Characterization of glycolytic pathway genes using RNA-Seq in developing kernels of Eucommia ulmoides

Authors: FENG, YANZHI - Paulownia Research And Development Center Of State Forestry Administration; ZHANG, LIN - Central South University Of Forestry And Technology; FU, JIANMIN - Paulownia Research And Development Center Of State Forestry Administration; LI, FANGDONG - Paulownia Research And Development Center Of State Forestry Administration; WANG, LU - Paulownia Research And Development Center Of State Forestry Administration; TAN, XIAOFENG - Central South University Of Forestry And Technology; MO, WENJUAN - Chinese Academy Of Forestry; Cao, Heping

Submitted to: Journal of Agricultural and Food Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/13/2016
Publication Date: 4/13/2016
Citation: Feng, Y., Zhang, L., Fu, J., Li, F., Wang, L., Tan, X., Mo, W., Cao, H. 2016. Characterization of glycolytic pathway genes using RNA-Seq in developing kernels of Eucommia ulmoides. Journal of Agricultural and Food Chemistry. 64(18):3712-3731. https://doi.org/10.1021/acs.jafc.5b05918.
DOI: https://doi.org/10.1021/acs.jafc.5b05918

Interpretive Summary: Eucommia ulmoides Oliver, the only member of the Eucommiaceae family, is a rare and valuable tree endemic to China. It is used to produce a highly valued traditional Chinese medicine and also famous as a source of gutta-percha. Oil from the seeds of E. ulmoides contains various fatty acids, especially rich in a-linolenic acid, which constitutes as much as 60% of the total fatty acids. Plant glycolytic pathway consists of ten continuous reactions regulated by ten families of enzymes. The importance of glycolysis in living organisms lies not only in its role in metabolism, but also in its links to complex network pathways regulated by diverse enzymes. Abnormalities in any of these enzymes will lead to alterations in downstream pathways. Fatty acid biosynthesis, and therefore the biosynthesis of oil as well, are affected directly by the glycolytic pathway with respect to the generation of essential metabolites and the rates at which they are produced; however, nothing was done at the molecular level of the glycolytic pathway in E. ulmoides seeds. Construction of cDNA libraries and expressed sequence tags constitute the efficient methods for large-scale gene discovery. Novel genes related to specific biological pathways can be identified by de novo transcriptome analyses, whereas the accuracy, sensitivity, high-throughput and low cost of next-generation RNA sequencing technologies facilitate studies of specific cellular pathways and gene expression patterns. The objectives of this study were to identify novel genes of E. ulmoides related to glycolytic metabolism and to analyze the expression patterns of selected genes in the seeds. We took advantages of the above-mentioned approaches and constructed four cDNA libraries for RNA sequencing. We analyzed its transcriptome and identified 120 unigenes involved in the glycolytic pathway of E. ulmoides. Finally, we characterized the expression of all 120 unigenes related to glycolytic pathway digitally and validated 10 genes encoding major enzymes in this pathway by quantitative real-time polymerase chain reaction. This study provides a complete picture for glycolytic pathway in E. ulmoides kernels, which is valuable genetic resource in glycolytic metabolism of the tree.

Technical Abstract: Eucommia ulmoides Oliver, the only member of the Eucommiaceae family, is a rare and valuable tree used to produce a highly valued traditional Chinese medicine and contains a-linolenic acid up to 60% of the total fatty acids in the seeds. Glycolysis provides both cellular energy and the intermediates for other biosynthetic processes. However, nothing was done on the molecular basis of the glycolytic pathway in E. ulmoides seeds. The purposes of this study were to identify novel genes of E. ulmoides related to glycolytic metabolism and to analyze the expression patterns of selected genes in the seeds. Transcriptome sequencing based on the Illumina platform generated 96,469 unigenes in four cDNA libraries constructed using RNAs from 70 and 160 days after flowering kernels of two varieties. We identified and characterized the digital expression of 120 unigenes coding for 24 protein families involved in seed glycolytic pathway including those encode enolase, fructose-bisphosphate aldolase, glucose-6-phosphate 1-epimerase, glucose-6-phosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, 6-phosphofructokinase I, phosphoglycerate kinase, pyruvate kinase, and triosephosphate isomerase. The expression of ten unigenes encoding key enzymes in the glycolytic pathway was validated by qPCR using RNAs from six seed stages of each variety. The qPCR analyses showed that expression profiles of nine out of the 10 genes were well consistent with their digital expression in transcriptomic analyses. These results provide valuable information on the genetic resources in glycolytic metabolism of E. ulmoides.