荔枝LcICE1的克隆及在低温胁迫下的功能

doi:10.16420/j.issn.0513-353x.2024-0875

摘要/Abstract

摘要：

冷响应转录因子ICE在植物的抗寒机制中发挥着重要作用。以‘怀枝’荔枝为材料，克隆获得LcICE1，基因全长共2 242 bp，开放阅读框共1 614 bp，编码537个氨基酸，包含bHLH结构和3个功能位点。通过进化树分析发现LcICE1与龙眼DlICE1相似度最高，说明二者亲缘关系较近；通过ProtParam预测、信号肽、蛋白预测和二级结构预测分析发现，LcICE1可能存在二硫键，为非分泌蛋白，定位于细胞核中，且不存在跨膜结构域，α-螺旋作为蛋白中的大量结构元件分布于整个肽链中；NetPhosk 3.1 Server预测显示LcICE1蛋白有4个糖基化位点和49个磷酸化位点。沉默LcICE1增加了低温胁迫下‘妃子笑’荔枝愈伤组织的褐化程度，即愈伤组织的抗寒性降低。进一步的低温处理结果显示，空白对照和 pCAMBIA1301-Empty 的荔枝愈伤组织比 LcICE1 过表达的愈伤组织积累更多的超氧阴离子和H₂O₂，即LcICE1 过表达可以增强荔枝愈伤组织的抗寒性。

关键词: 荔枝, LcICE1, 低温胁迫, 功能分析

Abstract:

The ICE plays an important role in the cold resistance mechanisms of plants. The gene LcICE1 was cloned from‘Huaizhi’litchi，and the gene is 2 242 bp in length，with an open reading frame of 1 614 bp，encoding 537 amino acids，including the bHLH structure and three functional sites. Evolutionary tree analysis revealed that LcICE1 had the highest similarity to DlICE1，indicating that litchi and longan are the closest relatives. Through ProtParam prediction，signal peptide，protein prediction and secondary structure prediction analysis，it was found that LcICE1 may have disulfide bonds，is a non-secretory protein，located in the nucleus，and does not have transmembrane domain，α-helix as a large number of structural elements in the protein distributed throughout the peptide chain；NetPhosk 3.1 server predicted that LcICE1 protein has 4 glycosylation sites and 49 phosphorylation sites. The VIGS treatment assay demonstrated that silencing LcICE1 promoted the browning degree of‘Feizixiao’litchi callus under low-temperature stress，indicating that the cold resistance of callus tissue decreased. Further，low temperature treatment results showed that the blank control and pCAMBIA1301-Empty litchi callus accumulated more and H₂O₂ than pCAMBIA1301-LcICE1 litchi callus，suggesting that over-expression of LcICE1 enhances the cold resistance of litchi callus.

Key words: litchi, LcICE1, cold stress, function analysis

张晓婷, 庄赟, 萧凯庭, 周碧燕. 荔枝LcICE1的克隆及在低温胁迫下的功能[J]. 园艺学报, 2025, 52(11): 2861-2875.

ZHANG Xiaoting, ZHUANG Yun, XIAO Kaiting, ZHOU Biyan. Cloning of Litchi LcICE1 Gene and Its Functional Analysis Under Low Temperature Stress[J]. Acta Horticulturae Sinica, 2025, 52(11): 2861-2875.

图/表 12

表1 人工气候室控温程序设置

Table 1 The temperature control program setting for artificial climate chambers

处理天数/d Treatment days	不同时刻温度/℃ Temperature at different o’clock
处理天数/d Treatment days	1：00	8：00	12：00	15：00	19：00	22：00
0	22	22	24	20	20	17
1	17	17	20	16	16	12
2	12	12	15	11	11	7
3	7	7	9	5	5	2
4	2	2	2	2	2	2
5 ~ 12	2	2	2	2	2	2

表 2 本研究中所用引物及序列

Table 2 Primers and sequences used in this study

用途 Purpose	基因名称 Name	上游引物（5′-3′） Sequence F	下游引物（5′-3′） Sequence R
基因克隆 Gene cloning	pCambia1301-LcICE1	caccaccaccaccacCACGTGATGCTCTCCAGAGTGAACGGTG	ggggaaattcgagctGGTCACCCTACATCATGCCATGGAAGCC
亚细胞定位 Subcellular localization	pGreen-C18-LcICE	cggtatcgatAAGCTTATGCTCTCCAGAGTGAACGGT	tttactcatactagtGGATCCCATCATGCCATGGAAGCC
VIGS	pTRV2-LcICE	gttaccgaattcTCTAGAGCGAAGAAT	cttcgggacatgCCCGGGTGAGCCAGGTGGGGTTGACT
qPCR	LcDREB1D	CAGCATCAAATGGCGGAAGGT	GCAACTAATTCCACAATGAAGC
	LcCBF5	ATGACTGATACTGAGTTGTCC	CTACTCATCATCTTTGTTCTTC
	LcAFP3	AACAACCCTGAGCCTCCCCACAATC	CAAGTCCCTCAAAATAGTCCTCCTC
	LcCOR413IM1	TCAGTCACCAGTGTCCAGGCAG	GAAGCAACTGGATTGGTGTGGC
	LcCOR413PM2	AGATGGATTGCTTTCATTGCTGTTG	AGCAAATAACAAGCAATGGCAAGAC
	LcCOR27	ATCTTCTGCCAACAACCACCTC	TTTTCTTGGTAGGTGCTCATCG
	Actin	AGTTTGGTTGATGTGGGAGAC	TGGCTGAACCCGAGATGAT

图1 过表达荔枝愈伤组织的获得 a：初筛；b：二筛；c：转化；d：培养。图a和图b中右上角分别为筛选合格的荔枝愈伤组织；不同阶段培养皿尺寸已在图中标记

Fig. 1 Obtaining over-expression litchi callus a：Initial screening；b：Second sieves；c：Transformation；d：Cultivate. The upper right corner in a and b respectively shows the litchi callus that can proceed to the next step after passing the screening process. The sizes of culture dishes at different stages have been marked in the figure

图2 低温处理下‘怀枝’荔枝中LcICE1的表达 ** 表示与处理前（0 d）有显著差异（P < 0.01）

Fig. 2 Expression of LcICE1 in litchi‘Huaizhi’under low-temperature treated ** indicate significant differences from 0 d（before low-temperature treated）（P < 0.01）

图3 LcICE1的保守结构域（a）及系统进化树构建（b） Ptr：枳；Ca：辣椒；Pist：阿月浑子；Mi：杧果；Dl：龙眼；Dz：榴莲；Jc：野核桃；Rc：砂藓；Vv：葡萄；Va：山葡萄

Fig. 3 Conservative structure domain（a）and system evolution tree construction（b）of LcICE1 Ptr：Poncirus trifoliata；Ca：Capsicum annuum；Pist：Pistacia vera；Mi：Mangifera indica；Dl：Dimocarpus longan；Dz：Durio zibethinus；Jc：Juglans cathayensis；Rc：Racomitrium canescens；Vv：Vitis vinifera；Va：Vitis amurensis

图4 LcICE1二级结构预测分析蓝色代表α-螺旋；绿色代表β-转角；红色代表延伸链；橙色代表无规则卷曲

Fig. 4 Analysis of secondary structure prediction in LcICE1 Blue represents α-spiral；green represents β-corner；red represents the extension chain；orange represents irregular curls

图5 LcICE1 蛋白的亚细胞定位

Fig. 5 Subcellular localization of LcICE1

图6 荔枝愈伤组织中的 LcICE1 的鉴定 a：愈伤组织GUS染色；b：愈伤组织中LcICE1的表达；c：单个愈伤组织的GUS染色。** 表示与空白对照（CK）有显著差异（P < 0.01）

Fig. 6 Identification of LcICE1 in litchi callus a：GUS staining of a large number of callus tissues；b：The relative expression level of LcICE1 in litchi callus tissue；c：GUS staining of individual callus tissue. ** indicate significant differences from the control（CK）（P < 0.01）

图7 低温相关基因在过表达LcICE1荔枝愈伤组织中的表达 ** 表示与处理前（0 h）有极显著差异（P < 0.01）

Fig. 7 Expression of the related genes in litchi callus of over-expressed LcICE1 ** indicate significant differences from 0 h（before low-temperature treated）（P < 0.01）

图8 低温处理前后以及恢复过程中荔枝愈伤组织的表型

Fig. 8 Phenotypes of litchi callus before and after low-temperature treatment and during recovery

图9 低温处理后荔枝愈伤组织的NBT（a）和DAB（b）化学染色

Fig. 9 NBT（a）and DAB（b）chemical staining of litchi callus under low-temperature treatment

图10 低温处理后VIGS和对照处理的荔枝愈伤组织表型

Fig. 10 Phenotypes of VIGS-treated and control litchi callus after low-temperature treatment

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