About 0.5 g of the surface-disinfected reed roots were frozen with liquid nitrogen and ground to a fine powder in a sterilized and precooled mortar. Then, the hot cetyltrimethylammonium bromide (CTAB) procedure (Xie et al., 1999) was used to extract the total DNA. The DNA was then resuspended in 25 μL of sterile Milli-Q water. The pair of primers 799f (5′-AACAGGATTAGATACCCTG-3′) and 1492r (5′-GGTTACCTTGTTACGACTT-3′) (Chelius & Triplett, 2001) was selected to amplify the DNA of reed endophytic bacteria. The 50-μL PCR mixture contained 100 ng of DNA extract, 5 μL 10 × Taq reaction buffer (including 1.5 mM MgCl2), 10 pmol of each primer, www.selleckchem.com/products/Rapamycin.html 200 μM each dNTP, and 1.5 U of Taq DNA polymerase (Takara
Co.). After initial denaturation at 94 °C for 5 min, each thermal cycling was as follows: denaturation at 94 °C for 1 min, annealing at 53 °C for 1 min, and elongation at 72 °C for 1 min. At the end of 30 cycles, the final extension step was at 72 °C for 15 min. Products of four parallel PCRs were combined and separated electrophoretically. A band approximately 700 bp in size in the electrophoresis pattern was excised from a 1% agarose gel and purified using the Gel Extraction Kit (Omega Co.) as described by the manufacturer. The purified PCR products were ligated into the pMD18-T vector (Takara Co.). Escherichia coli Top10 competent cells (Tiangen
Co.) were transformed with the ligation PI3K Inhibitor Library supplier products and spread onto LB agar plates with ampicillin (100 mg L−1) for standard blue and white screening (Sambrook et al., 1989). Randomly selected colonies were screened directly for inserts by performing colony PCR with Venetoclax chemical structure primers RV-M (5′-GAGCGGATAACAATTTCACACAGG-3′) and M13-47 (5′-CGCCAGGGTTTTCCCAGTCACGAC-3′) for the vector (Takara Co.). A total of 180 clones containing inserts of the correct size were sequenced
using an ABI PRISM 3730 automatic sequencer (Shanghai Sangon Co. Ltd). After being trimmed by removing the vector sequences using the editseq program in the dnastar package (Burland, 2000), clones with >97% sequence identity were grouped into one operational taxonomic unit (OTU) by sequencher 4.8 (Gene Codes, Ann Arbor, MI). All the nucleotide sequences, approximately 700 bases, were compared with the NCBI database using blastn or aligned by the identify analysis of EzTaxon server 2.1 (Chun et al., 2007). Sequences with >97% similarity were assigned to the same species and those with >95% similarity were assigned to the same genus. The sequences were aligned using clustal w (Thompson et al., 1994), and tree constructions were performed with the mega 3 program package (Kumar et al., 2004) using the neighbor-joining method. Bootstrap analysis was performed using data resampled 1000 times. The trees were constructed by calculating Kimura distances (Kimura, 1980).