菜心不同器官糖含量比较及代谢相关基因的表达分析

doi:10.3864/j.issn.0578-1752.2023.11.010

摘要/Abstract

摘要：

【目的】菜心口感脆嫩，营养价值高，是华南地区人们最喜爱的蔬菜之一，其中糖的种类和含量是决定菜心甜度和风味的主要因素。分析菜心不同器官糖含量及与糖代谢和转运相关基因的表达，以探明导致菜心不同器官糖含量差异的分子机制。【方法】采用高效液相色谱法测定菜心叶片、薹茎和花蕾中可溶性糖、蔗糖、葡萄糖和果糖的含量；通过转录组测序技术对菜心叶片、薹茎和花蕾基因的表达量进行全面系统的分析，并利用DESeq2软件鉴定3个不同器官间的差异表达基因，分析糖代谢通路及转运过程涉及的酶编码基因的表达，在基迪奥生信云平台将筛选到的与糖代谢转运相关的差异表达基因和糖含量进行相关性分析，同时利用实时荧光定量PCR（qRT-PCR）技术对部分基因的表达量进行验证。【结果】糖含量测定结果表明，菜心不同器官之间糖含量差异明显。可溶性糖、蔗糖、葡萄糖和果糖含量均呈现花蕾>薹茎>叶片的趋势。花蕾中葡萄糖含量分别为薹茎和叶片的1.3倍和1.6倍，果糖含量分别为薹茎和叶片的1.42倍和1.78倍。通过分析糖代谢关键酶和转运蛋白编码基因与糖含量变化趋势的一致性，共找出18个与糖含量变化趋势一致的基因。通过分析糖代谢转运中相关差异基因的表达与糖种类及含量的相关性和一致性，鉴定了14个与糖含量变化趋势一致的DEG。合并7个共有基因（Bra020096、Bra029914、Bra033419、Bra037980、Bra027398、Bra006129和Bra006130），共计25个基因与糖含量变化趋势一致。qRT-PCR结果分析表明筛选到的8个差异基因的表达量FPKM值与相对表达水平高度一致，说明测序结果准确可靠。【结论】菜心花蕾中的糖含量高于薹茎和叶片，是由合成相关酶编码基因的表达在薹茎和花蕾中高于叶片所致，研究结果为揭示菜心不同器官中糖代谢及转运的分子机制奠定了一定基础。

关键词: 菜心, 器官, 糖类, 转录组, 基因

Abstract:

【Objective】 Chinese flowering cabbage has become one of the most popular vegetables in southern China due to its crunchy taste and high nutritional value. The type and content of sugars are the main factors for determining the sweetness and flavour of Chinese flowering cabbage. Therefore, this paper analyzed the sugar content and the expression of genes related to sugar metabolism and transport in different organs, so as to investigate the molecular mechanisms responsible for the differences in sugar content in different organs of Chinese flowering cabbage. 【Method】 In this experiment, the content of soluble sugars, sucrose, glucose and fructose in leaves, stalks and flower buds were determined by high performance liquid chromatography (HPLC). The expression of genes in leaves, stalks and buds was analyzed comprehensively and systematically by transcriptome sequencing (RNA-seq), and the differentially expressed genes (DEGs) between three different organs were identified using DESeq2 software. In addition, the expression of genes encoding enzymes involved in the sugar metabolic pathway and transport process was analyzed, and the correlation with sugar content were analyzed on OmicShare. Moreover, the expression of some genes was verified using quantitative real-time fluorescence PCR (qRT-PCR).【Result】The sugar content varied significantly among the different organs of Chinese flowering cabbage. Soluble sugars, sucrose, glucose and fructose all showed a trend of buds>stalks>leaves. The glucose content in flower buds was 1.3 and 1.6 times of that in stalks and leaves; the fructose content was 1.42 and 1.78 times of that in stalks and leaves, respectively. By analyzing the relation and consistency between the expression of genes coding enzymes involving in sugar metabolism and transport and content of the sugar, a total of 18 genes were screened. By analyzing the correlation and consistency of the expression of DEGs among different organs with sugar species and content, 14 DEGs were identified. By merging 7 common genes (Bra020096, Bra029914, Bra033419, Bra037980, Bra027398, Bra006129 and Bra006130), expression of 25 genes in total were consistent with the sugar content. qRT-PCR results showed that the relative expression of the eight DEGs were highly consistent with their FPKM values from RNA-seq, indicating that the sequencing results were accurate and reliable.【Conclusion】 The sugar content in the flower buds of Chinese flowering cabbage was higher than that in stalks and leaves, because the expression of genes encoding sugar synthesis-related enzymes was higher in buds than in stalks and leaves. These results laid a certain foundation for revealing the molecular mechanism of sugar metabolism and transport in different organs of Chinese flowering cabbage.

Key words: Chinese flowering cabbage, organs, sugar, RNA-seq, gene

冯献君, 王丽, 王彤, 侯雷平, 李梅兰. 菜心不同器官糖含量比较及代谢相关基因的表达分析[J]. 中国农业科学, 2023, 56(11): 2158-2171.

FENG XianJun, WANG Li, WANG Tong, HOU LeiPing, LI MeiLan. Comparison of Sugar Content and Expression Analysis of Genes Related to Sugar Metabolism in Different Parts of Chinese Flowering Cabbage[J]. Scientia Agricultura Sinica, 2023, 56(11): 2158-2171.

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链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2023.11.010

https://www.chinaagrisci.com/CN/Y2023/V56/I11/2158

图/表 11

表1

图1

表2

图2

图3

图4

表3

糖代谢相关酶的基因表达"

酶 Enzyme	基因 Gene	表达量 FPKM			表达模式 Expression pattern
酶 Enzyme	基因 Gene	叶片 Leaf	薹茎 Stalk	花蕾 Bud	叶vs薹 L vs S	叶vs花 L vs B
SPS1F	Bra002289	30.62±1.16	39.12±0.27	40.34±4.08	上调 Up	上调 Up
SPS1F	Bra020096	2.20±0.17	3.49±1.30	10.96±0.92	上调 Up	上调 Up
SPS2F	Bra006090	2.25±0.21	5.81±0.10	5.76±0.47	上调 Up	上调 Up
SPS3F	Bra015364	0.22±0.02	0.26±0.14	16.96±4.35	上调 Up	上调 Up
SPS4F	Bra033195	101.31±4.16	2.8±0.72	51.68±10.47	下调 Down	下调 Down
SS1	Bra002332	7.71±0.52	269.62±15.50	116.37±27.63	上调 Up	上调 Up
SS1	Bra006578	2.38±0.09	53.80±6.15	15.93±2.47	上调 Up	上调 Up
SS2	Bra037432	0.23±0.02	0.01±0.01	0.24±0.02	下调 Down	上调 Up
SS3	Bra036282	0.17±0.01	0.06±0.01	8.20±0.32	下调 Down	上调 Up
SS5	Bra039502	3.10±0.34	13.00±0.94	2.17±0.41	上调 Up	下调 Down
SS6	Bra015995	2.56±0.09	7.55±0.16	2.09±0.05	上调 Up	下调 Down
SS6	Bra003845	1.98±0.06	8.73±0.42	1.91±0.16	上调 Up	下调 Down
VINV1	Bra036652	0.00	0.08±0.03	0.01±0.01	上调 Up	上调 Up
VINV1	Bra027030	3.16 ±0.33	3.85±0.64	7.66±3.72	上调 Up	上调 Up
VINV2	Bra019749	37.34±2.12	211.83±13.08	44.90±8.51	上调 Up	上调 Up
VINV2	Bra026984	31.37±3.10	203.28±28.08	62.53±5.39	上调 Up	上调 Up
CWINV1	Bra027397	0.81±0.14	0.02±0.02	1.18±0.13	下调 Down	上调 Up
CWINV1	Bra021508	2.84±0.51	0.01±0.01	1.18±0.56	下调 Down	下调 Down
CWINV2	Bra033419	0.08±0.01	0.52±0.06	24.25±0.84	上调 Up	上调 Up
CWINV3	Bra037980	4.81±0.61	16.25±2.04	17.97±1.55	上调 Up	上调 Up
CWINV4	Bra017257	0.64±0.14	0.05±0.01	74.68±17.23	下调 Down	上调 Up
CWINV4	Bra023053	1.51±0.32	0.12±0.04	3.68±0.11	下调 Down	上调 Up
CWINV5	Bra027398	0.00	0.00	4.36±0.08	—	上调 Up
CWINV6	Bra008926	0.09±0.02	0.13±0.02	0.14±0.06	上调 Up	上调 Up
	Bra006129	0.00	0.00	0.45±0.04	—	上调 Up
	Bra006130	0.03±0.02	0.04±0.01	2.81±0.11	上调 Up	上调 Up
SWEET10	Bra022761	0.00±0.00	0.02±0.01	127.4±6.32	上调 Up	上调 Up
SWEET11	Bra029914	69.68±7.78	15.86±2.32	6.01±0.37	下调 Down	下调 Down
SWEET15	Bra006185	0.68±0.12	0.23±0.07	4.54±0.64	下调 Down	上调 Up
	Bra008850	2.91±0.13	1.09±0.28	16.11±3.41	下调 Down	上调 Up
	Bra023394	0.00±0.00	0.01±0.01	6.47±2.92	上调 Up	上调 Up
SWEET16	Bra001638	0.00	0.00	0.34±0.02	—	上调 Up
SWEET16	Bra021190	0.56±0.33	0.79±0.14	0.17±0.03	上调 Up	下调 Down
SWEET17	Bra012752	4.23±0.47	16.64±1.94	7.72±0.04	上调 Up	上调 Up
SWEET17	Bra038060	4.29±0.48	16.57±3.61	2.97±0.03	上调 Up	下调 Down
SWEET1	Bra017916	28.71±0.41	8.40±0.20	220.91±21.96	下调 Down	上调 Up
SWEET1	Bra016421	23.32±0.81	4.08±0.27	23.49±1.83	下调 Down	上调 Up
SWEET2	Bra027312	16.42±3.49	0.98±0.11	0.93±0.44	下调 Down	下调 Down
	Bra027314	15.74±0.74	1.57±0.22	2.71±0.12	下调 Down	下调 Down
	Bra021577	20.43±1.51	7.01±0.69	6.95±0.04	下调 Down	下调 Down
SWEET3	Bra003075	0.03±0.01	0.00	55.95±0.93	下调 Down	上调 Up
SWEET3	Bra022636	0.00	0.00	54.66±0.30	—	上调 Up

表3

图5

图6

表4

与糖代谢密切相关的关键差异表达基因分析"

基因 Gene	编码酶 Coding enzyme	GO功能 GO function	表达量 FPKM			差异倍数 Log₂FC
基因 Gene	编码酶 Coding enzyme	GO功能 GO function	叶片 Leaf	薹茎 Stalk	花蕾 Bud	叶vs薹 L vs S	叶vs花 L vs B	薹vs花 S vs B
Bra019214	PGI1	糖酵解过程 Glycolytic process	88.78±2.27	49.07±1.30	47.39±3.31	-1.548	-1.928	—
Bra007210	UGP3	UDP葡萄糖代谢过程 UDP-glucose metabolic process	55.35±3.43	13.41±0.75	10.53±0.24	-2.741	-3.413	—
Bra015364	SPSB	蔗糖生物合成过程 Sucrose biosynthetic process	0.29±0.02	0.18±0.14	16.96±4.35	—	4.845	6.254
Bra020096	SPS1F		2.20±0.17	3.49±1.30	10.96±0.92	—	1.301	1.315
Bra014262	SPP1		0.32±0.01	1.60±0.45	3.68±0.34	1.645	2.535	—
Bra030439	SPP1		0.03±0.01	0.04±0.02	3.16±0.01	—	5.851	5.931
Bra036282	SS3	蔗糖代谢过程 Sucrose metabolic process	0.17±0.01	0.06±0.01	8.20±0.32	—	4.57	6.727
Bra033419	CWINV2	蔗糖分解代谢过程 Sucrose catabolic process	0.08±0.01	0.52±0.06	24.25±0.84	1.96	7.189	5.241
Bra037980	CWINV3		4.81±0.61	16.25±2.04	17.97±1.55	1.081	—	—
Bra017257	CWINV4		0.64±0.14	0.05±0.01	74.68±17.23	-4.42	5.84	10.282
Bra027398	CWINV5		0.00±0.00	0.00±0.00	4.36±0.08	—	9.398	10.28
Bra006129	CWINV6		0.00±0.00	0.00±0.00	0.45±0.04	—	6.833	7.717
Bra006130	CWINV6		0.03±0.02	0.04±0.01	2.81±0.11	—	5.892	5.791
Bra039025	HXK2	糖酵解过程 Glycolytic process	33.02±1.53	33.55±1.45	104.06±10.12	—	—	1.326
Bra015599	HXK3		1.29±0.16	3.71±0.56	12.48±0.16	—	2.246	1.419
Bra011789			0.00±0.00	0.00±0.00	4.07±0.82	—	9.226	10.11
Bra010616			0.00±0.00	0.00±0.00	1.80±0.56	—	8.127	9.01
Bra035821	HXK4		1.53±0.67	2.36±0.30	6.73±0.40	—	1.106	1.198
Bra007452	FRK4	果糖代谢过程 Fructose metabolic process	0.01±0.01	0.00±0.00	1.62±0.09	—	6.401	8.241
Bra029158	FRK1		0.10±0.01	0.90±0.09	4.75±0.56	2.475	4.54	2.074
Bra036482	GLK1		94.36±14.03	5.48±0.61	2.86±0.10	-4.51	-5.784	-1.268
Bra031129	GLK1		140.10±18.85	10.12±0.93	2.81±0.11	-4.482	-6.656	—
Bra033726	GLK2		67.67±1.71	13.42±1.13	10.29±1.60	-2.788	-3.543	—
Bra036015	GLK2		47.71±0.56	4.66±0.83	4.31±0.88	-4.293	-4.337	—
Bra022761	SWEET10	蔗糖转运 Sucrose transport	0.00±0.00	0.02±0.01	127.4±6.32	—	13.491	12.514
Bra029914	SWEET11		69.68±7.78	15.86±2.32	6.01±0.37	-2.796	-4.544	-1.75
Bra026487	SWEET12		0.26±0.1	18.27±5.08	18.24±0.98	5.478	5.178	—
Bra023394	SWEET15		0.00±0.00	0.01±0.01	6.47±2.92	—	9.215	9.131
Bra038242	STP4	葡萄糖输入 Glucose import	16.00±0.34	5.99±0.06	1.66±0.06	-2.1	-4.277	-2.173
Bra032018	VGT1	果糖跨膜转运；葡萄糖输入 Fructose transmembrane transport; glucose import	10.55±0.07	6.61±1.04	19.24±1.88	-1.356	—	1.219
Bra017916	SWEET1	糖跨膜转运 Sugar transmembrane transport	28.71±0.41	8.40±0.20	220.91±21.96	-2.476	1.927	4.425
Bra022636	SWEET3	糖跨膜转运 Sugar transmembrane transport	0.00±0.00	0.00±0.00	54.66±0.30	—	12.114	12.994

表4

图7

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引物名称 Primer name	正向引物 Forward primer (F)	反向引物 Reverse primer (R)
β-actin	GTTGCTATCCAGGCTGTTCT	AGCGTGAGGAAGAGCATAAC
Bra017257	AACGTCAAGAGTGTATCCAG	AGCTCCATTATTCATCATCTG
Bra033726	AAGGGATCATTACATGGCG	AAAGGTCGTCGTAGAAATCG
Bra038242	CACTGACAAGGCTAAGCAAA	ATCAACTGTGGTCTGTAACG
Bra033419	TCAACTCTCAAAGGAGACAC	ACAGCTTTGGTAGGATACAC
Bra036015	AGTGCCATCTCGGATTTTAG	CACGCGCTAGTAAATGTTTC
Bra015364	CTTAGATACTTGTTTGTGCGG	CTCTCTGATGGAACGATGTC
Bra016357	CTCACAAGAACAAGGATAGCA	TGCGTCTACATTCTCATAGTC
Bra031129	GAGACGAAGACGAGAGAGCTA	ATCCACCTTCTTCACCTTTCGC

样品名称 Sample	原始Reads Raw reads	GC含量 GC content (%)	Q30碱基百分比 ≥ Q30 (%)	可比对reads数 Mapped reads	唯一比对reads数 Unique mapped reads
叶片-1 Leaf-1	50121098	48.35	94.80	43670202 (89.75%)	42083742 (86.49%)
叶片-2 Leaf-2	44695888	48.33	94.80	38980000 (89.52%)	37644105 (86.45%)
叶片-3 Leaf-3	53080028	49.44	95.03	46199884 (90.48%)	44398661 (86.95%)
薹茎-1 Stalk-1	54125218	49.02	94.55	46442755 (89.01%)	45253057 (86.73%)
薹茎-2 Stalk-2	54650960	48.10	94.46	47363455 (88.62%)	46267582 (86.57%)
薹茎-3 Stalk-3	58405446	48.29	92.86	50627900 (88.55%)	49523709 (86.62%)
花蕾-1 Bud-1	54307592	47.53	94.09	46647934 (88.22%)	45488092 (86.02%)
花蕾-2 Bud-2	49747848	47.23	92.67	42692224 (88.05%)	41538120 (85.67%)
花蕾-3 Bud-3	61677872	46.87	94.11	53241893 (88.02%)	51887560 (85.78%)