大豆细胞质雄性不育弱恢复型杂种F1育性转变的转录组分析

doi:10.13560/j.cnki.biotech.bull.1985.2024-0066

摘要/Abstract

摘要：

【目的】 探索大豆细胞质雄性不育弱恢复型杂种F₁育性转变在RNA水平上的分子机制，以期从育性转变的角度为大豆细胞质雄性不育的分子机理提供有价值的信息。【方法】 以弱恢复型杂种F₁（H3A×SXTH3）为研究对象，设置苗期短光照（植株育性不育）和正常光照（植株育性可育）处理，在盛花期分别采集不同大小的混合花芽进行转录组测序和RT-qPCR分析。【结果】 筛选出3 917个差异表达基因，苗期短光照处理后，2 134个基因下调表达，1 783个基因上调表达。对差异表达基因进行生物信息学分析，GO显著富集分析表明，碳水化合物代谢过程、跨膜转运活性和细胞外围等功能在育性转变中行使着主要生物学功能；KEGG通路显著富集分析表明，戊糖葡萄糖醛酸的相互转化、淀粉蔗糖代谢和植物昼夜节律等通路为育性转变的主要代谢通路。11个基因的RT-qPCR分析发现，大豆细胞质雄性不育相关基因和PPR基因参与了大豆细胞质雄性不育弱恢复型杂种F₁育性转变过程。【结论】 推测大豆细胞质雄性不育弱恢复型杂种F₁育性转变与植物昼夜节律、PPR和大豆细胞质雄性不育相关的线粒体、花粉壁发育、碳水化合物代谢、糖转运和活性氧代谢等基因的异常表达相关，当昼夜节律通路的关键基因变化，引起PPR基因和大豆细胞质雄性不育相关基因的表达水平变化，将会发生育性转变。

关键词: 大豆, 细胞质雄性不育, 杂种F₁, 育性, 转录组测序

Abstract:

【Objective】 It is to explore the molecular mechanism of fertility transformation in weakly restoring hybrid F₁ of soybean cytoplasmic male sterility at the RNA level, then to provide valuable information on the molecular mechanism of male sterility in soybean from the perspective of fertility transition.【Method】 Using weakly restoring hybrid F₁（H3A× SXTH3）as the research object, and short light（plant fertility is sterile）and normal light（plant fertility is fertile）treatments were set up during the seedling stage. Mixed flower buds of different sizes were collected during the peak flowering period for transcriptome sequencing and quantitative real time PCR（RT-qPCR）analysis.【Result】 Total 3 917 differentially expressed genes were screened out. After short light treatment during the seedling stage, 2 134 genes were downregulated and 1 783 genes were upregulated. Bioinformatics analysis was conducted on differentially expressed genes, and gene ontology（GO）significant enrichment analysis showed that carbohydrate metabolic process, transmembrane transporter activity, and cell periphery functions played the main biological functions during fertility transition. The significant enrichment analysis of the kyoto encyclopedia of genes and genomes（KEGG）pathway showed that the main metabolic pathways for fertility transformation were pentose and glucuronate interconversions, starch and sucrose metabolism, and plant circadian rhythm. RT-qPCR analysis of 11 genes revealed that genes related to soybean cytoplasmic male sterility and pentatricopeptide repeats protein（PPR）were involved during fertility transformation of weakly restoring cytoplasmic male sterile hybrid F₁ in soybean.【Conclusion】 It was speculated that the fertility transformation of weakly restoring hybrid F₁ was related to abnormal expressions of genes related to plant circadian rhythm, PPR, and soybean cytoplasmic male sterility, such as mitochondrial, pollen wall development, carbohydrate metabolism, sugar transport, and reactive oxygen species metabolism. Key genes in the circadian rhythm pathway change cause changes in the expression levels of PPR genes and soybean cytoplasmic male sterility related genes, then fertility transformation will occur.

Key words: soybean, cytoplasmic male sterility, hybrid F₁, fertility, transcriptome sequencing

白志元, 徐菲, 杨午, 王明贵, 杨玉花, 张海平, 张瑞军. 大豆细胞质雄性不育弱恢复型杂种F₁育性转变的转录组分析[J]. 生物技术通报, 2024, 40(6): 134-142.

BAI Zhi-yuan, XU Fei, YANG Wu, WANG Ming-gui, YANG Yu-hua, ZHANG Hai-ping, ZHANG Rui-jun. Transcriptome Analysis of Fertility Transformation in Weakly Restoring Hybrid F₁of Soybean Cytoplasmic Male Sterility[J]. Biotechnology Bulletin, 2024, 40(6): 134-142.

图/表 6

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基因ID Gene_id	基因注释 Gene annotation	正向引物序列 Forward primer sequence（5'-3'）	反向引物序列 Reverse primer sequence（5'-3'）
AFR34332.1	COX2	TGGCTATTCCTCACGATTGCT	GCATCATAGGTGTTGCTGCG
AFR34381.1	ORF103c	AAACTCGAAAGGGTGGAAGTCT	TCCCCTGCATAGGGAATACCA
AFR34382.1	ORF178	GGCATCGGTCCGTAGAACTT	TCCATAACATAACGGGGCGG
Glyma.02G008300	果胶甲基酯酶Pectin methylesterase	CTCATTAGCAAGCAGGAGGCT	AGCATCGAAAGGAACGCCAG
Glyma.13G064700	果胶裂解酶Pectin lyase	CGACAACGCCAAGGAGATCA	ATGGGTGAGCCTGAAGTGAC
Glyma.07G246600	UDP葡萄糖醛酸脱羧酶 UDP glucuronic acid decarboxylase	AACCGCACCTCAGATTCACC	TGAGGAGCCGACACTATGGA
Glyma.20G103900	糖转运蛋白11 Sugar transporter protein 11	AGCTTGTTCTTGCAAGTGAGTC	CTGAGGCCTGTATTTTGGCG
Glyma.02G154400	谷胱甘肽S转移酶Glutathione S-transferase	CTGCATCACCTCCCAACGAT	ATATCAGCCACCCATGCACC
Glyma.07G225400	L抗坏血酸氧化酶L-ascorbic acid oxidase	GTGCCCCAGCAGGTTATTCT	CCCAATGGAAAAGCAAGGGC
Glyma.01G011300	含五肽重复序列的蛋白质At1g31920 PPR At1g31920	GACCGTAATCGCTTCCACCA	AAGATGCTCAACATACCACAATG
Glyma.08G038200	含五肽重复序列的蛋白质At5g21222异构体X1 PPR At5g21222 isoform X1	TGGGGATCACTTCTTGGTGC	TCAGTTTCCTGACCTGACGC

基因ID Gene_id	基因注释 Gene annotation	正向引物序列 Forward primer sequence（5'-3'）	反向引物序列 Reverse primer sequence（5'-3'）
AFR34332.1	COX2	TGGCTATTCCTCACGATTGCT	GCATCATAGGTGTTGCTGCG
AFR34381.1	ORF103c	AAACTCGAAAGGGTGGAAGTCT	TCCCCTGCATAGGGAATACCA
AFR34382.1	ORF178	GGCATCGGTCCGTAGAACTT	TCCATAACATAACGGGGCGG
Glyma.02G008300	果胶甲基酯酶Pectin methylesterase	CTCATTAGCAAGCAGGAGGCT	AGCATCGAAAGGAACGCCAG
Glyma.13G064700	果胶裂解酶Pectin lyase	CGACAACGCCAAGGAGATCA	ATGGGTGAGCCTGAAGTGAC
Glyma.07G246600	UDP葡萄糖醛酸脱羧酶 UDP glucuronic acid decarboxylase	AACCGCACCTCAGATTCACC	TGAGGAGCCGACACTATGGA
Glyma.20G103900	糖转运蛋白11 Sugar transporter protein 11	AGCTTGTTCTTGCAAGTGAGTC	CTGAGGCCTGTATTTTGGCG
Glyma.02G154400	谷胱甘肽S转移酶Glutathione S-transferase	CTGCATCACCTCCCAACGAT	ATATCAGCCACCCATGCACC
Glyma.07G225400	L抗坏血酸氧化酶L-ascorbic acid oxidase	GTGCCCCAGCAGGTTATTCT	CCCAATGGAAAAGCAAGGGC
Glyma.01G011300	含五肽重复序列的蛋白质At1g31920 PPR At1g31920	GACCGTAATCGCTTCCACCA	AAGATGCTCAACATACCACAATG
Glyma.08G038200	含五肽重复序列的蛋白质At5g21222异构体X1 PPR At5g21222 isoform X1	TGGGGATCACTTCTTGGTGC	TCAGTTTCCTGACCTGACGC

样品 Sample	Reads总数 Total reads	碱基总数 Total bases	Q20/%	Q30/%	过滤读数 Clean reads	过滤碱基数Clean bases	过滤数百分比 Clean percentage/%	比对总数 Total_ mapped	比对总数百分比Total_ mapped percentage/%	单个比对数 Uniquely_mapped	单个比对数百分比Uniquely_mapped percentage/%
A_T_1	51 129 282	7 669 392 300	98.01	94.68	47 150 672	7 072 600 800	92.21	45 134 613	95.72	44 178 583	97.88
A_T_2	51 432 310	7 714 846 500	98.03	94.73	47 431 430	7 114 714 500	92.22	45 474 740	95.87	44 424 415	97.69
A_T_3	43 683 610	6 552 541 500	97.93	94.56	40 201 490	6 030 223 500	92.02	38 478 493	95.71	37 620 308	97.77
A_CK_1	51 196 992	7 679 548 800	97.98	94.65	47 273 686	7 091 052 900	92.33	45 349 845	95.93	44 350 422	97.80
A_CK_2	46 737 644	7 010 646 600	98.18	94.95	43 318 284	6 497 742 600	92.68	41 632 540	96.11	40 757 276	97.90
A_CK_3	49 420 632	7 413 094 800	98.05	94.75	45 611 544	6 841 731 600	92.29	43 802 214	96.03	42 854 974	97.84
平均 Average	48 933 412	7 340 011 750	98.03	94.72	45 164 518	6 774 677 650	92.29	43 312 074	95.90	42 364 330	97.81

样品 Sample	Reads总数 Total reads	碱基总数 Total bases	Q20/%	Q30/%	过滤读数 Clean reads	过滤碱基数Clean bases	过滤数百分比 Clean percentage/%	比对总数 Total_ mapped	比对总数百分比Total_ mapped percentage/%	单个比对数 Uniquely_mapped	单个比对数百分比Uniquely_mapped percentage/%
A_T_1	51 129 282	7 669 392 300	98.01	94.68	47 150 672	7 072 600 800	92.21	45 134 613	95.72	44 178 583	97.88
A_T_2	51 432 310	7 714 846 500	98.03	94.73	47 431 430	7 114 714 500	92.22	45 474 740	95.87	44 424 415	97.69
A_T_3	43 683 610	6 552 541 500	97.93	94.56	40 201 490	6 030 223 500	92.02	38 478 493	95.71	37 620 308	97.77
A_CK_1	51 196 992	7 679 548 800	97.98	94.65	47 273 686	7 091 052 900	92.33	45 349 845	95.93	44 350 422	97.80
A_CK_2	46 737 644	7 010 646 600	98.18	94.95	43 318 284	6 497 742 600	92.68	41 632 540	96.11	40 757 276	97.90
A_CK_3	49 420 632	7 413 094 800	98.05	94.75	45 611 544	6 841 731 600	92.29	43 802 214	96.03	42 854 974	97.84
平均 Average	48 933 412	7 340 011 750	98.03	94.72	45 164 518	6 774 677 650	92.29	43 312 074	95.90	42 364 330	97.81

基因ID Gene ID	A_CK平均值 baseMean_A_CK	A_T平均值 baseMean_A_T	基因注释 Gene annotation	Log₂（变化倍数） log₂（FC）	P值 P value
Glyma.03G261800	9 951.556 4	55.757 5	MYB转录因子MYB114 MYB transcription factor MYB114	-7.479 6	4.09E-222
Glyma.19G260900	3 490.330 4	54.321 4	LHY蛋白 LHY protein	-6.005 7	2.61E-150
Glyma.16G017400	14 705.494 2	897.924 7	晚期伸长下胚轴/昼夜节律时钟相关蛋白 Late elongation hypocotyl/ circadian clock related proteins	-4.033 6	1.45E-98
Glyma.02G267800	662.352 9	165.158 2	E3泛素蛋白连接酶COP1 E3 ubiquitin-protein ligase COP1	-2.003 8	5.92E-20
Glyma.14G049700	1 034.788 7	263.650 1	E3泛素蛋白连接酶COP1 E3 ubiquitin-protein ligase COP1	-1.972 6	2.56E-22
Glyma.07G048500	1 425.609 8	402.471 1	LHY1蛋白 LHY1 protein	-1.824 6	5.89E-21
Glyma.07G013500	264.329 0	87.150 1	ELF3蛋白2亚型X1 ELF3 protein 2 isoform X1	-1.600 8	1.67E-09
Glyma.03G225000	801.462 1	309.803 6	转录因子PIF3 Transcription factor PIF3	-1.371 3	9.81E-12
Glyma.14G174200	959.094 4	386.228 3	隐色素-1 Cryptochrome-1	-1.312 2	2.63E-11
Glyma.16G092700	205.123 5	102.488 5	bZIP转录因子bZIP69 bZIP transcription factor bZIP69	-1.001 0	0.000 170 8
Glyma.04G228300	520.821 0	5 515.352 4	双组分响应调节器样APRR9亚型X2 Two component response regulator like APRR9 subtype X2	3.404 6	1.95E-23
Glyma.16G150700	1.939 3	16.919 3	磷脂酰乙醇胺结合蛋白FT2a Phosphatidyl ethanolamine binding protein FT2a	3.125 1	0.043 659 5
Glyma.06G136600	232.560 3	1 063.196 7	双组分响应调节器，如PRR95 Two component response regulator-like PRR95	2.192 7	3.16E-05
Glyma.07G058200	340.731 0	1 475.765 4	PHYA-105 1亚型X2的蛋白抑制剂 Protein inhibitor of PHYA-105 1 isoform X2	2.114 8	4.15E-05
Glyma.10G221500	2 158.991 6	6 635.026 4	GIGANTEA核蛋白 GIGANTEA nuclear protein	1.619 7	4.07E-10
Glyma.16G027200	342.509 0	880.504 9	PHYA-105蛋白抑制剂 Protein suppressor of PHYA-105	1.362 2	7.15E-12
Glyma.01G192100	426.999 5	1 089.072 6	酪蛋白激酶II亚单位α-2 Casein kinase II subunit α-2	1.350 8	2.99E-12
Glyma.04G050200	542.052 4	1 120.864 8	早花相关蛋白 Ealrly flower related proteins	1.048 1	0.002 186 9
Glyma.19G260400	586.539 7	1 207.006 2	双组分响应调节器样PRR95亚型X1 Two component response regulator-like PRR95 isoform X1	1.041 1	0.018 590 2
Glyma.13G135900	1 456.119 6	2 977.895 5	双组分响应调节器APRR7亚型X1 Two component response regulator-like APRR7 isoform X1	1.032 2	4.15E-09

大豆细胞质雄性不育弱恢复型杂种F₁育性转变的转录组分析

Transcriptome Analysis of Fertility Transformation in Weakly Restoring Hybrid F₁of Soybean Cytoplasmic Male Sterility

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