水稻苗期致死突变体的鉴定及其基因定位

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

摘要/Abstract

摘要：

肽链释放因子RF1通过识别终止密码子在翻译终止过程中起重要调控作用。获得水稻苗期致死突变体，明确OseRF1-1表达模式，为进一步研究OseRF1-1功能奠定基础。以粳稻花之舞和T-DNA插入突变体为材料，利用Tail-PCR、石蜡切片技术、Southern杂交和Northern杂交技术鉴定一个肽链释放因子eRF1-1的T-DNA单拷贝插入突变体ls。采用DNAMAN6.0软件和SMART对OseRF1-1进行生物信息学分析，利用RT-qPCR方法分析OseRF1-1的组织特异性表达。通过PEG介导方法转化拟南芥，明确OseRF1-1的亚细胞定位。结果显示，ls突变体植株在3-5叶分蘖期迅速死亡；ls突变体叶鞘与分蘖节连接区域褐化，分蘖节越往下部位症状较严重；Tail-PCR分析结果显示，ls突变体T-DNA插入时丢失了T-DNA的右边界和NOS的终止子序列导致GUS和OseRF1-1融合转录，而GUS的终止密码子仍然存在，进而引起OseRF1-1不能进行正常翻译；ls突变体转录出GUS和OseRF1-1的融合转录子；OseRF1-1在各组织中均检测到表达且在穗中高表达；该基因定位于细胞核和细胞质。ls突变体导致水稻苗期致死可能是由于T-DNA插入OseRF1-1导致该基因不能正常转录引起。

关键词: 水稻, 肽链释放因子1, ls突变体, 苗期致死

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

唐跃辉, 赵雨凡, 林锦, 王胤, 曹博远, 车怡帆, 杨文杰, 包欣欣, 杨同文. 水稻苗期致死突变体的鉴定及其基因定位[J]. 生物技术通报, 2022, 38(10): 124-131.

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.

图/表 9

表1 本研究所需引物

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

图1 野生型和ls突变体表型 a：3-5叶期的ls突变体表型；b：野生型和ls突变体植株苗期茎基部和叶鞘表型

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

图2 野生型和ls突变体叶鞘与分蘖节部位显微镜结构分析 a：WT；b-f：ls突变体。Bar=1 mm

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

图3 野生型和ls突变体横切面结构 I-IV：野生型和ls突变体分蘖节从下往上横切面

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

图4 ls突变体基因组DNA Southern杂交以潮霉素基因序列为探针，X：Xba I；Ev：EcoR V；EI：EcoR I

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

图5 ls突变体中T-DNA插入位置分析 a：T-DNA插入位点；b：T-DNA插入转录本分析；c：以GUS基因序列作为探针，Northern杂交；d：以eRF1-1基因序列作为探针，Northern杂交

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

图6 eRF1和其同源蛋白氨基酸序列分析 OseRF1-1：水稻，Os01g0939500；OseRF1-2：水稻，XP_015630898；OseRF1-3：水稻，XP_025881251；OseRF1-4：水稻，XP_015647295；AteRF1-1：拟南芥，AT5G47880；AteRF1-2：拟南芥，AT1G12920；AteRF1-3：拟南芥，AT3G26618；TaeRF1：小麦，KAF6986654；ZmeRF1：玉米，ACG27998；AleRF1-3：玉山筷子芥，XP_002875327；BneRF1-3：中华菊头蝠，XP_019577167

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

图7 OseRF1家族基因的表达模式分析

Fig. 7 Expression profile of OseRF1 family genes

图8 OseRF1-1的亚细胞定位

Fig. 8 Subcellular localization of OseRF1-1

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