Identification of MV-generated ROS responsive EST clones in floral buds of Litchi chinensis Sonn.

Abstract

A suppression subtractive hybridization library was constructed using inflorescence primordia of 'Nuomici' litchi to identify EST clones responsive to MV-generated ROS. 93 ESTs could be aligned as unique gene sequences in the inflorescence primordia of litchi. Litchi is an evergreen woody tree widely cultivated in subtropical and tropical regions. However, defective flowering is a pending problem of litchi production. Our previous study indicated that reactive oxygen species (ROS) induced by methyl viologen dichloride hydrate (MV) promotes flowering in litchi. In the present study, a suppression subtractive hybridization (SSH) library was constructed using inflorescence primordia of 'Nuomici' with the aim to find out ROS responsive clones during floral differentiation. 1856 Expressed sequence tag (EST) clones were randomly selected. Clones carrying single exogenous fragments were screened by reverse northern analysis to identify those responsive to MV-generated ROS. A total of 783 differentially expressed EST clones were identified as MV responsive cDNA and were subjected to sequencing. Among them, 26 clones were represented more than three times. 783 clones were aligned to 93 unique gene sequences. The unique genes were classified into 9 categories. 16 % of them were involved in transport facilitation, 11 % in transcription regulation, 4 % in stress response, 9 % in carbohydrate metabolism, 1 % in secondary metabolism, 14 % in intracellular signaling, and 25 % in other metabolism, while 9 % were genes with unknown functions and 11 % were genes with no match in the database.

Identification of nitric oxide responsive genes in the floral buds of Litchi chinensis

“…To investigate the expression of candidate NO responsive unique genes during normal floral tissue differentiation, we determined their relative expression in buds of the terminal shoots at the induction stage (25 December 2011), floral initiation stage (13 January 2012), rhachis developmental stages when apical and lateral axillary panicle began to develop (1, 5, and 12 February 2012), and floral organ developmental stage (20 February 2012) as described by Liu et al (2013). The results show that the relative expression of LcANR at the floral induction stage was low and increased by 121.0 and 162.6 % at the floral initiation stage and the early rhachis developmental stage, respectively.…”

“…We isolated total RNA from floral buds or panicle primordia, and it was used to generate the first-strand cDNA according to the method of Liu et al (2013). The expression analysis was performed at the transcript level which was determined by RT-qPCR using single-stranded cDNA as template.…”

“…To screen positive clones, a reverse Northern blot was performed according to the method of Liu et al (2013). Two cDNA probes, one driver and the other tester, were prepared using random primers and were labelled with digoxin (DIG).…”

“…Identification of the full spectrum of downstream target genes controlled by NO and ROS would be the first step to elucidate the mechanism of stress-induced flowering in litchi. Liu et al (2013) used the suppression subtractive hybridization (SSH) cDNA library screening to identify ROS responsive genes. This approach was also used to identify NO responsive genes in the litchi primordia.…”

Genome-Wide Transcriptomic Analysis Reveals a Regulatory Network of Oxidative Stress-Induced Flowering Signals Produced in Litchi Leaves

“…We have previously shown that reactive oxygen species (ROS) promoted flowering and decreased the chilling requirement for flowering [9] and obtained a few ROS-responsive Expressed sequence tag (EST) clones [10]. We also performed RNA-sequencing (RNA-Seq) to obtain a global transcriptome of the apical meristem, and to identify genes involved in the transformation from vegetative meristems to inflorescence meristems triggered by ROS and chilling [11].…”