小麦TaLCD基因的克隆及其对渗透胁迫的调节作用

doi:10.11983/CBB19109

摘要/Abstract

摘要： 半胱氨酸脱巯基酶(CDes)可催化降解半胱氨酸(Cys)生成硫化氢(H₂S)。通过克隆小麦(Triticum aestivum)中的L-半胱氨酸脱巯基酶基因TaLCD, 并将其在拟南芥(Arabidopsis thaliana)中过表达, 探讨TaLCD对渗透胁迫条件下种子萌发和根系生长的影响, 并分析其对干旱胁迫的调节作用。结果显示, 盐胁迫条件下, TaLCD过表达植株种子萌发率显著高于野生型; 甘露醇处理条件下, TaLCD过表达植株的根长也显著高于野生型, 且TaLCD过表达显著提高植株抗旱性。此外, TaLCD过表达植株对ABA更加敏感, ABA处理下TaLCD过表达植株的种子萌发率及根长均显著低于野生型。干旱胁迫下, TaLCD过表达植株胁迫响应基因(COR47、RD29A、RAB18和RD22)及ABA信号途径相关基因(NCED3、HAB1、HAB2、ABI1、ABI2和ABF2)的表达水平均显著高于野生型。因此推测, TaLCD增强植株抗旱和抗盐能力可能依赖于ABA信号途径。

关键词: TaLCD, 渗透胁迫, ABA, 萌发和生长

Abstract: Cysteine desulphydrase (CDes) can degrade cysteine (Cys) to form hydrogen sulfide (H₂S). In this study, L-cysteine desulphydrase TaLCD from wheat (Triticum aestivum) was cloned and overexpressed in Arabidopsis thaliana. The effects of TaLCD on seed germination and root growth under osmotic stress in TaLCD overexpressing plants were examined, and the response to drought stress was also investigated. The results showed that, the seed germination rate of TaLCD overexpressing plants under salt treatment was remarkably higher than the wild type (WT), and the root length of TaLCD overexpressing plants was obviously longer when exposed to mannitol. Moreover, overexpression of TaLCD distinctly increased plant drought resistance. In addition, TaLCD overexpressing plants were more sensitive to ABA, with decreased seed germination rate and root length under ABA treatment. Furthermore, the expression of stress-responsive genes (COR47, RD29A, RAB18 and RD22) and ABA signaling pathway-related genes (NCED3, HAB1, HAB2, ABI1, ABI2 and ABF2) was significantly higher in TaLCD overexpressing plants than that in WT under drought stress. These results suggest that TaLCD enhances the drought and salt tolerance of plants possibly by ABA signaling pathway.

Key words: TaLCD, osmotic stress, ABA, germination and growth

张扬,刘华杰,薛瑞丽,李海霞,李华. 小麦TaLCD基因的克隆及其对渗透胁迫的调节作用. 植物学报, 2020, 55(2): 137-146.

Yang Zhang,Huajie Liu,Ruili Xue,Haixia Li,Hua Li. Cloning of Wheat TaLCD Gene and Its Regulation on Osmotic Stress. Chinese Bulletin of Botany, 2020, 55(2): 137-146.

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链接本文: https://www.chinbullbotany.com/CN/10.11983/CBB19109

https://www.chinbullbotany.com/CN/Y2020/V55/I2/137

图/表 9

表1 qRT-PCR分析所用引物

Table 1 Primers used for qRT-PCR analysis

Gene name	Primer sequences
Gene name	Forward (5'-3')	Reverse (5'-3')
TaDCD	GAGGAAGGACGGAAGCCATAT	TCAGGGTCATCGCAAACAGAG
TaLCD	TCCATTACGCCTACGGAGCAG	CAAGCCGGACCTTACGACCA
ABF2	ATCAGAAGGGATAGGGAAGAGTAAT	TTGGTCTGCCGTGAATATCTGT
HAB1	GTGTTCTCGCCATGTCTAGGTC	CTATTTCGCAGACTTCTTGGTTG
HAB2	GCAGAAGTCCTTATTGCGAGTC	CTCAGAAGTTGCCACCTCCATA
ABI1	TGACGGCTGTGAAGAGAGTA	CCATCTCACACGCTTCTTCA
ABI2	ATTCAGACCATTCACTGACCCTC	GCTCCGTCGCCAGAACAAG
NCED3	TGGCTTCTTTCACGGCAAC	ACAACAATGGCGGGAGAGTTT
COR47	GAGGTTACGGATCGTGGAT	GAGCTGTTGGATCGGTGA
RAB18	ATGGCGTCTTACCAGAACCGTCCA	TACCCTTGGCCACCTGATCC
RD29A	GTTACTGATCCCACCAAAGAAGA	GGAGACTCATCAGTCACTTCCA
RD22	AGGGCTGTTTCCACTGAGG	CACCACAGATTTATCGTCAGACA
Actin2	TACCCGATGGGCAAGTCA	TGCTCATACGGTCAGCGATA

图1 TaLCD序列分析 (A) 小麦中LCD氨基酸序列与其它4个物种(粗山羊草、二穗短柄草、谷子和短花药野生稻)中LCD序列同源比对; (B) 5个物种间LCD进化树分析; (C) TaLCD的结构示意图, 黑框表示Aminotran_5结构域。

Figure 1 Sequence analysis of TaLCD (A) Comparison of the derived amino acid sequences of TaLCD in Triticum aestivum with other 4 species (Aegilops tauschii, Brachypodium distachyon, Setaria italica, and Oryza brachyantha); (B) Phylogenetic analysis of LCD in five species; (C) Schematic structures of TaLCD, black box indicates Aminotran_5 domain.

图2 NaCl和PEG处理下小麦TaLCD的表达及酶活变化 (A) NaCl处理下TaLCD的表达; (B) PEG处理下TaLCD的表达; (C) NaCl处理下TaLCD酶活变化; (D) PEG处理下TaLCD酶活变化。* 表示差异显著(P<0.05)。

Figure 2 The expression of TaLCD and enzyme activity of TaLCD in wheat under NaCl and PEG treatments (A) The expression of TaLCD under NaCl treatment; (B) The expression of TaLCD under PEG treatment; (C) The enzyme activity of TaLCD under NaCl treatment; (D) The enzyme activity of TaLCD under PEG treatment. * indicate significant differences (P<0.05).

图3 TaLCD转基因拟南芥植株中TaLCD的表达及酶活 (A) RT-PCR; (B) qRT-PCR; (C) NaCl处理下TaLCD的酶活变化。Marker: DNA分子量标准; WT: 野生型; 2-8、3-4、5-5和12-4: TaLCD转基因株系; * 表示差异显著(P<0.05)。

Figure 3 Expression of TaLCD and enzyme activity of TaLCD in transgenic Arabidopsis (A) RT-PCR; (B) qRT-PCR; (C) Enzyme activity of TaLCD under NaCl treatment. Marker: DNA marker; WT: Wild type; 2-8, 3-4, 5-5, and 12-4: TaLCD transgenic lines; * indicate significant differences (P<0.05).

图4 NaCl处理对TaLCD转基因拟南芥种子萌发率的影响 WT: 野生型; OX-LCD-1和OX-LCD-2: 转基因株系; * 表示差异显著(P<0.05)。

Figure 4 Seed germination rate of Arabidopsis lines expressing TaLCD under different concentrations of NaCl treatment WT: Wild type; OX-LCD-1 and OX-LCD-2: Transgenic lines; * indicate significant differences (P<0.05).

图5 不同浓度甘露醇处理下TaLCD转基因拟南芥的根长 (A) 表型变化(Bars=1 cm); (B) 根长变化数据统计。WT: 野生型; OX- LCD-1和OX-LCD-2: 转基因株系; * 表示差异显著(P<0.05)。

Figure 5 Root growth of Arabidopsis lines expressing TaLCD under different concentrations of mannitol treatment (A) Phenotype of root growth (Bars=1 cm); (B) Root elongation measurements. WT: Wild type; OX-LCD-1 and OX-LCD-2: Transgenic lines; * indicate significant differences (P<0.05).

图6 TaLCD转基因拟南芥的抗旱性 (A) 生长3周的野生型(WT)、OX-LCD-1和OX-LCD-2植株在干旱处理后的表型(Bars=2 cm); (B) 叶片失水率

Figure 6 Drought resistance of Arabidopsis lines expressing TaLCD (A) Phenotype of three week-old wild type (WT), OX-LCD-1 and OX-LCD-2 plants after drought treatment (Bars=2 cm); (B) Leaf water loss

图7 不同浓度ABA处理下TaLCD转基因拟南芥的种子萌发率及生长状况 (A) 生长10天后的表型(Bars=1 cm); (B) 培养4天后的种子萌发率; (C) 生长10天后的根长表型(Bars=0.4 cm); (D) 生长10天后的根长。WT: 野生型; ABA: 脱落酸; * 表示差异显著(P<0.05)。

Figure 7 Seed germination and growth of Arabidopsis lines expressing TaLCD under different concentrations of ABA treatment (A) Phenotype on the 10th day (Bars=1 cm); (B) Seed germination rate after 4 d incubation; (C) Phenotype of root growth on the 10th day (Bars=0.4 cm); (D) Root length on the 10th day. WT: Wild type; ABA: Abscisic acid; * indicate significant differences (P<0.05).

图8 干旱胁迫对TaLCD转基因拟南芥中胁迫响应基因及ABA信号途径相关基因表达的影响 (A)-(D) 胁迫响应基因; (E)-(J) ABA信号途径相关基因。WT: 野生型; OX-LCD-2: 转基因株系

Figure 8 The expression of stress response genes and ABA signaling related genes in Arabidopsis lines expressing TaLCD under drought stress (A)-(D) Stress response genes; (E)-(J) ABA signaling related genes. WT: Wild type; OX-LCD-2: Transgenic lines

参考文献 28

[1]	侯智慧, 刘菁, 侯丽霞, 李希东, 刘新 ( 2011). H₂S可能作为H₂O₂的下游信号介导茉莉酸诱导的蚕豆气孔关闭. 植物学报 46, 396-406.
[2]	刘菁, 侯智慧, 赵方贵, 刘新 ( 2011). H₂S介导ABA诱导蚕豆气孔运动的生理机制研究. 西北植物学报 31, 298-304.
[3]	单长卷, 周岩 ( 2011). 外源硫化氢对水分胁迫下玉米种子萌发和生长的影响. 广东农业科学 38(20), 28-30.
[4]	Chen J, Wang WH, Wu FH, You CY, Liu TW, Dong XJ, He JX, Zheng HL ( 2013). Hydrogen sulfide alleviates aluminum toxicity in barley seedlings. Plant Soil 362, 301-318.
[5]	Christou A, Manganaris GA, Papadopoulos I, Fotopoulos V ( 2013). Hydrogen sulfide induces systemic tolerance to salinity and non-ionic osmotic stress in strawberry plants through modification of reactive species biosynthesis and transcriptional regulation of multiple defence pathways. J Exp Bot 64, 1953-1966.
[6]	Dooley FD, Nair SP, Ward PD ( 2013). Increased growth and germination success in plants following hydrogen sulfide administration. PLoS One 8, e62048.
[7]	Fang HH, Liu ZQ, Long YP, Liang YL, Jin ZP, Zhang LP, Liu DM, Li H, Zhai JX, Pei YX ( 2017). The Ca ²⁺/CaM2- binding transcription factor TGA3 elevates LCD expression and H₂S production to bolster Cr ⁶⁺ tolerance in Arabidopsis. Plant J 91, 1038-1050.
[8]	García-Mata C, Lamattina L ( 2010). Hydrogen sulphide, a novel gasotransmitter involved in guard cell signaling. New Phytol 188, 977-984.
[9]	Guo HM, Xiao TY, Zhou H, Xie YJ, Shen WB ( 2016). Hydrogen sulfide: a versatile regulator of environmental stress in plants. Acta Physiol Plant 38, 16.
[10]	Jia HL, Hu YF, Fan TT, Li JS ( 2015). Hydrogen sulfide modulates actin-dependent auxin transport via regulating ABPs results in changing of root development in Arabidopsis. Sci Rep 5, 8251.
[11]	Jin ZP, Shen JJ, Qiao ZJ, Yang GD, Wang R, Pei YX ( 2011). Hydrogen sulfide improves drought resistance in Arabidopsis thaliana. Biochem Biophys Res Commun 414, 481-486.
[12]	Jin ZP, Xue SW, Luo YA, Tian BH, Fang HH, Li H, Pei YX ( 2013). Hydrogen sulfide interacting with abscisic acid in stomatal regulation responses to drought stress in Arabidopsis. Plant Physiol Biochem 62, 41-46.
[13]	Letunic I, Doerks T, Bork P ( 2015). SMART: recent updates, new developments and status in 2015. Nucleic Acids Res 43, D257-D260.
[14]	Li CJ, Liu ZJ, Zhang QR, Wang RZ, Xiao LT, Ma H, Chong K, Xu YY ( 2012). SKP1 is involved in abscisic acid signaling to regulate seed germination, stomatal opening and root growth in Arabidopsis thaliana. Plant Cell Environ 35, 952-965.
[15]	Li H, Gao MQ, Xue RL, Wang D, Zhao HJ ( 2015). Effect of hydrogen sulfide on D1 protein in wheat under drought stress. Acta Physiol Plant 37, 225.
[16]	Li H, Li M, Wei XL, Zhang X, Xue RL, Zhao YD, Zhao HJ ( 2017). Transcriptome analysis of drought-responsive genes regulated by hydrogen sulfide in wheat ( Triticum aestivum L.) leaves. Mol Genet Genomics 292, 1091-1110.
[17]	Ma DY, Ding HN, Wang CY, Qin HX, Han QX, Hou JF, Lu HF, Xie YX, Guo TC ( 2016). Alleviation of drought stress by hydrogen sulfide is partially related to the abscisic acid signaling pathway in wheat. PLoS One 11, e0163082.
[18]	Papenbrock J, Riemenschneider A, Kamp A, Schulz- Vogt HN, Schmidt A ( 2007). Characterization of cysteine-degrading and H₂S-releasing enzymes of higher plants—from the field to the test tube and back. Plant Biol 9, 582-588.
[19]	Shen JJ, Qiao ZJ, Xing TJ, Zhang LP, Liang YL, Jin ZP, Yang GD, Pei YX ( 2012). Cadmium toxicity is alleviated by AtLCD and AtDCD in Escherichia coli. J Appl Microbiol 113, 1130-1138.
[20]	Shi HT, Ye TT, Chan ZL ( 2013). Exogenous application of hydrogen sulfide donor sodium hydrosulfide enhanced multiple abiotic stress tolerance in bermudagrass (Cynodon dactylon(L). Pers.). Plant Physiol Biochem 71, 226-234.
[21]	Shi HT, Ye TT, Han N, Bian HW, Liu XD, Chan ZL ( 2015). Hydrogen sulfide regulates abiotic stress tolerance and biotic stress resistance in Arabidopsis. J Integr Plant Biol 57, 628-640.
[22]	Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S ( 2011). MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28, 2731-2739.
[23]	Wang YQ, Li L, Cui WT, Xu S, Shen WB, Wang R ( 2012). Hydrogen sulfide enhances alfalfa ( Medicago sativa) tolerance against salinity during seed germination by nitric oxide pathway. Plant Soil 351, 107-119.
[24]	Xie YJ, Lai DW, Mao Y, Zhang W, Shen WB, Guan RZ ( 2013). Molecular cloning, characterization, and expression analysis of a novel gene encoding L-cysteine desulfhydrase from Brassica napus. Mol Biotechnol 54, 737-746.
[25]	Zhang H, Hu LY, Hu KD, He YD, Wang SH, Luo JP ( 2008). Hydrogen sulfide promotes wheat seed germination and alleviates oxidative damage against copper stress. J Integr Plant Biol 50, 1518-1529.
[26]	Zhang H, Hu LY, Li P, Hu KD, Jiang CX, Luo JP ( 2010a). Hydrogen sulfide alleviated chromium toxicity in wheat. Biol Plantarum 54, 743-747.
[27]	Zhang H, Hu SL, Zhang ZJ, Hu LY, Jiang CX, Wei ZJ, Liu J, Wang HL, Jiang ST ( 2011). Hydrogen sulfide acts as a regulator of flower senescence in plants. Postharvest Biol Technol 60, 251-257.
[28]	Zhang H, Jiao H, Jiang CX, Wang SH, Wei ZJ, Luo JP, Jones RL ( 2010b). Hydrogen sulfide protects soybean seedlings against drought-induced oxidative stress. Acta Physiol Plant 32, 849-857.