西蒙得木HSP20基因家族的进化、表达和功能分析

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

摘要/Abstract

摘要：

【目的】 研究西蒙得木HSP20家族成员的结构和功能，为后续研究提供支撑。【方法】 从结构分析、系统发育、表达模式等方面对西蒙得木HSP20家族成员（ScHSP20s）进行系统分析，并对ScHSP20-17进行功能分析。【结果】 西蒙得木基因组中包含35个HSP20基因座位，其中8个基因涉及串联重复，5个涉及片段重复。系统发育分析表明，35个ScHSP20聚为12个亚家族，其中核质亚家族数量最多。启动子分析表明，多数ScHSP20家族成员启动子区域含有激素响应和非生物胁迫等相关顺式作用元件。转录组分析表明，ScHSP20-35和ScHSP20-17等部分热激蛋白基因在花和种子发育过程中表达量发生显著性改变，而ScHSP20-17和ScHSP20-28等基因受高温诱导。亚细胞定位分析表明，ScHSP20-17编码的蛋白定位于细胞核中，并且在大肠杆菌、酵母和烟草中过表达ScHSP20-17能显著提高它们对高温胁迫的耐受性。【结论】 部分ScHSP20家族成员参与了西蒙得木生殖发育和非生物胁迫应答。

关键词: 西蒙得木, HSP20, 亚细胞定位, 表达分析, 高温胁迫

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

李博静, 郑腊梅, 吴乌云, 高飞, 周宜君. 西蒙得木HSP20基因家族的进化、表达和功能分析[J]. 生物技术通报, 2024, 40(6): 190-202.

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.

图/表 14

表1 荧光定量PCR引物

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

表2 引物序列

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

图1 ScHSP20基因家族成员在染色体上的分布右括号代表串联重复

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

图2 ScHSP20基因的保守基序（A）及基因结构（B）

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

图3 拟南芥和西蒙得木HSP20基因的进化分析 C I、C II、C III、C IV、C V、C VI、C VII：细胞质或细胞核；MI、MII：线粒体；ER：内质网；P：质体；Po：过氧化物酶体

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

图4 ScHSP20家族成员的片段重复事件分布情况

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

表3 ScHSP20家族成员中片段重复基因对的Ka/Ks值

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

图5 ScHSP20基因在不同组织及不同胁迫下的表达 A：叶片和果实；B：雄花不同花期；C：雌花不同花期；D：种子不同发育时期及不同部位（1：早期发育种子；2：中期发育种子；3：后期发育种子；4：干种子；5：中期发育的种子子叶；6：中期发育种子胚轴；7：中期发育种子种皮）；E：ScHSP20基因在渗透（OS）、高温（HT）、低温（LT）胁迫下的表达

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

图6 ScHSP20基因启动子区域顺式作用元件的预测

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

图7 13个ScHSP20基因在高温胁迫下的RT-qPCR分析

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

图8 ScHSP-17蛋白的亚细胞定位

Fig. 8 Subcellular localization of ScHSP-17 protein

图9 ScHSP20-17异源表达对大肠杆菌高温胁迫耐受性的影响 A：高温胁迫处理下重组菌与对照菌的生长曲线；B：处理前后对照菌与重组菌的生长情况；C：高温胁迫处理前后对照菌与重组菌的存活率统计。*表示在P<0.05水平差异显著

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

图10 ScHSP20-17异源表达对酵母高温胁迫耐受性的影响

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

图11 ScHSP20-17异源表达对烟草高温胁迫耐受性的影响 A：荧光蛋白灯照射下未注射菌液的烟草成像；B：荧光蛋白灯照射下注射菌液的烟草成像；C：野生型烟草（WT）、注射空载（PRI）菌液的烟草和注射携带PRI-ScHSP20-17-GFP载体菌液的烟草叶片RT-qPCR分析；D-E：野生型烟草；F：瞬转空载（PRI）的烟草；G：瞬转ScHSP20-17的烟草；H：MDA含量测定

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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