Plant Gene Engineering Center
  Research Direction

The Plant Gene Engineering Centerwas formed in 2010 upon the arrival of David Ow. In 2012, the PGEC was merged into the newly formed Molecular Analysis & Genetic Improvement Center(MAGIC).

 

Research Direction

 

Site-specific gene stacking

In many crop species, transgenic traits are introduced into transformable varieties before introgressing them out to field cultivars.  For diploids or polyploids that behave as diploids, the ‘n’ number of unlinked transgenic loci can be assorted as homozygous into a single genome at a probability of (1/4)n.  However, along with the ‘x’ number of other nontransgenic traits that breeders need to assemble into the same genome, the (1/4)n+x probability for a ‘breeding stack’ makes line conversion difficult.  To minimize the number of segregating transgenic loci, the option of in vitro stacking prior to its introduction into the plant genome would mean the re-engineering and re-deregulating of previously introduced traits each time a new trait is introduced.  The option of bypassing introgression by directly transforming field cultivars is also not practical as most field cultivars are difficult to transform.  Moreover, each locale-specific cultivar would harbor an independent integration event that requires individual de-regulation. 

PhD student Hou Lili and colleagues demonstrated recombinase-mediated gene stacking in tobacco.  Through two rounds of integration, followed by deletion of unneeded DNA, precise structure and reproducible expression of the sequentially added traits were obtained.

Hou, L., Yau, Y-Y, Wei, J., Han, Z., Dong, Z., Ow, D.W.  2014. An open source system for in planta gene stacking by Bxb1 and Cre recombinases.  Molecular Plant 7:1756-1765.

Associate Researcher Li Ruyu and colleagues have developed a biolistic mediated method for site-specific integration and have demonstrated gene insertions into those target sites work efficiently.  To implementing this system in rice, a number of precise target sites in the rice genome were screened. 

Li, R., Han, Z., Hou, L., Kaur, G., Qian, Y., Ow, D.W.  2016.  Method for biolistic site-specific integration in rice and tobacco catalyzed by Bxb1 integrase.  In: Methods Mol Biol. 1469: 15-30 (Chromosome and Genomic Engineering in Plants, Ed. M. Murata), Humana Press, (Book Chapter).

PhD student Chen Weiqiang further developed a method to stack genes in in vitro that would be compatible with the recombinase-mediated gene stacking system in vivo.

Chen, W., Ow, D.W.  2016.  Protocol for in vitro stacked molecules compatible with in vivo recombinase mediated gene stacking.  In: Methods Mol Biol. 1469: 31-47 (Chromosome and Genomic Engineering in Plants, Ed. M. Murata), Humana Press, (Book Chapter).

  

PhD student Maryam Rajaee reported a new location to split the Cre recombinase for protein fragment complementation.  Plant lines producing the Cre recombinase, a DNA scanning protein, can be difficult to maintain stably.  By producing two inactive parts of the recombinase in separate plants, we can maintain stable lines that can be hybridized to produce functional recombinase.  

Rajaee, M., Ow, D.W. 2017. A new location to split Cre recombinase for protein fragment complementation.  Plant Biotechnology Journal 15: 1420-1428. 

Recent reviews on recombinase mediated gene stacking:

Ow, D.W.  2016.  The long road to recombinase-mediated plant transformation.  Plant Biotechnology Journal 14:441-117.

Chen, W., Ow, D.W.  2017.  Precise, flexible and affordable gene stacking for crop improvement.  Bioengineered 8:451-456.

 

Metal and oxidative stress tolerance

Environmental stresses reduce plant productivity.  Both abiotic and biotic stresses disrupt normal cellular homeostasis leading to elevated levels of reactive oxygen species that in turn leads to cellular damage and cell death.  We have been interested in the molecular mechanisms of plant tolerance to heavy metals, which also leads to oxidative stress. 

Previously, we described Arabidopsis Oxidative Stress 2 (OXS2) as a transcription factor for regulating stress escape in response to oxidative and metal stress.  Ph.D. student HE Lilong and colleagues have since extended this research to crop species and reported that members of the maize OXS2 family can activate transcription of a gene encoding a putative SAM-dependent methyltransferase, a new factor for enhanced Cd tolerance. 

He, L., M., X., Li, Z., Jiao, Z., Li, Y., Ow, D.W. 2016.  Maize OXIDATIVE STRESS 2 homologs enhance cadmium tolerance in Arabidopsis through activation of a putative SAM-dependent methyltransferase gene.  Plant Physiology 171:1675-1685.

 

Previously, we described Arabidopsis Oxidative Stress 3 (OXS3) as a putative histone modification factor in response to oxidative and metal stress.   Assistant Researcher Wang Changhu and colleagues have shown that overproducing certain rice OXS3 family member proteins can lower the cadmium content in rice grain.  Due to soil pollution problems in China, rice with high Cd content has been found in recent years.  As this problem cannot be solved easily through soil remediation, the engineering of low cadmium rice may provide a solution to minimize dietary intake of cadmium. 

Wang, C., Guo, W., Ye, S., Wei, P., Ow, D.W.  2016. Reduction of Cd in rice through expression of OXS3-like gene fragments.  Molecular Plant, 9:301-304.

Wang, C., Guo, W., Cai, X., Li, R., Ow, D.W. 2018. Engineering low-cadmium rice through stress-inducible expression of OXS3-family member genes.  New Biotechnology https://doi.org/10.1016/j.nbt.2018.04.004.

 

Postdoctoral fellow He Yumei and colleagues elucidated a new cadmium induced disulfide stress pathway in the fission yeast.   This new Oxs1-Pap1 regulatory pathway appears evolutionarily conserved, as heterologous (human, mouse and Arabidopsis) Oxs1 and Pap1-homologues can bind interchangeably with each other in vitro, and at least in the fission yeast, heterologous Oxs1 and Pap1-homologues can substitute for S. pombe Oxs1 and Pap1 to enhance stress tolerance.

He, Y., Chen, Y., Song, W., Zhu, L., Dong, Z., Ow, D.W.  2017. A Pap1-Oxs1 signaling pathway for disulfide stress in Schizosaccharomyces pombe.  Nucleic Acids Research 45: 106-114.

 

PhD student Chen Yan and colleagues found that the Oxs1 NES (nuclear export signal) can protect fission yeast against oxidative stress by serving as a competitive substrate for Crm1-mediated export of Pap1. Higher Pap1 concentration in the nucleus primes the expression of stress tolerance genes.  This method of blocking Crm1 mediated export of nuclear proteins may find medical relevance as an alternative to chemical drugs currently being tested to block the nuclear export of tumor suppressor p53 in cancer therapy, or proteins necessary for maturation of HIV-1 and influenza viruses. 

Chen, Y., Zhang, Y., Dong, Z., Ow, D.W.  2018. Protection from disulfide stress by inhibition of Pap1 nuclear export in Schizosaccharomyces pombe.  Genetics, early online September 4, 2018. 

   

Urban farming

By 2040, Chinese cities will house a billion residents, and the need to provide fresh food for this large population will become a greater challenge.  Urban development reduces arable land for traditional agriculture while urban industrialization pushes clean growing areas further away from urban centers.  Hence, not only is China facing the prospect of less capacity for food production, but also escalating costs (including environmental costs) for food transport and packaging.  Roof top agriculture can reclaim some of the land displaced by urban sprawl. Locally grown vegetables would save otherwise costly handling, packaging and transport that depend on fossil fuel.  Water and fertilizers are more efficient in closed hydroponics systems without causing eutrophication of scarce drinking water supplies.  Herbicides are not necessary, and pesticides can be substantially reduced.  Hydroponically grown vegetables can be healthier than soil-grown counterparts given the high soil pollution near most urban surrounds.  PhD student LIU Ting and colleagues experimentally tested roof top farming, and the data showed that rooftop grown leafy vegetables can be produced more cost effectively and with higher quality than market equivalents.

Liu, T., Yang, M, Han, Z., Ow, D.W.  2016. Rooftop production of leafy vegetables can be profitable and less contaminated than farm grown vegetables.  Agronomy for Sustainable Development 36:41 DOI 10.1007/s13593-016-0378-6.

See also News media report from:

Conservation Magazine (Healthier and fresher greens calling from the rooftop, July 22, 2016).  

Quartz News (Rooftop hydroponic systems in cities produce vegetables that are cheaper and healthier than rural farms, December 14, 2016).

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