菜薹BrNAP克隆及其在采后叶片衰老中的功能分析

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

摘要/Abstract

摘要：

在菜薹（Brassica rapa var. parachinensis）中克隆BrNAP1并分析其功能。BrNAP1编码区全长813 bp,编码270个氨基酸,具有NAP转录因子特有的保守结构域,属于NAP亚家族成员。BrNAP1表达量与叶片衰老程度呈正相关且受ABA诱导表达上调。亚细胞定位试验表明BrNAP1定位于细胞核。互补试验显示BrNAP1能使拟南芥atnap滞绿表型回复至野生型,过表达则能引起采后叶片早衰。双荧光素酶试验表明BrNAP1能够激活BrSAG113表达。这些说明BrNAP1是菜薹采后叶片衰老的正调控基因。

关键词: 菜薹, 采后, 叶片衰老, BrNAP1, 功能分析, 脱落酸

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

中图分类号:

S634.5

李钰卓, 刘柯, 袁璐, 曹丽雯, 王挺进, 甘苏生, 陈利萍. 菜薹BrNAP克隆及其在采后叶片衰老中的功能分析[J]. 园艺学报, 2021, 48(1): 60-72.

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.

图/表 13

表1 本研究中所用的引物

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

图1 AtNAP,Bra003998与Bra004385氨基酸序列比对图

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

图2 菜薹采后黑暗贮藏下叶片黄化表型及生理指标变化

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.

图3 菜薹采后叶片黑暗贮藏0 ~ 5 d气孔形态变化情况

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

图4 菜薹采后叶片黑暗贮藏下衰老相关基因相对表达量变化情况

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

图5 菜薹采后叶片黑暗贮藏下Bra003998、Bra004385表达模式分析

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

图6 菜薹BrNAP1蛋白在烟草叶片表皮中的亚细胞定位

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

图7 拟南芥BrNAP1互补（atnap + BrNAP1-1和atnap + BrNAP1-3）及过表达植株（OE-3和OE-4）叶片衰老表型分析

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

图8 拟南芥BrNAP1互补植株叶片叶绿素含量变化

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

图9 BrNAP1互补转基因株系叶片衰老相关基因表达量测定

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

图10 拟南芥BrNAP1过表达植株叶片叶绿素含量变化

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

图11 BrNAP1过表达转基因株系叶片衰老相关基因表达量测定

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

图12 BrNAP1转录激活BrSAG113表达

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

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