Effect of Cre-miR171 on Drought Stress Response in Citrus

FENG Jipeng; HU Zhou; ZHANG Manman; WANG Xiaoli; YI Qian; ZHU Shiping; ZHAO Xiaochun

doi:10.13718/j.cnki.xdzk.2023.07.011

2023 Volume 45 Issue 7

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FENG Jipeng, HU Zhou, ZHANG Manman, et al. Effect of Cre-miR171 on Drought Stress Response in Citrus[J]. Journal of Southwest University Natural Science Edition, 2023, 45(7): 123-137. doi: 10.13718/j.cnki.xdzk.2023.07.011

Citation:

FENG Jipeng, HU Zhou, ZHANG Manman, et al. Effect of Cre-miR171 on Drought Stress Response in Citrus[J]. Journal of Southwest University Natural Science Edition, 2023, 45(7): 123-137. doi: 10.13718/j.cnki.xdzk.2023.07.011

Effect of Cre-miR171 on Drought Stress Response in Citrus

1.
Citrus Research Institute, Southwest University/National Citrus Engineering Research Center, Chongqing 400712, China
2.
Integrative Science Center of Germplasm Creation in Western China(CHONGQING) Science City, Chongqing 401329, China

More Information

Corresponding author: ZHU Shiping ;
Received Date: 17/04/2023
Available Online: 20/07/2023
MSC: S666;Q344⁺.13

Abstract

Drought is an important environmental factor affecting the citrus production. In order to explore the role of Cre-miR171 in citrus drought tolerance, this study analyzed the cis-acting elements of its precursor upstream promoter, studied the expression characteristics in different citrus tissues, and growth and development stages, verified the target genes of Cre-miR171, and evaluated the expression patterns of Cre-miR171 and its target genes in different parts of roots under drought stress, as well as the drought tolerance of Cre-miR171 overexpression Arabidopsis and citrus plants. The results showed that the promoter region of Cre-miR171 precursor sequence has a variety of elements related to regulation of stress response. Cre-miR171 was highly expressed in the early stage of root development, and the expression level in the lateral root area was higher than that in the root tip. Through bioinformatics analysis and tissue expression correlation analysis, three target genes, CclSCL6, CclSCL22 and CclSCL26, were identified. The targeting relationship between these three target genes and Cre-miR171 was confirmed by tobacco transient expression experiments. In a 15-day drought stress treatment, the expression of Cre-miR171 in the lateral root zone and root tip showed a significant opposite trend from the 10th day of treatment with up-regulated expression in the root tip and down-regulated expression in the lateral root zone. The expression of the three target genes also showed corresponding changes. The sensitivity of Arabidopsis and citrus plants overexpressing Cre-miR171 to drought was significantly higher than that of the control, and the elongation growth of main roots and the generation of lateral roots in citrus were inhibited. The above results indicated that Cre-miR171 can affect the elongation growth and lateral root formation, and negatively regulate the drought tolerance in citrus.
- Cre-miR171,
- drought stress,
- root growth and development,
- citrus

References

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柑橘是我国最重要的水果，主要种植在南方各省市的山地和丘陵地区. 柑橘产区横跨几个气候带，许多柑橘产区的降雨量季节分配不均匀，因此季节性缺水造成的干旱是制约我国许多地区柑橘产业发展的一个主要因素^[1]. 植物有多种抗旱和耐旱机制应对干旱胁迫，以维持正常的生长和发育. 近年的研究发现，一些小分子RNA不仅与植物的生长发育密切相关，而且在植物的抗旱中也发挥着至关重要的作用^[2]. 小分子RNA主要有3种类型，siRNAs，miRNAs和piRNAs，其中miRNAs是目前生物体内存在最广泛的，也是研究最多的一类小RNA. 这些小RNA长度大约在20~24个核苷酸，它们通过碱基互补配对与靶基因结合，进而在转录后水平调控基因表达，促使mRNA降解或抑制其翻译，起负调控靶基因的作用^[3-4]. miRNA通过调控靶基因参与植物的生长发育和逆境响应^[5]，许多研究报道指出，有多种miRNA参与植物的干旱胁迫响应. miR196g是最早在水稻中被发现的与干旱相关的miRNA^[6]，豇豆中发现了有近20个miRNA以不同的方式响应干旱胁迫^[7]，干旱逆境诱导碧桃叶片及根部中262和368个miRNA差异表达^[8]，miR168和miR396在拟南芥和烟草中的表达也受干旱胁迫诱导^[9]. 在模式植物拟南芥中，miR171在干旱、低温、低氧、低磷、低硫等胁迫下表达量都会发生变化^[10]. 豆科植物中miR171也响应干旱胁迫^[11]. 在水稻中，过表达osa-miR171f可以通过调控类黄酮生物合成基因的表达来增强水稻的耐旱性^[12]，它通过调节SCL6-I和SCL6-II的转录水平在抗旱性中发挥作用. 在苹果中，敲除mdm-miR171i和过表达它的靶基因MsSCL26.1均可增强其抗旱性^[13]. 进一步研究发现，mdm-miR171通过调节抗氧化基因表达和抗坏血酸代谢来响应干旱胁迫. 以上研究结果表明，miR171家族成员响应植物的干旱胁迫. 目前的研究表明，miR171家族的功能高度保守. 因此可以推测柑橘中的Cre-miR171也可能响应干旱胁迫，但柑橘中Cre-miR171是否响应干旱胁迫以及调控的靶基因的研究还未见报道.

为明确Cre-miR171对柑橘耐旱的作用，本研究通过对Cre-miR171在不同柑橘砧木品种、组织及根系不同部位、发育时期的表达特征进行分析，对Cre-miR171过表达的柑橘砧木枳和模式植物拟南芥进行耐旱性及根系形态评价，探究Cre-miR171对植物响应干旱胁迫的影响. 并在转基因柑橘中对预测的靶基因进行表达分析，为深入研究Cre-miR171调控柑橘耐旱性的分子机理提供参考.

1. 材料与方法

1.1. 试验材料

柑橘砧木品种枳(Poncirus trifoliata Raf.)、扁平橘(Citrus depressa Hayata. ‘Shiikuwasha’)、红柠檬(C. limonia Osbeck. ‘Canton Lemon’)的种子采自国家果树种质柑橘圃(重庆，北碚). 本氏烟(Nicotiana benthamiana)、拟南芥(Arabidopsis thaliana)种子为本实验室自繁.

DNA提取试剂盒(植物基因组DNA快速提取试剂盒)，反转录试剂盒(Revert Aid First Strand cDNA Synthesis Kit)，RNA提取试剂盒(天根DP504)，限制性内切酶(Thermo Fisher scientific)，DNA Marker (Takara)，引物合成和测序由北京擎科生物科技有限公司完成.

1.2. 试验方法

将枳、扁平橘和红柠檬的种子去除种皮后于(28±1) ℃下保湿催芽. 挑选萌芽一致的种子播种于膨胀珍珠岩∶蛭石(体积比1∶1)的基质中，在温室(25±1) ℃中培养. 选取一月龄左右的不同砧木幼苗，取其根、茎、叶用于Cre-miR171的组织表达分析. 分别于枳种子播种后30 d，60 d和90 d，取全根用于根的不同发育时期Cre-miR171的表达分析. 于枳种子播种后45 d，取根尖和侧根发生区样品用于Cre-miR171在根不同部位的表达分析. 于枳种子播种后60 d，取茎、叶、侧根发生区以及根尖用于Cre-miR171靶基因的表达分析. 以上取样各5株，设5个生物学重复.

选取长势一致的1月龄枳的水培幼苗，分别用15%的PEG₆₀₀₀及100 μM的脱落酸(ABA)水溶液进行胁迫处理，在处理的0 h，3 h，6 h，12 h，24 h分别采集全根作为样品，用于胁迫处理下Cre-miR171的表达分析. 选取播种后2个月、长势一致的盆栽枳幼苗于温室内进行干旱处理，在干旱处理的0 d，5 d，10 d，15 d分别采集根尖区与侧根发生区的组织样品，用于干旱胁迫下Cre-miR171的表达分析.

1.3. 生物信息学分析

Cre-miR171的前体序列来自本实验室前期构建的小RNA文库. 利用Tbtools对Cre-miR171前体序列与从柑橘基因组数据库下载得到的克里曼丁橘(C. clementina)基因组数据进行序列比对，获得Cre-miR171前体序列上游3 000 bp启动子序列，其顺式作用元件通过PlantCARE(http://bioinformatics.psb.ugent.be/webtools/plantcare/html/)进行预测分析. 利用psRNATarget：A Plant Small RNA Target Analysis Server (2017 Update) 在线网站psRNATarget(2017 update)软件进行小RNA靶基因的互补配对，以miRNA的5′端为种子序列与参考基因组克里曼丁橘的转录本3'UTR区对Cre-miR171的靶基因进行预测. 靶基因序列从在线网站Phytozome(https://phytozome-next.jgi.doe.gov/pz/portal.html)上获取.

1.4. 过表达Cre-miR171载体的构建及遗传转化

采用Primer 3在线软件设计1对Cre-miR171前体序列的特异引物，分别在序列的两端加上SwaI，BamHI酶切位点(表 1)，以枳的DNA为模板，对Cre-miR171进行克隆. 用Solution I连接酶将Cre-miR171前体序列的全长与经SwaI，BamHI双酶切后的PGBi载体进行连接，转化到大肠杆菌DH5a感受态细胞中，挑选单克隆送往北京擎科生物科技有限公司进行测序. 经测序验证正确的过表达载体转入农杆菌EHA105感受态细胞，用枳上胚轴进行柑橘的遗传转化^[14]，采用花序侵染法进行拟南芥的遗传转化^[15].

1.5. 转基因植株的鉴定与其靶基因的筛选

利用PCR方法对Cre-miR171的过表达柑橘植株和拟南芥植株进行鉴定. 正向引物是35S启动子一段序列，反向引物为Cre-miR171前体序列(表 1)，以转基因植株的DNA为模板进行扩增. 采用茎环法qRT-PCR检测各转基因柑橘植株的Cre-miR171相对表达量，选择Cre-miR171表达量最高的3株进行嫁接扩繁. 同时对其靶基因的表达进行定量分析.

1.6. 烟草瞬时表达载体的构建与转化

构建两类烟草瞬时表达载体进行Cre-miR171与其靶基因的互作验证. 一类为Cre-miR171前体序列超表达的GUS载体，将Cre-miR171的前体序列与线性化的GUS载体连接；另一类为改造过的超表达载体pMS4载体，pMS4载体在GFP基因前后有两个酶切位点XhoI和XbaI，经双酶切之后用于插入miRNA靶基因位点. 靶基因位点是通过人工合成的两条寡核苷酸链退火后形成的双链，分为S Cclscl6，SCclscl22，SCclscl26以及经过改造的MTSCclscl6，MTSCclscl22，MTSCclscl26两类(表 2). 重组后的质粒经北京擎科生物科技有限公司测序验证正确后，转化至农杆菌GV3101菌株的感受态细胞，在烟草中利用农杆菌介导的侵染共同表达miRNA及其靶基因.

1.7. Cre-miR171及其靶基因的荧光定量分析

茎环qRT-PCR和qRT-PCR分别被用来分析miRNA及其靶基因的相对表达量. miRNA的内参基因为U6，靶基因的内参基因为柑橘CsActin. 用Primer5软件设计miRNA的反转录引物和靶基因、内参基因的实时荧光定量PCR引物(表 3). qRT-PCR反应体系为：2×NovoStart^Ⓒ SYBR qPCR SuperMix 5 μL，正、反向引物各0.2 μL，RNase Free Water 2.6 μL，cDNA 2 μL(500 ng). 扩增程序为：95 ℃预变性10 min，然后95 ℃ 15 s，58 ℃ 60 s，共设置40个循环. 所有的定量反应都在实时PCR System (Applied Biosystems 7 500 Fast，Applied Biosystems，USA)上完成. 每个qRT-PCR反应重复3次. miRNA及其靶基因的相对表达量采用2^-ΔΔCT法进行计算^[16].

1.8. 转基因植株的耐旱性评价

选择长势一致的过表达Cre-miR171的拟南芥植株进行26 d的干旱处理，并观察其在干旱情况下的表型变化. 在处理14 d时取叶片样，测定POD与脯氨酸的含量.

选择长势一致的枳野生型和转基因植株，剪取相同生长部位的叶片，放置在干燥的滤纸上，测定脱水处理60 min内的质量变化，计算相对失水率. 对脱水处理后的叶片进行电导率的测定，计算处理后叶片的相对电导率.

剪取对照组和Cre-miR171表达量最高的过表达枳的枝条，扦插于蛭石基质中，待其长出根系后，评价分析根的生长发育表现.

3. 结论与讨论

在植物中，miR171参与调节植物基本的发育和代谢过程^[18-19]. 在模式植物拟南芥中，miR171通过调控其靶基因AtSCL6-II，AtSCL6-III和AtSCL6-IV的表达影响茎尖分生组织^[20-21]. 过表达miR171前体可以抑制腋芽形成和花芽数量，从而改变拟南芥茎的结构^[22]. 拟南芥幼苗在高盐、低温和干旱胁迫下miR171的表达量都会上调^[9]，推测miR171参与了拟南芥非生物胁迫的调控. 番茄中miR171和SCL6通过调节激素信号去调控番茄的生长发育^[23]. 甘蔗中miR171响应盐胁迫^[24].

Cre-miR171启动子区有光信号、脱落酸、水杨酸、茉莉酸甲酯等激素和胁迫响应元件，同时miR171被证实响应光信号^[25]、茉莉酸甲酯^[26]、水杨酸、脱落酸^[27]诱导，表明Cre-miR171可能受光信号、茉莉酸甲酯、水杨酸、脱落酸等多条信号通路的影响，推测Cre-miR171可能参与逆境胁迫下的应答及调控. 表达Cre-miR171的拟南芥对干旱胁迫更敏感，脱水处理对过表达Cre-miR171枳叶片的膜系统的伤害显著高于对照，说明过表达Cre-miR171的枳转基因植株的耐旱能力降低. 这与在苹果中的研究结果相似^[13]，表明Cre-miR171对柑橘的耐旱性有负调控的作用.

有研究表明miR171在根的发育和生长中发挥重要作用，miR171参与苹果^[28]和葡萄^[29]的不定根形成，miR171还影响了水稻的根系发育^[30]，同时miR171被证实可以影响蒺藜苜蓿的侧根密度^[31]. 本研究中，Cre-miR171在柑橘根发育的早期高表达，而且在侧根发生区的表达量高于根尖，表明Cre-miR171对柑橘根系的发育及侧根的形成有重要影响.

本研究中预测并验证的3个Cre-miR171的靶基因(CclSCL6，CclSCL22，CclSCL26)是植物中特有的转录因子，其功能具有多样性，参与植物的生长发育^[32]、信号转导、生物胁迫和非生物胁迫相关的应答过程^[33]. Cre-miR171在响应干旱胁迫后在侧根发生区下调表达，在根尖上调表达，趋势相反，3个靶基因的表达也随之发生相应的显著变化，说明在干旱胁迫下，Cre-miR171能通过对靶基因表达的调控抑制根的伸长生长，促进侧根的发生. 可以推测，Cre-miR171可能调控柑橘根系在干旱胁迫下的可塑性.

Figure (8) Table (5) Reference (33)

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Effect of Cre-miR171 on Drought Stress Response in Citrus