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The Functional Identification Of A Grapevine Class Ⅲ Chitinase Gene VCH3 Promoter

Posted on:2005-10-07Degree:MasterType:Thesis
Country:ChinaCandidate:W WeiFull Text:PDF
GTID:2120360125952617Subject:Biophysics
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Efficient plant genetic engineering relies on designing appropriate gene expression system by using inducible and tissue-specific promoter to construct compound promoters to drive gene express at the required moment and in the appropriate tissues, and avoid its undesirably overexpression driving by the constitutive promoters. But more efficient and applicable tissue-specific promoters are not so much and the inducible ones are less than tissue-specific promoter. In this thesis, a series of deletion fragments of the Class III chitinase VCH3 promoter isolated from grapevine (Vitis amurensis Rubr.) were produced and studies have been conducted on inducible activitity and tissue-specific expression of VCH3 promoter in transgenic Nicotiana tobacum L cv. NC89. The main results are as follows:1. Four deletion fragments of VCH3 promoter were produced by polymerase chain reaction (PCR), and the constructed deletion fragments::GUS chimera were transferred to Nicotiana tobacum L cv. NV89 by Agrobacterium tumefaciens-mediated leaf discs transformation. The functional properties of each protomer fragment were examined by fluorometric analysis of GUS activity. The results show that the largest promoter fragment(-1187 ~ +7,contains two SA cis-motifs) directed the strongest GUS activity and are two folds equal to the second length fragment in transgenic tobacco roots treated by SA. The similarly higher level of GUS expression was observed in VCH3(-589)GUS and VCH3(-892)GUS transgenic plants with one cw-motif. VCH3(-276)GUS (not containing SA cis-motif) directed the lowest GUS activity, only a little higher than CK.2. After the mutation of two of the SA m-motifs, five deletion fragments of VCH3 promoter were correspondingly produced [VCH3*(-1187 II )GUS contains two mutant SA cw-motifs;VCH3*(-1187 I )GUS have one complete and one mutant SA cis-motif; VCH3*(-5S9)GUS and VCH3*(-892)GUS contain one mutant SA cw-motif; VCH3*(-276)GUS contains no SA cis-motif]. The functional properties of each mutant promoter fragment were also examined by fluorometric analysis of GUS activity, and the results showed that GUS activity directed by VCH3*(-1187I )GUS was almost the same as VCH3(-892)GUS and VCH3(-5S9)GUS which contain one SA cw-motifs. VCH3*(-1187 II)GUS, VCH3*(-892)GUS VCH3*(-589)GUS directed the similar GUS activity. The similarly higher level of GUS expression was observed in VCH3*(-216)GUS and VCH3(-276)GUS. These results indicated that the two SA cis-motifs were required for the maximal expression of the GUS reporter gene by SA induction, the SA-responsive cis-motifs are of functional importance for the expression of GUS induced by SA.3. The histochemical analysis of GUS in the transgenic tobacco roots treated by SA was performed. It was observed that, before and after mutation, the GUS activities were more active in vascular tissue than that in outer and inner cortexes in both the cross and longitudinal sections of transgenic tobacco roots treated by SA. TheGUS activity of the full-length promoter (-1187 ~ +7) with two SA cis-motifs is the most active among all the fragments. VCH3*(-1187 I )GUS ,VCH3(-589)GUS and VCH3(-892)GUS with one SA cis-acting motif are less active than VCH3(-1187)GUS. While the VCH3*(-5S9)GUS, VCH3*(-S92)GUS, VCH3*(-1187 II)GUS with mutant SA cis-acting motifs and VCH3(-276)GUS, VCH3(-276*)GUS with no SA cis-acting motif are less active than the fragments with one SA cis-motif. These results suggesting that VCH3 promoter is inducible highly expressed by SA in vascular tissue and the region involved in vascular tissue-specific expression of VCH3 promoter upon SA inducibility appears to be located between positions -276bp and +7bp relative to the transcriptional start site.
Keywords/Search Tags:VCH3 promoter, salicylic acid, cis-acting motif, vascular tissue-specific expression
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