蜡梅胚胎晚期丰富蛋白基因CpLEA的表达及抗性分析

doi:10.16420/j.issn.0513-353x.2021-0987

摘要/Abstract

摘要：

将蜡梅（Chimonanthus praecox）第3组LEA蛋白基因CpLEA在大肠杆菌中进行诱导表达，结果发现CpLEA重组蛋白在体外低温胁迫下能够保护乳酸脱氢酶的活性，且转CpLEA大肠杆菌的抗寒性有所提高；该基因在拟南芥中超表达，拟南芥的抗寒性和抗旱性均有所提高，且抗性的提高程度与基因过表达的积累量正相关，证实该基因能够参与植物的抗逆过程，在低温和渗透胁迫响应中发挥一定作用。通过qRT-PCR进一步分析花蕾期和露瓣期蜡梅瓶插花在低温（4 ℃）胁迫后CpLEA的表达特性，结果表明：与对照（22 ℃）相比，花蕾期CpLEA表达量在低温胁迫后逐渐升高，48 h达到峰值，约为未处理表达量的28倍，随后表达量下降，但仍然高于对照；露瓣期时该基因表达量随低温处理时间的延长逐渐升高，72 h后达到最高，约为未处理表达量的9倍。室温下蜡梅瓶插花中CpLEA的表达量基本恒定，而低温处理则可诱导该基因在蜡梅花发育早期表达量有不同程度的提高。

关键词: 蜡梅, LEA, 原核表达, 转基因, 低温胁迫

Abstract:

To characterize the function of CpLEA，a group three LEA protein gene of Chimonanthus praecox（wintersweet），CpLEA is transformed into Escherichia coli and Arabidopsis thaliana. Compared with the control strain，transgenic E. coli showed higher tolerance to low-temperature. In vitro，purified CpLEA protein could protect lactate dehydrogenase（LDH）from inactivation. Overexpression of the CpLEA gene in Arabidopsis also improved the resistance of transgenic Arabidopsis to cold and drought stresses and the degree of improved resistance was positively correlated with the accumulation of gene overexpression. The expression characteristics of CpLEA gene were further analyzed by qRT-PCR in wintersweet cutflower branch during flower-bud and display-petal stage respectively under 4 ℃ low-temperature treatment. CpLEA gene expression was found to gradually increase after low-temperature treatment in cutflower branch at flower-bud stage，reaching a peak at 48 h of treatment，which was about 28 times higher than expression of the untreated group，and then the expression decreased，but was still higher than the untreated group. The expression of CpLEA gradually increased with the extension of low-temperature treatment time in cutflower branch at display-petal stage，then reached the highest level at 72 h，which was nine times as much as the untreated group. The expression of CpLEA in cutflowers remained basically constant at room temperature，while the low-temperature treatment induced a different degree of expression increase of CpLEA in the early stage of flower development. These results indicated that the CpLEA would play a protective role in plant cells under low-temperature stress，especially in early stage of flower development.

Key words: Chimonanthus praecox, LEA, prokaryotic expression, transgenic, low-temperature stress

中图分类号:

S685.99

任菲, 卢苗苗, 刘吉祥, 陈信立, 刘道凤, 眭顺照, 马婧. 蜡梅胚胎晚期丰富蛋白基因CpLEA的表达及抗性分析[J]. 园艺学报, 2023, 50(2): 359-370.

REN Fei, LU Miaomiao, LIU Jixiang, CHEN Xinli, LIU Daofeng, SUI Shunzhao, MA Jing. Expression and Adversity Resistance Analysis of a Late Embryogenesis Abundant Protein Gene CpLEA from Chimonanthus praecox[J]. Acta Horticulturae Sinica, 2023, 50(2): 359-370.

图/表 10

表1 实时荧光定量引物序列

Table 1 Primer sequences used in real-time quantitative PCR

基因 Gene	正向引物（5′-3′） Forward primer	反向引物（5′-3′） Reverse primer
CpLEA	CAGGAGAAGACGAGTCAGATGGT	CCGAGTCCCTTGTAGATTGG
Atactin	CTTCGTCTTCCACTTCAG	ATCATACCAGTCTCAACAC
Actin	GTTATGGTTGGGATGGGACAGAAAG	GGGCTTCAGTAAGGAAACAGGA
Tublin	TAGTGACAAGACAGTAGGTGGAGGT	GTAGGTTCCAGTCCTCACTTCATC

图1 诱导大肠杆菌BL21/pET32a（+）-CpLEA获得CpLEA蛋白的SDS-PAGE电泳图 M：蛋白marker；1：未诱导；2：诱导后的总蛋白；3：诱导后的上清液；4：诱导后的沉淀；5：平衡缓冲液洗脱；6、7、8：20、50、300 mmol · L-1咪唑洗脱。

Fig. 1 SDS-PAGE electrophoresis of CpLEA protein obtained by induction of Escherichia coli BL21/pET32a（+）-CpLEA M：Protein marker；1：Total protein without induction；2：Total protein after induction；3：Supernatant protein after induction；4：Precipitation protein after induction；5：Buffering solution；6，7，8：20，50，300 mmol · L-1 imidazole elution.

图2 大肠杆菌转CpLEA菌株BL21/pET32a（+）-CpLEA和转空载体菌株BL21/pET32a（+）的菌落密度（A）和菌液生长曲线（B）

Fig. 2 Colony density（A）and bacterial growth curve（B）of Escherichia coli to CpLEA strain BL21/pET32a（+）-CpLEA and to empty vector strain BL21/pET32a（+）

图3 CpLEA蛋白在低温胁迫下对乳酸脱氢酶（LDH）活性的影响不同小写字母表示各反应体系间差异显著（P < 0.05）。

Fig. 3 Lactate dehydrogenase（LDH）activity by CpLEA protein under different stress conditions Different lowercase letters indicate significant differences among different reaction systems at 0.05 level.

图4 植物表达载体pCAMBIA2301G-CpLEA的PCR（A1）及酶切后的载体（B1） M：DL2000 marker；+：阳性对照质粒；-：阴性对照水。

Fig. 4 PCR（A1）and enzyme cutting verification（B1）electrophoresis of plant expression vector M：DL2000 marker；+：Positive controlar；-：Negative control，ddH2O.

图5 转CpLEA拟南芥株系PCR（A）、RT-PCR（B）及其CpLEA的表达（C） M：DL2000 marker；1 ~ 11、L-1 ~ L-9：转基因株系；+：质粒；-：阴性对照水；WT：野生型。

Fig. 5 PCR（A）and RT-PCR（B）of Arabidopsis thaliana overexpressing CpLEA lines and expression analysis of CpLEA（C） M：DL2000 marker；1-11，L-1-L-9：Transgenic lines；+：Positive controlar；-：Negative control，ddH2O；WT：Wild type.

图6 拟南芥过表达CpLEA株系（L-6、L-7、L-4）和野生型（WT）的抗旱性 A：10% PEG模拟干旱下的种子萌发；B：干旱胁迫后的幼苗表型；C：复水后的存活率；D：干旱胁迫后的细胞膜稳定性。不同小写字母表示材料间差异显著（P < 0.05）。

Fig. 6 Drought resistance in Arabidopsis thaliana overexpressing CpLEA lines（L-6，L-7，L-4）and wild type（WT） A：Seed germination under 10% PEG simulated drought；B：Seedling phenotype after drought stress；C：Survival rate after rewatering；D：CMS after drought stress. Different lowercase letters indicate significant differences among materials at 0.05 level.

图7 低温处理拟南芥过表达CpLEA株系（L-6、L-7、L-4）和野生型（WT）的表型

Fig. 7 Phenotype of Arabidopsis thaliana overexpressing CpLEA lines（L-6，L-7，L-4）and wild type（WT）after cold treatment

图 8 拟南芥过表达CpLEA株系（L-6、L-7、L-4）和野生型（WT）的的抗寒性相关指标不同小写字母表示相同处理不同材料间差异显著（P < 0.05）。

Fig. 8 Cold resistance in Arabidopsis thaliana overexpressing CpLEA lines（L-6，L-7，L-4）and wild type（WT） Different lowercase letters indicate significant differences among different materials with the same treatment at 0.05 level.

图9 CpLEA在蜡梅瓶插花不同花发育时期的表达分析不同小写字母表示相同处理不同处理时间差异显著（P < 0.05）。

Fig. 9 Expression analysis of CpLEA during flower stage Different lowercase letters indicate significant differences in the same treatment and different treatment times at 0.05 level.

参考文献 26

[1]	Babu R C, Zhang J X, Blum A, Ho D T, Wu R, Nguyen H. 2004. HVA1,a LEA gene from barley confers dehydration tolerance in transgenic rice(Oryza sativa L.)via cell membrane protection. Plant Science, 166 (4):855-862. doi: 10.1016/j.plantsci.2003.11.023 URL
[2]	Bao F, Du D L, An Y, Yang W R, Wang J, Cheng T R, Zhang Q X. 2017. Overexpression of Prunus mume dehydrin genes in tobacco enhances tolerance to cold and drought. Frontiers in Plant Science, 8:151-163.
[3]	Battaglia M, Olvera-Carrillo Y, Garciarrubio A, Campos F, Covarrubias A A. 2008. The enigmatic LEA proteins and other hydrophilins. Plant Physiol, 148 (1):6-24. doi: 10.1104/pp.108.120725 pmid: 18772351
[4]	Chen J, Fan L, Du Y, Zhu W N, Tang Z Q, Li N, Zhang D P, Zhang L S. 2016. Temporal and spatial expression and function of TaDlea3 in Triticum aestivum during developmental stages under drought stress. Plant Science, 252:290-299. doi: 10.1016/j.plantsci.2016.08.010 URL
[5]	Christian N, Jean D, Kenneth E, Tessa P, Norman H, Fathey S. 2002. Cold-regulated cereal chloroplast late embryogenesis abundant-like proteins. Molecular characterization and functional analyses. Plant Physiology, 129 (3):1368-1381. doi: 10.1104/pp.001925 pmid: 12114590
[6]	Cuevas-Velazquez C L, Rendon-Luna D F, Covarrubias A A. 2014. Dissecting the cryoprotection mechanisms for dehydrins. Frontiers in Plant Science, 5:1-6.
[7]	Fuxreiter M, Simon I, Friedrich P, Tompa P. 2004. Preformed structural elements feature in partner recognition by intrinsically unstructured proteins. Journal of Molecμlar Biology, 338 (5):1015-1026.
[8]	Gao T, Mo Y X, Huang H Y, Yu J M, Wang Y, Wang W D. 2021. Heterologous expression of Camellia sinensis Late Embryogenesis Abundant Protein Gene 1(CsLEA1)confers cold stress tolerance in Escherichia coli and yeast. Horticultural Plant Journal, 7 (1):89-96. doi: 10.1016/j.hpj.2020.09.005 URL
[9]	Ge Shibei, Jiang Xiaochun, Wang Lingyu, Yu Jingquan, Zhou Yanhong. 2020. Recent advances in the role and mechanism of arbuscular mycorrhizainduced improvement of abiotic stress tolerance in horticultural plan. Acta Horticulturae Sinica, 47 (9):1752-1776. (in Chinese)
	葛诗蓓, 姜小春, 王羚羽, 喻景权, 周艳虹. 2020. 园艺植物丛枝菌根抗非生物胁迫的作用机制研究进展. 园艺学报, 47 (9):1752-1776.
[10]	Grelet J, Benamar A, Teyssier E, Avelange-Macherel M H, Grunwald D, Macherel D. 2005. Identification in pea seed mitochondria of a late-embryogenes is abundant protein able to protect enzymes from drying. Plant Physiology, 137 (1):157-167. doi: 10.1104/pp.104.052480 URL
[11]	Hu Ting-zhang, Yang Jun-nian, Wu Ying-mei, Chen Zai-gang. 2012. Expression of OsLEA19a gene in Escherichia coli and its resistance analysis. Acta Agriculturae Boreali-Sinica, 27 (6):1-4. (in Chinese) doi: 10.3969/j.issn.1000-7091.2012.06.001
	胡廷章, 杨俊年, 吴应梅, 陈再刚. 2012. 水稻OsLEA19a基因在大肠杆菌中的表达和抗逆性分析. 华北农学报, 27 (6):1-4. doi: 10.3969/j.issn.1000-7091.2012.06.001
[12]	Hundertmark M, Hincha D K. 2008. LEA(late embryogenesis abundant)proteins and their encoding genes in Arabidopsis thaliana. BioMed Central, 9 (1):118.
[13]	Ingram J, Bartels D. 1996. The molecular basis of dehydration tolerance in plants. Annual Review of Plant Physiology and Plant Molecular Biology, 47:377-403. pmid: 15012294
[14]	Iturriaga G, Schneider K, Salamini F, Bartels D. 1992. Expression of desiccation-related proteins from the resurrection plant Craterostigma plantagineum in transgenic tobacco. Plant Molecular Biology, 20 (3):555-558. doi: 10.1007/BF00040614 pmid: 1421157
[15]	Liu Y, Liang J N, Sun L P, Yang X H, Li D Q. 2016. Group 3 LEA protein,ZmLEA3,is involved in protection from low temperature stress. Frontiers in Plant Science, 7:1011. doi: 10.3389/fpls.2016.01011 pmid: 27471509
[16]	Liu Y, Zheng Y Z. 2005. PM2,a group 3 LEA protein from soybean,and its 22-mer repeating region confer salt tolerance in Escherichia coli. Biochemical and Biophysical Research Communications, 331 (1):325-332. doi: 10.1016/j.bbrc.2005.03.165 URL
[17]	Liu Zi-cheng, Miao Li-li, Wang Jing-yi, Yang De-long, Mao Xin-guo, Jing Rui-lian. 2016. Cloning and characterization of transcription factor TaWRKY35 in wheat(Triticum aestivum). Scientia Agricultura Sinica, 49 (12):2245-2254. (in Chinese) doi: 10.3864/j.issn.0578-1752.2016.12.001
	刘自成, 苗丽丽, 王景一, 杨德龙, 毛新国, 景蕊莲. 2016. 普通小麦转录因子基因TaWRKY35的克隆及功能分析. 中国农业科学, 49 (12):2245-2254. doi: 10.3864/j.issn.0578-1752.2016.12.001
[18]	Ma Jing, Sun Wen-ting, Wang Jing, Sui Shun-zhao, Li Ming-yang. 2014. Cloning and expression analysis of a late embryogenesis abundant protein gene CpLEA from Chimonanthus praecox. Acta Horticulturae Sinica, 41 (8):1663-1672. (in Chinese)
	马婧, 孙文婷, 王晶, 眭顺照, 李名扬. 2014. 蜡梅胚胎晚期丰富蛋白基因CpLEA的克隆及表达分析. 园艺学报, 41 (8):1663-1672.
[19]	Mariana A, Juriaan R, Timothy J, Jill M, Wilco L, Henk H. 2019. Structural plasticity of intrinsically disordered lea proteins from Xerophyta schlechteri provides protection in vitro and in vivo. Frontiers in Plant Science, 10 (10):1272-1279. doi: 10.3389/fpls.2019.01272 URL
[20]	Sun M Z, Shen Y, Yin K D, Guo Y X, Cai X X, Yang J K, Zhu Y M, Jia B W, Sun X L. 2019. A late embryogenesis abundant protein GsPM30 interacts with a receptor like cytoplasmic kinase GsCBRLK and regulates environmental stress responses. Plant Science:an International Journal of Experimental Plant Biology, 283:70-82.
[21]	Tang Xiao-qian, Yu Li-xia, Wu Xiao-lu, Yan Bo. 2010. Research advance in the group three of late-embryogenesis-abundant proteins and genes. Chinese Bulletin of Life Sciences, 22 (6):551-555. (in Chinese)
	汤晓倩, 于丽霞, 武晓璐, 鄢波. 2010. 第三组胚胎晚期丰富蛋白lea3基因研究概述. 生命科学, 22 (6):551-555.
[22]	Wang L J, Li X F, Chen S Y, Liu G S. 2009. Enhanced drought tolerance in transgenic Leymus chinensis plants with constitutively expressed wheat TaLEA3. Biotechnology Letters, 31 (2):313-319. doi: 10.1007/s10529-008-9864-5 URL
[23]	Wang W G, Li R, Liu B, Li L, Wang S H, Chen F. 2012. Alternatively splicedtranscripts of group 3 late embryogenesis abundant protein from Pogonatherum paniceum confer different abiotic stress tolerance in Escherichia coli. Journal of Plant Physiology, 169 (15):1559-1564. doi: 10.1016/j.jplph.2012.06.017 URL
[24]	Wang X Y, Zhang L S, Zhang Y N, Bai Z Q, Liu H, Zhang D P. 2017. Triticum aestivum WRAB 18 functions in plastids and confers abiotic stress tolerance when overexpressed in Escherichia coli and Nicotiania benthamiana. PLoS ONE, 12 (2):320-340.
[25]	Yu J N, Zhang J S, Shan L, Chen S Y. 2005. Two new group 3 LEA genes of wheat and their functional analysis in yeast. Journal of Integrative Plant Biology, 47 (11):1372-1381. doi: 10.1111/jipb.2005.47.issue-11 URL
[26]	Zhao Bing, Zhang Qi-xiang. 2008. Variation analysis of floral traits in Chinese Chimonanthus praecox germplasm resources. Acta Horticulturae Sinica, 35 (3):383-388. (in Chinese)
	赵冰, 张启翔. 2008. 中国蜡梅种质资源花性状的变异分析. 园艺学报, 35 (3):383-388.