Genomics and Applied Biology 2019, Vol.10, No.1, 1-9
7
structural genes (Yang et al., 2012), and four new miRNAs were found to be associated with bHLH transcription
factor in the predicted miRNAs. In
Camellia sinensis
, miR156 regulated DFR gene by inhibiting the target gene
SPL, thus affected the accumulation of catechin. In
Arabidopsis thaliana
, miR156 regulated SPL transcription
factor to control the biosynthesis of sesquiterpenes. MYB transcription factor directly regulated the expression
level of key enzymes in anthocyanin biosynthesis (Kobayashi et al., 2002; Takos et al., 2006). In this study, 7
miRNA target genes associated with MYB transcription factors were obtained, including miR156, mi319, mi845,
mi156 and new miRNA novel_65 and novel_14. miRNA166 regulated HD-ZIP transcription factor in plants and
was involved in organ building and meristem formation in
Arabidopsis thaliana
(Green et al., 2005). WRKY
transcription factor has been proved to be widely involved in plant vegetative growth, organ development and
material metabolism (Zhou et al., 2011; Wei et al., 2015). In this study, the target genes HD-ZIP, SPL, NAC, MYB,
ARFs, bHLH and WRKY transcription factors of miR166a, miR156a, miR164a, miR845c, miR160c, novel_65
and novel_113 were analyzed and predicted. These predicted conserved transcription factors of target genes
suggested that they had the same function in the growth and development of “Zijuan” tea tree, especially in the
regulation of secondary metabolism.
Anthocyanin metabolism is a branch of flavonoid biosynthesis pathway. Anthocyanin accumulation in Zijuan tea
tree is a complex process involving some precursors and a series of gene regulation. Therefore, the association
between miRNA and their target needs further identification on the accumulation mechanism of anthocyanins in
“Zijuan”.
3 Materials and Methods
3.1 Plant materials
The tea tree “Zijuan” in the test base of Tea Research Institute of Academy of Sciences in Yunnan province was
selected as materials. The young buds, second leaves, open leaves and mature leaves were collected on October
15
th
, 2016. The samples were quickly fixed with liquid nitrogen and then put into -80°C for miRNA analysis.
3.2 Extraction of total RNA
Total RNA was extracted by using TRIzol (Invitrogen, Carlsbad, CA, USA) kit. The total RNA of young buds,
second leaves, open leaves, and mature leaves were extracted, and analyzed whether RNA was contaminated and
degradation degree by 1% agarose gel electrophoresis. The concentration of RNA was quantified by Qubit, the
purity of RNA was detected by Nanodrop, and the integrity of RNA was accurately detected by Agilent 2100.
3.3 Construction and sequencing of small RNA library
The Small RNA Sample Pre Kit was used to construct the library of young buds, second leaves, open leaves and
mature leaves, respectively. Using the 3’-end hydroxyl of small RNA and the special structure of 5’-end complete
phosphate groups, total RNA as the starting sample was directly added to the 3’-end and 5’-end of small RNA,
and then reverse transcription was conducted to synthesize cDNA. After PCR amplification, the target DNA
fragment was separated by polyacrylamide gel electrophoresis, and the cDNA library was obtained after gel
cutting and recovery. Then, Qubit 2.0 was used for preliminary quantification of the cDNA library, and the library
was diluted to 1 ng/uL, and then Agilent 2100 was used to detect the insert size of the cDNA library. The effective
concentration of the library was more than 2 nmol/L. After the library inspection was qualified, the gene
sequencing was performed by Illumina HiSeq 2500 (completed by Beijing Novogene).
3.4 Analysis of sequencing data
The original image data files obtained by illumina HiSeqTM2500 sequencing were transformed into Raw reads
through base calling analysis. The Raw reads obtained by sequencing were processed and the low-quality reads
with the joint were processed to obtain Clean reads. sRNA with a length range of 18~30 was screened for analysis.
sRNA with a length range of 18~30 was positioned on the reference sequence by bowtie (regarded as mapped
sRNA). The mapped sRNA was compared with the sequences in the grape miRBase library to obtain the detailed
information of the small RNA on each sample matching. If grape ncRNA annotation information was available,
the ncRNA sequences of the species were used to annotate the small RNA. If not, the tRNA, rRNA, snoRNA and
snRNA in Rfam were selected to annotate the small RNA. The analysis of new miRNA was conducted by
