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Isolation of α-Tubulin CpTUA Gene from Cucurbita pepo and It’s Application as An Internal Standard
Zhu Haisheng, Liu Jianting, Wen Wenxu, Ye Xinru, Wang Bin, Li Yongping, Chen Mindong, Lin Hui, Wen Qingfang*
Crops Research Institute, Fujian Academy of Agricultural Sciences, Vegetable Research Center, Fujian Academy of Agricultural Sciences, Fujian Engineering Research Center for Vegetables, Fuzhou 350013, China
Abstract: The study was to obtain the α-tubulin gene of zucchini and design the qRT-PCR primers. A 1 863 bp cDNA was obtained by transcriptome sequencing and RT-PCR. This gene was named CpTUA and the GenBank accession was MH310440. Bioinformatics analysis results indicated that the sequence contained a size of 1 353 bp open reading frame (ORF) encoding 450 amino acids， with a theoretical molecular weight of 49.57 kDa and a protein isoelectric point (PI) of 4.84. Motif Scan analysis showed that CpTUA protein had the Tubulin and Tubulin-C domains of conserved actin in the position of 49―246 and 248―393 sites， respectively. Homol ogy analysis revealed that CpTUA shared 99% identity with the homologous proteins from Cucurbita moschata and Cucurbita maxima which were also belong to Cucumber the same as Cucurbita pepo， proving that it was highly conservative. A pair of qRT-PCR primers were then derived from the CpTUA gene sequence which had the high specificity and repeatability. The RT-PCR and qRT-PCR indicated that the CpTUA gene was stable expression in different tissues and under different stress treatments， so it was suitable as a reference gene for the analysis of gene expression patterns in zucchini. The present study has provided an important reference for analysis the expression of critical genes zucchini.
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