Identification and Gene Mapping of a Seedling Lethal Mutant in Rice

doi:10.13560/j.cnki.biotech.bull.1985.2022-0069

Abstract

Abstract:

Peptide release factor 1（RF1）plays a key role in the regulation of the translation termination by recognizing stop codons. Rice seedling lethal mutant was obtained，the expression profile of OseRF1-1 was clarified，and a basis for further function analysis of OseRF1-1 was provided. Using the Japonica rice cv. Huazhiwu and T-DNA insertion mutant as materials，Tail-PCR，paraffin section technology，Southern blotting and Northern blotting technologies were used to identify a T-DNA single-copy insertion mutant of the peptide release factor eRF1-1，named ls. DNAMAN6.0 software and SMART were applied to analyze the OseRF1-1’s bioinformatics characteristics，and RT-qPCR was used to analyze the tissue-specific expression of OseRF1-1. Transforming Arabidopsis thaliana by a PEG-mediated approach was to clarify the subcellular localization of OseRF1-1. The results suggested that the ls mutant rapidly died at the 3rd to 5th leaf tillering stages. Paraffin section showed that the junction region between leaf sheath and tiller node in ls mutant was browning，and the symptoms were more serious in the lower part of tiller nodes. Tail-PCR analysis showed that the right border of the T-DNA and the NOS terminator sequence in ls mutant were lost，resulting in a fusion transcript of GUS and OseRF1-1 in ls mutant. However，the stop codon remained in GUS，which led to the OseRF1-1 not translated normally. OseRF1-1 was expressed in all tissues tested，especially highly expressed in panicles. This OseRF1-1 gene was localized in nucleus and cytoplasm. Above results indicate that ls mutant causing rice seedlings lethal may be resulted from the abnormal transcription of OseRF1-1 gene by T-DNA insertion.

Key words: Oryza sativa L., peptide release factor 1, ls mutant, seedling lethal

TANG Yue-hui, ZHAO Yu-fan, LIN Jin, WANG Yin, CAO Bo-yuan, CHE Yi-fan, YANG Wen-jie, BAO Xin-xin, YANG Tong-wen. Identification and Gene Mapping of a Seedling Lethal Mutant in Rice[J]. Biotechnology Bulletin, 2022, 38(10): 124-131.

Figures/Tables 9

Table 1 Primers used in the study

引物名称Primer name	引物序列Primer sequence（5'-3'）	产物大小Product size/bp	用途Purpose
GUSF	GATGTTGGCGACCTCGTATT	354	探针制备Probe preparation
GUSR	ACAGCGTCTCCGACCTGAT
eRF1-1F	GCTACTCCTACAAAACACCAGATG	368
eRF1-1R	GCATACCAAGAAGGCAAGAAC
RAD1	ACGATGGACTCCAGAGCGGCCGCVNVNNNGGAA		Tail-PCR
RAD2	ACGATGGACTCCAGAGCGGCCGCBNBNNNGGTT
RAD3	ACGATGGACTCCAGAGCGGCCGCHNVNNNCCAC
RAD4	ACGATGGACTCCAGAGCGGCCGCVVNVNNNCCAA
RAD5	ACGATGGACTCCAGAGCGGCCGCBDNBNNNCGGT
AC	ACGATGGACTCCAGAG
RB1	GTCGTCGGTGAACAGGTATGGAAT
RB2	ACGATGGACTCCAGTCCGGCCTGGCGGTAACAAG- AAAGGGATCTTCACT
RB3	AAACCGCAGCAGGGAGGCAAAC
OseRF1-1F	CGAGCACGACACGAGCTTT	179	定量表达分析Quantitative expression analysis
OseRF1-1R	GTGCAGCATCAAGTCCTTTAAT
OseRF1-2F	AATCGTTCGTCCTGCTGCTT	151
OseRF1-2R	TTGCCTCTAGCAGACTCCAGTG
OseRF1-3F	ACGAACATCACCGCAACCT	178
OseRF1-3R	CGAAATCATGCTCGTACCGTT
OseRF1-4F	AAGTTCAGAGGCGAGGGTTT	176
OseRF1-4R	CTGACTCCAGAGCCTTGATTAG
RUB1F	AGGGTTCACAAGTCTGCCTATT	165
RUB1R	TTCCATGCTGCTCTACCACAG
OseRF1-1F OseRF1-1R	CGAGCACGACACGAGCTTT AGATCCAACGTCACAGGGTACAT	1 342	亚细胞定位载体构建引物 Subcellular localization vector construction primers

Fig. 1 Phenotype of wild type and ls mutant a：Phenotype of ls mutants at 3-5 leaf stage. b：Phenotype of stem base and sheath at seedling stage in wild type and ls mutant plants

Fig. 2 Microscopic structure analysis of wild type and ls mutants leaf sheaths and tiller node sites a：WT；b-f：ls mutants. Bar=1 mm

Fig. 3 Cross-sections of wild-type and ls mutant I-IV：Cross-section of tiller nodes of wild type and ls mutants

Fig. 4 Southern blot of genome DNA in the ls mutant Hygromycin gene sequence was used as probe. X：Xba I；Ev：EcoR V；EI：EcoR I

Fig. 5 T-DNA insertion site analysis of ls mutant a：T-DNA insertion site；b：T-DNA insert transcript analysis；c：GUS gene sequence was used as probe. Northern hybridization；d：eRF1-1 gene sequence was used as probe. Northern hybridization

Fig. 6 Analysis of amino acid sequences of eRF1 and its homologous proteins OseRF1-1：Oryza sativa Japonica，Os01g0939500；OseRF1-2：O. sativa，XP_015630898；OseRF1-3：O. sativa，XP_025881251；OseRF1-4：O. sativa，XP_015647295；AteRF1-1：Arabidopsis thaliana，AT5G47880；AteRF1-2：A. thaliana，AT1G12920；AteRF1-3：A. thaliana，AT3G26618；TaeRF1：Triticum aestivum，KAF6986654；ZmeRF1：Zea mays L.，ACG27998；AleRF1-3：Arabidopsis lyrata subsp. Lyrate，XP_002875327；BneRF1-3：Rhinolophus sinicus，XP_019577167

Fig. 7 Expression profile of OseRF1 family genes

Fig. 8 Subcellular localization of OseRF1-1

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