基于转录组分析挖掘罗氏沼虾新内参基因

doi:10.3969/j.issn.1004-1524.20240514

浙江农业学报 ›› 2025, Vol. 37 ›› Issue (7): 1424-1429.DOI: 10.3969/j.issn.1004-1524.20240514

基于转录组分析挖掘罗氏沼虾新内参基因

任晋东(), 陈红林, 牛宝龙, 许晓军(), 楼宝

浙江省农业科学院水生生物研究所,浙江杭州 310021

收稿日期:2024-06-13 出版日期:2025-07-25 发布日期:2025-08-20
作者简介:任晋东(1983—),男,河南淇县人,博士,助理研究员,研究方向为分子育种。E-mail:renjindong@126.com
通讯作者: ^*许晓军,E-mail:xuxj@zaas.ac.cn
基金资助:
宁波市重点技术研发项目(2024Z278)

Mining new housekeeping genes of Macrobrachium rosenbergii based on transcriptome analysis

REN Jindong(), CHEN Honglin, NIU Baolong, XU Xiaojun(), LOU Bao

Institute of Hydrobiology, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China

Received:2024-06-13 Online:2025-07-25 Published:2025-08-20

摘要/Abstract

摘要： 内参基因挖掘、鉴定是基因表达定量分析的基础。为挖掘罗氏沼虾各组织稳定表达的内参基因,利用转录组相对表达量数据,通过变异系数分析和基因表达稳定性参数(M值)等分析、鉴定罗氏沼虾新的内参基因。结果表明:有1 902个基因在罗氏沼虾腮腺、眼柄、心脏、肝胰腺、胃、大螯肌肉、腹部肌肉、精巢、促雄性腺、卵巢这10个组织内的相对表达量(FPKM值)达到5以上,基于各基因在不同组织间的平均变异系数,随机筛选获得10个平均变异系数均低于常用内参基因β-actin的候选基因。进一步的M值筛选发现,这10个候选基因在各组织的M值都低于β-actin,并以RNF10基因的M值最低。在10个候选基因中,选择M值最低的RNF10基因和M值仅次于β-actin的S/TPP2A基因进行定量聚合酶链反应(qPCR),结果表明,这二者在10个组织中的M值均低于β-actin基因,其中,RNF10在10个组织中展现出最优的表达稳定性。研究结果表明,利用转录组数据可以获得具有表达稳定性的内参基因,罗氏沼虾RNF10基因在不同组织内表达稳定,可用作罗氏沼虾基因表达定量分析的内参基因。

关键词: 罗氏沼虾(Macrobrachium rosenbergii), 内参基因, 表达稳定性参数

Abstract:

The identification of housekeeping genes in organisms is important for quantitative analysis of gene expression. To identify stable housekeeping genes in various tissues of the giant freshwater prawn Macrobrachium rosenbergii, the relative expression data in multiple tissues, including gill, eye stalk, heart, hepatopancreas, stomach, big claw muscle, abdominal muscle, testis, androgenic gland and ovary, was used to identify new candidate housekeeping genes for quantitative analysis of gene expression in various tissues. The results showed that 1 902 genes were obtained with average FPKM values above 5 in the 10 tissues. Based on the average variation coefficients of these genes, 10 candidate genes were screened out randomly with smaller average variation coefficients than the common housekeeping gene β-actin. Further selection with gene expression stability parameter (M value) showed that, the M value of the selected 10 candidate genes in 10 tissues were all smaller than that of β-actin. Among the 10 candidate genes, the RNF10 gene exhibited the smallest M value, while the S/TPP2A gene showed the biggest M value except the β-actin gene. Quantitative PCR test on RNF10, S/TPP2A and β-actin gene varified that the M values of RNF10, S/TPP2A were smaller than that of β-actin, and the RNF10 gene exhibited the best expression stability. Thus, it was feasible to screen out housekeeping genes by using transcriptome data, variability, and the RNF10 gene could be used as a housekeeping gene with good expression stability in 10 tissues of of Macrobrachium rosenbergii.

Key words: Macrobrachium rosenbergii, housekeeping gene, expression stability parameter

中图分类号:

S917
Q953.1

任晋东, 陈红林, 牛宝龙, 许晓军, 楼宝. 基于转录组分析挖掘罗氏沼虾新内参基因[J]. 浙江农业学报, 2025, 37(7): 1424-1429.

REN Jindong, CHEN Honglin, NIU Baolong, XU Xiaojun, LOU Bao. Mining new housekeeping genes of Macrobrachium rosenbergii based on transcriptome analysis[J]. Acta Agriculturae Zhejiangensis, 2025, 37(7): 1424-1429.

图/表 7

参考文献 14

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引物名称 Primer name	序列Sequences(5’→3’)	GC含量 GC content/%	解链温度 Melting temperature/℃
RNF10-F	ATAATGGCTTGCTAAACTGGTCA	39.1	58.9
RNF10-R	TTCCATAATCTTTAAGCGGAGG	40.9	58.6
S/TPP2A-F	CAGTGTGGGGTCTTGTTTGACT	50.0	59.0
S/TPP2A-R	TGACGGATTAGTAGAAGGTGGC	50.0	58.8
β-actin-F	GTGCCCATCTACGAGGGTTAT	52.4	58.3
β-actin-R	ATCTCCTGCTCGAAGTCCAAT	47.6	58.0

引物名称 Primer name	序列Sequences(5’→3’)	GC含量 GC content/%	解链温度 Melting temperature/℃
RNF10-F	ATAATGGCTTGCTAAACTGGTCA	39.1	58.9
RNF10-R	TTCCATAATCTTTAAGCGGAGG	40.9	58.6
S/TPP2A-F	CAGTGTGGGGTCTTGTTTGACT	50.0	59.0
S/TPP2A-R	TGACGGATTAGTAGAAGGTGGC	50.0	58.8
β-actin-F	GTGCCCATCTACGAGGGTTAT	52.4	58.3
β-actin-R	ATCTCCTGCTCGAAGTCCAAT	47.6	58.0

组织 Tissue	基因数 Gene quantity	FPKM 平均值 Average of FPKM	FPKM标准误 Standard error of FPKM
肌肉Abdominal muscle	1 902	230.3	37.23
大鳌肌肉Big claw muscle	1 902	328.1	61.12
眼柄Eye stalk	1 902	215.7	46.97
腮Gill	1 902	512.6	126.80
心脏Heart	1 902	600.3	170.00
肝胰腺Hepatopancreas	1 902	231.9	54.21
胃Stomach	1 902	454.2	103.70
卵巢Ovary	1 902	341.6	56.43
促雄性腺Androgenic gland	1 902	286.7	43.97
精巢Testis	1 902	263.3	34.90

组织 Tissue	基因数 Gene quantity	FPKM 平均值 Average of FPKM	FPKM标准误 Standard error of FPKM
肌肉Abdominal muscle	1 902	230.3	37.23
大鳌肌肉Big claw muscle	1 902	328.1	61.12
眼柄Eye stalk	1 902	215.7	46.97
腮Gill	1 902	512.6	126.80
心脏Heart	1 902	600.3	170.00
肝胰腺Hepatopancreas	1 902	231.9	54.21
胃Stomach	1 902	454.2	103.70
卵巢Ovary	1 902	341.6	56.43
促雄性腺Androgenic gland	1 902	286.7	43.97
精巢Testis	1 902	263.3	34.90

基因 Gene	在不同组织中的M值M value in different tissues
基因 Gene	Ag	Am	Bc	Es	Gi	Hat	Hp	Ov	Sm	Tt
RNF10	0.137 3	0.186 7	0.164 4	0.158 3	0.188 6	0.170 1	0.152 6	0.166 8	0.235 2	0.186 9
S/TPP2A	0.170 4	0.209 7	0.336 3	0.273 1	0.216 7	0.257 1	0.260 9	0.240 3	0.361 8	0.243 8
β-actin	0.591 8	0.504 6	0.659 0	0.558 4	0.671 0	0.463 2	0.489 0	0.309 0	0.721 8	0.540 5
Rab11A	0.144 7	0.208 4	0.159 2	0.156 9	0.182 6	0.167 5	0.147 6	0.163 4	0.241 0	0.182 2
eIF4a	0.148 0	0.205 7	0.171 7	0.163 4	0.206 9	0.177 3	0.146 9	0.144 8	0.289 1	0.177 9
DCN1	0.164 9	0.177 2	0.170 7	0.190 9	0.201 6	0.187 6	0.200 8	0.157 4	0.390 7	0.176 6
NDUFA13	0.150 5	0.201 4	0.199 4	0.165 5	0.214 3	0.252 9	0.178 7	0.159 0	0.287 1	0.185 5
MEF2b	0.198 5	0.229 0	0.214 1	0.193 9	0.245 8	0.192 5	0.196 4	0.180 9	0.254 2	0.178 8
COXII	0.208 4	0.242 0	0.182 9	0.188 7	0.258 8	0.236 9	0.196 9	0.252 7	0.261 6	0.217 8
gC1qR	0.159 8	0.283 5	0.160 8	0.192 5	0.298 1	0.183 5	0.226 1	0.248 5	0.270 1	0.341 0
IAP	0.167 6	0.209 7	0.234 7	0.167 0	0.267 0	0.331 1	0.188 6	0.163 6	0.291 5	0.392 9

基于转录组分析挖掘罗氏沼虾新内参基因

Mining new housekeeping genes of Macrobrachium rosenbergii based on transcriptome analysis

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