Cloning And Functional Analyses Of BrNAP In Postharvest Leaf Senescence In Chinese Flowering Cabbage

As one of the most important leafy vegetables in China,Chinese flowering cabbage(Brassica rapa var.parachinensis)is popular among consumers with its crispy and tender texture and rich nutrition,and has high economic value.Postharvest leaf senescence(yellowing)significantly reduces quality and shelf life of Chinese flowering cabbage.Therefore,it is of great theory and application value to deeply explore the postharvest leaf senescence mechanism of Chinese flowering cabbage.The NAP subfamily is a member of the NAC transcription factor family and is widely involved in the regulation of leaf senescence in plants and participates in ABA-induced leaf senescence.The transcription factor AtNAP inhibits stomata closure,accelerates water loss and triggers leaf senescence through promoting the expression of target gene AtSAG113.In this study,dark-induced senescence and exogenous ABA treatment were performed on postharvest leaves of Chinese flowering cabbage.It was found that the ABA treatment affected the stomatal conductance,the expression of gene annotated as NAP was up-regulated,and the leaf senescence accelerated.On this basis,functional studies were conducted on AtNAP orthologous genes in Chinese flowering cabbage,and further function was explored in postharvest leaf senescence.The main results of the study are as follows:(1)After the Chinese flowering cabbage leaves were harvested,they were stored 0 d,1 d,3 d,5 d in dark and stored in dark after ABA treatment.After ABA treatment,the changes in leaf chlorophyll content and relative conductivity intensified,and the degree of stomatal opening first decreased and then increased.The expression of BrSGR1 and other senescence-associated genes were up-regulated.ABA treatment can accelerate the postharvest leaf senescence process of Chinese flowering cabbage.The expression of Bra003998,a gene annotated as NAP in Brassica rapa Chiifu-401 gene library,increased initially and then decreased as the aging process progresses.Another annotated gene,Bra004385,was positively correlated with leaf senescence,and ABA treatment could upregulate its expression,which is consistent with the expression pattern of NAP in other species.It is believed that Bra004385 may have similar gene function as AtNAP.(2)Bra003998 and Bra004385 were successfully cloned and the coding regions of the two genes are 720 bp and 813 bp respectively.Bioinformatics analysis showed that Bra003998 and Bra004385 have high homology with NAP in many species such as AtNAP.Bra004385 has structural characteristics of the NAP subfamily,while Bra003998 lacks the highly conserved A domain.It is speculated that Bra004385 belongs to the NAP subfamily and named BrNAP1.Subcellular localization experiment revealed that BrNAP1 is a nuclear protein.Therefore,BrNAP1 is candidate a positive regulator of leaf senescence in Chinese flowering cabbage.(3)Successful obtained Arabidopsis thaliana BrNAP1 complementary(in atnap)and overexpressing(in Col-0)transgenic plants.Complementary experiments show that BrNAP1 can restore the Arabidopsis atnap phenotype to the wild type.Overexpression of BrNAP1 causes precocious leaf senescence,the expression level of AtNAP target gene AtSAG113 was about twice that of the control.BrNAP1 transcriptionally induced the activity of BrSAG113 in dual-luciferase assay.These results suggest that BrNAP1 has AtNAP-like functions and is a positive regulator of leaf senescence in Chinese flowering cabbage.(4)The target site was designed according to the sequence of BrNAP1 and the CRISPR/Cas9 vector was successfully constructed.Using cotyledon-cotyledon petiole as the infectious material,the vector was transferred into Chinese flowering cabbage through Agrobacterium-mediated method and successfully obtained the edited plant.