枳PtSARD1的特征分析及其抗旱功能初探

doi:10.16420/j.issn.0513-353x.2024-0478

摘要/Abstract

摘要： 为了探究枳[Poncirus trifoliata（L.）Raf.]水杨酸合成相关转录因子系统获得抗性缺陷1（systemic acquired resistance deficient 1，PtSARD1）在干旱应答中的作用，克隆了其1 425 bp的开放阅读框，通过序列分析、亚细胞定位、表达量分析、过表达、RNA干扰（RNAi）、抗性生理指标测定等方法对PtSARD1进行了初步研究。结果表明：PtSARD1含有3个钙调素结合蛋白结构域，且亚细胞定位表明该蛋白会从细胞核转移到细胞膜。PtSARD1在根、茎、叶中均有表达，叶片中较高，其转录表达受干旱和水杨酸诱导。经转化获得RNAi枳植株和PtSARD1过表达烟草。PtSARD1-RNAi枳离体叶片失水更快，而PtSARD1过表达烟草（A7和F7）离体叶片的相对失水率更低，在控水条件下盆栽苗生长状态更好，脯氨酸含量更高，电导率和丙二醛含量更低。以上结果表明，PtSARD1在枳干旱响应中起正向调控作用。

关键词: 柑橘, 抗旱, 基因功能, 水杨酸, SARD1

Abstract:

Systemic Acquired Resistance Deficient 1（SARD1）is a transcription factor related to salicylic acid synthesis. In order to investigate its role in drought response，the 1 425 bp open reading frame of SARD1 in Poncirus trifoliata（named as PtSARD1）was obtained. The characterization of PtSARD1 was preliminarily studied by sequence analysis，subcellular localization，relative expression level，overexpression（OE），RNA interference（RNAi）and tolerance-related physiological indicators measurement. The results revealed that PtSARD1 contained three calmodulin binding protein domains and the subcellular localization of this protein will change over time，transferring from the nucleus to the cell membrane. The expression of PtSARD1 was detected in root，stem and leaf，with higher expression in leaf. Meanwhile，PtSARD1 was induced by drought and exogenous application of salicylic acid. PtSARD1-RNAi P. trifoliata and PtSARD1-OE tobacco were obtained to study its function in drought response. The detached leaves of two PtSARD1-RNAi P. trifoliata lines showed higher relative water loss at each time point. Relative water loss in detached leaves of two PtSARD1-OE tobacco lines（A7 and F7）were lower than that of the wild type tobacco（WT）. When water was withheld，potted seedlings of A7 and F7 showed better growth status，higher proline content，but lower electrolyte leakage and malondialdehyde content. Overall，these results indicated that PtSARD1 may play a positive regulatory role in the drought response of P. trifoliata.

Key words: citrus, drought tolerance, gene function, salicylic acid, SARD1

莫锦夏, 李芳, 熊欣婷, 钟灶发, 彭婷. 枳PtSARD1的特征分析及其抗旱功能初探[J]. 园艺学报, 2025, 52(6): 1399-1411.

MO Jinxia, LI Fang, XIONG Xinting, ZHONG Zaofa, PENG Ting. Characterization of PtSARD1 and Its Preliminary Role in Drought Tolerance[J]. Acta Horticulturae Sinica, 2025, 52(6): 1399-1411.

图/表 11

表1 本研究所用引物

Table 1 Primers used in this study

引物用途 Primer usage	引物名称 Primer name	引物序列（5′-3′） Primer sequence
全长扩增 Full-length amplification	PtSARD1-F	ATGGCTGCTAAACGGTTTCTT
全长扩增 Full-length amplification	PtSARD1-R	TTAATTCCCATTGACGAAGGAC
亚细胞定位 Subcellular localization	PtSARD1-101LYFP-Kpn I-F	GGGGTACCCCATGGCTGCTAAACGGT
亚细胞定位 Subcellular localization	PtSARD1-101LYFP-Sma I-R	TCCCCCGGGGGAATTCCCATTGACGAAG
实时荧光定量PCR RT-qPCR	PtSARD1-qPCR-F	GCCTTTGCTTAGACGAGTGG
实时荧光定量PCR RT-qPCR	PtSARD1-qPCR-R	TTTGCTTCCGGTAAAGATGG
过表达 Overexpression	PtSARD1-pBI121-Xba I-F	GCTCTAGAATGGCTGCTAAACGGTTT
过表达 Overexpression	PtSARD1-pBI121-Sma I-R	TCCCCCGGGTTAATTCCCATTGACGAAGGACC
RNA干涉 RNA interference	PtSARD1-pHELLSGATE2-F	GGGGACAAGTTTGTACAAAAAAGCAGGCTAGCCGCAAATTCAGAATTGGT
RNA干涉 RNA interference	PtSARD1-pHELLSGATE2-R	GGGGACCACTTTGTACAAGAAAGCTGGGTCTTTCTAAGTCTTTGGGGCTCC
卡那霉素抗性基因 NPT II	NPT II-F	CGCAGAAGGCAATGTCATACCA
卡那霉素抗性基因 NPT II	NPT II-R	CCTTTGCTCGGAAGAGTATGAA
内参基因 Reference gene	Actin -F	CATCCCTCAGCACCTTCC
内参基因 Reference gene	Actin -R	CCAACCTTAGCACTTCTCC

图1 枳（Pt）、甜橙（Cs）和拟南芥（At）SARD1蛋白的序列比对

Fig. 1 Amino acid sequence alignment of SARD1 proteins in Poncitrus trifoliata（Pt），Citrus sinensis（Cs） and Arabidopsis thaliana（At）

图2 PtSARD1蛋白的磷酸化位点（A）和N-糖基化位点预测（B）

Fig. 2 Predication of phosphorylation sites（A）and N-glycosylation sites（B）in PtSARD1

图3 PtSARD1的亚细胞定位分析

Fig. 3 Subcellular localization analysis of PtSARD1 in Arabidopsis protoplast

图4 PtSARD1在不同组织（A）和干旱（B）及外源SA处理下（C）的表达模式不同小写字母表示不同材料间差异显著（P < 0.05）

Fig. 4 Expression patterns of PtSARD1 in different tissues（A）and in response to drought（B）as well as exogenous application of SA（C） Different lowercase letters indicate significant differences among various tested samples（P < 0.05）

图5 PtSARD1-RNAi枳的遗传转化（A）和阳性植株鉴定（B） WT：野生型；D-1和D-2：PtSARD1-RNAi系。下同

Fig. 5 Generation（A）and identification（B）of PtSARD1-RNAi Poncirus trifoliata WT：Wild type；D-1 and D-2：PtSARD1-RNAi lines. The same below

图6 PtSARD1-RNAi枳（D-1和D-2）和野生型（WT）离体脱水叶片的表型（A）和相对失水率（B） *、**分别表示转基因植株与野生型在P < 0.05、P < 0.01水平上差异显著。下同

Fig. 6 Morphological comparison（A）and relative water loss（B）of two PtSARD1-RNAi Poncirus trifoliata lines（D-1 and D-2）and the wild type（WT） *and ** indicate that there are significant differences at the levels of P < 0.05 and P < 0.01 between transgenetic plants and wild type. The same below

图7 烟草的遗传转化（A）和PtSARD1过表达系鉴定（B） WT：野生型；EV：空载；A7和F7：过表达烟草系。下同

Fig. 7 Generation（A）and identification（B）of PtSARD1-overexpression tobacco lines WT：Wide type；EV：Empty vector；A7 and F7：PtSARD1-overexpression tobacco lines. The same below

图8 野生型烟草（WT）和PtSARD1过表达烟草（A7和F7）离体叶片的表型（A）和相对失水率（B）

Fig. 8 Morphological comparison（A）and relative water loss（B）of the wild type tobacco（WT） and two PtSARD1-overexpression tobacco lines（A7 and F7）

图9 控水处理下野生型烟草（WT）和PtSARD1过表达烟草（A7和F7）的表型（A）、脯氨酸含量（B）、电导率（C）和丙二醛含量（D）不同小写字母表示3个材料在同一处理时间点差异显著（P < 0.05）

Fig. 9 Comparisons of morphology（A），proline content（B），electrolyte leakage（C）and malondialdehyde（MDA）content（D）in the wild type tobacco（WT）PtSARD1-overexpression tobacco lines（A7 and F7）under water withholding treatment Different lowercase letters indicate significant differences among the three lines at the same time point（P < 0.05）

图10 野生型烟草（WT）和PtSARD1过表达烟草（A7和F7）的水杨酸含量不同小写字母表示不同材料在不同时间点的SA含量差异显著（P < 0.05）

Fig. 10 Salicylic acid contents of wild type tobacco and PtSARD1-overexpression tobacco lines（A7 and F7） Different lowercase letters indicate significant differences among different treatment time（P < 0.05）

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