Effects of peroxidase gene <i>PagPRX</i>19 on salt tolerance of poplar ‘84K’

HUANG Qingchen; LAI Jianxin; HUANG Lichao; LU Mengzhu

doi:10.11833/j.issn.2095-0756.20220387

Volume 39 Issue 6

Nov. 2022

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HUANG Qingchen, LAI Jianxin, HUANG Lichao, LU Mengzhu. Effects of peroxidase gene PagPRX19 on salt tolerance of poplar ‘84K’[J]. Journal of Zhejiang A&F University, 2022, 39(6): 1163-1172. doi: 10.11833/j.issn.2095-0756.20220387

Citation:

HUANG Qingchen, LAI Jianxin, HUANG Lichao, LU Mengzhu. Effects of peroxidase gene PagPRX19 on salt tolerance of poplar ‘84K’[J]. Journal of Zhejiang A&F University, 2022, 39(6): 1163-1172. doi: 10.11833/j.issn.2095-0756.20220387

Effects of peroxidase gene PagPRX19 on salt tolerance of poplar ‘84K’

doi: 10.11833/j.issn.2095-0756.20220387

HUANG Qingchen^{1, 2
,},
LAI Jianxin^{1, 2},
HUANG Lichao^{1, 2
,
,},
LU Mengzhu^{1, 2}

1.
College of Forestry and Biotechnology, Zhejiang A&F University, Hangzhou 311300, Zhejiang, China
2.
State Key Laboratory of Subtropical Silviculture, Zhejiang A&F University, Hangzhou 311300, Zhejiang, China

Received Date: 2022-06-09
Accepted Date: 2022-09-19
Rev Recd Date: 2022-09-14

Available Online: 2022-11-21

Publish Date: 2022-12-20

Abstract

Objective Salt damage, as an abiotic stress factor affecting plant growth and development, seriously threatens tree growth. Under salt stress, the level of endogenous reactive oxygen species (ROS) in plants increases, resulting in oxidative stress and affecting the normal growth and development of plants. By enhancing the expression level of PRX family members and changing ROS levels, the salt tolerance ability of poplar can be enhanced, and the mechanism of PRX members involved in regulating salt stress response can be revealed. Method Poplar ‘84K’ (Populus alba × P. glandulosa ‘84K’) was used as material, and PagPRX19, a member of PRX family, was selected for cloning and construction of overexpression vector by bioinformatics analysis. Overexpressed plants were obtained by Agrobacterium tumefaciens mediated leaf disk transformation. The tissue culture seedlings of non-transgenetic plants (the control) and PagPRX19 overexpressed plants growing for 45 days and the soil culture seedlings growing for 2 months were used as experimental materials for salt stress treatment. The plant phenotype was observed and the physiological indexes such as proline, malondialdehyde and electrolyte permeability were detected and analyzed. Result (1) PagPRX19 gene was cloned, overexpression vector was constructed, and transgenic positive plants were obtained. After molecular identification, 2 overexpressed lines OE#1 and OE#2 were used for further analysis. (2) Preliminary phenotypic observation showed that PagPRX19 overexpressed plants decreased in height and increased in ground diameter, compared with the control. (3) Compared with the control, the overexpressed plants showed lower leaf shrinkage and plant growth inhibition under salt stress. Tissue culture seedlings showed similar results under salt stress. (4) ROS level of transgenic plants decreased, and ROS level of leaves and roots of overexpressed plants remained lower than that of the control under salt stress. The content of proline in overexpressed plants increased, the water holding capacity of leaves increased, and the contents of malondialdehyde and electrolyte permeability were lower than those of wild type. The transgenic plants showed high salt tolerance from the physiological aspect. Conclusion Overexpression of PagPRX19 can reduce ROS level of transgenic poplar ‘84K’ plants under salt stress, alleviate oxidative stress, and enhance salt tolerance. [Ch, 11 fig. 23 ref.]
- Populus alba × P. glandulosa ‘84K’,
- peroxidase,
- reactive oxygen species (ROS),
- salt stress,
- salt tolerance

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土地盐碱化增大了土壤渗透压，导致植物吸水困难，对植物造成了生理干旱^[1]。过多钠离子(Na⁺)、氯离子(Cl⁻)的积累，导致膜结构破坏，对植物造成渗透胁迫^[2−3]。盐胁迫还导致植物内源活性氧(ROS)增加，引起细胞膜损伤甚至细胞死亡，抑制植株生长发育^[4]。ROS作为响应盐胁迫的关键因子，在低水平下，诱导增强抗氧化酶活性，抵御盐胁迫；在高水平下，过量积累造成氧化胁迫，导致生物大分子产生不可逆的损伤，改变细胞形态结构，抑制植株生长发育^[5]。为缓解ROS积累引起的氧化胁迫，植物通过增强抗氧化酶系统相关酶活性来降低体内的ROS，从而提高抗逆性^[6]。植物的抗氧化酶系统主要包括超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、抗坏血酸过氧化物酶(APX)、过氧化物酶(PRX)等^[7]。

在小麦Triticum aestivum中过表达TaPRX-2A，植株抗氧化能力增强，ROS下降，耐盐性增加^[8]。拟南芥Arabidopsis thaliana AtPRX19参与胁迫(盐害、干旱、病虫害等)后的氧化应激反应，使得ROS增加，对植物造成氧化胁迫。盐胁迫下，拟南芥AtPRX19表达量上调，ROS减少，抵御胁迫能力增强^[9]。在胡萝卜Daucus carota中异源表达OsPRX114同样降低过氧化氢(H₂O₂)水平，提高植株的耐盐性^[10]。玉米Zea mays的PRX家族成员ZmPRX26、ZmPRX42、ZmPRX71、ZmPRX75和ZmPRX78参与了对包括盐胁迫在内多种非生物胁迫的响应^[11]。部分PRX家族成员通过协调水杨酸(SA)、茉莉酸(JA)和乙烯(ET)等激素水平发挥作用^[12]。

研究林木对盐胁迫的响应，揭示耐盐性相关机理，对培育耐盐性更强林木品种具有重要意义。为研究杨树Populus PRX家族对林木耐盐性的影响，本研究以银腺杨‘84K’ Populus alba × P. glandulosa ‘84K’ (84K杨)为材料，通过构建PagPRX19的过表达转基因株系，改变杨树H₂O₂水平，并分析了杨树耐盐相关生理指标，以期揭示PagPRX19参与调控杨树盐胁迫响应的机制，为杨树的分子育种提供理论依据。

1. 材料与方法

1.1. 材料

研究材料为84K杨，过表达PagPRX19株系为本研究获得。

1.2. 方法

1.2.1. 生物信息学分析

Phytozome v13数据库中获取毛果杨Populus trichocarpa和拟南芥的PRX家族的蛋白序列、蛋白编码区(CDS)序列、启动子序列。利用P1ant CARE (http://bioinformatics.psb.u-gent.be/webtooLs/pLantcare/html/)在线软件对PRX家族启动子顺式作用元件预测。使用MEGA v7软件，构建拟南芥和杨树的PRX家族的系统发育树，分析杨树和拟南芥PRX基因家族成员在进化关系上的同源性。利用MEGE v5.4.1 (https://meme-suite.org/meme/tooLs/meme)在线软件分析PRX家族基因保守结构域。

1.2.2. 基因表达模式分析

选用生长一致的84K杨树苗，分别从茎尖(SAM)、茎段3~10节间(IN₃~IN₁₀)、形成层(Ca)、幼叶(YL)、成熟叶(ML)、根(R)、木质部(Xy)取样，所有材料取3个生物学重复。采用实时荧光定量PCR (RT-qPCR)检测该基因在不同组织内的表达模式。

1.2.3. 转基因苗获得

在84K杨基因组数据库中，通过Blast获取毛果杨PRX19同源基因PagPRX19，设计引物扩增获得目的基因(F端引物：ATGTATACAACAATCATGCCT，R端引物：TTATGTATGCATACTGCAAAC)，使用Gateway技术构建过表达载体35S::PagPRX19，利用叶盘转化法转化84K杨获得过表达PagPRX19转基因苗^[13]。

1.2.4. 盐胁迫处理

①组培盐胁迫处理。以组培培养45 d的过表达株系为材料，以非转基因植株为对照。盐胁迫组接顶芽于100 mmol·L⁻¹氯化钠(NaCl)生根培养基，对照组接顶芽于0 mmol·L⁻¹NaCl生根培养基。每组处理12瓶，每瓶接2株顶芽。培养温度为23~25 ℃，光周期为8 h/16 h(黑暗/光照)。处理25 d，观察植株在长期盐胁迫下耐盐能力。②土培盐胁迫处理。以土培生长2个月的过表达株系为实验材料，以非转基因植株为对照，每组处理5株。盐胁迫组隔2 d浇灌1次100 mmol·L⁻¹的NaCl溶液，对照组隔2 d浇灌1次0 mmol·L⁻¹的NaCl溶液。培养温度为23~25 ℃，光周期为8 h/16 h(黑暗/光照)。处理7 d，取样测定各项生理指标，处理12 d后拍照^[14]。

1.2.5. 生理指标测定

①脯氨酸质量分数测定。采用脯氨酸测试盒(南京建成，A107-1-1)测定脯氨酸质量分数。②相对含水量测定。取新鲜叶片记录鲜质量(W_F)，超纯水(ddH₂O)浸泡24 h记录质量(W_T)，65 ℃烘箱干燥3 d，记录干质量(W_D)。叶片相对含水量=(W_F−W_D)/(W_T−W_D)×100%^[15]。③丙二醛(MDA)质量摩尔浓度测定。称取植物叶片0.2 g，加入4 mL质量浓度为10%的三氯乙酸(TCA)溶液和石英砂研磨至匀浆，4 000 r·min⁻¹离心10 min，取上清液2 mL，加入2 mL硫代巴比妥酸(TAB)溶液，对照为2 mL ddH₂O。摇匀后沸水浴15 min，−20 ℃迅速冷却后4 000 r·min⁻¹离心2 min，取上清液，在532和600 nm波长下测量吸光度。MDA浓度C_MDA(μmol·L⁻¹)=[D(532)−D(600)]/155×1 000，MDA质量摩尔浓度(nmol·g⁻¹)=(C_MDA×V_{提取液体积})/F_样品质量^[16]。④电解质渗透率测定。采用五点取样法取样，加入6 mL ddH₂O，28 ℃摇床震荡1 h。取出测第1次电导值(G₁)。沸水浴30 min，冷却至室温，摇匀测量第2次电导值(G₂)。电解质渗透率=第1次电导值/第2次电导值×100%^[17]。⑤ROS水平定性测量。DAB染色剂试剂盒(索莱宝，DA1010)染色检测H₂O₂；氯化硝基四氮唑蓝(NBT)粉末(索莱宝，N8140)染色检测超氧阴离子(O₂ ^.−)。

3. 讨论

过氧化酶PRX可催化H₂O₂，降低ROS水平，减轻胁迫对细胞的伤害^[3]。因此，通过抗氧化酶清除盐胁迫产生的ROS，以抵御胁迫带来的细胞损伤，提高林木的抗性，可作为抗逆分子育种手段。本研究通过过表达过氧化酶基因，改变84K杨内源ROS水平，从而探究ROS水平对杨树耐盐性的影响。

生物信息学分析发现：PagPRX19存在多个与IAA、ABA、或MeJA相关的顺式作用元件，这些激素与盐胁迫密切相关，参与植物耐盐调控。基因表达模式分析发现：PagPRX19在植株各个组织都有表达。因此选取该基因作为研究对象，创制过表达植株。与对照植株相比，过表达植株叶片和茎段ROS水平降低。过基因植株H₂O₂降低，O₂ ^.−也随之降低。可能是过表达加速了H₂O₂的清除，植物自身调控SOD酶加速分解O₂ ^.−产生H₂O₂，从而导致了O₂ ^.−减少^[9]。对获得的转基因植株进行盐胁迫处理，在100 mmol·L⁻¹NaCl处理下，对照植株出现了胁迫症状，但过表达植株症状轻或没有出现症状。过表达植株可能通过降低盐胁迫下植株氧化胁迫程度，增强耐盐性。ROS水平检测结果验证了这一推论，盐胁迫下，过表达植株与对照相比，ROS水平仍保持较低水平。表明过表达PagPRX19可降低盐胁迫下ROS的伤害，从而增强了转基因植株的抗性。

研究表明：盐胁迫下脯氨酸质量分数增加^[17]，葡萄Vitis vinifera砧木在一定程度内的耐盐性越强，脯氨酸等有机渗透物质就越多^[18]。本研究表明：盐胁迫下，过表达PagPRX19植株脯氨酸质量分数高于对照植株，使得植株的耐盐能力增强。此外，盐胁迫造成植物叶片含水量下降，植物生理活动会受到影响^[19]。丙二醛和电解质渗透率反映了细胞膜损坏程度^[20]。盐胁迫下，植株细胞膜被破坏，细胞膜内物质外渗，电解质渗透率升高，丙二醛增加^[21−22]，而耐盐品种一般表现为叶片相对含水量高、电解质渗透率和丙二醛低的特点^{[19, 23]}。本研究中，盐胁迫下PagPRX19过表达植株叶片相对含水量比对照高，丙二醛、电解质渗透率降低，表明转基因植株细胞膜完整性较好，耐盐性增强。

4. 结论

过表达过氧化物酶基因PagPRX19可降低84K杨植株内源ROS水平，且在盐胁迫下过表达植株ROS仍保持低水平，细胞膜的破坏程度降低、叶片的持水能力增强。表明在杨树中过表达PagPRX19，能促进盐胁迫下植株内源ROS的清除，从而减缓盐胁迫造成的氧化胁迫，提高植株的耐盐性。

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