Identification of Critical Genes Involved in Postharvest Sprouting of Potato Tubers Based on RNA-seq Analysis

WU Lin; YANG Suyu; YANG Yang; LIAO Ximei; ZHU Xuedong; ZHU Feifei; LIANG Fengming; LI Yong; LYU Dianqiu

doi:10.13718/j.cnki.xdzk.2023.11.008

2023 Volume 45 Issue 11

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WU Lin, YANG Suyu, YANG Yang, et al. Identification of Critical Genes Involved in Postharvest Sprouting of Potato Tubers Based on RNA-seq Analysis[J]. Journal of Southwest University Natural Science Edition, 2023, 45(11): 70-80. doi: 10.13718/j.cnki.xdzk.2023.11.008

Citation:

WU Lin, YANG Suyu, YANG Yang, et al. Identification of Critical Genes Involved in Postharvest Sprouting of Potato Tubers Based on RNA-seq Analysis[J]. Journal of Southwest University Natural Science Edition, 2023, 45(11): 70-80. doi: 10.13718/j.cnki.xdzk.2023.11.008

Identification of Critical Genes Involved in Postharvest Sprouting of Potato Tubers Based on RNA-seq Analysis

1.
Integrative Science Center of Germplasm Creation in Western China (CHONGQING) Science City, Southwest University, Chongqing 400715, China
2.
College of Agronomy and Biotechnology, Southwest University, Chongqing 400715, China
3.
Southeast Chongqing Academy of Agricultural Sciences, Fuling Chongqing 408000, China
4.
Wuxi County Shuguang Agricultural Science and Technology Development Co. Ltd., Wuxi Chongqing 405899, China
5.
College of Agriculture, Anshun University, Anshun Guizhou 561000, China
6.
Chongqing Key Laboratory of Biology and Genetic Breeding for Tuber and Root Crops, Chongqing 400715, China

More Information

Corresponding author: LYU Dianqiu
Received Date: 12/08/2023
Available Online: 20/11/2023
MSC: S532

Abstract

The potato is the fourth main food crops worldwide with high nutritional value. However, potato tuber postharvest sprouting significantly affects the quality of product and seed potatoes. Through phenotype observation, comparison of the histological structures, analysis of physiological and biochemical indexes, measurement of contents of phytohormones, and monitoring of the marker gene expression, we confirmed the three main transition stages of postharvest sprouting including dormancy, dormancy releasing, and bud growth. Additionally, RNA-seq was performed to identify the critical genes involved in potato tuber sprouting. The results exhibited that the time points of tuber dormancy releasing and bud growth were at 30 d and 45 d after postharvest, respectively. During tuber dormancy to dormancy releasing, expression levels of 1 008 genes (252 upregulated and 756 downregulated) were significantly changed. During dormancy releasing to bud growth, 4 576 genes differentially expressed (DE), including 2 800 upregulated and 1 696 downregulated. Among these DE genes, expressions of 156 transcription factor (TF) genes were greatly changed, most of these TFs belonging to AP2, MYB, and bHLH TF families. Meanwhile, several genes participated in ABA and GA biosynthesis, metabolism, and signaling, also observably changed their expression levels, such as NCED4, ZEP, KS KO2, ABI5, etc. KEEG enrichment analysis showed that those DE genes mainly enriched in the pathways of phenylpropanoid biosynthesis (ko00940), phenylalanine metabolism (ko00360), carotenoid biosynthesis (ko00100), etc.
- potato tuber,
- postharvest sprouting,
- transcriptome,
- critical genes

References

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马铃薯是种植面积仅次于小麦、水稻和玉米的第4大粮食作物^[1]. 中国是全球马铃薯第一大生产国. 据国家统计局数据，2021年我国马铃薯种植面积约460.6万hm²，产量达1 830.9万t. 马铃薯块茎采后发芽不仅显著降低商品价值，而且还严重威胁种薯的质量安全，造成巨大的经济损失^[2].

为抑制马铃薯块茎采后发芽，国内外已开发出多种方法^[3]. 如使用化学抑芽剂，包括氯苯胺灵(Chlorpropham，CIPC)^[4]、过氧化氢(Hydrogen Peroxide Plus，HPP)^[5]、精油^[6]等. 其中，CIPC应用最为广泛^[4]. 但是，CIPC的抑芽作用不可逆，会严重影响种薯芽的活性；加之CIPC具有致癌性和毒性，2020年起欧盟已撤销其产品登记^[7]. 此外，紫外(Ultraviolet，UV)辐射处理也可以有效降低马铃薯采后发芽率^[8].

以往的研究发现，马铃薯块茎从休眠到发芽过程受植物激素严格调控. 脱落酸(Abscisic acid，ABA)是促进马铃薯块茎休眠的主要物质，通过抑制细胞分裂及降低块茎中能量代谢抑制发芽^[9]. 与ABA作用相反，赤霉素(Gibberellins，GAs)促进马铃薯块茎发芽^[10]. 此外，细胞分裂素、生长素、茉莉酸等植物激素也具有调控马铃薯块茎采后休眠的作用^[11].

近年来，随着测序技术和高通量筛选技术的发展，部分马铃薯休眠调节相关基因已被挖掘. Faivre-Rampant等^[12]利用休眠和休眠解除的马铃薯块茎构建抑制性消减杂交文库(Suppression Subtractive Hybridization，SSH)，筛选到385个差异表达基因；功能富集分析表明参与转录的相关基因表达发生显著变化. Liu等^[13]利用休眠与发芽块茎构建SSH文库，鉴定到300个差异基因；与转录调控、信号转导、胁迫响应相关基因在马铃薯解除休眠时显著上调表达. Li等^[14]利用转录组测序分析了樟脑抑制马铃薯采后休眠的差异基因和蛋白，鉴定到约4 000个差异表达转录本；Kyoto Encyclopedia of Genes and Genomes (KEGG)分析表明，樟脑可能通过调控马铃薯块茎中内源植物激素合成与信号转导抑制发芽.

马铃薯块茎休眠和发芽的分子调控机制已有少量报道. Farre等^[15]利用马铃薯块茎特异表达基因Patatin的启动子，在块茎中特异表达大肠杆菌无机焦磷酸酶基因PPase，发现转基因马铃薯块茎较野生型提前6~7周发芽. Carrera等^[16]发现超表达马铃薯赤霉素合成酶关键基因GA₂₀-oxidasse，转基因马铃薯块茎出现提前发芽表型. Pasare等^[17]证明，抑制马铃薯独脚金内脂(Strigolactone，SLs)生物合成关键酶基因CCD8会显著缩短转基因马铃薯休眠期. Li等^[18]发现异源过表达马铃薯StHSP26.5基因，转基因烟草种子发芽被显著提前. 相反，在马铃薯中沉默alpha-amylase基因StAmy23则会显著抑制块茎发芽^[19]. 最近的研究证明，转录因子基因StTCP15通过介导ABA与GA的动态平衡调控马铃薯块茎发芽^[20].

本研究通过马铃薯块茎采后发芽过程中的表型观察、细胞学特性分析、生理生化指标检测及发芽marker基因(dUTPase)表达变化等，明确了块茎休眠期、休眠解除期及芽生长期3个关键时间点. 通过Illumina测序平台对3个时期的块茎进行转录组测序分析，挖掘马铃薯采后发芽关键基因，并从植物激素合成、信号转导角度系统分析了植物激素在调控马铃薯块茎发芽中的功能，为马铃薯块茎发芽关键基因功能解析和作用机制研究奠定了分子学基础.

1. 材料与方法

1.1. 试验材料

2022年秋季将马铃薯短休眠期栽培品种‘费乌瑞它’种植于重庆市渝东南农业科学院实验基地，常规田间管理，成熟时挑选300个大小均匀(约40 g/个)、健康、无损伤的块茎，洗净、自然干燥后，置于暗黑环境(室温22 ℃±2 ℃、相对湿度75%)中愈合1周，开始进行发芽统计. 期间，每隔15 d进行拍照记录，并参考Li等^[14]的取样方法进行取样，每个时间点设置3个生物学重复. 样品经液氮速冻后，置于-80 ℃保存，用于后续试验.

1.2. 块茎发芽过程的细胞学观察

块茎发芽过程的细胞学观察主要参考Liu等^[13]的取样和切片制作方法. 以块茎顶芽为中心，用直径5 mm的打孔器取样，样品置于福尔马林-醋酸-酒精固定液(FAA)(70%酒精∶冰醋酸∶甲醛=90∶5∶5)中，真空处理10 min，于4 ℃保存. 切片采用莱卡RM2016切片机，成像采用尼康Eclipse E100光学显微镜+尼康DS-U3成像系统.

1.3. 块茎发芽过程的ABA和GA₃检测

ABA和GA₃检测分别采用江苏酶免实业有限公司(http://www.mmbio.cn/)的植物脱落酸(ABA)ELISA试剂盒(MM1185-01)与植物赤霉素(GA₃)ELISA试剂盒(MM-3587201)，参照说明书进行检测.

1.4. 块茎发芽过程中的淀粉、可溶性糖、蛋白检测

淀粉、可溶性糖、蛋白含量检测分别采用苏州格锐思生物科技有限公司(http://www.geruisi-bio.com/)的淀粉含量测定试剂盒(蒽酮比色法，G0507W)、可溶性糖含量(SS)测定试剂盒(蒽酮比色法，G0501W)、蛋白含量(SP)测定试剂盒(双缩脲法，G0432W)，参照说明书进行检测.

1.5. Total RNA提取与转录组测序

马铃薯块茎Total RNA提取采用RNeasy Plant Mini Kit(QIAGEN，德国). Total RNA纯度检测采用NanoDrop 2000((Invitrogen，美国)；完整性检测采用Agient2100(Agilent Technologies，美国). 核糖体RNA去除采用Epicenter Ribo-Zero^TM rRNA Removal Kit (Epicenter，Madison，美国). cDNA文库构建采用NEB Next Ultra Directional RNA Library Prep Kit(NEB，美国). 转录组测序在百迈客生物科技有限公司Illumina Novaseq6000测序平台进行. 为提高分析质量，带接头和低质量序列被去除. 利用HISAT2软件将Clean Reads与马铃薯DM参考基因组(http://spuddb.uga.edu/dm_v6_1_download.shtml)进行精确比对，再利用String Tie软件对比对上的Reads进行组装，用于后续分析^[21-22]. 转录组原始数据已上传至美国国家生物技术信息中心(NCBI)数据库(Accession number：PRJNA1003271).

1.6. 差异表达基因筛选与KEGG富集分析

利用DESeq2软件对测序数据进行差异表达基因分析，筛选标准设置为|Fold Chang|≥2且p-value＜0.01. 将KOBAS 2.0(http://www.biostars.org/p/200126/)用于KEGG富集分析，显著富集筛选标准为p-value＜0.05.

1.7. 实时荧光定量PCR(qRT-PCR)分析

根据转录组测序结果，随机挑选8条差异表达基因，利用NCBI网站(http://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?LINK_LOC=BlastHome)设计特异引物(表 1). 采用FastPure© Plant Total RNA Isolation Kit (Polysaccharides & Polyphenolics-rich)(Vazyme，中国)提取马铃薯发芽样品总RNA. 利用HiScritpt© Ⅲ 1st Strand cDNA Synthesis Kit (+gDNA wiper) (Vazyme，中国)反转录获得cDNA. Quantitative real time polymerase chain reaction (qPCR-PCR) 采用Bio-Rad CFX Connect Real-Time System with iTaq Universal SYBR^© Green Supermix(Bio-Rad，美国)进行处理. 程序设置为95 ℃变性3 min，(95 ℃变性15 s，60 ℃退火30 s，72 ℃延伸45 s)× 40循环，并进行溶解曲线分析. 相对表达计算采用2-^ΔΔCt[1]. 马铃薯Ef1α为内参基因^[19-20].

3. 讨论

为明确‘费乌瑞它’马铃薯采后发芽过程的关键变化时期，本研究对采后贮藏过程中的块茎顶芽表型及细胞学特性进行了分析. 结果表明，‘费乌瑞它’马铃薯采后发芽可分为3个关键时期，分别为休眠期(0 d)，休眠解除期(30 d)和芽生长期(45 d)，该结果与Li等^[14]的报道相一致. Li等发现，采后贮藏30~70 d，‘费乌瑞它’马铃薯的芽可长至2~10 mm. 然而，Liu等^[13]报道，‘费乌瑞它’马铃薯采后第8周为休眠解除期，第9周芽长才长至2 mm. 引起这种差异的主要原因可能是Liu等^[13]发芽试验采用的是试管薯(0.5 g/个)，而本研究与Li等^[14]均采用大田收获的马铃薯(约40 g/个). Ebrahim等^[23]证明，马铃薯休眠期与块茎大小成反比，通常更小的薯采后发芽更慢.

在马铃薯采后发芽过程中，可溶性糖含量显著增加、蛋白与淀粉含量显著减少. 本研究结果与张路^[24]的结果一致. 在贮藏过程中，马铃薯块茎内淀粉和还原性糖相互转化，为马铃薯块茎发芽提供能量. 而巩慧玲等^[25]研究发现，马铃薯块茎中蛋白含量在贮藏期间比较稳定，变化不大. 同时，本研究发现在马铃薯块茎发芽过程中，内源植物激素ABA含量显著减少，GA₃含量显著增加. 结果与前人的报道一致^[9-10].

RNA-Seq分析发现，‘费乌瑞它’马铃薯块茎从休眠期(D0)到休眠解除期(D30)共有1 008个基因表达发生显著变化. 休眠解除期(D30)到芽生长期(D45)共有4 576个基因表达发生显著变化. 其中，355个基因表达在贮藏过程中持续地发生显著变化. 差异变化基因数量较之前Li等^[14]的报道更多，Li等^[14]发现，‘费乌瑞它’马铃薯块茎休眠期到芽生长期共有3925个基因表达发生显著变化. 因此，本研究的数据进一步补充了马铃薯块茎发芽相关基因.

马铃薯块茎发芽过程显著变化基因被富集到苯丙烷生物合成通路(ko00940)、苯丙氨酸代谢通路(ko00360)、类胡萝卜素生物合成通路(ko00100)、DNA复制(ko03030)和糖代谢通路(ko00520)等. 研究结果表明，苯丙烷生物合成通路、苯丙氨酸代谢、类胡萝卜素生物合成相关基因可能参与马铃薯块茎发芽调控，这与前人的报道一致. 例如，Conrad等^[26]发现杏树的芽休眠解除可能与苯丙氨酸代谢高度相关. Deng等^[27]报道，马铃薯的Snakin-2(SN2)基因通过负调控木质素生物合成，显著抑制块茎采后发芽.

本研究还发现从马铃薯休眠期(D0)到休眠解除期(D30)，共有46个转录因子基因表达发生显著变化；从休眠解除期(D30)到芽生长期(D45)共有156个转录因子基因表达发生显著变化. 这些转录因子基因主要属于AP2转录因子家族、MYB转录因子家族及bHLH转录因子家族. 同时，ABA与GA生物合成、代谢及信号转导相关基因表达也发现了显著变化，说明AP2，MYB，bHLH转录因子家族基因，ABA与GA合成、代谢、信号转导基因可能在调控马铃薯采后发芽过程中发挥了重要作用，与前人报道一致. 例如，Wang等^[28]发现，大豆的一个AP2转录因子基因GmSGR通过减少ABA的敏感性显著促进种子发芽. Himi等^[29]发现，大麦的一个MYB转录因子基因Ant28通过调节花青苷合成显著抑制种子萌发. Liu等^[30]报道，拟南芥中过表达ABA代谢酶基因CYP707A导致ABA含量显著减少，种子发芽显著提前.

4. 结论

马铃薯‘费乌瑞它’块茎贮藏30 d时芽休眠解除；贮藏45 d时芽开始生长. 在贮藏过程中，可溶性糖含量显著增加、淀粉和蛋白含量显著减少. 从芽休眠到休眠解除，休眠解除到芽生长分别有1 008个、4 576个基因表达发生显著变化，其中355个基因持续地发生显著变化. 在这些差异表达基因中，包括156个转录因子基因，大多属于AP2，MYB，bHLH转录因子家族. 这些差异表达基因被富集到苯丙烷生物合成通路、苯丙氨酸代谢通路、类胡萝卜素生物合成通路、DNA复制和糖代谢通路.

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Identification of Critical Genes Involved in Postharvest Sprouting of Potato Tubers Based on RNA-seq Analysis