无患子雄性不育品种‘琦蕊’不同发育时期雄花转录组分析

doi:10.13560/j.cnki.biotech.bull.1985.2024-0039

摘要/Abstract

摘要：

【目的】 探究无患子（Sapindus mukorossi Gaertn.）雄性不育品种‘琦蕊’雄花不同发育时期的细胞学特征及差异表达基因，为深入解析无患子雄性不育发生的分子机制提供理论依据。【方法】 在观察雄花花药不同发育过程的细胞学特点基础上，进一步利用RNA-Seq技术分别对小孢子母细胞时期（T1）、四分体时期（T2）、单核小孢子时期（T3）的雄花进行转录组比较分析，筛选关键差异表达基因。【结果】 ‘琦蕊’绒毡层和内层细胞的持续膨大与增殖使得药室结构混乱，药室空间不足，最终导致无可育花粉产生。内源激素测定发现，茉莉酸水平在‘琦蕊’雄花发育过程中呈下降趋势。3个时期的转录组分析共筛选到2 990个差异表达基因（differentially expressed genes, DEGs），其中T2_vs_T1中722个（516个上调，206个下调），T3_vs_T2中1 741个（765个上调，976个下调）。GO和KEGG分析表明，这些DEGs主要富集在代谢过程、细胞分裂、花粉壁合成、激素信号转导等方面，共鉴定到32个花粉发育相关DEGs、27个激素合成及信号转导DEGs。随机选取9个DEGs进行RT-qPCR分析，结果与RNA-Seq数据趋势一致，证明转录组数据的准确性。【结论】 绒毡层细胞持续增殖和内层细胞延迟退化不断挤压小孢子是导致无患子‘琦蕊’雄性不育发生的主要原因，物质代谢、细胞分裂、激素水平、花粉发育等相关基因在雄性不育发生过程中发挥重要作用。

关键词: 无患子, 雄性不育, 转录组, 差异表达基因, 绒毡层, 细胞分裂, 茉莉酸

Abstract:

【Objective】 The objective of this work is to observe the cytological characteristics of the anther development in the male sterile Sapindus mukorossi ‘Qirui’, identify differentially expressed genes（DEGs）during the critical period of anther development，which provides a theoretical basis for deeply analyzing the molecular mechanism of the male sterility. 【Method】 By examining the cytological features of male anther development stages, RNA-Seq technology was used to compare the transcriptome of male buds at different stages: microspore mother cell stage（T1）, tetrad stage（T2）and mononuclear microspore stage（T3）, to identify key DEGs. 【Result】 The study revealed that the continuous expansion and proliferation of ‘Qirui’ tapetum and endothecium led to a chaotic anthecium structure and insufficient anthecium space, resulting in abortive pollen. Additionally, a decrease in the level of jasmonic acid（JA）from endogenous hormone determination was observed during male bud development. A total of 2 990 DEGs were detected through transcriptome analysis at the three anther development stages, of which 722 in T2_vs_T1（516 up-regulated and 206 down-regulated）, and 1 741 in T3_vs_T2（765 up-regulated, 976 down-regulated）. GO and KEGG analysis showed that these DEGs were primarily enriched in metabolic processes, cell division, pollen wall synthesis, hormone signal transduction, etc. Specifically, 32 DEGs were associated with pollen development, and 27 DEGs were linked to hormone synthesis and signal transduction. Nine DEGs were randomly selected for RT-qPCR analysis, and the results were consistent with the trend of RNA-Seq data, which proved the accuracy of transcriptome data. 【Conclusion】 The continuous proliferation of tapetum, delayed degeneration of endothecium, and continuous extrusion of microspores are the key factors leading to male sterility in S. mukorossi ‘Qirui’. Overall, genes related to material metabolism, cell division, hormone level and pollen development play crucial roles in the mechanism of male sterility.

Key words: Sapindus mukorossi Gaertn., male sterility, transcriptome, differential expressed genes（DEGs）, tapetum, cell division, jasmonic acid

廖杨梅, 赵国春, 翁学煌, 贾黎明, 陈仲. 无患子雄性不育品种‘琦蕊’不同发育时期雄花转录组分析[J]. 生物技术通报, 2024, 40(7): 197-206.

LIAO Yang-mei, ZHAO Guo-chun, WENG Xue-huang, JIA Li-ming, CHEN Zhong. Transcriptome Sequencing of Male Sterile Buds at Different Developmental Stages in Sapindus mukorossi ‘Qirui’[J]. Biotechnology Bulletin, 2024, 40(7): 197-206.

图/表 9

表1 RT-qPCR引物信息

Table 1 Primers used in the RT-qPCR

基因编号Gene ID	基因名称Gene name	正向引物序列Forward primer sequence（5'-3'）	反向引物序列Reverse primer sequence（5'-3'）
Samuk01G0221000	SmDYT1	CACTTACATAGAGGTGCTG	CTCAATCCCACTATTCTTC
Samuk09G0006500	SmMYB35	ACATACCTCACCACCACCT	TTGACCATCTTCTGCCTAT
Samuk03G0239600	SmbHLH091	ACGGCAGGAAGATGTCAGT	GTCGTGAATCAAATGGGTG
Samuk02G0196100	SmCAT2	AACCCAAAGTCTCACATCC	CACCCACATCATCAAAGAG
Samuk12G0082500	SmMIOX1	TGCTGAGGCTATTAGGAAG	CGAAGTTAGGCAGAAGAAG
Samuk07G0073400	SmCYP704B1	AAAAGCAGGAGGGATGGTG	TGTATGAGGCTGCGTCAGG
Samuk07G0100300	SmMYB62	AACAGCCAAATCACAGACG	GCTCCTAGATTGAACCCAC
Samuk03G0033700	SmTCF1	TTAAGTCCAATATGCGTGTC	GCCCAAACTGATTCCCAC
Samuk04G0015800	SmCER1	GGCATCCAGGTTACCACAT	CATCTCCCACCACCCATAC
Samuk07G0011500	SmRPL1	CTGACCACCCCACCACTCTC	TCCTCCTCATCTGCCACCTC

图1 ‘琦蕊’花药早期发育显微结构特征 T1、T2、T3分别指无患子花发育的第8期（S8）、第9期（S9）、第10b期（S10b），S12为无患子花发育的第12期；T：绒毡层；Tds：四分体；E：表皮；En：内层；ML：中层；比例尺=100 μm。下同

Fig. 1 Microstructure of early development of ‘Qirui’ anther T1, T2 and T3 refer to the 8th（S8）, 9th（S9）and 10b（S10b）stages of S. mukorossi Gaertn. flower development respectively, and S12 refers to the 12th stage. T: Tapetum; Tds: tetrad; E: epidermis; En: endothecium; ML: middle layer. Bar = 100 μm. The same below

图2 ‘琦蕊’雄花发育过程内源激素含量变化 JA：茉莉酸；SA：水杨酸；IAA：生长素；ET：乙烯

Fig. 2 Changes of endogenous hormone content during male buds development of ‘Qirui’ JA: Jasmonic acid; SA：salicylic acid; IAA: auxin; ETH: ethylene

表2 转录组测序数据评估

Table 2 Data evaluation of sample sequencing

样本名称 Sample name	原始数据 Raw reads	过滤后数据 Total clean reads	总映射率 Total mapping ratio/%	单一映射率 Uniq mapped reads/%	Q20/%	Q30/%
T1-1	88 252 858	85 911 910	95.86	72.67	98.37	95.02
T1-2	91 157 116	88 898 424	95.83	82.08	98.46	95.22
T1-3	88 984 514	86 405 806	95.85	75.65	98.10	94.31
T2-1	88 937 894	86 784 106	95.91	80.89	98.28	94.76
T2-2	100 745 016	98 376 588	95.87	82.46	98.42	95.11
T2-3	107 473 734	105 653 102	95.97	84.99	98.44	95.12
T3-1	95 388 566	93 391 938	95.77	86.11	98.36	94.95
T3-2	89 881 480	87 909 282	96.08	80.18	98.43	95.17
T3-3	96 582 048	95 072 204	96.00	84.08	98.39	95.00

图3 差异表达基因的RT-qPCR验证

Fig. 3 RT-qPCR verification of differentially expressed genes

图4 不同时期的差异表达基因

Fig. 4 Numbers of differentially expressed genes in different periods

图5 差异表达基因GO功能富集和KEGG通路富集分析 A、C：T2_vs_T1中差异表达基因的GO功能注释（A）和KEGG通路富集（C）；B、D：T3_vs_T2中差异表达基因的GO功能注释（B）和KEGG通路富集（D）

Fig. 5 GO functional enrichment and KEGG pathway enrichment of differentially expressed genes A, C: GO functional annotation（A）and KEGG pathway enrichment（C）of differentially expressed genes in T2_vs_T1; B, D: GO functional annotation（B）and KEGG pathway enrichment（D）of differentially expressed genes in T3_vs_T2

图6 雄性不育关键候选差异基因表达分析 A：花粉发育基因；B：细胞分裂CYCD家族3个基因；C：茉莉酸合成及信号转导基因

Fig. 6 Expression analysis of key candidate differential genes for male sterility A: Pollen development genes. B: Cell division CYCD family of 3 genes. C: Jasmonic acid synthesis and signal transduction genes

图7 差异表达转录因子家族分析

Fig. 7 Analysis of differentially expressed transcription factor family

参考文献 41

[1]	Xu YS, Yu D, Chen J, et al. A review of rice male sterility types and their sterility mechanisms[J]. Heliyon, 2023.
[2]	李翔, 李涛, 宫超, 等. 番茄雄性不育研究现状与展望[J]. 广东农业科学, 2023, 50(02): 49-58.
	Li X, Li T, Gong C, et al. Current situation and prospect of research on male sterility of tomato[J]. Guangdong Agricultural Sciences, 2023, 50(02): 49-58.
[3]	Scott RJ, Spielman M, Dickinson HG. Stamen structure and function[J]. The Plant Cell, 2004, 16(suppl_1): S46-S60.
[4]	Kaul MLH. Male sterility in higher plants[M]. Springer Science & Business Media, 2012.
[5]	Chen L, Liu YG. Male sterility and fertility restoration in crops[J]. Annual review of plant biology, 2014, 65: 579-606. doi: 10.1146/annurev-arplant-050213-040119 pmid: 24313845
[6]	De Storme N, Geelen D. The impact of environmental stress on male reproductive development in plants: biological processes and molecular mechanisms[J]. Plant Cell Environ, 2014, 37(1): 1-18.
[7]	Dukowic-Schulze S, van der Linde K. Oxygen, secreted proteins and small RNAs: mobile elements that govern anther development[J]. Plant Reproduction, 2021, 34(1): 1-19. doi: 10.1007/s00497-020-00401-0 pmid: 33492519
[8]	吴智明, 胡开林, 符积钦, 等. 辣椒胞质雄性不育与花蕾内源激素含量的关系[J]. 华南农业大学学报, 2010, 31(2): 1-4.
	Wu ZM, Hu KL, Fu JQ, et al. Relationships between cytoplasmic male sterility and endogenous hormone content of pepper bud[J]. Journal of South China Agricultural University, 2010, 31(2): 1-4.
[9]	Liu H, Xie WF, Zhang L, et al. Auxin biosynthesis by the YUCCA6 flavin monooxygenase gene in woodland strawberry[J]. Journal of Integrative Plant Biology, 2014, 56(4): 350-363.
[10]	Zhang ZB, Zhu J, Gao JF, et al. Transcription factor AtMYB103 is required for anther development by regulating tapetum development, callose dissolution and exine formation in Arabidopsis[J]. The Plant Journal, 2007, 52(3): 528-538.
[11]	Thorstensen T, Grini PE, Mercy IS, et al. The Arabidopsis SET-domain protein ASHR3 is involved in stamen development and interacts with the bHLH transcription factor ABORTED MICROSPORES(AMS)[J]. Plant Molecular Biology, 2008, 66(1): 47-59.
[12]	Xiong SX, Lu JY, Lou Y, et al. The transcription factors MS 188 and AMS form a complex to activate the expression of CYP703A2 for sporopollenin biosynthesis in Arabidopsis thaliana[J]. The Plant Journal, 2016, 88(6): 936-946.
[13]	刘济铭, 孙操稳, 何秋阳, 等. 国内外无患子属种质资源研究进展[J]. 世界林业研究, 2017, 30(6): 12-18.
	Liu JM, Sun CW, He QY, et al. Research progress in Sapindus L. germplasm resources[J]. World Forestry Research, 2017, 30(6): 12-18.
[14]	邵文豪, 刁松锋, 董汝湘, 等. 无患子种实形态及经济性状的地理变异[J]. 林业科学研究, 2013, 26(5): 603-608.
	Shao WH, Diao SF, Dong RX, et al. Study on geographic variation of morphology and economic character of fruit and seed of Sapindus mukorossi[J]. Forest Research, 2013, 26(5): 603-608.
[15]	赵国春. 无患子花发育及性别分化机理研究[D]. 北京: 北京林业大学, 2023.
	Zhao GC. Study on the mechanism of flower development and sex differentiation of Sapindus mukorossi[D]. Beijing: Beijing Forestry University, 2023.
[16]	Xu YY, Zhao GC, Ji XQ, et al. Metabolome and transcriptome analysis reveals the transcriptional regulatory mechanism of triterpenoid saponin biosynthesis in soapberry(Sapindus mukorossi Gaertn.)[J]. Journal of Agricultural and Food Chemistry, 2022, 70(23): 7095-7109.
[17]	徐圆圆, 赵国春, 郝颖颖, 等. 无患子RT-qPCR内参基因的筛选与验证[J]. 生物技术通报, 2022, 38(10): 80-89. doi: 10.13560/j.cnki.biotech.bull.1985.2021-1616
	Xu YY, Zhao GC, Hao YY, et al. Reference genes selection and validation for RT-qPCR in Sapindus mukorossi[J]. Biotechnology Bulletin, 2022, 38(10): 80-89.
[18]	Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2^{- ΔΔCT} method[J]. Methods, 2001, 25(4): 402-408. doi: 10.1006/meth.2001.1262 pmid: 11846609
[19]	Jiang J, Zhang Z, Cao J. Pollen wall development: the associated enzymes and metabolic pathways[J]. Plant Biology, 2013, 15(2): 249-263. doi: 10.1111/j.1438-8677.2012.00706.x pmid: 23252839
[20]	Vu KV, Nguyen NT, Jeong CY, et al. Systematic deletion of the ER lectin chaperone genes reveals their roles in vegetative growth and male gametophyte development in Arabidopsis[J]. The Plant Journal, 2017, 89(5): 972-983.
[21]	Yang K, Zhou XJ, Wang YY, et al. Carbohydrate metabolism and gene regulation during anther development in an androdioecious tree, Tapiscia sinensis[J]. Annals of Botany, 2017, 120(6): 967-977. doi: 10.1093/aob/mcx094 pmid: 28961748
[22]	Wang R, Dobritsa AA. Exine and aperture patterns on the pollen surface: their formation and roles in plant reproduction[J]. Annual Plant Reviews Online, 2018: 589-628.
[23]	Mondol PC, Xu D, Duan L, et al. Defective pollen wall 3(DPW3), a novel alpha integrin-like protein, is required for pollen wall formation in rice[J]. New Phytologist, 2020, 225(2): 807-822.
[24]	Li FS, Phyo P, Jacobowitz J, et al. The molecular structure of plant sporopollenin[J]. Nature plants, 2019, 5(1): 41-46.
[25]	Tidy AC, Ferjentsikova I, Vizcay-Barrena G, et al. Sporophytic control of pollen meiotic progression is mediated by tapetum expression of ABORTED MICROSPORES[J]. Journal of Experimental Botany, 2022, 73(16): 5543-5558.
[26]	Flores-Tornero M, Anoman AD, Rosa-Téllez S, et al. Lack of phosphoserine phosphatase activity alters pollen and tapetum development in Arabidopsis thaliana[J]. Plant Science, 2015, 235: 81-88. doi: 10.1016/j.plantsci.2015.03.001 pmid: 25900568
[27]	Chen W, Yu XH, Zhang K, et al. Male Sterile2 encodes a plastid-localized fatty acyl carrier protein reductase required for pollen exine development in Arabidopsis[J]. Plant Physiology, 2011, 157(2): 842-853.
[28]	Aarts MG, Keijzer CJ, Stiekema WJ, et al. Molecular characterization of the CER1 gene of arabidopsis involved in epicuticular wax biosynthesis and pollen fertility[J]. The Plant Cell, 1995, 7(12): 2115-2127.
[29]	Suzuki T, Narciso JO, Zeng W, et al. KNS4/UPEX1: a type II arabinogalactan β-(1, 3)-galactosyltransferase required for pollen exine development[J]. Plant Physiology, 2017, 173(1): 183-205.
[30]	Li JJ, Han SH, Ding XL, et al. Comparative transcriptome analysis between the cytoplasmic male sterile line NJCMS1A and its maintainer NJCMS1B in soybean(Glycine max(L.)Merr.)[J]. PLoS One, 2015, 10(5): e0126771.
[31]	Hua MY, Yin WZ, Fernández Gómez J, et al. Barley TAPETAL DEVELOPMENT and FUNCTION1(HvTDF1)gene reveals conserved and unique roles in controlling anther tapetum development in dicot and monocot plants[J]. New Phytologist, 2023, 240(1): 173-190.
[32]	Li H, Zhang DB. Biosynthesis of anther cuticle and pollen exine in rice[J]. Plant signaling & behavior, 2010, 5(9): 1121-1123.
[33]	Zhao YN, Luo LL, Xu JS, et al. Malate transported from chloroplast to mitochondrion triggers production of ROS and PCD in Arabidopsis thaliana[J]. Cell Research, 2018, 28(4): 448-461.
[34]	Liu J, Xia C, Dong HX, et al. Wheat male-sterile 2 reduces ROS levels to inhibit anther development by deactivating ROS modulator 1[J]. Molecular Plant, 2022, 15(9): 1428-1439.
[35]	Zhang DD, Liu D, Lv XM, et al. The cysteine protease CEP1, a key executor involved in tapetal programmed cell death, regulates pollen development in Arabidopsis[J]. The Plant Cell, 2014, 26(7): 2939-2961.
[36]	De Veylder L, Beeckman T, Inzé D. The ins and outs of the plant cell cycle[J]. Nature Reviews Molecular Cell Biology, 2007, 8 (8): 655-665. doi: 10.1038/nrm2227 pmid: 17643126
[37]	Zhang TB, Yuan SH, Liu ZH, et al. Comparative transcriptome analysis reveals hormone signal transduction and sucrose metabolism related genes involved in the regulation of anther dehiscence in photo-thermo-sensitive genic male sterile wheat[J]. Biomolecules, 2022, 12(8): 1149
[38]	He MT, Wang XX, Bu YN, et al. Gibberellin confers to the expression of TaGA-6D and negatively regulates the fertility of wheat with Aegilops juvenalis cytoplasm[J]. Plant Science, 2023: 111771.
[39]	Park JH, Halitschke R, Kim HB, et al. A knock-out mutation in allene oxide synthase results in male sterility and defective wound signal transduction in Arabidopsis due to a block in jasmonic acid biosynthesis[J]. The Plant Journal, 2002, 31(1): 1-12.
[40]	Li Z, Luo X, Ou Y, et al. JASMONATE-ZIM DOMAIN proteins engage polycomb chromatin modifiers to modulate jasmonate signaling in Arabidopsis[J]. Molecular Plant, 2021, 14(5): 732-747.
[41]	Fang YX, Guo DS, Wang Y, et al. Rice transcriptional repressor OsTIE1 controls anther dehiscence and male sterility by regulating JA biosynthesis[J]. The Plant Cell, 2024: koae028.