Transcriptome and expression analysis of gibberellin-related genes in large-fruit type bud mutant of Zhuosexiang grape

【Objective】The study aimed to explore the difference of morphological structure affecting large-fruit type grape bud mutant, and the relationship between gibberellins (GAs) and large-fruit type grape bud mutant. Mutants of fruit trees can produce new traits so they can be used foe variety selection. GAs play key role in regulating cell elongation and promoting fruit enlargement. The increase of endogenous GAs content is positively correlated with the size of fruit and the number of peel cells. 【Methods】Zhuosexiang grape and its large-fruit type bud mutant were used as experimental materials. Through the determination of fruit transcriptome at 15 days after flowering and the qRT-PCR expres- sion analysis of gibberellin-related genes VvKO, VvGA2ox2, VvGA2ox4 and VvGA3ox4, combined with the measurement of fruit size and morphology at 15 days after flowering and at fruit maturity stage, the paraffin section was made to compare the peel thickness, and the enzyme activity of the screened GAs enzyme was detected. The correlation analysis of the vertical and horizontal diameters of the fruits, the key genes VvKO, VvGA2ox2, VvGA2ox4 and VvGA3ox4, and the screened GAs enzyme activity was invetigated.【Results】The results showed that the fruit grains and ears of the large-fruit type bud mutant of Zhuosexiang were significantly higher than those of at 15 days after flowering and fruit maturity. There were some differences in peel morphological structure between the two test materials at 15 days after flowering and fruit maturity. At 15 days after flowering, the epidermal cells of the large-fruit type bud mutant were three layers, and the epidermal cells of the Zhuosexiang were only one layer. There was no significant difference in the morphology of subepidermal cells between the two, both were irregularly oval, and there was no significant difference in peel thickness between the two. At fruit maturity stage, the peel thickness of large-fruit type bud mutant grapes was significantly larger than that of Zhuosexiang, and the corky epidermal cells were thicker (black area). The subepidermal cells of the largefruit type bud mutant were larger and mostly irregularly vesicular, while the subepidermal cells of the Zhuosexiang were smaller and mostly conical or long spindle- shaped. At 15 days after flowering, the flesh cells of Zhuosexiang were irregular oval, and the flesh cells of the large- fruit type bud mutant were irregular oval. The subepidermal cells of the large-fruit type bud mutant were larger and mostly irregularly vesicular, while the subepidermal cells of the Zhuosexiang were relatively smaller and mostly conical or long spindle- shaped. At the mature stage, the flesh cells of Zhuosexiang were irregularly oval, and the flesh cells of large- fruit type bud mutant were irregularly vesicle-shaped. The transcriptome sequencing results of the young fruits at 15 days after flowering showed that the number of differentially expressed genes was 2242, with 1167 up-regulated genes and 1075 down-regulated genes. In order to explore the biological functions and properties of differentially expressed genes between Zhuosexiang and its large-fruit type bud mutant, GO enrichment analysis was performed on differentially expressed genes between Zhuosexiang and large-fruit type bud mutant. The results showed that differentially expressed genes (DEGs) were mainly annotated into three functional groups: the first was biological process, which was enriched in phosphorylation, regulation of DNA-templated transcription and protein phosphorylation; the second was biological process, which was enriched in phosphorylation and regulation of DNA-templated transcription and protein phosphorylation. The second is the cell component, and the more enriched is the membrane-related gene; the third is molecular function, which is enriched in enzyme activity and binding- related genes. The results of KEGG enrichment showed that there were more differential genes in the following pathways: 59 genes were up-regulated and 33 genes were down-regulated in the biosynthesis of secondary metabolites pathway; there were 16 up-regulated genes and 3 down-regulated genes in the biosynthesis of amino acids pathway. There were 9 up-regulated genes and 0 down-regulated genes in the carbon fixation by Calvin cycle pathway. Phagosome pathway up- regulated 11 genes and down- regulated 0; Glycolysis/Gluconeogenesis pathway up- regulated 12 genes, down- regulated 0; Phenylpropanoid biosynthesis pathway up- regulated 10 genes and downregulated 4 genes. GAs is an important factor for grape fruit enlargement. In order to study the relationship between GAs and the mutant of Zhuosexiang, the expression of four gibberellin-related genes was analyzed by qRT-PCR. The results showed that the expression levels of VvKO, VvGA3ox4, VvGA2ox4 and VvGA2ox2 genes in Zhuosexiang were significantly higher than those in large-fruit type bud mutant (P＜0.05), which were 2.33 times, 5.13 times, 30.67 times and 751 times higher than those in large-fruittype bud mutant. GA2ox enzyme is an important enzyme for the degradation of GAs in plants. In order to further clarify the role of GA2ox gene in the large-fruit type bud mutant, the GA2ox enzyme activity in young fruit at 15 days after flowering was determined. The results showed that the GA2ox enzyme activity in the young fruit of Zhuosexiang and large-fruit type bud mutant were 17.58 U · g- 1 and 13.54 U · g- 1 . The GA2ox enzyme activity in the fruit of Zhuosexiang was 1.3 times as high as that of largefruit type bud mutant, which was significantly higher than that of large-fruit type bud mutant (P＜0.05). The correlation analysis between four key differential genes, one enzyme activity and fruit longitudinal and transverse diameter was carried out. The VvGA2ox2, VvGA3ox4 and GA2ox enzyme, VvGA2ox4 and VvGA3ox4, fruit transverse diameter and longitudinal diameter were significantly and positively correlated, and the VvGA2ox2, VvGA2ox4, VvGA3ox4 and GA2ox enzyme were significantly and negatively correlated with fruit longitudinal and transverse diameter. The VvGA2ox4, VvGA3ox4 and VvGA2ox2, VvGA2ox4 and GA2ox were significantly positively correlated. The results showed that the GA2ox enzyme activity of VvGA2ox2, VvGA2ox4 and VvGA3ox4 increased when the expression of the three genes was up-regulated, thereby inhibiting the increase of the vertical and horizontal diameters of the fruit. The contents of common GA3, GA4 and GA7 in the young fruit of large-fruit type bud mutant were significantly higher than those of Zhuosexiang.【Conclusion】There were significant differences in pericarp morphological structure between the two test materials. The number of epidermal cell layers of large-fruit type bud mutant was more at 15 days after flowering. During the ripening period, the peel of large-fruit type bud mutant grape was thicker, and the cork-like epidermal cells were thicker, and the subepidermal cells were larger and mostly irregularly vesicle-like, while the subepidermal cells of Zhuosexiang were smaller and mostly conical or long spindle-shaped. The number of significantly different genes between the two was 2242, involving multiple membrane-related genes, transcription factors and enzyme activity genes, of which the number of membrane-related differential genes was the largest. In terms of metabolic pathways, there were many differentially expressed genes involved in amino acid biosynthesis, amphetamine biosynthesis, biosynthesis of secondary metabolites, Calvin cycle carbon fixation, phagosome and glycolysis/glucose production. The expression levels of the VvKO, VvGA2ox2, VvGA2ox4 and VvGA3ox4 genes in Zhuosexiang grape were significantly higher than those in largefruit type bud mutant, especially the VvGA2ox2 and VvGA2ox4, and their corresponding GA2ox enzyme activities were also significantly higher than those in large-fruit type bud mutant. The correlation analysis results were also consistent with the research results, indicating that gibberellin- related genes were important factors affecting the development of Zhuosexiang grape large-fruit type bud mutant.