草棉不同倍性材料叶片转录组差异表达分析

doi:10.3724/SP.J.1006.2022.14168

Abstract

Abstract:

To investigate the basis for the relative expression level of genes between the phenotypic changes of autopolyploid and the changes of genetic material during chromosome doubling, the differentially expressed genes between autopolyploids and diploids were screened by transcriptome sequencing of G. herbaceum. In this study, the diploid G. herbaceum (CK) and autopolyploid progenies were used as materials, autotriploid (T) and autotetraploid (M) plants were identified by flow cytometry. After the physiological indexes of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), proline, and soluble protein contents of autotetraploids and diploids were compared, and transcriptome sequencing were analyzed. The contents of SOD, POD, CAT, proline, and soluble protein in the leaves of autotetraploids were significantly higher than those of diploids. DESeq difference analysis showed that there were 4166 (2832 up-regulated and 1334 down-regulated), 4037 (1766 up-regulated and 2271 down-regulated), and 4184 (2679 up-regulated and 1505 down-regulated) DEGs, in CK vs T, T vs M, and CK vs M, respectively, and 205 DEGs were common expressed among three groups of pairwise comparison of different ploidy plants. GO function revealed that DEGs had significant differences in intrinsic component of membrane, integral component of membrane, catalytic activity. KEGG enrichment indicated that DEGs was more active than other pathways in flavonoid biosynthesis, phenylpropane biosynthesis, and photosynthesis-antenna protein. Through enrichment analysis, the DEGs related to ploidy in CK vs T and CK vs M were screened, and the results demonstrated that polyploidy were detected mostly up-regulated, in response to stimulus (62.46% and 54.84%), stress (57.42% and 53.85%), cell (70.16% and 66.22%), metabolism (72.73% and 66.14%), biologically active substances and resistance (60.33% and 63.37%), photosynthesis (58.06% and 63.16%), which may be the key factor leading to polyploid advantage. A total of 27 resistance-related genes (19 up-regulated and 8 down-regulated) and 11 photosynthetic-related genes (9 up-regulated and 2 down-regulated) were obtained by Nr annotation CK vs M. Cluster analysis of 27 DEGs related to photosynthesis in different ploidy of G. herbaceum showed that the relative expression of 18 genes (66.67%) had the highest expression abundance in autotetraploids. We performed Nr annotation and expression analysis on 205 DEGs common expressed among three groups, indicating that 30 genes were up-regulated, and the relative expression was the highest in autotetraploids. While 7 genes were down-regulated, and the expression was the lowest in autotetraploids. The 37 genes may be influenced by DNA gene-dose or related to plant ploidy level. The results showed that polyploid plants are superior to diploid plants in terms of resistance, growth vigor, metabolism, and photosynthesis rate.

Key words: Gossypium herbaceum, autopolyploids, flow cytometry, physiological indexs, transcriptome sequencing, differentially expressed genes

YANG Ya-Jie, LI Yu-Ying, SHEN Zhuang-Zhuang, CHEN Tian, RONG Er-Hua, WU Yu-Xiang. Differential expressed analysis by transcriptome sequencing in leaves of different ploidy Gossypium herbaceum[J].Acta Agronomica Sinica, 2022, 48(11): 2733-2748.

Figures/Tables 10

Table 1

Table 2

Table 3

Fig. 1

Fig. 2

Table 4

Functional classification of differentially expressed genes in CK vs M"

功能分类 Functional characterization	GO条目/代谢通路 GO term/pathway	上调基因数量 Number of up-regulated genes	下调基因数量 Number of down-regulated genes	差异表达基因数量 Number of DEGs	FDR
抗逆性 Stress resistance	应激反应 Response to stimulus (GO:0050896)	153 (54.84%)	126	279	1
抗逆性 Stress resistance	逆境反应 Response to stress (GO:0006950)	77 (53.85%)	66	143	1
细胞 Cell	细胞生长 Cell growth (GO:0016049)	16 (80.00%)	4	20	0.4033192
	细胞周期 Cell cycle (GO:0007049)	43 (91.49%)	4	47	0.0009799
	细胞壁 Cell wall (GO:0005618)	55 (83.33%)	11	66	6.599E-06
	细胞壁组织及生物发生 Cell wall organization or biogenesis (GO:0071554)	82 (82.83%)	17	99	4.835E-07
	细胞器 Organelle (GO:0043226)	371 (64.63%)	203	574	1
	生物调节 Biological regulation (GO:0065007)	255 (56.79%)	194	449	1
	细胞过程 Cellular process (GO:0009987)	926 (63.21%)	539	1465	1
	细胞膜 Membrane (GO:0016020)	920 (70.28%)	389	1309	4.826E-10
代谢 Metabolism	碳水化合物代谢 Carbohydrate metabolism (-)	140 (64.81%)	76	216	0.6612823
	氨基酸代谢 Amino acid metabolism (-)	80 (62.50%)	48	128	0.5819777
	核苷酸代谢 Nucleotide metabolism (-)	22 (66.67%)	11	33	0.5692334
	脂质代谢 Lipid metabolism (-)	96 (71.64%)	38	134	0.4141365
活性物质和抗性 Active substances and resistance	苯丙烷生物合成 Phenylpropanoid biosynthesis (ko00940)	33 (78.57%)	9	42	0.0492074
	黄酮和黄酮醇生物合成 Flavone and flavonol biosynthesis (ko00944)	2 (100%)	0	2	0.1934463
	类黄酮生物合成 Flavonoid biosynthesis (ko00941)	19 (100%)	0	19	0.0007179
功能分类 Functional characterization	GO条目/代谢通路 GO term/pathway	上调基因数量 Number of up-regulated genes	下调基因数量 Number of down-regulated genes	差异表达基因数量 Number of DEGs	FDR
活性物质和抗性 Active substances and resistance	单萜生物合成 Monoterpenoid biosynthesis (ko00902)	2 (66.67%)	1	3	0.3228966
	单萜生物合成 Monoterpenoid biosynthesis (ko00902)	2 (66.67%)	1	3	0.3228966
	二萜生物合成 Diterpenoid biosynthesis (ko00904)	5 (71.43%)	2	7	0.4795232
	倍半萜和三萜生物合成 Sesquiterpenoid and triterpenoid biosynthesis (ko00909)	6 (85.71%)	1	7	0.1255444
	萜类主链生物合成 Terpenoid backbone biosynthesis (ko00900)	6 (85.71%)	1	7	1
	过氧化物酶体 Peroxisome (ko04146)	4 (33.33%)	8	12	1
	半胱氨酸和蛋氨酸代谢 Cysteine and methionine metabolism (ko00270)	14 (66.67%)	7	21	0.4112855
	植物-病原相互作用 Plant-pathogen interaction (ko04626)	15 (42.86%)	20	35	0.9309574
	MAPK信号通路-植物 MAPK signaling pathway-plant (ko04016)	14 (45.16%)	17	31	0.4434954
	植物激素信号转导 Plant hormone signal transduction (ko04075)	34 (59.65%)	23	57	0.4795232
光合 Photosynthesis	光合作用-天线蛋白 Photosynthesis-antenna proteins (ko00196)	10 (100%)	0	10	0.034253
	类胡萝卜素生物合成 Carotenoid biosynthesis (ko00906)	1 (16.67%)	5	6	0.6849565
	卟啉与叶绿素代谢 Porphyrin and chlorophyll metabolism (ko00860)	3 (100%)	0	3	1
	光合合成碳的固定 Carbon fixation in photosynthetic organisms (ko00710)	5 (45.45%)	6	11	1
	光合作用 Photosynthesis (ko00195)	5 (62.50%)	3	8	1

Table 4

Table 5

Differentially expressed genes related to partial resistance and photosynthesis in CK vs M"

分类 Classify	代谢通路 Pathway	基因ID Gene ID	Nr 注释 Nr annotation	FDR
抗性 Resistance	MAPK信号通路-植物 MAPK signaling pathway-plant	gene-LOC108472111↑	EIN3结合F-box 1-样蛋白 EIN3-binding F-box 1-like protein	0.011587493
		gene-LOC108453563↑	EIN3结合F-box 1-样蛋白 EIN3-binding F-box 1-like protein	0.044251564
		gene-LOC108467504↑	蛋白磷酸酶2C 77-样蛋白 Protein phosphatase 2C 77-like protein	0.014589885
		gene-LOC108478294↑	丝裂原活化蛋白激酶9 Mitogen-activated protein kinase kinase 9	0.0008591
		gene-LOC108457272↑	乙烯受体2-样蛋白 Ethylene receptor 2-like protein	0.024368941
		gene-LOC108476169↑	内切几丁质酶1 Endochitinase 1	0.215277812
		gene-LOC108483169↓	乙烯受体Ethylene receptor	0.000750193
		gene-LOC108463936↓	丝裂原活化蛋白激酶17 Mitogen-activated protein kinase 17	0.016328221
		gene-LOC108450183↓	丝裂原活化蛋白激酶3 Mitogen-activated protein kinase 3	0.233022156
	植物激素信号转导 Plant hormone signal transduction	gene-LOC108484000↑	DELLA蛋白DWARF8-类 DELLA protein DWARF8-like	0.077283861
		gene-LOC108472111↑	EIN3结合F-box 1-样蛋白 EIN3-binding F-box 1-like protein	0.011587493
		gene-LOC108450344↑	生长素反应基因IAA27-样蛋白 Auxin-responsive IAA27-like protein	0.006431987
		gene-LOC108482284↑	蛋白TIFY 9-样蛋白 Protein TIFY 9-like protein	0.003835613
		gene-LOC108467504↑	蛋白磷酸酶2C 77-样蛋白 Protein phosphatase 2C 77-like protein	0.014589885
		gene-LOC108470619↑	生长素诱导的AUX28 Auxin-induced AUX28	2.81365E-06
		gene-LOC108466602↑	细胞周期蛋白D3-2-样蛋白 Cyclin-D3-2-like protein	0.134508825
		gene-LOC108453563↑	EIN3结合F-box 1-样蛋白 EIN3-binding F-box 1-like protein	0.044251564
		gene-LOC108461714↑	双组分反应调节剂ARR12-样蛋白 Two-component response regulator ARR12-like protein	0.170570504
		gene-LOC108485375↑	对脱落酸不敏感的5-样蛋白4 Abscisic acid-insensitive 5-like protein 4	0.002462993
		gene-LOC108469335↑	生长素反应基因IAA30-样蛋白 Auxin-responsive IAA30-like protein	0.1026197
		gene-LOC108457272↑	乙烯受体2-样蛋白 Ethylene receptor 2-like protein	0.024368941
抗性 Resistance	植物激素信号转导 Plant hormone signal transduction	gene-LOC108457634↑	生长素反应基因IAA9-样蛋白 Auxin-responsive IAA9-like protein	0.211599147
		gene-LOC108451896↓	IAA16蛋白IAA16 protein	0.01579825
		gene-LOC108457881↓	转录因子PIF3 Transcription factor PIF3	0.013753921
		gene-LOC108488454↓	双组分反应调节剂ARR9-样蛋白 Two-component response regulator ARR9-like protein	0.041768036
		gene-LOC108456238↓	调节剂NPR5-样蛋白 Regulatory NPR5-like protein	0.02941149
		gene-LOC108474109↓	转录因子PIF3-样蛋白 Transcription factor PIF3-like protein	6.26018E-05
光合 Photosynthesis	光合作用-天线蛋白 Photosynthesis-antenna proteins	gene-LOC108480333↑	叶绿素a-b结合13, 叶绿体 Chlorophyll a-b binding 13, chloroplastic	0.002356614
		gene-LOC108476043↑	叶绿素a-b结合CP24 10A, 叶绿体 Chlorophyll a-b binding CP24 10A, chloroplastic	0.000187662
		gene-LOC108489181↑	LHCII型叶绿素a-b结合蛋白-类 Chlorophyll a-b binding protein of LHCII type 1-like	1.58539E-05
	类胡萝卜素生物合成 Carotenoid biosynthesis	gene-LOC108469787↑	八氢番茄红素合成酶Phytoene synthase	1.59539E-05
	类胡萝卜素生物合成 Carotenoid biosynthesis	gene-LOC108461129↓	Zeta胡萝卜素去饱和酶, 叶绿体 Zeta-carotene desaturase, chloroplastic/chromoplastic	0.000454432
	卟啉与叶绿素代谢 Porphyrin and chlorophyll metabolism	gene-LOC108450311↑	原叶绿素还原酶, 叶绿体 Protochlorophyllide reductase, chloroplastic	0.000599834
	卟啉与叶绿素代谢 Porphyrin and chlorophyll metabolism	gene-LOC108461585↑	原叶绿素还原酶, 叶绿体 Protochlorophyllide reductase, chloroplastic	0.004268314
	光合合成碳的固定 Carbon fixation in photosynthetic organisms	gene-LOC108483247↑	磷酸烯醇式丙酮酸羧化酶 Phosphoenolpyruvate carboxylase, housekeeping isozyme	0.003229881
	光合合成碳的固定 Carbon fixation in photosynthetic organisms	gene-LOC108453781↑	果糖二磷酸醛缩酶, 细胞质同工酶 Fructose-bisphosphate aldolase, cytoplasmic isozyme	0.097210442
	光合作用 Photosynthesis	gene-LOC108488482↑	光系统I反应中心亚单位IV, 叶绿体 Photosystem I reaction center subunit IV, chloroplastic	0.012650004
	光合作用 Photosynthesis	gene-GoarCp007↓	ATP合成酶CF0 B亚单位 ATP synthase CF0 B subunit	0.159437765

Table 5

Table 6

Fig. 3

Table 7

Functional characteristics of common differentially expressed genes in different groups"

基因ID Gene ID	Nr注释 Nr annotation	GO条目 GO term
gene-LOC108450929↑	果胶甲基酯酶 Pectin methylesterase (gi\|377824753)	果胶分解代谢过程, 催化活性的负调节, 细胞壁降解 Pectin catabolic process (GO:0045490), negative regulation of catalytic activity (GO:0043086), cell wall modification (GO:0042545)
gene-LOC108489968↑	蛋白酪氨酸磷酸酶基因Pten (gi\|728820218)	肽基酪氨酸去磷酸化 Peptidyl-tyrosine dephosphorylation (GO:0035335)
gene-LOC108468194↑	65-kD微管相关4-样蛋白 65-kD microtubule-associated 4-like protein (gi\|728815965)	微管细胞骨架组织, 胞质分裂 Microtubule cytoskeleton organization (GO:0000226), cytokinesis (GO:0000910)
gene-LOC108473756↑	信号肽肽酶-样2B Signal peptide peptidase-like 2B (gi\|728825570)	蛋白质水解 Proteolysis (GO:0006508)
gene-LOC108450751↑	帕他汀A-3组 Patatin group A-3 (gi\|728835268)	脂质分解代谢过程 Lipid catabolic process (GO:0016042)
gene-LOC108489958↑	肽转运蛋白PTR1-样蛋白 Peptide transporter PTR1-like protein (gi\|728848860)	寡肽运输 Oligopeptide transport (GO:0006857)
gene-LOC108464066↑	类枯草杆菌蛋白酶 Subtilisin-like protease (gi\|728836560)	蛋白质水解 Proteolysis (GO:0006508)
gene-LOC108454777↑	BAG家族分子伴侣调节因子3 BAG family molecular chaperone regulator 3 (gi\|728828609)	—
基因ID Gene ID	Nr注释 Nr annotation	GO条目 GO term
gene-LOC108474367↑	膜蛋白B Envelope glycoprotein B (gi\|728812650)	—
gene-LOC108474374↑	多聚半乳糖醛酸酶, 非催化亚单位蛋白 Polygalacturonase non-catalytic subunit protein (gi\|728845004)	—
gene-LOC108467652↑	3-酮酰基辅酶A合酶48 3-ketoacyl-CoA synthase 48 (gi\|939191559)	脂肪酸生物合成过程 Fatty acid biosynthetic process (GO:0006633)
gene-LOC108463792↑	蛋白IQ-31结构域-样蛋白 Protein IQ-DOMAIN 31-like protein (gi\|728817716)	—
gene-LOC108482485↑	RNA聚合酶相关rapA RNA polymerase-associated rapA (gi\|728833883)	—
gene-LOC108488520↑	扩张蛋白-A5-样蛋白 Expansin-A5-like protein (gi\|728838775)	维细胞生长, 植物型细胞壁组织, 初生根发育 Unidimensional cell growth (GO:0009826), plant-type cell wall organization (GO:0009664), primary root development (GO:0080022)
gene-LOC108470064	束状阿拉伯半乳糖蛋白7 Fasciclin-like arabinogalactan protein 7 (gi\|728814486)	—
gene-LOC108457041↑	抗氧化1 Oxidation resistance 1 (gi\|728849019)	—
gene-LOC108454471↑	非特异性脂质转移3 Non-specific lipid-transfer 3 (gi\|728831038)	脂质转运 Lipid transport (GO:0006869)
gene-LOC108453673↑	微管相关TORTIFOLIA1-样蛋白 Microtubule-associated TORTIFOLIA1-like protein (gi\|728816012)	—
gene-LOC108450464↑	溶酶体β-葡萄糖苷酶 Lysosomal beta glucosidase (gi\|728850760)	葡聚糖分解代谢过程 Glucan catabolic process (GO:0009251)
gene-LOC108471291↑	蛋白透明种皮12-样蛋白 Protein transparent testa 12-like protein (gi\|728842256)	药物跨膜转运 Drug transmembrane transport (GO:0006855)
gene-LOC108450235↑	微管相关TORTIFOLIA1-样蛋白 Microtubule-associated TORTIFOLIA1-like protein (gi\|728831405)	—
gene-LOC108452970↑	孢子衣SA Spore coat SA (gi\|728832383)	—
gene-LOC108488617↑	脂质转移蛋白前体 Lipid transfer protein precursor (gi\|403391463)	脂质转运 Lipid transport (GO:0006869)
gene-LOC108483479↑	微管相关TORTIFOLIA1-样蛋白 Microtubule-associated TORTIFOLIA1-like protein (gi\|728812036)	—
gene-LOC108461575↑	葡甘聚糖4-β-甘露糖基转移酶9-样蛋白 Glucomannan 4-beta-mannosyltransferase 9-like protein (gi\|728840378)	—
基因ID Gene ID	Nr注释 Nr annotation	GO条目 GO term
基因ID Gene ID	Nr注释 Nr annotation	GO条目 GO term	gene-LOC108451848↑	纤维素合酶A催化亚基6[UDP形成]-样蛋白 Cellulose synthase A catalytic subunit 6 [UDP-forming]-like protein (gi\|728849775)	纤维素生物合成过程, 细胞壁组织 Cellulose biosynthetic process (GO:0030244), cell wall organization (GO:0071555)
gene-LOC108452375↑	双功能单脱氢抗坏血酸还原酶和碳酸酐酶-3 Bifunctional monodehydroascorbate reductase and carbonic anhydrase nectarin-3 (gi\|728829994)	—
gene-LOC108465289↑	葡甘聚糖4-β-甘露糖基转移酶2-样蛋白 Glucomannan 4-beta-mannosyltransferase 2-like protein (gi\|728841581)	甘露糖基化, 黏质生物合成过程, 与种皮发育有关的黏质代谢过程 Mannosylation (GO:0097502), mucilage biosynthetic process (GO:0010192), mucilage metabolic process involved in seed coat development (GO:0048359)
gene-LOC108457166↑	葡甘聚糖4-β-甘露糖基转移酶2-样蛋白 Glucomannan 4-beta-mannosyltransferase 2-like protein (gi\|728809263)	甘露糖基化, 黏质生物合成过程, 与种皮发育有关的黏质代谢过程 Mannosylation (GO:0097502), mucilage biosynthetic process (GO:0010192), mucilage metabolic process involved in seed coat development (GO:0048359)
gene-LOC108471046↑	磷酸核糖氨基咪唑琥珀酰胺合酶 Phosphoribosylaminoimidazole-succinocarboxamide synthase (gi\|728831447)	—
gene-LOC108489598↓	(+)-新薄荷醇脱氢酶-样蛋白 (+)-neomenthol dehydrogenase-like protein (gi\|7288377490)	氧化还原过程 Oxidation-reduction process (GO:0055114)
gene-LOC108472997↓	光调节Light-regulated (gi\|728817354)	—
gene-LOC108459335↓	光调节Light-regulated (gi\|728842555)	—
gene-LOC108476977↓	β-淀粉酶3, 叶绿体-样蛋白 Beta-amylase 3, chloroplastic-like protein (gi\|728849255)	多糖分解代谢过程 Polysaccharide catabolic process (GO:0000272)
gene-LOC108475943↓	丝氨酸/苏氨酸蛋白激酶CTR1 Serine/threonine-protein kinase CTR1 (gi\|728830021)	蛋白质磷酸化 Protein phosphorylation (GO:0006468)
gene-LOC108476209↓	γ-谷氨酰连接酶Gamma-glutamyl ligase (gi\|728810338)	—
gene-LOC108472513↓	戊二醛基二磷酸合酶, 叶绿体 Ent-copalyl diphosphate synthase, chloroplastic (gi\|728815153)	代谢过程Metabolic process (GO:0008152)

Table 7

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材料代号 Material code	平均荧光强度 X-mean	倍性 Ploidy
1	89.68	二倍体Diploid
2	139.51	三倍体Triploid
3	125.93	三倍体Triploid
4	135.29	三倍体Triploid
5	185.39	四倍体Tetraploid
6	181.19	四倍体Tetraploid
7	179.46	四倍体Tetraploid
8	178.46	四倍体Tetraploid
9	187.80	四倍体Tetraploid
10	157.11	三倍体-四倍体 Triploid-tetraploid
11	159.34	三倍体-四倍体 Triploid-tetraploid

光合指标 Photosynthetic index	二倍体 Diploid	三倍体 Triploid	四倍体 Tetraploid
净光合速率P_n (μmol m^-2 s^-1)	13.07±0.89 c	15.40±0.73 b	19.15±0.86 a
相对叶绿素含量 SPAD	33.23±1.77 c	36.05±0.66 b	38.07±0.81 a

材料 Material	SOD活性 SOD activity (U g^-1 min^-1)	POD活性 POD activity (U g^-1 min^-1)	CAT活性 CAT activity (U g^-1 min^-1)	脯氨酸含量 Proline content (μg g^-1)	可溶性蛋白含量 Soluble sugar content (mg g^-1)
二倍体Diploid	138.96±4.10	76.22±7.90	9.33±0.67	8.14±0.23	8.70±0.55
同源四倍体Autotetraploid	186.14±10.86^**	99.56±7.00^*	17.22±2.36^**	12.74±0.36^**	14.92±0.35^**

Differential expressed analysis by transcriptome sequencing in leaves of different ploidy Gossypium herbaceum

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功能分类 Functional characterization	CK vs T DEGs		T vs M DEGs		CK vs M DEGs
功能分类 Functional characterization	Up	DEGs	Up	DEGs	Up	DEGs
应激反应 Response to stimulus	178 (62.46%)	285	116 (42.34%)	274	153 (54.84%)	279
逆境反应 Response to stress	89 (57.42%)	155	69 (46.62%)	148	77 (53.85%)	143
细胞 Cell	2586 (70.16%)	3686	1621 (45.22%)	3585	2668 (66.22%)	4029
代谢 Metabolism	360 (72.73%)	495	164 (38.32%)	428	338 (66.14%)	511
活性物质和抗性 Active substances and resistance	181 (60.33%)	300	124 (47.69%)	260	154 (63.37%)	243
光合 Photosynthesis	18 (58.06%)	31	21 (43.75%)	48	24 (63.16%)	38

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