荷花NnCYP707A1的克隆及功能分析

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

摘要/Abstract

摘要：

目的利用荷花具有较强的重金属吸附能力这一特性，为其参与铜污染防治的植物修复提供优质的基因资源。方法基于课题组前期对荷花铜胁迫转录组数据的分析，筛选并克隆荷花铜胁迫的关键基因NnCYP707A1，利用在线网站及软件分析其序列特征和进化关系，通过农杆菌注射烟草观察亚细胞定位情况，通过转化酿酒酵母、瞬时转化荷花，分析其对铜胁迫的响应。结果 NnCYP707A1的ORF全长1 065 bp，编码354个氨基酸，含有细胞色素P450家族的保守结构域PFGXGXHXCPG；NnCYP707A1蛋白为稳定的亲水性蛋白，分子量为40.592 kD，等电点数为8.68；其属于CYP707A亚族，与毛果杨的PtCYP707A1遗传距离较近；亚细胞定位结果表明，该蛋白定位于细胞质；过表达NnCYP707A1增强了酿酒酵母对铜胁迫的敏感性；过表达荷花植株（OE-CYP707A1）的相关生理指标未得到改善，而沉默荷花植株（cyp707a1）可以缓解铜胁迫对其的抑制作用。结论过表达NnCYP707A1对铜胁迫耐受性减弱，降低了荷花的抗铜性。

关键词: 荷花, 铜胁迫, NnCYP707A1, 本源转化, 基因功能

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

刘红利, 马一丹, 王婉茹, 杨娅茹, 贺丹, 刘艺平, 孔德政. 荷花NnCYP707A1的克隆及功能分析[J]. 生物技术通报, 2025, 41(5): 197-207.

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.

图/表 11

表1 本研究所用引物

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

图1 NnCYP707A1的PCR产物电泳检测

Fig. 1 Electrophoresis of PCR products of NnCYP707A1 gene

图2 NnCYP707A1核苷酸序列及其翻译的氨基酸序列绿色框为起始密码子和终止密码子，蓝色框为P450家族保守结构域

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

图3 NnCYP707A1蛋白的生物信息学分析A：结构域预测；B：亲疏水性分析；C：蛋白质跨膜结构域分析；D：二级结构预测；E：磷酸化位点分析；F：三级结构预测

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

图4 NnCYP707A1蛋白序列及其他12种植物同源序列的多重比对

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

图5 NnCYP707A1蛋白与其他12种植物CYP707A蛋白的系统进化分析

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

图6 NnCYP707A1的亚细胞定位35S：：GFP：携带空载pC2300-GFP的农杆菌菌株；35S：：NnCYP707A1：：GFP：携带重组载体pC2300-NnCYP707A1-GFP的农杆菌菌株。比例尺=10 μm

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

图7 NnCYP707A1在酵母体系中的耐铜性鉴定

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

图8 NnCYP707A1的瞬时载体构建A：过表达载体PIR-NnCYP707A1的PCR鉴定；B：沉默载体IR-NnCYP707A1-RI的PCR鉴定；M：DL2000 marker；1-3：重组菌液

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

图9 NnCYP707A1的过表达效率及沉默效率Yanyangtian：对照植株；OE-CYP707A1：过表达荷花植株；cyp707a1：沉默荷花植株。不同小写字母表示存在显著性差异（P<0.05）。下同

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

图10 NnCYP707A1转基因荷花在铜胁迫下的生理指标

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

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