Cloning of orfH522 from CMS Sunflower (Helianthus annuzis L.) for Anther Specific Expression
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Date
2004
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Professor Jayashankar Telangana State Agricultural University
Abstract
The present investigation was carried out with the aim of developing plant transformation vectors suitable for induction of male sterility in crop plants. This research intended to clone a mitochondrial, male stedhy inducing gene orfl522 from PET1 CMS line of sunflower under a tapetum specific promoter, TA29 promoter fiom tobacco in fiame with a mitochondria1 target peptide coxIV pre-sequence fiom yeast, so that ORFH522 would be targeted into mitochondria in the target plants, evethough the protein is synthesised as a nuclear encoded protein in the transgenic plants. nos terminator fiom Agrobacterium was proposed to be used as termination signal in the gene cassette. As appropriate controls gene cassettes were developed where orfH522 was cloned under a constitutive promoter, CaMV 35s in frame with or without mitochondria1 target sequence so that the effect of expressing ORFH522 constitutively could be determined in comparison to tapeturn specific expression. The development of the proposed gene cassettes was carried out using a three step strategy. In the first step, the component sequences TA29 promoter fkom tobacco, coxIV pre-sequence from yeast, 0@22 fiom sunflower and terminator sequence fiom Agrobacterium were amplified hm their respective sources using appropriate PCR techniques and cloned in TIA cloning vector. The authenticity of the protein encoding . sequences (coxIV preaequence and or-522) was confirmed by sequencing. In the second step, these sequences were cloned in plasmid vector pUC18 in appropriate/correct orientation by following serial directional cloning using the restriction enzymes sites incorporated in forward and reverse primers employed for amplifying each of the component sequences in the first step. The orientation of cloning of the component sequences in pUC18 was confirmed by appropriate restriction digestion analysis using cloning enzymes and PCR analysis using component sequence specific primers. Also, these cassettes were completely sequenced to obtain the ultimate level of confirmation of orientation of cloning and genetic 'information of the gene cassettes. In the third step, these cassettes were excised out fiom pUC18 vectors and were cloned in binary vector pCAMBIA 1305.2. The cloning was confirmed hy restriction digestions. orfH522 with or without coxIV pre-sequence was also cloned under CaMV 35s promoter. For this, orfl522, 'coxN presequ&ce-orjH522' were amplified and cloned in TIA cloning vector and later cloned in pRTlOO (which has CaMV 35s promoter and CaMV poly A signal flanking the MCS) with appropriate restriction enzymes. The two gene cassettes were excised out from pRTlOO with Hindu1 and cloned in pCAMBIA 1305.2 :. Thus, in the present investigation four gene cassettes were developed which when successfully introduced into crop plants, could induce male sterility. These constructs could unequivocally establish the role of om522 in causing male sterility in PET1 CMS line of sunflower.
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