| 570 | 9 | 7 |
| 下载次数 | 被引频次 | 阅读次数 |
为探明桑树AP2/ERF转录因子MnERF2基因对桑树耐盐胁迫能力的影响，利用构建好的植物过表达和抑制表达载体瞬时转化桑树，并比较分析NaCl胁迫前后瞬时转化桑树中的活性氧(ROS)含量、抗氧化酶活性、抗氧化物质含量以及抗氧化酶基因的表达量，以综合评定MnERF2基因的耐盐功能。通过荧光定量RT-PCR对盐胁迫后MnERF2表达量的分析显示，成功获得MnERF2基因瞬时过表达(OE)、抑制表达(RNAi)转基因植株。进一步的生理指标分析结果显示，不同时间盐胁迫下瞬时过表达MnERF2植株中的ROS■、H_2O2、·OH)和丙二醛(MDA)含量明显低于对照和RNAi株系，过氧化物歧化酶(SOD)、过氧化物酶(POD)、过氧化氢酶(CAT)、谷胱甘肽S-转移酶(GST)活性以及抗坏血酸(AsA)、还原型谷胱甘肽(GSH)、脯氨酸含量高于对照和RNAi株系，且48 h处理下SOD、POD、CAT三种酶基因表达量显著高于对照和RNAi株系。说明MnERF2基因响应桑树的盐胁迫，通过提高植株的保护酶活性和抗氧化物质含量、降低活性氧氧化及细胞损伤程度提高桑树的耐盐能力。
Abstract：To investigate the effects of AP2/ERF transcription factor MnERF2 on salt resistance of mulberry, the plant overexpression vector and knockdown vector were constructed and transient transformed into mulberry. The ROS content, antioxidant enzymes activities, antioxidant substances contents and expression level of antioxidant enzyme genes in transient transformed mulberry were analyzed before and after NaCl stress, and the function of MnERF2 in salt tolerance was comprehensively evaluated. The analysis of MnERF2 expression after salt stress by fluorescence quantitative RT-PCR showed that the MnERF2 gene transiently overexpressed(OE) and inhibited(RNAi) transgenic plants had been successfully obtained. Further analysis of physiological indicators showed that the contents of ■、H_2O2、·OH and MDA in OE plants under salt stress at different times were significantly lower than those in wild and RNAi plants, while the activities of SOD, POD, CAT and GST were higher as well as the contents of AsA, GSH and proline. The expression level of the three enzyme genes was significantly higher than those of wild and RNAi plants after 48 h treatment. It indicated that MnERF2 gene responded to the salt stress of mulberry, and the salt tolerance of mulberry could be improved by increasing the activity of protective enzymes and the content of osmotic regulatory substances, and reducing the degree of reactive oxygen oxidation and cell damage.
[1] 热依麦阿依·阿布都艾尼，陈静，陈芸，等.盐胁迫下棉花根系的转录组分析及耐盐基因筛选[J].华南师范大学学报(自然科学版),2020,52(5):85-92.
[2] 许盼云，吴玉霞，何天明.植物对盐碱胁迫的适应机理研究进展[J].中国野生植物资源，2020,39(10):41-49.
[3] Sakuma Y,Liu Q,Dubouzet J G,et al.DNA-binding specificity of the ERF/AP2 domain of Arabidopsis DREBs,transcription factors involved in dehydration- and cold-inducible gene expression[J].Biochemical and Biophysical Research Communications,2002,290(3):998-1009.
[4] 何宁佳，向仲怀.桑树基因组[M].北京：中国林业出版社，2016:147-148.
[5] Jofuku K D,Denboer B G W,Vanmontagu M,et al.Control of Arabidopsis flower and seed development by the homeotic gene Apetala2[J].Plant Cell,1994,6(9):1211-1225.
[6] 张麒，陈静，李俐，等.植物AP2/ERF转录因子家族的研究进展[J].生物技术通报，2018,34(8):1-7.
[7] Najafi S,Sorkheh K,Nasernakhaei F.Characterization of the APETALA2/Ethylene-responsive factor (AP2/ERF) transcription factor family in sunflower[J].Scientific Reports,2018,8(9):11576.
[8] Gilmour S J,Sebolt A M,Salazar M P,et al.Overexpression of the Arabidopsis CBF3 transcriptional activator mimics multiple biochemical changes associated with cold acclimation[J].Plant Physiology,2000,124:1854-1865.
[9] Cheng M C,Liao P M,Kuo W W,et al.The Arabidopsis ET-HYLENE RESPONSE FACTOR1 regulates abiotic stress-responsive gene expression by binding to different cis-acting elements in response to different stress signals[J].Plant Physiology,2013,162(3):1566-1582.
[10] 覃利萍.刚毛柽柳AP2/ERF转录因子ThCRF1响应盐胁迫的调控机理研究[D].乌鲁木齐：新疆大学，2018.
[11] Yao Y,He R J,Xie Q L,et al.ETHYLENE RESPONSE FACTOR 74 (ERF74) plays an essential role in controlling a respiratory burst oxidase homolog D (RbohD)-dependent mechanism in response to different stresses in Arabidopsis[J].New Phytologist,2017,213(4):1667-1681.
[12] Dou H O,Xv K P,Meng Q W,et al.Potato plants ectopically expressing Arabidopsis thaliana CBF3 exhibit enhanced tolerance to high-temperature stress[J].Plant Cell and Environment,2015,38(1):61-72.
[13] Ji X Y,Nie X G,Liu Y J,et al.A bHLH gene from Tamarix hispida improves abiotic stress tolerance by enhancing osmotic potential and decreasing reactive oxygen species accumulation[J].Tree Physiology,2016,36(2):193-207.
[14] Elstner E F,Heupel A.Inhibition of nitrite formation from hydroxylammoniumchloride:a simple assay for superoxide dismutase[J].Analytical Biochemistry,1976,70(2):616-620.
[15] Patterson B D,Macrae E A,Ferguson I B.Estimation of hydrogen peroxide in plant extracts using titanium (IV)[J].Analytical Biochemistry,1984,139(2):487-492.
[16] Pandey P,Srivastava R K,Rajpoot R,et al.Water deficit and aluminum interactive effects on generation of reactive oxygen species and responses of antioxidative enzymes in the seedlings of two rice cultivars differing in stress tolerance[J].Environmental Science and Pollution Research,2016,23(2):1516-1528.
[17] Singh N,Ma L Q,Srivastava M,et al.Metabolic adaptations to arsenic-induced oxidative stress in Pteris vittata L.and Pteris ensiformis L.[J].Plant Science,2006,170(2):274-282.
[18] Devos C H R,Vonk M J,Vooijs R,et al.Glutathione depletion due to copper-induced phytochelatin synthesis causes oxidative stress in silene-Cucubalus[J].Plant Physiology,1992,98:853-858.
[19] Fuerst E P,Irzyk G P,Miller K D.Partial characterization of glutathione S-transferase isozymes induced by the herbicide safener benoxacor in maize[J].Plant Physiology,1993,102(3):795-802.
[20] 董亚茹，张艳波，赵东晓，等.外源24-表油菜素内酯对NaCl胁迫下桑树幼苗的缓解效应[J].核农学报，2021,35(6):1466-1475.
[21] Liu Y J,Ji X Y,Nie X G,et al.Arabidopsis AtbHLH112 regulates the expression of genes involved in abiotic stress tolerance by binding to their E-box and GCG-box motifs[J].New Phytologist,2015,207(3):692-709.
[22] Xu Z S,Chen M,Li L C,et al.Functions and application of the AP2/ERF transcription factor family in crop improvement[J].Journal of Integrative Plant Biology,2011,53(7):570-585.
[23] Klay I,Gouia S,Lu M,et al.Ethylene Response Factors (ERF) are differentially regulated by different abiotic stress types in tomato plants[J].Plant Science,2018,274:137-145.
[24] Zhang G Y,Chen M,Li L C,et al.Overexpression of the soybean GmERF3 gene,an AP2/ERF type transcription factor for increased tolerances to salt,drought,and diseases in transgenic tobacco[J].Journal of Experimental Botany,2009,60(13):3781-3796.
[25] Zhang J,Shi S Z,Jiang Y N,et al.Genome-wide investigation of the AP2/ERF superfamily and their expression under salt stress in Chinese willow (Salix matsudana)[J].Peerj,2021,9(6):e11076.
[26] Abogadallah G M,Nada R M,Malinowski R,et al.Overexpression of HARDY,an AP2/ERF gene from Arabidopsis,improves drought and salt tolerance by reducing transpiration and sodium uptake in transgenic Trifolium alexandrinum L.[J].Planta,2011,233(6):1265-1276.
[27] 张群，及晓宇，贺子航，等.白桦BpGRAS1基因的克隆及耐盐功能分析[J].南京林业大学学报(自然科学版),2021,45(5):38-46.
[28] Qin L P,Wang L Q,Guo Y,et al.An ERF transcription factor from Tamarix hispida,ThCRF1,can adjust osmotic potential and reactive oxygen species scavenging capability to improve salt tolerance[J].Plant Science,2017,265:154-166.
[29] Moller I M.Plant mitochondria and oxidative stress:electron transport,NADPH turnover,and metabolism of reactive oxygen species[J].Annual Review of Plant Physiology and Plant Molecular Biology,2001,52:561-591.
[30] Gill S S,Tuteja N.Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants[J].Plant Physiology and Biochemistry,2010,48(12):909-930.
[31] 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[J].Plant Cell,2015,27(5):1445-1460.
[32] Davey M W,Stals E,Panis B,et al.High-throughput determination of malondialdehyde in plant tissues[J].Analytical Biochemistry,2005,347(2):201-207.
[33] Hasan M S,Singh V,Islam S,et al.Genome-wide identification and expression profiling of glutathione S-transferase family under multiple abiotic and biotic stresses in Medicago truncatula L.[J].PLoS ONE,2021,16 (2):1-17.
[34] Xu J,Tian Y S,Xing X J,et al.Over-expression of AtGSTU19 provides tolerance to salt,drought and methyl viologen stresses in Arabidopsis[J].Physiologia Plantarum,2016,156:164-175.
[35] Yang G Y,Wang Y C,Xia D A,et al.Overexpression of a GST gene (ThGSTZ1) from Tamarix hispida improves drought and salinity tolerance by enhancing the ability to scavenge reactive oxygen species[J].Plant Cell Tissue and Organ Culture,2014,117:99-112.
[36] Wang F B,Tong W J,Zhu H,et al.A novel Cys(2)/His(2) zinc finger protein gene from sweet potato,IbZFP1,is involved in salt and drought tolerance in transgenic Arabidopsis[J].Planta,2016,243(3):783-797.
[37] Li H,Liu J X,Wang Y,et al.The ascorbate peroxidase 1 regulates ascorbic acid metabolism in fresh-cut leaves of tea plant during postharvest storage under light/dark conditions[J].Plant Science,2020,296:110500.
[38] Ahammed G J,Wang Y Q,Mao Q,et al.Dopamine alleviates bisphenol A-induced phytotoxicity by enhancing antioxidant and detoxification potential in cucumber[J].Environmental Pollution,2020,259:113957.
[39] Ma Y,Liu H H,Wu J X,et al.The adverse health effects of bisphenol A and related toxicity mechanisms[J].Environmental Research,2019,176:108575.
[40] Geu-Flores F,Moldrup M E,Bottcher C,et al.Cytosolic gamma-glutamyl peptidases process glutathione conjugates in the biosynthesis of glucosinolates and camalexin in Arabidopsis[J].Plant Cell,2011,23:2456-2469.
[41] Stein H,Honig A,Miller G,et al.Elevation of free proline and proline-rich protein levels by simultaneous manipulations of proline biosynthesis and degradation in plants[J].Plant Science,2011,181(2):140-150.
基本信息:
DOI：10.14083/j.issn.1001-4942.2022.04.002
中图分类号:S888.2
引用信息:
[1]董亚茹,聂玉霞,李云芝,等.瞬时过表达MnERF2基因对桑树耐盐性的影响[J].山东农业科学,2022,54(04):9-16.DOI:10.14083/j.issn.1001-4942.2022.04.002.
基金信息:
山东省自然科学基金项目(ZR2019PC043); 烟台市科技创新发展计划项目(2021XDHZ057); 山东省科技特派员行动计划项目(2020KJTPY070); 山东省现代农业产业技术体系蚕桑产业创新团队项目(SDAIT-18-03)
2021-11-11
2021
2022-04-08
2022
2
2022-04-13
2022-04-13
2022-04-13