番茄转录因子SlNAC29在调控植株衰老中的作用及机理

doi:10.3864/j.issn.0578-1752.2021.24.009

摘要/Abstract

摘要：

【背景】番茄（Solanum lycopersicum）作为连续发芽分化和坐果的重要园艺作物,早衰是限制其生长期长短、产量和品质的重要因素。NAC（NAM、ATAF1/2和CUC2）转录因子家族参与调控拟南芥、水稻等多种植物衰老进程,但在番茄中的研究尚不深入。目前已知,SlNAP2（NAC-like, activated by apetala3/pistillata）参与番茄植株衰老进程。【目的】SlNAC29为SlNAP2的同源基因,对其在番茄植株衰老中的功能及调控机制进行研究,以期为园艺栽培中番茄的衰老调控及种质创新提供科学依据。【方法】以野生型番茄（Condine Red,CR）为背景,采用qRT-PCR技术明确SlNAC29在不同衰老阶段叶片中的相对表达量,并分别利用CRISPR/Cas9基因编辑技术和基因过表达技术构建Slnac29纯合突变体及OE: SlNAC29稳定过表达植株。在此基础上,在自然生长状态下和黑暗处理诱导衰老条件下,对野生型、Slnac29突变体和OE:SlNAC29过表达植株的生长、叶绿素含量、光合作用、叶片衰老和叶绿素降解相关基因的相对表达量等参数进行分析,明确SlNAC29转录因子在调控番茄植株衰老中的生物学功能;进一步利用聚类热图分析过表达植株OE: SlNAC29中29个衰老相关基因、叶绿素降解基因以及ABA合成/信号转导相关基因的相对表达量。并选取在黑暗诱导衰老条件下不同株系植株中表达差异明显的4个基因进行凝胶迁移阻滞分析（electrophoretic mobility shift analysis,EMSA）,以鉴定SlNAC29直接转录调控的靶标基因及其与衰老调控的关系。【结果】SlNAC29在初老叶和衰老叶片中的相对表达量较嫩叶和成熟叶显著上升。自然生长状态下,突变体材料Slnac29与野生型长势以及光合速率无明显差异,而过表达材料OE: SlNAC29株高则显著低于野生型植株,叶绿素含量和光合速率分别是野生型植株的25%和50%。在黑暗诱导衰老的条件下,野生型植株叶片明显变黄,叶绿素含量显著下降。Slnac29突变缓解了叶片衰老程度,叶片无明显变黄,叶绿素含量是野生型的3倍,衰老相关基因（senescence-associated genes,SAGs）和叶绿素降解基因的表达量均较低。OE: SlNAC29则相反,叶片衰老程度比野生型和Slnac29突变体均明显严重。基因聚类分析表明多个衰老相关基因和叶绿素降解基因在OE: SlNAC29植株中显著上调表达。EMSA鉴定到SlNAC29能够直接与衰老相关基因家族SAGs成员SlAGT1（Glyoxylate aminotransferase）启动子绑定,且SlAGT1在OE: SlNAC29中的相对表达量较野生型和Slnac29突变体显著增加。【结论】转录因子SlNAC29调控番茄植株的衰老,促进番茄叶片在黑暗诱导条件下的衰老进程。SlNAC29直接绑定衰老相关基因SlAGT1启动子区域调控其转录表达。

关键词: 番茄, SlNAC29, 转录调控, 衰老, SlAGT1

Abstract:

【Background】 Tomato（Solanum lycopersicum）is an important horticultural crop with continuous flower bud differentiation and fruiting. Premature senescence seriously limits tomato plants growth period, crop yield and fruit quality. NAC (NAM, ATAF1/2 and CUC2) transcription factor family regulates leaf senescence process in Arabidopsis, rice and other plants. Nevertheless, the roles of tomato NAC transcription factor in the regulation of leaf senescence have not been well understood. SlNAP2 (NAC-like, activated by apetala3/pistillata) is known to be involved in the regulation of tomato leaf senescence. 【Objective】 SlNAC29 transcription factor is the homologous gene of SlNAP2 in tomato, while its function remains largely unclear. In this study, the role of SlNAC29 and its underlying mechanism in leaf senescence was investigated, which can provide some scientific basis for tomato senescence regulation and germplasm innovation. 【Method】Condine Red (CR) was used as the wild-type background in this study. qRT-PCR was used to analyze the relative expression of SlNAC29 in tomato leaves at different senescence stages. Slnac29 homozygous mutant lines and the OE: SlNAC29 stable overexpression lines were generated through CRISPR/Cas9 gene editing and over-expression approaches, respectively. Using these lines, plant growth phenotypes, chlorophyll content, leaf photosynthesis, transcription of senescence- and chlorophyll degradation- related genes were analyzed under both natural and dark-induced senescence conditions. The clustering heat map was used to analyze the relative expression of 29 genes, including senescence-, chlorophyll degradation- and ABA biosynthesis/signaling-associated genes. Based on gene expression profiles, four of them were selected to electrophoretic mobility shift analysis (EMSA) to identify the SlNAC29-target gene during senescence process. 【Result】The relative expression of SlNAC29 was significantly up-regulated in early senescent and senescent leaves, as compared with young and mature leaves. Under natural growth condition, the Slnac29 mutant lines showed no differences with the wild-type in terms of plant growth phenotypes and photosynthetic rate. By contrast, the height of OE: SlNAC29 plant was shorter than wild-type plants, OE: SlNAC29 plants also showed lower chlorophyll content and photosynthetic rate, which were only 25% and 50% of the wild-type control, respectively. Under dark-induced senescence condition, the leaves of wild-type plants turned yellow and the chlorophyll content decreased significantly. The senescent phenotypes were alleviated in Slnac29 mutant lines, which not only have significant higher chlorophyll content, but also showed higher transcript level of senescence-associated genes (SAGs) and chlorophyll degradation-related genes. On the contrary, the dark-induced senescence effect was aggravated in OE: SlNAC29. Cluster analysis showed that several genes, especially SAGs and chlorophyll degradation-related genes SlSAG12, SlAGT1, SlSGR1 and SlNYC1, were significantly up-regulated in OE: SlNAC29 plants. The EMSA analysis showed that SlNAC29 could directly bind to the promoter of SlAGT1 (Glyoxylate aminotransferase), a member of SAGs. Moreover, the relative expression of SlAGT1 in OE: SlNAC29 was significantly higher than that of wild-type and Slnac29 plants. 【Conclusion】 SlNAC29 transcription factor is involved in the regulation of leaf senescence in tomato plants, which promotes the senescence process under dark conditions. SlNAC29 may directly bind to the promoter region of the senescence-related gene SlAGT1 to regulate its transcriptional expression.

Key words: tomato, SlNAC29, transcription regulation, senescence, SlAGT1

王萍,郑晨飞,王娇,胡璋健,邵淑君,师恺. 番茄转录因子SlNAC29在调控植株衰老中的作用及机理[J]. 中国农业科学, 2021, 54(24): 5266-5276.

WANG Ping,ZHENG ChenFei,WANG Jiao,HU ZhangJian,SHAO ShuJun,SHI Kai. The Role and Mechanism of Tomato SlNAC29 Transcription Factor in Regulating Plant Senescence[J]. Scientia Agricultura Sinica, 2021, 54(24): 5266-5276.

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引物名称 Primer name	基因编号 Accession number	正向引物 Forward primer (5′-3′)	反向引物 Reverse primer (5′-3′)	用途 Amplification
E-SlNAC29	Solyc05g007770	TTggcgcgccATGGTTGGAAAAATTAGCTC	CGGggtaccCTGAGATTGAAATGTTGG	基因扩增 Gene amplification
CRISPR-Slnac29	Solyc05g007770	GATTgTTCGATCCCTGGGTATTACC	AAACGGTAATACCCAGGGATCGAAc
SlNAC29-GFP	Solyc05g007770	CgagctcATGGTTGGAAAAATTAGCTC	TCCcccgggtCTGAGATTGAAATGTTGGATTC
RT- SlNAC29	Solyc05g007770	CCAAATAGAGCAGCTGTGTCA	TTGGTGGTTTGCCTTTGTAA	qRT-PCR
RT- SlAGT1	Solyc10g076250	TGCCATAAACCCTGACCCAT	CTATCCCACCAACACCCTGT
RT- SlSAG12	Solyc02g076910	GTGGCTAATCAACCGGTGTC	CACTTGCAGCACCATATCCC
RT- SlSGR1	Solyc08g080090	TTCTCAGTTGCAAGGTTGGT	CCTTGAGAACCACAGGGAGT
RT- SlNYC1	Solyc07g024000	CAGGCAGCTTCAATCATCCC	TAGGTCGGTTAGGACCATGC
RT- SlACTIN	Solyc03g078400	TGTCCCTATTTACGAGGGTTATGC	CAGTTAAATCACGACCAGCAAGAT
SlAGT1	Solyc10g076250	AATTGAAATTACGTAAATGGAACGTAACCAAAATTATATC	GATATAATTTTGGTTACGTTCCATTTACGTAATTTCAATT	EMSA
SlSAG12	Solyc02g076910	GTTTTGAAGATGCACACACGAAAATACATGTACAATTGAT	ATCAATTGTACATGTATTTTCGTGTGTGCATCTTCAAAAC
SlSGR1	Solyc08g080090	CAATCAATTTATACACGCCTTAACTTATTTTTAAAAATGC	GCATTTTTAAAAATAAGTTAAGGCGTGTATAAATTGATTG
SlNYC1	Solyc07g024000	CTAGCATTTTAATATATGAATACACGTGAACAAAATTTCC	GGAAATTTTGTTCACGTGTATTCATATATTAAAATGCTAG

目标基因 Target gene	登录号 Accession number	靶标位点个数 NAC core binding site number
SlAGT1	Solyc10g076250	2
SlSAG12	Solyc02g076910	3
SlSGR1	Solyc08g080090	3
SlNYC1	Solyc07g024000	5