Cloning and identification of drought tolerance function of the LjbHLH34 gene in Lotus japonicus

doi:10.11686/cyxb2022008

Abstract

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

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.

Figures/Tables 10

References 40

1	Szczyglowski K， Stougaard J. Lotus genome： Pod of gold for legume research. Trends in Plant Science， 2008， 13（10）： 515-517.
2	Choi H K， Mun J H， Kim D J， et al. Estimating genome conservation between crop and model legume species. Proceedings of the National Academy of Sciences， 2004， 101（43）： 15289-15294.
3	Calzadilla P I， Signorelli S， Escaray F J， et al. Photosynthetic responses mediate the adaptation of two Lotus japonicus ecotypes to low temperature. Plant Science， 2016， 250： 59-68.
4	Signorelli S， Corpas F J， Borsani O， et al. Water stress induces a differential and spatially distributed nitro-oxidative stress response in roots and leaves of Lotus japonicus. Plant Science， 2013， 201/202： 137-146.
5	Díaz P， Monza J， Márquez A. Drought and saline stress. Springer Netherlands， 2005， DOI： 10.1007/1-4020-3735-X_3.
6	Gong Z Z， Xiong L M， Shi H Z， et al. Plant abiotic stress response and nutrient use efficiency.Science China （Life Sciences）， 2020， 63（5）： 635-674.
7	Xie Y P， Chen P X， Yan Y， et al. An atypical R2R3 MYB transcription factor increases cold hardiness by CBF-dependent and CBF-independent pathways in apple. New Phytologist， 2018， 218（1）： 201-218.
8	Samira R， Li B H， Kliebenstein D， et al. The bHLH transcription factor ILR3 modulates multiple stress responses in Arabidopsis. Plant Molecular Biology， 2018， 97（4/5）： 297-309.
9	Govind G， Harshavardhan T G， Patricia J K， et al. Identification and functional validation of a unique set of drought induced genes preferentially expressed in response to gradual water stress in peanut. Molecular Genetics and Genomics， 2009， 281（6）： 607.
10	Jin J P， Zhang H， Kong L， et al. PlantTFDB 3.0： A portal for the functional and evolutionary study of plant transcription factors. Nucleic Acids Research， 2014， 42： D1182-D1187.
11	Heim M A， Jakoby M， Werber M， et al. The basic helix-loop-helix transcription factor family in plants： A genome-wide study of protein structure and functional diversity. Molecular Biology and Evolution， 2003， 20（5）： 735-747.
12	Feller A， Machemer K， Braun E L， et al. Evolutionary and comparative analysis of MYB and bHLH plant transcription factors. The Plant Journal： For Cell and Molecular Biology， 2011， 66（1）： 94-116.
13	Zhu L L， Zhou B. Regulation of bHLH protein in plant development and abiotic stress. Molecular Plant Breeding， 2021， http：//kns.cnki.net/kcms/detail/46.1068.S.20210222.1744.012.html.
	朱璐璐，周波. bHLH蛋白在植物发育及非生物胁迫中的调控. 分子植物育种， 2021， http：//kns.cnki.net/kcms/detail/46.1068.S.20210222.1744.012.html.
14	Ren Y R， Yang Y Y， Zhao Q， et al. MdCIB1， an apple bHLH transcription factor， plays a positive regulator in response to drought stress. Environmental and Experimental Botany， 2021， 188： 1-11.
15	Pillitteri L J， Torii K U. Breaking the silence： Three bHLH proteins direct cell-fate decisions during stomatal development. Bioessays， 2010， 29（9）： 861-870.
16	Zhao M， Morohashi K， Hatlestad G， et al. The TTG1-bHLH-MYB complex controls trichome cell fate and patterning through direct targeting of regulatory loci. Development， 2008， 135（11）： 1991-1999.
17	Abe H. Arabidopsis AtMYC2 （bHLH） and AtMYB2 （MYB） function as transcriptional activators in abscisic acid signaling. Plant Cell， 2003， 15（1）： 63-78.
18	Liu W W， Tai H H， Li S S， et al. bHLH122 is important for drought and osmotic stress resistance in Arabidopsis and in the repression of ABA catabolism. New Phytologist， 2014， 201（4）： 1192-1204.
19	Li H G， Sun J Q， Xu Y X， et al. The bHLH-type transcription factor AtAIB positively regulates ABA response in Arabidopsis. Plant Molecular Biology， 2007， 65（5）： 655-665.
20	Min J H， Ju H W， Yoon D， et al. Arabidopsis basic helix-loop-helix 34 （bHLH34） is involved in glucose signaling through binding to a GAGA Cis-element. Frontiers in Plant Science， 2017， 8： 1-14.
21	Seo J S， Joo J， Kim M J， et al. OsbHLH148， a basic helix-loop-helix protein， interacts with OsJAZ proteins in a jasmonate signaling pathway leading to drought tolerance in rice. The Plant Journal， 2011， 65（6）： 907-921.
22	Chen C， Liu F， Zhang K X， et al. MeJA-responsive bHLH transcription factor LjbHLH7 regulates cyanogenic glucoside biosynthesis in Lotus japonicus. Journal of Experimental Botany， 2022， 73（8）： 2650-2665.
23	Hou Y Y， Li X， Long R C， et al. Effect of overexpression of the alfalfa MsHB7 gene on drought tolerance of Arabidopsis. Acta Prataculturae Sinica， 2021， 30（4）： 170-179.
	候怡谣，李霄，龙瑞才，等. 过量表达紫花苜蓿MsHB7基因对拟南芥耐旱性的影响. 草业学报， 2021， 30（4）： 170-179.
24	Murre C， McCaw P S， Baltimore D. A new DNA binding and dimerization motif in immunoglobulin enhancer binding， daughterless， MyoD， and myc proteins. Cell， 1989， 56（5）： 777-783.
25	Atchley W R， Fitch W M. A natural classification of the basic helix-loop-helix class of transcription factors. The Proceedings of the National Academy of Sciences， 1997， 94（10）： 5172-5176.
26	Toledo-Ortiz G， Huq E， Quail P H. The Arabidopsis basic/helix-loop-helix transcription factor family. The Plant Cell， 2003， 15（8）： 1749-1770.
27	Hao Y Q， Zong X M， Ren P， et al. Basic helix-loop-helix （bHLH） transcription factors regulate a wide range of functions in Arabidopsis. International Journal of Molecular Sciences， 2021， 22（13）： 1-20.
28	Min J H， Park C R， Jang Y H， et al. A basic helix-loop-helix 104 （bHLH104） protein functions as a transcriptional repressor for glucose and abscisic acid signaling in Arabidopsis. Plant Physiology and Biochemistry， 2019， 136： 34-42.
29	Liu Y J， Ji X Y， Nie X G， et al. AtbHLH112 regulates the expression of genes involved in abiotic stress tolerance by binding to their E-box and GCG-box motifs. New Phytologist， 2015， 207（3）： 692-709.
30	Jiang Y， Yang B， Deyholos M K. Functional characterization of the Arabidopsis bHLH92 transcription factor in abiotic stress. Molecular Genetics and Genomics， 2009， 282（5）： 503-516.
31	Zhao Q， Fan Z H， Qiu L， et al. MdbHLH130， an apple bHLH transcription factor， confers water stress resistance by regulating stomatal closure and ROS homeostasis in transgenic tobacco. Frontiers in Plant Science， 2020， 11： 1-16.
32	Li Z， Liu C， Zhang Y， et al. The bHLH family member ZmPTF1 regulates drought tolerance in maize by promoting root development and abscisic acid synthesis. Journal of Experimental Botany， 2019， 70（19）： 5471-5486.
33	Zhang H， Wang P H. Determination of relative water， content in plant leaves in vivo. Plant Physiology Communications， 1991， 27（3）： 217-219.
34	Lanceras J C. Quantitative trait loci associated with drought tolerance at reproductive stage in rice. Plant Physiology， 2004， 135（1）： 384-399.
35	Sudhakar C， Lakshmi A， Giridarakumar S. Changes in the antioxidant enzyme efficacy in two high yielding genotypes of mulberry （Morus alba L.） under NaCl salinity. Plant Science， 2001， 161（3）： 613-619.
36	Pan L. Multi-Omics analysis reveal molecular mechanisms of drought resistance in annual ryegrass （Lolium multiflorum L.）. Chengdu： Sichuan Agricultural University， 2018.
	潘玲. 多组学联合分析揭示多花黑麦草抗旱响应机制研究. 成都：四川农业大学， 2018.
37	Frugoli J. Catalase is encoded by a multigene family in Arabidopsis thaliana （L.） Heynh. Plant Physiology， 1996， 112（1）： 327-336.
38	Velinov V， Vaseva I， Zehirov G， et al. Overexpression of the NMig1 gene encoding a NudC domain protein enhances root growth and abiotic stress tolerance in Arabidopsis thaliana. Frontiers in Plant Science， 2020， DOI： 10.3389/fpls.2020.00815.
39	Zou J J， Li X D， Ratnasekera D， et al. Arabidopsis calcium-dependent protein kinase8 and catalase3 function in abscisic acid-mediated signaling and H₂O₂ homeostasis in stomatal guard cells under drought stress. The Plant Cell， 2015， 27（5）： 1445-1460.
40	Wang R S， Pandey S， Li S， et al. Common and unique elements of the ABA-regulated transcriptome of Arabidopsis guard cells. BMC Genomics， 2011， 12（1）： 1-24.

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

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

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