Plant molecular farming is simply defined as the production of proteins therapeutics (PT) in plants, which involves transient gene expression in plants and purification of expressed protein to a great scale for diagnosis, treatment and other applications. This is the rapid, economical, safe and reproducible approach for the production of PT as compared to bacterial and mammalian systems. Protein yield and post-translational modifications are the major roadblocks that can be overcome by high expression strategies includes over expression constructs, suitable plant host systems and glycoengineering of proteins. The inherent ability of ideally producing safe, functional protein is the most striking phenomenon recognised by the pharmaceutical industries and developed many therapeutic products within few weeks to meet escalating demands during pandemic/epidemic outbreaks recently.
The seed oil of Jatropha curcas is considered a potential bioenergy source that could replace fossil fuels. However, the seed yield of Jatropha is low and has yet to be improved. We previously reported that e xogenous cytokinin treatment increased the seed yield of Jatropha. Cytokinin levels are directly regulated by isopentenyl transferases (IPT), cytochrome P450 monooxygenase (CYP735A) and cytokinin oxidase/dehydrogenase (CKX). In this study, we cloned six IPT genes, one JcCYP735A gene, and seven JcCKX genes. The expression patterns of these 14 genes in various organs were determined using real-time quantitative PCR. JcIPT1 was primarily expressed in roots and seeds , JcIPT2 was expressed in roots, apical meristems, and mature leaves, JcIPT3 was expressed in stems and mature leaves, JcIPT5 was expressed in roots and mature leaves, JcIPT6 was expressed in seeds at 10 days after pollination, and JcIPT9 was expressed in mature leaves. JcCYP735A was mainly expressed in roots, flower buds, and seeds. The seven JcCKX genes also showed different expression patterns in different organs of Jatropha. In addition, CK levels were detected in flower buds and seeds at different stages of development. The concentration of iP, iPR and tZ increased with flower development, and the concentration of iP decreased with seed development, while that of tZ increased. We further analyzed the function of JcCYP735A using the CRISPR-Cas9 system, and found that the concentrations of tZ and tZR decreased significantly in the Jccyp735a mutants, which showed severely retarded growth. These findings will be helpful for further studies of the functions of cytokinin metabolic genes and understanding the roles of cytokinins in Jatropha growth and development.
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