Cloning And Construction Of Rna Interfere Vector Of Ice Recrystallization Inhibition Gene Form Lolium Perenne

Plant cold-stress associate-proteins are classified as special proteins participate in frost-resistant life events, in which ones in regulation of plant physiological and biochemical activities and against cold environmental stress,it is called antifreeze protein. Antifreeze proteins binding to the surface of ice crystals and prevent ice crystal growth, non-reducing point other than the freezing point,so it creates the gap between the melting point and freezing point, and that is charactered as thermal hysteresis activity.It control the growth of ice crystals and inhibit ice recrystallization, compared with insects and fishes antifreeze proteins, plant antifreeze proteins have lower thermal hysteresis activity, but they have a very high recrystallization inhibition activity, so plant antifreeze proteins also be called recrystallization inhibition proteins in the research.Recrystallization inhibition activities of plant antifreeze proteins are different among various species, and the homology between different sequences is very low. Although the researchs about ice recrystallization inhibition proteins have done in vitro,and made a number of models to explain its mechanism, but so far still have not been scientifically confirmed. The major works of this paper is about ryegrass because of its special properties and it have heavily attract me,the content are list as follows:1. The cloning of ryegrass recrystallization inhibition proteins gene family.First,i get the informations about ice recrystallization inhibition Proteins gene family from the Genbank,designed four pairs primers and cloned the special sequences from the DNA templates and cDNAtemplates.According to the analysis of alignment of sequences,we find the LpIRIP sequences may proceeded splicing post of the transcripting,and there maybe exist intron in the inner of IRIP genes.2. The research about expression of IRIP genes in E.coli. First,we succeed in construction of the four IRIP genes prokaryotic expression vector,subclone the genes coding sequences into pET-28a and pET-30a vectors.Next,after the identification of restriction enzyme digestion, transformed the vectors into the expression cell BL32 (λDE3). At the concentration of 1mM IPTG induced,the recombination pET-30a-IRIP3 successfully express the target protein,it is about 26kDa.3. The construction of Ryegrass binary silence vector. First, we design special primers according to conserved region by the result of alignment among four IRIP genes sequences and antifreeze protein LpAFP(AJ277399). Next,we clone the slience target sequences, and load the fragments into the RNA interfere intermediate vector pBlueActin, and then transform them into binary vector pCAMBIA2301,made the plant slience binary vectors.And after that works,transfer the binary silencing vector into agrobacterium LBA4404.