Cloning and Sequence Analysis of OsSDR1 Gene from Restorer Line of Hybrid Rice Fuihui 7185
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摘要: 植物种子从休眠到萌发的转变是其生命周期中一个关键的生理过程，其中，植物生长调节剂发挥了重要的调控作用，而SDR1（short-chain dehydrogenase/reductase 1）是调控营养信号转导和植物生长调节剂合成的关键酶。本研究克隆获得了福恢7185、福恢653和云恢290中SDR1基因的CDS序列，比对发现其在3个不同休眠品种并无差异，但与NCBI上日本晴的基因序列相比，缺失了9个碱基，3个丙氨酸；结合qRT-PCR技术分析SDR1基因在不同休眠水稻品种种胚中的表达情况，探索其与种子休眠的关系，同时利用GST pull-down技术找到SDR1的1个互作蛋白，结果表明，该蛋白与休眠相关，初步表明SDR1基因可能通过与其它结构原件的互作来调控种子休眠。Abstract: The transition from dormancy to germination in seeds is a key physiological process during the lifecycle of plants. Hormones play an important regulatory role on it, While, short-chain dehydrogenase/reductase (SDR1) is a molecular link between nutrient signaling and plant hormone biosynthesis. The coding sequences (CDS) of SDR1 were cloned from different dormancy varieties FH7185, FH653 and YH290, respectively. The results of sequences blast indicated that the coding regions of OsSDR1 among the three varieties were homologous, but there were 9 bases deletion, coding three alanine compared to Nipponbare. Meanwhile, the results of real-time quantitative PCR (qRT-PCR)of SDR1 expression level in seed embryo among different dormancy varieties indicated that the expression level of SDR1 was significantly different among them. Moreover, the corresponding interaction proteins of OsSDR1 were screened by GST pull-down experiments and an interaction protein of OsSDR1 was found and its Genbank serial number designated as Os05g0140800, it was verified related to seed dormancy. The preliminary results showed that SDR1 gene might regulate seed dormancy through interaction with other structural elements.
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Key words:
- rice /
- Fuhui 7185 /
- OsSDR1 /
- cloning /
- sequence analysis /
- interaction protein
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图 1 不同水稻材料休眠性分析
注：A为种子发芽试验；B为种子发芽率情况。
Figure 1. Seed dormancy of different rice varieties
图 2 FH7185、FH653和YH290中OsSDR1编码区序列的克隆
Figure 2. CDS cloning of OsSDR1gene from FH7185, FH653 and YH290
图 3 FH7185、FH653、YH290和日本晴中OsSDR1编码区的序列比对
Figure 3. The sequence blast of OsSDR1 CDS between FH7185, FH653, YH290 and Nipponbare with target sequence
图 4 OsSDR1基因在不同水稻材料中的差异表达分析
Figure 4. The expression analysis of OsSDR1 gene in different rice varieties by qRT-PCR
图 5 OsSDR1蛋白和空载体的诱导表达
注：M为180 kDa；1为诱导沉淀；2、3为诱导上清；4为对照沉淀；5、6为对照上清。
Figure 5. Induction expression of OsSDR1protein and empty vector
图 6 OsSDR1蛋白和空载体的纯化
注：A：M为180 kDa；1~4为纯化蛋白；B：M为180 kDa；1~3为对照纯化蛋白。
Figure 6. Purification of OsSDR1 protein and empty vector
图 7 OsSDR1蛋白和空载体的GST pull-down
注：M为180kDa；1、3为FH7185种子总蛋白；2为目的蛋白；4为对照蛋白。
Figure 7. GST pull-down of OsSDR1 protein and empty vector
表 1 SDR1基因实时定量PCR与分子克隆引物信息
Table 1. Primers used in SDR1 qRT-PCR and molecular cloning
引物 序列 OsSDR1-qRT-PCR-F ATGGATGTCCGCCGCCGCCGCC OsSDR1-qRT-PCR-R TTAATCTTCAAATGCTCTCAAATTGTGA Actin-F CCTCGTCTGCGATAATGGAACTG Actin-R CCCTGGGCGCATCATCTC OsSDR1-F ATGGATGTCCGCCGCCGCCGCC OsSDR1-R TTAATCTTCAAATGCTCTCAAATTGTGA OsSDR1-F(带酶切位点) GGATCC ATGTCCGCCGCCGC OsSDR1-R(带酶切位点) CTCGAG TTAATCTTCAAATGCTCT 
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表 2 OsSDR1蛋白的质谱分析结果
Table 2. The results ofmass spectrometry analysis of OsSDR1 protein
Locus Name Gene Description Os05g0140800 Similar to Dormancy related protein Os02g0718900 ADP, ATP carrier protein, mitochondrial precursor Os12g0632700 Similar to Malate dehydrogenase, glyoxysoma Os07g0609000 Derived by automated computational analysis using gene prediction method: Gnomon Os04g0404400 Derived by automated computational analysis using gene prediction method: Gnomon Os05g0438800 Similar to Actin 1 Os08g0434300 Similar to Malate dehydrogenase precursor
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