de Andrade Larissa Mara, Dos Santos Brito Michael, Fávero Peixoto Junior Rafael, Marchiori Paulo Eduardo Ribeiro, Nóbile Paula Macedo, Martins Alexandre Palma Boer, Ribeiro Rafael Vasconcelos, Creste Silvana
Centro de Cana, Instituto Agronômico (IAC), P.O. Box 206, Ribeirão Preto, SP 14001-970 Brazil.
Departamento de Genética, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP 14049-900 Brazil.
Plant Methods. 2017 Apr 17;13:28. doi: 10.1186/s13007-017-0178-2. eCollection 2017.
Sugarcane ( spp.) is the main raw material for sugar and ethanol production. Among the abiotic stress, drought is the main one that negatively impact sugarcane yield. Although gene expression analysis through quantitative PCR (qPCR) has increased our knowledge about biological processes related to drought, gene network that mediates sugarcane responses to water deficit remains elusive. In such scenario, validation of reference gene is a major requirement for successful analyzes involving qPCR.
In this study, candidate genes were tested for their suitable as reference genes for qPCR analyses in two sugarcane cultivars with varying drought tolerance. Eight candidate reference genes were evaluated in leaves sampled in plants subjected to water deficit in both field and greenhouse conditions. In addition, five genes were evaluated in shoot roots of plants subjected to water deficit by adding PEG8000 to the nutrient solution. NormFinder and RefFinder algorithms were used to identify the most stable gene(s) among genotypes and under different experimental conditions. Both algorithms revealed that in leaf samples, and genes were more suitable as reference genes, whereas was the best reference one in shoot roots.
Reference genes suitable for sugarcane under water deficit were identified, which would lead to a more accurate and reliable analysis of qPCR. Thus, results obtained in this study may guide future research on gene expression in sugarcane under varying water conditions.
甘蔗（甘蔗属）是糖和乙醇生产的主要原料。在非生物胁迫中，干旱是对甘蔗产量产生负面影响的主要因素。尽管通过定量PCR（qPCR）进行的基因表达分析增加了我们对与干旱相关的生物学过程的了解，但介导甘蔗对水分亏缺反应的基因网络仍然难以捉摸。在这种情况下，验证内参基因是成功进行涉及qPCR分析的主要要求。
在本研究中，对两个耐旱性不同的甘蔗品种中作为qPCR分析内参基因的适用性进行了测试。在田间和温室条件下对遭受水分亏缺的植物叶片中采集的样本评估了8个候选内参基因。此外，通过向营养液中添加聚乙二醇8000对遭受水分亏缺的植物的茎根中的5个基因进行了评估。使用NormFinder和RefFinder算法在不同基因型和不同实验条件下鉴定最稳定的基因。两种算法均显示，在叶片样本中，基因和更适合作为内参基因，而在茎根中是最佳内参基因。
鉴定出了水分亏缺条件下适合甘蔗的内参基因，这将使qPCR分析更加准确可靠。因此，本研究获得的结果可能会指导未来在不同水分条件下甘蔗基因表达的研究。
