According to the manufacturer's instructions of the pMD19-T vector kit (Takara Bio, Inc.), the TFF3-encoding gene and 6×his tag were linked to the T vector to generate pMD19-T-TFF3-6×his and transformed into E. coli DH5α. Positive clones were identified by colony PCR, where a single bacterium is used as a template, and can quickly identify whether the colony is a positive colony with the target gene. Plasmids extracted from the positive clones were sequenced by Takara Bio, Inc. The primers were forward, 5′-GCgtcgacATGGAGGCCAGGATGT-3′ and reverse, 5′-CGGggtaccTTAGTGATGTGATGGTGATG GAAGGTGCATTCT-3′; lower-case letters denote the enzyme restriction sites for SalI and KpnI and the underlined bases denote the nucleotide sequence encoding the 6×his tag. To construct the sus scrofa TFF3 expression vector pNCMO2-TFF3-6×his, the fused TFF3-6×his combined fragment from pMD19-T-TFF3-6×his was amplified by PCR. The following thermocycling conditions were used for the PCR: initial denaturation at 95°C for 10 min; followed by 35 cycles of 95°C for 30 sec, 56°C for 30 sec and 72°C for 30 sec; and a final extension 72°C for 10 min. Digestion with SalI and KpnI followed by ligation at 16°C overnight was used to clone the amplified fragment into the pNCMO2 vector to generate pNCMO2-TFF3-6×his. DNA sequencing was performed to confirm the cloned DNA sequence (Takara Bio, Inc.).
Pmd19 t vector kit
Manufactured by Takara Bio
Sourced in Japan
The PMD19-T vector kit is a plasmid-based cloning system designed for the efficient insertion and expression of target genes in a variety of host cells. The core function of this product is to provide a versatile platform for the subcloning and expression of DNA sequences.
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3 protocols using pmd19 t vector kit
1
Cloning and Expression of Sus scrofa TFF3
2
Validation of Gene Expression by qRT-PCR
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qRT-PCR analysis was performed to validate the expression pattern of selected genes identified by the digital expression analysis. The RNA separately extracted from all of the 16 petal samples was used. First strand cDNA was synthesized from 1 μg of total RNA using PrimeScript RT (Perfect Real Time) kit (TAKARA, Japan). The correctness of the gene sequences in the reference transcriptome was validated by reverse transcription PCR and TA cloning using PMD19-T vector kit (TAKARA), followed by sequencing. The qRT-PCR reactions with gene-specific primers (Supplementary Table 1 ) were performed using a Real-time PCR Detection Systems (RocheCycler 480, Roche, Germany) and SYBRGreen PCR Master Mix (Roche, Germany) according to the manufacturer's instructions. Three independent biological replicates were performed for each reaction. Expression levels of the selected unigenes were normalized to that of an internal reference gene, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), which was revealed as a stably expressed gene by our Illumina sequencing data and previous studies (Wang et al., 2012 (link)). As the PCR efficiency for all the gene-specific primers ranged between 93 and 107% over 1000-fold of cDNA dilution (Supplementary Figure 1 and Supplementary Table 1 ), relative expression levels were calculated using the 2−ΔΔCt method (Livak and Schmittgen, 2001 (link)).
3
Molecular Cloning and Phylogenetic Analysis of BcNRT Genes
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We isolated total RNA using TRIzol. Reverse transcriptase reaction was performed with a reverse transcription kit (TaKaRa Bio, Tokyo, Japan) to obtain cDNA. The specific primers of three BcNRT genes (BcNRT1.1, BcNRT1.2, and BcNRT2.1) are designed based on the sequence of Arabidopsis (AtNRT1.1 Gene ID: 837763, AtNRT1.2 Gene ID: 843321, AtNRT2.1 Gene ID: 837327) (Table S1). The CDS sequence of BcNRT genes was amplified using PCR with specific primers. The NRT cloned products were recovered with a DNA gel recovery kit (Shanghai Shenggong) and NRT gene fragments were connected to the T-vector using the pMD19-T Vector Kit (TaKaRa Bio, Tokyo, Japan). We obtained BcNRT1.1, BcNRT1.2, and BcNRT2.1 gene sequences for subsequent analysis after they were sent to Invitrogen (Shanghai, China) Corporation for sequencing. A phylogenetic tree was constructed using Mega7.0 software, and DNAMAN software was used for homology comparison [36] (link).
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