Improving Foreign Gene Expression Through Ubil Intron Modification In Monocotyledons

Intron-mediated enhancement (IME) of gene expression in plants was first demonstrated in1987.Ithas being payed attention to its function of gene expression. IME has been observed in a wide range ofeukaryotes, including vertebrates, invertebrates, fungi, and plants. Many introns have been found inenhancement of gene expression.The mechanism of IME is largely unknown. Although the underlying mechanisms have not beenidentified, several features are known.The enhancement of gene expression is different in variant introns.There are many factors relate to the mechanism, such as the direction and position of intron, its own sequence,and the flake sequence of exon and so on.It has been demonstrated that a construct containing the ubi1intron was five-fold higher thanthat of the intronless control construct in maize calli. To address the mechanisms of intron-mediatedenhancement, we used reporter gene fusions to identify features of the ubi1first intron required forenhancement in maize calli.In this research, parts of sequence were deleted to analysis potential different enhancement. Eightplant transient expression vectors-pSG(13i-P1)N、pSG(13i-P2)N、pSG(13i-P3)N、pSG(13i-P4)N、pSG(13i-P5)N、pSG(13i-P6)N、pSG(13i-A)N and pSG(13i-B)N containing eight different deletion ofubi1sequence. GUS activity in maize calli bombarded with these constructs was detected. The resultshowed that the deletions in5’ terminal were enhancer than3’ terminal, in other words, the3’ end ismore important than5’ end. A142-bp deletion (P5sequence) conferred approximately the same20to50folds stimulation typical for the full-length intron in this transient expression system. Then weconstructed a series of intron mutations based on P5sequence. We got some base mutant to the motifTCGATC at the11bp、78bp、282bp and510bp. Seven mutation construct pSG(13i-M1)N (orpSG(13i-M78)N)、pSG(13i-M2)N、pSG(13i-M3)N、pSG(13i-M5)N pSG(13i-M11)N、pSG(13i-M282)Nand pSG(13i-M510)N containing different position and number of mutations. GUS activity in maizecalli showed that most of the vectors reduce the enhancement of reporter gene but thepSG(13i-M5)N.So we got the P5and M5artificial introns to stimulate gene more expression thanfull-length ubi1.We tried to get a general vector by inserting the P5or M5intron to enhance exogenous geneexpression in monocots. These vectors including pSAiGN(full-length ubi1)、pSAP5GN、pSAM5GN、pSA12iGN、pSA12P5GN and pSA12M5GN were constructed. GUS activity in maize and rice callibombarded was detected. The results showed these vectors are all play an important role inenhancement of gene expression, at the same time we found the enhancement is conservative inmonocots.