蜜橘CcHY5的克隆及其对果实转色功能的研究

doi:10.16420/j.issn.0513-353x.2023-0441

摘要/Abstract

摘要：

蜜橘果实在25 ℃贮藏可加速转色，而在32 ℃则抑制转色。以这两个温度下贮藏的蜜橘果皮为材料，挖掘到对叶绿素代谢具有显著影响的关键转录因子CcHY5。对CcHY5的结构特征进行了分析，并利用农杆菌EHA105介导的瞬时过表达体系，分别在烟草叶片和蜜橘果实上对该转录因子基因功能进行了深入研究。结果显示，CcHY5全长为507 bp，编码168个氨基酸，与拟南芥AtHY5同源性较高，其启动子区域具有光响应元件、脱落酸响应元件及生长素响应元件等各类响应元件，亚细胞定位于细胞核，具有转录激活活性。烟草叶片瞬时过表达CcHY5后出现了明显黄化现象，且在25 ℃下黄化速度较32 ℃更快。蜜橘果皮瞬时过表达CcHY5后在注射孔附近发生了明显转色，而阴性对照依然保持绿色，与阴性对照相比，不同温度下果皮中CcHY5均显著上调表达，但32 ℃下表达量较25 ℃显著降低。瞬时过表达后的烟草叶片和蜜橘果皮中叶绿素及其代谢产物（叶绿素a、叶绿素b、脱镁叶绿酸甲酯a、脱镁叶绿酸甲酯b及脱镁叶绿素a）含量显著降低，而蜜橘果皮中类胡萝卜素及其代谢产物含量与阴性对照无显著差异。瞬时过表达后的蜜橘果皮中叶绿素循环基因CcCHL1、CcCHL2和CcCHL3的表达受到不同程度抑制，叶绿素降解代谢基因CcNYC和CcRCCR的表达显著上调。综上，适宜温度（25 ℃）下CcHY5可能通过促进蜜橘果皮中叶绿素降解，加速其转色进程。

关键词: 蜜橘, HY5转录因子, 叶绿素, 功能, 转色, 贮藏温度

Abstract:

25 ℃ storage accelerated the coloration of mandarin fruits，while 32 ℃ storage inhibited their coloring. Using mandarin peel stored at these two temperatures as material，the key transcription factor CcHY5 was discovered that has a significant impact on chlorophyll metabolism. The structural characteristics of CcHY5 and conducted in-depth research on the gene function of this transcription factor were analyzed using the transient overexpression system mediated by Agrobacterium EHA105 on tobacco leaves and mandarin fruits，respectively. The results showed that the total length of CcHY5 was 507 bp，coded 168 amino acids，and had high homology with the AtHY5 of the model crop Arabidopsis. Its promoter region contains various response elements such as light responsive elements，abscisic acid responsive elements，and auxin responsive elements；subcellular localization in the nucleus with transcriptional activation activity. Tobacco leaves showed significant yellowing after transient overexpression of CcHY5，and the yellowing rate was faster at 25 ℃ than at 32 ℃. After transient overexpression of CcHY5 in the peel of mandarin fruit，a significant coloration occurred near the injection well，while the negative control remained green. CcHY5 was significantly up-regulated in the peel at different temperatures compared to the negative control，but the expression level was significantly reduced at 32 ℃ compared to 25 ℃. The content of chlorophyll and its metabolites（chlorophyll a，chlorophyll b，pheophorbide a，pheophorbide b and pheophytin a）in tobacco leaves and mandarin fruits peel significantly decreased，however，there was no significant difference in the content of carotenoids and their metabolites in the peel of mandarin fruits compared to the negative control. After transient overexpression，the expression of chlorophyll cycle genes CcCHL1，CcCHL2，and CcCHL3 in mandarin peel was inhibited to varying degrees，while the expression of chlorophyll degradation metabolism genes CcNYC and CcRCCR was significantly up-regulated. In summary，at an appropriate temperature（25 ℃），CcHY5 may accelerate the coloration process of mandarin peel by promoting chlorophyll degradation.

Key words: mandarin fruit, HY5 transcription factor, chlorophyll, function, coloration, storage temperature

邓淑芳, 刘倩, 刘玲, 陈鸥, 王文军, 曾凯芳, 邓丽莉. 蜜橘CcHY5的克隆及其对果实转色功能的研究[J]. 园艺学报, 2024, 51(5): 939-955.

DENG Shufang, LIU Qian, LIU Ling, CHEN Ou, WANG Wenjun, ZENG Kaifang, DENG Lili. Cloning of Mandarin Fruit CcHY5 and Its Function in Fruit Coloration[J]. Acta Horticulturae Sinica, 2024, 51(5): 939-955.

图/表 15

表1 qRT-PCR中所用的引物序列

Table 1 Primers used in qRT-PCR

基因名称 Gene name	上游引物（5′-3′） Forward primer	下游引物（5′-3′） Reverse primer
Actin	TGGATTCTGGTGATGGTGTG	TGGATTCTGGTGATGGTGTG
CcHY5	AAGGCGTACTTGAATGAGTT	AGGTAACTTTCAACCAGCAA
CcNYC	GGCACGGTTTTCCTTTACAGATG	TGTTGTAGTTCTGACGCTTTCTG
CcRCCR	CAAACGTTGCAGGTCTTCGG	CAAACGTTGCAGGTCTTCGG
CcCHL1	CCCGTCTCCGTTGAAGCTAA	GCAGTGCCGTGGAAAAACAA
CcCHL2	CGTTCTCGCGCGGAAAATAA	TTTGGAGGAGGTGGTGAGGA
CcCHL3	CCTTGTGCTCCGAAAGGAGT	CCTTGTGCTCCGAAAGGAGT

图1 不同贮藏温度对蜜橘果皮CcHY5表达量的影响 A：RNA-seq数据中的FPKM值，不同小写字母表示差异显著，P < 0.05；B：RT-qPCR测定的表达量。 * α = 0.05，** α = 0.01。

Fig. 1 Effect of different storage temperatures on the expression of CcHY5 in mandarin fruit peel A：FPKM values from RNAseq data，different lowercase letters indicate significant difference at 0.05 level；B：Gene quantification by RT-qPCR. * α = 0.05，** α = 0.01.

图2 CcHY5的克隆

Fig. 2 Cloning of CcHY5

图3 蜜橘CcHY5与拟南芥AtbZIP系统进化树

Fig. 3 Phylogenetic tree analysis of citrus CcHY5 and Arabidopsis AtbZIP

表2 CcHY5启动子区域顺式作用元件分析

Table 2 Analysis of cis-elements in CcHY5 promoter region

顺式作用元件 cis-Element	序列 Sequence	预测功能 Predictive function	数量 Quantity
ABRE	ACGTG	脱落酸响应元件 Response element for abscisic acid	2
	CACGTG	脱落酸响应元件 Response element for abscisic acid	1
Box 4	ATTAAT	光响应元件 Response element for light	4
G-box	CACGTG	光响应元件 Response element for light	2
	TACGTG	光响应元件 Response element for light	1
ACE	CTAACGTATT	光响应元件 Response element for light	1
CAT-box	GCCACT	分生组织表达元件 Expression element of meristem tissue	2
AuxRR-core	GGTCCAT	生长素响应元件 Response element for auxin	1
CGTCA-motif	CGTCA	茉莉酸甲酯响应元件 Response element for methyl jasmonate	1
TGACG-motif	TGACG	茉莉酸甲酯响应元件 Response element for methyl jasmonate	1
ARE	AAACCA	厌氧诱导响应元件 Anaerobic induced response element	3
CCAAT-box	CAACGG	MYB结合位点 MYB binding site	1

图4 CcHY5在烟草叶片细胞中的亚细胞定位

Fig. 4 Subcellular localization of CcHY5 in tobacco leaf cells

图5 CcHY5在酵母中的转录激活活性分析将pGBKT7-CcHY5转化酵母细胞，以pGBKT7-53 + pGADT7-T为阳性对照、pGBKT7为阴性对照，在单缺（SD/-Trp）及三缺（SD/-Trp-His-Ade）培养基上培养，根据酵母生长状态及滴加X-α-Gal后变蓝色表示具转录激活活性。

Fig. 5 Analysis of transcriptional activation activity of CcHY5 in yeast The pGBKT7-CcHY5 transformed yeast cells were cultured in single-deficient and triple-deficient medium with pGBKT7-53 + pGADT7-T as positive control and pGBKT7 as negative control. And transcriptional activation activity was analyzed based on yeast growth status and color change with addition of X-α-Gal.

图6 烟草叶片CcHY5瞬时过表达4 d后的外观变化

Fig. 6 Changes in appearance of tobacco leaves after transient overexpression of CcHY5 for four days

图7 烟草叶片CcHY5瞬时过表达后色素含量的变化（25 ℃）

Fig. 7 Changes in pigment content of tobacco leaves after transient overexpression of CcHY5（25 ℃） Student’s t-test，* α = 0.05，** α = 0.01.

图8 不同贮藏温度对瞬时过表达后蜜橘果皮CcHY5表达量的影响差异显著性分析采用Duncan’s检验，不同小写字母表示同一时间不同处理间差异显著，P < 0.05。

Fig. 8 Effect of different storage temperatures on the expression of CcHY5 in mandarin fruit peel after transient overexpression Significant difference analysis used Duncan’s test，different lowercase letters indicate significant difference between different treatments at the same time，P < 0.05.

图9 蜜橘果实CcHY5瞬时过表达后果皮外观及色差值的变化（25 ℃）

Fig. 9 Changes in appearance and color index of citrus peel after transient overexpression of CcHY5（25 ℃） Student’s t-test，* α = 0.05，** α = 0.01.

图10 瞬时过表达CcHY5蜜橘果实果皮类胡萝卜素及其代谢产物含量（25 ℃）

Fig. 10 Effects of transient overexpression of CcHY5 on the content of carotenoid and its metabolites in the peel of mandarin fruit（25 ℃） Student’s t-test，* α = 0.05.

图11 CcHY5瞬时过表达对蜜橘果实果皮叶绿素及其代谢产物含量的影响（25 ℃）

Fig. 11 Effects of transient overexpression of CcHY5 on the content of chlorophyll and its metabolites in the peel of mandarin fruit（25 ℃） Student’s t-test，* α = 0.05，** α = 0.01.

图12 CcHY5瞬时过表达对蜜橘果皮CcHY5及叶绿素代谢途径相关基因相对表达的影响（25 ℃）

Fig. 12 Effects of transient overexpression of CcHY5 on the relative expression of CcHY5 and chlorophyll metabolism pathway related genes in mandarin fruit peel（25 ℃） Student’s t-test，* α = 0.05，** α = 0.01.

图13 CcHY5瞬时过表达后蜜橘果皮叶绿素及其代谢产物含量与叶绿素代谢基因表达量的皮尔逊相关性分析

Fig. 13 Pearson correlation analysis of chlorophyll and its metabolites and chlorophyll metabolism genes in mandarin fruit peel after transient overexpression of CcHY5 *P < 0.05，** P < 0.01.

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