大蒜生物钟基因AsRVE1和AsRVE2及其在渗透胁迫下的表达分析

doi:10.16420/j.issn.0513-353x.2020-0961

园艺学报 ›› 2021, Vol. 48 ›› Issue (9): 1706-1716.doi: 10.16420/j.issn.0513-353x.2020-0961

大蒜生物钟基因AsRVE1和AsRVE2及其在渗透胁迫下的表达分析

卞诗村¹, 陆雅妮¹, 许吴俊¹, 陈伯清², 王广龙²^,^*(), 熊爱生³^,^*()

¹淮阴工学院应用技术学院,江苏淮安 223003
²淮阴工学院生命科学与食品工程学院,江苏淮安 223003
³南京农业大学园艺学院,作物遗传与种质创新国家重点实验室,农业农村部华东地区园艺作物生物学与种质创制重点实验室,南京 210095

收稿日期:2021-05-13 修回日期:2021-07-31 出版日期:2021-09-25 发布日期:2021-09-30
通讯作者: 王广龙,熊爱生 E-mail:guanglongwang@hyit.edu.cn;xiongaisheng@njau.edu.cn
基金资助:
江苏省杰出青年基金项目(BK20130027);教育部新世纪优秀人才支持计划项目(NCET-11-0670);江苏省高等学校大学生创新创业训练计划项目(201911049022Y)

Garlic Circadian Clock Genes AsRVE1 and AsRVE2 and Their Expression Analysis Under Osmotic Stress

BIAN Shicun¹, LU Yani¹, XU Wujun¹, CHEN Boqing², WANG Guanglong²^,^*(), XIONG Aisheng³^,^*()

¹Faculty of Applied Technology,Huaiyin Institute of Technology,Huaian,Jiangsu 223003,China
²School of Life Science and Food Engineering,Huaiyin Institute of Technology,Huaian,Jiangsu 223003,China
³College of Horticulture,State Key Laboratory of Crop Genetics and Germplasm Enhancement,Ministry of Agriculture and Rural Affairs Key Laboratory of Biology and Germplasm Enhancement of Horticultural Crops in East China,Nanjing Agricultural University,Nanjing 210095,China

Received:2021-05-13 Revised:2021-07-31 Online:2021-09-25 Published:2021-09-30
Contact: WANG Guanglong,XIONG Aisheng E-mail:guanglongwang@hyit.edu.cn;xiongaisheng@njau.edu.cn

摘要/Abstract

摘要：

为了解大蒜生物钟相关基因REVEILLEs（RVEs）的序列特征及其在渗透胁迫下的功能,从大蒜中克隆得到AsRVE1和AsRVE2基因,并对其在盐胁迫和模拟干旱胁迫下的表达特征进行了分析。序列分析表明,AsRVE1和AsRVE2分别含有1 050和975 bp的开放阅读框,编码349和324个氨基酸。在进化关系上,大蒜AsRVE和AsRVE2与玉米ZmRVE2和凤梨AcRVE2的较近。不同植物RVE氨基酸序列同源性较低,但N端保守结构域序列一致性较高。AsRVE1和AsRVE2均能响应昼夜节律的变化,在大蒜不同组织中均能表达,在不同组织间AsRVE1的表达差异不大,AsRVE2在根中表达相对较高。干旱胁迫和盐胁迫在不同组织内均诱导了AsRVE1和AsRVE2的表达。结果表明,AsRVE1和AsRVE2可能参与了大蒜植株抵御盐胁迫和干旱胁迫的过程,可进一步鉴定其生物学功能。

关键词: 大蒜, 生物钟, RVE转录因子, 渗透胁迫, 基因克隆, 表达分析

Abstract:

In order to understand the sequence characteristics of circadian clock-related genes REVEILLEs（RVEs）in garlic and their roles in osmotic stress response,AsRVE1 and AsRVE2 genes were isolated from garlic,and their expression patterns under salt stress and simulated drought stress were analyzed by qRT-PCR. Sequence analysis showed that the open reading frames of AsRVE1 and AsRVE2 genes were 1 050 and 975 bp in length,encoding 349 and 324 amino acids,respectively. In terms of evolutionary relationship,AsRVE1 and AsRVE2 were relatively close to ZmRVE2 in corn and AcRVE2 in pineapple. The RVE amino acid sequences of different plants had low homology,but showed high consistency in the conserved domain at the N-terminus. Both AsRVE1 and AsRVE2 genes can respond to changes in circadian rhythms,and their expression can be detected in different tissues;the expression of AsRVE1 in different tissues was not obviously different,the expression of AsRVE2 in the roots was relatively high. Both drought stress and salt stress induced the altered expression of AsRVE1 and AsRVE2 genes in different tissues. These results indicated that AsRVE1 and AsRVE2 genes may be involved in drought- and salt-stress response in garlic. The AsRVE1 and AsRVE2 genes cloned in this study laid a foundation for further identification of the biological functions and regulatory mechanisms of RVE.

Key words: garlic, circadian rhythm, RVE transcription factor, osmotic stress, gene cloning, expression analysis

中图分类号:

S633.4

卞诗村, 陆雅妮, 许吴俊, 陈伯清, 王广龙, 熊爱生. 大蒜生物钟基因AsRVE1和AsRVE2及其在渗透胁迫下的表达分析[J]. 园艺学报, 2021, 48(9): 1706-1716.

BIAN Shicun, LU Yani, XU Wujun, CHEN Boqing, WANG Guanglong, XIONG Aisheng. Garlic Circadian Clock Genes AsRVE1 and AsRVE2 and Their Expression Analysis Under Osmotic Stress[J]. Acta Horticulturae Sinica, 2021, 48(9): 1706-1716.

图/表 10

表1 用于基因克隆和荧光定量PCR的引物

Table 1 Primer sequences used for gene cloning and quantitative real time fluorescence PCR

用途 Use	基因名称 Gene name	引物（上游/下游） Primer（Forward/Reverse）
克隆 Gene cloning	AsRVE1	ATGACAAGTGATCCTTTATACTTGG/TCATCTCTTGTATGGAAGAAATCCT
克隆 Gene cloning	AsRVE2	ATGCAGATCAGTAGTACATCACCTG/TCACCTCCTGTACGGAACAAATCCC
荧光定量PCR Quantitative real time fluorescence PCR	AsRVE1	TCAGTGGATGTGGAGAACAATACCT/TGATGAGTAAGGAGGCAACAGATGA
	AsRVE2	ACAGCGTGAGAGGTGGACTGAT/GAGATGCAGACGGCGAACCAA
	AsSAND	GCGTCAACGAATGTTCCAATTACCA/TCTCTTCAGTCTCAACTTCATCAGCAT

图1 大蒜AsRVE1和AsRVE2的RT-PCR扩增

Fig. 1 AsRVE1 and AsRVE2 genes amplified from garlic by RT-PCR

图2 大蒜AsRVE1（左）和AsRVE2（右）的核苷酸序列及其编码的氨基酸序列

Fig. 2 Nucleotide sequences and amino acid sequences of garlic AsRVE1（left）and AsRVE2（right）genes

图3 大蒜与其他植物RVE氨基酸序列的系统进化树

Fig. 3 Phylogenetic tree of RVE amino acid sequences from garlic and other plants

图4 大蒜与其他物种RVE氨基酸序列比对分析 As：大蒜;At：拟南芥;Ls：莴苣;Zm：玉米;Rs：萝卜;Vr：绿豆;Ac：凤梨。* 表示DNA结合位点。

Fig. 4 Alignment of RVE amino acid sequences from garlic and other plant As：Allium sativum;At：Arabidopsis thaliana;Ls：Lactuca sativa;Zm：Zea mays;Rs：Raphanus sativus;Vr：Vigna radiata;Ac：Ananas comosus. * indicates DNA binding sites.

图5 大蒜AsRVE1和AsRVE2二级结构预测蓝色代表α螺旋,红色代表延伸链,绿色代表β转角,紫色代表无规则卷曲。

Fig. 5 Prediction of secondary structure of AsRVE1 and AsRVE2 of garlic Blue,red,green,and purple respectively represent alpha helix,extended strand,beta turn,and random coil.

图6 大蒜AsRVE1和AsRVE2的蛋白相互作用网络

Fig. 6 Protein-protein interaction network of garlic AsRVE1 and AsRVE2

图7 大蒜AsRVE1和AsRVE2对光周期的响应不同小写字母表示在0.05水平上差异显著。

Fig. 7 Response of garlic AsRVE1 and AsRVE2 to light period Different lowercase letters represent significant differences at 0.05 level.

图8 大蒜AsRVE1和AsRVE2在干旱胁迫下的表达相同组织内不同字母表示在0.05水平上差异显著。

Fig. 8 Expression level of garlic AsRVE1 and AsRVE2 under drought stress Different lowercase letters in the same tissue represent significant differences at 0.05 level.

图9 大蒜AsRVE1和AsRVE2在盐胁迫下的表达相同组织内不同字母表示在0.05水平上差异显著。

Fig. 9 Expression level of garlic AsRVE1 and AsRVE2 under salt stress Different lowercase letters in the same tissue represent significant differences at 0.05 level.

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大蒜生物钟基因AsRVE1和AsRVE2及其在渗透胁迫下的表达分析

Garlic Circadian Clock Genes AsRVE1 and AsRVE2 and Their Expression Analysis Under Osmotic Stress

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大蒜生物钟基因AsRVE1和AsRVE2及其在渗透胁迫下的表达分析

Garlic Circadian Clock Genes AsRVE1 and AsRVE2 and Their Expression Analysis Under Osmotic Stress

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