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目的克隆刺五加的液泡膜内在蛋白(tonoplast intrinsic proteins,TIP)基因,并对其进行生物信息学和表达分析。方法采用cDNA末端快速扩增(RACE)技术克隆刺五加TIP基因cDNA的全长序列。以GAPDH为内参照基因,通过RT-PCR法检测TIP基因在不同生长发育时期和器官中的表达情况。结果刺五加TIP基因cDNA的全长1 080 bp,开放阅读框长756 bp,编码251个氨基酸的蛋白,该蛋白包含TIP家族的标志性序列。刺五加的TIP蛋白具有6个跨膜螺旋,定位于液泡膜。表达分析结果显示,刺五加TIP基因在不同生长发育时期和不同器官中均有表达,但表达量具有显著差异(P<0.05)。其中盛花期的表达量最高,是最低量果实快速生长期的2.07倍;各器官中,叶片的表达量最高,是最低量根的1.73倍。结论首次分离到刺五加TIP基因的cDNA全长序列,并证实其在盛花期的叶中表达量最高,为进一步研究TIP基因对刺五加水分代谢的影响奠定基础。
Objective To clone the tonoplast intrinsic proteins (TIP) gene of Acanthopanax senticosus and analyze its bioinformatics and expression profile. Methods The full length cDNA of TIP gene of Acanthopanax senticosus was cloned by rapid amplification of cDNA ends (RACE). Using GAPDH as an internal reference gene, the expression of TIP gene in different growth stages and organs was detected by RT-PCR. Results The full-length cDNA of TIP gene was 150 bp in length and 756 bp in length. It encoded a 251 amino acid protein containing the TIP family marker sequence. Acanthopanax senticosus TIP protein has six transmembrane helices, located in the tonoplast. Expression analysis showed that the TIP gene of Acanthopanax senticosus was expressed in different growth stages and organs, but the expression level was significantly different (P <0.05). Among them, the highest expression was in full flowering stage, which was 2.07 times faster than the lowest fruit growth stage. Among all organs, leaf expression was the highest, which was 1.73 times of the lowest. Conclusion The full-length cDNA sequence of TIP gene from Acanthopanax senticosus was isolated for the first time and its expression level was the highest in full leaf during flowering. It laid the foundation for further study on the effect of TIP gene on the water metabolism of Acanthopanax.