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采用RT-PCR结合RACE法,通过T/A克隆测序,获得龙眼胚性愈伤组织磷酸丙糖异构酶基因(TPI)两个类型的全长序列TPIⅠ(GenBank登录号:FJ595244)和TPIⅡ(GenBank登录号:GU073294),序列全长分别为1084bp和1113bp,其ORF都由765bp核苷酸组成,编码254个氨基酸,与其它植物的TPI在核苷酸序列和推导的氨基酸序列的相似性较高。qRT-PCR结果分析表明,在龙眼体胚发生过程中,TPI在松散型胚性愈伤组织阶段的转录水平比较低,在胚性愈伤组织Ⅱ阶段转录水平急剧升高,并一直持续到胚性紧实球形结构阶段,球形胚阶段大幅下调,之后至子叶形胚阶段都维持在较低水平,初步证明TPI及其编码蛋白在龙眼启动体胚发生和早期体胚发育中行使重要的功能。
The full-length TPIⅠ (GenBank accession number: FJ595244) and TPIⅡ (TPJ) were cloned by RT-PCR and RACE from T / A clones. GenBank accession number: GU073294). The full length of the sequence was 1084bp and 1113bp, respectively. The ORFs consisted of 765bp nucleotides and encoded 254 amino acids. Compared with the nucleotide sequences and deduced amino acid sequences of other plant TPIs, high. Analysis of qRT-PCR results showed that during the process of longan somatic embryogenesis, the transcript level of TPI was relatively low at the stage of loose embryogenic callus, and the level of transcript in phase Ⅱ of embryogenic callus increased rapidly and continued until embryo At the stage of sexual compact spherical structure, the stage of globular embryo was significantly reduced, and then the stage of cotyledon embryo was maintained at a low level, which initially demonstrated that TPI and its encoded protein played an important role in the initiation of somatic embryogenesis and early somatic embryogenesis in longan.