JIA-2018-09

1935 HAN Jiao et al. Journal of Integrative Agriculture 2018, 17(9): 1932–1945 Pure Plant RNA Kit (TransGen Biotech, China), and RNA concentrations were determined by ND2000 spectrophotometer (Thermo Scientific Co., USA). Total 1 µg of RNA was reverse-transcripted into the cDNA using TransScript One-Step gDNARemoval and cDNASynthesis SuperMix (TransGen Biotech, China) according to the manufacturer’s manual. Quantitative PCR procedures were performed in a 20-μL of reaction solution containing 10 μL of 2×TransStart ® Top Green qPCR Super Mix, 0.4 μL of sense and antisense primers (Table 1), 1 μL of cDNA template and 8.2 μL of RNA-free water using the TransStart Top Green qPCR SuperMix (TransGen Biotech, China). Quantitative PCR procedures were performed with the IQ5 Multicolor Real-Time PCR Detection System (Bio-Rad, Richmond, CA) under the following circles: 94°C for 30 s, 45 cycles of 94°C for 5 s, 55°C for 15 s, and 72°C for 10 s according to the manufacturer’s instructions. The relative expression levels of targets genes in the transgenic rice were calculated by the 2 –∆∆C T formula (Livak and Schmittgen 2001) using OsActin as an internal control. The relative expression levels of the McPht gene in M. crystallinum L. were calculated by the 2 –∆∆C T formula using McActin as an internal control. All nucleotides sequences of the specific primers for qRT-PCR analyses were shown in Table 1. 2.4. Subcellular localization analysis The McPht cDNA fragments were amplified by RT-PCR using a pair of specific primers with Pst I /Nco I sites (Table 1), and inserted into the 16318hGFP expression vector under the control of the 35S promoter with Pst I /Nco I-specific sites (Wang et al . 2013). Both the McPht::GFP fusion construct and the 16318hGFP empty vector were transformed into the leave protoplasts of the 3-wk-old Arabidopsis by a PEG- mediated method (Locatelli et al . 2003). The transformants were placed at room temperature over 18 h, and the images of the transgenic protoplasts were photographed by a LSM700 Confocal Laser Microscope (Carl Zeiss AG, Oberkochen, Germany). 2.5. Roots recording and physiological measure- ments Roots scanning was performed by a scanner of EPSONLA, UK, and the data were analyzed by WINRHIZO Pro software of 2004c version (WINRHIZO, Regent Co., Canada). Roots fresh weights were weighed under natural dehydration. Root activities were measured at 485 nm by triphenyltetrazolium chloride (TTC) method using a spectrophotometer (HACH DR/4000U, USA) (Lindström and Nyström1987). Total 0.2 g of dry matter were weighted, and digested in the solution containing 5 mL of concentrated H 2 SO 4 and 2 mL of H 2 O 2 , and then the generating colorless digestion solutions were cooled and diluted to the volume of 100 mL by distilled water for determination of total phosphate in plants. 2.6. cDNA library construction and transcriptome sequencing Fresh plant leaves were sampled and stored in liquid nitrogen, and approximately 1 µg of RNA was extracted for constructing the cDNA library, which was generated using NEBNext ® Ultra™ RNA Library Prep Kit for Illumina ® (NEB, USA) following the manufacturer’s recommendations. The mRNA preparation was performed by poly-Toligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in NEB Next First Strand Synthesis Reaction Buffer (5×). The first strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNase H – ). The second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease and polymerase. All adaptors were ligated after adenylation of DNA fragments at 3´-end. The library fragments were purified withAMPure XP System (Beckman Coulter, Beverly, USA). Agilent 2100 Bioanaylzer (Agilent Technologeis Co., USA) and ABI StepOnePlus Real-Time PCR System (Applied Biosystems ABI Co., USA) are used for qualifying the sample library and transcriptome sequencing. 2.7. Sequencing data filtering Sequencing data were gained by Illumina HiSeq TM2000 platform with PE125 method. The original images were transformed into the raw reads by base recognition, and the raw reads were filtered to obtain clean reads, which further were assembled using Software Trinity Program (Grabherr et al . 2011). All contigs were generated by the assembly of the information between overlap sequences, and then partially data were assembled, and the unigenes were produced using non-redundant protein sequences (Nr), clusters of orthologous groups of proteins (COG), euKaryotic ortholog groups (KOG), gene ontology (GO), and Kyoto encyclopedia of genes and genomes (KEGG). 2.8. Differentially expressed gene, GO enrichment, and KEGG analyses The reads of gene number were counted by the HTSeq software. The gene expression levels were calculated by the fragment per kb per million fragments (FPKM) method (Trapnell et al . 2010). Both DESeq and DESeq2 were used to detect the differentially expressed genes between the