磷胁迫下羊草的响应及磷响应相关基因表达分析

doi:10.3864/j.issn.0578-1752.2019.23.003

Abstract

Abstract:

【Objective】Phosphorus is the nutrient elements that is essential for plant growth. It would provide the basic data for molecular mechanism investigation of Leymus chinensis responding to inorganic phosphate (Pi) treatment, via analyzing the response under Pi stress in L. chinensis, and selection of the reference genes for qRT-PCR analysis, along with relative expression analysis of Pi-responsive genes. 【Method】Using L. chinensis seedlings as materials, the length of shoots and roots were measured, and the contents of Pi were examined via vanadium molybdenum yellow colorimetric method following different concentration of Pi treatments. Based on transcriptome data of L. chinensis, as well as NCBI nucleic acid sequences database, 7 and 1 candidate reference genes were selected, respectively. Total RNA was isolated from each sample after different concentration of Pi treatment. Expression level of candidate reference genes was examined through qRT-PCR, and the stability was calculated by geNorm, NormFinder and Bestkeeper. According to the most stable reference genes selected, the relative expression of Pi-responsive genes was analyzed according to the qRT-PCR results. 【Result】The phenotype observation showed that both lower and higher concentration of Pi stresses reduced the growth of L. chinensis, and the shoot growth was more sensitive to low Pi (or Pi deficiency) stress, whereas root length was more sensitive to high Pi stress. In addition, the accumulation of Pi was enhanced along with the increased concentration of Pi treatments. The melting curve analysis showed that single peak of every candidate reference gene was observed, and gene expression profiles indicated that their CT values ranged from 17.16 to 26.61, among which LcGAPDH exhibited the highest expression abundance with CT values ranged form 17.16 to 20.22, and Lc18SrRNA showed the lowest expression level with CT values ranged form 23.28 to 26.61. LcARPT (2.09%) showed the smallest expression variation, and LcTUA (6.8%) had the largest expression variation. Finally, according to the stability ranking of geNorm, NormFinder and Bestkeeper, the comprehensive stability ranking of 8 candidate reference genes were obtained via calculation geometric mean, among which the top three stable genes were Lc18SrRNA, LcCAP and LcEF1α, and the most unstable two genes were LcTUA and LcTUB. Using Lc18SrRNA, LcCAP and LcEF1α as reference genes, qRT-PCR results showed that, compared with the control, the expression of LcPHO1-2 was induced by low Pi or complete deprive of Pi, LcPAP2 was induced by high Pi, while the expressions of LcPAP27 was induced by both low and high Pi treatments.【Conclusion】Both lower and higher concentration of Pi stresses blocked the L. chinensis growth, and different responsive patterns were observed between the shoots and roots. Three relatively stable expression genes Lc18SrRNA, LcCAP and LcEF1α were selected and could be used as reference genes for qRT-PCR analysis under Pi stress. LcPHO1-2 and LcPAP2 were involved in the response of L. chinensis to low Pi and high Pi treatment, respectively, while LcPAP27 was participated in the responsive process of both low Pi and high Pi treatments.

Key words: Leymus chinensis, Pi stress, qRT-PCR, reference gene, Pi-responsive gene

WAN DongLi,HOU XiangYang,DING Yong,REN WeiBo,WANG Kai,LI XiLiang,WAN YongQing. Response and the Expression of Pi-Responsive Genes in Leymus chinensis Under Inorganic Phosphate Treatment[J].Scientia Agricultura Sinica, 2019, 52(23): 4215-4227.

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References 39

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基因名称 Gene name	基因描述 Gene description	引物序列 Primer sequences (5′-3′)	引物扩增效率 Amplification efficiency (%)	产物大小 Amplicon length (bp)	溶解温度 Product melting temperature (℃)
LcTUA	α-微管蛋白Alpha-tubulin	F: ACGAGGCCATCTACGACATCT	95.0	135	87.6
		R: CCACGTTCAGGGCACCAT
LcTUB	β-微管蛋白Beta-tubulin	F: TCCCTCGCCTCCACTTCTT	107.2	197	88.8
		R: CTCCTTTGTGCTCATCTTCCC
LcGAPDH	甘油醛3-磷酸脱氢酶Glyceraldehyde-3-phosphate dehydrogenase	F: AAACGCAACGAAGCAACTACA	97.7	105	82.2
	甘油醛3-磷酸脱氢酶Glyceraldehyde-3-phosphate dehydrogenase	R: AGAGCACCAAGCCGCAAG
LcEF1α	延伸因子1-α Elongation factor 1-alpha	F: CCGCCATCAAGAAGAAATAGG	103.9	98	82.6
	延伸因子1-α Elongation factor 1-alpha	R: CGCCGAAGCAGGAATAAAA
Lc18SrRNA	18S核糖体RNA 18S ribosomal RNA	F: TTGAAGCTGATAGGAGGGAGG	96.7	98	82.8
	18S核糖体RNA 18S ribosomal RNA	R: CTGAAGGTTGGGCGGAATA
LcCAP	腺苷酰环化酶相关蛋白 Adenylyl cyclase-associated protein	F: AGGACGGGCAGTTCACCA	103.6	114	83.1
	腺苷酰环化酶相关蛋白 Adenylyl cyclase-associated protein	R: ACAAGGCAAAGGCAGCATC
LcAPRT	腺嘌呤磷酸核糖转化酶 Adenine phosphoribosyl transferase	F: GAATCCTCTGGCTTCAACACC	101.4	118	83.7
	腺嘌呤磷酸核糖转化酶 Adenine phosphoribosyl transferase	R: GGACATCACAACCTTGCTTCTT
LcACT	肌动蛋白Actin	F: ATTGTGCTCAGTGGTGGGTCA	106.5	137	85.5
		R: CCAATCCAAACACTGTACTTCCTC
LcPHO1-2	磷酸盐1-2 Phosphate1-2	F: CTCGCCTACTGGATTTCTCCC	-	97	82.5
	磷酸盐1-2 Phosphate1-2	R: CCAACATTGTTCAAGTGTTCGTTC
LcPAP2	紫色酸性磷酸酶2 Purple acid phosphatase2	F: CTCGCGTCCGCACACATAAT	-	83	82.9
	紫色酸性磷酸酶2 Purple acid phosphatase2	R: TCCGCTTCCAGCCACTTATA
LcPAP27	紫色酸性磷酸酶27 Purple acid phosphatase27	F: CGACTCCTTCACCATCCACA	-	172	88.3
	紫色酸性磷酸酶27 Purple acid phosphatase27	R: TTCACGCACACGAGATTCTTT

基因 Gene	平均数 Mean	标准差 SD	变异系数 CV(%)
LcACT	20.10	0.97	4.82
LcTUA	23.15	1.58	6.80
LcTUB	20.75	1.37	6.60
LcAPRT	23.32	0.49	2.09
LcEF1α	21.11	0.92	4.35
Lc18SrRNA	25.27	0.93	3.68
LcCAP	22.56	0.89	3.95
LcGAPDH	18.61	0.92	4.96

排名 Rank	geNorm		NormFinder		Bestkeeper
排名 Rank	基因Gene	M值M value	基因Gene	稳定值Stability value	基因Gene	标准差Std dev (± CP)
1	LcCAP	0.42	Lc18SrRNA	0.138	LcAPRT	0.40
2	Lc18SrRNA	0.42	LcCAP	0.231	Lc18SrRNA	0.73
3	LcEF1α	0.49	LcEF1α	0.255	LcCAP	0.75
4	LcACT	0.52	LcACT	0.342	LcEF1α	0.76
5	LcGAPDH	0.54	LcGAPDH	0.390	LcGAPDH	0.76
6	LcAPRT	0.60	LcTUB	0.457	LcACT	0.81
7	LcTUB	0.68	LcAPRT	0.470	LcTUB	1.04
8	LcTUA	0.78	LcTUA	0.692	LcTUA	1.16

排名Rank	基因Gene	几何平均数Geomean
1	Lc18SrRNA	1.260
2	LcCAP	1.817
3	LcEF1α	3.302
4	LcAPRT	3.476
5	LcACT	4.579
6	LcGAPDH	4.642
7	LcTUB	6.649
8	LcTUA	8.000

Response and the Expression of Pi-Responsive Genes in Leymus chinensis Under Inorganic Phosphate Treatment

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