黄瓜二倍体及其同源四倍体果实转录组分析

doi:10.13560/j.cnki.biotech.bull.1985.2022-0974

摘要/Abstract

摘要：

果形和糖含量分别是与瓜类蔬菜作物外观、营养及风味密切相关的重要品质性状。为探究黄瓜倍性差异导致果形及糖含量变化的分子机制，以果形和糖含量差异显著的黄瓜二倍体（长果/低糖）和四倍体（短果/高糖）为试材，分别取开花当天和花后9 d的果实进行转录组分析。结果表明，在二、四倍体花后9 d果实中共鉴定到1 874个差异表达基因（DEGs），其中上调基因818个和下调基因1 056个；GO分析显示，DEGs显著富集在“DNA复制”（GO:0006260）、“DNA代谢过程”（GO:0006259）、“蛋白质复合体”（GO:0032993）、“染色体”（GO:0005694）、“蛋白质二聚活性”（GO:0046983）等GO条目上；KEGG分析表明，DEGs富集在“植物激素信号转导”（csv04075）、“淀粉与蔗糖代谢”（csv00500）等代谢通路上；参与“植物激素信号转导”相关基因AUX1、DELLA、B-ARR、TCH4上调表达，GH3、GID1、PP2C、CYCD3下调表达，以及参与“淀粉与蔗糖代谢”相关基因SPS、SUSY上调表达，可能与果形指数减小和果实糖含量增加有关。荧光定量PCR（RT-qPCR）分析显示，除ARF4外，所选定DEGs的表达模式与RNA-Seq中表达趋势相一致，其中SUSY在二、四倍体黄瓜果实中差异表达极显著，推测该基因在调控黄瓜四倍体果形及糖含量形成过程中起到重要作用。

关键词: 黄瓜, 同源四倍体, 转录组, 差异表达基因

Abstract:

Fruit shape and sugar content are important quality traits closely related to the appearance, nutrition and flavor of melon vegetables, respectively. To investigate the molecular mechanism of fruit shape and sugar content in cucumber caused by ploidy differences, the diploid（long fruit/low sugar）and autotetraploid（short fruit/high sugar）cucumber with significant differences of shape and sugar content were selected as the study materials, and RNA-seq analysis on the fruits at 0 and 9 d after flowering were performed. The results showed that a total of 1 874 differentially expressed genes（DEGs）were identified from diploid and autotetraploid at 9 d after flowering, including 818 up-regulated genes and 1 056 down-regulated genes. GO analysis demonstrated that the DEGs were significantly enriched in DNA replication（GO:0006260）, DNA metabolic process（GO:0006259）, protein-DNA complex（GO:0032993）, chromosome（GO:0005694）, and protein dimerization activity（GO:0046983）. KEGG analysis revealed that the DEGs were enriched in “plant hormone signal transduction”（csv04075）, “starch and sucrose metabolism”（csv00500）and other metabolic pathways. The up-regulated expressions of AUX1, DELLA, B-ARR and TCH4 and down-regulated expression of GH3, GID1, PP2C and CYCD3 related to “plant hormone signal transduction”, as well as the up-regulated expression of SPS and SUSY related to “starch and sucrose metabolism”, may correlate with fruit shape index decreasing shortening and sugar content increasing in the cucumber. Quantitative reverse transcription-PCR（RT-qPCR）analysis showed that the expression pattern of selected DEGs was consistent with the expression trend of RNA-Seq except for ARF4, among which SUSY was highly differentially expressed between diploid and autotetraploid cucumber fruits, suggesting that SUSY plays a critical role in regulating fruit shape and sugar content in cucumber autotetraploid.

Key words: cucumber, autotetraploid, transcriptome, differentially expressed genes

谢洋, 邢雨蒙, 周国彦, 刘美妍, 银珊珊, 闫立英. 黄瓜二倍体及其同源四倍体果实转录组分析[J]. 生物技术通报, 2023, 39(3): 152-162.

XIE Yang, XING Yu-meng, ZHOU Guo-yan, LIU Mei-yan, YIN Shan-shan, YAN Li-ying. Transcriptome Analysis of Diploid and Autotetraploid in Cucumber Fruit[J]. Biotechnology Bulletin, 2023, 39(3): 152-162.

图/表 9

图1 黄瓜二倍体及其同源四倍体果形及可溶性糖含量分析 A：黄瓜二、四倍体不同发育时期果实形态学观察；B：黄瓜二、四倍体不同发育时期果实细胞学观察；C：黄瓜二、四倍体不同发育时期果实果形指标统计；D：黄瓜二、四倍体果实可溶性糖含量分析。不同小写字母表示在0.05水平差异显著

Fig. 1 Analysis of diploid and autotetraploid fruit shapes as well as their soluble sugar contents in cucumber A: Morphological observation of cucumber fruits at different developmental stages of diploid and tetraploid. B: Fruit cytology of cucumber diploid and tetraploid at different developmental stages. C: Statistics of fruit shape index of cucumber diploid and tetraploid at different developmental stages. D: Analysis of soluble sugar content in diploid and tetraploid fruits of cucumber. Different lower letters indicate significant difference at 0.05 level

表1 转录组测序数据质量分析

Table 1 Quality analysis of transcriptome sequencing data

样品名称 Sample name	Raw reads/Mb	Clean reads/Mb	Unique map/Mb	Q30/%	GC/%
CK_S1_1	45.32	43.78	40.87	92.94	40.88
CK_S1_2	48.02	45.29	43.08	94.72	40.85
CK_S2_1	44.23	43.35	40.80	93.02	42.14
CK_S2_2	44.89	43.22	40.68	92.63	42.28
T_S1_1	46.21	43.69	40.35	92.56	40.82
T_S1_2	47.89	45.85	42.61	92.98	41.09
T_S2_1	45.83	44.53	42.08	92.9	42.43
T_S2_2	47.69	45.88	43.46	93.49	42.43

图2 Clean reads在基因组区域分布情况黄瓜二、四倍体果实不同发育时期样本测序的clean reads在基因组区域分布情况

Fig. 2 Percents of clean reads mapped in genome regions Distribution of clean reads sequenced in different developmental stages of cucumber diploid and tetraploid fruits in genomic regions

图3 样本基因表达量分布及相关性分析 A：黄瓜二、四倍体果实不同发育时期样本基因表达量分布盒形图；B：黄瓜二、四倍体果实不同发育时期样本皮尔逊相关性分析；C：黄瓜二、四倍体果实不同发育时期样本基因表达量PCA分析

Fig. 3 Gene expression distribution and correlation analysis of samples A: Box diagram of gene expression distribution in cucumber diploid and tetraploid fruits at different developmental stages. B: Pearson correlation analysis of cucumber diploid and tetraploid fruit samples at different developmental stages. C: PCA analysis of gene expression levels in diploid and tetraploid cucumber fruits at different developmental stages

图4 差异基因数目统计及维恩图分析 A：黄瓜二、四倍体果实不同发育时期不同比较组差异基因统计柱状图；B：黄瓜二、四倍体果实不同发育时期不同比较组差异基因维恩图

Fig. 4 Analysis of DEGs statistical number and Venn diagram A: Bar chart of DEGs statistics of different comparison groups in different developmental stages of cucumber diploid and tetraploid fruits. B: Venn diagram of DEGs in different comparison groups at different developmental stages of cucumber diploid and tetraploid fruits

图5 GO与KEGG富集散点图 A：黄瓜二、四倍体果实（T_S2vsCK_S2）差异基因GO富集散点图；B：黄瓜二、四倍体果实（T_S2vsCK_S2）差异基因KEGG富集散点图

Fig. 5 Rich distribution point diagram for GO and KEGG A: Map of GO enrichment of DEGs in T_S2vsCK_S2 of cucumber fruit. B: Map of KEGG enrichment of DEGs in T_S2vsCK_S2 of cucumber fruit

表2 KEGG富集通路上关键候选基因

Table 2 Key candidate genes in KEGG enrichment pathway

基因名称Gene name	基因 ID Gene ID	log₂FC	基因注释Gene annotation
AUX1	Csa_3G731880	0.511028525	Auxin influx carrier（AUX1 LAX family）
B-ARR	Csa_2G350290	1.199233905	Uncharacterized protein LOC101203501 isoform X1
DELLA	Csa_5G569350	0.641993478	DELLA protein
SIMKK	Csa_3G651720	0.521551332	Mitogen-activated protein kinase kinase 4/5
EIN2	Csa_6G445230	0.401357885	Ethylene-insensitive protein 2
EIN3	Csa_6G051520	0.672607401	Ethylene-insensitive protein 3
BRI1	Csa_6G486870	0.529640378	Protein brassinosteroid insensitive 1
BIN2	Csa_6G157100	0.536467357	Protein brassinosteroid insensitive 2
BZR1/2	Csa_2G361450	0.587660284	Brassinosteroid resistant 1/2
TCH4	Csa_1G422470	1.185601828	Xyloglucan:xyloglucosyl transferase TCH4
TGA	Csa_6G152350	1.955263915	Transcription factor TGA
GH3	Csa_6G493310	-0.840958686	Auxin responsive GH3 gene family
GID1	Csa_7G391240	-1.403103089	Gibberellin receptor GID1
PP2C	Csa_1G574880	-1.528004155	Protein phosphatase 2C
ETR	Csa_2G070880	-0.799320186	Ethylene receptor
BAK1	Csa_7G060160	-0.875573337	Brassinosteroid insensitive 1-associated receptor kinase 1
BKI1	Csa_2G359970	-1.610933985	BRI1 kinase inhibitor 1
CYCD3	Csa_6G491650	-1.00709524	Cyclin D3, plant
JAZ	Csa_1G042920	-0.913564833	Jasmonate ZIM domain-containing protein
AGLU1	Csa_1G716250	0.650665663	Alpha-glucosidase
SPS	Csa_2G401440	0.977042232	Sucrose-phosphate synthase
SUSY	Csa_5G322500	2.088838655	Sucrose synthase
FRK	Csa_3G345390	-0.650140834	Fuctokinase
AGPase	Csa_3G149890	-1.393581098	Glucose-1-phosphate adenylyltransferase
SS	Csa_6G497160	-0.899393235	Starch synthase
GTase	Csa_5G606600	-0.684711983	4-alpha-glucanotransferase
GBE1	Csa_7G213190	-1.304743764	1,4-alpha-glucan branching enzyme
BMY	Csa_3G017020	-3.271484788	Beta-amylase
TRE	Csa_5G149880	-1.411978481	Alpha,alpha-trehalase

表2 KEGG富集通路上关键候选基因

Table 2 Key candidate genes in KEGG enrichment pathway

基因名称Gene name	基因 ID Gene ID	log₂FC	基因注释Gene annotation
AUX1	Csa_3G731880	0.511028525	Auxin influx carrier（AUX1 LAX family）
B-ARR	Csa_2G350290	1.199233905	Uncharacterized protein LOC101203501 isoform X1
DELLA	Csa_5G569350	0.641993478	DELLA protein
SIMKK	Csa_3G651720	0.521551332	Mitogen-activated protein kinase kinase 4/5
EIN2	Csa_6G445230	0.401357885	Ethylene-insensitive protein 2
EIN3	Csa_6G051520	0.672607401	Ethylene-insensitive protein 3
BRI1	Csa_6G486870	0.529640378	Protein brassinosteroid insensitive 1
BIN2	Csa_6G157100	0.536467357	Protein brassinosteroid insensitive 2
BZR1/2	Csa_2G361450	0.587660284	Brassinosteroid resistant 1/2
TCH4	Csa_1G422470	1.185601828	Xyloglucan:xyloglucosyl transferase TCH4
TGA	Csa_6G152350	1.955263915	Transcription factor TGA
GH3	Csa_6G493310	-0.840958686	Auxin responsive GH3 gene family
GID1	Csa_7G391240	-1.403103089	Gibberellin receptor GID1
PP2C	Csa_1G574880	-1.528004155	Protein phosphatase 2C
ETR	Csa_2G070880	-0.799320186	Ethylene receptor
BAK1	Csa_7G060160	-0.875573337	Brassinosteroid insensitive 1-associated receptor kinase 1
BKI1	Csa_2G359970	-1.610933985	BRI1 kinase inhibitor 1
CYCD3	Csa_6G491650	-1.00709524	Cyclin D3, plant
JAZ	Csa_1G042920	-0.913564833	Jasmonate ZIM domain-containing protein
AGLU1	Csa_1G716250	0.650665663	Alpha-glucosidase
SPS	Csa_2G401440	0.977042232	Sucrose-phosphate synthase
SUSY	Csa_5G322500	2.088838655	Sucrose synthase
FRK	Csa_3G345390	-0.650140834	Fuctokinase
AGPase	Csa_3G149890	-1.393581098	Glucose-1-phosphate adenylyltransferase
SS	Csa_6G497160	-0.899393235	Starch synthase
GTase	Csa_5G606600	-0.684711983	4-alpha-glucanotransferase
GBE1	Csa_7G213190	-1.304743764	1,4-alpha-glucan branching enzyme
BMY	Csa_3G017020	-3.271484788	Beta-amylase
TRE	Csa_5G149880	-1.411978481	Alpha,alpha-trehalase

表3 RT-qPCR引物序列信息

Table 3 Information of RT-qPCR primer sequences

基因名称Gene name	注释Annotation	引物序列Primer sequence（5'-3'）
SUSY	蔗糖合酶 Sucrose synthase	F：TGAGAACGATGAGCATATAG
SUSY	蔗糖合酶 Sucrose synthase	R：TCCAACCACAACAAGATT
K-box	转录因子K-box Transcription factor K-box	F：CAAGAACAGGAAGAGGAA
K-box	转录因子K-box Transcription factor K-box	R：GAGGAGAAGACGATAAGAG
WUS	WUSCHEL相关homeobox WUSCHEL-related homeobox	F：GTGAGCCATTGATGACAT
WUS	WUSCHEL相关homeobox WUSCHEL-related homeobox	R：GGAACCAGTTATAGACATTAGA
SUT	蔗糖转运蛋白 Sucrose transporter	F：CTGAGACGGTTACTAAGAG
SUT	蔗糖转运蛋白 Sucrose transporter	R：TTGAATATAAGGAGTGAGAAGA
GLU8	葡萄糖转运蛋白8 Glucose transporter protein 8	F：CCTCACAACACTCAATCA
GLU8	葡萄糖转运蛋白8 Glucose transporter protein 8	R：CACAGACCAACAATCAGA
DNAH	DNA解旋酶 DNA helicase	F：GCTCAAGTCGTTACCATT
DNAH	DNA解旋酶 DNA helicase	R：CATCAACTGAACTCACCTT
ARF4	激素响应因子4 Auxin response factor 4	F：GTCTACCATACTCGTGTT
ARF4	激素响应因子4 Auxin response factor 4	R：CTAATGCCAGTGATTGTG
GIR	赤霉素调控蛋白 Gibberellin-regulated protein	F：TCCTCTCTTCTCGTTCTT
GIR	赤霉素调控蛋白 Gibberellin-regulated protein	R：GCTCCTCCACAATCAATA
LRK	蛋白激酶家族蛋白 Protein kinase family protein	F：CGGAGATGACTGTTGTAA
LRK	蛋白激酶家族蛋白 Protein kinase family protein	R：TCCAATCAGCAATAATAACG
CsActin	内参基因 Actin gene	F：ATTGTTCTCAGTGGTGGTTCTAC
CsActin	内参基因 Actin gene	R：CCTTTGAGATCCACATCTGCT

图6 候选基因RT-qPCR分析不同小写字母代表同一基因在二、四倍体黄瓜果实中表达量存在显著差异（P<0.05）

Fig. 6 RT-qPCR analysis of candidate genes Different lowercase letters indicate significant differences in the expression levels of the same gene in diploid and tetraploid cucumber fruits at 0.05 level

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