牡丹<i>PsDHN1</i>基因克隆及转基因拟南芥的耐涝性分析

doi:10.3785/j.issn.1008-9209.2020.09.031

浙江大学学报（农业与生命科学版）

2021, Vol. 47

Issue (3): 335-346 DOI: 10.3785/j.issn.1008-9209.2020.09.031

园艺学

牡丹PsDHN1基因克隆及转基因拟南芥的耐涝性分析

刘慧春(

),张加强,马广莹,周江华,许雯婷,朱开元(

)

浙江省园林植物与花卉研究所，杭州 311251

Cloning of PsDHN1 gene of Paeonia suffruticosa and waterlogging tolerance analysis of transgenic Arabidopsis with PsDHN1 gene

Huichun LIU(

),Jiaqiang ZHANG,Guangying MA,Jianghua ZHOU,Wenting XU,Kaiyuan ZHU(

)

Zhejiang Institute of Landscape Plants and Flowers, Hangzhou 311251, China

全文: PDF(8294 KB) HTML

摘要：

在前期研究的基础上，从牡丹叶片中获得了脱水素基因PsDHN1的中间序列，利用cDNA末端快速扩增（rapid-amplification of cDNA ends, RACE）技术克隆获得了该基因的全长序列。通过生物信息学分析及转化拟南芥进行功能验证，探讨牡丹脱水素基因PsDHN1的特性及其响应涝害胁迫的功能。生物信息学分析结果表明：PsDHN1基因全长cDNA为864 bp，包括471 bp的开放阅读框，104 bp的5′非编码区和289 bp的3′非编码区。PsDHN1蛋白含有156个氨基酸，其分子质量为16.39 kDa，理论等电点为8.87，亲水性指数为－1.274，为亲水性蛋白，不稳定性指数为44.05，为不稳定性蛋白。PsDHN1蛋白包含3个α-螺旋和1个β-折叠，以及2个Y片段、1个S片段和2个K片段，属于典型的Y₂SK₂型脱水素。牡丹的PsDHN1蛋白与其他9个物种有一定的同源性，其中与苦瓜、麻风树和胡桃DHN1蛋白的相似度分别达到了58%、54%和53%。亚细胞定位结果表明，PsDHN1主要定位于细胞核和细胞膜上。耐涝性试验结果显示：从表型上看，转PsDHN1基因的拟南芥植株耐涝能力及恢复生长的情况均优于野生型植株；涝害相关的酶如蔗糖合成酶、丙酮酸脱羧酶、α-淀粉酶活性及可溶性蛋白含量均为转基因拟南芥总体高于野生型植株。该研究结果为牡丹耐涝基因的挖掘和进一步研究牡丹耐涝的分子机制提供了理论依据。

关键词： 牡丹; PsDHN1基因; 基因克隆; 耐涝性

Abstract:

Based on the previous studies, an intermediate sequence of dehydrin gene PsDHN1 was obtained from the leaves of Paeonia suffruticosa and the full-length sequence of PsDHN1 gene was cloned by the rapid-amplification of cDNA ends (RACE) method. In order to explore the characteristics of PsDHN1 and its function in response to waterlogging stress, bioinformatic analysis and transformation into Arabidopsis for functional verification were performed. The results of bioinformatic analysis showed that the full length cDNA of PsDHN1 was 864 bp, containing an open reading frame (ORF) of 471 bp, and the lengths of 5′ and 3′ noncoding regions were 104 bp and 289 bp, respectively. The protein encoded by PsDHN1 gene contained 156 amino acids with the molecular mass of 16.39 kDa, theoretical isoelectric point of 8.87, hydrophilic index of －1.274, belonging to hydrophilic protein, and instability index of 44.05, belonging to unstable protein. The PsDHN1 protein contained three α-helixes and one β-flod, and two Y fragments, one S fragment and two K fragments that belonged to the typical Y₂SK₂ dehydratin. In addition, the PsDHN1 protein of P. suffruticosa had some homologies with other nine species, among which the similarities between P. suffruticosa and Momordica charantia, Jatropha curcas, Juglans regia were 58%, 54% and 53%, respectively. Subcellular localization results showed that PsDHN1 was mainly localized in the nucleus and cell membrane. The results of waterlogging tolerance test showed that the phenotype of transgenic Arabidopsis plants with PsDHN1 gene had better waterlogging resistance and growth recovery than the wild-type plants. Moreover, the physiological indexes that related to waterlogging tolerance such as sucrose synthase, pyruvate decarboxylase, α-amylase activities and soluble protein content reflected that transgenic Arabidopsis had higher waterlogging tolerance than wild-type plants. The results provide a theoretical basis for mining waterlogging tolerance genes and further research on the molecular mechanisms of P. suffruticosa.

Key words: Paeonia suffruticosa PsDHN1 gene gene cloning waterlogging tolerance

收稿日期: 2020-09-03 出版日期: 2021-06-25

CLC:

S 685.11

基金资助: 浙江省农业科学院公共发展专项(617CF0101G);浙江省公益技术应用研究计划(LGN18C150007)

通讯作者: 朱开元 E-mail: lhuichun@163.com;52011648@qq.com

作者简介: 刘慧春（https://orcid.org/0000-0003-4714-2359)，Tel：+86-571-82704530，E-mail：lhuichun@163.com

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引用本文:

刘慧春,张加强,马广莹,周江华,许雯婷,朱开元. 牡丹PsDHN1基因克隆及转基因拟南芥的耐涝性分析[J]. 浙江大学学报（农业与生命科学版）, 2021, 47(3): 335-346.

Huichun LIU,Jiaqiang ZHANG,Guangying MA,Jianghua ZHOU,Wenting XU,Kaiyuan ZHU. Cloning of PsDHN1 gene of Paeonia suffruticosa and waterlogging tolerance analysis of transgenic Arabidopsis with PsDHN1 gene. Journal of Zhejiang University (Agriculture and Life Sciences), 2021, 47(3): 335-346.

链接本文:

http://www.zjujournals.com/agr/CN/10.3785/j.issn.1008-9209.2020.09.031 或 http://www.zjujournals.com/agr/CN/Y2021/V47/I3/335

表1 引物序列

图1 PsDHN1基因全长cDNA序列及其推测的氨基酸序列和结构域下划线依次代表Y片段、Y片段、S片段、K片段和K片段.

表2 PsDHN1蛋白的氨基酸组分

图2 PsDHN1蛋白的二级结构预测

表3 磷酸化位点预测结果

图3 PsDHN1蛋白的三级结构预测

图4 牡丹PsDHN1蛋白的多序列比对

图5 PsDHN1蛋白与其他物种DHN1蛋白的聚类分析

图6 PsDHN1在本氏烟草叶片中的亚细胞定位GFP：明场下显微镜显示的照片；DIC：暗场下显微镜显示的照片；GFP＋DIC：GFP和DIC 2张照片重叠后的效果。

图7 转基因拟南芥PsDHN1基因的实时荧光定量表达分析WT：野生型植株；P1～P3：转基因植株。

图8 转基因拟南芥涝害处理的表型变化A～B.淹水处理5 d后；C～D.恢复生长7 d后。

图9 涝害胁迫下转基因拟南芥的生理指标测定短栅上的不同小写字母表示在相同处理时间内不同植株间在P＜0.05水平差异有统计学意义；n=3.

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