柑橘CsNAC2在果实绿霉病抗病性中的功能和机制研究

doi:10.16420/j.issn.0513-353x.2022-1208

摘要/Abstract

摘要：

为探究柑橘转录因子CsNAC2对果实绿霉病抗病性的影响，以‘锦橙447'为材料，克隆了CsNAC2的编码序列。通过生物信息分析、酵母单杂交、RNA-Seq、凝胶迁移实验和qRT-PCR，探究柑橘转录因子CsNAC2对果实绿霉病抗性的调控作用。结果表明，CsNAC2编码区序列为909 bp，编码302个氨基酸，其蛋白质分子量为34.40 kD。CsNAC2具备NAC家族转录因子的结构特点，在N端含有保守的NAC结构域。在柑橘果实上瞬时过表达CsNAC2能够有效降低果实绿霉病的发病率和病斑直径。转录组分析发现CsNAC2能够参与果实中多个与抗病相关的代谢途径，对木质素合成途径以及氨基酸和核苷酸糖代谢途径进行深入研究发现，CsNAC2能够分别结合这两个途径中的CsLAC7和CsEP3的启动子，瞬时过表达CsNAC2能够提高CsLAC7和CsEP3的表达量。上述结果表明，CsNAC2可能通过激活CsLAC7和CsEP3的表达，从而提高柑橘果实对绿霉病的抗性。

关键词: 柑橘, 绿霉病, 转录因子CsNAC2, 抗病性, 分子机制

Abstract:

In order to investigate the effect of citrus transcription factor CsNAC2 on resistance to green mold，the coding sequence of CsNAC2 was cloned using‘Jincheng 447'as study material. Bioinformatics analysis，yeast one-hybrid assay，RNA-Seq，EMSA and qRT-PCR were employed to explore the regulatory function of CsNAC2 on fruit green mold resistance. The results showed that the coding sequence of CsNAC2 is 909 bp，encoding 302 amino acids，and the molecular weight of the protein is 34.40 kD. CsNAC2 possesses the structural characteristics of the NAC family and contains conserved NAC domain in the N terminal. Transient overexpression of CsNAC2 on citrus fruit could effectively reduce the incidence of green mold in fruit，with a considerable decrease in incidence and spot diameter. Transcriptomic analysis showed that CsNAC2 can participate in several metabolic pathways related to disease resistance in fruits. Further studies on lignin synthesis pathway，amino acid and nucleotide sugar metabolism pathway showed that CsNAC2 can bind the CsLAC7 and CsEP3 gene promoters in these two pathways，respectively. Transient overexpression of CsNAC2 increased the expression of CsLAC7 and CsEP3 genes. The comprehensive results indicated that CsNAC2 may activates the expression of CsLAC7 and CsEP3 genes to enhance the resistance to green mold in citrus fruits.

Key words: citrus, green mold, transcription factor CsNAC2, disease resistance, molecular mechanism

陈琪, 李婷, 陈佳琳, 陈鸥, 王文军, 姚世响, 曾凯芳. 柑橘CsNAC2在果实绿霉病抗病性中的功能和机制研究[J]. 园艺学报, 2023, 50(12): 2701-2712.

CHEN Qi, LI Ting, CHEN Jialin, CHEN Ou, WANG Wenjun, YAO Shixiang, ZENG Kaifang. Studies on Function and Mechanism of CsNAC2 Transcription Factor in Resistance to Green Mold in Citrus[J]. Acta Horticulturae Sinica, 2023, 50(12): 2701-2712.

图/表 9

表1 试验中所用引物序列

Table 1 Specific primer sequences used in experiments

引物名称 Primer name	引物序列（5′-3′） Sequence
CsNAC2-For	ATGACGGCTGAGTTACAGTTACCA
CsNAC2-Rev	TTAAAAGGGTTTCGGAAGGTACATGAA
CsNAC2-pGBKT7-For	AGGCCGAATTCCCGGGGATCCATGACGGCTGAGTTACAGTTACCA
CsNAC2-pGBKT7-Rev	CTAGTTATGCGGCCGCTGCAGTTAAAAGGGTTTCGGAAGGTACATGAA
CsNAC2-pEAQ-For	CAAATTCGCGACCGGTATGACGGCTGAGTTACAGTTACCA
CsNAC2-pEAQ-Rev	AGTTAAAGGCCTCGAGAAAGGGTTTCGGAAGGTACATGAA
CsNAC2-GST-For	GGTTCCGCGTGGATCCATGACGGCTGAGTTACAGTTACCA
CsNAC2-GST-Rev	AGTCACGATGCGGCCGCAAAGGGTTTCGGAAGGTACATGAA
CsLAC7-probe-For	AATTTTTCACGGCACTGCTTCAATTGTAATGCGTGTGTCAGTAG
CsLAC7-probe-Rev	TGCTGACAAGCTACTGACACACGCATTACAATTGAAGCAGTG
CsLAC7-mutant probe-For	AATTTTTAAAAGCACTAAAACAATTGTAATGAAAATGTCAGTAAAAAGTCAGCA
CsLAC7-mutant probe-Rev	TGCTGACTTTTTACTGACATTTTCATTACAATTGTTTTAGTGCTTTTAAAAATT
CsEP3-probe-For	TACTCGCTCGACAATAGATCCGTAGTCAACACGGAGGACAAGAA
CsEP3-probe-Rev	TTCTTGTCCTCCGTGTTGACTACGGATCTATTGTCGAGCGAGTA
CsEP3-mutant probe-For	TACTCGCTCGACAATAGATCAAAAGTCAAAAAAGAGGACAAGAA
CsEP3-mutant probe-Rev	TTCTTGTCCTCTTTTTTGACTTTTGATCTATTGTCGAGCGAGTA
CsLAC7-For	CCTGCAAAACACAGCGTTGA
CsLAC7-Rev	GTAGGGATCCCGTCCTGAAA
CsEP3-For	TGTGTCAAAACTCCGTTGCC
CsEP3-Rev	AGCACAATGAGACGGAGAACT
Action-For	ATCTGCTGGAAGGTGCTGAG
Action-Rev	CCAAGCAGCATGAAGATCAA

图1 CsNAC2与其他物种中NAC转录因子的氨基酸序列比对分析 Mi：杧果;Cc：克莱门柚;Me：木薯;Cp：番木瓜。

Fig. 1 Comparison of amino acid sequences between CsNAC2 and NAC transcription factors in other species Mi：Mangifera indica;Cc：Citrus clementina;Me：Manihot esculenta;Cp：Carica papaya.

图2 CsNAC2与其他物种中NAC转录因子的进化关系结点处数值表示自展值，值越大该节点置信度越高。

Fig. 2 The evolutionary relationship between CsNAC2 and NAC transcription factors in other species The value at the node represents the bootstrap value，and the larger the value，the higher the confidence of the node.

图3 CsNAC2在酵母中的转录激活活性

Fig. 3 Transcriptional activation activity of CsNAC2 in yeast

图4 CsNAC2 瞬时过表达对柑橘果实接种指状青霉引起的绿霉病发病率与病斑直径的影响 ** P < 0.01.

Fig. 4 Effect of transient overexpression of CsNAC2 on the disease incidence and lesion diameter of citrus green mold caused by inoculation of Penicillium digitalum in citrus fruits

表2 CsNAC2可能与抗病相关的下游靶基因

Table 2 Possible downstream target genes of CsNAC2 associated with disease resistance

途径Pathway	基因 Gene symbol	log₂(FC)	功能注释 Description	基因编号 Citrus sinensis ID
苯丙烷生物合成 Phenylpropanoid biosynthesis	COMT1	1.07	咖啡酸3甲基转移酶 Caffeic acid 3-O-Methyltransferase-like	Cs5g13580
	COMT	1.29	咖啡酸3甲基转移酶 Caffeic acid 3-O-Methyltransferase-like	Cs5g18050
	LAC7	1.18	漆酶 Laccase-7-like	Cs6g07400
类胡萝卜素生物合成 Carotenoid biosynthesis	NCED1	1.18	9-顺式环氧类胡萝卜素双加氧酶 9-cis-Epoxycarotenoid dioxygenase	Cs2g03270
类胡萝卜素生物合成 Carotenoid biosynthesis	NCED3	1.42	9-顺式环氧类胡萝卜素双加氧酶 9-cis-Epoxycarotenoid dioxygenase	Cs5g14370
氨基酸和核苷酸糖代谢 Amino sugar and nucleotide sugar metabolism	EP3	1.38	几丁质酶 Chitinase	Cs5g21870
角质素、软木脂和蜡质生物合成 Cutin, suberine and wax biosynthesis	CER1	1.22	乙醛脱羧酶 Aldehyde decarbonylase	Cs1g02750
半胱氨酸和蛋氨酸代谢 Cysteine and methionine metabolism	CAS2	1.29	半胱胺酸合成酶 Cysteine synthase	orange1.1t00386
谷胱甘肽代谢 Glutathione metabolism	HSP26-A	1.06	谷胱甘肽S转移酶 Glutathione S-transferase	Cs7g15760
植物激素信号转导 Plant hormone signal transduction	IAA29	1.25	植物激素响应蛋白IAA Auxin-responsive protein IAA	Cs4g18240

图5 CsNAC2对下游靶基因CsLAC7和CsEP3启动子的结合能力分别以只添加GST蛋白和不添加任何蛋白作为阴性对照，试验组均添加GST-CsNAC2蛋白。标红部分表示NAC下游靶基因启动子区的顺式作用元件NACRS，突变探针是将NACRS的核心基序全部替换成“AAAA”碱基序列。“+”表示添加探针，“−”表示不添加探针，“++”表示加入高浓度的探针。

Fig. 5 The binding ability of CsNAC2 to promoters of downstream target genes CsLAC7 and CsEP3 GST protein and no protein were used as negative controls，respectively，and all experimental groups were added with GST-CsNAC2 protein. The marked red part represents the cis-acting elements NACRS in the promoter of downstream target genes of NAC，and the mutant probe replaced the core motif of NACRS with the“AAAA”base sequence. The symbols“+”and“−”represent presence or absence of the probe and the symbol“++”represent high concentration probe.

图6 CsNAC2瞬时过表达对下游靶基因CsLAC7和CsEP3表达量的影响 ** P < 0.01.

Fig. 6 Effects of transient overexpression of CsNAC2 on the expression levels of downstream target genes CsLAC7 and CsEP3

图7 柑橘中CsNAC2 瞬时过表达对果皮中木质素含量的影响 ** P < 0.01.

Fig. 7 Effects on lignin content in citrus peel after instantaneous overexpression of CsNAC2

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