMS (ethyl methanesulfonate) induced library of Jinhui 10 (WT). The dwarfism of rgs1 originated from the shortened internodes and the characteristic of multi-tiller was caused by the rapid growth of tiller buds. Genetic analysis showed that the mutational traits of rgs1 was controlled by a single recessive nuclear gene. LOC_Os12g21710, encoding a chloroplast ζ-carotene isomerase in the mapping region, had a base substitution (G in rgs1 and A in wild type) in the third exon, resulting in the earlier termination of protein translation, which was regarded as the candidate gene of RGS1. The qRT-PCR analysis showed that, compared with WT, the relative expression levels of HTD1, D27, and D10 controlling strigolactone (SLs) synthesis and of D14 and TB1 regulating SLs signal transduction decreased significantly and the relative expression level of D53 acting as a repressor of SLs signaling increased significantly, suggesting that the mutational characteristics of rgs1 were related with the SLs pathway. The transcriptional level of OsNCED1, encoding a key enzyme of abscisic acid (ABA) synthesis, was almost absent in rgs1, while the relative expression levels of MOC3 and FON1 were significantly enhanced, indicating that the rapid seed germination of rgs1 was associated with the defect of ABA synthesis. Therefore, we predicted that RGS1 should be a key gene regulating the cooperation of SL and ABA pathway, thus regulating seed germination, plant height, and tiller number in rice.

Key words: rice (Oryza sativa L.), tillering dwarf, seed germination, chloroplast ζ-carotene isomerase (T20/MIT1), abscisic acid (ABA)

表1

基因定位及相关基因定量引物序列"

引物名称
Primer name		 正向引物
Forward primer (5°-3°)		 反向引物
Reverse primer (5°-3°)
In12-1 GCTATGTCAAACACGGTCTTATT CTGGTGTATCCAACGCTTGT
In12-2 AGAGAGAGGAGAGGCTAGAG AAGTAGTTTGATTCTTTCATCCC
ACTIN GACCCAGATCATGTTTGAGACCT CAGTGTGGCTGACACCATCAC
HTD1 TCAAGCTGCTCCTACCAGTGGTT TGGTTGGCGTAGCTTGGGTTT
D27 GATGGCTACGAGAGCCTGATAGAT AGATTCCATAACCTCACACGGC
D10 CGTTCGTGACGTTCCACTTCATC TGTTGGCGTTGTGCTCGCA
D14 TTCTTGAACGACAGCGACTACCAC TTGAAGAGGGTGCGGCTGAA
D53 AAACGGTCACCTCTTGCTGCA TGTCATTCCTGTTAGGACCACGG
D3 TCAACCCACGGTTCAGCGATT TCCAGCACATTGTGCTGGAGAT
TB1 ATGGACATACCGCTTTACCAACAG GGTGGTAGTAGAAGAAGGTGGAATG
MOC3 GATCAAGATCCTGCGGGAGCTGTAC GAGGCGCTTCTTCTGGCGCTC
FON1 TGCGTGAAGGAGGACAACATCATC AGCAGCAGGTTCGTCTCCCTGTT

图1

野生型(WT)和突变体(rgs1)的种子萌发及脱落酸合成关键基因的形态特征和qRT-PCR分析 A~D: WT和rgs1种子播种在芽床上萌发1 d (Fig. A)、2 d (Fig. B)、3 d (Fig. C)和4 d (Fig. D)的形态特征, Bar: 5 mm; E: WT和rgs1种子播种后胚根长统计; D2: 种子萌发48 h; D3: 种子萌发72 h; D4: 种子萌发96 h; F~H: ABA合成途径关键基因在WT和rgs1胚根中的表达; *: 在P < 0.05水平差异显著; **: 在P < 0.01水平差异显著。"

图2

野生型(WT)和突变体(rgs1)的形态学和细胞学观察 A: WT和rgs1分蘖期植株, 标尺为20.00 cm; B: WT和rgs1成熟期植株, 标尺为20.00 cm; C: WT和rgs1的穗及倒1~5节间(从左至右), 标尺为5.00 cm; D: WT和rgs1株高构成因素统计分析(PL、1st、2nd、3rd、4th、5th分别代表穗长、倒1节间、倒2节间、倒3节间、倒4节间和倒5节间); E: WT和rgs1的有效穗数; F、G: WT和rgs1的四叶期茎秆基部石蜡切片, 红色箭头所指处为分蘖芽, 标尺为200 μm; *在P < 0.05水平差异显著; **在P < 0.01水平差异显著。"

图3

RGS1目的基因的分子定位及鉴定 A: RGS1在12号染色体上的分子定位; B: RGS1候选基因LOC_Os12g21710, 黑框代表外显子; C: 野生型WT和rgs1突变位点测序验证, *为突变位点, 红色字母为突变碱基。"

图4

水稻分蘖发育调控基因在野生型(WT)和突变体rgs1中的表达分析 A: RGS1在野生型缙恢10号中的表达模式; B~H: 分蘖调控相关基因在WT和rgs1中的表达分析; **: 在P < 0.01水平差异显著。"

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