Functional Characterization of Major Regulators of Lipid Accumulation in Camelina sativa, a Promising Bioenergy Crop.

Cooper Kuess & Dr. Haiying Liang

Abstract

Camelina sativa is an oilseed crop capable of producing vegetable oil. The species has high oil production that can be used for a variety of commercial practices such as biofuel. Additionally, C. sativa has a short life cycle, produces many seeds, can be readily transformed, and has an already mapped genome. Ultimately, these factors make C. sativa a promising species for lipid metabolism research in addition to its economic values. This summer’s research focused on targeting four transcription factors ( LEC1, LEC2, ABI3, and FUS3) using an RNA interfering approach to create transgenic plants. Using Agrobacterium, the RNAi constructs can ultimately be inserted into C. sativa’s genome to create transgenic plants with target gene down-regulated. In the future, transgenic plants will ultimately be grown and characterized to analyze the loss-of-function effect of the above-mentioned transcription factors on lipid accumulation and metabolism.

Background Information

RNAi is a mechanism by which double-stranded RNA segments interfere with mRNA by splicing RNA segments apart. The sense and antisense sequences used in this study bind to form double-stranded RNAi. Dicer proteins will then cut RNAi, allowing an RISC complex to form. The RISC complex will then target mRNA of interest, resulting in cleavage of mRNA and inhibition of translation. The RNAi  mechanism is illustrated in Figure. 1.

Fig. 1. RNAi mechanism.

Materials and Methods

  1. Amplify target sequences from C. sativa genomic DNA using PCR and insert into TOPO pCR2.1 plasmid (Fig. 2).
  2. Digest insert from TOPO pCR2.1, and place into the second plasmid  pBS-KAN, which is used to hold the sense and antisense inserts in place. The XhoI site was used for the sense sequence while ClaI was used for the antisense (with the FUS3 antisense using  HindIII).
  3. Digest sense and antisense sequences along with a 35S promoter and a terminator from an octopine synthase gene from pBS-KAN with Pstl and SacI and ligate into the final pCAMBIA plasmid, a  vector that can be used for Agrobacterium-mediated transformation of C. sativa.
  4. Transform C. sativa plants with RNAi constructs.

 

Fig. 2. TOPO plasmid used for cloning and duplicating sequences. 

Fig. 3. pBS-KAN plasmid used for holding sense and antisense sequences in place. 

Fig. 4. pCAMBIA plasmid used for Agrobacterium transformation. 

Results

All sense and antisense sequences have been cloned into TOPO pCR2.1. The sense and antisense of FUS3 are currently in pBAS-KAN, ready for pCAM2301 cloning. The cloning of sense sequence of LEC1, LEC2, and ABI3 into pBS-KAn has been complete. The cloning progress is shown in Table 1.

Table 1. RNAi cloning progress of various genes. +: sense sequence; -: antisense sequence.

TOPO pCR2.1 pBS-KAN
FUS3 +- +-
LEC1 +- +
LEC2 +- +
ABI3 +- +

Fig. 5. Amplified target gene sequences from C. sativa genomic DNA. 

Fig. 6. PCR confirmation of presence of insert in TOPO pCR2.1 with M13 primers.

Fig. 7. PCR confirmation of presence of insert in pBS-KAN with gene-specific primers.

Future Work

Future work will focus on getting each gene’s sense and antisense sequence into pCAMBIA plasmids in preparation for final transformation into Camellia sativa. Upon transformation, transgenic plants will be analyzed for differences in lifecycles, growth, and lipid characteristics.