基于转录组测序筛选鸡蛋绿壳性状相关基因

doi:10.3864/j.issn.0578-1752.2023.08.014

中国农业科学 ›› 2023, Vol. 56 ›› Issue (8): 1594-1605.doi: 10.3864/j.issn.0578-1752.2023.08.014

基于转录组测序筛选鸡蛋绿壳性状相关基因

肖涛¹^,³(), 李辉¹(), 罗韦¹, 叶涛¹, 余欢¹, 陈友波¹, 石钰仕¹, 赵德鹏¹, 吴芸²

¹ 高原山地动物遗传育种与繁殖教育部重点实验室/贵州省动物遗传育种与繁殖重点实验室/贵州大学动物科学学院，贵阳 550025
² 遵义师范学院生物与农业科技学院，贵州遵义 563006
³ 安顺市畜牧技术推广站，贵州安顺 561000

收稿日期:2021-12-13 接受日期:2022-11-16 出版日期:2023-04-16 发布日期:2023-04-23
联系方式: 肖涛，E-mail：798061734@qq.com。
基金资助:
贵州省地方家禽产业联合攻关项目(黔财农[2020]175号); 贵州省科技支撑计划(黔科合支撑[2019]2285号); 赤水河流域资源保护与开发研究院项目(CSH-[2019]-005)

Screening of Candidate Genes for Green Shell Egg Shell Color Traits in Chishui Black Bone Chicken Based on Transcriptome Sequencing

XIAO Tao¹^,³(), LI Hui¹(), LUO Wei¹, YE Tao¹, YU Huan¹, CHEN YouBo¹, SHI YuShi¹, ZHAO DePeng¹, WU Yun²

¹ Key Laboratory of Animal Genetics, Breeding and Reproduction in the Plateau Mountainous Region, Ministry of Education/ Guizhou Provincial Key Laboratory of Animal Genetics, Breeding and Reproduction/College of Animal Science, Guizhou University, Guiyang 550025
² College of Biology and Agriculture Technology, Zunyi Normal University, Zunyi 563006, Guizhou
³ Anshun City Animal Husbandary Technology Extension Station, Anshun 561000, Guizhou

Received:2021-12-13 Accepted:2022-11-16 Published:2023-04-16 Online:2023-04-23

摘要/Abstract

摘要：

【背景】绿壳蛋深受消费者喜爱，蛋壳绿色的深浅在市场上是影响绿壳蛋定价销售的重要参考指标。绿壳蛋的形成受多基因共同调控，蛋壳绿色深浅不一，其分子机理尚不清楚。通过对赤水乌骨鸡蛋壳腺组织进行转录组测序，挖掘其调控绿壳蛋蛋壳颜色深浅的候选基因以及关键信号通路，探究影响蛋壳颜色遗传性，以期发展绿壳蛋的选种选育和提高经济效益。【目的】探讨赤水乌骨鸡的遗传基础，并通过SLCO1B3基因分型对其进行鉴定和筛选，以期通过分子标记在青壳鸡育种规划中提供新的见解，并在后期的选择策略中帮助控制和提高赤水乌骨鸡蛋壳品质的同质性。【方法】以纯系的 280 日龄赤水乌骨鸡为研究对象，屠宰产浅绿色蛋（QL）和深绿色蛋（SL）的母鸡各3只，采集蛋壳腺以RNA-seq技术检测分析，筛选与蛋壳颜色密切相关的差异表达基因（differentially expressed genes, DEGs），并对这些 DEGs进行 GO 和 KEGG 富集分析。利用qRT-PCR技术检测与蛋壳颜色相关的 6 个候选基因的转录水平变化以验证转录组数据可靠性。【结果】在深绿组与浅绿组中共筛选到93个显著DEGs，有 59 个基因在深绿组中显著上调，34个显著下调。DEGs注释到 GO 数据库进行比对，主要显著性富集的是钠离子转运、负离子结合、肌质网膜等。KEGG 分析富集程度最显著的是醛固酮调节钠的重吸收，以及富集的矿物质吸收、亚油酸的新陈代谢等信号通路。通过qRT-PCR验证，结果显示这些基因的表达趋势与转录组的测序结果一致。【结论】经功能分析，TF 基因、SCNN1 家族基因、CYP450 家族基因、SLC 家族基因和FAM 家族基因，以及醛固酮调节钠的重吸收信号通路参与蛋壳色素合成、转运和沉积。上述基因和信号通路可能是影响赤水乌骨鸡蛋壳绿色深浅不一的候选基因和关键信号通路。

关键词: 转录组测序, 赤水乌骨鸡, 绿壳蛋, 蛋壳颜色, qRT-PCR

Abstract:

【Background】Green shell eggs are loved by consumers, and the green color of eggshell is an important reference index affecting the pricing and sales of green shell eggs in the market. The formation of green shell eggs is regulated by multiple genes, and the color of green shell varies. However, the molecular mechanism of green shell eggs is still unclear. In this study, transcriptomic sequencing was conducted on the eggshell gland tissue of Chishui black bone chicken, and the candidate genes and key signal pathways that regulated the depth and depth of eggshell color of green shell eggs were excavated, so as to explore the heritability of eggshell color, and to develop the seed selection and breeding of green shell eggs and improve economic benefits. 【Objective】The aim of this study was to investigate the genetic basis of Chishui black bone chicken and to identify and then screen them by SLCO1B3 genotyping, in order to provide new insights through molecular markers in the breeding planning of green-shelled hens, and to help control and improve the homogeneity of eggshell quality of Chishui black bone chicken in the later selection strategy. 【Method】A pure 280-day-old Chishui black bone chicken was used as the research object. Three hens were slaughtered to produce light green eggs (QL) and dark green eggs (SL), and the eggshell glands were collected and analyzed by RNA-SEQ technology. Selected differentially expressed genes (DEGs) closely related to eggshell color, and analyzed for GO and KEGG enrichment. Qrt-pcr was used to detect the transcriptional level changes of six candidate genes related to eggshell color to verify the reliability of transcriptomic data. 【Result】A total of 93 DEGs were screened in SL group and QL group, among which 59 genes were up-regulated and 34 genes were down-regulated in SL group. DEGs was annotated to GO database for comparison, and sodium ion transport, negative ion binding, and sarcoplasmic reticulum were mainly enriched significantly. KEGG analysis showed that aldosterone regulated sodium reabsorption, enriched mineral absorption, linoleic acid metabolism and other signal pathways. The results of QRT-PCR showed that the expression trend of these genes was consistent with the transcriptome sequencing results. 【Conclusion】By functional analysis, TF gene, SCNN1 gene, CYP450 gene, SLC gene and FAM gene, as well as the sodium reabsorption signaling pathway regulated by aldosterone, might be involved in eggshell pigment synthesis, transport and deposition. These genes and signal pathways might be candidate genes and key signal pathways affecting the different shades of green in eggshell of Chishui black bone chicken.

Key words: transcriptome sequencing, Chishui black bone chicken, green egg, eggshell color, qRT-PCR

肖涛, 李辉, 罗韦, 叶涛, 余欢, 陈友波, 石钰仕, 赵德鹏, 吴芸. 基于转录组测序筛选鸡蛋绿壳性状相关基因[J]. 中国农业科学, 2023, 56(8): 1594-1605.

XIAO Tao, LI Hui, LUO Wei, YE Tao, YU Huan, CHEN YouBo, SHI YuShi, ZHAO DePeng, WU Yun. Screening of Candidate Genes for Green Shell Egg Shell Color Traits in Chishui Black Bone Chicken Based on Transcriptome Sequencing[J]. Scientia Agricultura Sinica, 2023, 56(8): 1594-1605.

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基因 Gene	引物序列 Primer sequence (5'-3')	产物大小 Amplicon length (bp)	退火温度 The annealing temperature (℃)
SLC34A2	F: TCGGTCCGTTCACTCTGTTG R: GCCACGTTGCCTTTGTGATT	164	60.0
SLC20A1	F: CAGAAAGGCGTCAAATGGTC R: CACGGTGCAGGCGTAGAA	180	54.0
CYP27A1	F: AGGACTTTCGTCTGGCTCT R: CTCCGCATCGGGTATTT	185	58.6
TF	F: AGATGCCATTAGCTTGGAC R: GGTTGTGGAGCCTTCAGTA	109	60.0
FAM136A	F: GCACGCTTCACTGCTCTGACA R: ACCCGTCCCGCATCCTCTT	153	57.0
SLC6A9	F: CGTACCTCTGCTACCGCAAT R: CACGCGTCATGGACACAAAG	261	60.0
β-actin	F: CAGCAAGCAGGAGTACGATG R: ATAAAGCCATGCCAATCTCG	145	60.0

样本 Sample	△L*	△a*
SL-1	56.48±0.52	-6.01±0.36B
SL-2	57.28±1.71	-6.40±0.88B
SL-3	55.45±2.32	-7.01±0.84B
QL-1	59.85±1.19	-3.93±0.29A
QL-2	59.18±2.85	-4.27±0.17A
QL-3	60.61±0.61	-4.16±0.07A

样本 Sample	浓度 Concentration(ng·μL^-1)	体积 Volume(μL)	总量 Total amount(μg)	RIN RIN	OD_260/280 OD_260/280	OD_260/230 OD_260/230	等级 Level
SL-1	651.5	20	13.0	9.5	1.99	1.93	A
SL-2	1729.2	20	34.6	9.5	2.02	1.63	A
SL-3	487.0	20	9.70	9.4	2.00	1.83	A
QL-1	993.2	20	19.9	8.9	2.01	1.87	A
QL-2	850.8	20	17.0	8.5	2.02	1.67	A
QL-3	1002.0	20	20.0	9.0	1.98	1.72	A

样品 Sample	原始读数 Raw reads	质控后序列 Clean reads	质控后序列量 Clean bases (G)	Q20 (%)	Q30 (%)	GC (%)
QL-1	57502212	56416296	7.75	97.26	92.45	47.99
QL-2	48412576	47722420	6.58	97.21	92.39	47.50
QL-3	42574086	42043646	5.79	97.41	92.83	47.56
SL-1	52836592	51864304	7.14	97.26	92.52	47.83
SL-2	57938312	57099094	7.87	97.23	92.39	47.07
SL-3	58071340	56266562	7.75	97.20	92.37	47.45

样品 Sample	质控序列条数 Total reads	比对率 Total mapped (%)	多比对率 Multiple mapped (%)	单一比对率 Unique mapped (%)	正链比对率 Reads map to '+' (%)	负链比对率 Reads map to '-' (%)	基因组全比对率 Non-splice reads (%)	外显子单比对率 Splice reads (%)
QL-1	56416296	91.54	2.29	89.25	44.59	44.66	60.74	28.51
QL-2	47722420	92.65	1.84	90.81	45.37	45.44	60.99	29.82
QL-3	42043646	92.13	2.91	89.22	44.57	44.64	60.29	28.93
SL-1	51864304	91.82	2.35	89.47	44.68	44.79	61.17	28.30
SL-2	57099094	92.45	2.39	90.06	44.99	45.07	61.28	28.78
SL-3	56266562	91.72	2.05	89.68	44.81	44.87	61.88	27.80

基于转录组测序筛选鸡蛋绿壳性状相关基因

Screening of Candidate Genes for Green Shell Egg Shell Color Traits in Chishui Black Bone Chicken Based on Transcriptome Sequencing

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	TF	CYP27A1	CYP3A5	SLC6A9	SLC34A2	SLC20A1	FAM136A	SCNN1A	SCNN1G	*ESC(△a)**
TF	1
CYP27A1	0.599	1
CYP3A5	-0.219	-0.577	1
CLC6A9	0.099	0.738	0.557	1
SLC34A22	-0.511	-0.883^*	-0.44	-0.767	1
SLC20A1	-0.489	-0.904^*	-0.527	-0.776	0.665	1
FAM136A	0.277	0.899^*	0.692	0.926^**	-0.907^*	-0.813^*	1
SCNN1A	-0.618	-0.921^**	-0.550	-0.609	0.655	0.943^**	-0.756	1
SCNN1G	-0.370	-0.937^**	-0.610	-0.893^*	0.913^*	0.857^*	-0.964^**	0.773	1
ESC (△a*)	0.561	0.953^**	0.465	0.830^*	-0.820^*	-0.965^**	0.882^*	-0.898^*	-0.938^**	1

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[4]	郝树琳,陈宏伟,廖芳丽,李莉,刘昌燕,刘良军,万正煌,沙爱华. 基于盐胁迫转录组信息的蚕豆F-box基因家族分析[J]. 中国农业科学, 2020, 53(17): 3443-3454.
[5]	丁艳娟,刘永康,罗雨嘉,邓颖梅,徐红星,唐斌,徐彩娣. 褐飞虱GSK-3调控糖原与海藻糖代谢的潜在功能[J]. 中国农业科学, 2019, 52(7): 1237-1246.
[6]	万东莉,侯向阳,丁勇,任卫波,王凯,李西良,万永青. 磷胁迫下羊草的响应及磷响应相关基因表达分析[J]. 中国农业科学, 2019, 52(23): 4215-4227.
[7]	薛红丽，杨军军，汤沙，智慧，王蕊，贾冠清，乔治军，刁现民. 谷子穗顶端败育突变体sipaa1的表型分析和基因定位[J]. 中国农业科学, 2018, 51(9): 1627-1640.
[8]	刘宏祥,徐文娟,朱春红,陶志云,宋卫涛,章双杰,李慧芳. 鸭胚胎发育中后期胸肌发育阻滞的RNA-seq分析[J]. 中国农业科学, 2018, 51(22): 4373-4386.
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[10]	严莉，王翠平，陈建伟，乔改霞，李健. 基于转录组信息的黑果枸杞MYB转录因子家族分析[J]. 中国农业科学, 2017, 50(20): 3991-4002.
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[12]	彭馥芝，冉茂良，翁波，李智，董莲花，陈斌. 猪睾丸组织定量PCR分析中内参基因的选择[J]. 中国农业科学, 2017, 50(15): 3033-3041.
[13]	孙明岳，周君，谭秋平，付喜玲，陈修德，李玲，高东升. 苹果bZIP转录因子家族生物信息学分析及其在休眠芽中的表达[J]. 中国农业科学, 2016, 49(7): 1325-1345.
[14]	高坤，尚梦珂，钱荷英，覃光星，郭锡杰. 家蚕感染二分浓核病毒（镇江株）的数字基因表达谱分析[J]. 中国农业科学, 2016, 49(17): 3453-3464.
[15]	杨瑞先, 范晓静, 邱思鑫, 蔡学清, 胡方平. 利用cDNA-AFLP分析内生解淀粉芽胞杆菌 Fy11诱导辣椒差异表达基因[J]. 中国农业科学, 2013, 46(12): 2449-2458.