Woraathasin Natthakorn, Nakkanong Korakot, Nualsri Charassri
Department of Plant Science, Faculty of Natural Resource, Prince of Songkla University, Hat Yai Campus, 90112 Songkhla, Thailan.
Center of Excellence on Agricultural Biotechnology: (AG-BIO/PERDO-CHE), 10900 Bangkok,Thailand.
Pak J Biol Sci. 2017;20(5):233-243. doi: 10.3923/pjbs.2017.233.243.
Pathogenesis-related (PR) proteins are dramatically accumulated after pathogen infection. Innate defense response through increasing PR-proteins is important for rubber rootstock selection that is tolerant to the white root disease caused by Rigidoporus microporus. This study was aimed to investigate the expression levels of PR-1 and PR-3 genes in tolerant (PB5/51) and susceptible (BPM24 and RRIM600) rubber clones after R. microporus infection.
The mRNA of HbPR-1b and HbPR-3 was isolated and characterized from rubber leaves. Gene expression levels of HbPR-1b and HbPR-3 were compared among three rubber clones (PB5/51, BPM24 and RRIM600) after R. microporus infection at 0, 12, 24, 48, 72 and 96 h using quantitative real-time PCR. The relative transcript abundances between inoculated and control plants were compared using the means of gene expression between time points and by Tukey's HSD test. A probability value (p<0.05) was used to determine the significance of difference between time points.
The open reading frame of HbPR-1b is 492 bp with deduced 163 amino acid residues and the phylogenetic analysis showed it shared significant evolutionary history and clustering into group I of PR-protein. Moreover, the partial HbPR-3 was isolated with 390 bp. Gene expression levels of HbPR-1b and HbPR-3 showed marked differences in both transcripts depending on the rubber clones. Two genes demonstrated up-regulation of both tolerance and susceptibility in response to attack by R. microporus. The highest expression levels were found in seedlings of PB5/51 after inoculation. In RRIM600, low expression levels of HbPR-1b and HbPR-3 were initially observed but gradually increased at 24 h post inoculation. The transcription profile of HbPR-1b was stable expression in BPM24.
The results demonstrated that the level ofHbPR-1b and HbPR-3 transcription can distinguish between tolerant and susceptible clones. The candidate defense genes to the white root disease were observed in PB5/51 seedlings, particularly HbPR-1b.
病程相关（PR）蛋白在病原体感染后会大量积累。通过增加PR蛋白来启动防御反应，对于筛选耐微小多孔硬皮马勃引起的白根病的橡胶砧木很重要。本研究旨在调查微小多孔硬皮马勃感染后，耐病（PB5/51）和感病（BPM24和RRIM600）橡胶品种中PR-1和PR-3基因的表达水平。
从橡胶叶片中分离并鉴定了HbPR-1b和HbPR-3的mRNA。在微小多孔硬皮马勃感染后0、12、24、48、72和96小时，使用定量实时PCR比较了三个橡胶品种（PB5/51、BPM24和RRIM600）中HbPR-1b和HbPR-3的基因表达水平。使用时间点之间的基因表达平均值并通过Tukey's HSD检验，比较接种植物和对照植物之间的相对转录丰度。使用概率值（p<0.05）来确定时间点之间差异的显著性。
HbPR-1b的开放阅读框为492 bp，推导的氨基酸残基为163个，系统发育分析表明它具有显著的进化历史，属于PR蛋白的第一组。此外，分离出了390 bp的部分HbPR-3。HbPR-1b和HbPR-3的基因表达水平在两个转录本中均表现出明显差异，这取决于橡胶品种。两个基因在微小多孔硬皮马勃攻击后，在耐病和感病品种中均表现出上调。接种后，PB5/51幼苗中的表达水平最高。在RRIM600中，最初观察到HbPR-1b和HbPR-3的表达水平较低，但在接种后24小时逐渐增加。HbPR-1b的转录谱在BPM24中表达稳定。
结果表明，HbPR-1b和HbPR-3的转录水平可以区分耐病和感病品种。在PB5/51幼苗中观察到了对白根病的候选防御基因，尤其是HbPR-1b。
