玉米<i>ZmRAV1</i>基因的克隆分析与功能验证

doi:10.7668/hbnxb.20195345

摘要/Abstract

摘要：

前期转录组学分析发现,ZmRAV1为玉米响应干旱胁迫的候选基因,为了深入研究该基因的功能,克隆了ZmRAV1基因,应用生物信息学分析了ZmRAV1的编码序列,并在拟南芥中过表达该基因,通过干旱条件下过表达拟南芥株系表型、生理生化指标的测定验证其功能。结果表明:ZmRAV1基因全长1 176 bp,共编码389个氨基酸,二级结构中无规则卷曲占比最多,是一种不含信号肽、非跨膜的亲水性蛋白。亚细胞定位显示,该基因编码蛋白位于细胞核。ZmRAV1基因在不同物种间具有较高的保守性。从系统发育树上看,ZmRAV1与五节芒同源基因亲缘关系最强,同源性较高。干旱胁迫处理后,萌发期过表达拟南芥株系的根长显著高于野生型(WT)拟南芥,幼苗期野生型经过干旱胁迫后出现枯萎甚至死亡,存活率低于过表达株系,且干旱处理后ZmRAV1过表达株系过氧化物酶、超氧化物歧化酶活性均高于野生型,表明过表达ZmRAV1基因可提高拟南芥对干旱胁迫的抗性。综上所述,通过ZmRAV1基因编码序列的生物信息学分析以及转基因拟南芥株系的生理生化指标测定明确了ZmRAV1在响应干旱胁迫的过程中发挥正向调控的作用。

关键词: 玉米, 干旱胁迫, ZmRAV1, 亚细胞定位, 功能验证

Abstract:

Preliminary transcriptomic analysis identified ZmRAV1 as a candidate gene involved in maize's response to drought stress. To further investigate its function, this study cloned the ZmRAV1 gene, conducted bioinformatics analysis of its coding sequence, and overexpressed this gene in Arabidopsis thaliana. The function of ZmRAV1 was validated by assessing the phenotypes and physiological and biochemical indices of the transgenic Arabidopsis lines under drought conditions. The results showed that the ZmRAV1 gene had a total length of 1 176 bp and encoded 389 amino acids.It had the highest proportion of irregular coils in its secondary structure and was a hydrophilic protein that did not contain signal peptides and was non transmembrane.Subcellular localization indicated that the protein was located in the nucleus.ZmRAV1 exhibited high conservation across different species.Phylogenetic analysis indicated that ZmRAV1 shares the closest evolutionary relationship with its homolog in Miscanthus sinensis, showing a high degree of homology. After drought stress treatment,the root length of Arabidopsis thaliana lines overexpressing ZmRAV1 during germination was significantly higher than that of wild-type (WT)lines.In the seedling stage,WT showed withering or even death after drought stress,while the survival rate was lower than that of overexpressing lines.Moreover,the POD and SOD activities of ZmRAV1 overexpressing lines were higher than those of WT after drought treatment,indicating that overexpression of ZmRAV1 gene could enhance Arabidopsis thaliana's resistance to drought stress.

Key words: Zea mays L., Drought treatment, ZmRAV1, Subcellular localization, Functional verification

翟晓婷, 王鑫伟, 郝喜英, 沈俊睿, 高旭宏, 刘桂雪, 刘松涛. 玉米ZmRAV1基因的克隆分析与功能验证[J]. 华北农学报, 2025, 40(2): 18-26. doi: 10.7668/hbnxb.20195345.

ZHAI Xiaoting, WANG Xinwei, HAO Xiying, SHEN Junrui, GAO Xuhong, LIU Guixue, LIU Songtao. Cloning,Analysis and Functional Verification of ZmRAV1 Gene[J]. Acta Agriculturae Boreali-Sinica, 2025, 40(2): 18-26. doi: 10.7668/hbnxb.20195345.

http://www.hbnxb.net/CN/Y2025/V40/I2/18

图/表 8

图1 ZmRAV1基因全长扩增结果 M.DL2000 Marker;1,2.目的片段。

Fig.1 Results of full-length amplification of ZmRAV1 gene M.DL2000 Marker;1,2.Target fragment.

图2 ZmRAV1蛋白的二级结构预测(A)和三级结构预测(B)

Fig.2 The secondary structure(A) and tertiary structure(B) of ZmRAV1 protein

图3 ZmRAV1蛋白序列分析 A.蛋白跨膜结构预测;B.蛋白信号肽预测;C.蛋白质亲/疏水性预测;D.磷酸化位点预测。

Fig.3 Protein sequence analysis of ZmRAV1 A.Protein transmembrane domain prediction; B.Protein signal peptide prediction; C.Protein hydrophilicity/hydrophpbicity prediction; D.Phosphorylation site prediction.

图4 不同植物RAV1蛋白序列比对

Fig.4 Sequence alignment of RAV1 proteins in different plants

图5 不同植物RAV1蛋白的系统进化树

Fig.5 Phylogenetic tree for RAV1 proteins in different plants

图6 玉米原生质体中GFP荧光定位观察 A.pBWA (V) HS-1-GLosgfp-ZmRAV1侵染玉米原生质体GFP荧光观察;B.对照组原生质体GFP荧光观察。a、e.GFP绿色荧光;b、f.叶绿体自发荧光;c、g.明场;d、h.叠加后的结果。

Fig.6 Observation of GFP fluorescence localization in maize protoplasts A.Observation on GFP fluorescence of maize protoplasts infected with pBWA (V) HS-1-GLosgfp-ZmRAV1;B.GFP fluorescence of protoplasts in the control group was observed.a,e.GFP green fluorescence;b,f.Chloroplast autofluorescence;c,g.Bright;d,h.Overlapping.

图7 ZmRAV1过表达幼苗对甘露醇胁迫的敏感性 A.野生型和L1、L2、L4株系在不同浓度甘露醇的生长情况;B.野生型和过表达株系根长统计图。不同小写字母表示差异显著(P<0.05)。图8同。

Fig.7 Sensitivity of ZmRAV1 overrxpressing seedlings to mannitol stress A.Growth of wild-type and overexpressing seedlings L1,L2,L4 lines at different concentrations of mannitol; B.Statistical graph of root length of wild-type and overexpressing seedlings.Different lowercase letters indicated significant difference(P<0.05).The same as Fig.8.

图8 转基因材料苗期抗旱性鉴定 A.转基因拟南芥和野生型干旱处理前后表型变化;B.干旱处理前后拟南芥存活率;C.干旱处理前后POD活性;D.干旱处理前后SOD活性。

Fig.8 Identification of drought resistance during seedling stage of transgenic materials A.Phenotypic changes in transgenic Arabidopsis and WT before and after drought treatment; B.The change of survival rate before and after drought treatment; C.POD activity before and after drought treatment; D.SOD activity before and after drought treatment.

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玉米ZmRAV1基因的克隆分析与功能验证

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