Molecular cloning, bioinformatics characterization, and gene expression analysis of full-length expansin gene cDNA from Tetrastigma hemsleyanum / 中草药

ABSTRACT

Objective: To clone the sequence and characterize gene expression of an expansin gene from Tetrastigma hemsleyanum, and to predict its probable function. Methods: According to the conservative region sequences of expansin gene in GenBank database, one pair of degenerate primers were designed for RT-PCR. The conservative region fragment was first amplified by RT-PCR from cDNA template of T. hemsleyanum. The full-length sequence of expansin gene cDNA (named as Th-exp) was extended by RACE (rapid-amplification of cDNA ends). Th-exp expression levels in different organs were conducted by semi-quantitative RT-PCR and fluorescence quantitative PCR. Results: The full-length cDNA sequences of Th-exp gene were obtained in 782 bp. Th-exp gene containsed a 630 bp open reading frame, which encoded a protein of 209 amino acids (GenBank accession number KP693606). The protein encoded by Th-exp habours a typical expansin structure, including eight cysteine domains in N terminal region, four conserved tryptophan domain in C terminal region, and histidine function domain in intermediate region. Blast alignment showed that Th-exp was similar to expansin genes of Vitis vinifera and Tarenaya hassleriana. Semi-quantitative RT-PCR and fluorescence quantitative PCR indicated that Th-exp could express in the leaves, stems, ordinary fine roots, and calabash-shaped roots, while the expression level in calabash-shaped roots was higher than those in other organs. Conclusion: The expansin gene (Th-exp) is cloned from T. hemsleyanum, and it could be involved in the development of root tubers in T. hemsleyanum.