Evolution, Expression, and Functional Analysis of the HSP20 Gene Family from Simmondisa chinensis

doi:10.13560/j.cnki.biotech.bull.1985.2024-0012

Abstract

Abstract:

【Objective】 Clarifying the structure and function of members of the jojoba HSP20 family（ScHSP20）will provide support for subsequent study.【Method】 A systematic analysis was conducted on the ScHSP20 family members in jojoba from structural, phylogenetic, and expression perspectives, and functional analysis of ScHSP20-17 was performed.【Result】 The genome of jojoba encompassed 35 HSP20 gene loci, with 8 genes involved in tandem duplications and 5 in segmental duplications. Phylogenetic analysis showed that 35 ScHSP20 clusters into 12 subfamilies, with the highest number of nuclear cytoplasmic subfamilies. Promoter analysis revealed that the majority of ScHSP20 family members possessed cis-acting elements in their promoter regions associated with the responses to hormone and abiotic stress. Transcriptome analysis indicated that the expression levels of some ScHSP20 genes, such as ScHSP20-35 and ScHSP20-17, underwent significant changes during flower and seed development, and genes like ScHSP20-17 and ScHSP20-28 were induced by high temperatures. Subcellular localization analysis showed that the protein encoded by ScHSP20-17 was located in the nucleus, and the overexpression of ScHSP20-17 in Escherichia coli, Saccharomyces cerevisiae, and Nicotiana benthamiana markedly improved their tolerance to high temperature stress.【Conclusion】 Some members of the ScHSP20 family are involved in the reproductive development and response of jojoba to abiotic stress.

Key words: jojoba, HSP20, subcellular localization, expression analysis, heat stress

LI Bo-jing, ZHENG La-mei, WU Wu-yun, GAO Fei, ZHOU Yi-jun. Evolution, Expression, and Functional Analysis of the HSP20 Gene Family from Simmondisa chinensis[J]. Biotechnology Bulletin, 2024, 40(6): 190-202.

Figures/Tables 14

Table 1 Primers used in RT-qPCR

基因名称Gene name	上游引物 Forward primer（5'-3'）	下游引物 Reverse primer（5'-3'）
18S	cgttaacgaacgagacctca	cccagaacatctaagggcat
ScHSP20-1	gttgcggtcgcatttgttg	cgtccacggaagccatagaa
ScHSP20-3	gccctgaaaatgaagagggtac	cctttgaacttggcttggatg
ScHSP20-4	aatcaaaggagaaggggagaaa	aggaaccaccaccttcaacact
ScHSP20-13	cgtcaagagggactcaccaaa	caccagaaaccgaaggagga
ScHSP20-14	tcaagagggactcaccaaacag	agccacgtaagccccaatc
ScHSP20-16	acacccttcatcacatcctcg	agctgttggggtactccttcac
ScHSP20-17	accgtgtagagcggagcagt	cagtgacagtcaggaccccat
ScHSP20-20	cagtaagagggcggaggatg	aagggaaagacgacgaggc
ScHSP20-25	atggcgagcaaggcgttgacatgca	tgcatgtcaacgccttgctcgccat
ScHSP20-28	tggcctctgtacgtgctgatt	caccctgttctcctccacttct
ScHSP20-23	tgaggaacctccgacaaagac	cctcctgatgttgacgatgaaa
ScHSP20-26	tccaggtcttagcaaggagca	tgtctttccctgtgggttcg
ScHSP20-32	ctccctctgactgggtttcg	gggcgttctccacttatcctta

Table 2 Primer sequences

用途 Purpose	基因名称 Gene name	上游引物 Forward primer（5'-3'）	下游引物 Reverse primer（5'-3'）
原核表达载体 Prokaryotic expression vector	ScHSP20-17	cgcgtggatccccggATGTCGCTTATTCCAAGTAGGT	agtcagtcacgatgcACCAGAGATTTCAATGGCTTTG
酵母表达载体 Yeast expression vector	ScHSP20-17	ctatagggaatattaATGTCGCTTATTCCAAGTAGGT	cggccgttactagtgACCAGAGATTTCAATGGCTTTG
亚细胞定位载体 Subcellular localization vector	ScHSP20-17	agtccggagctagctATGTCGCTTATTCCAAGTAGGT	cccttgctcaccatgACCAGAGATTTCAATGGCTTTG
内参基因 Internal reference gene	Actin3	GAGGGCCGTGTTCCCCAGCATCGTC	TCTTTTTGATTGAGCCTCATCCCCT
基因表达分析 Gene expression analysis	ScHSP20-17	AAGAATGATACCTGGCACCG	TGACCTCCTCCTTAGGCACA

Fig. 1 Chromosomal distribution of ScHSP20 gene family members Right parentheses indicate concatenated repeats

Fig. 2 Conservative motifs（A）and gene structure（B）of ScHSP20 gene

Fig. 3 Evolutionary analysis of the HSP20 gene in Arabidopsis and S. chinensis C I, C II, C III, CIV, C V, C VI, C VII: Cytoplasm or nucleus. Ml, MII: Mitochondria. ER: Endoplasmic reticulum. P: Plastid. Po: Peroxisome

Fig. 4 Distribution of fragment repetitive events among ScHSP20 family members

Table 3 Ka/Ks values of fragment repeat gene pairs in ScHSP20 family members

重复基因对 Repeated gene pairs	Ka	Ks	Ka/Ks
ScHSP20-2 vs ScHSP20-17	0.383 89	2.622 38	0.146 39
ScHSP20-5 vs ScHSP20-6	0.003 77	0.027 34	0.137 86
ScHSP20-17 vs ScHSP20-24	0.137 24	1.495 32	0.091 78

Fig. 5 Expressions of ScHSP20 gene in different tissues and under different stresses A: Leaves and fruits of Simondwood. B: Male flowers at different flowering stages. C: Different flowering stages of female flowers in Simondmu. D: Different stages and parts of seed development in Simondwood（1: Early developmental seeds. 2: Mid stage developmental seeds. 3: Late developmental seeds. 4: Dry seeds. 5: Middle stage development of seed cotyledons. 6: Mid developmental seed hypocotyl. 7: Mid stage development of seed coat）. E: The expression of ScHSP20 gene under osmotic（OS）, high temperature（HT）, and low temperature（LT）stress

Fig. 6 Prediction of cis-acting elements in the promoter region of ScHSP20 gene

Fig. 7 RT-qPCR analysis of 13 ScHSP20 genes under high temperature stress

Fig. 8 Subcellular localization of ScHSP-17 protein

Fig. 9 Effects of heterologous expression of ScHSP20-17 gene on the tolerance of Escherichia coli to high temperature stress A: rowth curves of recombinant bacteria and control bacteria under high temperature stress. B: Growth of control and recombinant bacteria before and after treatment.C: Statistics of survival rate of control bacteria and recombinant bacteria before and after high temperature stress treatment. * indicate significant differences at P<0.05

Fig. 10 Effect of heterologous expression of ScHSP20-17 gene on yeast high-temperature stress tolerance

Fig. 11 Effects of heterologous expression of ScHSP20-17 gene on the tolerance of tobacco to high temperature stress A: Image of tobacco without injection of bacterial liquid under fluorescent protein lamp irradiation. B: Image of tobacco with injection of bacterial liquid under fluorescent protein lamp irradiation. C: Wild type tobacco（WT）, tobacco with injection of empty bacterial liquid（PRI）, and tobacco leaves with injection of bacterial liquid carrying PRI-ScHSP20-17-GFP vector qRT PCR analysis. D-E: Wild type tobacco. F: Tobacco with instantaneous rotation of empty liquid（PRI）. G: Tobacco with instantaneous rotation of ScHSP20-17. H: MDA content determination

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