Cloning of Litchi LcICE1 Gene and Its Functional Analysis Under Low Temperature Stress

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

Acta Horticulturae Sinica ›› 2025, Vol. 52 ›› Issue (11): 2861-2875.doi: 10.16420/j.issn.0513-353x.2024-0875

• Genetic & Breeding·Germplasm Resources·Molecular Biology • Previous Articles Next Articles

Cloning of Litchi LcICE1 Gene and Its Functional Analysis Under Low Temperature Stress

ZHANG Xiaoting¹^,^*(), ZHUANG Yun², XIAO Kaiting³, and ZHOU Biyan⁴

¹ High Latitude Crops Institute， Shanxi Agricultural University，Datong， Shanxi 037004， China
² The People’s Government of Wushi Town， Leizhou，Zhanjiang， Guangdong 524255， China
³ College of Education， Education Guangzhou Huaxia Vocational College， Guangzhou 510900， China
⁴ College of Horticulture， South China Agricultural University， Guangzhou 510642， China

Received:2025-07-07 Revised:2025-10-20 Online:2025-11-26 Published:2025-11-26
Contact: ZHANG Xiaoting

Abstract

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

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

Figures/Tables 12

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

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

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

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）

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

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

Fig. 5 Subcellular localization of LcICE1

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）

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）

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

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

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

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Cloning of Litchi LcICE1 Gene and Its Functional Analysis Under Low Temperature Stress

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