J Integr Plant Biol. ›› 1989, Vol. 31 ›› Issue (8): -.

• Research Articles •    

Isolation of Dual Promoter and Research on the Optimal Construction of Chimaeric Genes

Zhu Qun, Bai Yong-yan and Loo Shi-wei   

Abstract: Agrobacterium tumefaciens induces crown gall tumout formation on many dicotyledonous plants. Tumour formation is accompanied by transfer and integration of a specific part of the tumout inducing (Ti) plasmid into the nuclei of the host cells. This part of the Ti plasmid is termed T region in tile bacteria and T-DNA in the plant cells. Crown gall tissues synthesize unusual T-DNA specific compounds known as opines. The Ti plasmids are classified according to the types of opine. The octopine and nopaline Ti plasmids have been studied most extensively. Apart from TL-DNA, TR-DNA of octopine Ti plasmid can be expressed in transformed plant cells. Transcription analysis indicated that there are five transcripts mapped to the TR-DNA, of the five transcripts, three of them including the most abundant mRNA (1′ and 2′) have been linked with mannopine production. The 1′ (1.6 kb) and 2′ (1.45 kb) transcripts code for the second and first enzymes in the manopine biosynthetic pathway respectively, experiments show that the 1′ and 2′ mRNA levels are significantely greater than that of the OCS transcript which is the most abundant one among TL-DNA transcripts. The two mRNAs are divergently transcribed and both originate within about 500 bp DNA segment of the TR-DNA. From pAR17, a clone carrying a 1.5 kb fragment of TR-DNA, we subcloned a 490 bp fragment. It contains two promoters located in diverse orientation and ended just at ATG codon of 2′ promoter (the ATG codon is abolished) and several bp from 1′ ATG codon. There are several restriction endonuclease sites in the recombinant plasmids pBZ741 and pBZ742 which are pWR13 carrying the 490 bp fragment in different orientation. DNA sequencing shows that the promoters of gene 1′ and gene 2′ contain typical TATA boxes, but no ideal CAAT box. We have modified pBZ741 and pBZ742 to get pBZ741 m and pBZ742 m respectively, in pBZ741m, EcoRl site is just at ATG codon position of 2′ promoter; in pBZ742m, BamHl site is just at ATG codon. DNA sequencing confirmed the structures. In order to get improved gene expression, we constructed three different firefly luciferase chimeric genes: pBZ7610: promoter 2′+ luciferase structure gene+Nos terminator. pBZ81tl: promoter 2′ (modified) + luciferase structure gene +Nos terminator. pBZ8110: promoter 2′ (modified) +luciferase structure gene + Nos terminator (in reverse orientation). We set up three intermediate vectors pBZ7621:pBZ8117 and pBZ8116 by insertion of tmr gene of T-DNA into pBZ7610, pBZ8lll and pBZ8110 respectively. We incorporated pP, Z7621 pBZ8117 and pBZ8ll6 into plant genetic engineering vector pGV3850 individualy to get pBZ7629. pBZ8127 and pBZ8126. These three chimaeric genes have been transfered into tobacco plant. Results show that three genes can be expressed in transformed tobacco plants. The luciferase activities in terms of light production are as following: pBZ7629, 2.6×10–9; pBZS127, 3.8× 10–9; pBZ8126, 8.1× 10–10, These results suggest that both 5′ promoter and 3′ terminator are important for the chimeric gene expression in transformed plant. The 3′ end structure which we used is an 1.2 kb fragment of the T'DNA of the pTiC58. It contains the 5′ upstream structure of gene 613, so the reduced expression of luciferase chimeric gene carrying the 1.2 kb fragment in reverse orientation may be effect of a special siliencer element of 6b gene, it is worth to study further. The presumed translation start codon of luciferase structure gene used is 81 bp downstream from the fusion site. The modified dual promoter has a stronger activity in promotion of luciferase gene expression which is 1.5 times higher than that by normal dual promoter, it is possible that there are another TATA box and transcription start site of insect gene in the 81 bp fragment. This suggests that the distance between the promoter and gene translation initiation codon is very important for plant gene expression.

Key words: Dual promoter, Chimaeric gene, Luciferase, Intermediate vector, Trans- formation, Gene expression

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