不同组织及干旱胁迫下黄薇内参基因的筛选与验证

Screening and validation of reference genes in Heimia myrtifolia in different tissues and under drought stress

【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。

[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.]