Screening Of Reference Genes And Identification Of Nbs-Lrr Gene Analogs In Pepper (Capsicumannuum L.)

Pepper (Capsicum annuum L.) is one of the most important vegetable worldwhile. However, yield and quality of pepper are severely threatened by numbers of pathogens, including bacteria, fungi, viruses, and nematodes. Cloning resistance gene is important for breeding disease resistance cultivars. In this study, NBS-LRR gene analogs (CaRGAs) were obtained in pepper by PCR amplification. Their phylogenetic relationships were analzed, and expression levels of these genes under abiotic stresses and hormones treatment were also studied. In addition, in order to choose the suitable reference genes for qRT-PCR, we evaluated expression stability of seven commonly reference genes in pepper though real-time quantitative PCR (qRT-PCR) technique.Results were showed as follows:1. Identification of reference genes for reverse transcription quantitative real-time PCR normalization in pepperIn the present paper, we assessed stabilities of expression levels of seven potential reference genes in pepper "PBC631B" in order to find the optimal reference genes using qRT-PCR analysis in this important vegetable crop. These reference genes were evaluated in different pepper tissues, abiotic stress and hormones treatment samples. Three common statistical algorithms, geNorm, Norm-Finder and BestKeeper, were used to identify their expression stability and provided the accurate choice of reference genes. The results revealed that two reference genes, β-TUB and UBI-3, showed high stability in sample pools with abiotic stresses and hormone treatments. In different tissues and total sample pools tested, the expression levels of UBI-3and GAPDH were the most stable. Therefore, these reference genes should be used for qRT-PCR analysis under the experimental conditions tested in pepper. By contrast, UBI-1and ACT were identified as the least stable reference genes in all groups tested and should be discouraged for normalization. In conclusion, the validation of these candidate genes could provide useful guideline for reference gene selection in qRT-PCR studies in pepper.2. Cloning and expression analysis of NBS-LRR gene analogs in pepper (Capsicum annuum L.)To study structure and function of NBS-encoding resistance genes in pepper, two pairs of degenerate primer published previously, which were designed based on conserved domains in resistance genes, were used to isolate NBS-encoding sequences from pepper "PBC631B". Expression levels of four CaRGAs were further evaluated by RT-PCR and qRT-PCR in different pepper tissuesand hormones treatment samples. In this study, seventeen resistance gene analogues (RGAs) with uninterrupted open reading frames (ORFs) were obtained from pepper(Capsicum annuum L.)(designated CaRGA01～CaRGA17). Multiple sequence alignments among these CaRGAs revealed six conserved motifs (P-loop, RNBS-A-nonTIR/RNBS-A-TIR, Kinase-2, RNBS-B, RNBS-C and GLPL). Phylogenetic tree analysis between these CaRGAs and six known resistance genes (RPM1, Gpa2, L6, M, N and Prf) showed that they were grouped into nonTIR-NBS-LRR and TIR-NBS-LRR families and further seperated into four subgroups (CaRGA I-CaRGA IV). Among them, CaRGA10and CaRGA13showed high sequence identity (90%and91%, respectively) with bacterial spot disease resistance protein gene Bs4and Mi-1.4disease resistance protein gene from tomato, which suggested that these two genes might be a part of related resistance genes in pepper. RT-PCR and qRT-PCR analysise releaed that four CaRGAs showe different expression in pepper samples. In addition, both salicylic acid and abscisic acid can induce the expression of the CaRGA genes.This result suggests that they might play a potential role in mediating cross-talk between defense-signaling pathways.