Gene cloning and drought resistance identification of the gene HgS5 in Halogeton glomeratus

doi:10.11686/cyxb2024106

Abstract

Abstract:

To address environmental challenges involving escalating frequency and severity of drought， it is of utmost importance to develop a deep understanding of drought-resistance genes in plant genomes. Based on our initial laboratory analysis of transcriptome sequencing data from Halogeton glomeratus， the HgS5 gene exhibited the highest expression level and differential fold change following salt stress. This study focused on the HgS5 gene， conducting bioinformatics analysis and subcellular localization of the protein encoded by this target gene. We employed qRT-PCR to assess the relative expression of the target gene in the leaves and roots of Arabidopsis thaliana， and achieved heterologous expression in A. thaliana using Agrobacterium as a vector. In this experiment， we cloned the HgS5 gene from H. glomeratus and validated its drought resistance in A. thaliana. It was found that the HgS5 gene comprises 1738 base pairs， encoding 370 amino acids. The encoded protein is acidic and hydrophilic， lacking a transmembrane region. Featuring 116 promoter cis-acting elements， the HgS5 gene shares a A_thal_3526 conserved domain with homologous genes related to Carnegiea gigantea， Amaranthus tricolor， and Beta vulgaris. Subcellular localization indicated that the HgS5 gene is primarily expressed on the cell membrane. Fluorescence quantitative analysis showed that the HgS5 gene is predominantly expressed in the roots of A. thaliana， with a significantly increased expression level compared to other groups at 6 days （P<0.05）. The drought resistance assessment revealed a notable enhancement in drought tolerance in A. thaliana overexpressing the HgS5 gene， evident in a slower wilting rate of the plants. The HgS5 gene conferred resistance to dry conditions by influencing enzyme activity， initially increasing and subsequently decreasing the activities of superoxide dismutase， peroxidase， and catalase in the roots of A. thaliana. To summarize， the gene HgS5 plays a pivotal role in the process of drought resistance. The aim of this study was to provide a theoretical basis for further exploration of the molecular response mechanism of the HgS5 gene to drought stress.

Key words: Halogeton glomeratus, gene families, bioinformatics analysis, gene cloning, drought resistance identification

Xin-yao WANG, Ya-ping PENG, Li-rong YAO, Jun-cheng WANG, Er-jing SI, Hong ZHANG, Ke YANG, Xiao-le MA, Ya-xiong MENG, Hua-jun WANG, Bao-chun LI. Gene cloning and drought resistance identification of the gene HgS5 in Halogeton glomeratus[J]. Acta Prataculturae Sinica, 2025, 34(2): 184-195.

Figures/Tables 11

References 35

1	Tian L X， Yang Y， Song Y H， et al. Rehydration under extreme drought conditions affected rhizosphere microorganisms more than bulk soil in broomcorn millet farmland. Agricultural Water Management， 2024， 295（1）： 108781-108794.
2	Corwin D L. Climate change impacts on soil salinity in agricultural areas. European Journal of Soil Science， 2020， 72（2）： 842-862.
3	Sun M， Peng F， Xiao Y， et al. Exogenous phosphatidylcholine treatment alleviates drought stress and maintains the integrity of root cell membranes in peach. Scientia Horticulturae， 2020， 259（6）： 108821-108829.
4	Zhang H O. An analysis of the distribution and evolutionary characteristics of saline soils in China. Agriculture and Technology， 2022， 42（5）： 104-107.
5	Yang S S， Gao J F. Influence of active oxygen and free radicals on plant senescence. Acta Botanica Boreali-Occidentalia Sinica， 2001， 21（2）： 36-41.
	杨淑慎，高俊凤. 活性氧、自由基与植物的衰老. 西北植物学报， 2001， 21（2）： 36-41.
6	Zhang J， Mao C L， khan A， et al. Enhanced methane production by using phytoremediated Halogeton glomeratus as substrate via anaerobic digestion. Renewable Energy， 2022， 194（1）： 28-39.
7	Wang J C， Yang K， Yao L R， et al. Metabolomics analyses provide insights into nutritional value and abiotic stress tolerance in halophyte Halogeton glomeratus. Frontiers in Plant Science， 2021（12）： 703255-703264.
8	Eggers E J， Burgt A， Heusden S， et al. Neofunctionalisation of the Sli gene leads to self-compatibility and facilitates precision breeding in potato. Nature Communications， 2021（12）： 4141-4151.
9	Cheng Q， Gan Z， Wang Y， et al. The soybean gene J contributes to salt stress tolerance by up-regulating salt-responsive genes. Frontiers in Plant Science， 2020（11）： 272-281.
10	Gálvez R L. The application of metabolomics for the study of cereal corn （Zea mays L.）. Metabolites， 2020（10）： 300-308.
11	Ren X M， Hu Z R， Jiang X Z， et al. Analysis of physiological characteristics and related gene expression in response to low-temperature stress in different tobacco varieties. Molecular Plant Breeding， 2024（1）： 10-15.
	任晓敏，户正荣，姜习振，等. 低温胁迫对不同烟草品种的生理特性及相关基因表达分析. 分子植物育种， 2024（1）： 10-15.
12	Wang Q， Tang J， Han B， et al. Advances in genome-wide association studies of complex traits in rice. Theoretical and Applied Genetics， 2020， 133（1）： 1415-1425.
13	Zafar M M， Rehman A， Razzaq A， et al. Genome-wide characterization and expression analysis of Erf gene family in cotton. BMC Plant Biology， 2022， 134（1）： 22-43.
14	Patel J， Mishra A. Plant aquaporins alleviate drought tolerance in plants by modulating cellular biochemistry， root-architecture， and photosynthesis. Physiologia Plantarum， 2021， 172（2）： 1030-1044.
15	Xu X L. Cloning of tonoplast and plasma membrane Na⁺/H⁺ antiporter gene and isolating of 5′ flanking sequence of HgNHX1 from Halogeton glomeratus. Lanzhou： Gansu Agricultural University， 2014.
	徐先良. 盐生草Na⁺/H⁺逆向转运蛋白基因的克隆及HgNHX1基因5′端侧翼序列的分离. 兰州：甘肃农业大学， 2014.
16	Zhang Y， Li B C， Hu Y L， et al. Functional verification of HgNHX1 gene derived from Halogeton glomeratus in barley. Journal of Triticeae Crops， 2018， 38（8）： 929-934.
	张燕，李葆春，胡有良，等. 盐生草HgNHX1基因在大麦株系中的功能验证. 麦类作物学报， 2018， 38（8）： 929-934.
17	Zou L， Yang K， Xu X L， et al. Cloning and functional analysis of halophyte Halogeton glomeratus HgNHX1 promoter. Acta Prataculturae Sinica， 2017， 26（11）： 57-68.
	邹兰，杨轲，徐先良，等. 盐生草HgNHX1基因启动子的克隆及功能验证. 草业学报， 2017， 26（11）： 57-68.
18	Ma Y H， Xu X L， Wang J C， et al. Cloning and expression analysis of Actin gene fragment from halophyte Halogeton glomeratus. Pratacultural Science， 2015， 32（9）： 1432-1437.
	马艳红，徐先良，汪军成，等. 盐生草Actin基因片段的克隆及表达. 草业科学， 2015， 32（9）： 1432-1437.
19	Hu S Q， Wang J C， Yao L R， et al. Cloning and preliminary functional analysis of the root gene HgAKR6C of Halogeton glomeratus. Acta Prataculturae Sinica， 2024， 33（1）： 61-74.
	胡尚钦，汪军成，姚立蓉，等. 盐生草根系基因HgAKR6C的克隆与初步功能分析. 草业学报， 2024， 33（1）： 61-74.
20	Wang J C， Li B C， Meng Y X， et al. Transcriptomic profiling of the salt-stress response in the halophyte Halogeton glomeratus. BMC Genomics， 2015， 16（1）：169.
21	Wang J C， Wang H J， Yao L R， et al. Salt-tolerant gene HgS3 of Halogeton glomeratus and its application： CN107287212B. 2020-10-30.
	汪军成，王化俊，姚立蓉，等. 盐生草耐盐基因HgS3及其应用： CN107287212B. 2020-10-30.
22	Zhou M. Effects of exogenous calcium on physiological characteristics of Rhododendron ovatum Planch seed germination under drought stress. Beijing Agriculture， 2014（21）： 11-12.
	周敏. 干旱胁迫下外源钙对马缨杜鹃种子萌发生理特性的影响. 北京农业， 2014（21）： 11-12.
23	Ma J W， Ma Z K， Yao L R， et al. Regulating effect of exogenous melatonin on root growth of barley seedling under phosphorus stress. Journal of Triticeae Crops， 2023， 43（8）： 1020-1028.
	马静玮，马增科，姚立蓉，等. 低磷胁迫下外源褪黑素对大麦幼苗根系发育的调控作用. 麦类作物学报， 2023， 43（8）： 1020-1028.
24	Yao H. Cloning of DFR， FLS promoters and RNAi vector construction of LYCE and LYCB in Narcissus tazetta var. chinensis. Fuzhou： Fujian Agriculture and Forestry University， 2024.
	姚红. 中国水仙DFR和FLS启动子克隆及LYCE和LYCB RNAi表达载体的构建. 福州：福建农林大学， 2024.
25	Li G， Bai Y， Jia Z Y， et al. Phosphorus altered the response of ionomics and metabolomics to drought stress in wheat seedlings. Scientia Agricultural Sinica， 2022， 55（2）： 280-294.
	李刚，白阳，贾子颖，等.两种磷素水平下小麦苗期对干旱胁迫的离子组和代谢组响应. 中国农业科学， 2022， 55（2）： 280-294.
26	Zeng F L， Li Y F. Generation of active oxygen free radicals and its injury to microsome membranes in wheat leaves under drought stress. Chinese Bulletin of Botany， 1997， 39（12）： 1105-1109.
	曾福礼，李玉峰.干旱胁迫下小麦叶片微粒体活性氧自由基的产生及其对膜的伤害. 植物学报， 1997， 39（12）： 1105-1109.
27	Li D， Peng S， Chen S， et al. Identification and characterization of 5 walnut MYB genes in response to drought stress involved in ABA signaling. Physiology and Molecular Biology of Plants， 2021， 27（6）： 1323-1335.
28	Yang Y Y， Ren Y P， Su Y M， et al. Cloning and analysis of two promoters of stress-related genes in Medicago varia Xinmu-1. Pratacultural Science， 2012， 29（12）： 1887-1893.
	杨云尧，任燕萍，苏豫梅，等. 新牧1号苜蓿两个抗逆相关基因启动子的克隆及分析. 草业科学， 2012， 29（12）： 1887-1893.
29	Wang K， Nan L L， Guo Q E， et al. Effects of drought stress on root architecture of different root-type alfalfa. Acta Ecologica Sinica， 2022， 42（20）： 8365-8373.
	汪堃，南丽丽，郭全恩，等. 干旱胁迫对不同根型苜蓿根系构型的影响. 生态学报， 2022， 42（20）： 8365-8373.
30	Chen Q， Xu X Y， Wang J C， et al. Identification of a WRKY gene family based on full-length transcriptome sequences and analysis of response patterns under salt stress in Halogeton glomeratus. Acta Prataculturae Sinica， 2022， 31（12）： 146-157.
	陈倩，徐晓芸，汪军成，等. 基于全长转录组的盐生草WRKY基因家族的鉴定及其盐胁迫响应模式分析. 草业学报， 2022， 31（12）： 146-157.
31	Wu Y H， Liu W H， Liu K Q， et al. Effects of drought stress on leaf senescence and the active oxygen scavenging system of oat seedlings. Acta Prataculturae Sinica， 2022， 31（10）： 75-86.
	吴雨涵，刘文辉，刘凯强，等. 干旱胁迫对燕麦幼苗叶片光合特性及活性氧清除系统的影响. 草业学报， 2022， 31 （10）： 75-86.
32	Bogati K， Walczak M. The impact of drought stress on soil microbial community， enzyme activities and plants. Agronomy， 2022（12）： 189-199.
33	Wang W B， Kim Y H， Lee H S， et al. Analysis of antioxidant enzyme activity during germination of alfalfa under salt and drought stresses. Plant Physiology and Biochemistry， 2009， 47（7）： 570-577.
34	Han G， Dang Q， Zhao Z， et al. Responses of antioxidation protective system of Caragana korshinskii Kom. to drought stress. Acta Agrestia Sinica， 2010， 18（4）： 528-532.
	韩刚，党青，赵忠，等. 柠条抗氧化保护系统对干旱胁迫的响应. 草地学报， 2010， 18（4）： 528-532.
35	Jia H T， Hu X J， Qiu F T， et al. The effects of compound anti-drought seed soaking agent and seed coating agent on SOD， POD and CAT isozyme expression in wheat seedlings. Journal of Triticeae Crops， 2016， 36（5）： 647-652.
	贾洪涛，胡晓君，邱奉同，等. 小麦专用复方抗旱型浸种剂和包衣剂对小麦幼苗SOD、POD和CAT同工酶表达的影响. 麦类作物学报， 2016， 36（5）： 647-652.

引物名称Name of primers	引物序列Sequence of primers （5′-3′）	用途Function
HgS5-F1	CGTTGTTTCCAGCCCACTTC	克隆基因 Gene cloning
HgS5-R1	AATGTTAAACACTTTACAATAC	克隆基因 Gene cloning
HgS5-F1 HgS5-R1	GGGGTACCGACGTTGTTTCCAGCCCACTTC CGGGATCCAGAATGTTAAACACTTTACAATAC	表达载体 Expression vector
35s-F HgS5-R	GACGCACAATCCCACTATCC CGGGATCCAGAATGTTAAACACTTTACAATAC	鉴定引物 Identification primer
HgS5-F1	GCTCTAGAGACGTTGTTTCCAGCCCACT	亚细胞定位 Subcellular localization
HgS5-R1	GGGGTACCAGAATGTTAAACACTTTACAATAC	亚细胞定位 Subcellular localization
HgS5-F1	ACCCTGTCCTACAACCACCT	荧光定量PCR Real-time PCR
HgS5-R1	TGGGAGCAGGGACTCCATTA
Actin-F1	GGTGATGGTGTGTCTCACACTG
Actin-R1	GAGGTTTCCATCTCCTGCTCGTAG
Actin-F2	ACACAAGGTCTATGTCGGAAAT
Actin-R2	TAACACTCTCGTGCTTACGATT

引物名称Name of primers	引物序列Sequence of primers （5′-3′）	用途Function
HgS5-F1	CGTTGTTTCCAGCCCACTTC	克隆基因 Gene cloning
HgS5-R1	AATGTTAAACACTTTACAATAC	克隆基因 Gene cloning
HgS5-F1 HgS5-R1	GGGGTACCGACGTTGTTTCCAGCCCACTTC CGGGATCCAGAATGTTAAACACTTTACAATAC	表达载体 Expression vector
35s-F HgS5-R	GACGCACAATCCCACTATCC CGGGATCCAGAATGTTAAACACTTTACAATAC	鉴定引物 Identification primer
HgS5-F1	GCTCTAGAGACGTTGTTTCCAGCCCACT	亚细胞定位 Subcellular localization
HgS5-R1	GGGGTACCAGAATGTTAAACACTTTACAATAC	亚细胞定位 Subcellular localization
HgS5-F1	ACCCTGTCCTACAACCACCT	荧光定量PCR Real-time PCR
HgS5-R1	TGGGAGCAGGGACTCCATTA
Actin-F1	GGTGATGGTGTGTCTCACACTG
Actin-R1	GAGGTTTCCATCTCCTGCTCGTAG
Actin-F2	ACACAAGGTCTATGTCGGAAAT
Actin-R2	TAACACTCTCGTGCTTACGATT

项目 Item	0 d	3 d	6 d	9 d	12 d
WT-1	50.52	45.99	30.22	7.13	5.98
WT-2	50.94	43.08	30.98	7.15	5.87
WT-3	50.99	44.44	30.45	7.12	5.88
OE-1	50.87	45.35	30.45	7.14	5.56
OE-2	50.83	44.42	30.35	7.33	5.78
OE-3	50.76	43.55	30.57	7.25	5.88

项目 Item	0 d	3 d	6 d	9 d	12 d
WT-1	50.52	45.99	30.22	7.13	5.98
WT-2	50.94	43.08	30.98	7.15	5.87
WT-3	50.99	44.44	30.45	7.12	5.88
OE-1	50.87	45.35	30.45	7.14	5.56
OE-2	50.83	44.42	30.35	7.33	5.78
OE-3	50.76	43.55	30.57	7.25	5.88

[1]	Long-yi HE, Meng-meng TAN, Hai-tao CHE, Hong-ying ZHANG, Yu-xin ZHU, Yan-ni ZHANG. Cloning and analysis of drought tolerance function of the LpDREB9 in Lilium pumilum [J]. Acta Prataculturae Sinica, 2025, 34(1): 161-173.
[2]	Yi WU, Ya-lan FENG, Tian-ning WANG, Ji-hao JU, Hui-shu XIAO, Chao MA, Jun ZHANG. Genome-wide identification and expression analysis of the Hsp70 gene family in wheat and its ancestral species [J]. Acta Prataculturae Sinica, 2024, 33(7): 53-67.
[3]	Zhen-huan ZHANG, Li-rong YAO, Jun-cheng WANG, Er-jing SI, Hong ZHANG, Ke YANG, Xiao-le MA, Ya-xiong MENG, Hua-jun WANG, Bao-chun LI. Identification of AKR gene family members in Halogeton glomeratus and salt tolerance analysis of the root salt stress response gene HgAKR42639 [J]. Acta Prataculturae Sinica, 2024, 33(7): 68-83.
[4]	Hai-ming KONG, Jia-xing SONG, Jing YANG, Qian LI, Pei-zhi YANG, Yu-man CAO. Identification and transcript profiling of the CAMTA gene family under abiotic stress in alfalfa [J]. Acta Prataculturae Sinica, 2024, 33(5): 143-154.
[5]	Ze-bin LI, Yong-zheng QIU, Yan-jie LIU, Jin-qiu YU, Bai-ji WANG, Qian-ning LIU, Yue WANG, Guo-wen CUI. Identification of the BZR gene family in alfalfa and analysis of its transcriptional responses to abiotic stress [J]. Acta Prataculturae Sinica, 2024, 33(11): 106-122.
[6]	Xin-yue ZHOU, Qing-xue JIANG, Hui-li JIA, Lin MA, Lu FAN, Xue-min WANG. Cloning and salt-tolerance functional analysis of alfalfa MsBBX20 gene [J]. Acta Prataculturae Sinica, 2024, 33(10): 55-73.
[7]	Shang-qin HU, Jun-cheng WANG, Li-rong YAO, Er-jing SI, Xiao-le MA, Ke YANG, Hong ZHANG, Ya-xiong MENG, Hua-jun WANG, Bao-chun LI. Cloning and preliminary functional analysis of the root gene HgAKR6C of Halogeton glomeratus [J]. Acta Prataculturae Sinica, 2024, 33(1): 61-74.
[8]	Zhen-fen ZHANG, Rong HUANG, Xiang-yang LI, Bo YAO, Gui-qin ZHAO. Seed-borne bacterial diversity of oat and functional analysis based on Illumina MiSeq high-throughput sequencing [J]. Acta Prataculturae Sinica, 2023, 32(7): 96-108.
[9]	Yuan WANG, Jing WANG, Shu-xia LI. Cloning of MsBBX24 from alfalfa （Medicago sativa） and determination of its role in salt tolerance [J]. Acta Prataculturae Sinica, 2023, 32(3): 107-117.
[10]	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.
[11]	Qian CHEN, Xiao-yun XU, Jun-cheng WANG, Li-rong YAO, Er-jing SI, Ke YANG, Xiao-ling WEI, Xiao-le MA, Bao-chun LI, Xun-wu SHANG, Ya-xiong MENG, Hua-jun WANG. Identification of a WRKY gene family based on full-length transcriptome sequences and analysis of response patterns under salt stress in Halogeton glomeratus [J]. Acta Prataculturae Sinica, 2022, 31(12): 146-157.
[12]	HOU Jie-ru, DUAN Xiao-yue, LI Zhou, PENG Yan. Cloning and expression analysis of TrSAMDC1 in white clover [J]. Acta Prataculturae Sinica, 2020, 29(8): 170-178.
[13]	LUO Wei, SHU Jian-hong, LIU Xiao-xia, WANG Zi-yuan, MU Qiong, WANG Xiao-li, WU Jia-hai. Cloning, subcellular localization and expression analysis of the RVE8 gene from Festuca arundinacea [J]. Acta Prataculturae Sinica, 2020, 29(7): 60-69.
[14]	YANG Ting, ZHANG Jian-ping, LIU Zi-gang, QI Yan-ni, LI Wen-juan, XIE Ya-ping. Molecular cloning and expression of heteromeric ACCase subunit genes from flax [J]. Acta Prataculturae Sinica, 2020, 29(4): 111-120.
[15]	XIA Zeng-run, WANG Wen-ying, LIU Ya-qi, WANG Suo-min. Cloning and expression analysis of the K⁺ channel gene AvAKT1 in Apocynum venetum [J]. Acta Prataculturae Sinica, 2019, 28(8): 180-189.