The Construction Of Expression Vector Of Three Genes And CDNA Library Of Grimmia Pilifera Under Rehydration

Drought is the major environmental factor that influences the growth of plant, theinhibition of plants growth or even death can be caused by lack of moisture. So the study ofthe mechanism of the drought-tolerance plant and the application of drought-resistant geneshas become the hotspots in our society. In this experiment, Grimmia pilifera which isbelonging to the Grimmiaceae was used as material, and the function of its three genes whichrelated to drought-resistance were studied, and a cDNA library were constructed which basedon Grimmia pilifera rehydrated by12h.(1) Ascorbate peroxidase gene (GpAPX), Cys peroxiredoxins (GpPER) and ubiquitin-likeproteins (GpUBP) cDNA were obtained from total RNA extracted from Grimmia pilifera byRT-PCR; and the recombination expression vector pRI101-GpAPX、pRI101-GpPER andpRI101-GpUBP were constructed; the recombination plasmid was transformed into tobaccoby Agrobacterium-mediated genetic transformation.(2) The bioinformatics and expression analysis of GpUBP gene of Grimmia pilifera;(3) The Grimmia pilifera were treated by different rehydrated time. The results showedthat the timepoint of the highest content of free proline occurred are at the12thhour.(4) The cDNA library was constructed based on SMART technology. The Grimmiapilifera was rehydrated by12h, and the titer of the library was3.78×106pfu/mL, PCRamplification showed that the insert cDNA fragments mostly arranged from500-2500bp, withan average length of700bp, the recombinant rate was93.7%. The number of successfulsequencing clones was1001which including971optimal genes.432unigenes wete obtainedincluding96contigs and336singlets. The unigenes can be divided into16kinds according toGO assignments: cell (13.86%), extracellular region (0.37%), organelle (10.41%), proteincomplex (9.41%), antioxidant activity (0.87%), binding (9.9%), catalytic activity (9.16%),enzyme regulator activity (0.12%), signal transducer activity (0.24%), structural moleculeactivity (7.92%), transcription regulator activity (0.37%), translation regulator activity(0.12%), transporter activity (1.86%), cellular process (15.22%), development (0.12%),physiological process (18.07%), regulation of biological process (0.99%), response tostimulus (0.99%).(5)Genes of Grimmia pilifera cDNA library which related to the plant’s droughtresistance were contained with the ability of maintaining, biological activities under thecondition of extreme drought. It lays a foundation for the further research of the molecular mechanism of Grimmia pilifera development and the utilization of the drought-resistantgenetic.