Cloning and Functional Analyses of BrNAP in Postharvest Leaf Senescence in Chinese Flowering Cabbage

doi:10.16420/j.issn.0513-353x.2020-0295

Acta Horticulturae Sinica ›› 2021, Vol. 48 ›› Issue (1): 60-72.doi: 10.16420/j.issn.0513-353x.2020-0295

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Cloning and Functional Analyses of BrNAP in Postharvest Leaf Senescence in Chinese Flowering Cabbage

LI Yuzhuo¹, LIU Ke¹, YUAN Lu¹, CAO Liwen¹, WANG Tingjin¹, GAN Susheng²^,^**(), CHEN Liping¹^,^**()

¹Department of Horticulture,College of Agriculture and Biotechnology,Zhejiang University,Hangzhou 310058,China
²Plant Biology Section,School of Integrative Plant Science,Cornell University,Ithaca,NY 14853,USA

Received:2020-06-08 Revised:2020-07-27 Online:2021-01-25 Published:2021-01-29
Contact: GAN Susheng,CHEN Liping E-mail:sg288@cornell.edu;chenliping@zju.edu.cn

Abstract

Abstract:

This research revealed the cloning and functional analyses of AtNAP orthologous gene named BrNAP1 in Chinese flowering cabbage（Brassica rapa var. parachinensis）. The coding region of BrNAP1 is 813 bp in length and encodes 270 amino acids. It has a unique conserved domain of NAP transcription factors and belongs to the NAP subfamily. BrNAP1 expression increases with the progression of leaf senescence and can be induced by ABA treatment. Subcellular localization experiment revealed that BrNAP1 is a nuclear protein. Complementary experiments showed that BrNAP1 can restore the Arabidopsis atnap phenotype to the wild type. Overexpression of BrNAP1 causes precocious leaf senescence. BrNAP1 transcriptionally induced the activity of BrSAG113 in dual-luciferase assay. These results suggested that BrNAP1 has the AtNAP-like function and is a positive regulator gene of leaf senescence in Chinese flowering cabbage.

Key words: Chinese flowering cabbage, postharvest, leaf senescence, BrNAP1, functional analysis, abscisic acid

CLC Number:

S634.5

LI Yuzhuo, LIU Ke, YUAN Lu, CAO Liwen, WANG Tingjin, GAN Susheng, CHEN Liping. Cloning and Functional Analyses of BrNAP in Postharvest Leaf Senescence in Chinese Flowering Cabbage[J]. Acta Horticulturae Sinica, 2021, 48(1): 60-72.

Figures/Tables 13

Table 1 All primers used in this study

名称	上游引物（5′-3′）	下游引物（5′-3′）
Name	Forward primer	Reverse primer
Bra003998	CTCCAATTCTACTACACTCCCTCCAGGATT	TCATACGCTGAAACAGCTTCATTTTATAAT
Bra004385	TTTCTAACATTTCATAAACCTTGAAAACAC	TACGTTTGAACATGCTGAGCAGCTTAATAT
BrNAP1-GFP	AGCTCGGTACCCGGGATGGACATGAATCC CAACAC	CATGTCGACTCTAGAAAACTGAAACATAC TAGCCT
BrActin1-qPCR	CGCTTAACCCGAAAGCTAAC	TACGCCCACTAGCGTAAAG
Bra003998-qPCR	CGCTGTTTCGGGTTATTGG	CCTCATTTCTTCTATCTGCTTCGT
Bra004385-qPCR	CCCAACACTACACTCCCTCCA	GACGCCTACGTTACTCGAACC
BrSAG113-qPCR	TTACGAGTTAGCCAAGACCGAA	TAAACGCCGCAGAAGTGATAT
BrSGR1-qPCR	GTTGGGGTCCGCTTTGGGAA	AATCGAGCTAACCTGCGGGA
BrABI5-qPCR	ACGTTTGGGGAGATGACGCT	CCACCGCCGTAACAAACACC
BrPAO-qPCR	CAGCTTCAGCGACACTCACC	TCGCCGTGCTCTTCTTCGAT
AtActin2-qPCR	TCTCCCGCTATGTATGTCGC	GTCACGTCCAGCAAGGTCAA
AtSAG113-qPCR	CTCTGTCGTAACGGCAAAGC	GTCTGCTGATTACATACGGCTTC
AtSGR1-qPCR	AACCAACTCAATACGCATCACA	CAACAGAACAAAACGCCACTAA
AtPAO-qPCR	CTTTTCCACAACCCTTTACGC	GCAAGTGTCCATTCTCATCCAA
BrNAP1-SK	GCGGCCGCTCTAGAACTAGTGATGGAC ATGAATCCCAACAC	GGTCGACGGTATCGATAAGCTCTAAAACT GAAACATACTAG
Pro-BrSAG113-LUC	GTCGACGGTATCGATAAGCTTTCCAAA CTCAAAAATAATAGTTGT	CGCTCTAGAACTAGTGGATCCGCGATCGT TCAAACCGATCAACAC

Fig. 1 Amino acid sequence alignment of AtNAP,Bra003998 and Bra004385

Fig. 2 Phenotypical and physiological changes in leaves of Chinese flowering cabbage after harvest or ABA treatment in the dark * P < 0.05;** P < 0.01.

Fig. 3 Changes of stomatal aperture of Chinese flowering cabbage after harvest or ABA treatment in the dark

Fig. 4 Changes of relative expression of SAGs in postharvest leaves of Chinese flowering cabbage in the dark * P < 0.05;** P < 0.01.

Fig. 5 The postharvest expression patterns and ABA-inducibility of Bra003998 and Bra004385 in the dark * P < 0.05;** P < 0.01.

Fig. 6 Subcelluar localization of BrNAP1 in tobacco leaf epidermis cells

Fig. 7 Leaf senescence in transgenic Arabidopsis plants that either are complemented with BrNAP1（in atnap）or overexpressing BrNAP1 （in wild type）

Fig. 8 Chlorophyll contents changes in BrNAP1 complementary Arabidopsis lines* P < 0.05;** P < 0.01.

Fig. 9 Expression of some SAGs in BrNAP1 complementary Arabidopsis lines * P < 0.05;** P < 0.01.

Fig. 10 Chlorophyll contents changes in in transgenic Arabidopsis overexpressing BrNAP1 * P < 0.05;** P < 0.01.

Fig. 11 Expression of some SAGs in transgenic Arabidopsis overexpressing BrNAP1 * P < 0.05;** P < 0.01.

Fig. 12 BrNAP1 transcriptionally induced the activity of BrSAG113 in dual-luciferase assay * P < 0.05;** P < 0.01.

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Cloning and Functional Analyses of BrNAP in Postharvest Leaf Senescence in Chinese Flowering Cabbage

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