Cloning and Functional Analysis of NnCYP707A1 Gene from Lotus

doi:10.13560/j.cnki.biotech.bull.1985.2024-1008

Abstract

Abstract:

Objective To provide high quality genetic resources for phytoremediation of lotus with strong heavy metal adsorping capacity for polluted copper. Method Based on our previous analysis of transcriptomic data of copper stress in lotus, we screened and cloned the key gene NnCYP707A1 in lotus, classified its sequence characteristics and evolutionary relationship by using online websites and software, observed the subcellular localization by injecting Agrobacterium into tobacco, and analyzed its response to copper stress by transforming Saccharomyces cerevisiae and transient transforming lotus. Result NnCYP707A1 gene ORF was 1 065 bp long, encoded 354 amino acids, and contained the conserved domain PFGXGXHXCPG of the cytochrome P450 family. NnCYP707A1 protein was a stable hydrophilic protein with a molecular weight of 40.592 kD and an isoelectric number of 8.68. It belonged to CYP707A subfamily and was genetically close toPtCYP707A1 of Populus pilopus. The results of subcellular localization showed that the protein was located in cytoplasm. The overexpression of NnCYP707A1 gene enhanced the sensitivity of S. cerevisiae to copper stress. The related physiological indexes of overexpressing lotus plants (OE-CYP707A1) were not improved, while silencing lotus plants (cyp707a1) alleviated the inhibition effect of copper stress. Conclusion The overexpression of NnCYP707A1 weakens the tolerance to copper stress and reduces the copper resistance of lotus.

Key words: Nelumbo nucifera, copper stress, NnCYP707A1, original transformation, gene function

LIU Hong-li, MA Yi-dan, WANG Wan-ru, YANG Ya-ru, HE Dan, LIU Yi-ping, KONG De-zheng. Cloning and Functional Analysis of NnCYP707A1 Gene from Lotus[J]. Biotechnology Bulletin, 2025, 41(5): 197-207.

Figures/Tables 11

Table 1 Primers used in this study

引物名称Primer name	引物序列Primer sequence（5′‒3′）	引物用途Primer use
CYP707A1-F	ATGATTTCAAACCCAGAAGCTG	基因克隆
CYP707A1-R	TTACCTGTACTTTGTGGTCAGATGA	Gene cloning
2300-CYP-F	ACGGGGGACGAGCTCGGTACCATGATTTCAAACCCAGAAGCTG	载体构建
2300-CYP-R	CTTGCTCACCATGGTGTCGACCCTGTACTTTGTGGTCAGATGA	Vector construction
pYES2-CYP707A1-F	GCCGCCAGTGTGCTGGAATTCATGATTTCAAACCCAGAAGCTG
pYES2-CYP707A1-R	ACATGATGCGGCCCTCTAGACCTGTACTTTGTGGTCAGATGA
p-F	GCATAACCACTTTAACTAATAC
p-R	TCGGTTAGAGCGGATGTG
PIR-CYP-F	GCAGAATCTGAATTCGTCGACATGATTTCAAACCCAGAAGCTG
PIR-CYP-R	GCTCGAGAAGCTTGTCGACCCTGTACTTTGTGGTCAGATGA
RI-CYP-F	GCAGAATCTGAATTCGTCGACAGAGATACCACAGCCAGTGTTTTG
RI-CYP-R	GCTCGAGAAGCTTGTCGACCCTGTACTTTGTGGTCAGATG
qPCYP-F	ATTCACCACAGCCCAGACAACTTC	基因表达分析
qPCYP-R	CATTCCCAGGGCATGAGTGTATCC	Gene expression analysis
qPCYP707A1-F	CATTCACCACAGCCCAGACA
qPCYP707A1-R	CAGGGCATGAGTGTATCCCA

Fig. 1 Electrophoresis of PCR products of NnCYP707A1 gene

Fig. 2 NnCYP707A1 nucleotide sequence and its translated amino acid sequenceThe green boxes are the start and stop codons, and the blue boxes are the P450 family conserved domains

Fig. 3 Bioinformatics analysis of NnCYP707A1 proteinA: Domain prediction. B: Hydrophilic analysis. C: Protein transmembrane domain analysis. D: Secondary structure prediction. E: Phosphorylation site analysis. F: Tertiary structure prediction

Fig. 4 Multiple alignment of NnCYP707A1 protein sequences and homologous sequences of 12 other species of plants

Fig. 5 Phylogenetic analysis of NnCYP707A1 protein and CYP707A protein in 12 other plants

Fig. 6 Subcellular localization of NnCYP707A1 protein35S::GFP: Strains of Agrobacterium carrying empty pC2300-GFP. 35S::NnCYP707A1::GFP: Agrobacterium strain carrying recombinant vector pC2300-NnCYP707A1-GFP. Scale=10 μm

Fig. 7 Identification of resistance of NnCYP707A1 in yeast system to copper

Fig. 8 Transient vector construction of NnCYP707A1A: PCR identification of the overexpressed vector PIR-NnCYP707A1. B: PCR identification of silencing vector IR-NnCYP707A1-RI. M: DL2000 marker. 1-3: Recombinant bacterial solution

Fig. 9 Overexpression efficiency and silencing efficiency of NnCYP707A1 geneYanyangtian: Control plant. OE-CYP707A1: Overexpressed lotus plants. cyp707a1: Silenced lotus plants. Different lowercase letters indicate significant difference (P<0.05). The same below

Fig. 10 Physiological indexes of NnCYP707A1 transgenic lotus under copper stress

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