Establishment Of Two-dimensional Gel Electrophoresis Of Total Proteins From Kenaf Leaves And Leaf Proteomic Analysis Of Kenaf Cytoplasmic Male Sterility And Maintainer Lines

Kenaf(Hibiscus cannabinus L.),which belongs to the Malvaceae,is a short-day, annual, herbaceous plant cultivated for the soft bast fiber in the stem. It has the advantages of excellent environment-stress tolerance, wide adaptability,high fiber yield and good quality.In this reseach, we have selected kenaf CMS line L23A and maintainer L23B as experimental materials, and have used functional leaves surrounding the flower of three different periods ,such as young bud, large bud, blossom.We have got the differences in physiological and biochemical indicators. In order to investigate the biological function of special proteins,reveal the molecular mechnisms of infertility, adaptability in kenaf and increase the promotion of kenaf .The main results of this study are as follows:1.We have got proline content between maintainer line L23B and CMS line L23A in young bud, large bud, blossom three different periods . Proline at different periods are highly variable.The highest lies in young bud, and CMS line L23A higher than the maintainer line L23B.Soluble sugar at the three times is basicly stable , and maintainer line L23B is slightly higher than CMS lines L23A. Soluble protein content of maintainer line L23B is slightly higher than CMS lines L23A.2.We have established protein extraction system from kenaf leaves ,TCA-acetone extraction from kenaf leaves is more efficient than the method of phenol extraction and the protein spots were significantly increased. We have got more different points after we added thiourea to the lysis buffer .We stained 2-D gel with Coomassie Brilliant Blue G-250,the R-250 and silver ,and we have found Coomassie Brilliant Blue G-250 is the best, although the silver stain is sensitivethe , it ,s reproducibility is low.In the end ,we have chosen 300μg as the optimal quantity of sample . At the same time,we have used PDQUEST software to analyze 2-DE gel, we have got better resualt.We have layed experimental basis on kenaf proteomics. 3.We compared different proteins between the maintainer line L23B and CMS line L23A , and we have got 12 protein spots through MALDI-TOF-MS/MS tandem mass spectrometry analysis , of which six protein spots can be identified. Six kenaf infertility-related protein and its functions are as follows: a large subunit of Rubisco, o for all photosynthetic organisms as a key enzyme in photosynthetic carbon assimilation,Two Rubisco activating enzyme, for a wide range exists in the regulation of photosynthetic organisms Rubisco activity of enzymes,a transcription factor; a malate dehydrogenase, involved in the process of citric acid cycle key enzyme, a ribosomal protein, and ribosomal RNA constitute the ribosome.The results obtained in this study could be further use of genetic engineering techniques for the cultivation of kenaf and layed the theoretical basis on infertility.