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作物学报 ›› 2025, Vol. 51 ›› Issue (4): 863-872.doi: 10.3724/SP.J.1006.2025.44134

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

天山雪莲质膜水孔蛋白基因SiPIP1;3在番茄中的抗寒功能分析

张晓丽(), 刘晓燕, 夏雯雯(), 李锦()   

  1. 石河子大学生命科学学院农业生物技术重点实验室, 新疆石河子 832000
  • 收稿日期:2024-08-21 接受日期:2024-12-12 出版日期:2025-04-12 网络出版日期:2024-12-19
  • 通讯作者: 夏雯雯, E-mail: 365689825@qq.com; 李锦, E-mail: lijin@shzu.edu.cn
  • 作者简介:E-mail: 3317866967@qq.com
  • 基金资助:
    国家自然科学基金项目(32260726);“天池英才”引进计划项目(CZ001617);石河子大学高层次人才科研启动项目(RCZK202470)

Functional analysis of the plasma membrane intrinsic protein gene SiPIP1;3 from Saussurea involucrata in tomato

ZHANG Xiao-Li(), LIU Xiao-Yan, XIA Wen-Wen(), LI Jin()   

  1. Key Laboratory of Agricultural Biotechnology, College of Life Sciences, Shihezi University, Shihezi 832000, Xinjiang, China
  • Received:2024-08-21 Accepted:2024-12-12 Published:2025-04-12 Published online:2024-12-19
  • Contact: E-mail: 365689825@qq.com; E-mail: lijin@shzu.edu.cn
  • Supported by:
    National Natural Science Foundation of China(32260726);Tianchiyingcai Foundation(CZ001617);Shihezi University High Level Talent Research Launch Project(RCZK202470)

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摘要：

水孔蛋白(Aquaporin, AQPs)作为细胞水分跨膜运输的主要通道蛋白, 参与植物的生长及逆境适应过程。前期通过构建极端低温耐受植物天山雪莲(Saussurea involucrata)低温表达文库, 获得质膜水孔蛋白基因SiPIP1;3。为分析SiPIP1;3基因在低温适应中的功能, 构建植物表达载体并转化于冷敏感植物番茄。 结果发现,SiPIP1;3基因表达通过促进番茄在低温处理下的可溶性蛋白、可溶性糖和脯氨酸积累, 降低膜脂过氧化, 从而显著增强番茄的冷耐受性; 此外, 大田种植结果表明, SiPIP1;3基因表达可通过提高番茄叶片气孔导度以增加胞间CO2浓度, 从而显著提高番茄净光合效率和水分利用效率, 导致平均果实体积和平均单株果实数显著增加, 显著提高单株番茄果实产量。因此, SiPIP1;3基因表达可显著增强番茄的抗寒性和提高番茄果实产量, 为后期番茄抗寒新种质培育提供重要的基因资源和奠定理论基础。

关键词: SiPIP1, 3基因, 抗寒性, 光合效率, 果实产量

Abstract:

Aquaporins (AQPs), as key facilitators of water transport across cellular membranes, play an essential role in plant growth and adaptation to environmental stresses. Previously, the plasma membrane intrinsic protein gene SiPIP1;3 was cloned from a low-temperature expression library of Saussurea involucrata, a cold-tolerant herbaceous plant. To investigate the function of SiPIP1;3 under low-temperature stress, a plant expression vector containing SiPIP1;3 was constructed and transformed into cold-sensitive tomato plants. The results demonstrated that SiPIP1;3 expression significantly enhanced tomato tolerance to low-temperature treatment by promoting the accumulation of soluble proteins, soluble sugars, and proline, while reducing membrane lipid peroxidation. Moreover, field cultivation results revealed that SiPIP1;3 expression improved intercellular CO2 concentration by increasing stomatal conductance in tomato leaves. This led to a marked improvement in net photosynthetic efficiency and water use efficiency, ultimately resulting in a significant increase in both the average fruit size and fruit number per plant. In conclusion, the expression of SiPIP1;3 significantly enhances low-temperature tolerance and fruit yield in tomato plants. This study provides a valuable genetic resource and establishes a theoretical foundation for breeding cold-resistant tomato varieties.

Key words: SiPIP1, 3 gene, cold resistance, photosynthetic efficiency, fruit yield

图1

SiPIP1;3基因克隆"

图2

水孔蛋白SiPIP1;3蛋白序列分析 A: SiPIP1;3多序列比对; B: SiPIP1;3二级结构分析; C: SiPIP1;3亲疏水性分析; D: SiPIP1;3进化树。"

图3

转SiPIP1;3基因番茄株系鉴定 A: 转基因番茄株系DNA PCR鉴定; B: 转基因番茄株系RT-PCR鉴定。M: MARK III; WT: 野生型番茄株系。"

图4

野生型和转基因番茄低温胁迫下的表型和生理数据 A: 野生型和转基因番茄4℃处理8 h和-2℃处理2 h后的生长状态; B: 相对含水量; C: 相对电导率; D: 丙二醛含量; E: 可溶性蛋白含量; F: 可溶性糖含量; G: 脯氨酸含量。数据为3个重复的平均值±标准差。*、**、***、****分别表示在0.05、0.01、0.001、0.0001水平上差异显著。ns表示差异不显著。"

图5

野生型和转基因番茄的农艺性状分析 A: 果实成熟过程中番茄植株的整体生长状况; B: 野生型和转基因番茄产量比较; C: 2020年和2021年野生型和转基因番茄单株果实数量; D: 2020年和2021年野生型和转基因番茄果实的横经和纵经; E: 2020年和2021年野生型和转基因番茄单株果重。数据为21个重复的平均值±标准差。*、**、***、****分别表示在0.05、0.01、0.001、0.0001水平上差异显著。ns表示差异不显著。"

图6

野生型和转基因番茄光合指标 数据为21个重复的平均值±标准差。*、**、***、****分别表示在0.05、0.01、0.001、0.0001水平上差异显著。ns表示差异不显著。"

[1] Guy C. Cold acclimation and freezing stress tolerance: role of protein metabolism. Annu Rev Plant Physiol Plant Mol Biol, 1990, 41: 187-223.
[2] Pearce R. Plant freezing and damage. Ann Bot, 2001, 87: 417-424.
[3] Ou-Yang Q Q, Zhang Y W, Yang X Y, Yang C, Hou D Y, Liu H, Xu H W. Overexpression of OsPIN9 impairs chilling tolerance via disturbing ROS homeostasis in rice. Plants (Basel), 2023, 12: 2809.
[4] 刘文英, 张永芳, 张东旭. 植物抗寒基因研究进展. 山西大同大学学报(自然科学版), 2012, 28(6): 52-55.
Liu W Y, Zhang Y F, Zhang D X. Study on cold resistant genes in plants. J Shanxi Datong Univ (Nat Sci Edn), 2012, 28(6): 52-55 (in Chinese with English abstract).
[5] Wang G D, Liu Q, Shang X T, Chen C, Xu N, Guan J, Meng Q W. Overexpression of transcription factor SlNAC35 enhances the chilling tolerance of transgenic tomato. Biol Plant, 2018, 62: 479-488.
[6] 王冬梅, 李晓蓉, 李静, 张帅, 黄乐平. 转blti2抗寒基因棉花的抗寒性分析. 分子植物育种, 2011, 9: 1632-1636.
Wang D M, Li X R, Li J, Zhang S, Huang L P. Analysis of freezing resistance on transgenic cotton carrying blti2 gene. Mol Plant Breed, 2011, 9: 1632-1636 (in Chinese with English abstract).
[7] Wolter F P, Schmidt R, Heinz E. Chilling sensitivity of Arabidopsis thaliana with genetically engineered membrane lipids. EMBO J, 1992, 11: 4685-4692.
doi: 10.1002/j.1460-2075.1992.tb05573.x pmid: 1464304
[8] Artus N N, Uemura M, Steponkus P L, Gilmour S J, Lin C, Thomashow M F. Constitutive expression of the cold-regulated Arabidopsis thaliana COR15a gene affects both chloroplast and protoplast freezing tolerance. Proc Natl Acad Sci USA, 1996, 93: 13404-13409.
doi: 10.1073/pnas.93.23.13404 pmid: 11038526
[9] Fraysse L C, Wells B, McCann M C, Kjellbom P. Specific plasma membrane aquaporins of the PIP1 subfamily are expressed in sieve elements and guard cells. Biol Cell, 2005, 97: 519-534.
doi: 10.1042/BC20040122 pmid: 15898953
[10] Preston G M, Carroll T P, Guggino W B, Agre P. Appearance of water channels in Xenopus oocytes expressing red cell CHIP28 protein. Science, 1992, 256: 385-387.
doi: 10.1126/science.256.5055.385 pmid: 1373524
[11] Johanson U, Karlsson M, Johansson I, Gustavsson S, Sjövall S, Fraysse L, Weig A R, Kjellbom P. The complete set of genes encoding major intrinsic proteins in Arabidopsis provides a framework for a new nomenclature for major intrinsic proteins in plants. Plant Physiol, 2001, 126: 1358-1369.
doi: 10.1104/pp.126.4.1358 pmid: 11500536
[12] Maurel C, Boursiac Y, Luu D T, Santoni V, Shahzad Z, Verdoucq L. Aquaporins in plants. Physiol Rev, 2015, 95: 1321-1358.
doi: 10.1152/physrev.00008.2015 pmid: 26336033
[13] 尚珂含, 杨舒婷, 卞诗村, 刘梦婷, 安亚虹, 王广龙, 熊爱生. 芹菜水孔蛋白基因AgPIP2;1的克隆及其对非生物胁迫的响应. 核农学报, 2020, 34: 231-239.
doi: 10.11869/j.issn.100-8551.2020.02.0231
Shang K H, Yang S T, Bian S C, Liu M T, An Y H, Wang G L, Xiong A S. Cloning of an aquaporin gene AgPIP2;1 from celery and its response to abiotic stresses. J Nucl Agric Sci, 2020, 34: 231-239 (in Chinese with English abstract).
[14] Lee S H, Chung G C, Jang J Y, Ahn S J, Zwiazek J J. Overexpression of PIP2;5 aquaporin alleviates effects of low root temperature on cell hydraulic conductivity and growth in Arabidopsis. Plant Physiol, 2012, 159: 479-488.
[15] Jang J Y, Lee S H, Rhee J Y, Chung G C, Ahn S J, Kang H. Transgenic Arabidopsis and tobacco plants overexpressing an aquaporin respond differently to various abiotic stresses. Plant Mol Biol, 2007, 64: 621-632.
[16] Li G W, Zhang M H, Cai W M, Sun W N, Su W A. Characterization of OsPIP2;7, a water channel protein in rice. Plant Cell Physiol, 2008, 49: 1851-1858.
[17] Peng Y H, Lin W L, Cai W M, Arora R. Overexpression of a Panax ginseng tonoplast aquaporin alters salt tolerance, drought tolerance and cold acclimation ability in transgenic Arabidopsis plants. Planta, 2007, 226: 729-740.
[18] 李晨, 沈海涛, 张煜星, 王爱英, 祝建波. 利用Gateway技术构建天山雪莲cDNA表达文库. 石河子大学学报(自然科学版), 2010, 28: 534-536.
Li C, Shen H T, Zhang Y X, Wang A Y, Zhu J B. Construction of a cDNA library of Saussurea involucrata Kar. et kir using Gateway technology. J Shihezi Univ (Nat Sci), 2010, 28: 534-536 (in Chinese with English abstract).
[19] 焦天奇, 孙辉, 刘瑞娜, 王爱英, 祝建波. 转天山雪莲sikPIP3基因烟草的获得及抗逆性鉴定. 西北植物学报, 2012, 32: 431-438.
Jiao T Q, Sun H, Liu R N, Wang A Y, Zhu J B. Transformation of Saussurea involucrata sikPIP3 gene into tobacco and evaluation of transgenic plant stress-resistance. Acta Bot Boreali-Occident Sin, 2012, 32: 431-438 (in Chinese with English abstract).
[20] 黎玉顺, 刘逸泠, 刘步仓, 穆建强, 祝建波. 新疆雪莲水孔蛋白sikPIP1基因的克隆与功能分析. 生物技术通报, 2015, 31(9): 97-105.
doi: 10.13560/j.cnki.biotech.bull.1985.2015.09.013
Li Y S, Liu Y L, Liu B C, Mu J Q, Zhu J B. Cloning and function analysis of aquaporin protein gene sikPIP1 from Saussurea involucrata Kar. et Kir. Biotechnol Bull, 2015, 31(9): 97-105 (in Chinese with English abstract).
[21] Li J, Xia W W, Zang H X, Dai B, Zhang Y, Feng Y J, Wang A Y, Lin Z P, Liu H L, Zhu J B. Expression analysis of aquaporin genes in Saussurea involucrata rosette leaves and functional analysis of upregulated SiPIP1;5A under low-temperature stress. Environ Exp Bot, 2020, 171: 103958.
[22] Li J, Liu H L, Xia W W, Mu J Q, Feng Y J, Liu R N, Yan P Y, Wang A Y, Lin Z P, Guo Y, Zhu J B, Chen X F. De novo transcriptome sequencing and the hypothetical cold response mode of Saussurea involucrata in extreme cold environments. Int J Mol Sci, 2017, 18: 1155.
[23] Liu X Y, Xia W W, Zhang X L, Li A W, Qin J W, Sun H L, Li J, Zhu J B. Overexpression of the SiLEA5gene in Saussurea involucrata increases the low-temperature tolerance of transgenic tomatoes. Horticulturae, 2022, 8: 1023.
[24] Dong S K, Jiang Y Z, Dong Y C, Wang L B, Wang W J, Ma Z Z, Yan C, Ma C M, Liu L J. A study on soybean responses to drought stress and rehydration. Saudi J Biol Sci, 2019, 26: 2006-2017.
doi: 10.1016/j.sjbs.2019.08.005 pmid: 31889786
[25] Dai L L, Feng Z Z, Pan X D, Xu Y S, Li P, Lefohn A S, Harmens H, Kobayashi K. Increase of apoplastic ascorbate induced by ozone is insufficient to remove the negative effects in tobacco, soybean and poplar. Environ Pollut, 2019, 245: 380-388.
doi: S0269-7491(18)34199-X pmid: 30448508
[26] Cheng L Q, Li X X, Huang X, Ma T, Liang Y, Ma X Y, Peng X J, Jia J T, Chen S Y, Chen Y, et al. Overexpression of sheepgrass R1-MYB transcription factor LcMYB1 confers salt tolerance in transgenic Arabidopsis. Plant Physiol Biochem, 2013, 70: 252-260.
[27] 谢聪颖, 钟雪峰, 乌兰, 张馨予, 田霖, 杜国英. 低温胁迫下条斑紫菜的生长和光合生理生化响应. 中国海洋大学学报, 2024, 54(8): 33-42.
Xie C Y, Zhong X F, Wu L, Zhang X Y, Tian L, Du G Y. Growth performance and photosynthetic physiological and biochemical responses of Neopyropia yezoensis under low temperature stress. Period Ocean Univ China, 2024, 54(8): 33-42 (in Chinese with English abstract).
[28] Gilmour S J, Sebolt A M, Salazar M P, Everard J D, Thomashow M F. Overexpression of the Arabidopsis CBF3 transcriptional activator mimics multiple biochemical changes associated with cold acclimation. Plant Physiol, 2000, 124: 1854-1865.
doi: 10.1104/pp.124.4.1854 pmid: 11115899
[29] Gilmour S J, Zarka D G, Stockinger E J, Salazar M P, Houghton J M, Thomashow M F. Low temperature regulation of the Arabidopsis CBF family of AP2 transcriptional activators as an early step in cold-induced COR gene expression. Plant J, 1998, 16: 433-442.
doi: 10.1046/j.1365-313x.1998.00310.x pmid: 9881163
[30] He Y K, Tang R H, Hao Y, Stevens R D, Cook C W, Ahn S M, Jing L F, Yang Z G, Chen L E, Guo F Q, et al. Nitric oxide represses the Arabidopsis floral transition. Science, 2004, 305: 1968-1971.
[31] Hooijmaijers C, Rhee J Y, Kwak K J, Chung G C, Horie T, Katsuhara M, Kang H. Hydrogen peroxide permeability of plasma membrane aquaporins of Arabidopsis thaliana. J Plant Res, 2012, 125: 147-153.
doi: 10.1007/s10265-011-0413-2 pmid: 21390558
[32] Yaneff A, Sigaut L, Marquez M, Alleva K, Pietrasanta L I, Amodeo G. Heteromerization of PIP aquaporins affects their intrinsic permeability. Proc Natl Acad Sci USA, 2014, 111: 231-236.
doi: 10.1073/pnas.1316537111 pmid: 24367080
[33] Xia W W, Liu X Y, Xin H L, Wu X Y, Liu R N, Li J, Zhu J B. Saussurea involucrata PIP2;7 improves photosynthesis and drought resistance by decreasing the stomatal density and increasing intracellular osmotic pressure. Environ Exp Bot, 2021, 191: 104605.
[34] Xin H L, Li Q Q, Wang S S, Zhang Z X, Wu X Y, Liu R N, Zhu J B, Li J. Saussurea involucrata PIP2;4improves growth and drought tolerance in Nicotiana tabacum by increasing stomatal density and sensitivity. Plant Sci, 2023, 326: 111526.
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