Overexpression of CaCP1 Enhances Salt Stress Sensibility in Transgenic Tobacco

doi:10.13560/j.cnki.biotech.bull.1985.2022-0945

Abstract

Abstract:

Cysteine proteinases（CPs）play significant roles in salt stress responses of plants, thus the function of CaCP1 in salt resistance was studied by cloning CaCP1, which may provide reference for the breeding of pepper salt resistant varieties. First, the CaCP1 promoter of pepper（Capsicum annuum L.）was cloned, then recombinant vector with different length promoter fragments was constructed based on cis-regulatory elements associated with salt stress, and transient transformation of tobacco was performed. Salt treatment was carried out on T₃ generation tobacco transgenic lines overexpressing CaCP1, phenotype symptoms were observed, physiological indexes were measured, and the expressions of stress-related genes were analyzed. The results revealed that in response to salt treatment, the GUS expression in -410 to -1 bp region was the strongest. The leaves of CaCP1 transgenic plants wilted and yellowed earlier. The chlorophyll and relative water content were significantly lower than those of wild type, while MDA and electrolyte leakage rates were significantly higher than those of wild-type. ROS scavenger enzymes CAT and SOD activities decreased, while POD activity increased. Compared to wild-type, the expressions of ROS-scavenging genes（NtSOD, NtPOD, NtCAT, and NtAPX）and salt stress-related genes（NtLEA5, NtNHX1, NtP5CS1, and NtSOS1）significantly decreased. CaCP1 acts as a negative regulator to mediate the plant defense response to salt stress.

Key words: pepper, cysteine proteinase, transgenic, tobacco, salt stress

DU Qing-jie, ZHOU Lu-yao, YANG Si-zhen, ZHANG Jia-xin, CHEN Chun-lin, LI Juan-qi, LI Meng, ZHAO Shi-wen, XIAO Huai-juan, WANG Ji-qing. Overexpression of CaCP1 Enhances Salt Stress Sensibility in Transgenic Tobacco[J]. Biotechnology Bulletin, 2023, 39(2): 172-182.

Figures/Tables 9

References 49

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引物Primer	序列Sequence（5'-3'）	用途Purpose
GUS-F	TAGATCTGACTAGTTTACGTCCTGT	构建载体检测
GUS-R	TAGTCTGCCAGTTCAGTTCGT
CaMV35S-F	AAGACTGGCGAACAGTTCAT	载体构建
CaMV35S-R	ATAGTGGGATTGTGCGTCAT
1381-P1F	CCGGAATTCGCGAAGGTAGTATAATTTAAAAC	克隆启动子片段
1381-P2F	CCGGAATTCTGAATGAAATGATTTGTATTTTG
1381-P3F	CCGGAATTCTATGAAATGATCTAATATAATTG
1381-P4F	CCGGAATTCATAGAAATCATTAAGATTTTCCG
1381-P1R	CGCGTCGACTATAGAACAACTATATAGTATTATG
2307-CaCP1-F	TCTAGAATATAGTTGTTCTATAATGGCCTTT	克隆CaCP1
2307-CaCP1-R	GGTACCTTAAGGATAAATTTTCTTTTAGGC
GUS-QPCR-F	CAGTGAAGGGCCAACAGTTC	GUS基因的定量分析
GUS- QPCR-R	CATGTTCATCTGCCCAGTCG
NtCAT-F	AGGTACCGCTCATTCACACC	NtCAT基因的定量分析
NtCAT-R	AAGCAAGCTTTTGACCCAGA
NtSOD-F	AGCTACATGACGCCATTTCC	NtSOD基因的定量分析
NtSOD-R	CCCTGTAAAGCAGCACCTTC
NtAPX-F	CCATTTCCAGTGCTTGTGGTCTC	NtAPX基因的定量分析
NtAPX-R	ATAGGTACCAGCAGAGTGCCA
NtLEA5-F	CATCAGCTAGTGTGCCAGGT	NtLEA5基因的定量分析
NtLEA5-R	TGGCACCCATGATGTTGTCT
NtNHX1-F	CAACTGGTCTTCTTAGTGCT	NtNHX1基因的定量分析
NtNHX1-R	GCCTTGTAGTGACTCTTGAA
NtPOX2-F	CATCTTCACGGCTGTTCGAG	NtPOX2基因的定量分析
NtPOX2-R	TGTTGGGTGGTGAGGTCTTT
NtP5CS1-F	TTGCAAACTCTGTCCGTGTG	NtP5CS1基因的定量分析
NtP5CS1-R	TTGGCCTCCTTTCCTCCTTT
NtSOS1-F	CAAATGTTATCCCCCGAAAGC	NtSOS1基因的定量分析
NtSOS1-R	CGGAGAACCTGAGGAAATGTGA
NtActin-F	TGGCATCACACTTTCTACAA	RT-qPCR试验的内参基因
NtActin-R	CAACGGAATCTCTCAGCTCC

引物Primer	序列Sequence（5'-3'）	用途Purpose
GUS-F	TAGATCTGACTAGTTTACGTCCTGT	构建载体检测
GUS-R	TAGTCTGCCAGTTCAGTTCGT
CaMV35S-F	AAGACTGGCGAACAGTTCAT	载体构建
CaMV35S-R	ATAGTGGGATTGTGCGTCAT
1381-P1F	CCGGAATTCGCGAAGGTAGTATAATTTAAAAC	克隆启动子片段
1381-P2F	CCGGAATTCTGAATGAAATGATTTGTATTTTG
1381-P3F	CCGGAATTCTATGAAATGATCTAATATAATTG
1381-P4F	CCGGAATTCATAGAAATCATTAAGATTTTCCG
1381-P1R	CGCGTCGACTATAGAACAACTATATAGTATTATG
2307-CaCP1-F	TCTAGAATATAGTTGTTCTATAATGGCCTTT	克隆CaCP1
2307-CaCP1-R	GGTACCTTAAGGATAAATTTTCTTTTAGGC
GUS-QPCR-F	CAGTGAAGGGCCAACAGTTC	GUS基因的定量分析
GUS- QPCR-R	CATGTTCATCTGCCCAGTCG
NtCAT-F	AGGTACCGCTCATTCACACC	NtCAT基因的定量分析
NtCAT-R	AAGCAAGCTTTTGACCCAGA
NtSOD-F	AGCTACATGACGCCATTTCC	NtSOD基因的定量分析
NtSOD-R	CCCTGTAAAGCAGCACCTTC
NtAPX-F	CCATTTCCAGTGCTTGTGGTCTC	NtAPX基因的定量分析
NtAPX-R	ATAGGTACCAGCAGAGTGCCA
NtLEA5-F	CATCAGCTAGTGTGCCAGGT	NtLEA5基因的定量分析
NtLEA5-R	TGGCACCCATGATGTTGTCT
NtNHX1-F	CAACTGGTCTTCTTAGTGCT	NtNHX1基因的定量分析
NtNHX1-R	GCCTTGTAGTGACTCTTGAA
NtPOX2-F	CATCTTCACGGCTGTTCGAG	NtPOX2基因的定量分析
NtPOX2-R	TGTTGGGTGGTGAGGTCTTT
NtP5CS1-F	TTGCAAACTCTGTCCGTGTG	NtP5CS1基因的定量分析
NtP5CS1-R	TTGGCCTCCTTTCCTCCTTT
NtSOS1-F	CAAATGTTATCCCCCGAAAGC	NtSOS1基因的定量分析
NtSOS1-R	CGGAGAACCTGAGGAAATGTGA
NtActin-F	TGGCATCACACTTTCTACAA	RT-qPCR试验的内参基因
NtActin-R	CAACGGAATCTCTCAGCTCC