长茎葡萄蕨藻胁迫条件下RT-qPCR内参基因的筛选与验证

doi:10.13560/j.cnki.biotech.bull.1985.2020-1585

摘要/Abstract

摘要：

为了筛选出长茎葡萄蕨藻中稳定表达的内参基因用于实时荧光定量PCR的分析,以不同胁迫条件下长茎葡萄蕨藻的匍匐茎和直立枝为材料,使用比较Ct值法、BestKeeper、geNorm、NormFinder软件综合比较了常用的5个候选内参基因的稳定性,并对筛选出的内参基因进行了验证。不同内参基因在长茎葡萄蕨藻中的表达稳定性差异较大,ClACT和ClGAPDH在长茎葡萄蕨藻不同组织、不同胁迫条件下的表达稳定性均较好,而ClTUB表达稳定性则较差。在使用RT-qPCR对胁迫条件下长茎葡萄蕨藻进行基因表达分析时,采用ClACT和ClGAPDH组合作为内参基因可得出较为准确的结果。

关键词: 长茎葡萄蕨藻, 内参基因, 实时荧光定量PCR, 胁迫

Abstract:

In order to screen internal reference genes stably expressing in Caulerpa lentillifera for the analysis of real-time fluorescence quantitative PCR,the △Ct,BestKeeper,geNorm and NormFinder software were used to comprehensively compare the expression stabilities of 5 candidate reference genes,using the stolons and erect branches of C. lentillifera under various stress conditions,and the selected reference genes are verified. The expression stabilities of different reference genes in C. lentillifera differed largely. The expression stabilities of ClACT and ClGAPDH were quite good in different tissues and under different stress conditions,while the expression stability of ClTUB was poor. The combination of ClACT and ClGAPDH can be used as the internal reference gene and more accurate results can be acquired when RT-qPCR is used to analyze the gene expression of C. lentillifera under stress.

Key words: Caulerpa lentillifera, reference genes, RT-qPCR, stress

李恬静薇, 邹潇潇, 朱军, 鲍时翔. 长茎葡萄蕨藻胁迫条件下RT-qPCR内参基因的筛选与验证[J]. 生物技术通报, 2021, 37(10): 266-276.

LI Tianjingwei, ZOU Xiao-xiao, ZHU Jun, BAO Shi-xiang. Selection and Validation of Reference Genes for Quantitative Real-time PCR in Caulerpa lentillifera Under Stress Conditions[J]. Biotechnology Bulletin, 2021, 37(10): 266-276.

图/表 10

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处理组 Treatment group	处理条件 Treatment conditions	组织部位Tissues
对照组	温度27℃,光照40 μmol/（s · m²）,盐度30‰	直立枝
对照组	温度27℃,光照40 μmol/（s · m²）,盐度30‰	匍匐茎
温度胁迫	温度15℃,光照40 μmol/（s · m²）,盐度30‰	直立枝
	温度15℃,光照40 μmol/（s · m²）,盐度30‰	匍匐茎
	温度37℃,光照40 μmol/（s · m²）,盐度30‰	直立枝
	温度37℃,光照40 μmol/（s · m²）,盐度30‰	匍匐茎
光照胁迫	温度27℃,光照120 μmol/（s · m²）,盐度30‰	直立枝
	温度27℃,光照120 μmol/（s · m²）,盐度30‰	匍匐茎
	温度27℃,光照360 μmol/（s · m²）,盐度30‰	直立枝
	温度27℃,光照360 μmol/（s · m²）,盐度30‰	匍匐茎
盐度胁迫	温度27℃,光照360 μmol/（s · m²）,盐度15‰	直立枝
	温度27℃,光照360 μmol/（s · m²）,盐度15‰	匍匐茎
	温度27℃,光照360 μmol/（s · m²）,盐度40‰	直立枝
	温度27℃,光照360 μmol/（s · m²）,盐度40‰	匍匐茎

处理组 Treatment group	处理条件 Treatment conditions	组织部位Tissues
对照组	温度27℃,光照40 μmol/（s · m²）,盐度30‰	直立枝
对照组	温度27℃,光照40 μmol/（s · m²）,盐度30‰	匍匐茎
温度胁迫	温度15℃,光照40 μmol/（s · m²）,盐度30‰	直立枝
	温度15℃,光照40 μmol/（s · m²）,盐度30‰	匍匐茎
	温度37℃,光照40 μmol/（s · m²）,盐度30‰	直立枝
	温度37℃,光照40 μmol/（s · m²）,盐度30‰	匍匐茎
光照胁迫	温度27℃,光照120 μmol/（s · m²）,盐度30‰	直立枝
	温度27℃,光照120 μmol/（s · m²）,盐度30‰	匍匐茎
	温度27℃,光照360 μmol/（s · m²）,盐度30‰	直立枝
	温度27℃,光照360 μmol/（s · m²）,盐度30‰	匍匐茎
盐度胁迫	温度27℃,光照360 μmol/（s · m²）,盐度15‰	直立枝
	温度27℃,光照360 μmol/（s · m²）,盐度15‰	匍匐茎
	温度27℃,光照360 μmol/（s · m²）,盐度40‰	直立枝
	温度27℃,光照360 μmol/（s · m²）,盐度40‰	匍匐茎

候选基因Candidate gene	基因登录号 Accession No.	引物序列（上游/下游,5'-3'） Primer sequence（forward/revers, 5'-3'）	片段大小 Fragment length/bp	Tm/℃	E/%
ClACT	MW039598	GGTGGTGAACGATTCCGAT TTCTGACATCTCTTTACTGACTCTC	203	50	93.76
Cl18S	JN034412.1	ATCTAAGGAAGGCAGCAGG TCTATCGCCAGAAGTCCAAC	241	50	94.80
ClGAPDH	MW036245	GAAGAAGGACTGGAGAGGA GTAGGAACTCTAAACGCCA	135	50	95.36
ClEF1a	MW021590	AGCTGGAATTTCAAAAGACG AGAGATAGGAACAAAGGGGA	211	50	102.00
ClTUB	MW039599	ATTGGGAAGGAGATGGTGGA GACTTGCGGAGACGGATAAA	204	54	90.36

候选基因Candidate gene	基因登录号 Accession No.	引物序列（上游/下游,5'-3'） Primer sequence（forward/revers, 5'-3'）	片段大小 Fragment length/bp	Tm/℃	E/%
ClACT	MW039598	GGTGGTGAACGATTCCGAT TTCTGACATCTCTTTACTGACTCTC	203	50	93.76
Cl18S	JN034412.1	ATCTAAGGAAGGCAGCAGG TCTATCGCCAGAAGTCCAAC	241	50	94.80
ClGAPDH	MW036245	GAAGAAGGACTGGAGAGGA GTAGGAACTCTAAACGCCA	135	50	95.36
ClEF1a	MW021590	AGCTGGAATTTCAAAAGACG AGAGATAGGAACAAAGGGGA	211	50	102.00
ClTUB	MW039599	ATTGGGAAGGAGATGGTGGA GACTTGCGGAGACGGATAAA	204	54	90.36

基因 Gene	基因登录号 Accession No.	引物序列（上游/下游,5'-3'） Primer sequence (forward/reverse, 5'-3')	片段大小 Fragment length/bp	Tm/℃
ClHSP90	MW088997	ATGGAGGCATTGTCCGCTGTTGCTCATCGTCGTTATGGTGT	123	55
ClSSD	MW088998	CACTCGTGATGAGGATGGAAAGCTGGCTCCTTGCTCTTATCGTA	98	55