绢毛委陵菜PsWRKY40的克隆与耐镉功能分析

doi:10.16420/j.issn.0513-353x.2022-0213

摘要/Abstract

摘要：

以绢毛委陵菜（Potentilla sericea）为材料，克隆得到PsWRKY40的cDNA全长序列。该基因的开放阅读框长978 bp，编码325个氨基酸，属于WRKY家族Group Ⅱ亚族。荧光定量PCR分析表明，PsWRKY40被NaCl、CdCl₂和甘露醇显著诱导表达。将PsWRKY40异源转化拟南芥，转基因植株在镉处理后，其对Cd的抗性显著高于野生型植株，AtSOD、AtPOD和AtCAT的表达量显著提高。上述结果表明PsWRKY40可被镉诱导，可能参与绢毛委陵菜抗镉调控。

关键词: 绢毛委陵菜, WRKY, 镉胁迫, 基因克隆, 功能分析

Abstract:

The full-length cDNA sequence of PsWRKY40 was cloned from Potentilla sericea. The open reading frame of this gene was 978 bp in length，which encoded 325 amino acids，and belonged to the GroupⅡsubfamily of the WRKY family. The expression level of PsWRKY40 significantly increased by NaCl，CdCl₂ and mannitol treatments. PsWRKY40 was transformed into Arabidopsis thaliana heterologously，and transgenic plant was obtained. Under the treatment of Cd²⁺，it was found that the resistance of the transgenic lines was significantly higher than that of the wild type，and transgenic lines had a significantly higher expression level of AtSOD，AtPOD and AtCAT. The results showed that PsWRKY40 can be induced by Cd²⁺，so it may participate in the regulation of Cd²⁺ resistance in Potentilla sericea.

Key words: Potentilla sericea, WRKY, cadmium stress, gene cloning, functional analysis

中图分类号:

S682

高鹏飞, 高冰, 冯郑红, 吴建慧. 绢毛委陵菜PsWRKY40的克隆与耐镉功能分析[J]. 园艺学报, 2023, 50(6): 1269-1283.

GAO Pengfei, GAO Bing, FENG Zhenghong, WU Jianhui. Cloning and Cd-resistant Analysis of PsWRKY40 in Potentilla sericea[J]. Acta Horticulturae Sinica, 2023, 50(6): 1269-1283.

图/表 12

表1 引物碱基序列

Table 1 List of primer sequences

引物名称 Primer name	上游引物（5′-3′） Forward primer	下游引物（5′-3′） Reverse primer
PsWRKY40	TAGGTTCGTTTCGGGTTCAT	CTGGAGAGTTAGGCTACAAGTCAC
PsWRKY40-qPCR	GCTGCAAGTGCTTTGGTGAA	CCCTGCTCAACCTCTCTTGG
β-Actin	GCGACAATGGAACTGGAATGG	GACAATTTCCCGTTCAGCAGTG
pBI121-PsWRKY40	GCTCTAGAATGGACTACTCA	TTGCCAAATGTTTGAACGATC
Actin8	TCAGCACTTTCCAGCAGATG	CTGTGGACAATGCCTGGAC
AtSOD	AGGAAACATCACTGTTGGAGAT	GAGTTTGGTCCAGTAAGAGGAA
AtPOD	CGTGCCCTTCATATTGTTGG	GACGCCATCAACAACGAGTC
AtCAT	AGGATCAAACTTTGAGGGGTAG	CTTGTGGTTCCTGGAATCTACT

图1 PsWRKY40全长的PCR结果

Fig. 1 PCR results of full-length PsWRKY40

图2 PsWRKY40系统进化树数字代表可信度。

Fig. 2 Phylogenetic tree analysis of PsWRKY40 The number represents credibility.

图3 绢毛委陵菜PsWRKY40在不同非生物胁迫下的表达模式不同小写字母表示差异显著（P < 0.05）。

Fig. 3 Expression patterns of PsWRKY40 under different abiotic stresses Different lowercase letters indicate that there is a significant difference (P < 0.05).

图4 转基因拟南芥T0 ~ T3代种子卡那霉素抗性筛选

Fig. 4 Screening of transgenic Arabidopsis seeds T0-T3 for kanamycin resistance

图5 拟南芥PsWRKY40转基因株系的PCR（A）和qPCR（B）鉴定结果 CK为阳性对照，ddH2O为阴性对照，WT为野生型植株，OE-1 ~ OE-6为转基因植株。不同小写字母表示差异显著（P < 0.05）。

Fig. 5 PCR（A）and qPCR（B）identification of PsWRKY40 transgenic Arabidopsis thaliana CK is agrobacterium positive control, WT is wild-type Arabidopsis thaliana, ddH2O is negative control，OE-1-OE-6 are transgenic lines. Different lowercase letters indicate that there is a significant difference（P < 0.05）.

图6 PsWRKY40转基因拟南芥（OE）及其野生型（WT）在CdCl2处理下的种子萌发（A）和植株生长状态（B）

Fig. 6 Seed germination（A）and plant growth status（B）of PsWRY40 transgenic Arabidopsis thaliana（OE）and its wild type（WT）under CdCl2 treatments

表2 不同浓度CdCl2处理下PsWRKY40转基因拟南芥（OE）及其野生型（WT）的种子萌发率

Table 2 Seed germination rate of PsWRKY40 transgenic Arabidopsis thaliana (OE) and its wild tye (WT) treated with different concentrations of CdCl2 %

株系 Plant line	CdCl₂/（μmol · L^-1）
株系 Plant line	0	50	100	150
WT	97.33 ± 2.67 a	66.67 ± 3.53 b	21.63 ± 1.21 d	3.92 ± 2.15 f
OE-1	98.61 ± 1.39 a	96.14 ± 2.14 a	47.79 ± 3.26 c	15.17 ± 2.13 de
OE-2	100.00 ± 0.00 a	97.53 ± 2.47 a	49.95 ± 2.86 c	16.69 ± 2.61 de
OE-6	97.38 ± 1.31 a	96.00 ± 2.31 a	45.01 ± 4.03 c	11.71 ± 2.56 e

图7 PsWRKY40转基因拟南芥（OE）及其野生型（WT）在CdCl2处理下的氧化胁迫情况不同小写字母表示差异显著（P < 0.05）。下同。

Fig. 7 Oxidative stress condition of PsWRY40 transgenic Arabidopsis thaliana（OE）and its wild type（WT）under CdCl2 treatment Different lowercase letters indicate that there is a significant difference（P < 0.05）. The same below.

图8 PsWRKY40转基因拟南芥（OE）及其野生型（WT）在CdCl2处理下的抗氧化酶活性和叶绿素含量

Fig. 8 Antioxidative enzyme activity and chlorophyll content of PsWRY40 transgenic Arabidopsis thaliana（OE）and its wild type（WT）under CdCl2 treatment

图9 PsWRKY40转基因拟南芥（OE）及其野生型（WT）在CdCl2处理下的抗氧化相关基因的表达

Fig. 9 Antioxidant-related gene expression of PsWRY40 transgenic Arabidopsis thaliana（OE）and its wild type（WT）under CdCl2 treatment

图10 转基因拟南芥CdCl2处理下PsWRKY40表达量与相关基因表达量、酶活性、叶绿素等含量间的相关系数 “*”表示在0.05水平上差异显著，“**”表示在0.01水平上差异显著。

Fig. 10 Correlation coefficient between PsWRKY40 expression level and related gene expression, enzyme activity and chlorophyll content in transgenic Arabidopsis under CdCl2 treatment “*”represents significant at P < 0.05，“**”represent significant at P < 0.01.

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