龙眼TPI基因的克隆及其在体胚发生过程中的表达分析

 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1 084 bp和1 113 bp,其ORF都由765 bp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。

Cloning of TPI Gene from Embryogenic Callus and Its Expression Analysis During Somatic Embryogenesis in Longan

The complete cDNA sequence of triosephosphate isomerase gene(TPI)of longan embryogenic callus was obtained by reverse transcription polymerase chain reaction(RT-PCR)with rapid amplification of cDNA ends(RACE). And the mRNA transcription level of this gene was surveyed by real-time reverse transcription PCR(qRT-PCR)during somatic embryogenesis in longan. The results showed that the full-length cDNA sequences of two type longan embryogenic callus TPI were cloned, namely TPIⅠ(GenBank accession number FJ595244)and TPIⅡ(GenBank accession number GU073294). The complete nucleotide sequences of two TPI were 1 084 bp and 1 113 bp respectively. And they all contained a 765-nucleotides-long open reading fram(eORF)which encoded a protein of 254 amino acid residues. The results showed that the mRNA transcription levels of longan TPI were different at the different stages of longan somatic embryogenesis. In conclusion,it was preliminarily revealed that two type TPI genes and their encoded functional proteins may play an important role during the early somatic embryogenesis in longan.