Cloning Of Fructan Biosynthesis Pathway

Vander Meer and coworkers (115) isolated two full size cDNA clones encoding 1-SST and 1-FFT. Both these enzymes were isolated in high purity state and partial amino acid sequence was elucidated. From the amino acid sequence of several peptides, oligonucleotide primers were designed and used to generate fragments of both cDNA and RT-PCR. The PCR fragments were used to screen a tuber specific cDNA library of Helianthus tuberosus from which two complete cDNA encoding 1-sst and 1-fft were isolated. The deduced amino acid sequences of two cDNA were highly similar to each other (61%) and to vacuolar plant invertase especially tomato acid invertase (59%). The homology between 1-SST, 1-FFT and invertase was very high.

The two enzymes appear to be encoded by single-copy genes. Expression studies of 1-sst and 1-fft in Helianthus tuberosus revealed that the steady state mRNA level of 1-sst seems to be higher than that of 1-fft. Both genes were coordinately expressed in the same tissues and during same stage of development. Expression is observed in tissues where fructan synthesis takes place such as tubers and stems.

The functional identity of the two cloned genes was verified by expressing them in transgenic petunia plants. Expression of plant fructosyl transferase genes in plants that donot accumulate fructans can lead to the synthesis of fructans in vivo. Expression of H. tuberosus 1-sst driven by the CaMVc35S

promoter in transgenic petunia plants led to the production of oligofructans GF2 and GF3. The observation that only a limited amount of fructan accumulates in transgenic leaves harbouring either sst gene or combination of sst and fft genes may be related to the presence of other enzymes like invertase competing for sucrose. SST and FFT also compete with hydrolytic enzymes at the level of fructans as invertase can hydrolyze low molecular weight fructans (46).

Although the plants transformed with 35S-fft construct did not accumulate fructans because the oligofructans substrate for 1-FFT are not present in petunia, the transgenic plants did produce a functional 1-FFT enzyme which could synthesize fructans in vitro using GF3 as a substrate (115).

Vander Meer et al (115) combined two chimeric constructs by crossing transgenic 1-sst plants with 1-fft plants. They were successful for the first time to introduce a complete plant fructan synthesizing pathway into petunia which is naturally unable to synthesize fructans resulting in accumulation of high molecular weight fructans in old leaves. Based on the comparison of the predicted amino acid sequence of 1-sst and 1-fft with those of other plant fructosyl transferase genes, they postulated that both plant genes had evolved from invertase gene (115).

7. HIGH LEVEL FRUCTAN ACCUMULATION IN TRANSGENIC SUGAR

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