Gene Cloning and Characterization - Genetics, Genomics and Breeding of Crop Plants - page 202

with a higher affinity for saturated acyl-ACPs. During sunflower seed
development, both FatA and FatB thioesterases have been shown to be
expressed (Martinez-Force et al. 2000). For cloning the sunflower thioesterase
gene FatA , cDNA from developing sunflower endosperm was prepared
(Serrano-Vega et al. 2005). FatA protein sequences from public databases
were aligned to identify regions of homology using the CLUSTAL v1.8
program (Thompson et al 1997). A PCR fragment was amplified with two
degenerate primers designed from two highly conserved regions of FatA:
FatA1 (5'-GARATHTAYARRTAYCCNGC-3') and FatAB (5'-CAYTTCNCKN
CKRTANTC-3'). Using these primers, a 459 bp fragment from developing
sunflower seed cDNA was amplified by PCR, corresponding to the internal
part of a FatA mRNA. Subsequently, the full-length HaFatA1 cDNA clone of
1,095 bp was obtained by RACE using additional primers (Serrano-Vega et
al. 2005). The full-length cDNA was predicted to generate a pre-protein of
365 amino acids, with a molecular mass of 41.2 kDa. Functional studies of
this gene were performed in E. coli (Serrano-Vega et al. 2005).

6.3.5.2 Cloning Genes Involved in Tocopherol Biosynthesis in
Sunflower

Tocopherols are the most powerful natural fat-soluble antioxidants. The
four naturally occurring tocopherols ( -tocopherol) widely

differ in their relative in vivo (Vitamin E) and in vitro antioxidant properties
(Sattler et al. 2004). Sunflower seeds (wild type) normally accumulate 92-
98%

-, -, -, and

-tocopherol.
Demurin (1993) postulated two non-allelic unlinked genes, Tph1 and
Tph2 , controlling tocopherol composition in sunflower seeds. Tph1 gene
controls the ratio of -, and -tocopherol, and Tph2 gene affects that of the

-homologues. Recessive alleles were found by wide-scale screening
and selfing of spontaneous mutations. Vera-Ruiz et al. (2006) mapped the
Tph1 gene by SSR markers (Tang et al. 2002) and developed a linkage map of
the Tph1 -encompassing region. The Tph1 was mapped to the upper end of
the LG 1. The two map-based markers linked to this gene can be used to
assist selection for increased

- and

-tocopherol content.
Three mutant loci (m = Tph1 , g = Tph2 , and d ) have been shown to
disrupt the synthesis of -T) and produce novel tocopherol

(vitamin E) profiles in sunflower seeds (Tang et al. 2006). The m ( Tph1 )
mutation partially disrupts the synthesis of

-tocopherol (

-tocopherol (vitamin E) in
sunflower seeds and was predicted to disrupt a methyltransferase activity
necessary for synthesis of -tocopherol. A candidate gene approach

was undertaken to isolate two corresponding genes for sunflower, MT-1
and MT-2 , encoding for two 2-methyl-6-phytyl-1,4-benzoquinone/2-methyl-
6-solanyl-1,4-benzoquinone methyltransferase paralogs (MPBQ/ MSBQ-

- and