| 摘要: |
| 【目的】从常用苹果砧木楸子中克隆MpSnRK2.4基因，并将其转化番茄（Lycopersicon esculentum）品种Micro-Tom，为研究其功能奠定基础。【方法】以楸子幼叶为材料，通过RT-PCR扩增获得MpSnRK2.4基因的完整开放阅读框序列；利用Gateway技术构建pGWB411-SnRK2.4植物过表达载体，通过根癌农杆菌介导法将此载体转入Micro-Tom番茄中，通过卡那霉素筛选和转基因番茄RT-PCR鉴定，获得阳性转基因株系，利用qRT-PCR技术研究不同转基因株系中MpSnRK2.4基因的表达水平。【结果】成功克隆到长度为1 220 bp的MpSnRK2.4基因片段，该基因包含长1 026 bp的完整开放阅读框，编码341个氨基酸残基。通过在线软件分析发现，该基因的gDNA序列包含8个内含子，9个外显子；预测编码蛋白MpSnRK2.4的分子质量为 38.475 ku，理论等电点（pI）为 6.06。系统进化树分析表明，MpSnRK2.4与水稻OsSAPK3蛋白的亲缘关系最近，序列比对显示MpSnRK2.4蛋白与已知的其他植物SnRK2s蛋白序列具有较高的同源性。转基因植株PCR鉴定结果表明，MpSnRK2.4基因已成功转入番茄植株中，经qRT PCR发现，不同转基因株系的MpSnRK2.4基因表达量存在差异，其中株系1的表达水平最高。【结论】克隆获得了MpSnRK2.4基因全长序列，并得到了10个含有该基因的Micro-Tom番茄转基因株系。 |
| 关键词: 楸子 SnRK2 基因克隆 转基因 |
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| 基金项目:“十二五”国家科技计划项目“早中熟优质多抗苹果、梨新品种、砧木选育研究”（2013BAD02B01-2） |
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| Cloning and transformation of MpSnRK2.4 gene from Malus prunifolia |
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QIN Yuan,SHAO Yun,LIANG Dong,et al
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| Abstract: |
| 【Objective】MpSnRK2.4 gene cloned from Malus prunifolia was transformed into Lycopersicon esculentum cultivar Micro Tom to provide basis for understanding its functions.【Method】 The full-length ORF sequence of MpSnRK2.4 was cloned from young leaves of Malus prunifolia.The overexpression vector of pGWB411-SnRK2.4 was constructed by Gateway technique and transformed into Micro-Tom by the Agrobacterium mediated transformation method.The transgenic lines were identified by qRT-PCR after kanamycin screening and the expression of MpSnRK2.4 was analyzed in different transgenic lines.【Result】A sequence of 1 220 bp was cloned for MpSnRK2.4 gene.This sequence contained a full-length of ORF sequence (1 026 bp) encoding 341 amino acids.The gDNA sequence contained 8 introns and 9 extons,the predict molecular weight was 38.475 ku and isoelectric point was 6.06.Phylogenetic tree indicated that MpSnRK2.4 was most homologous with Oryza sativa OsSAPK3 and amino acid sequence alignment showed that MpSnRK2.4 was highly homologous with other known SnRK2s protein sequences.PCR test indicted that MpSnRK2.4 expressed in tomato successfully and qRT-PCR showed that MpSnRK2.4 expressed differently in transgenic lines with the highest expression in line 1.【Conclusion】MpSnRK2.4 gene was cloned and 10 transgenic Micro-Tom lines were obtained. |
| Key words: Malus prunifolia SnRK2 gene clone transgene |
