Analysis of Heat-tolerant Function of Pepper Vesicle Formation Related Gene CaSec16

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

Acta Horticulturae Sinica ›› 2023, Vol. 50 ›› Issue (11): 2387-2400.doi: 10.16420/j.issn.0513-353x.2022-0996

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

Analysis of Heat-tolerant Function of Pepper Vesicle Formation Related Gene CaSec16

YAN Bentao, JIAO Kexin, ZHAO Yue, YANG Qiaomin, CHEN Tao, LU Minghui^*()

College of Horticulture，Northwest Agriculture and Forestry University，Yangling，Shaanxi 712100，China

Received:2023-04-21 Revised:2023-07-17 Online:2023-11-25 Published:2023-11-28
Contact: LU Minghui

Abstract

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

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.

Figures/Tables 12

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

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

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

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.

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.

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.

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

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.

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

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

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

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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Analysis of Heat-tolerant Function of Pepper Vesicle Formation Related Gene CaSec16

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