日本百脉根LjbHLH34基因克隆及耐旱功能鉴定

doi:10.11686/cyxb2022008

摘要/Abstract

摘要：

干旱是影响植物生长发育的重要环境因素。本研究分析了日本百脉根抗旱相关基因LjbHLH34的耐旱功能，初步解析其响应干旱胁迫的分子机制，以期为百脉根抗旱分子育种提供理论基础。本研究克隆得到的LjbHLH34基因大小为711 bp、编码236个氨基酸，属bHLH转录因子家族成员。系统进化树分析显示，LjbHLH34蛋白与拟南芥bHLH Ⅳ亚家族中AtbHLH34和AtbHLH104亲缘关系较近。实时荧光定量分析表明LjbHLH34在日本百脉根的根中表达量最高，叶中次之，茎中最少，暗示其在日本百脉根多个组织中发挥作用；同时LjbHLH34基因也受聚乙二醇（PEG）和脱落酸（ABA）诱导表达。在酵母中检测发现LjbHLH34具有转录激活活性；亚细胞定位试验表明LjbHLH34蛋白定位于细胞核中。将LjbHLH34基因转入拟南芥获得过表达株系。在200 mmol·L^-1甘露醇胁迫下，LjbHLH34转基因拟南芥的根长明显长于野生型。干旱处理后，野生型拟南芥比转基因拟南芥萎蔫程度更加明显，而转基因株系的相对含水量和超氧化物歧化酶（SOD）活性显著高于野生型，丙二醛（MDA）积累显著低于野生型。qRT-PCR检测发现在干旱处理之后，与抗逆相关的基因AtCAT1、AtCAT3和AtRD22在转基因株系中的表达量显著升高。上述结果表明LjbHLH34正调控植物的抗旱性。

关键词: bHLH转录因子, 干旱胁迫, 基因克隆, 日本百脉根

Abstract:

Drought is an important environmental factor affecting plant growth and development. In this paper， we studied the drought tolerance function of the gene LjbHLH34， and conducted a preliminary analysis of its molecular mechanism in response to drought stress， in order to provide a theoretical basis for molecular breeding of Lotus japonicus drought resistance. In this study， the size of LjbHLH34 was found to be 711 bp and to encode 236 amino acids， belonging to the bHLH transcription factor family. Phylogenetic tree analysis showed that LjbHLH34 was closely related to AtbHLH34 and AtbHLH104 in the bHLH Ⅳ subfamily of Arabidopsis thaliana. The expression level of LjbHLH34 was the highest in the roots， the second highest in the leaves， and the lowest in the stems， suggesting that LjbHLH34 plays a role in multiple tissues of L. japonicus.LjbHLH34 gene expression was also induced by polyethylene glycol （PEG） and abscisic acid （ABA）. A yeast assay showed that LjbHLH34 had transcriptional activation activity， and a subcellular localization assay showed that LjbHLH34 was located in the nucleus. Under 200 mmol·L^-1 D-Mannitol stress， the root length of LjbHLH34 transgenic A. thaliana was significantly longer than that of the wild type. After drought treatment， the degree of withering of the wild-type was more obvious than transgenic Arabidopsis. At the same time， the relative water content and superoxide dismutase （SOD） activity of transgenic Arabidopsis were significantly higher than those of the wild-type， while malondialdehyde （MDA） accumulation was significantly lower than in the wild-type. qRT-PCR showed that the expression levels of stress-related genes AtCAT1， AtCAT3 and AtRD22 in transgenic lines were significantly increased after drought treatment. These results indicated that LjbHLH34 positively regulates drought resistance of plants.

Key words: bHLH transcription factors, drought stress, gene cloning, Lotus japonicus

刘福, 陈诚, 张凯旋, 周美亮, 张新全. 日本百脉根LjbHLH34基因克隆及耐旱功能鉴定[J]. 草业学报, 2023, 32(1): 178-191.

Fu LIU, Cheng CHEN, Kai-xuan ZHANG, Mei-liang ZHOU, Xin-quan ZHANG. Cloning and identification of drought tolerance function of the LjbHLH34 gene in Lotus japonicus[J]. Acta Prataculturae Sinica, 2023, 32(1): 178-191.

图/表 10

表1 引物序列

Table 1 Primer list

引物名称Primer name	引物序列Primer sequence （5′-3′）
T-LjbHLH34-F	ATGGTTTCCGCGGAAAACACC
T-LjbHLH34-R	TTAGGCAGCTGGTGGTCGGAG
M13-F	TGTAAAACGACGGCCAGT
pCAMBIA1307-LjbHLH34-F	ACTTGAACTCGGTATCTATGGTTTCCGCGGAAAAC
pCAMBIA1307-LjbHLH34-R	AGCTTGATATCGAATTCCTGGGCAGCTGGTGGTCGGAGTTG
pCAMBIA1307-F	AGGAAGTTCATTTCATTTGGA
pAN580-LjbHLH34-F	CCCAGATCAACTAGTATGGTTTCCGCGGAAAAC
pAN580-LjbHLH34-R	CTTGCTCACCATGGAGGCAGCTGGTGGTCGGAGTTG
PAN580-F	ATGACGCACAATCCCACTATCC
pGBKT7-LjbHLH34-F	TCAGAGGAGGACCTGCATATGATGGTTTCCGCGGAAAACA
pGBKT7-LjbHLH34-R	CTAGTTATGCGGCCGCTGCAGGCAGCTGGTGGTCGGAG
T7-F	TAATACGACTCACTATAGG
qLj-actin3-F	GTATTGTTGGCCGACCTCGT
qLj-actin3-R	AGCCTCAGTTAGAAGCACCG
qLj-bHLH34-F	ATGCAGTTCGAGTGGTGACG
qLj-bHLH34-R	AGACGGCAAAAATTGCCACA
qAt-actin7-F	TCCATGAAACAACTTACAACTCCATCA
qAt-actin7-R	CATCGTACTCACTCTTTGAAATCCACA
qAtCAT1-F	GAGATCCCCGTGGTTTTGCT
qAtCAT1-R	TGTGCAAACTCTCTGGGTGG
qAtCAT3-F	AGCTTCCAGTCAATGCTCCC
qAtCAT3-R	GTGAGACGTGGCTCCGATAG
qAtRD22-F	TTCGTCTTCCTCTGATCTGTCTTC
qAtRD22-R	TTTACTCCGCCTTTACCTACTTGG

图1 LjbHLH34蛋白序列分析

Fig.1 Sequence analysis of LjbHLH34

图2 LjbHLH34的亚细胞定位

Fig.2 Subcellular localization of LjbHLH34

图3 LjbHLH34在日本百脉根中的表达模式*： P<0.05； **： P<0.01. 下同The same below.

Fig.3 Expression pattern of LjbHLH34 in L. japonicus

图4 LjbHLH34转录因子转录激活活性

Fig.4 Transcriptional activation activity of LjbHLH34 transcription factor

图5 转LjbHLH34基因拟南芥的鉴定M： DNA分子量标准DNA marker； -：阴性对照Negative control； +：阳性对照Positive control； ***表示转基因拟南芥与WT相比差异极显著（P<0.001），WT、OE-1、OE-2和OE-3分别为野生型、过表达株系1、过表达株系2和过表达株系3，下同。*** indicates that the difference among transgenic A. thaliana and WT is extremely significant （P<0.001）， WT， OE-1， OE-2 and OE-3 were wild type， overexpressed line 1， overexpressed line 2 and overexpressed line 3， the same below.

Fig.5 Identification of transgenic A. thaliana with LjbHLH34

图6 甘露醇胁迫下拟南芥的根长**表示转基因拟南芥与WT相比差异显著（P<0.01）。** indicates that the difference among transgenic A. thaliana and WT is significant （P<0.01）. 下同The same below.

Fig.6 Root length of A. thaliana under D-Mannitol stress

图7 干旱胁迫后拟南芥表型

Fig.7 Phenotypic of A. thaliana under drought stress

图8 干旱胁迫下转基因拟南芥的生理指标*表示转基因拟南芥与WT相比差异显著（P<0.05）。* indicates that the difference among transgenic A. thaliana and WT is significant （P<0.05）. 下同The same below.

Fig.8 Physiological indexes of transgenic A. thaliana under drought stress

图9 干旱处理下抗性相关基因的表达

Fig.9 Expression of resistance related genes under drought treatment

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