| Blueberry (Vaccinium spp.) is a perennial bush shrub fruit tree belong to Ericaceae family. Fruits are rich in anthocyanins, flavonoids and other bioactive substances, which contributes to its bio-functions such as anti-cancer, free radicals scavenging and anti-aging capacities, etc. But blueberry cultivation requires critical rhizospheric conditions, which limited its large-scale cultivation arround the country. Non-acid soil is one of the major factors that influence the growth and development of blueberry. It is widely acknowledged that pH4.5-5.5 is the optimum pH condition for the growths of blueberry, and a non-acid rhizospheric condition would result in stunted growth, leaf chlorosis and other abnormal phenomena. Quite a lot researches have been reported about the mechanism of non-acid rhizosphere negatively affection blueberry. But most of the researches were limited in physiological analysis and the molecular mechanism was still rarely reported.The present study mainly includes four parts. Firstly, five major cultivars in Zhejiang province,’Sharpblue’,’O’Neal’,’Star’,’Brigitta’ and ’Earliblue’ were screened of the adaptability to the non-acid soil, based on the physiological analysis. Secondly, transcriptome of blueberry roots under different pH treatments were sequenced by the technology of RNA-Seq, and a large number of transcript sequences were obtained. Thirdly, three expression profile libraries (ie, pH5.0, pH7.0-3d and pH7.0-6d library) were respectively constructed, then the differentially expressed genes were screened to elucidate the digital gene expression profiles. Finally, the effects pH and different nitrogen sources on expression levels of nitrogen, iron metabolism-related genes. The main results were summarized as followings:1. Two-year-old blueberry seedlings of Sharpblue, O’Neal, Star, Brigitta and Earliblue were subject to pH5.0, pH7.0 and pH7.5 rhizospheric treatments for six months. The results showed that seedlings treated by pH7.0 and pH7.5 rhizosphere exhibited chlorosis in leaves, roots were blackening and reduced in biomass, the general decreases were also observed in Fv/Fm, chlorophyll content and root activity, meanwhile, the FCR activities were adaptively increased in roots and leaves. However, among the five cultivars, based on the above physiological analysis, the northern highbush blueberry cultivars (Earliblue and Brigitta) showed higher sensitivity to non-acid conditions, as the southern highbush cultivars (Sharpblue, O’Neal and Star) exhibited a relatively endurances to non-acid rhizosphere. Furthermore, O’Neal and Earliblue were deemed as the most endurable and most sensitive cultivars.2. A total of 110,098,570 Clean reads derived from transcriptome sequencing of blueberry roots and 287,800 Mixed-Unigenes were obtained through de nove transcriptome assembly and stitching. Average length and N50 of Mixed-Unigene were 526 bp and 997 nt. Then the Mixed-Unigene sequences were aligned with several public databases, the results showed that 127,216,77,897 and 81,531 sequences respectively were annotated by NR (NCBI non-redundant protein database), NT (NCBI non-redundant nucleotide database) and Swiss-Prot (Redundant protein database). According to the COG (Cluster of orthologous groups of proteins) and KEGG (The kyoto encyclopedia of genes and genomes database) analysis, the 59,776 sequences were divided into 25 categories and 79,995 sequences were involved in 129 KEGG pathways, respectively. Based on NR annotation information,86,806 sequences were categorized into three GO (Gene ontology) categories of biological process, cellular component and molecular function. By comparison with public protein databases,129,972 proteins encoded frame (CDS) were obtained, whereas most of the amino acid length were less than 500 nt. Through simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) analysis, it was found that the largest number of SSR di-nucleotide had 18,938 and transition variation were occupied about 2/3 in SNP.3. Through multiple comparison on the differentially expressed genes in pH5.0, pH7.0-3d and pH7.0-6d libraries, the 7,336 DEGs were found differently expressed in pH7.0-3d treatment compared with acidic control (pH5.0), among which,4,669 genes were significantly down-regulated and 2,667 were up-regulated. A total of 7,973 differently expressed DEGs were identified in comparing pH5.0 with pH7.0-6d libraries, with 3,650 being up-regulated and 4,323 being down-regulated. KEGG function analysis indicated that 1,836 (pH5.0-Vs-pH7.0-3d),2,182 (pH5.0-Vs-pH7.0-6d) and 1,054 (pH7.0-3d-Vs-pH7.0-6d) were significantly enriched in 6,7 and 8 KEGG pathways. The transcription factors belonged to 24 different families were also exploited from the transcriptome libraries,which mainly involved transcription factor family of ERF, NAC, WRKY, bHLH, MYB and so on. Hierarchical clustering analysis on expression patterns between three treatments found that 486 DEGs were grouped into six patterns, which was the largest expression pattern of up-up-down. Furthermore, six unigenes were randomly selected to confirm expression tendency of RNA-Seq differentially expressed genes using qRT-PCR technology. The results showed that DEGs expression patterns obtained from qRT-PCR analysis were significantly consistent with the RPKM results, with the correlation coefficient R values reached 0.8210.4. Blueberries were respectively transferred to hydroponics of pH5.0-NH4+, pH5.0-NO3-, pH7.5-NH4+ and pH7.5-NO3- treatments for 15 days. After treatments, decreased Fv/Fm were observed in treatments of pH7.5-NH4+ and pH7.5-NO3-, and the roots in both treatments exhibited a darker feature. The chlorophyll content, root activity and total nitrogen content were highest under pH5.0-NH4+ treatment, followed by pH5.0-NO3-, the lowest were found in pH7.5-NH4+ treatment. In comparison to the NH4+-N source treatment, nitrate contents in roots were gradually increased under the NO3--N condition, which had no correlation with pH treatments. Meanwhile, compared with the acid treatment, the activity of FCR were significantly increased by alkali treatment, and hardly correlated with different forms of nitrogen source.5. The qRT-PCR analysis revealed that, in contrast to the pH5.0-NH4+ treatment, the expression levels of transcription factor (FER), ferritin 2 (Fer2) under the other three conditions were relatively decreased, while up-regulated expression were observed in ferric reduction oxidase 2 (FR02), natural resistance-associated macrophage protein 3 (Nramp3) and plasma membrane H+-ATPase (HA). The expression levels of ammonium transporters (AMTs) was significantly enhanced when supplying with NH4+-N source. However, higher expressions of nitrate transporter (NRT1.5 and NRT2), nitrate reductase (NR) and nitrite reductase (NiR) were induced when being fed with NO3- as the sole source, which implied the expressions of genes related to the reduction and transportation of nitrate/nitrite were greatly affected by the nitrogen sources. Meanwhile, the transcript level of glutamine synthetases (GSs) and glutamate synthases (GOGATs) were declined when grown on pH7.5 mediums, it may deduced the genes involved ammonion assimilation of blueberry were mainly regulated pH values and not significantly related to nitrogen sources. |
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