Cloning and bioinformatics analysis of full-length cDNA of RuEG genes in blackberry and molecular identification of transgenic tomato
Using blackberry fruit cDNA as a template, the obtained fragments were spliced by DNAStar software after PCR amplification, and the full-length ORFs of RuEG genes were obtained. Bioinformatics analysis showed that the ORF sequence of RuEG1 was 1578 bp in length, encoding 525 amino acids, and that of RuEG2 was 1863 bp in length, encoding 620 amino acids.
The target gene was inserted into the expression vector (Fig. 1a), which was transferred into tomato by Agrobacterium-mediated method, and several transgenic lines were obtained after screening with Kan (Fig. 1b). We randomly selected eight independent transgenic lines with better growth status for testing. The RNA was extracted from tomato leaves, and single-stranded cDNA was synthesized by reverse transcription. The PCR amplification was carried out using specific primers for RuEG1 and RuEG2 (Fig. S1). Unique bands of 599 and 527 bp were amplified, respectively, indicating that RuEG1 and RuEG2 were successfully embedded into the tomato genome. This also showed that transgenic tomato plants were successfully obtained.
Analysis of Cx gene expression characteristics
The EG genes play an important role in plant growth and development, such as organ shedding, fruit ripening, and softening, and different members of the EG family have different expressions [27, 28]. Gene PpEG4 was cloned from peach fruit, and its expression began at the young fruit stage, reached a peak before respiratory saltation, and then gradually decreased [20]. Cold stress severely inhibited the transcript and protein levels of EBF1, ABI5-like, and fruit softening-related genes in banana. The ectopic and transient overexpression of EBF1 and ABI5-like genes in tomato and Fenjiao banana accelerated fruit ripening and softening by promoting ethylene production, starch and cell wall degradation, and decreasing fruit firmness [29]. In the present study, exogenous overexpression of RuEG1 and RuEG2 was used to study the effect of RuEG genes in transgenic plants, using non-transgenic tomato as control. In order to analyze expression of RuEG genes in tomato, we determined the transcription levels of tomato fruit at different developmental stages using qRT-PCR (Fig. 1c, d, e, f). The qRT-PCR results showed that RuEG1 and RuEG2 were expressed in large quantities in transgenic tomato, RuEG1 was expressed in large quantities in yellow fruit stage, RuEG2 was expressed in large quantities in broken color stage, and no expression was detected in wild type, indicating that the key gene specific to blackberry was transferred. During the storage period, RuEG1 and RuEG2 were expressed in large quantities at 21 and 7 days, respectively. Combined with the results of the Cx activity and cellulose content in transgenic tomato, this indicated that the RuEG genes cloned from blackberry significantly improved the Cx activity and then reduced the cellulose content.
Analysis of physiological index of fruit
Fruit firmness can measure the degree of softening, storage-resistance of berry fruits, and the texture change of fruit tissues after harvest, which is related to many factors such as cell wall structure and decay [30]. This not only has an important impact on the edible quality of fruits, but also on the processing quality of fruits and resistance to diseases after harvest. Fruit firmness decreased continuously with development stage, the firmness of transgenic fruits in the immature fruit stage did not differ from that of the control, and that of RuEG1 was slightly lower in the green ripe fruit stage (Fig. 2). Firmness of transgenic fruit was significantly lower than that of the control group from the broken color fruit stage to the red fruit stage, and firmness of RuEG2 fruit was the lowest. We speculate that overexpression of EG genes might lead to changes in the content of cell wall components. At 7 days of storage, the firmness of wild-type tomato decreased from 2.24 ± 0.10 to 1.04 ± 0.10 N, and that of transgenic RuEG1 and RuEG2 decreased from 1.63 ± 0.20 and 1.26 ± 0.26 to 0.73 ± 0.02 and 0.63 ± 0.07 N, respectively. At 14 days of storage, the firmness of wild-type tomatoes decreased significantly but did not significantly differ with that of transgenic tomatoes. At 21 days, the firmness of all tomatoes decreased slightly, with no significant difference between wild-type and transgenic tomatoes.
Phenotypic identification and microstructural observation of fruit
The fruit development was divided into five stages (Fig. S2): immature fruit, green ripe fruit, broken colored fruit, yellow ripe fruit, and red ripe fruit. In order to investigate the internal morphological differences of tomato plants after overexpression of EG genes, the phenotype and micromorphological structure of fruit were observed by scanning electron microscopy (Fig. 3). The fruit cross-sections in different periods were compared. In the immature fruit stage, the palisade tissue of all fruit was arranged neatly and tightly, with full shape, no collapse, and small cells. There was no difference between transgenic and control fruit. In the green ripe fruit stage, the cells increased with development, but the structure was still clear and there was no collapse. There was no significant difference between transgenic and control fruit. At the breaking stage, the fruit tissues of both wild-type and transgenic tomato began to collapse. The cells of RuEG1 were not clear but still intact, while the tissues of RuEG2 were loose and partially broken, and the cross-section flesh began to fall off, indicating that the structure of RuEG2 fruit had begun to change during this period, possibly because overexpression of RuEG2 affected the contents of the cell wall. At the yellow ripe fruit stage, wild-type tomato tissues were more complete than genetically modified ones, and the outline was clearer. Some RuEG1 tissues were loose and broken, while RuEG2 tissues collapsed completely, and the structure was no longer obvious. In the red ripe fruit stage, some tissues of wild-type tomato were broken, but the structure was still complete and the outline was clear. Most of the RuEG1 fruit structures collapsed, a few were intact, and some of the outline remained; whereas the RuEG2 fruit structures completely collapsed and the three-dimensional structure disappeared. In conclusion, the RuEG1 fruit tissue began to collapse at the yellow fruit stage, while tissue rupture of RuEG2 began at the earlier color breaking stage, suggesting that the two genes played their roles at different periods. The results suggest that there may be a direct correlation between RuEG1 and RuEG2 of blackberry and tomato morphogenesis.
Determination of cell wall composition
Structure of the fruit cell wall is the key factor determining fruit firmness. It is generally believed that breakdown of the internal structure of the cell wall is the initial cause of fruit softening. Cellulose, hemicellulose, and pectin mainly exist in the cell wall of fruit, forming an important material basis for fruit firmness. The content and decomposition of cellulose and hemicellulose are closely related to the ripening and softening of fruit [31]. Cellulose molecules gather to form microfilaments, which form the skeleton of the cell wall [32]. In this study, the cellulose content of transgenic tomato decreased significantly at yellow and red fruit stages, and was significantly lower than that of wild-type tomato, indicating that RuEG1 and RuEG2 overexpression enhanced the degradation of cellulose in the fruit cell wall. After 7 days of storage, the cellulose content was significantly lower in transgenic than wild-type tomato (Fig. 4). Hemicellulose is a polysaccharide whose main chain is composed of β-1,4-D-polysaccharide. Hemicellulose can bind microfibers and play a role in maintaining the firmness and flexibility of tissues in the cell wall [33]. The results of this study showed that the hemicellulose content of wild-type tomato initially increased and then decreased in the development stage, reached the maximum value in the yellow fruit stage, and decreased in the red fruit stage. The hemicellulose content of transgenic RuEG1 fruit decreased to the lowest value in the red fruit stage, while that of transgenic RuEG2 fruit increased first and then decreased. The hemicellulose content was lower in transgenic than wild-type fruit. During the storage period, the hemicellulose content decreased significantly after 7 days of storage, the RuEG2 content was the lowest in transgenic fruits, and reached a very low level after 21 days of storage (Fig. 4). During the ripening and softening process, the pectin in the cell wall continues to break down, causing fruit to become less rigid and gradually soften [34]. During fruit development, the pectin content of wild-type and transgenic RuEG1 fruit initially increased and then decreased, reaching their maximum values at the breaking and green ripe stages, respectively. The pectin content of transgenic fruit showed a downward trend and, from the breaking stage onward, was much lower than that of wild-type fruit. During storage, the pectin content decreased continuously, but was higher in transgenic than wild-type fruit, but firmness did not significantly differ among the two groups. We speculate that the proportion of soluble pectin increased, coupled with the degradation of cellulose and semi-fiber, and so cell wall structure could not be maintained.
Determination of Cx activity and PME
It is generally believed that fruit softening is caused by a variety enzymes working together to hydrolyze the cell wall, thus reducing the connections between cells, leading to cell dispersion and gradual softening [35]. The enzyme PG is one key enzyme in fruit softening, mainly responsible for decomposition of pectin and the action of polygalactose aldehyde in the cell wall to degrade it into galacturonic acid, thus leading to structural disintegration of the fruit cell wall and accelerating fruit softening [36]. The PME mainly acts as an assistant to PG, which is demethylated by acting on galacturonic acid residues in cell wall pectin, while galacturonic acid residues are the substrate of PG action. The PME makes the polysaccharide uronic acid more vulnerable to PG degradation, and mainly acts on pectin substances in the cell wall to promote pectin hydrolysis, thus accelerating disintegration of the cell wall and promoting fruit softening [37, 38]. In this study, at the red fruit stage, the PME activity of both transgenic tomatoes reached maximum values of 65.3 ± 1.5 and 72.4 ± 3.6 U·g FW− 1, respectively, both significantly higher than that of wild type (Fig. 5). Compared with wild type, the PME activity of transgenic RuEG1 tomato was 18.9% higher and that of transgenic RuEG2 tomato was 31.9% higher. The Cx is an enzyme complex that mainly degrades cellulose in cell walls, and a variety of cell wall degradation enzymes work together to participate in the disintegration of cell wall structure [39]. The Cx activity in both transformed tomatoes showed no significant difference at the immature fruit stage with the wild type, but was significantly higher than the wild type in other stages (Fig. 5). The Cx acts on cellulose in the cell wall, consistent with the cellulose content results. The maximum values were 286.0 ± 13.3 and 337.4 ± 12.7 U·g FW− 1 at the red fruit stage. Compared with wild-type tomato, the Cx activity of transgenic RuEG1 tomato was 22.8%~69.9% higher at the green ripening stage, and that of transgenic RuEG2 tomato was 36.4–73.1% higher.
Correlation analysis
The correlations among firmness, gene expression, Cx activity, pectinase activity, and cell wall components (cellulose, hemicellulose, and pectin) were analyzed using SPSS software (Fig. 6). Firmness was extremely significantly negatively correlated (P < 0.01) with Cx and pectinase activities, with correlation coefficients of − 0.77 and − 0.99, respectively. Firmness was also extremely significantly positively correlated with the cellulose, hemicellulose, and pectin, with correlation coefficients of 0.63, 0.70, and 0.88, respectively. The Cx activity with cellulose content, and pectinase activity with pectin content, were extremely significantly negatively correlated with correlation coefficients of − 0.71 and − 0.74, respectively. It should be noted that Cx activity and corresponding gene expression level was positively correlated, but the correlation (0.20) was not significant. This was because, although gene expression in a certain period caused the increase in Cx activity, the expression level was lower in the later part of this period, but the activity did not decrease. For example, the sudden increase of RuEG2 expression level in the breaking stage caused the increase of Cx activity, but the expression level was lower in the yellow fruit than in the breaking stage, while the Cx activity did not decrease.
Transcriptome analysis
Identification and comparison of DEGs
The two RuEGs played the roles in different ripening stages of fruit, and the RNA-seq technology was further used to explore the effect of overexpressed genes on transcription. Overexpressed RuEG1 fruit was compared with the control group (RuEG1/CK), and 96 DEGs (2 times, Fig. 7a) were screened, of which 46 were up-regulated and 50 were down-regulated in condition of P-adjust < 0.05. And there were 76 annotated genes and are listed in Table S2. The DEGs of overexpressed RuEG2 fruit compared with the control group (RuEG2/CK) was 170, of which 76 were up-regulated and 94 were down-regulated (2 times, Fig. 7b). And there were 124 annotated genes and are listed in Table S3.
Bioinformatics analysis of differential genes
The GO analysis results of DEGs in the RuEG1/CK and RuEG2/CK groups are shown in Fig. 8, which shows information about BP, CC, and MF. RuEG1/CK and RuEG2/CK showed the similar trend in all three categories. In the BP category, the genes were mainly involved in cellular process and metabolic process. The cell part was the largest proportion, followed by membrane part and organelle in the CC category. In the MF category, the number of catalytic activity was most, followed by binding in RuEG1/CK group, but in group RuEG2/CK, the situation was opposite.
Analysis of the KEGG metabolic pathways
KEGG is a major public database for the systematic analysis of genes expression pathways in cells. A KEGG analysis was conducted to further understand the DEGs functions, which is shown in Fig. 9. Transcriptome data found that genes in the RuEG1/CK and RuEG2/CK groups were partially differential genes involved in cell energy metabolism processes, such as the pentose phosphate pathway, starch and sucrose metabolism, the glycolysis process, amino acid metabolism and fatty acid oxidation. Among the differential genes in the RuEG1/CK group, 6 were involved in metabolism of cofactors and vitamins pathway, which was the most common gene pathway, followed by the lipid metabolism and carbohydrate metabolism pathway 5 genes, respectively. In the RuEG2/CK group, there was 27 genes on the pathway of carbohydrate metabolism, which also had the most genes, the second was amino acid metabolism pathway with 22 genes, followed by the energy metabolism pathway with 20 genes.
qRT-PCR verification of the DEGs
The expression levels of some cell wall degrading enzyme genes were high and significantly different compared with the control group when the expression P-value < 0.05, but they were not listed in the screening condition when P-adjust < 0.05. The cell wall degrading enzyme genes of XTH3, Cel2, Cel8 and Man4 and some key genes in metabolic pathways were selected to analyze the amount of expression by qRT-PCR. As shown in Fig. 11, the expression of XTH3, Cel2, CPA, AR and G6P-1-E genes was up regulated in RuEG1 compared control group, respectively. And the expression of genes XTH3, Cel2, Cel8, α-Glu, GADPH and G6P-1-E was up regulated in RuEG2 compared control group. The results showed that the gene expression data from the transcriptome analysis and the reverse transcription quantitative PCR results had a similar trend, which suggested that DEGs were credible.
Altered genes in metabolic pathways
To further understand the molecular changes in the regulated genes involved in biometabolic processes, the gene expression patterns of metabolic pathways members in RuEG1/CK and RuEG2/CK groups were analyzed, after combining the GO enrichment and KEGG metabolic pathway analysis results. (Fig. 10). The DEGs mainly involved HMP, EMP, amino acid and fat metabolism, electron transport chain and so on. Glucose-6-phosphate 1-epimerase (G6P-1-E) and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in EMP pathway was up-regulated gene in RuEG2/CK group. Glucosidase is a large class of enzymes in the glycoside hydrolase family. The main function of α- Glucosidase(α-Glu) is to hydrolyze α-1,4 glycosidic bonds and release glucose as a product, which is an indispensable enzyme in the glucose metabolism pathway of organisms[40]. The expression levels of α-Glu in transgenic fruits were all up-regulated, and the RuEG2/CK group was higher than the RuEG1/CK group. Therefore, it is speculated that the up-regulation expression of α-Glu in transgenic fruits promotes the degradation of cellulose. G6P-1-E converts glucose 6-phosphate into fructose 6-phosphate in EMP pathway[41].GAPDH is a constitutively expressed protein during intracellular glucose metabolism, is involved in the glycolysis pathway, and is closely related to ATP synthesis [42]. The up-regulated expression of the above three genes indicated that cellulose decomposition and energy metabolism accelerated in RuEG2/CK group. Aldose reductase (AR), 6-phosphogluconolactonase (PGLS) and glutathione S-transferase (GST) were up-regulated in RuEG1/CK group. Aldose reductase belongs to AKR1B1 in aldosterone reductase family, which is a NADPH-dependent enzyme and can catalyze the reduction of hydrophilic and hydrophobic aldehydes[43]. Glutathione S- transferase is the key enzyme of glutathione binding reaction, and the initial step of catalyzing glutathione binding reaction mainly exists in cytosol[44]. Peroxiredoxins are enzymes that catalyze the reduction of hydrogen peroxide and alkyl hydroperoxides[45]. The up-regulated gene 1-Cys peroxiredoxin A (CPA) catalyzed the conversion of glutathione to glutathione disulfide in RuEG1/CK group. PGLS participates in HMP and catalyzes 6-phospholutenolide to produce 6-phospholuconate. Therefore, up-regulated genes were mainly involved in HMP and amino acid metabolism pathway, thereby reducing fruit defense capability and accelerating softening. In addition, glycerophosphodiester phosphodiesterase(GDPD1) was down-regulated in RuEG1/CK group and 3-ketoacyl-CoA synthase 6 (KDS6) is up-regulated in RuEG2/CK group in the fatty acid metabolism pathway. In terms of electron chain transfer, NADH dehydrogenase (ndhD) was down-regulated in both groups, which indicated that respiration had weakened in transgenic fruits. Therefore, overexpression of RuEG1 and RuEG2 genes partly speeds up cellular metabolism, making the fruit in the yellow fruit stage softer than the control. XTH enzymes are thought to play a key role in fruit ripening by loosening the cell wall in preparation for further modification by other cell wall-associated enzymes and through disassembly of xyloglucan[46]. The results of transcriptome and qRT-PCR in two groups of transgenic fruits showed that the expression levels of XET3 were higher than those of the control, indicating that the overexpression of RuEG1 and RuEG2 genes enhanced the expression level of XET3, which was speculated to have a positive effect on the enhancement of enzyme activity, the degradation of cell wall, and the promotion of fruit softening process. Cellulose is degraded by the actions of cellobiohydrolases(CBH), endo-1,4-β-glucanases (EG), and β-glucosidases. The CBH and EG worked synergistically to degrade cellulose. The cel2 and cel8 are predicted to encode EGs [47]. In this study, we found that the expression levels of cel2 and cel8 determined by transcriptome and qRT-PCR in the transgenic fruit were higher than those of the control fruit, and the relative expression level of cel8 in the RuEG2 transgenic fruit was 68.87 ± 6.95. Therefore, it was speculated that the high expression of these two genes played a positive role in the degradation process of cell wall, and thus promoted the fruit softening.