|
Phase 5 (2012 Deadline)
Targeted Engineering of Brassica Juncea Seed Biochemistry to Produce Reduced-Viscosity Plant Oils for Direct Use as Biofuel Timothy P. Durrett, Kansas State University Iqbal Munir, Agricultural University Peshawar Project Overview
Pakistan is burdened by a huge oil import bill which will only increase as the population continues to grow. Furthermore, the cost of oil is anticipated to rise, exacerbating the situation. Biodiesel production can be a viable solution to this challenge and can offer possibilities to gradually arrive at the required additional production level in a manner acceptable to Pakistan’s society.
Brassica juncea is a desirable biofuel target species as it can resist extreme temperatures and requires lower moisture for reasonable production in marginal land. The overall goal of this project is to alter the synthesis of vegetable oil in the seeds of B. juncea to produce high levels of reduced viscosity oil (acTAGs). The altered structure of acTAGs with lower viscosity and improved cold temperature properties make them potentially useful as an improved straight vegetable oil (SVO) biofuel. In addition to their renewable nature, SVO fuels are appealing because they are simpler and cheaper to produce than biodiesel, making them valuable for isolated rural communities where the delivery of liquid fuels is difficult.
Progress Reports 
Transgenic B. Juncea
2014: As little is known about the specific genes responsible for the synthesis of vegetable oil in B. juncea the team needed to obtain a gene expression profile of developing seeds. RNA transcripts indicating which genes were active were extracted from seeds at stages with active lipid synthesis. This RNA was submitted for analysis using next-generation sequencing technologies. In order to create transgenic B. juncea plants the team produced EaDAcT enzyme gene constructs. These were successfully used for transformation and the first generation of transgenic B. juncea plants is currently growing. The team will then analyze the seed from this transgenic plant. In Pakistan, a total of two PhD students and two laboratory technicians are currently involved in the project. Additionally, 30 undergraduate and five M. Phil students received practical training in methods pertinent to the project. On his end, Dr. Durrett helped train two different PhD students on this project. One PhD student from Dr. Munir’s group spent six months in Dr. Durrett’s lab learning different aspects of lipid extraction and analysis. Another Pakistani PhD. student will visit the Durrett lab in 2015 to conduct part of her research work and obtain additional training in aspects of lipid analysis. The project team helped organize the National Conference on Innovative Technologies and Sustainable Developments in Agriculture in Baragali, Pakistan in August. Both Drs. Munir and Durrett and other colleagues spoke about the use of plant oil for biofuel purposes while at the conference. A training workshop will be organized at IBGE, the University of Agriculture Peshawar. The team also plans to publish a manuscript describing the transcriptomic profiling of developing B. juncea seeds.
2015: In the previous reporting period, the team extracted RNA from three developmental stages with active seed oil synthesis. This high quality RNA was sent for sequencing. During this reporting period the sequencing data was received and using bioinformatics tools a transcriptomic profile of B. juncea seeds during development was generated. Importantly, the team was able to identify sequences with high homology to DGAT1 and PDAT genes in other Brassicaceae. DGAT1 and PDAT are enzymes known to synthesize regular TAGs and whose activity competes with the EaDAcT enzyme we are transforming into B. juncea.
Based off the transcript profiling data, the team designed constructs that would silence the DGAT1 and PDAT genes in B. juncea. Specifically, the sequence was designed to produce RNA that folds back to form a hairpin loop, a common method called RNA interference (RNAi) to target genes for silencing. Given their complexity, the “hairpin” portion of the constructs were to be created via direct DNA synthesis. The PDAT-RNAi sequence was successfully synthesized and then cloned into the EaDAcT transformation vectors developed in the previous reporting period.
In the previous reporting period, a construct expressing EaDAcT was transformed into B. juncea and two transgenic plants generated. Unfortunately, analysis of the seed derived from these plants revealed that no acetyl-TAGs were present in the oil.
2016: The overall goal of this project is to alter the synthesis of vegetable oil in the seeds of the crop plant Brassica juncea to produce high levels of a reduced viscosity oil. We refer to these unusual oil molecules as acetyl-TAGs because they have a slightly different structure compared to the typical triacylglycerols (TAGs) that constitute regular vegetable oils. The altered structure of acetyl-TAGs means that they possess lower viscosity and improved cold temperature properties, making them potentially useful as an improved straight vegetable oil (SVO) biofuel. In addition to their renewable nature, SVO fuels are appealing because they are simpler and cheaper to produce than biodiesel, making them valuable for isolated rural communities where the delivery of liquid fuels is difficult. B. juncea is a desirable biofuel target species as it can resist extreme temperatures and requires lower moisture for reasonable production. Acetyl-TAGs are synthesized through the action of an enzyme called EaDAcT isolated from the seeds of the ornamental shrub Euonymus alatus (more commonly known as the Burning Bush). To produce acetyl-TAGs in B. juncea, we will create transgenic plants expressing EaDAcT. Additionally, to produce high levels of acetyl-TAGs, we will simultaneously suppress the synthesis of regular vegetable oil in these seeds. As little is known about the specific genes responsible for the synthesis of vegetable oil in B. juncea, we need to obtain a gene expression profile of developing seeds. RNA transcripts indicating which genes were active were extracted from seeds at stages with active lipid synthesis. This RNA was sequenced and the reads assembled to obtain the sequences of genes expressed in B. juncea seeds, as well as information about the level of expression. By comparing these sequences to the genes known to be important for the synthesis of oil in related plant species we were able to identify similar genes in B. juncea. We then designed and synthesized DNA sequences to silence these genes. These sequences were combined with the expression of EaDAcT, so that acetyl-TAGs will be synthesized while the production of regular TAGs will be reduced. These DNA constructs have been successfully transformed into B. juncea plants. In the near future we will analyze the oil composition of the seeds of these plants to confirm the production of acetyl-TAGs.
2017: The overall goal of this project is to alter the synthesis of vegetable oil in the seeds of the crop plant Brassica juncea to produce high levels of a reduced viscosity oil. We refer to these unusual oil molecules as acetyl-TAGs because they have a slightly different structure compared to the typical triacylglycerols (TAGs) that constitute regular vegetable oils. The altered structure of acetyl-TAGs means that they possess lower viscosity and improved cold temperature properties, making them potentially useful as an improved straight vegetable oil (SVO) biofuel. In addition to their renewable nature, SVO fuels are appealing because they are simpler and cheaper to produce than biodiesel, making them valuable for isolated rural communities where the delivery of liquid fuels is difficult. B. juncea is a desirable biofuel target species as it can resist extreme temperatures and requires lower moisture for reasonable production. Acetyl-TAGs are synthesized through the action of an enzyme called EaDAcT isolated from the seeds of the ornamental shrub Euonymus alatus (more commonly known as the Burning Bush). To produce acetyl-TAGs in B. juncea, we will create transgenic plants expressing EaDAcT. Additionally, to produce high levels of acetyl-TAGs, we will simultaneously suppress the synthesis of regular vegetable oil in these seeds. As little is known about the specific genes responsible for the synthesis of vegetable oil in B. juncea, we need to obtain a gene expression profile of developing seed. RNA transcripts indicating which genes were active were extracted from seeds at stages with active lipid synthesis. This RNA was sequenced and the reads assembled to obtain the sequences of genes expressed in B. juncea seeds, as well as information about the level of expression. By comparing these sequences to the genes known to be important for the synthesis of oil in related plant species we were able to identify similar genes in B. juncea. We then designed and synthesized DNA sequences to silence these genes. These sequences were combined with the expression of EaDAcT, so that acetyl-TAGs will be synthesized while the production of regular TAGs will be reduced. While initial results suggested the successful transformation of B. juncea, analysis of subsequent generations failed to detect the presence of the EaDAcT gene. Seeds therefore did not contain acetyl-TAG. We are attempting to improve transformation efficiency with more reliable methods and new DNA constructs. Some of these constructs contain improved versions of EaDAcT that will hopefully result in higher production of low viscosity seed oil in B. juncea.
| 
