| As an important fruit crop,grape is planted world.The planting area of table grape in China ranks first in the world.Grape berries have thin skin,rich juice and high sugar content.After harvest,they are prone to decay,peduncle browning and grain shattering,which have a great impact on the shelf quality of grapes.Color is an important index affecting the appearance quality of grapes.HardnessFirmness is an important factor affecting the storage performance of grapes.Fruit softening will make fruits susceptible to pathogens,reduce their shelf quality,and greatly shorten the storage period and shelf life.Promoting grape fruit coloring,inhibiting fruit softening and prolonging grape storage time are one of the important problems in the grape industry.Therefore,it is of great significance to improve fruit quality and prolong the shelf life of grapes by analyzing the effect of ethylene on the ripening and color change of ’Summer Black’ grapes as well as the function mechanism of VvEXPLA1 gene in grape fruit ripening and softening.In this study,ethylene treatment was carried out one week before the verasion of’Summer Black ’ grape fruit,and water treatment was used as control.The effects of ethylene treatment on the growth,color change,ethylene release and related gene expression of ’Summer Black’ grape fruit were analyzed.The expression of EXP family genes during fruit development of ’Shine Muscat’ and ’Hanxiangmi’ was analyzed,and the gene VvEXPLA1 that may be involved in fruit firmness change was screened.The role of VvEXPLAl in fruit softening was preliminarily analyzed,and its expression was regulated by transcription factor VvbZIP60 by yeast one-hybrid and dual luciferase experiments.The main results are as follows:1、In order to study the effect of ethylene on the ripening of Summer Black’grape fruit,the grape cluster was treated with 500 mg/L ethylene(ETH)and 0.05%(v/v)Tween 20 one week before verasion of the fruit.The distilled water was added with 0.05%(v/v)Tween 20 was used as control(CK).Samples were collected at 0 d,5 d,10 d and 15 d after treatment,single grain weight,vertical and horizontal diameter,hardness,ethylene release,anthocyanin and chlorophyll content in the skin were quantified.The relative expression levels of genes related to chlorophyll degradation pathway and ethylene synthesis pathway were analyzed.The results showed that the single fruit weight,fruit longitudinal diameter,transverse diameter and soluble solids content increased gradually,while the titratable acid content decreased gradually.The 10 d and 15 d after treatment were significantly lower than the control.The fruit firmness after ETH treatment was lower than that of the control.Compared with the control,it was significantly lower than the control at 10 d and 15 d.After ethylene treatment,fruit verasion significantly advanced,the ethylene release increased significantly,and the chlorophyll content in the berry skin decreased significantly.At 5 days after treatment,the chlorophyll content in the skin decreased by 1.13 times compared with the control.At 10 days after ethylene treatment,the chlorophyll content in the skin decreased by 1.4 times compared with the control.At 15 days after ethylene treatment,the chlorophyll content in the berries decreased by 1.3 times compared with the control.After ETH treatment,the anthocyanin content in the skin increased significantly.In the expression of chlorophyll degradation pathway genes,VvNYC,VvCLH2 and VvRCCR were significantly reduced;in contrast,the three VvNOL,VvPAO,VvPPH,VvPAO1,CLH1,and VvNYC1 genes increased to varying degrees.There was no significant change in the ethylene key gene VvACOs,and VvACS8 was significantly increased.It indicated that ethylene treatment could promote the degradation of chlorophyll in the skin,thereby promoting fruit ripening.2、In the RNA-seq data of ’Shine Muscat’ and ’Hanxiangmi’ fruits at different developmental stages,20 EXP genes were deferentially expressed.Among them,the expression of VvEXPLA1 gene in the two varieties gradually increased with the ripening and softening of the fruit.The plant overexpression vector of VvEXPLA1 was constructed and transformed into tomato by Agrobacterium-mediated method.According to the results of qRT-PCR,VvEXPLA1-1,VvEXPLA1-2 and VvErXPLA1-3 were selected for further functional analysis.The fruit firmness of VvEXPLA1 transgenic plants was significantly reduced.In the fruit,the content of protopectin decreased and the content of soluble pectin(WSP)increased,indicating that VvEXPLA1 played an important role in fruit softening.3、In order to further study the mechanism of VvEXPLA1 in fruit softening,this study screened its upstream regulatory gene VvbZIP60 by yeast one-hybrid experiment,and negatively regulation of VvbZIP60 on the expression of VvEXPLA1 gene was confirmed by dual luciferase experiment. |
