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筛选74个花生种质材料,最后以发芽率在80%以上的9650作为试验材料。对9650分别进行2℃和25℃(对照)处理,分别提取2、6和8h花生样品的总RNA。利用GeneFishing随机引物,克隆测序,获得34个独特的基因序列。经BLAST2GO注释,包括参与细胞合成的组分、应激蛋白反应、代谢过程中的转运与运输、蛋白质的合成、以及耐低温相关的基因序列。进一步利用定量RT-PCR证实了4个基因的相对表达量,选择其中亲环蛋白基因相关序列,通过5’-RACE获得cDNA全长,为通过转基因技术对其进行功能分析奠定了基础。
74 peanut germplasm materials were screened, and the 9650 with the germination rate above 80% was used as the test material. The total RNA of peanut samples of 2, 6 and 8 h were extracted from 9650 at 2 ℃ and 25 ℃ (control) respectively. Using GeneFishing random primers, cloned and sequenced to obtain 34 unique gene sequences. The BLAST2GO annotation includes components involved in cell synthesis, stress protein reactions, transport and trafficking during metabolism, protein synthesis, and cryogenic-related gene sequences. The relative expression levels of four genes were confirmed by quantitative RT-PCR. The sequences of the cyclophilin gene were selected and the full-length cDNA was obtained by 5’-RACE, which laid the foundation for the functional analysis of the gene by transgenic technology.