Cloning and Tissue Expressions of Sucrose Phosphate Synthase Gene in Cassava
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摘要: 通过同源克隆从木薯品种辐选01（RS01）中获得SPS基因保守序列，利用RACE扩增技术获得全长cDNA序列并使用相关软件进行生物信息学分析，并采用实时荧光定量PCR分析木薯SPS基因在不同时期、不同组织中的相对表达量。结果表明：克隆获得的木薯SPS基因cDNA序列全长3857 bp，开放阅读框（ORF）长3228 bp，编码1076个氨基酸，命名为MeSPS（GenBank登录号KX822780）；同源性分析表明，MeSPS基因与麻风树、蓖麻和荔枝的SPS基因序列同源性较高，分别为89%、89%和87%；其氨基酸序列与其他植物SPS氨基酸同源性在77%~92%；经qRT-PCR分析结果表明，MeSPS在苗期的相对表达量较高；在同一生长时期，MeSPS在叶片中相对表达量高于茎段和块根。Abstract: Sucrose phosphate synthase (SPS) gene from cassava RS01 was cloned to study its biological functions. The conserved fragment of the gene was first obtained by homologous cloning, and its full length cDNA by rapid amplification of cDNA ends (RACE). The characteristics, gene sequence and other biological information retrieved were analyzed. Expressions of the genes from various tissues of the plants at different times were compared by RT-qPCR. The results showed that the cDNA of SPS gene had a full length of 3, 857 bp, and an ORF of 3, 228 bp encoding a protein of 1, 076 amino acids. It was subsequently named MeSPS with a GenBank number of KX822780. Its homologous alignments with the nucleotide and protein databases were highly homologs with those of Jatropha carcas L., Ricinus communis L., and Litchi chinensis Sonn. at 89%, 89%, and 87%, respectively. Its amino acid sequence was 77%-92% in similarity with the SPS genes of other plants. The qRT-PCR analysis showed that MeSPS gene was highly expressed during seeding, and, was more expressed in the leaves than the stems or roots on a plant in a same growth period.
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Key words:
- cassava /
- sucrose phosphate synthase /
- gene cloning /
- expression analysis
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图 1 木薯总RNA
注：M为DL2000 DNA Maker; 1~2为木薯总RNA conservative region。
Figure 1. Total RNA of cassava
图 2 MeSPS保守区PCR扩增结果
注：DL5000 DNA Maker; 1为PCR产物。
Figure 2. Amplification results on MeSPS
图 4 3′末端RACE扩增结果
注：M为DL2000 DNA Maker; 1~2为3′RACE产物。
Figure 4. 3′RACE amplified results
图 5 MeSPS基因核苷酸序列及其编码的氨基酸序列
Figure 5. Nucleotide sequence of MeSPS gene and its deduced amino acid sequence
图 7 MeSPS在不同组织、不同时期的相对表达量
Figure 7. Relative expressions of MeSPS in various tissues at different times
表 1 基因克隆及实时荧光定量PCR引物序列
Table 1. Primers for MeSPS gene cloning and qPCR
引物名称 引物序列(5′-3′) MeSPS-F ATGGCTGGCAACGATTGGATT MeSPS-R CTATCCCTTGACACATGCTAATTGC MeSPS-5A CGACGAAATAGCGAGTGGGACTAAACCTT MeSPS-5B GACGACTTAGCATCATCCAGGC MeSPS-3F ATGCCAACAGAAACTACCCGCTCGC 3′RACE通用引物 GGCCACGCGTCGACTAGTACTTTTTTTTTTTTTTTTTT MeSPS-qF TGATACGGGTGGTCAGGTT MeSPS-qR CTGTGGGCTCACCATAACTC Actin-F CGATGGTCGTACAACTGGTAT Actin-R ATCCTCGAATCCAGACACTGT 
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