The Role Of Citrus CsARF21 Gene In Fruit Ripening

Citrus(Citrus reticulata Blanco)fruit has high nutritional value and is the most important fruit tree in southern China with the widest cultivation area.However,due to the excessive number of mid-ripening varieties and the small number of early and late ripening varieties,the concentration of citrus fruits in the mature stage in China is a major industrial problem.Excess seasonal output,especially the centralized marketing of mid-ripening varieties,has brought serious impact on the market supply and demand relationship and price,and greatly affected the economic benefits of the industry.Adjusting the structure of ripening period of citrus varieties is one of the key links to solve this problem.The study of fruit ripening mechanism can provide theoretical support and molecular genetic resources for the improvement of ripening period of citrus varieties and the breeding of new varieties.In our previous study,it was found that high concentration of free IAA could delay the color transformation of orange fruit,and transcriptome RNA Seq analysis revealed that the Auxin response factor gene Cs ARF21 might be involved in this process.In this study,the expression of auxin response factor Cs ARF21 in citrus fruits during fruit development and ripening was analyzed.The expression of Cs ARF21 gene was analyzed in response to treatment with IAA.Cs ARF21 gene was cloned and an overexpression vector was constructed.Cs ARF21 gene was heterologously expressed in tomato(Micro-Tom),and the potential function of this gene in regulating fruit ripening was analyzed.The specific research results were as follows:1.The expression level of Cs ARF21 gene increased continuously from the expansion stage to the color-breaking stage,and the relative expression level of the color-breaking stage was more than 2 times that of the expansion stage.The expression level of Cs ARF21 gene decreased to the level of expansion after entering the transchromatic phase.The high expression of Cs ARF21 gene at coloration stage suggests that it may be involved in the formation and accumulation of carotenoids in citrus fruits and fruit ripening.2.Cs ARF21 gene expression reached its peak at 3 h after treatment.The expression level of Cs ARF21 gene in fruits treated with 100μM IAA was about 3.5 times that of the control,and that in fruits treated with 1000μM IAA was about 4.5 times that of the control.The expression level of Cs ARF21 gene in fruits treated with 100μM IAA for 6 h was not significantly different from that in fruits treated with 3 h.IAA induced Cs ARF21 expression with concentration effect,and the higher the concentration,the more intense and longer the induced expression.The expression level of Cs ARF21 was still more than twice that of the control after treatment with 1000μM IAA for 12 h,while the expression level of Cs ARF21 in the fruits treated with 100μM IAA decreased to the control level.The results showed that Cs ARF21 gene was a key member of the auxin response factor family in response to exogenous IAA treatment in citrus fruits after full development.The high expression of Cs ARF21 gene was closely related to the delayed color turning of citrus fruits after IAA treatment.3.Research function Cs ARF21 heterologous expression gene in tomato:the CDS sequence of Cs ARF21 gene was cloned and its overexpression vector was constructed.The micro-Tom tomato was transformed by Agrobacterium-mediated method.Through PCR detection of hygromycin resistance gene and verification of target gene,11 transgenic tomato strains with target gene were obtained,and could determine the Cs ARF21 gene expression in transgenic tomatoes.Differences in appearance and ripening stage between transgenic tomatoes and controls are being observed.