辣椒囊泡形成相关基因CaSec16耐热功能分析

doi:10.16420/j.issn.0513-353x.2022-0996

摘要/Abstract

摘要：

囊泡运输在植物抗逆过程中发挥着重要作用，但辣椒囊泡形成相关基因CaSec16（COPII coat assembly protein Sec16-like）在耐热中的功能尚不清楚。辣椒CaSec16定位于细胞质和细胞核，并在各个组织中均有表达，其表达量在热胁迫过程中呈上调趋势。与对照植株相比，CaSec16沉默植株的耐热性降低，表现为热胁迫下生长点死亡，叶片萎蔫程度加重，相对电导率升高，总叶绿素含量降低，死细胞和H₂O₂积累增多，丙二醛和脯氨酸含量增多，并且耐热标记基因的上调表达受到抑制。相反，与对照植株相比，CaSec16瞬时过表达植株的耐热性升高，表现为热胁迫下叶片萎蔫程度减轻，丙二醛和脯氨酸含量减少，并且耐热标记基因的上调表达得到加强。推测CaSec16对辣椒的耐热性具有正向调控作用。

关键词: 辣椒, CaSec16, 囊泡运输, 耐热性

Abstract:

Vesicle transport plays important roles in plant tolerance to abiotic stress. However，the function of pepper CaSec16（COPII coat assembly protein Sec16-like），a gene related to vesicle formation，remains unclear in heat tolerance. CaSec16 was localized in cytoplasm and nucleus，and was expressed in various tissues of pepper. The expression of CaSec16 was up-regulated during heat stress. Compared with the control plants，the heat tolerance of pepper plants with CaSec16-silenced was compromised，which was shown by the death of growing point，the aggravated leaf wilting，the higher increment in relative electric conductivity，the lower contents of total chlorophyll，the higher accumulation of dead cells，H₂O₂，MDA and proline increased，and the weakened up-regulation of heat-tolerance marker genes. On the contrary，compared with the control plants，the heat tolerance of CaSec16 transient- overexpression pepper plants was improved，which was presented the mitigated leaf wilting，the lower contents of MDA and proline，and the strengthened up-regulation of heat-tolerance marker genes. Taken together，it was speculated that CaSec16 positively regulates the heat tolerance of pepper.

Key words: pepper, CaSec16, vesicle transport, heat tolerance

阎本涛, 焦可心, 赵悦, 杨巧敏, 陈涛, 逯明辉. 辣椒囊泡形成相关基因CaSec16耐热功能分析[J]. 园艺学报, 2023, 50(11): 2387-2400.

YAN Bentao, JIAO Kexin, ZHAO Yue, YANG Qiaomin, CHEN Tao, LU Minghui. Analysis of Heat-tolerant Function of Pepper Vesicle Formation Related Gene CaSec16[J]. Acta Horticulturae Sinica, 2023, 50(11): 2387-2400.

图/表 12

表1 PCR引物信息

Table 1 Information of PCR primers

用途 Purpose	名称 Name	序列（5′-3′） Sequence
亚细胞定位/瞬时过表达载体构建 Construction of subcellular localization/ transient overexpression vector	pART27-CaSec16-F	GATGAACTATACAAAGAATTCATGGCGGACATTCAGAGGTGCAG
	pART27-CaSec16-R	CAGGACTCTAGATTAGGTACCCTACAGCCAAAAGATACTACTACTAACAC
基因表达分析 Analysis of gene expression	CaUbi3-F	TGTCCATCTGCTCTCTGTTG
	CaUbi3-R	CACCCCAAGCACAATAAGAC
	qCaSec16-F	CCCTGTCGCTGCTACATTCT
	qCaSec16-R	GCTGGCAACACAGATTTCGG
	qCaHsfA2-F	GTAGCATCAGTAGCCACAGC
	qCaHsfA2-R	CAAGCAACTCTTCCCAAATA
	qCaHsp70.1-F	CAGGTGTGCTAGTTCAGGTGT
	qCaHsp70.1-R	TGACCTGAGGCACTCCTCTT
基因沉默载体构建	TRV2：CaSec16-F	CGGAATTCATGGCGGACATTCAGAGGTG
Construction of gene-silencing vector	TRV2：CaSec16-R	CCCTCGAGTGATGGTGGATTTGCTGATG

图1 辣椒CaSec16的保守结构域（A）及与其他物种Sec16的系统进化树（B）

Fig. 1 The conserved domain of capsicum CaSec16（A）and its phylogenetic tree wigh Sec16 of other species（B）

表2 CaSec16启动子顺式作用元件预测

Table 2 Predicted cis-acting elements in CaSec16 promoter

元件 Component	序列 Sequence	数量 Quantity	功能 Function
CGTCA-motif	CGTCA	1	响应茉莉酸信号Involved in the MeJA-responsiveness
ARE	AAACCA	3	响应厌氧信号Responsed to anaerobic signals
GARE-motif	TCTGTTG	1	响应赤霉素信号Involved in the gibberellin responsiveness
TCT-motiff	TCTTAC	2	响应光信号Involved in the light responsiveness
TGACG-motif	TGACG	1	响应茉莉酸信号Involved in the MeJA-responsiveness
chs-Unit 1 m1	ACCTAACCCGC	1	响应光信号Involved in the light responsiveness
MBS	CAACTG	2	响应干旱信号Involved in the drouoght responsiveness
GT1-motif	GGTTAA	3	响应光信号Involved in the light responsiveness
GT1-motif	GGTTAAT	1	响应光信号Involved in the light responsiveness

图2 CaSec16的亚细胞定位 A：CaSec16亚细胞定位；B：细胞核和细胞质的荧光光密度；C：pART27-CaSec16与细胞核染料DAPI共定位；D：GFP（绿色）和DAPI（蓝色）共定位的密度图。pART27：空载体；pART27-CaSec16：CaSec16基因表达载体；** P < 0.01。

Fig. 2 Subcellular localization of CaSec16 A：Subcellular localization of CaSec16；B：The optical density of fluorescence in the nucleus and cytoplasm；C：pART27-CaSec16 co-locates with nuclear dye DAPI；D：Density of GFP（green）and DAPI（blue）co-location；pART27：empty vector；pART27-CaSec16：CaSec16 expression vector. ** P < 0.01.

图3 CaSec16基因在辣椒不同组织（A）和热胁迫不同时间（B）的表达特性 * P < 0.05，** P < 0.01。下同。

Fig. 3 Expression characteristics of CaSec16 gene in different tissues（A）and at different time of heat stress（B）of capsicum * P < 0.05，** P < 0.01. The same below.

图4 辣椒CaSec16基因沉默植株表型（A）及沉默效率（B） TRV2:CaSec16：CaSec16沉默植株；TRV2:CaPDS：基因沉默阳性对照；TRV2:00：基因沉默阴性对照。下同。

Fig. 4 Capsicum CaSec16 silencing plant phenotype（A）and silencing efficiency（B） TRV2:CaSec16：CaSec16-silenced plants；TRV2:CaPDS：Positive control；TRV2:00：Negative control. The same below.

图5 CaSec16基因沉默辣椒植株的热害表型（A）及相对电导率（B）

Fig. 5 Heat damage phenotype（A）and relative conductivity（B）of CaSec16 gene silenced pepper plants

图6 热处理前后CaSec16沉默和对照植株的组织化学染色及MDA和Pro的含量 A：叶片死细胞积累（台盼蓝染色）；B：叶片H2O2积累（DAB染色）；C：叶片丙二醛含量；D：叶片脯氨酸含量。

Fig. 6 Histochemical staining and content of MDA and Pro in CaSec16-silenced and control plants before and after heart treatment A：Accumulation of dead cells in leaves（Trypan blue staining）；B：Leaf H2O2 accumulation（DAB staining）；C：Malondialdehyde content in leaves；D：Leaf proline content.

图7 CaSec16沉默和对照植株的耐热标记基因CaHsp70.1和CaHsfA2相对表达量

Fig. 7 The relative expression levels of heat-resistant marker genes CaHsp70.1 and CaHsfA2 in CaSec16-silenced and control plants

图8 CaSec16沉默和对照植株的总叶绿素含量

Fig. 8 Total chlorophyll content of CaSec16-silenced and control plants

图9 CaSec16瞬时过表达和对照植株的CaSec16相对表达量（A）和热害表型（B）

Fig. 9 Relative expression of CaSec16（A）and heat damage phenotype（B）of CaSec16 in transient overexpression and control plants

图10 热处理前后CaSec16瞬时过表达和对照植株的MDA和Pro的含量及耐热标记基因的相对表达量

Fig. 10 The content of MDA and Pro and the relative expression levels of heat-resistant marker genes in CaSec16 transient overexpression and control plants before and after heart treatment

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