Superscript 3 rnase h reverse trancrip tase

Total RNAs of leaves were extracted by Easy Pure Plant RNA Kit (TransGene Biotech, Beijing) and then determined using Nano-Drop2000 spectrophotometer. First-strand cDNA was prepared using SuperScript III RNase H–Reverse Trancrip-tase (Invitrogen, USA) using 2 μg of total RNA. Quantitative RT-PCR procedures were carried out by the Light Cycler System (Bio-Rad, Richmond, CA) with the SYBR^® Premix Ex TaqTM Kit (TaKaRa, Dalian, China), in a 15 μL reaction solution containing 7.5 μL of SYBR^® Premix Ex TaqTM 2×, 0.3 μmol·L^–1 of each forward and reverse primers (Table S1), and 1.5 μL of cDNA template, and appropriate amounts of sterile ddH₂O. All qRT-PCR amplification conditions were standardized according to the manufacturer’s instructions. Three independent replicates were performed on each cDNA sample. The relative expression level was calculated using the 2^−△△Ct formula^{39 (link)} with the reference genes β-actin gene (GenBank ID: AB181991.1) as internal standard.

To analyze the
expression of genes related to the synthesis of Moco and molybdenum
enzymes including CNX1, CNX2, CNX3, AO, XDH, and NR, fresh roots and
shoots were ground to a fine powder in liquid nitrogen. Total RNA
was extracted using TRIzol Reagent (Invitrogen, USA). The RNA concentration
was determined by a Nano-Drop2000 spectrophotometer (Thermo, USA)
and cDNA was synthesized using 20 μg of RNA and SuperScript
III RNase H–Reverse Trancrip-tase (Invitrogen, USA) according
to the manufacturer instructions. The SYBR Premix Ex TaqTM Kit (TaKaRa,
Dalian, China) and the Light Cycler System (Bio-Rad, Richmond, CA)
were used for RT-PCR. The actin gene was used as the internal standard.
Primer sequences for each gene are shown in Table S6.