| Blueberry (Vaccinium spp.) is a perennial bush shrub fruit tree belonging to Ericaceae family. Its fruit is rich in bioactive substances such as anthocyanins, and flavonoids, which named it as one of the’Five healthiest fruits in the world’. Recently, the planting areas extended rapidly in South China, comparatively, the researches on biological and physiological characteristics of the plant species greatly lagged behind the increasing scales. It was widely acknowledged that blueberry were a chloride-sensitive plant despite the theoretical mechanism still being far from clear. Although the chlorine is one of the 16 necessary elements for plants, the stress of excessive chloride always induces leaf burn and brown spots, early leaf drop or defoliation occurs along with the toxicity prolonged or strengthened. However, the mechanism of chloride toxicity in plants still lacks of the research.Blueberry seedlings of relatively Chloride-endurable cultivar (’Sharpblue’) and chloride-sensitive cultivar (’Brijjita’) were used as plant materials to study the effects of chloride stress on the photosynthesis and expressions of genes related to nitrogen metablism. Then, the transcriptome of blueberry leave under chloride stress was sequenced by the technology of RNA-Seq, and a large number of transcript sequences were obtained. Based on the transcriptome data, two expression profile libraries (ie, the control and 150 mM Cl- 2d treatment library) were respectively constructed, and the differentially expressed genes were screened to elucidate the digital gene expression profiles and molecular mechanism of chloride toxicity in blueberry. The main results were summarized as followings:1. After 150 mM Cl- 2d treatment, the burn and yellow spots occurred in the leaves, meanwhile, the chlorine concentration were 20.07 and 21.48 mg/L in Brijitta and Sharpblue, respectively. The symptoms became more severe as the toxicity prolonged or strengthened. Comparatively, the chlorophyll contents significantly deduced at 75 mM chloride 2d treatment. The chloride stress also generally caused the decreases in photosynthetic rate, the gene expression levels of proteins in PSII. The abnormal chlorophyll fluorescence parameters were also observed, all of these indicated the severe damages occurred in PSII, which may result from the decreases in the gene expressing levels of antioxidative enzymes such as SODs, POD and CAT. In contrast to Brijitta, the chloride toxicity symptoms appeared more lately in leaves of Sharpblue, and the decrease percentages of genes were overall lower in Sharpblue. This may be an important reason for the relatively higher chloride endurance in Sharpblue.2. The quantitive RT-PCR was applied to study the expression patterns of the genes related to the nitrogen metabolism in leaves of blueberry. Compared with the control, the expression of Ammonium Transporters 2 (AMT2) were significantly increased by the chloride treatments. However, the expressions of AMT1 and AMT3, which indicated that expressions of AMTs were differently affected by chloride stress and involved in different functions. Overall, the increased expressions of nitrate transporter (NRT1.5 and NRT2), nitrate reductase (NR) were observed in chloride treatments, especially at 75 mM Cl- 2d treatment. These increased expression levels may be a adaptable response to the chloride stress.3. Transcriptome of blueberry leaves were accomplished by Illumina Hiseq technique. A toltal of 103,560 Unigene were obtained by de novo assembly of the clean data. The average length of All-Unigene is 1027.95 bp, which ranged from 13,843 to and 201 bp in length. Most of Unigenes (57.58%) were longer than 400 bp. Then we aligned the Unigenes to the public database such as NR, COG and KEGG.It was found 6,151 and 3,489 All-unigene can be mapped onto GO database and KEGG database. But there are still 12.9% of the All-Unigene lack annotate. It may result from the unavailability of the whole genome database in blueberry and the number of reported EST also be limited. It aslo may be because of the high repeat sequence in some short fragments, which resluted in the unvalid alignment results.4. Based on the KEGG dabatase, the gene expression patterns were analyzed between the control treatment and 150mM Cl- 2d libraries. According to the functional classification and the pathways involved of the Unigenes, the differentially expressed genes were classified in different pathways. It were found that 1,177,700 and 45 Unigenes were respectively enriched in Metabolic pathways, Biosynthesis of secondary metabolites and Photosynthesis. A total of 10,025 Unigenes which differentially expressed in the control and 150mM Cl- 2d treatment were further classified on the base of the GO annotations. The results showed 2,825 genes were significantly down-regulated and 7,200 were up-regulated,123 genes were related to DNA recombination,123 genes functions as base mismatch repairing and 235 were involed in DNA reprications. |
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