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Transcription profiles of boron-deficiency-responsive genes in citrus rootstock root by suppression subtractive hybridization and cDNA microarray.

Zhou GF, Liu YZ, Sheng O, Wei QJ, Yang CQ, Peng SA - Front Plant Sci (2015)

Bottom Line: Boron (B) deficiency has seriously negative effect on citrus production.All these results indicated that CC was more tolerant than TO to B-deficiency stress.The B-deficiency responsive genes identified in this study could provide further information for understanding the mechanisms of B-deficiency tolerance in citrus.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Horticultural Plant Biology (Ministry of Education), College of Horticulture and Forestry Science, Huazhong Agricultural University Wuhan, China ; National Navel Orange Engineering Research Center, College of Navel Orange, Gannan Normal University Ganzhou, China.

ABSTRACT
Boron (B) deficiency has seriously negative effect on citrus production. Carrizo citrange (CC) has been reported as a B-deficiency tolerant rootstock. However, the molecular mechanism of its B-deficiency tolerance remained not well-explored. To understand the molecular basis of citrus rootstock to B-deficiency, suppression subtractive hybridization (SSH) and microarray approaches were combined to identify the potential important or novel genes responsive to B-deficiency. Firstly four SSH libraries were constructed for the root tissue of two citrus rootstocks CC and Trifoliate orange (TO) to compare B-deficiency treated and non-treated plants. Then 7680 clones from these SSH libraries were used to construct a cDNA array and microarray analysis was carried out to verify the expression changes of these clones upon B-deficiency treatment at various time points compared to the corresponding controls. A total of 139 unigenes that were differentially expressed upon B-deficiency stress either in CC or TO were identified from microarray analysis, some of these genes have not previously been reported to be associated with B-deficiency stress. In this work, several genes involved in cell wall metabolism and transmembrane transport were identified to be highly regulated under B-deficiency stress, and a total of 23 metabolic pathways were affected by B-deficiency, especially the lignin biosynthesis pathway, nitrogen metabolism, and glycolytic pathway. All these results indicated that CC was more tolerant than TO to B-deficiency stress. The B-deficiency responsive genes identified in this study could provide further information for understanding the mechanisms of B-deficiency tolerance in citrus.

No MeSH data available.


Related in: MedlinePlus

Verification of microarray results by quantitative real-time RT-PCR. (A) Expression ratios of the selected genes from microarray analysis results; (B–K) Quantitative real-time RT-PCR analysis of the selected genes. The relative expression level of Beta-1,3-glucanase gene (B) Elongation factor 1-alpha gene (C) Proline-rich cell wall protein gene (D) Xyloglucan endotransglucosylase/hydrolase gene (E) Vinorine synthase gene (F) Hypothetical protein gene (G) Isoflavone 2′-hydroxylase gene (H) Isoflavone reductase gene (I) Peroxidase gene (J) Elongation factor EF-1 gene (K). RNA of seedling root was extracted at three time points and gene expression levels were analyzed by Quantitative real-time RT-PCR assays Data presented are means ± SE of three biological replicates (n = 6). REL, Relative expression level.
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Figure 5: Verification of microarray results by quantitative real-time RT-PCR. (A) Expression ratios of the selected genes from microarray analysis results; (B–K) Quantitative real-time RT-PCR analysis of the selected genes. The relative expression level of Beta-1,3-glucanase gene (B) Elongation factor 1-alpha gene (C) Proline-rich cell wall protein gene (D) Xyloglucan endotransglucosylase/hydrolase gene (E) Vinorine synthase gene (F) Hypothetical protein gene (G) Isoflavone 2′-hydroxylase gene (H) Isoflavone reductase gene (I) Peroxidase gene (J) Elongation factor EF-1 gene (K). RNA of seedling root was extracted at three time points and gene expression levels were analyzed by Quantitative real-time RT-PCR assays Data presented are means ± SE of three biological replicates (n = 6). REL, Relative expression level.

Mentions: To confirm the results obtained using cDNA microarray analysis, 10 genes were randomly selected to analyze their expression profiles by quantitative real-time PCR. Quantitative real-time PCR was performed using total RNA isolated from 6 to 24 h in both CC and TO plants root, respectively. The gene-specific primer pairs are listed in Table S1. Quantitative real-time PCR data agreed with the microarray data for 27 out of 30 (90%) data points (Figure 5). These results confirmed the differential expression of all 10 selected genes.


Transcription profiles of boron-deficiency-responsive genes in citrus rootstock root by suppression subtractive hybridization and cDNA microarray.

Zhou GF, Liu YZ, Sheng O, Wei QJ, Yang CQ, Peng SA - Front Plant Sci (2015)

Verification of microarray results by quantitative real-time RT-PCR. (A) Expression ratios of the selected genes from microarray analysis results; (B–K) Quantitative real-time RT-PCR analysis of the selected genes. The relative expression level of Beta-1,3-glucanase gene (B) Elongation factor 1-alpha gene (C) Proline-rich cell wall protein gene (D) Xyloglucan endotransglucosylase/hydrolase gene (E) Vinorine synthase gene (F) Hypothetical protein gene (G) Isoflavone 2′-hydroxylase gene (H) Isoflavone reductase gene (I) Peroxidase gene (J) Elongation factor EF-1 gene (K). RNA of seedling root was extracted at three time points and gene expression levels were analyzed by Quantitative real-time RT-PCR assays Data presented are means ± SE of three biological replicates (n = 6). REL, Relative expression level.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4309116&req=5

Figure 5: Verification of microarray results by quantitative real-time RT-PCR. (A) Expression ratios of the selected genes from microarray analysis results; (B–K) Quantitative real-time RT-PCR analysis of the selected genes. The relative expression level of Beta-1,3-glucanase gene (B) Elongation factor 1-alpha gene (C) Proline-rich cell wall protein gene (D) Xyloglucan endotransglucosylase/hydrolase gene (E) Vinorine synthase gene (F) Hypothetical protein gene (G) Isoflavone 2′-hydroxylase gene (H) Isoflavone reductase gene (I) Peroxidase gene (J) Elongation factor EF-1 gene (K). RNA of seedling root was extracted at three time points and gene expression levels were analyzed by Quantitative real-time RT-PCR assays Data presented are means ± SE of three biological replicates (n = 6). REL, Relative expression level.
Mentions: To confirm the results obtained using cDNA microarray analysis, 10 genes were randomly selected to analyze their expression profiles by quantitative real-time PCR. Quantitative real-time PCR was performed using total RNA isolated from 6 to 24 h in both CC and TO plants root, respectively. The gene-specific primer pairs are listed in Table S1. Quantitative real-time PCR data agreed with the microarray data for 27 out of 30 (90%) data points (Figure 5). These results confirmed the differential expression of all 10 selected genes.

Bottom Line: Boron (B) deficiency has seriously negative effect on citrus production.All these results indicated that CC was more tolerant than TO to B-deficiency stress.The B-deficiency responsive genes identified in this study could provide further information for understanding the mechanisms of B-deficiency tolerance in citrus.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Horticultural Plant Biology (Ministry of Education), College of Horticulture and Forestry Science, Huazhong Agricultural University Wuhan, China ; National Navel Orange Engineering Research Center, College of Navel Orange, Gannan Normal University Ganzhou, China.

ABSTRACT
Boron (B) deficiency has seriously negative effect on citrus production. Carrizo citrange (CC) has been reported as a B-deficiency tolerant rootstock. However, the molecular mechanism of its B-deficiency tolerance remained not well-explored. To understand the molecular basis of citrus rootstock to B-deficiency, suppression subtractive hybridization (SSH) and microarray approaches were combined to identify the potential important or novel genes responsive to B-deficiency. Firstly four SSH libraries were constructed for the root tissue of two citrus rootstocks CC and Trifoliate orange (TO) to compare B-deficiency treated and non-treated plants. Then 7680 clones from these SSH libraries were used to construct a cDNA array and microarray analysis was carried out to verify the expression changes of these clones upon B-deficiency treatment at various time points compared to the corresponding controls. A total of 139 unigenes that were differentially expressed upon B-deficiency stress either in CC or TO were identified from microarray analysis, some of these genes have not previously been reported to be associated with B-deficiency stress. In this work, several genes involved in cell wall metabolism and transmembrane transport were identified to be highly regulated under B-deficiency stress, and a total of 23 metabolic pathways were affected by B-deficiency, especially the lignin biosynthesis pathway, nitrogen metabolism, and glycolytic pathway. All these results indicated that CC was more tolerant than TO to B-deficiency stress. The B-deficiency responsive genes identified in this study could provide further information for understanding the mechanisms of B-deficiency tolerance in citrus.

No MeSH data available.


Related in: MedlinePlus