Abstract

Research Article

Stability determination of candidate reference genes in cucumber plants subjected to stresses from Phytophthora melonis

Tingquan Wu*, Rui Wang, Hu Du, Qingmin Jin, Yujuan Zhong, , Chunpeng Yao, Xiaomei Xu and Yu’e Lin

Published: 15 April, 2019 | Volume 3 - Issue 1 | Pages: 028-035

Stable reference genes are indispensable for ensuring the fidelity of determined gene expression levels. However, the expression levels of reference genes are unable to remain constant under all possible experimental conditions. Therefore, the stability determination of reference genes is necessary in an experimental system set. In the preset study, the stability of nine cucumber candidate reference genes (CsACT, CsUBQ, CsEF1α, CsCYP, CsαTU, CsCACS, CsTIP41, CsYSL8 and CsHEL) subjected to stresses from Phytophthora melonis(P. melonis) were determined using four different analysis methods, including Delta Ct, BestKeeper, NormFinder and GeNorm. The study results revealed that CsUBQ and CsCYP were the most stable genes suitable as internal control in cucumber plants under attack by P. melonis condition.

Read Full Article HTML DOI: 10.29328/journal.jpsp.1001028 Cite this Article Read Full Article PDF

Keywords:

Cucumber; Reference gene; P. melonis; Gene stability

References

  1. Jain M, Kaur N, Garg R, Thakur JK, Tyagi AK, et al. Structure and expression analysis of early auxin-responsive Aux/IAA gene family in rice (Oryza sativa). Funct Integr Genomics. 2006; 6: 47-59. Ref.: https://tinyurl.com/y4pl23bf
  2. Gantasala NP, Papolu PK, Thakur PK, Kamaraju D, Sreevathsa RU. Selection and validation of reference genes for quantitative gene expression studies by real-time PCR in eggplant (Solanum melongena L). BMC Res Notes. 2013; 6: 312. Ref.: https://tinyurl.com/y4xoe9cz
  3. Thellin O, Zorzi W, Lakaye B, De-Borman B, Coumans B, et al. Husekeeping genes as internal standards: use and limits. J. Biotechnol. 1999; 75: 291-295. Ref.: https://tinyurl.com/y4cltzsy
  4. Huggett J, Dheda K, Bustin S, Zumla A. Real-time RT-PCR normalization: strategies and considerations. Genes Immun. 2006; 6: 279-84. Ref.: https://tinyurl.com/y4wlytm4
  5. Tian C, Jiang Q, Wang F, Wang GL, Xu ZS, et al. Selection of suitable reference genes for qPCR normalization under abiotic stresses and hormone stimuli in carrot leaves. Plos One. 2015; 10: e0117569. Ref.: https://tinyurl.com/y6e4nqcs
  6. Santi S, Schmidt W. Laser microdissection-assisted analysis of the functional fate of iron deficiency-induced root hairs in cucumber. J Exp Bot. 2008; 59:697-704. Ref.: https://tinyurl.com/y3u8yhj3
  7. Xia XJ, Zhang Y, Wu JX, Wang JT, Zhou YH, et al. Brassinosteroids promote metabolism of pesticides in cucumber. Agric Food Chem. 2009; 57: 8406-8413. Ref.: https://tinyurl.com/y3mxyjnd
  8. Robinson TL, Sutherland IA, Sutherland J. Validation of candidate bovine reference genes for use with real-time PCR. Vet Immunol Immunopathol. 2007; 115: 160-165. Ref.: https://tinyurl.com/y6hpdq7j
  9. Migocka M, Papierniak A. Identification of suitable reference genes for studying gene expression in cucumber plants subjected to abiotic stress and growth regulators. Molecular Breeding. 2011; 28: 343-357. Ref.: https://tinyurl.com/y5af57ng
  10. Chandna R, Augustine R, Bisht NC. Evaluation of candidate reference genes for gene expression normalization in Brassica juncea using real time quantitative RT-PCR. PLoS One. 2012; 7: e36918. Ref.: https://tinyurl.com/y3n85cka
  11. Hu R, Fan C, Li H, Zhang Q, Fu YF. Evaluation of putative reference genes for gene expression normalization in soybean by quantitative real-time RT-PCR. BMC Mol Biol. 2009; 10:93. Ref.: https://tinyurl.com/y2dzlz8y
  12. Warzybok A, Migocka M. Reliable reference genes for normalization of gene expression in cucumber grown under different nitrogen nutrition. Plos One. 2013; 8: e72887 Ref.: https://tinyurl.com/y67f7g8m
  13. Wan H, Zhao Z, Qian C, Sui Y, Chen J. Selection of appropriate reference genes for gene expression studies by quantitative real-time polymerase chain reaction in cucumber. Anal Biochem. 2009; 399: 257-261. Ref.: https://tinyurl.com/y3ayy86k
  14. Bustin SA. Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems. J Mol Endocrinol. 2002; 29: 23-39. Ref.: https://tinyurl.com/y3nrrjmo
  15. Silver N, Best S, Jiang J, Thein SL. Selection of housekeeping genes for gene expression studies in human reticulocytes using real-time PCR. BMC Mol Biol. 2006; 7: 33. Ref.: https://tinyurl.com/y6zlcchr
  16. Pfaffl MW, Tichopad A, Prgomet C, Neuvians TP. Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper-excel-based tool using pair-wise correlations. Biotechnol Lett. 2004; 26: 509-515. Ref..: https://tinyurl.com/y4y3frkb
  17. Andersen CL, Jensen JL, Orntoft TF. Normalization of real-time quantitative reverse transcription-PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets. Cancer Res. 2004; 64: 5245-5250. Ref.: https://tinyurl.com/yya9s2cj
  18. Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, et al. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 2012; 18: 3 (RESEARCH0034). Ref.: https://tinyurl.com/yy2kw3pu
  19. Song Y, Du D, Chu M, Li X, Xu H, et al. Cloning and characterization of a cucumber phospholipase D gene in response to excess nitrate and other abiotic stresses. Scientia Horticulturae. 2012; 135:128-136. Ref.: https://tinyurl.com/y3f92yns
  20. Wang R, Chen M, Liao F, Jiang F, Ma B, et al. Cloning of the gene encoding cucumber lumazine synthase and an analysis of its promoter activity in cucumber. Canadian Journal of Plant Science. 2010; 90: 809-818. Ref.: https://tinyurl.com/yy97ybos
  21. Wang M, Bi H, Liu P, Ai X. Molecular cloning and expression analysis of the gene encoding sedoheptulose-1,7-bisphosphatase from Cucumis sativus. Scientia Horticulturae. 2011; 129: 414-420. Ref.: https://tinyurl.com/y5937kcp
  22. Qi J, Liu X, Shen D, Miao H, Xie B, et al. A genomic variation map provides insights into the genetic basis of cucumber domestication and diversity. Nat Genet. 2013; 45: 1510-1515. Ref.: https://tinyurl.com/y3hn9wys
  23. Chandrasekaran J, Brumin M, Wolf D, Leibman D, Klap C, et al. Development of broad virus resistance in non-transgenic cucumber using crispr/cas9 technology. Mol Plant Pathol. 2016; 17: 1140-1153. Ref.: https://tinyurl.com/y6rngnpp
  24. Huang S, Li R, Zhang Z, Li L, Gu X, Fan W, et al (2009) The genome of the cucumber, Cucumis sativus L. Nat Genet. 2009; 475: 1275-1281. Ref.: https://tinyurl.com/y2xzsvp9
  25. Wu T, Wang R, Xu X, He X, Sun B, et al. Cucumis sativus L-type lectin receptor kinase (CsLecRK) gene family response to Phytophthora melonis, Phytophthora capsici and water immersion in disease resistant and susceptible cucumber cultivars. Gene. 2014; 549: 214-222. Ref.: https://tinyurl.com/yxsmlxse
  26. Xu X, Wang R, Chao J, Lin Y, Jin Q, et al. The expression patterns of Cucumis sativus WRKY (CsWRKY) family under the condition of inoculation with Phytophthora melonisin disease resistant and susceptible cucumber cultivars. Canadian Journal of Plant Science. 2015; 95: 1121-1131. Ref.: https://tinyurl.com/yycqkswq

Figures:

Figure 1

Figure 1

Figure 1

Figure 2

Figure 1

Figure 3

Similar Articles

Recently Viewed

Read More

Most Viewed

Read More

Help ?