Field Detection Of Cold Tolerant Transgenic Cassava&Isolation And Identification Of Cold Related Gene From Manihot Esculenta Crantz

Cold stress is one of the severe environmental stresses which limits plant development and growth. The loss caused by cold and freezing injury can be high up to hundreds of billion dollars a year. Cassava (Manihot esculenta) originated in tropics is a cold sensitive crop. It can be used as staple food, animal feed and for producing starch and bioethanol, which maks it to be a potential tropical bioenergy plant. However, cassava is sensitive to cold and freezing, conditiond, only10℃can injure it. Thus, it has significant meaning of improving cold resistance of cassava. The high heterozygosis, long selection time, heavy workload and uncontrollable variation are the disadvantages of traditional breeding for cassava, which is abandoned gradually. Instead of the traditional breeding, modern molecular biological technique is used to transfer genes into cassava to increase the quility of cassava. Former researchers found that CBF (C-repeat Binding factor) can bind with COR (cold-regulated) gene to activate the expression of a series cold resistance gene, and then improve the cold hardiness. In this research, agronomic characteristics and cold resistance physiological indices of transgenic and control cassava were initially observed and determined. Some of the transgenic lines showed cold resistant improvement with no changed phenotype and yeild. And a CBF3homologous gene was cloned from cassava, named as MeCBF1(GenBank accession number: JQ339740). A plant express vector pVKH-35S-MeCBF1-pA was constructed, then transferred into Arabidopsis for genetic transformation study. Realtime fluorescence quantitative PCR (RTFQ PCR) was used for studying the temporal-spatial expression analysis of MeCBFl. The results showed that:1. From both the determinations of all indices and the observations of agronomic characteristics, transgenic lines, except84(2), showed improved cold hardiness and no significant differences with control in yield and phenotype.2. The results of the agronomic characteristics and cold resistance physiological indices of transgenic plants showed that the height and the stem diameter of transgenic lines were lower and thinner than those of control, while the leaf area were larger, the chlorophyll content were higher than control, the solube protein, free proline were higher than control in cold months. Thus, it could be concluded that the cold hardinesses of transgenic plants were improved to some extent.3. A pair of primers was designed according to the genomic information of cassava in website http://www.phytozome.net, with the restriction enzyme cutting site BamH I and Sal I, the CBF homologous gene in cassava was cloned and named MeCBFl. The plant expression vector pVKH-35S-MeCBF1-pA was constructed. 4. Plant expression vector pVKH-35S-MeCBF1-pA was transferred into Agrobacterium tumefaciens, and Arabidopsis by agroinfection.5. MeCBF1gene was sequenced and analyzed by bioinformatics. The result showed that, the length of MeCBF1was1037bp, the Open Reading Frame starts in102bp, stops in798, and total length is696bp, which encods231amino acids. Sequence alignment with CBF genes of other species provided on GenBank showed that the similarity among MeCBFl, HbCBFl in rubber and DREBF1in castor was high up to91%and89%, respectively.6. Cassava plants were treated under4℃, then RNA of root, stem and leaf were extracted at different treated time points:0h,1h,2h,4h,6h,8h. cDNA was obtained by reverse transcription, realtime fluorescence quantitative PCR was used to analyze MeCBF1expression under cold stress. The result showed that, a peak of MeCBF1expression occurred at2h after cold stress, then returned to low level. Moreover, the expression of MeCBF1was different in root, stem and leaf, the maximum expression value of MeCBF1was in stem, then in leaf, and the expression level was lowest in root.