菜豆PvLEA基因的克隆及耐盐功能鉴定

doi:10.11924/j.issn.1000-6850.casb2024-0284

中国农学通报 ›› 2025, Vol. 41 ›› Issue (5): 44-49.doi: 10.11924/j.issn.1000-6850.casb2024-0284

菜豆PvLEA基因的克隆及耐盐功能鉴定

刘畅(), 张宇, 李梦妤, 冯国军, 刘大军, 杨晓旭, 闫志山

黑龙江大学现代农业与生态环境学院，哈尔滨 150080

收稿日期:2024-04-30 修回日期:2024-08-16 出版日期:2025-02-15 发布日期:2025-02-11
作者简介:
刘畅，女，1989年出生，辽宁沈阳人，副研究员，博士研究生，研究方向：蔬菜遗传育种与生物技术。通信地址：150080 黑龙江省哈尔滨市南岗区学府路74号，E-mail：lcky0909@126.com。
基金资助:
黑龙江省现代农业协同创新推广体系“油豆角协同创新推广体系”; 黑龙江省种业创新发展项目“东北油豆角种质资源创新及新品种选育（菜豆联合育种攻关）”; 黑龙江省重点研发计划项目专项课题计划“主要蔬菜作物分子辅助育种决策与新品系培育研究”(SC2022ZX02C0202); 黑龙江省优秀青年教师基础研究支持计划“菜豆豆荚纤维突变基因pv-bfm定位与候选基因功能验证”(YQJH2024198)

Cloning of PvLEA Gene in Snap Bean and Identification of Salt Tolerance Function

LIU Chang(), ZHANG Yu, LI Mengyu, FENG Guojun, LIU Dajun, YANG Xiaoxu, YAN Zhishan

College of Modern Agriculture and Ecological Environment, Heilongjiang University, Harbin 150080

Received:2024-04-30 Revised:2024-08-16 Published:2025-02-15 Online:2025-02-11

摘要/Abstract

摘要：

为了应对土壤盐渍化对全球农业带来的新挑战，本研究旨在挖掘菜豆的耐盐基因以增强其耐盐性。以菜豆耐盐品种P19023为试材，对PvLEA(Phvul.008G228100)基因进行克隆、表达特性分析、创制以野生型拟南芥Col-0为背景的PvLEA过表达株系，并对转基因株系进行耐盐相关生理指标的鉴定。结果表明PvLEA基因能够在盐胁迫下诱导表达，盐胁迫下拟南芥PvLEA过表达植株与野生相比根长增长95.83%、生物量增加34.07%、SOD酶活性升高99.47%、POD酶活性上升112.58%、CAT酶活性上升28.57%、脯氨酸含量上升811.02%、ASA含量上升113.33%，MDA含量下降55.4%。这说明PvLEA在耐盐能力中起到了正向调控的作用，为后续深入研究及菜豆耐盐新品种培育提供了理论基础。

关键词: 菜豆, 耐盐基因, 晚期胚胎发生丰度(LEA)蛋白, 克隆, 过表达, 拟南芥, 功能鉴定, 活性氧

Abstract:

The aims are to meet the new challenge of soil salinization to global agriculture, explore salt tolerance genes in snap beans, and improve their salt tolerance. Using the salt tolerant variety P19023 of snap beans as the test material, the PvLEA (Phvul. 008G228100) gene was cloned and its expression characteristics were analyzed, and a PvLEA overexpression strain was created on the background of wild-type Arabidopsis thaliana Col-0. The salt tolerance related physiological indicators of the transgenic strain were identified. The results showed that PvLEA gene could be induced to express under salt stress. Compared with wild plants, PvLEA overexpression plants increased root length by 95.83%, biomass by 34.07%, SOD activity by 99.47%, POD activity by 112.58%, CAT activity by 28.57%, proline content by 811.02%, ASA content by 113.33%, MDA content by 55.4% under salt stress. These results indicated that PvLEA played a positive regulatory role in salt tolerance, which provided a theoretical basis for further study and breeding of new salt tolerant varieties of snap beans.

Key words: snap bean, salt tolerance gene, late embryogenesis abundance (LEA) protein, clone, overexpression, Arabidopsis thaliana, functional identification, ROS

刘畅, 张宇, 李梦妤, 冯国军, 刘大军, 杨晓旭, 闫志山. 菜豆PvLEA基因的克隆及耐盐功能鉴定[J]. 中国农学通报, 2025, 41(5): 44-49.

LIU Chang, ZHANG Yu, LI Mengyu, FENG Guojun, LIU Dajun, YANG Xiaoxu, YAN Zhishan. Cloning of PvLEA Gene in Snap Bean and Identification of Salt Tolerance Function[J]. Chinese Agricultural Science Bulletin, 2025, 41(5): 44-49.

图/表 7

基因名称	引物序列(5'-3')
PvLEA-F(qPCR)	CGGTGGTAAACGAAGCAACG
PvLEA-R(qPCR)	CACCACCAGAGGAAGTGACG
β-actin	CCAAGGCAAGGTACGATGAAA
β-actin	AGAGATGGGAACGAAGGGGAT

表1. 实时荧光定量PCR引物

图1. 盐胁迫下PvLEA基因在菜豆各组织中不同时间的相对表达量

图2. PvLEA过表达载体菌落PCR琼脂糖凝胶电泳结果

图3. PvLEA过表达株系T1-T3代PCR验证 +，-，O，M分别代表阳性对照，阴性对照，水和Marker

图4. 不同盐浓度处理下拟南芥野生型和PvLEA过表达株系主根长

图5. 不同盐浓度处理下拟南芥野生型和PvLEA过表达株系生物量测定

图6. 盐胁迫下拟南芥野生型和PvLEA过表达株系相关生理指标测定

参考文献 26

[1]	ZHU J K, LIU J, XIONG L. Genetic analysis of salt tolerance in Arabidopsis: evidence for a critical role of potassium nutrition[J]. The plant cell, 1998, 10(7):1181-1191.
[2]	CHINNUSAMY V, SCHUMAKER K, ZHU J K. Molecular genetic perspectives on cross-talk and specificity in abiotic stress signalling in plants[J]. Journal of experimental botany, 2004, 55(395):225-236. doi: 10.1093/jxb/erh005 pmid: 14673035
[3]	DANQUAH A, DE ZELICOURT A, COLCOMBET J, et al. The role of ABA and MAPK signaling pathways in plant abiotic stress responses[J]. Biotechnol adv, 2014, 32(1):40-52. doi: 10.1016/j.biotechadv.2013.09.006 pmid: 24091291
[4]	PIETERSE C M, VAN DER DOES D, ZAMIOUDIS C, et al. Hormonal modulation of plant immunity[J]. Annual review of cell and developmental biology, 2012,28:489-521.
[5]	GRELET J, BENAMAR A, TEYSSIER E, et al. Identification in pea seed mitochondria of a late-embryogenesis abundant protein able to protect enzymes from drying1[J]. Plant physiology, 2005,137:157-167.
[6]	BRAY E A. Molecular responses to water deficit[J]. Plant physiol, 1993, 103(4):1035-1040. pmid: 12231998
[7]	UMEZAWA T, FUJITA M, FUJITA Y, et al. Engineering drought tolerance in plants: discovering and tailoring genes to unlock the future[J]. Current opinion in biotechnology, 2006, 17(2):113-122. doi: 10.1016/j.copbio.2006.02.002 pmid: 16495045
[8]	HINCHA D K, THALHAMMER A. LEA proteins: IDPs with versatile functions in cellular dehydration tolerance[J]. Biochemical society transactions, 2012, 40(5):1000-1003. pmid: 22988854
[9]	张晓敏. 园林绿化植物沙棘的耐盐性研究及耐盐品种筛选[D]. 沈阳: 沈阳农业大学, 2021.
[10]	GIANNOPOLITIS C N, RIES S K. Superoxide dismutases: i. occurrence in higher plants 1 2[J]. Plant physiology, 1977, 59(2):309. doi: 10.1104/pp.59.2.309 pmid: 16659839
[11]	THOMAS R L, JEN J J, MORR C V. Changes in solule and ound peroxidase, IAA oxidase during tamato fruit development[J]. Journal of food science, 1981,47:158-161.
[12]	CAKMAK I, MARSCHNER H. Magnesium deficiency and high light intensity enhance activities of superoxide dismutase, ascorbate peroxidase, and glutathione reductase in bean leaves 1[J]. Plant physiology, 1992, 98(4):1222-1227. doi: 10.1104/pp.98.4.1222 pmid: 16668779
[13]	DHINDSA R S, PAMELA P D, THORPE T A. Leaf senescence: correlated with increased levels of membrane permeability and lipid peroxidation, and decreased levels of superoxide dismutase and catalase[J]. Journal of experimental botany, 1981(1):93-101.
[14]	高俊凤. 植物生理学实验指导[M]. 植物生理学实验指导, 2006:219.
[15]	LIANG W, MA X, WAN P, et al. Plant salt-tolerance mechanism: a review[J]. Biochemical and biophysical research communications, 2018, 495(1):286-291. doi: S0006-291X(17)32220-9 pmid: 29128358
[16]	MUNNS R, TESTER M. Mechanisms of salinity tolerance[J]. Annual review of plant biology, 2008, 59:651-681. doi: 10.1146/annurev.arplant.59.032607.092911 pmid: 18444910
[17]	HAJIHASHEMI S, SKALICKY M, BRESTIC M, et al. Effect of sodium nitroprusside on physiological and anatomical features of salt-stressed Raphanus sativus[J]. Plant physiology and biochemistry, 2021,169:160-170.
[18]	DURE L, GREENWAY S C, GALAU G A. Developmental biochemistry of cottonseed embryogenesis and germination: changing messenger ribonucleic acid populations as shown by in vitro and in vivo protein synthesis[J]. Biochemistry, 1981, 20(14):4162-4168. doi: 10.1021/bi00517a033 pmid: 7284317
[19]	ZHOU Y, HE P, XU Y, et al. Overexpression of CsLEA11, a Y 3 SK 2 -type dehydrin gene from cucumber (Cucumis sativus), enhances tolerance to heat and cold in Escherichia coli[J]. Amb express, 2017, 7(1):182.
[20]	ALTUNOGLU Y C, BALOGLU M C, BALOGLU P, et al. Genome-wide identification and comparative expression analysis of LEA genes in watermelon and melon genomes[J]. Physiology & molecular biology of plants an international journal of functional plant biology, 2017, 23(1):5.
[21]	XU M, TONG Q, WANG Y, et al. Transcriptomic analysis of grapevine LEA gene family in response to osmotic and cold stress, and functional analyses of VamDHN3 gene[J]. Plant and cell physiology, 2020, 61(4):775-786.
[22]	KOVACS D, AGOSTON B, TOMPA P. Disordered plant LEA proteins as molecular chaperones[J]. Plant signaling & behavior, 2008, 3(9): 710-713.
[23]	JIN X, CAO D, WANG Z, et al. Genome-wide identification and expression analyses of the LEA protein gene family in tea plant reveal their involvement in seed development and abiotic stress responses[J]. Scientific reports, 2019, 9(1): 14123.
[24]	LIU H, XING M, YANG W, et al. Genome-wide identification of and functional insights into the late embryogenesis abundant (LEA) gene family in bread wheat (Triticum aestivum)[J]. Scientific reports, 2019, 9(1): 13375.
[25]	MA L, ZHU T, WANG H, et al. Genome-wide identification, phylogenetic analysis and expression profiling of the late embryogenesis-abundant (LEA) gene family in Brachypodium distachyon[J]. Functional plant biology, 2021, 48(4):386-401.
[26]	LIU Y, XIE L, LIANG X, et al. CpLEA5, the late embryogenesis abundant protein gene from chimonanthus praecox, possesses low temperature and osmotic resistances in prokaryote and eukaryotes[J]. International journal of molecular sciences, 2015, 16(11): 26978-26990. doi: 10.3390/ijms161126006 pmid: 26569231

菜豆PvLEA基因的克隆及耐盐功能鉴定

Cloning of PvLEA Gene in Snap Bean and Identification of Salt Tolerance Function

RichHTML

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

图/表 7

参考文献 26

相关文章 15

编辑推荐

Metrics

本文评价

关于本刊

作者中心

审者中心

在线期刊

联系我们

[1]	陈虞超, 郭生虎, 田莉, 钟楠, 杨建国, 甘晓燕. 芦竹独脚金内酯受体基因克隆及功能验证[J]. 中国农学通报, 2025, 41(5): 50-55.
[2]	李淑佳, 孙来军, 蒙亚浩, 王祎辰, 李晓旭, 冯国军, 杨凤艳. 基于机器视觉的菜豆种子品种智能识别与分类[J]. 中国农学通报, 2025, 41(4): 156-164.
[3]	肖鸿勇, 王丽红, 阴长发, 黄建华, 陈洪凡, 兰波, 杨迎青. 芝麻茎点枯病综合防控技术研究进展[J]. 中国农学通报, 2024, 40(8): 119-125.
[4]	李学庆, 冯国军, 刘大军, 刘畅, 闫志山, 杨晓旭. 细菌性疫病对菜豆光合特性和生理指标的影响[J]. 中国农学通报, 2024, 40(4): 132-139.
[5]	李海东, 纪聪聪, 徐一萍, 康涛, 陈建生, 张艳艳, 张玲, 李文金. 锌对花生活性氧代谢、衰老和产量的影响[J]. 中国农学通报, 2024, 40(4): 20-25.
[6]	李昕然, 刘畅, 冯国军, 刘大军, 安普南, 杨晓旭. 木质素沉积对种皮开裂的影响[J]. 中国农学通报, 2024, 40(36): 51-56.
[7]	张泰劼, 郭文磊, 田兴山, 张纯. 香豆素对白花鬼针种子萌发及生长的影响[J]. 中国农学通报, 2024, 40(31): 111-118.
[8]	孙靖雯, 张光辉, 刘畅, 冯国军, 刘大军, 闫志山, 杨晓旭. 菜豆种子硬实分析和破除研究[J]. 中国农学通报, 2024, 40(30): 33-39.
[9]	韩进财, 罗丁峰, 柏浩东, 李祖任. 稗草EcHPPD基因的克隆及生物信息学分析[J]. 中国农学通报, 2024, 40(3): 87-94.
[10]	仲维婷, 张琼, 兴旺, 刘大丽. 异源表达甜菜BvHSP18.2基因增强拟南芥对镉胁迫的耐受性研究[J]. 中国农学通报, 2024, 40(29): 14-20.
[11]	刘晓敏, 孙志广, 迟铭, 邢运高, 徐波, 李景芳, 刘艳, 卢百关, 王宝祥, 徐大勇. 一个新的水稻黄绿叶突变体ygl-9108的鉴定与基因定位[J]. 中国农学通报, 2024, 40(26): 22-29.
[12]	朱晨宇, 谢榕榕, 王月敏, 徐志豪, 柯玉琴, 郑朝元, 李文卿. 钾镁肥优化对烤烟后期活性氧代谢和激素含量的影响[J]. 中国农学通报, 2024, 40(25): 24-29.
[13]	沈祺, 冯国军, 刘大军, 卫梦丹, 刘畅, 杨晓旭. CaCl₂引发对菜豆幼苗生长、光合参数及生理生化的影响[J]. 中国农学通报, 2024, 40(24): 21-27.
[14]	韩济民, 孙璐, 冯国军, 刘大军, 刘畅, 杨晓旭. 超干处理对菜豆种子活力及生理生化活性的影响[J]. 中国农学通报, 2024, 40(15): 66-72.
[15]	吴冉迪, 葛丽清, 张付斗, 申时才, 杨云海, 杨韶松, 郑凤萍, 范泽文, 高家乐, 徐高峰. 2种农作物对外来入侵植物空心莲子草茎节克隆繁殖与生长的影响[J]. 中国农学通报, 2024, 40(14): 104-111.