Cloning Full-length Gene from Oenanthe japonica

Flowers of O. japonica were selected for RNA extraction by using RNA pure Plant Kit (CWBIO, China) according to the instructions. Subsequently, one μg total RNA was used to synthesize the cDNA through the method described previously (Sun et al., 2019 (link)). Specific primers for cloning the full-length gene coding sequence were designed based on the assembled transcriptomic information. After PCR, the products of appropriate length were sub-cloned into the pMD18-T vector (Takara, Japan) and verified by sequencing. The list of primers used for gene cloning is provided in Table S1. Multisequence alignment of deduced protein sequences was analyzed using DNAMAN 6.0. And the Neighbor-Joining phylogenetic tree was constructed with MEGA version 7.0 software using 1,000 bootstrap replicates.

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Sun W., Zhou N., Feng C., Sun S., Tang M., Tang X., Ju Z, & Yi Y. (2021). Functional analysis of a dihydroflavonol 4-reductase gene in Ophiorrhiza japonica (OjDFR1) reveals its role in the regulation of anthocyanin. PeerJ, 9, e12323.

Publication 2021

RNA pure Plant Kit pMD18-T vector

Cdna Coding sequence Flowers Gene Plant Primers Protein Sequences alignment Transcriptomic Vector

Corresponding Organization :

Other organizations : Guizhou Normal University, Guiyang College of Traditional Chinese Medicine

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Variable analysis

independent variables

Flowers of O. japonica were selected for RNA extraction

dependent variables

RNA extraction
CDNA synthesis
Cloning the full-length gene coding sequence
Multisequence alignment of deduced protein sequences
Neighbor-Joining phylogenetic tree construction

control variables

RNA pure Plant Kit (CWBIO, China) instructions were followed for RNA extraction
The method described previously (Sun et al., 2019) was used for cDNA synthesis
Primers were designed based on the assembled transcriptomic information
PCR products of appropriate length were sub-cloned into the pMD18-T vector (Takara, Japan) and verified by sequencing
DNAMAN 6.0 was used for multisequence alignment of deduced protein sequences
MEGA version 7.0 software using 1,000 bootstrap replicates was used for Neighbor-Joining phylogenetic tree construction

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