青杄转录共激活因子PwMBF1c的功能研究与验证

doi:10.13560/j.cnki.biotech.bull.1985.2022-0812

摘要/Abstract

摘要：

克隆通过青杄干旱转录组筛选到的差异候选基因PwMBF1c，对其生物学功能进行分析和验证，以期能够解析青杄的耐旱机理，并为培育优良抗逆林木品种提供基因资源。利用RT-qPCR技术分析PwMBF1c对干旱、高温、低温及激素处理的响应。瞬时转化烟草叶片和洋葱表皮细胞检测PwMBF1c的亚细胞定位。利用酵母单杂验证PwMBF1c的转录活性。在拟南芥和马铃薯体系中验证了PwMBF1c抗旱应答的功能。PwMBF1c主要在成熟叶中表达，其转录水平受干旱、高温、水杨酸（SA）诱导上调；PwMBF1c的C端具有转录激活活性，而N端和全长不具有转录活性；PwMBF1c定位于细胞膜、细胞质和细胞核；过表达PwMBF1c显著提高了拟南芥和马铃薯的耐旱性。干旱胁迫下，与对照组Col-0和VC相比，过表达拟南芥株系PwMBF1c-L1和PwMBF1c-L2存活率更高，叶绿素含量较高，MDA含量较低；在PEG处理后，与野生型马铃薯Y5相比，过表达PwMBF1c显著提高了苗高。PwMBF1c的表达水平受干旱等多种逆境和激素的诱导，PwMBF1c主要定位于细胞膜、细胞质和细胞核，且该蛋白的C端具有转录激活活性。PwMBF1c能够显著提高转基因拟南芥和马铃薯的耐旱性。

关键词: 青杄, MBF1c共激活因子, 胁迫响应, 基因表达, 耐旱性

Abstract:

We cloned PwMBF1c, a candidate gene in response to drought stress based on the transcriptomes of Picea wilsonii under drought stress and verified its biological function in order to explore the drought tolerance mechanism of P. wilsonii and to provide genetic resources for breeding excellent resistant forest varieties. RT-qPCR was used to analyze the expression profile of PwMBF1c to drought, high temperature, low temperature and hormone treatments. Transiently transformed tobacco leaf and onion epidermal cells were used to detect the subcellular localization of PwMBF1c. The transcriptional activity of PwMBF1c was verified by yeast one-hybrid. The function of PwMBF1c was verified in Arabidopsis thaliana and potato systems. PwMBF1c was mainly expressed in mature leaves, and its transcription level was strongly up-regulated by drought, high temperature and salicylic acid（SA）treatment. The C-terminus of PwMBF1c showed transcriptional activation activity, but not for the N-terminus or full-length of PwMBF1c. PwMBF1c was localized in cell membrane, cytoplasm and nucleus, and overexpression of PwMBF1c significantly increased drought tolerance in both Arabidopsis and potato. Under drought stress, compared with the control（Col-0 and VC）, PwMBF1c-L1 and PwMBF1c-L2 lines showed higher survival rate, higher chlorophyll content and lower MDA content. After PEG treatment, PwMBF1c significantly increased seedling height compared with wild-type potato（Y5）. The expression of PwMBF1c significantly increased by multiple abiotic stresses such as drought. PwMBF1c is mainly localized in cell membrane, cytoplasm and nucleus, and the C-terminus of PwMBF1c showed transcriptional activation activity. PwMBF1c significantly enhanced the drought tolerance of A. thaliana and potato.

Key words: Picea wilsonii, MBF1c coactivator, stress response, gene expression, drought tolerance

刘奎, 李兴芬, 杨沛欣, 仲昭晨, 曹一博, 张凌云. 青杄转录共激活因子PwMBF1c的功能研究与验证[J]. 生物技术通报, 2023, 39(5): 205-216.

LIU Kui, LI Xing-fen, YANG Pei-xin, ZHONG Zhao-chen, CAO Yi-bo, ZHANG Ling-yun. Functional Study and Validation of Transcriptional Coactivator PwMBF1c in Picea wilsonii[J]. Biotechnology Bulletin, 2023, 39(5): 205-216.

图/表 9

表1 引物序列

Table 1 Primer sequences

用途Use	引物名称Primer name	序列Sequence
RT-qPCR	AtActin-F	GGTAACATTGTGCTCAGTGGTGG
	AtActin-R	AACGACCTTAATCTTCATGCTGC
	PwEF1α-RT-F	AACTGGAGAAGGAACCCAAG
	PwEF1α-RT-R	AACGACCCAATGGAGGATAC
	PwMBF1c-RT-F	ATGCCGAGCCGAACGAA
	PwMBF1c-RT-R	AACCGCCTTGGGATCACG
载体构建 Vector construction	pEASY-T1-PwMBF1c-F	GAAGGATGCCGAGCCGAA
	pEASY-T1-PwMBF1c-R	GATGACGACAAGCAATTAGTGAGA
	pGBKT7-PwMBF1c-F	CCGGAATTCATGCCGAGCCGAACGAAC
	pGBKT7-PwMBF1c-R	TCCCCCGGGGGTGGACTGATGACGACAAGCAATTA
	pGBKT7-PwMBF1c-C-F	CCGGAATTCGATTCGACATGCTATACAGAAGGC
	pGBKT7-PwMBF1c-C-R	TCCCCCGGGGGTGGACTGATGACGACAAGCAATTA
	pGBKT7-PwMBF1c-N-F	CCGGAATTCATGCCGAGCCGAACGAAC
	pGBKT7-PwMBF1c-N-R	TCCCCCGGGGGGCCTTCTGTATAGCATGTCGAATC

图1 PwMBF1c蛋白序列比对与系统进化树

Fig. 1 PwMBF1c protein sequence alignment and phylog-enetic tree

图2 不同逆境及激素处理下PwMBF1c表达水平检测

Fig. 2 Expressions of PwMBF1c under different stress conditions and hormone treatments

图3 PwMBF1c的亚细胞定位 GFP: pCM1205空载体；PwMBF1c-GFP: PwMBF1c与GFP标签的融合蛋白。A：PwMBF1c在烟草叶片的亚细胞定位；B：PwMBF1c在洋葱中的亚细胞定位。标尺Bar：50 µm

Fig. 3 Subcellular localization of PwMBF1c GFP: pCM1205 empty vector. PwMBF1c-GFP: Fusion protein of PwMBF1c and GFP tag. A: Subcellular localization of PwMBF1c in tobacco leaves. B: Subcellular localization of PwMBF1c in onion. The bar in the image: 50 µm

图4 PwMBF1c转录活性鉴定

Fig. 4 Identification of PwMBF1c transcriptional activity

图5 过表达PwMBF1c显著增强了拟南芥的干旱耐受性 Col-0：野生型；VC：空载对照；L1、L2：PwMBF1c过表达株系。A：干旱处理下，不同PwMBF1c转基因拟南芥株系的表型；B：PwMBF1c在不同株系中的相对表达量；C：干旱处理后的存活率；D、E：干旱处理下，叶片的MDA和叶绿素含量

Fig. 5 Overexpression of PwMBF1c significantly improved the drought tolerance of Arabidopsis Col-0: Wild type; VC: empty vector; L1, L2: overexpressing lines of PwMBF1c. A: Phenotypes of different PwMBF1c transgenic lines under drought treatment. B: Relative expressions of PwMBF1c in different lines. C: Statistics of survival rates after drought treatment. D, E: MDA content and chlorophyll content of isolated leaves under drought treatment

图6 不同株系拟南芥在干旱胁迫下的萌发率 A：0、100、200、300 mmol/L甘露醇处理下不同株系的萌发状况；B-E：0、100、200、300 mmol/L甘露醇处理下不同株系的萌发率

Fig. 6 Germination rates of different lines of Arabidopsis under drought stress A: Germination status of different lines treated with 0/100/200/300 mmol/L mannitol. B-E: Germination rates of different lines treated with 0,100,200, and 300 mmol/L mannitol.

图7 过表达PwMBF1c拟南芥的根长 A：甘露醇处理下不同株系的根长生长状况；B：甘露醇处理下不同株系的根长统计

Fig. 7 Root length of overexpressing PwMBF1c in Arabidopsis A: Root length growths of different lines treated with mannitol. B: Root length statistics of different lines treated with mannitol

图8 不同株系马铃薯的干旱表型 Y5：马铃薯野生型。A：在PEG处理下，不同株系的生长状况；B：在PEG处理下，不同株系苗高的统计

Fig. 8 Drought phenotypes of different potato lines Y5: Wild type. A: Growth status of different lines under PEG treatment. B: Seedling heights of different lines under PEG treatments

参考文献 35

[1]	Hommel M, Khalil-Ahmad Q, Jaimes-Miranda F, et al. Over-expression of a chimeric gene of the transcriptional co-activator MBF1 fused to the EAR repressor motif causes developmental alteration in Arabidopsis and tomato[J]. Plant Sci, 2008, 175(1-2): 168-177. doi: 10.1016/j.plantsci.2008.01.019 URL
[2]	Ray S, Dansana PK, Giri J, et al. Modulation of transcription factor and metabolic pathway genes in response to water-deficit stress in rice[J]. Funct Integr Genomics, 2011, 11(1): 157-178. doi: 10.1007/s10142-010-0187-y URL
[3]	Guo WL, Chen RG, Du XH, et al. Reduced tolerance to abiotic stress in transgenic Arabidopsis overexpressing a Capsicum annuum multiprotein bridging factor 1[J]. BMC Plant Biol, 2014, 14: 138. doi: 10.1186/1471-2229-14-138
[4]	Zhao Q, He L, Wang B, et al. Overexpression of a multiprotein bridging factor 1 gene DgMBF1 improves the salinity tolerance of Chrysanthemum[J]. Int J Mol Sci, 2019, 20(10): 2453. doi: 10.3390/ijms20102453 URL
[5]	Suzuki N, Rizhsky L, Liang HJ, et al. Enhanced tolerance to environmental stress in transgenic plants expressing the transcriptional coactivator multiprotein bridging factor 1c[J]. Plant Physiol, 2005, 139(3): 1313-1322. doi: 10.1104/pp.105.070110 pmid: 16244138
[6]	Wang XL, Du Y, Yu DQ. Trehalose phosphate synthase 5-dependent trehalose metabolism modulates basal defense responses in Arabidopsis thaliana[J]. J Integr Plant Biol, 2019, 61(4): 509-527. doi: 10.1111/jipb.v61.4 URL
[7]	Qin DD, Wang F, Geng XL, et al. Overexpression of heat stress-responsive TaMBF1c, a wheat(Triticum aestivum L.) Multiprotein Bridging Factor, confers heat tolerance in both yeast and rice[J]. Plant Mol Biol, 2015, 87(1-2): 31-45. doi: 10.1007/s11103-014-0259-9 URL
[8]	Tian XJ, Qin Z, Zhao Y, et al. Stress Granule-associated TaMBF1c confers thermotolerance through regulating specific mRNA translation in wheat(Triticum aestivum)[J]. New Phytol, 2022, 233(4): 1719-1731. doi: 10.1111/nph.v233.4 URL
[9]	Saleh O, Harb J, Karrity A, et al. Identification of differentially expressed genes in two grape varieties cultivated in semi-arid and temperate regions from West-Bank, Palestine[J]. Agri Gene, 2018, 7: 34-42. doi: 10.1016/j.aggene.2017.11.001 URL
[10]	Xu Y, Huang BR. Transcriptomic analysis reveals unique molecular factors for lipid hydrolysis, secondary cell-walls and oxidative protection associated with thermotolerance in perennial grass[J]. BMC Genomics, 2018, 19(1): 70. doi: 10.1186/s12864-018-4437-z pmid: 29357827
[11]	李小冬, 尚以顺, 武语迪, 等. 紫花苜蓿MsMBF1c基因在拟南芥中表达提高转基因植株的耐热性[J]. 草业学报, 2019, 28(10): 187-198. doi: 10.11686/cyxb2018739
	Li XD, Shang YS, Wu YD, et al. Overexpression of Medicago sativa Multi protein Bridging Factor 1c(MsMBF1c)enhances thermotolerance of Arabidopsis[J]. Acta Prataculturae Sin, 2019, 28(10): 187-198.
[12]	Yan Q, Hou HM, Singer SD, et al. The grape VvMBF1 gene improves drought stress tolerance in transgenic Arabidopsis thaliana[J]. Plant Cell Tissue Organ Cult, 2014, 118(3): 571-582. doi: 10.1007/s11240-014-0508-2 URL
[13]	齐瑞, 郭星, 赵阳, 等. 白龙江珍稀濒危植物大果青杄群落物种多样性特征分析[J]. 西北林学院学报, 2017, 32(2): 161-164.
	Qi R, Guo X, Zhao Y, et al. Species diversity characteristics of the community of rare and endangered Picea neoveitchii in the Bailongjiang River[J]. J Northwest For Univ, 2017, 32(2): 161-164.
[14]	Zhang H, Shi Y, Liu XR, et al. Transgenic creeping bentgrass plants expressing a Picea wilsonii dehydrin gene(PicW)demonstrate improved freezing tolerance[J]. Mol Biol Rep, 2018, 45(6): 1627-1635. doi: 10.1007/s11033-018-4304-7 pmid: 30105551
[15]	Li LL, Yu YL, Wei J, et al. Homologous HAP5 subunit from Picea wilsonii improved tolerance to salt and decreased sensitivity to ABA in transformed Arabidopsis[J]. Planta, 2013, 238(2): 345-356. doi: 10.1007/s00425-013-1894-0 URL
[16]	苗雅慧, 鞠丹, 梁珂豪, 等. 青杄转录因子基因PwNF-YB8的克隆与功能分析[J]. 林业科学, 2021, 57(5): 77-92.
	Miao YH, Ju D, Liang KH, et al. Cloning and functional analysis of transcription factor gene PwNF-YB8 from Picea wilsonii[J]. Sci Silvae Sin, 2021, 57(5): 77-92.
[17]	刘峻玲, 梁珂豪, 苗雅慧, 等. 青杄PwUSP1基因特征及对干旱和盐胁迫的响应[J]. 北京林业大学学报, 2020, 42(10): 62-70.
	Liu JL, Liang KH, Miao YH, et al. Characteristics of PwUSP1 in Picea wilsonii and its response to drought and salt stress[J]. J Beijing For Univ, 2020, 42(10): 62-70.
[18]	崔潇月. 青杄盐胁迫响应基因筛选及功能验证体系的建立[D]. 北京: 北京林业大学, 2018.
	Cui XY. Screening of salt stress response genes in Picea wilsonii and establishment of gene function verification system[D]. Beijing: Beijing Forestry University, 2018.
[19]	刘亚静, 马齐军, 李浩浩, 等. 苹果MdCBL3基因的克隆与功能鉴定[J]. 园艺学报, 2017, 44(1): 1-10. doi: 10.16420/j.issn.0513-353x.2016-0489
	Liu YJ, Ma QJ, Li HH, et al. Molecular cloning and functional characterization of MdCBL3 in apple[J]. Acta Hortic Sin, 2017, 44(1): 1-10.
[20]	Zhang FJ, Xie YH, Jiang H, et al. The ankyrin repeat-containing protein MdANK2B regulates salt tolerance and ABA sensitivity in Malus domestica[J]. Plant Cell Rep, 2021, 40(2): 405-419. doi: 10.1007/s00299-020-02642-9
[21]	Li FQ, Ueda H, et al. Multiprotein bridging factor 1(MBF1)is an evolutionarily conserved transcriptional coactivator that connects a regulatory factor and TATA element-binding protein[J]. Proc Natl Acad Sci USA, 1997, 94(14): 7251-7256. doi: 10.1073/pnas.94.14.7251 URL
[22]	Brendel C, Gelman L, Auwerx J. Multiprotein bridging factor-1(MBF-1)is a cofactor for nuclear receptors that regulate lipid metabolism[J]. Mol Endocrinol, 2002, 16(6): 1367-1377. pmid: 12040021
[23]	Wang YY, Wei XL, Huang J, et al. Modification and functional adaptation of the MBF1 gene family in the lichenized fungus Endocarpon pusillum under environmental stress[J]. Sci Rep, 2017, 7(1): 16333. doi: 10.1038/s41598-017-16716-4
[24]	Alavilli H, Lee H, Park M, et al. Antarctic moss multiprotein bridging factor 1c overexpression in Arabidopsis resulted in enhanced tolerance to salt stress[J]. Front Plant Sci, 2017, 8: 1206. doi: 10.3389/fpls.2017.01206 pmid: 28744295
[25]	Uji T, Sato R, Mizuta H, et al. Changes in membrane fluidity and phospholipase D activity are required for heat activation of PyMBF1 in Pyropia yezoensis(Rhodophyta)[J]. J Appl Phycol, 2013, 25(6): 1887-1893. doi: 10.1007/s10811-013-0006-7 URL
[26]	Zhang Y, Zhang G, Dong YL, et al. Cloning and characterization of a MBF1 transcriptional coactivator factor in wheat induced by stripe rust pathogen[J]. Acta Agronomica Sinica, 2009, 35(1): 11-17. doi: 10.3724/SP.J.1006.2009.00011 URL
[27]	Sugikawa Y, Ebihara S, Tsuda K, et al. Transcriptional coactivator MBF1s from Arabidopsis predominantly localize in nucleolus[J]. J Plant Res, 2005, 118(6): 431-437. pmid: 16283071
[28]	Kim GD, Cho YH, Yoo SD. Regulatory functions of evolutionarily conserved AN1/A20-like Zinc finger family proteins in Arabidopsis stress responses under high temperature[J]. Biochem Biophys Res Commun, 2015, 457(2): 213-220. doi: 10.1016/j.bbrc.2014.12.090 URL
[29]	赵楠. 菊花CmMBF1c基因调控耐涝性的分子机制研究[D]. 南京: 南京农业大学, 2019.
	Zhao N. Molecular mechanisms of CmMBF1c involved in chrysanthemum waterlogging tolerance[D]. Nanjing: Nanjing Agricultural University, 2019.
[30]	Hao YJ, Wei W, Song QX, et al. Soybean NAC transcription factors promote abiotic stress tolerance and lateral root formation in transgenic plants[J]. Plant J, 2011, 68(2): 302-313. doi: 10.1111/j.1365-313X.2011.04687.x URL
[31]	Rizhsky L, Liang HJ, Shuman J, et al. When defense pathways collide. the response of Arabidopsis to a combination of drought and heat stress[J]. Plant Physiol, 2004, 134(4): 1683-1696. doi: 10.1104/pp.103.033431 pmid: 15047901
[32]	Sah SK, Reddy KR, Li JX. Abscisic acid and abiotic stress tolerance in crop plants[J]. Front Plant Sci, 2016, 7: 571. doi: 10.3389/fpls.2016.00571 pmid: 27200044
[33]	Li YM, Sun XR, You JL, et al. Transcriptional analysis of the early ripening of ‘Summer Black’ grape in response to abscisic acid treatment[J]. Sci Hortic, 2022, 299: 111054. doi: 10.1016/j.scienta.2022.111054 URL
[34]	Baloglu MC, Inal B, Kavas M, et al. Diverse expression pattern of wheat transcription factors against abiotic stresses in wheat species[J]. Gene, 2014, 550(1): 117-122. doi: 10.1016/j.gene.2014.08.025 pmid: 25130909
[35]	Katano K, Kataoka R, Fujii M, et al. Differences between seedlings and flowers in anti-ROS based heat responses of Arabidopsis plants deficient in cyclic nucleotide gated channel 2[J]. Plant Physiol Biochem, 2018, 123: 288-296. doi: 10.1016/j.plaphy.2017.12.021 URL