Screening and validation of reference genes in Heimia myrtifolia in different tissues and under drought stress
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摘要:
目的 为黄薇Heimia myrtifolia不同组织及不同干旱胁迫下基因表达分析筛选最适内参基因。 方法 选取黄薇盛花期的根、茎、叶、花，以及5种不同干旱处理的叶片作为实验材料，借助RT-qPCR技术对黄薇转录组数据筛选的9个候选内参基因进行分析，并利用软件geNorm、BestKeeper、NormFinder和RefFinder综合评价候选基因的表达稳定性。最后选取2个与胁迫相关的基因CSLD和SOD，对所选内参基因进行验证。 结果 geNorm、BestKeeper和NormFinder分析得出的候选内参基因排序存在一定差异。利用在线软件RefFinder对上述3个软件的结果综合分析得出：在不同组织中，最稳定的内参基因为GAPDH，最不稳定的内参基因为TUA；在干旱胁迫中，最稳定的内参基因为GAPDH，最不稳定的内参基因为TUB。在全部样本中，最稳定的内参基因为GAPDH，最不稳定的内参基因为18S RNA。对不同组织和干旱胁迫下的CSLD和SOD基因表达模式进行验证表明：上述2个基因与筛选所得内参基因的表达量和变化趋势均较为一致。 结论 在不同组织和干旱处理后，GAPDH是最适合黄薇基因表达的内参基因。图4表4参33 Abstract:Objective The objective is to study the gene expression of Heimia myrtifolia in different tissues and under different drought stress. Method The root, stem, leaf, and flower of H. myrtifolia at full flowering stage and 5 leaves under different drought treatments were selected as experimental materials, and 9 candidate internal reference genes screened from the transcriptome data were analyzed by RT-qPCR. The software geNorm, BestKeeper, NormFinder, and RefFinder were used to comprehensively evaluate the expression stability of candidate reference genes. Finally, 2 stress-related genes CSLD and SOD were selected to verify the selected internal reference genes. Result GeNorm, BestKeeper, and NormFinder showed some differences in the ranking of candidate internal parameters. By using the online software RefFinder to comprehensively analyze the results of the above 3 software, it was concluded that GAPDH was the most stable internal reference gene and TUA was the most unstable in different tissues. In drought stress, GAPDH was the most stable internal reference gene and TUB was the least stable. In all samples, the most stable reference gene was GAPDH, and the most unstable one was 18S RNA. The expression patterns of CSLD and SOD genes in different tissues and under drought stress were verified, which showed that the expression levels and change trends of the above 2 genes were consistent with those of the selected internal reference genes. Conclusion GAPDH is the best internal reference gene for gene expression of H. myrtifolia in different tissues and under drought treatment. [Ch, 4 fig. 4 tab. 33 ref.] -
图 1 黄薇总RNA琼脂糖凝胶电泳分析(A)和RT-qPCR扩增产物特异性(B)
Figure 1 Agarose gel electrophoresis analysis of total RNA (A) and specificity of products amplified by RT-qPCR (B) in H. myrtifolia
图 3 geNorm分析9个候选内参基因的表达稳定值和配对变异值
Figure 3 Stable expression values and paired variation values of nine candidate reference genes were analyzed by geNorm
图 4 不同组织和干旱胁迫下CSLD (A)和SOD基因(B)利用不同内参基因的表达分析
Figure 4 Expression analysis for CSLD gene (A) and SOD gene (B) using different reference genes in different tissues and drought stress
表 1 黄薇候选内参基因引物信息
Table 1. Primer information of candidate reference gene in H. myrtifolia
基因 正向引物序列(5′→3′) 反向引物序列(5′→3′) 扩增大小/bp 退火温度/℃ 扩增效率/% R2 EF-1α TGGTTTTGAGGCTGGTATCTCC TTTGCTTGACACCAAGGGTGA 80 56.5 95.6 0.998 TUA TCTCTGCCTTGACCGAGTGA ACCACCAACGGCACTGAAAA 82 56.5 96.6 0.998 CYP ACCCCGACTCGTCCTACAAG TCGGTGTTCCGCTCCAAATG 130 58.0 106.8 0.999 GAPDH AGAAGGTCGTCATTTCTGCCC TGGTTGTGCAGCTAGCGTTG 114 57.5 108.4 0.999 18S RNA CAGGGCCTAGGATTTCGTCC GCCTTCAATCTTAGTCGTGGC 113 58.5 100.5 0.992 UBC GACCTGATGACACTCCCTGG TCACAGTTGGTGGTTTGTTCG 87 57.5 99.1 0.999 TUB GGGTGCTGAGCTTATTGATGC TGAGCAATGTCCCCATGCCT 131 57.5 96.8 0.996 ACT AGGGAATGCCTTTTGATTGATCC AAACATAAGCTCCACTGCCCTC 102 57 109.7 0.999 DNA J CGGAGCTATCACCCCGATG CGGCCTCACCATACCTGTCA 127 59.5 100.2 0.997 CSLD TACCTTGTCCCTTTCGGCG TCAGCGTCCTCATCCCGATA 149 57.4 95.1 0.997 SOD GTTGACGCAAGACGAGGGA CCGTTGGTCGTGTCACCAT 108 57.3 96.0 0.997 
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表 2 NormFinder分析内参基因的表达稳定值
Table 2. Expression stability values of nine candidate reference genes calculated by the NormFinder
基因 不同组织 干旱胁迫 全部样品 表达稳定值 排序 表达稳定值 排序 表达稳定值 排序 GAPDH 0.069 2 0.142 1 0.359 1 UBC 0.593 5 0.452 3 0.578 2 EF-1α 0.904 6 0.800 5 0.862 3 TUA 1.483 9 0.239 2 0.910 4 CYP 0.069 1 0.611 4 0.911 5 DNA J 0.579 4 1.479 6 1.094 6 18S RNA 0.494 3 1.542 8 1.366 7 ACT 1.095 7 1.507 7 1.390 8 TUB 1.294 8 1.816 9 1.505 9
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表 3 BestKeeper分析内参基因的表达稳定值
Table 3. Expression stability values of nine candidate reference genes calculated by the BestKeeper
基因 不同组织 干旱胁迫 全部样品 CV±SD 排序 CV±SD 排序 CV±SD 排序 EF-1α 3.46 ± 0.95 2 1.97 ± 0.53 1 2.58 ± 0.71 1 GAPDH 2.65 ± 0.69 1 3.78 ± 0.94 4 3.59 ± 0.91 2 UBC 3.73 ± 1.04 4 4.32 ± 1.18 5 4.21 ± 1.16 3 TUB 3.47 ± 1.06 3 6.14 ± 1.86 9 4.98 ± 1.51 4 TUA 6.65 ± 1.89 8 3.52 ± 0.95 3 5.26 ± 1.45 5 ACT 5.03 ± 1.46 5 6.11 ± 1.84 8 5.72 ± 1.69 6 CYP 6.36 ± 1.99 6 3.34 ± 0.96 2 6.11 ± 1.83 7 DNA J 7.31 ± 2.18 9 6.11 ± 1.81 7 6.81 ± 2.03 8 18S RNA 6.57 ± 2.05 7 5.68 ± 1.64 6 7.61 ± 2.28 9
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表 4 RefFinder分析内参基因的表达稳定值
Table 4. Expression stability of candidate reference genes ranked by RefFinder
排序 不同组织 干旱胁迫 全部样品 基因 表达稳
定值基因 表达稳
定值基因 表达稳
定值1 GAPDH 1.41 GAPDH 1.19 GAPDH 1.19 2 CYP 2.11 TUA 1.86 UBC 2.11 3 18S RNA 3.57 EF-1α 3.34 EF-1α 2.45 4 UBC 4.76 UBC 3.41 TUA 4.28 5 TUB 4.76 CYP 4.00 CYP 5.23 6 EF-1α 5.05 ACT 6.70 DNA J 6.45 7 ACT 5.12 DNA J 6.96 ACT 6.73 8 DNA J 5.44 18S RNA 7.20 TUB 6.84 9 TUA 9.00 TUB 9.00 18S RNA 7.45
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