黄瓜脱镁螯合酶基因CsMDC表达分析及其调控叶绿素降解的功能验证

doi:10.16420/j.issn.0513-353x.2024-0160

摘要/Abstract

摘要：

本研究以‘中农12号’黄瓜（Cucumis sativus L.）为材料，分析黄瓜CsMDC的表达情况，进一步验证其在叶绿素降解过程中的作用。对CsMDC蛋白的理化性质进行分析预测，发现其与同科同属植物甜瓜（Cucumis melo L.）相似度最高，其次是同科异属植物冬瓜（Benincasa hispida Cogn.）、南瓜（Cucurbita moschata Duch.）、笋瓜（Cucurbita maxima Duch.）等，与其他科植物的相似度较低，表明MDC具有明显的种属特征；qRT-PCR分析不同外源生长调节剂处理与非生物胁迫下CsMDC的表达模式，其具有不同的应答特征，该基因在衰老组织中相对表达量最高；构建CsMDC过表达烟草进行功能验证，过表达烟草叶绿素含量、干鲜比、净光合速率等均显著低于野生型，活性氧含量显著高于野生型，qRT-PCR结果显示过表达烟草在受到黑暗胁迫后光系统蛋白基因表达量显著下降，叶绿素降解相关基因表达量显著提高。以上说明黄瓜CsMDC与叶绿素降解密切相关，受不同激素和非生物胁迫诱导，正向调控叶绿素降解及植物衰老。

关键词: 黄瓜, 脱镁螯合酶, CsMDC, 表达模式, 叶绿素降解, 衰老, 非生物胁迫

Abstract:

In this study，cucumber cultivar‘Zhongnong 12’（Cucumis sativus L.）was used to analyze the expression of CsMDC in cucumber and further verify its role in the process of chlorophyll degradation. The analysis and prediction of the physicochemical properties of the CsMDC protein revealed that it has the highest similarity to melon（Cucumis melo L.）from the same family and genus，followed by plants from the same family but different genera such as wax gourd（Benincasa hispida Cogn.），pumpkin（Cucurbita moschata Duch.），and squash（Cucurbita maxima Duch.）. Its similarity to plants from other families is relatively low，indicating that MDC has distinct species-specific characteristics. qRT-PCR analysis of the expression patterns of CsMDC under different exogenous growth regulator treatments and abiotic stresses showed that the gene has the highest relative expression in senescent tissues and exhibits different response characteristics to various conditions. Functional analysis through the construction of CsMDC-overexpressing tobacco plants demonstrated that the overexpression of CsMDC resulted in significantly lower chlorophyll content，dry-to-fresh weight ratio，and net photosynthetic rate compared to wild-type plants，while the reactive oxygen species content was significantly higher. qRT-PCR results showed that the expression of photosystem protein genes in the CsMDC-overexpressing tobacco plants significantly decreased after dark stress，while the expression of genes related to chlorophyll degradation significantly increased. These findings suggest that CsMDC in cucumber is closely related to chlorophyll degradation，induced by different hormones and environmental stresses，and positively regulates chlorophyll degradation and plant senescence.

Key words: cucumber, Mg-dechelatase, CsMDC, expression pattern, chlorophyll degradation, senescence, abiotic stress

李媚晴, 罗思菲, 贾曜豪, 王维贵, 孙锦. 黄瓜脱镁螯合酶基因CsMDC表达分析及其调控叶绿素降解的功能验证[J]. 园艺学报, 2025, 52(6): 1463-1476.

LI Meiqing, LUO Sifei, JIA Yaohao, WANG Weigui, and SUN Jin. Expression Analysis of the Cucumber Mg-Dechelatase Gene CsMDC and Functional Verification of Its Role in Regulating Chlorlorophyll Degradation[J]. Acta Horticulturae Sinica, 2025, 52(6): 1463-1476.

图/表 11

表1 CsMDC基因PCR及qRT-PCR引物

Table 1 The primers of CsMDC gene of PCR or qRT-PCR

用途Application	引物名称Primer name	引物序列（5′-3′）Primer sequence
CsMDC全长PCR PCR of CsMDC full length	CsMDC-F	TTAAACCGATCACAAACC
CsMDC全长PCR PCR of CsMDC full length	CsMDC-R	AGGGCAGTCAATAAAATG
CsMDC实时荧光定量PCR qRT-PCR of CsMDC	qPCR-CsMDC-F	CGCAAAGAACTCCCTGTGGT
CsMDC实时荧光定量PCR qRT-PCR of CsMDC	qPCR-CsMDC-R	AATGCAAGTAATTTGCTGATAGTGT
CsAction实时荧光定量PCR qRT-PCR of CsActin	CsActin-F	CAGGAATCCACGAAACTACT
CsAction实时荧光定量PCR qRT-PCR of CsActin	CsActin-R	AGACCCTCCAATCCAAACAC

表2 叶绿素降解相关基因qPCR引物

Table 2 qPCR primers for the Chl. degredation genes of tobacco

基因Gene	正向引物（5′-3′）Forward primer	反向引物（5′-3′）Reverse primer
NtRbcL	GTCCCCTGTTGGGATGTACTATT	TGTGAGTTCACGTTCTCATCATC
NtRbcS	TCATTGGATTCGACAACGTG	CACAACCCCTAAAGACAAGACA
NtLhcb1	GCTGCTACAATGGCTCTTT	TGGCGACAGTCTTTCTCA
NtLhcb2	TCCGAGCAAACTCCATCT	CAGTGTCCCATCCGTAA
NtLhcb4	GAGATGGGCTATGTTGGC	TGGAGAATGGGAGTGGTT
NtNYC	TAAACAACGCTGGGACAA	TAAACAACGCTGGGACAA
NtRCCR	CTGTGGAGAATCGGCTTGG	ACCTGGGAAGAGGAGTGGC
NtActin	CATTGGCGCTGAGAGATTCC	GCAGCTTCCATTCCGATCA

图1 黄瓜CsMDC克隆结果

Fig. 1 Cucumber CsMDC cloning result

图2 19个物种MDC氨基酸序列系统进化关系位于横线上方的小数代表进化分支长度，节点附近的整数为自展值

Fig. 2 Evolutionary relationships of MDC amino acid sequences of 19 species The decimal above the horizontal line represents the length of the evolutionary branch，and the integer near the node is the bootstrap value

图3 黄瓜叶片CsMDC在不同外源植物生长调节剂处理与非生物胁迫下的qRT-PCR分析对照：叶面喷施蒸馏水；外源ABA、GA3、SA和MeJA处理：分别在叶面喷施100 μmoL · L-1的ABA、GA3、SA和MeJA；干旱处理：20 %（w/v）聚乙二醇（PEG-6000）；高温处理：42 ℃；低温处理：4 ℃；黑暗处理：光照强度0 μmoL · m-2 · s-1。使用2-ΔΔCT方法计算基因的相对表达量（Livak & Schmittgen，2001），经正态性检验证明数据符合正态性，采用独立样本t检验进行显著性分析，* P < 0.05，** P < 0.01，*** P < 0.001

Fig. 3 qRT-PCR analysis of CSMDC in cucumber leaves under different exogenous plant growth regulator treatments and abiotic stresses Control group：foliar spray with distilled water；Exogenous ABA，GA3，SA，and MeJA treatments：foliar spray with 100 μmoL · L⁻¹ ABA，GA3，SA，and MeJA，respectively；Drought treatment：20%（w/v）polyethylene glycol（PEG-6000）；High temperature treatment：42 ℃；Low temperature treatment：4 ℃；Darkness treatment：Light intensity 0 μmoL · m-2 · s-1. Relative expression was calculated using the 2-∆∆CTmethod（Livak et al.，2001）. Independent samples t-test was applied for significance analysis after confirming normality of the data through normality tests. * P < 0.05，** P < 0.01，*** P < 0.001

图4 黄瓜不同组织中CsMDC的相对表达量使用2-∆∆CT方法计算基因的相对表达量（Livak & Schmittgen，2001），经正态性检验证明数据符合正态性，采用单因素ANOVA和Duncan’s检验进行显著性分析，不同小写字母表示有显著差异，P < 0.05，下同

Fig. 4 Tissue-specific expression of CsMDC in cucumber Relative expression was calculated using the 2-∆∆CT method. Normality tests confirmed that the data met the normality assumption. Statistical significance was analyzed by one-way ANOVA followed by Duncan’s multiple range test and different lowercase letters indicate significant differences，P < 0.05，the same below

图5 黄瓜CsMDC转基因烟草的PCR鉴定（A）、qRT-PCR鉴定（B）、黑暗处理前后的烟草表型图（C）和干鲜比（D）

Fig. 5 The PCR analysis（A），qRT-PCR analysis（B），phenotype images of tobacco before and after dark treatment（C），and dry weight to fresh weight ratio（D）of cucumber CsMDC transgenic tobacco

图6 黑暗处理前后黄瓜CsMDC转基因烟草植株的叶绿素含量、净光合速率、叶片最大光化学效率[Fv/Fm]和非调节性能量耗散的量子产量[Y（NO）]

Fig. 6 Chlorophyll content，net photosynthetic rate，maximum photochemical efficiency of leaves，and quantum yield of non-regulated energy dissipationin cucumber CsMDC transgenic tobacco plants before and after dark treatment

图7 黑暗处理前后黄瓜CsMDC转基因烟草的过氧化氢含量、超氧阴离子含量和超氧阴离子产生速率

Fig. 7 H2O2 content，O2- content，and O2- production rate in cucumber CsMDC transgenic tobacco before and after dark treatment

图8 黑暗处理前后黄瓜CsMDC转基因烟草叶绿素降解相关基因的相对表达量野生型烟草不含CsMDC基因，因此图中不显示

Fig. 8 Relative expression of chlorophyll degradation-related genes in cucumber CsMDC transgenic tobacco before and after dark treatment Wild type tobacco lacks the CsMDC gene，resulting in no detectable signal in this figure

图9 黑暗处理前后黄瓜CsMDC转基因烟草光系统相关基因的相对表达量 Lhcb1、Lhcb2、Lhcb4、RbcL和RbcS：光系统蛋白基因；GLN1：谷氨酰胺合成酶基因，该基因编码蛋白通过氮同化和光呼吸调控间接支持光合作用（Ji et al.，2019）

Fig. 9 Relative expression of photosystem-related genes in cucumber CsMDC transgenic tobacco before and after dark treatment Lhcb1、Lhcb2、Lhcb4、RbcL and RbcS：Photosystem protein genes；GLN1：Glutamine synthetase gene，which encodes a protein that indirectly supports photosynthesis through nitrogen assimilation and photorespiration regulation

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