快速冷驯化条件下稻水象甲内参基因的筛选及<i>LoTPS</i>基因表达

doi:10.7668/hbnxb.20193545

摘要/Abstract

摘要：

为筛选出适合快速冷驯化条件下稻水象甲成虫荧光定量PCR的内参基因,并分析快速冷驯化低温胁迫下海藻糖合成酶基因(LoTPS)的表达模式,从稻水象甲转录组数据库中筛选出10个具有完整开放阅读框的待选内参基因,以稻水象甲成虫为试验材料,通过实时荧光定量PCR测定内参基因在不同快速冷驯化条件下的表达水平,通过常用的内参基因稳定性分析方法ΔCt法和geNorm、NormFinder和BestKeeper等软件综合评价待选的10个内参基因表达的稳定性,测定稻水象甲海藻糖合成酶基因在不同快速冷驯化条件下的表达变化,分析其与低温的相关性。试验结果表明,在ΔCt法分析中RPS18表达丰度最高,geNorm软件分析中较稳定表达的内参基因是RPS18和G6DPH,G6DPH在NormFinder软件分析中是表达最稳定的内参基因,BestKeeper软件分析表明,RPL13和α-tubulin表达较稳定,综合以上各分析方法得出RPS18基因在快速冷驯化条件下的稳定性较高。以RPS18为内参基因进一步分析LoTPS基因在快速冷驯化条件下的表达模式发现,LoTPS基因表达量在快速冷驯化条件下有所增加,各冷驯化处理均高于对照。因此,内参基因RPS18在稻水象甲成虫体内表达相对稳定,可以作为稻水象甲不同快速冷驯化条件下的内参基因,低温胁迫促进了在快速冷驯化条件下海藻糖合成酶基因的上调表达,且随着冷驯化时间的延长而表达量增加。

关键词: 稻水象甲, 快速冷驯化, 内参基因, 海藻糖合成酶, 基因表达

Abstract:

To screen out the stable reference gene for Real-time quantitative PCR under rapid cold hardening conditions,ten candidate reference genes from the transcriptome sequencing of Lissorhoptrus oryzophilus Kuschel were selected.Real-time quantitative PCR was used to obtain the expression levels of the selected candidate reference genes in adults under different rapid cold hardening conditions.The expression stability of the selected candidate reference genes was analyzed by ΔCt method,geNorm,NormFinder and BestKeeper softwares.ΔCt predicted the most stable candidate reference gene was RPS18.It was revealed that RPS18 and G6DPH were the most stable reference genes based on the geNorm analysis,while G6DPH, RPL13 and α-tubulin were the most stable reference genes based on the NormFinder analysis and BestKeeper analysis,respectively.The stability of RPS18 gene was relatively high based on comprehensive analysis. It was found that the expression level of LoTPS gene increased under rapid cold acclimation when RPS18 was used as an internal reference gene, and it was higher in all the cold acclimation treatments than control. Therefore, RPS18 was relatively stable and could be used as an internal reference gene under different rapid cold acclimation conditions. Low temperature stress promoted the up-regulation of LoTPS gene under rapid cold acclimation, and the expression level increased with the prolongation of cold acclimation time.

Key words: Lissorhoptrus oryzophilus Kuschel, Rapid cold hardening, Reference genes, Trehalose synthetase, Gene expression

祁立中, 王晓, 王庆泰, 王尚, 张炬红. 快速冷驯化条件下稻水象甲内参基因的筛选及LoTPS基因表达[J]. 华北农学报, 2023, 38(3): 176-183. doi: 10.7668/hbnxb.20193545.

QI Lizhong, WANG Xiao, WANG Qingtai, WANG Shang, ZHANG Juhong. Screening of Reference Genes and Analysis of LoTPS Gene Expression in Lissorhoptrus oryzophilus under Rapid Cold Hardening[J]. Acta Agriculturae Boreali-Sinica, 2023, 38(3): 176-183. doi: 10.7668/hbnxb.20193545.

http://www.hbnxb.net/CN/Y2023/V38/I3/176

图/表 9

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基因 Gene	基因全称 Gene name	引物序列(5'-3') Primer sequence(5'-3')	扩增长度/bp Amplification length
RPL18	核糖体蛋白L18基因	F: CCCCGAAAAGTCAAGATG	135
		R: CAGTGCCTACGATAACAGC
G6DPH	葡萄糖-6-磷酸脱氢酶基因	F: TGATATGGAAGAAACTGAGC	175
		R: TTGATGTAGTAGGGGCGT
RPS27	核糖体蛋白S27基因	F: TATTCCCCCAGACCAACA	125
		R: CGCCACCTCTCAACCTAA
RPS11	11S核糖体蛋白基因	F: TCCTTATGCAGCTATGTTAG	118
		R: CCAGGGGTTTTAGTTTTG
TBP	TATA盒结合蛋白基因	F: GAAATGCCGAATACAACC	184
		R: TTAGCAGGAAAACCCAAC
RPL13	核糖体蛋白L13基因	F: TTCTTATCCTTCCTCCGC	196
		R: TTCTTATCCTTCCTCCGC
RPS18	18S核糖体蛋白基因	F: GTAATGTTTGCCTTGACTG	121
		R: TTTCTACTTCCTCTTCGG
RPS3	3S核糖体蛋白基因	F: GCCTGCTATGGTGTGCTGA	255
		R: TTTCTTGGGGCCGTTCTT
α-tubulin	α-微管蛋白基因	F: TGTAGATGAAGTAAGACGAGG	232
		R: TGATGTGAAACCAGAGCC
GADPH	甘油醛-3-磷酸脱氢酶基因	F: GTGTGTGGTGTCAATTTGG	159
		R: AGTAGCAGTAGTTGCGTGG
AK	精氨酸激酶基因	F: AAAACCAAAAAGACACCCAC	106
		R: ATACGCTGACATCACCCC
TPS	海藻糖合成酶基因	F:GCGTTTGGTGTGGATTGG	182
		R:GATGATGTGCGAGGAGGT

基因 Gene	基因全称 Gene name	引物序列(5'-3') Primer sequence(5'-3')	扩增长度/bp Amplification length
RPL18	核糖体蛋白L18基因	F: CCCCGAAAAGTCAAGATG	135
		R: CAGTGCCTACGATAACAGC
G6DPH	葡萄糖-6-磷酸脱氢酶基因	F: TGATATGGAAGAAACTGAGC	175
		R: TTGATGTAGTAGGGGCGT
RPS27	核糖体蛋白S27基因	F: TATTCCCCCAGACCAACA	125
		R: CGCCACCTCTCAACCTAA
RPS11	11S核糖体蛋白基因	F: TCCTTATGCAGCTATGTTAG	118
		R: CCAGGGGTTTTAGTTTTG
TBP	TATA盒结合蛋白基因	F: GAAATGCCGAATACAACC	184
		R: TTAGCAGGAAAACCCAAC
RPL13	核糖体蛋白L13基因	F: TTCTTATCCTTCCTCCGC	196
		R: TTCTTATCCTTCCTCCGC
RPS18	18S核糖体蛋白基因	F: GTAATGTTTGCCTTGACTG	121
		R: TTTCTACTTCCTCTTCGG
RPS3	3S核糖体蛋白基因	F: GCCTGCTATGGTGTGCTGA	255
		R: TTTCTTGGGGCCGTTCTT
α-tubulin	α-微管蛋白基因	F: TGTAGATGAAGTAAGACGAGG	232
		R: TGATGTGAAACCAGAGCC
GADPH	甘油醛-3-磷酸脱氢酶基因	F: GTGTGTGGTGTCAATTTGG	159
		R: AGTAGCAGTAGTTGCGTGG
AK	精氨酸激酶基因	F: AAAACCAAAAAGACACCCAC	106
		R: ATACGCTGACATCACCCC
TPS	海藻糖合成酶基因	F:GCGTTTGGTGTGGATTGG	182
		R:GATGATGTGCGAGGAGGT

基因 Gene	基因长度/bp Gene size	最佳匹配项 Best hit
基因 Gene	基因长度/bp Gene size	物种 Species	GenBank登录号 GenBank accession No.	E值 E value	序列一致性/% Sequence identity
TBP	660	米象	XP_030767118.1	0.0	93.42
RPL13	609	米象	XP_030748989.1	5e-142	95.57
RPS18	459	米象	XP_030761149.1	2e-106	99.34
RPS3	573	亚洲玉米螟	XP_028167501.1	2e-137	100.00
α-tubulin	807	芒果横线尾夜蛾	APX43972.1	3e-171	88.60
GADPH	1 002	大灰象	QCO71350.1	0.0	93.39
RPL18	567	米象	XP_030766187.1	2e-125	94.15
G6DPH	1 581	米象	XP_030748345.1	0.0	99.00
RPS27	468	米象	XP_030762344.1	6e-108	98.08
RPS11	456	米象	XP_030750232.1	3e-105	100.00
AK	1 071	稻水象甲	ASV50718.1	0.0	100.00

基因 Gene	基因长度/bp Gene size	最佳匹配项 Best hit
基因 Gene	基因长度/bp Gene size	物种 Species	GenBank登录号 GenBank accession No.	E值 E value	序列一致性/% Sequence identity
TBP	660	米象	XP_030767118.1	0.0	93.42
RPL13	609	米象	XP_030748989.1	5e-142	95.57
RPS18	459	米象	XP_030761149.1	2e-106	99.34
RPS3	573	亚洲玉米螟	XP_028167501.1	2e-137	100.00
α-tubulin	807	芒果横线尾夜蛾	APX43972.1	3e-171	88.60
GADPH	1 002	大灰象	QCO71350.1	0.0	93.39
RPL18	567	米象	XP_030766187.1	2e-125	94.15
G6DPH	1 581	米象	XP_030748345.1	0.0	99.00
RPS27	468	米象	XP_030762344.1	6e-108	98.08
RPS11	456	米象	XP_030750232.1	3e-105	100.00
AK	1 071	稻水象甲	ASV50718.1	0.0	100.00

内参基因 Reference genes	几何平均数 Geo mean	算数平均数 Ar mean	最小值 Min	最大值 Max	标准差 s	变异系数/% CV
TBP	27.57	27.84	22.48	33.61	3.46	12.41
RPS18	17.66	17.93	13.80	21.23	2.74	15.28
α-tubulin	25.64	25.75	22.91	29.93	2.17	8.43
RPL13	21.91	22.03	18.67	25.08	2.02	9.18
GADPH	22.10	22.27	17.87	26.57	2.18	9.77
AK	23.71	24.02	17.99	29.40	3.24	13.49
G6DPH	23.80	23.94	20.33	25.98	2.30	9.60
RPS11	34.00	34.18	29.36	37.41	3.17	9.26
RPS27	18.94	19.11	15.62	21.83	2.25	11.79
RPL18	20.87	21.12	16.37	24.19	2.84	13.43

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快速冷驯化条件下稻水象甲内参基因的筛选及LoTPS基因表达

Screening of Reference Genes and Analysis of LoTPS Gene Expression in Lissorhoptrus oryzophilus under Rapid Cold Hardening

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排名 Rank	方法Method
	ΔCt	geNorm	NormFinder	BestKeeper
	ΔCt	geNorm	NormFinder	标准差s	变异系数CV
1	RPS18	G6DPH	G6DPH	RPL13	α-tubulin
2	RPS27	RPS18	RPS18	α-tubulin	RPL13
3	RPL18	RPL18	RPS27	GADPH	G6DPH
4	RPL13	RPS27	RPL18	RPS27	GADPH
5	GADPH	GADPH	GADPH	G6DPH	RPS27
6	AK	α-tubulin	α-tubulin	RPS18	TBP
7	G6DPH	AK	AK	RPL18	RPL18
8	α-tubulin	RPL13	RPL13	AK	AK
9	TBP	TBP	TBP	TBP	RPS18

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快速冷驯化条件下稻水象甲内参基因的筛选及LoTPS基因表达

Screening of Reference Genes and Analysis of LoTPS Gene Expression in Lissorhoptrus oryzophilus under Rapid Cold Hardening

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