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Acta Agronomica Sinica ›› 2023, Vol. 49 ›› Issue (8): 2288-2295.doi: 10.3724/SP.J.1006.2023.22056

• RESEARCH NOTES • Previous Articles     Next Articles

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 Online:2023-08-12 Published:2022-12-02
  • Contact: SANG Xian-Chun,and WANG Xiao-Wen E-mail:632063809@qq.com;sangxianchun@163.com;xwwang78@126.com
  • 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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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)

Table 1

Primer sequences used for gene mapping and qPCR analysis"

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

Fig. 1

Morphological characteristics and the relative expression levels of key genes in seed germination and abscisic acid synthesis between wild type (WT) and mutant rgs1 A-D: morphological characteristics of WT and rgs1 seeds that were seeded and germinated on the bud bed for 1 d (Fig. A), 2 d (Fig. B), 3 d (Fig. C), and 4 d (Fig. D), respectively. Bar: 5 mm; E: radicle length of WT and rgs1 seeds after sowing; D2: seed germination for 48 h; D3: seed germination for 72 h; D4: seed germination for 96 h; F-H: the qRT-PCR analysis of key genes regulating ABA synthesis at the radicle section; *: P < 0.05; **: P < 0.01."

Fig. 2

Comparison of morphology and cytology between wild type (WT) and mutant rgs1 A: the plant of WT and rgs1 at tillering stage, bar: 20.00 cm; B: the plant of WT and rgs1 at maturity stage, bar: 20.00 cm; C: the panicle and internodes of WT and rgs1 (listing from left to right), bar: 5.00 cm; D: plant height components of WT and rgs1 (PL presents panicle length, 1st, 2nd, 3rd, 4th, and 5th indicate the corresponding internode length, respectively); E: the number of effective panicles of WT and rgs1; F, G: paraffin section of WT and rgs1 based on the tiller bud position at four-leaf stage, the red arrow points to the tiller buds, bar: 200 μm; *: P < 0.05; **: P < 0.01."

Fig. 3

Gene mapping and identification of RGS1 candidate genes A: gene mapping of the RGS1 on chromosome 12; B: frame diagram of LOC_Os12g21710, the candidate of RGS1, and the black rectangles represent exons; C: the sequence of mutational position in wild-type and mutant rgs1. * represents the mutational loci, and red letter represents the mutant base."

Fig. 4

Relative expression level of genes controlling rice tiller development between wild type (WT) and mutant rgs1 A: the relative expression pattern of RGS1 genes in wild type Jinhui 10; B-H: the relative expression level of genes regulating tiller growth in WT and rgs1; **: P < 0.01."

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