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Analysis of the Molecular Motif for Inducing Response to Ethylene and Jasmonic Acid in Pib Promoter Via Rice Transformation

Author: YuLi
Tutor: YangShiHu
School: Nanjing Agricultural College
Course: Crop genetics
Keywords: Rice Pib gene Promoter gus Ethylene Jasmonic acid
CLC: S511
Type: Master's thesis
Year: 2009
Downloads: 20
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Abstract

Plant gene promoter contains a number of important cis-acting elements, which involved in the regulation of downstream of the corresponding gene expression under the transcription level, is the center of transcriptional regulation and an important aspect of studying the mechanism of gene expression and regulation. The deletion fragment and reporter gene were constructed fusion gene, and transfor it to rice. Then comparison of the expression leve of reporter gene to study the function of promoter elements, and this is an important method to study the promoter structure and function.Pib gene is the first major blast resistant gene cloned from rice using map-based clone technique and belongs to NBS-LRR resistant disease gene family. Previous studies show that the expression of rice blast resistance of Pib gene donor plant induced by ethylene, jasmonic acid, salicylic acid, sodium chloride and dark factors without the induction of pathogen inoculation. the relationship between induced factors such as salicylic acid, sodium chloride and dark etal and Pib promoter elements has been transgenic reportion. Ethylene and jasmonic factors are important signal molecule of plant defense response, which play an important role in the resistance of biological and non-biological factors and the gene regulation in process of plant growth and development. However, whether the ethylene and jasmonic acid induced responsion of Pib gene is the promoter’s responsion, as well as the relationship between ethylene and jasmonic acid-induced factors and molecular components need further experimental verification.For analyzing the acid molecular mechanism of Pib promoter in response to ethylene and jasmonic and determine the necessary sequence region for ethylene and jasmonic acid induction, In this paper, By using transgenic rice plants of 5’deletion pib promoter fragment-gus constructions to study nitial the biological function of pib promoter of ethylene and jasmonic acid-induced response component though transgenic technology platform and the reporter gene system, which it will offer important theoretical basis for gene expression-regulation studies and resistant disease breeding.1. The full length promoter of Pib (-3572～2bp) and three different 5’deletion fragments of Pib promoter (-2692～2bp,-1335～2bp,-761～2bp) were synthesized by PCR and then were substituted for 35S upstream gus in binary plasmid pCAMBIA1301 to construct recombined plasmids of Pib promoter-gus fusions. The recombined plasmids are pNAR901, pNAR902, pNAR903 and pNAR904. All recombined binary vectors were confirmed by restricted enzyme digestion and transferred into Agrobacterium tumefaciens strain EHA101 by freeze-thaw method.2. By Agrobacterium-mediated method, four recombined plasmids of 5’deletion pib promoter fragment-gus constructions pNAR901, pNAR902, pNAR903and pNAR904 were transferred into japonica rice R109. pCAMBIA1301 was also transferred at the same time. By Agrobacterium infection, hygromycin selection, differentiation, strong sprout, pNAR901 transformed plants was 22, pNAR902 transformed plants was 27, pNAR903 transformed plants was20, pNAR904 transformed plants was 30, pCAMBIA1301 transformed plants was 5, PCR analysis revealed that the transform frequency is 74.8%. PCR and Southern blot tests confirmed the integration of foreign gene.3. By using transgenic rice plants of 5’deletion Pib promoter fragment-gus constructions, Quality and quantum analysis of gus activities in transgenic plants at both protein and mRNA levels revealed that promotion activity of full length promoter of Pib (-3572～2bp, pNAR901)was highest in four recombinants and the GUS activities in its transgenic plant organs enhanced obviously at 6h after inducing of ethylene or jasmonic acid. But when the-3572～-2692bp sequence was knocked out from Pib promoter, the deleted Pib promoters showed decreased promotion activity and loss of inducing response to ethylene and jasmonic acid. Comparing the pNAR902(-2692～2bp) with pNAR903(-1335～2bp) and pNAR904(-761～2bp), the disparity in lengths of deleted Pib promoter sequences was more than 2 or 3 times but there was no difference for inducing response to ethylene or jasmonic acid among their transgenic plants. All these experimental results indicated that shared deleted sequences (-3572bp--2692 bp) in the three deleted Pib promoter constructs was the essential region to the inducing response of ethylene and jasmonic acid. Soft ware searching for Pib promoter sequence indicated that there was only one GCCGCC motif at-2722bp of this shared deleted segment in the Pib promoter sequence. Our rice transgenic experimental results confirmed the GCCGCC may be a cis-motif for inducing response of ethylene and jasmonic acid for Pib gene.

Analysis of the Molecular Motif for Inducing Response to Ethylene and Jasmonic Acid in Pib Promoter Via Rice Transformation

Abstract

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