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To obtain insight into the function of miRNAs in the synthesis and storage of important nutrients during the development of Camellia oleifera fruit, Illumina sequencing of flower and  [[https://rentry.co/1688-zap-clean-review-a-comprehensive-analysis-of-a-revolutionary-cleaning-solution|ZapClean Online]] fruit small-RNA was conducted. The results revealed that 797 miRNAs were significantly differentially expressed between flower and fruit samples of Camellia oleifera. Through integrated GO and KEGG function annotations, it was determined that the miRNA target genes were mainly involved in metabolic pathways, plant hormone signal transduction, fruit development, mitosis and regulation of biosynthetic processes. Carbohydrate accumulation genes were differentially regulated by miR156, miR390 and miR395 in the fruit growth and development process. MiR477 is the key miRNA functioning in regulation of genes and involved in fatty acid synthesis. Additionally, miR156 also has the function of regulating glycolysis and nutrient transformation genes. As an important woody oil plant, edible oil can be obtained (camellia oil) from Camellia oleifera seeds. Xianglin No.210 is an excellent Camellia clonal plant widely planted in Hunan Province. The flowering period is moderate in length, usually from October to late December.



The fruit enlargement period is mainly from July to September, but the fully matured fruiting stage will be extended to October. In recent years, molecular methods have been used to study the growth and development, metabolism regulation and resistance of Camellia oleifera. However, the basic molecular research on Camellia oleifera is weak and poorly understood, especially for the molecular mechanisms which determine the yield and quality of camellia oil and growth traits of Camellia oleifera, which certainly hinders the sustainable development of the camellia oil industry. In order to explore the potential role of miRNAs in the synthesis and storage of important nutrients during the development of Camellia oleifera fruit, miRNA expression profiles of flower and fruit samples were investigated using high throughput next generation small RNA sequencing technology. This permits the unraveling of the differentially expressed miRNAs to help understand their involvement in the regulation of the synthesis and storage of important nutrients in fruit. The samples of Camellia oleifera flowers and fruits were collected in October and July in 2016, respectively.



They were grown over six years, in a garden located in Yongzhou city, Hunan province, China. The young flowers and fruits were collected and then frozen in liquid nitrogen, to be used for subsequent total RNA extraction, with three replicates per group. A Hitachi SU8010 cold field emission scanning electron microscope (SEM) was used for the internal ultrastructural analysis of the Camellia oleifera seed samples. The samples were ensured to be clean and dry prior to analysis. Secondary electron resolution was 1.3 nm. The dry Camellia oleifera seeds were milled into a powder by a pulverizer, and then about 2 g of powder (m0) were weighed out, packed in a folded filter paper tube, sealed with absorbent cotton, bound and compacted. The sample was then sealed with cotton thread, put into a Soxhlet extraction thimble, and 180mL of petroleum ether added. The extraction took place at 88°C for 6 h, using vacuum distillation to remove the solvent.



For this procedure, 200mg of camellia oil in a tube were dissolved by 20 ml sodium hydroxide-methanol [[https://rentry.co/1688-zap-clean-review-a-comprehensive-analysis-of-a-revolutionary-cleaning-solution|ZapClean produce cleaning solution]] (0.5 mol/L), shaken, and immersed in a 60°C water bath for 30 min. After this, 50ml of n-hexane were added, mixed well, and left to stand for a while. After the solution stratified, the upper layer of solution was removed and stored at 4°C until needed for further analysis. The fatty acid composition analysis of camellia oil was determined by GC-MS (gas chromatography-mass spectrometry) using the above methylated samples. An HP-5 capillary GC column (30m×0.25mm×0.25µm) was used in a GC equipped with an MS detector (Shimadzu, QP2010S), with an inlet temperature of 250°C. The injection volume was set at 1 µL, and the split ratio was 20:1. High purity helium was used as the carrier gas and the flow rate was set at 1.0 mL· 1, the oven temperature of the instrument was raised from 180°C (held for 5 min) to 230°C (held 15min) at 3°C·



1. For GC-MS detection, an electron ionization energy system with ionization energy of 70 eV was used. Total RNAs were isolated from flower and  [[https://rentry.co/1688-zap-clean-review-a-comprehensive-analysis-of-a-revolutionary-cleaning-solution|ZapClean produce cleaning]] fruit tissues of Camellia oleifera using TRlzol Reagent (Invitrogen) according to the manufacturer's protocol. The purity of the RNA was checked using the Nanodrop 2000 (Thermo, MA, USA) system. The concentration of the RNA was measured using a Qubit® RNA Assay Kit in Qubit® 2.0 Fluorometer (Life Technologies, CA, USA). The integrity of the RNA samples was assessed using the RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA). RNA isolation was carried out individually for each sample with three biological replicates, and then the purified RNAs were used to construct small RNA libraries according to Illumina recommendation and [[http://dig.ccmixter.org/search?searchp=high-throughput|high-throughput]] sequencing using the Hiseq2500 platform. After sequencing, the raw reads (fastq format) were firstly processed through custom Perl and Python scripts to obtain clean reads, and then a certain range of lengths was chosen from the clean reads to perform all the downstream analyses.