蚬壳花椒种子萌发时期内参基因的筛选与验证

Selection and validation of reference genes for quantitative real-time PCR during seed germination of Zanthoxylum dissitum

关键词:  蚬壳花椒, 种子萌发, 实时荧光定量 PCR, 内参基因, 基因表达

Key words:  Zanthoxylum dissitum, seed germination, quantitative real-time PCR(qRT-PCR), reference gene, gene expression

摘要:

为了解蚬壳花椒种子萌发的分子机制,需要筛选蚬壳花椒种子萌发时期表达稳定的内参基因。该研究通过赤霉素处理种子促进萌发,以不同萌发阶段的蚬壳花椒种子为材料,采用实时荧光定量PCR技术分析了6个候选内参基因GAPDH、ACT、18SrRNA、UBQ5、TUA和CYP在蚬壳花椒种子萌发时期的表达稳定性。结果表明:(1)α-淀粉酶基因、DELLA基因和异柠檬酸裂解酶基因分别反应了种子萌发阶段糖、激素和脂肪的代谢活动,因此选择蚬壳花椒异柠檬酸裂解酶基因(Unigene0032088)、α-淀粉酶基因(Unigene0033597)和DELLA基因(Unigene0058868)作为验证基因进行相对表达量验证。(2)综合geNorm、NormFinder和BestKeeper的分析结果显示在蚬壳花椒种子萌发过程中ACT表达稳定性最好,UBQ5次之。(3)以ACT、UBQ5基因为内参基因的结果显示验证基因的表达量与种子萌发生理状态一致,初步揭示了GA处理的种子易于萌发而清水处理的种子在萌发第3天容易腐败这一现象出现的可能原因。综上所述,ACT是蚬壳花椒种子萌发时期最合适的内参基因,其次是UBQ5。

Abstract:

In order to understand the molecular mechanism of the seed germination, we need to screen the reference genes of stable expression during the seed germination of Zenthoxylum dissitum. The seeds of Z. dissitum were treated with the gibberellins(GA)to promote germination. The samples were collected in 0, 1, 2, 3 and 5 d of germination. The quantitative real-time PCR(qRT-PCR)was performed to analyze the expression stabilities of six candidates such as Glyceraldehyde-3-phosphate(GAPDH), Actin(ACT), 18S ribosomal RNA(18SrRNA), ubiquitin-40S ribosomal(UBQ5), α-tubulin(TUA) and cyclophilin(CYP)among the seeds of the different germination stages. The α-amylase gene, DELLA gene and isocitrate lyase gene respectively reflected the metabolic activities of the sugars, hormones and lipids during the germination of Z. dissitum seeds. Therefore, isocitrate lyase (Unigene0032088), α-amylase(Unigene0033597)and DELLA (Unigene0058868)of Z. dissitum were selected as the validation genes for the relative expression verification during the seed germination. The comprehensive analysis of three softwares including geNorm, NormFinder and BestKeeper indicated that the expression stablility of ACT was the best and the second was UBQ5. The expressions of the validation genes were consistent with the states of seeds germination when ACT and UBQ5 were selected as the reference genes. Besides, when the ACT and UBQ5 genes were selected as the reference genes, the relative expression levels of isocitrate lyase, α-amylase and DELLA of zanthoxylum revealed the possible reasons that the seeds treated with GA were able to germinate successfully but the seeds treated with water easily spoiled in the 3rd day of germination. In conclusion, ACT is the preferred reference gene during the seed germination of Z. dissitum, followed by UBQ5.