Cloning And Function Analysis Of Promoter Of A Zinc-finger Gene Preferentially Expressed In Stem And Leaf In Rice

The temporal and spatial expressions of genes are depended on the cooperative interactions with the cis-elements of the promoter and the corresponding transcriptional factors. It is useful, that an appropriate promoter (such as composed,tissue-specific and inducible promoter) is selected or constructed,for studying biological function and regulatory mechanism of a new gene and for producing some useful proteins or other metabolites at the special positions or the special development phases of the plant and really realizing the three-dimensionally precise regulation of the transgenes in target gene expression time ,position and quantity. Promoters of some genes specially-expressed in rice stem and leaf might be important in rice transgenes for diseases and pests resistance, because they were only expressed in (partly) vegetal organs and not in rice seed, especially edible part of seed.l.By RT-PCR technique, expression patterns of 354 zinc finger genes were analysed during ripening phase in rice in our lab.The results were found that only 170 genes were detected products of gene expression. It was found that some zinc finger genes were specially transcribed in seed, stem, leaf or/and root. We selected one zinc finger gene ZF153 expressed specially in rice stem and leaf for further study.2．On the basis of the cloning of ZF153 gene and its promoter, the sequence of the ZF153 promoter was analyzed by PLACE. A lot of kept sequences of the high plant promoters were found, it is implied that this sequence has potential promoter function. Furthermore, A lot of specially elements were also found in this sequence, such as YACT-a key component of Mem 1 (mesophyll expression module 1) found in the cis-regulatory element in the distal region of the phosphoenolpyruvate carboxylase (ppcA1) of the C4 dicot F.It is implied that expression of the gene would be regulated specially by this sequence.3．The expression pattern of ZF153 was analysed again in different tissues (organs) and seed development stages in rice by RT-PCR. To elucidate the transcription pattern and biologica function of the gene ZF153, we cloned and constructed fusing vector pC153P-GUS with promoter sequence of the gene and coding sequence of GUS gene,while cDNA sequence of ZF153 was cloned to construct sense and anti-sense expression vectors pCA153gS and pCA153gA, respectively.Rice transgenic plants of the vectors pC153P-GUS and pCA153gS above were generated by Agrobacterium-mediated. PCR tests of marker gene Hyg (hygromycin) indicated the recombinant segments of pC153P-GUS and pCA153gS had been integrated into rice genome. Detection of the GUS activity showed that GUS of pC153P-GUS transgenic plants was only expressed in stem, leaf, hull and root. Activation of GUS was very high in stem and leaf, and weak in hull and root of rice.While activation of GUS was not detected in rice stamen, pistill, and embryo and endosperm of seed. It was implied that ZF153 might be a zinc finger gene that were preferentially expression in stem and leaf and its promoter may be useful for rice transgene for diseases and pests resistance in rice.