Identification of cis-elements and evaluation of upstream regulatory region of a rice anther-specific gene, OSIPP3, conferring pollen-specific expression in Oryza sativa (L.) ssp indica

Promoters comprise of various cis-regulatory elements which control development and physiology of plants by regulating gene expression. To understand the promoter specificity and also identification of functional cis-acting elements, progressive 50 deletion analysis of the promoter fragments is widely used. We have evaluated the activity of regulatory elements of 50 promoter deletion sequences of anther-specific gene OSIPP3, viz. OSIPP3D1 (1504 bp), OSIPP3-D2 (968 bp), OSIPP3-D3 (388 bp) and OSIPP3-D4 (286 bp) through the expression of transgene GUS in rice. In silico analysis of 1504-bp sequence harboring different copy number of cis-acting regulatory elements such as POLLENLELAT52, GTGANTG10, enhancer element of LAT52 and LAT56 indicated thatthey were essential for high level of expression in pollen. Histochemical GUS analysis of the transgenic plants revealed that 1504-and 968-bp fragments directed GUS expression in roots and anthers, while the 388-and 286-bp fragments restricted the GUS expression to only pollen, of which 388 bp conferred strong GUS expression. Further, GUS staining analysis of different panicle development stages (P1-P6) confirmed that the GUS gene was preferentially expressed only at P6 stage (late pollen stage). The qRT-PCR analysis of GUS transcript revealed 23-fold higher expression of GUS transcript in OSIPP3-D1 followed by OSIPP3-D2 (eightfold) and OSIPP3-D3 (threefold) when compared to OSIPP3-D4. Based on our results, we proposed that among the two smaller fragments, the 388-bp upstream regulatory region could be considered as a promising candidate for pollen-specific expression of agronomically important transgenes in rice.