Prokaryotic and tissue-specific expression of RglKAT gene from Rehmannia glutinosa

Y Q ZHOU; Y H ZHANG; W S WANG; X N WANG; H Y DUAN

doi:10.56093/ijas.v85i11.53762

Authors

Y Q ZHOU Professor, College of Life Sciences, Henan Normal University, Xinxiang Henan, 453 007, China; and Key Laboratory for Microorganisms and Functional Molecules, University of Henan Province, No. 46 Jianshe Road, Xinxiang Henan, 453 007, P R China
Y H ZHANG Graduates, Plant Genetics, College of Life Science, College of Life Sciences, Henan Normal University, Xinxiang Henan, 453 007, China; and Key Laboratory for Microorganisms and Functional Molecules, University of Henan Province, No. 46 Jianshe Road, Xinxiang Henan, 453 007, P R China
W S WANG College of Life Sciences, Henan Normal University, Xinxiang Henan, 453 007, China; and Key Laboratory for Microorganisms and Functional Molecules, University of Henan Province, No. 46 Jianshe Road, Xinxiang Henan, 453 007, P R China
X N WANG
H Y DUAN College of Life Sciences, Henan Normal University, Xinxiang Henan, 453 007, China; and Key Laboratory for Microorganisms and Functional Molecules, University of Henan Province, No. 46 Jianshe Road, Xinxiang Henan, 453 007, P R China

https://doi.org/10.56093/ijas.v85i11.53762

Keywords:

Cloning, Expression, 3-Ketoacyl-CoA thiolase (KAT), Rehmannia glutinosa, Real Time quantitative PCR

Abstract

A 1640-bp full-length cDNA of the 3-ketoacyl-CoA thiolase gene was cloned from Rehmannia glutinosa L. leaf using RT-PCR and RACE. Bioinformatics analyses indicated that it contained one 1395-bp open reading frameencoding a thiolase of 464 amino acids with the predicted molecular weight of 49 kDa, and had a similarity of 73-82% to other species at the nucleotide level and 63-88% to other species at the amino acid level, respectively. The recombinant RglKAT was expressed in a bacterial system in an active form, analyzed by PAGE-CBB staining and purified using Ni-agarose chromatography. Real Time quantitative PCR analysis showed that relative expression levels of the RglKAT
gene differed between R.glutinosa tissues, with the highest levels in the petals and the lowest in the early senescing stems. These results revealed that RglKAT gene could be cloned and heterogeneously expressed, and related to R. glutinosa growth and development.

Downloads

Download data is not yet available.

Author Biography

X N WANG

Graduates, Plant Genetics, College of Life Science,

References

Carrie C, Monika W, Murcha A, Millar H, Smith S M, Whelan J. 2007. Nine 3-ketoacyl-CoA thiolases (KATs) and acetoacetyl- CoA thiolases (ACATs) encoded by five genes in Arabidopsis thaliana are targeted either to peroxisomes or cytosol but not to mitochondria. Plant Molecular Biology 63: 97–108. DOI: https://doi.org/10.1007/s11103-006-9075-1

Dyer J H, Maina A, Gomez I D, Cadet M, Oeljeklaus S and Schiedel A C. 2009. Cloning, expression and purification of an acetoacetyl-CoA thiolase from sunflower cotyledon. International Journal of Bioogical Sciences 5: 736–44. DOI: https://doi.org/10.7150/ijbs.5.736

Footitt S, Cornah J E, Pracharoenwattana I, Bryce J H, Smith S M. 2007. The Arabidopsis 3-ketoacyl-CoA thiolase-2, kat2-1, mutant exhibits increased flowering but reduced reproductive success. Journal Experimental Botany 58: 2 959–68. DOI: https://doi.org/10.1093/jxb/erm146

Germain V, Rylott E L, Larson T R, Sherson S M, Bechtold N and Carde J P. 2011. Requirement for 3-ketoacyl-CoA thiolase-2 in peroxisome development, fatty acid -oxidation and breakdown of triacylglycerol in lipid bodies of Arabidopsis seedlings. Plant Journal 28: 1–12. DOI: https://doi.org/10.1046/j.1365-313X.2001.01095.x

Hou WH, Sun P, Chen QJ and Li XE. 2011. Selection of the reference genes for gene expression studies in Rehmannia glutinosa by Real-time quantitative PCR. Chinese Agricultural Science Bulletin 27: 76–82. Chinese.

Kim Y S, Ryuk J A, Ko B S. 2012. Discrimination of Korean Rehmannia glutinosa from Chinese Rehmannia glutinosa using sequence- characterized amplified region marker. Journal of the Korean Society for Applied Biological Chemistry 55: 1– 6. DOI: https://doi.org/10.1007/s13765-012-0001-8

Kumar S, Arasan T, Park J I, Ahmed N U, Jung H J and Hur Y. 2013. Characterization and expression analysis of dirigent family genes related to stresses in Brassica. Plant Physiology and Biochemistry 67: 144–53. DOI: https://doi.org/10.1016/j.plaphy.2013.02.030

Livak K J and Schmittgen T D 2001.Analysis of relative gene expression data using real time quantitative PCR and 2(-Delta Delta C(T) method. Method 25: 402–8. DOI: https://doi.org/10.1006/meth.2001.1262

Record E, Moukha S and Asther M 2001. Cloning and expression in phospholipid containing cultures of the gene encoding the specific phosphatidylglycerol/ phosphatidylinositol transfer protein from Aspergillus oryzae: evidence that the pg/pi-tp is tandemly arrangedwith the putative3-ketoacyl-CoA thiolase gene. Gene 262: 61–72. DOI: https://doi.org/10.1016/S0378-1119(00)00514-X

Sun P, Zhou L L L and Xianen Li. 2012. Transcriptome filing and dsicovery of terpenoid biosynthetic genes in Rehmannia glutinosa. http://www. paper.edu.cn/ releasepaper/ content/ 201207-99

Sun P, Guo Y H, Qi J J, Zhou L L and Li X E. 2008. Cloning and analysis of the actin gene from Rehmannia glutinosa.L. Journal of Anhui Agricultural Sciences 36: 8470-8471-8474, Chinese.

Sun P, Guo Y H, Qi J J, Zhou L L and Li X E. 2010. Isolation and expression analysis of tuberous root development related genes in Rehmannia glutinosa.Molecular biology Reports 37: 1 069– 79. DOI: https://doi.org/10.1007/s11033-009-9834-6

Sun P, Guo Y H, Qi J J, Zhou L L, Zhang M M and Li X E. 2009. Clonning and expression of RgPR-10 gene from Rehmannia glutinosa. Acta Agriculturae Boreali-occidentalis Sinica 18: 300–304, Chinese.

Sundaramoorthy R, Micossi E, Magnus S, Germain A V, James H and Bryce S M.2006. The crystal structure of a plant 3-ketoacyl- CoA thiolase reveals the potential for Redox control of peroxisomal fatty acid -oxidation. Journal of Moecular Biology 359: 347–57. DOI: https://doi.org/10.1016/j.jmb.2006.03.032

Zimmermann P, Hirsch-Hoffmann M, Hennig L.2004.Gruissem W. GENEVESTIGATOR. Arabidopsis microarray database and analysis toolbox. Plant Physiology 136: 2 621–32. DOI: https://doi.org/10.1104/pp.104.046367

Zhou Y Q, Yao H L, Duan H Y, Zhou C E, Zhang Y H, Chen Y M.2012. Cloning and sequencing of one DNA fragment and its internal transposase gene in Rehmannia glutinosa f. hueichingensis (Chan et Sehih) Hsiao. Journal of Henan Agricultural Sciences 41: 106–9. Chinese.

Zang Y C, Sun P, Yang T X and Li X E 2012. Cloning and expression analysis of RgExpA1 in Rehmannia glutinosa. Biotechnology Bulletin 2: 420–5, Chinese.

Zhou Y Q, Zhang Y H, Chen Y M, Zhang Y, Li J Y, Chen J J. 2013. Cloning and bioinformatics analysis of one vacuolar H+– Pyrophosphatase gene in Rehmannia glutinosa, f. hueichingensis (Chan et Sehih) Hsiao. Journal Henan Agricultural Sciences 42: 107–11ÿ114. Chinese.