Evaluating promoters for putative root specific expression of transgenes in cassava storage roots

Abstract

Cassava has been recognized as an important crop to more than 600 million people around the world prompting scientists to look for ways to improve the crop’s nutritive qualities, enhance its resistance to diseases and pests; reduce its levels of cyanide toxicity and reduce the crop’s rate of postharvest physiological deterioration (PPD). To develop improved cassava, genetic engineering is being used as a tool to deliver genes of interest directly into farmer preferred varieties. However, only a small number of promoters have been shown to drive transgenes in cassava and none of them has been shown to drive transgenes in the crop in a root specific manner. This necessitates research to reveal and characterize additional promoters. In this work the efficiency of expression of eight promoters; Cauliflower mosaic virus 35S, Patatin from potato, Sporamin from sweet potato, Isoflavone synthase (IFS) from soybean, rol B from Agrobacterium rhizogenes and three promoters that drive the granule bound starch synthase (GBSS) gene; cassava GBSS, potato GBSS and sweetpotato GBSS, to drive expression of the Eschericia coli uidA gene in 60444 cassava variety. The β- glucuronidase (GUS) activity directed by each promoter was assessed using histochemical, 4-Methylumbelliferyl β-D-glucuronide and reverse transcriptase quantitative PCR assays. The ability of patatin to drive a single carotene gene; crt-B carotene was compared with its ability to drive two genes; DXS and crt-B in 60444 and TME7 cassava varieties in field and greenhouse environments. Carotene levels were measured using standard spectrophotometric method. The expression levels for both uidA and the carotene genes were analyzed using GeneStat software. The constitutive 35S control promoter, drove expression in all the plant tissues tested. Patatin and sporamin, when used with their homologous 3’UTR drove significantly higher (P<0.05) GUS activity in storage roots, than that driven by the control 35S promoter and by the other test promoters. When used with its homologous 3’UTR patatin driven expression was significantly higher (P<0.05) than for the patatin construct carrying a heterologous 3’UTR from nopaline synthase (NOS). However, the patatin /NOS 3’ UTR and cassava GBSS / NOS 3’ UTR constructs also directed high transgene activity to cassava storage roots which was not significantly different (P>0.05) from that driven by the 35S control. Activity directed to the storage roots by the other promoters; rol B, potato GBSS, IFS and sweet potato GBSS were significantly lower (P>0.05) than that directed by the 35S. In the stems and leaves, all the promoters directed significantly lower levels of transgene activity (P<0.05) compared to the 35S. The levels of carotenes were significantly higher (P<0.05) when the DXS and crt-B gene were used together than when the crt-B was used alone. In conclusion, patatin, sporamin and cassava GBSS, though not purely root specific, are good promoters for driving transgenes to improve cassava. Additionally, while targeting to raise provitamin A in cassava, a combination of two carotene genes is recommended. Further investigations should be done to identify promoters which are more root specific for cassava.