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Genetic modification of Crambe abyssinica for better understanding of the biosynthesis pathways of erucic acid

Wang, Danni (2012) MOBT16 20112
Degree Projects in Molecular Biology
Abstract
Abstract

Replacing fossil oil with renewable resources perhaps is the most urgent challenge that the human being now is facing. Bioenergy has been considered as one of the best alternatives to fossil oil. Diverse non-edible applications of plant oil have been explored in the chemical industry recent years. However, both quantity and quality need to be improved from different oil crops for diverse industrial uses. Genetic engineering offers a good opportunity for improving plant oil qualities and quantities in a more precise and efficient way compared with the conventional breeding. Crambe abyssinica is regarded to be an important oil seed crop as it contains a high proportion of erucic acid (22:1) in its seeds, which has extensive... (More)
Abstract

Replacing fossil oil with renewable resources perhaps is the most urgent challenge that the human being now is facing. Bioenergy has been considered as one of the best alternatives to fossil oil. Diverse non-edible applications of plant oil have been explored in the chemical industry recent years. However, both quantity and quality need to be improved from different oil crops for diverse industrial uses. Genetic engineering offers a good opportunity for improving plant oil qualities and quantities in a more precise and efficient way compared with the conventional breeding. Crambe abyssinica is regarded to be an important oil seed crop as it contains a high proportion of erucic acid (22:1) in its seeds, which has extensive applications for industrial purposes. In this study, genetic engineering was employed to develop transgenic lines with modified oil composition for evaluating the important enzymes involved in the erucic acid biosynthesis. The integrations of the PDAT-RNAi and LPCAT-PDCT-RNAi genes into the crambe genome were confirmed by PCR analysis, Southern blot and qRT-PCR. In transgenic line with PDAT-RNAi, the average level of 18:1 was decreased while 18:2 was increased compared with non-transgenic control, indicating more 18:1 fluxed into 18:2. In transgenic line with LPCAT-PDCT-RNAi, the obvious decrease of 18:2 and increase of 18:1 were observed while 22:1 decreased compared with control group. The results facilitate the better understanding of acyl-CoA independent pathway of seed oil biosynthesis and provide new perspectives on how to improve the 22:1 content.

Popular science summary:

Insights into erucic acid biosynthesis in Crambe abyssinica

Crambe abyssinica is considered to be an important oilseed crop for industrial oil production. Crambe contains55-60%of erucicacidin its seed oil. Erucicacidhas extensive industrial applications, especially widely used as a slipping agent in plastic production for better separation of plastics. It is estimated that the purification cost of erucic acid from other fatty acids is to be halved by each 10 % increase of erucic acid in its seed oil. However, the biosynthesispathways of erucic acid have not been elucidated clearly in crambe.

Fatty acids are converted into triacylglycerol (TAG), commonly called storage or seed oil mainly through the Kenney pathway, but also through the acyl-CoA-independent pathway. Erucic acid (22:1) is a very long chain fatty acid and is elongated from 18:1-CoA through Kennedy pathway. However, the acyl-CoA-independent pathwaycouldpresumablydecrease the22:1 content bydiminishingthe 18:1-CoA pool.Thus, blocking the acyl-CoA-independent pathway could possibly increase the erucic acid content. Three genes named PDAT, PDCT and LPCAT have been identified in this pathway. In this project, we evaluated the functions of these three genes on the pathway of erucic acid biosynthesisthrough using the RNA interference technology. This will help us to further increase erucic acid contents through modification of these genes in combination with other important genes in the future.

Two gene constructs containing either PDAT-RNAi or LPCAT-PDCT-RNAi were introduced into the crambe genome through Agrobacterium-mediated transformation to down regulate the corresponding enzymes. Transgenic lines were confirmed by PCR and Southern blot analyses. The fatty acid composition and erucic acid contents in crambe transgenic lines were analyzed by gas chromatography. Transgenic lines with down-regulated PDAT and LPCAT-PDCT genes exhibited drastic changes in the fatty acid composition. Down regulation of PDAT led to a decreased level in 18:1 and 22:1, indicating 18:1 was inhibited to form TAG via acyl-CoA-independent way, but other genes need to be modulated for allocating more 18:1 into the Kennedy pathway. Down regulation of LPCAT and PDCT simultaneously resulted in an increased level in 18:1 and the decreased 22:1 content, indicating the blocking of the two genes would enlarge the 18:1 pool size which is a prerequisite to approach higher 22:1 levels. This result is interesting because increased 18:1 pool should in theory lead to unchanged or increased 22:1. We assume that the inhibition of the two genes might employ other synthesis pathways and led to pleiotropic effects on lipid metabolism. Retransformation or crossing with existing transgenic lines with high erucic acid (73%) obtained from previous studies is expected to achieve ultra-higher erucic acid contents (over 80%).

Supervisors: Li-Hua Zhu & Rui Guan
Master´s Degree Project 60 credits in Plant Biology 2012
Department of Plant Breeding and Biotechnology, Swedish University of Agricultural Sciences, Alnarp. (Less)
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author
Wang, Danni
supervisor
organization
course
MOBT16 20112
year
type
H2 - Master's Degree (Two Years)
subject
language
English
id
3633582
date added to LUP
2013-04-16 14:15:49
date last changed
2013-04-16 14:15:49
@misc{3633582,
  abstract     = {Abstract

Replacing fossil oil with renewable resources perhaps is the most urgent challenge that the human being now is facing. Bioenergy has been considered as one of the best alternatives to fossil oil. Diverse non-edible applications of plant oil have been explored in the chemical industry recent years. However, both quantity and quality need to be improved from different oil crops for diverse industrial uses. Genetic engineering offers a good opportunity for improving plant oil qualities and quantities in a more precise and efficient way compared with the conventional breeding. Crambe abyssinica is regarded to be an important oil seed crop as it contains a high proportion of erucic acid (22:1) in its seeds, which has extensive applications for industrial purposes. In this study, genetic engineering was employed to develop transgenic lines with modified oil composition for evaluating the important enzymes involved in the erucic acid biosynthesis. The integrations of the PDAT-RNAi and LPCAT-PDCT-RNAi genes into the crambe genome were confirmed by PCR analysis, Southern blot and qRT-PCR. In transgenic line with PDAT-RNAi, the average level of 18:1 was decreased while 18:2 was increased compared with non-transgenic control, indicating more 18:1 fluxed into 18:2. In transgenic line with LPCAT-PDCT-RNAi, the obvious decrease of 18:2 and increase of 18:1 were observed while 22:1 decreased compared with control group. The results facilitate the better understanding of acyl-CoA independent pathway of seed oil biosynthesis and provide new perspectives on how to improve the 22:1 content. 

Popular science summary:

 Insights into erucic acid biosynthesis in Crambe abyssinica 

Crambe abyssinica is considered to be an important oilseed crop for industrial oil production. Crambe contains55-60%of erucicacidin its seed oil. Erucicacidhas extensive industrial applications, especially widely used as a slipping agent in plastic production for better separation of plastics. It is estimated that the purification cost of erucic acid from other fatty acids is to be halved by each 10 % increase of erucic acid in its seed oil. However, the biosynthesispathways of erucic acid have not been elucidated clearly in crambe. 

Fatty acids are converted into triacylglycerol (TAG), commonly called storage or seed oil mainly through the Kenney pathway, but also through the acyl-CoA-independent pathway. Erucic acid (22:1) is a very long chain fatty acid and is elongated from 18:1-CoA through Kennedy pathway. However, the acyl-CoA-independent pathwaycouldpresumablydecrease the22:1 content bydiminishingthe 18:1-CoA pool.Thus, blocking the acyl-CoA-independent pathway could possibly increase the erucic acid content. Three genes named PDAT, PDCT and LPCAT have been identified in this pathway. In this project, we evaluated the functions of these three genes on the pathway of erucic acid biosynthesisthrough using the RNA interference technology. This will help us to further increase erucic acid contents through modification of these genes in combination with other important genes in the future. 

Two gene constructs containing either PDAT-RNAi or LPCAT-PDCT-RNAi were introduced into the crambe genome through Agrobacterium-mediated transformation to down regulate the corresponding enzymes. Transgenic lines were confirmed by PCR and Southern blot analyses. The fatty acid composition and erucic acid contents in crambe transgenic lines were analyzed by gas chromatography. Transgenic lines with down-regulated PDAT and LPCAT-PDCT genes exhibited drastic changes in the fatty acid composition. Down regulation of PDAT led to a decreased level in 18:1 and 22:1, indicating 18:1 was inhibited to form TAG via acyl-CoA-independent way, but other genes need to be modulated for allocating more 18:1 into the Kennedy pathway. Down regulation of LPCAT and PDCT simultaneously resulted in an increased level in 18:1 and the decreased 22:1 content, indicating the blocking of the two genes would enlarge the 18:1 pool size which is a prerequisite to approach higher 22:1 levels. This result is interesting because increased 18:1 pool should in theory lead to unchanged or increased 22:1. We assume that the inhibition of the two genes might employ other synthesis pathways and led to pleiotropic effects on lipid metabolism. Retransformation or crossing with existing transgenic lines with high erucic acid (73%) obtained from previous studies is expected to achieve ultra-higher erucic acid contents (over 80%). 

Supervisors: Li-Hua Zhu & Rui Guan 
Master´s Degree Project 60 credits in Plant Biology 2012 
Department of Plant Breeding and Biotechnology, Swedish University of Agricultural Sciences, Alnarp.},
  author       = {Wang, Danni},
  language     = {eng},
  note         = {Student Paper},
  title        = {Genetic modification of Crambe abyssinica for better understanding of the biosynthesis pathways of erucic acid},
  year         = {2012},
}