A Preliminary Study On Two Genes Encoding Rna-binding Protein In Malus Prunifolia

Drought stress has been becoming one of the most important abiotic stresses that affectplant growth and agricultural production today. China has almost the biggest cultivation andproduction. However, most of apple cultivation in our country is in arid and semi-arid areas,which are often influenced by water stress. So studying the response mechanism to droughtstress of apple is of important theoretical guidance for actual production.The responses of plants to the drought are multi-level, including differences in geneexpression, physiological and biochemical reactions and visually anatomical andmorphological changes. Based on the gene encoding product, these genes can be divided totwo classes, one type is functional genes, and another is regulatory genes. Genes encodingRNA-binding protein are one type of regulatory genes.This experiment was mainly based on two genes in Malus prunifolia（Willd）Borkh. leafcDNA library. MpGR-RBP was a full length gene and had a ORF of516bp. The experimentwas conducted to construct the gene over-expression vector, and transforme it into a modeltomato plant Micro-Tom via Agrobacterium-mediated transformation. MpYTH is anapproximately1400bp fragment in the library. In this experiment we analysed the differentialgene expression in drought and NaCl treatment and designed primiers to get full-lengthsequence, and finally performed subcellular localization and some structural analysis. Themain results are as follows:1. MpGR-RBP1gene with restriction enzymes site Sal I and Xba I was cut from thecloning vector pMD-19T-simple, and was directionally inserted into the plant expressionvector pWR306which carry the GUS reporter gene and the hygromycin marker gene. Thenthe plant over-expression vector pWR-GR-RBP was successfully constructed.2. Based on tomato Micro-Tom regeneration system and hygromycin screening systemestablished by our laboratory, the MpGR-RBP1was transformed into tomato Micro-Tom viaAgrobacterium-mediated transformation. After hygromycin selection and GUS staining, threeof25seedlings are considered to be transgenic plants. PCR detection is still in progress.3. Based on the fragment in the cDNA library and apple genome prediction of ORFsequence, we designed primers YTHF5’ ATG GCG ACC ACC GTT TCC3’，YTHR5’ TTT TGT TAC AAT CAT CTT GGT CAC AGC3’ to get MpYTH gene full-length sequence with a2034bp open reading frame which encoding677amino acids. The theoretical molecularweight is73.9KD and theoretical isoelectric point is8.15. The encoding protein contains aYT521-B homology domain, and its secondary structure prediction contains six α-helices andfive β-sheets, so it is a mixed protein.4. The subcellular localization vector of MpYTH was constructed using pBI-221vectorcarrying the GFP protein. The subcellular localization vector was introduced into onionepidermal cells using particle bombardment. The fluorescent microscope observation showedthat GFP fusion protein exists only in the nucleus and therefore MpYTH gene is located in thenucleus of the cell.5. RNA was extracted from Malus prunifolia（Willd）Borkh leaves after drought andNaCl treatment. According to sequence the primers for the quantitative Real-Time PCR weredesigned. The results showed that MpYTH expression levels gradually increased underdrought treatment. The maximum occurred on the fourth day, and it was almost59-foldgreater than the control. With the aggravation of drought stress, the expression levelsgradually declined. After NaCl treatment the MpYTH expression level gradually increased,and peaked on the fourth hour, and it was almost10-fold greater than the control. After asharp decline on the sixth hour, it rose again on the eighth hour and then gradually declined.