Genetic Transformation Of A Fusion Gene TPSP For Trehalose Synthesis And Cloning Of A Water Sress Induced Gene TaSNAC1 In Wheat

Drought is a major factor which limited the high and stable yield of wheat,the cloning of drought-resistance related genes and the transgentic study is an important branch of crop molecular breeding for drought tolerance.In this study,based on the constructed plant expression vector,which harboring a fusion gene TPSP for trehalose synthesis,the genetic transformation of several wheat varieties was performed and some transgenic wheat materials were obtained.In addition,based on the gene expression profile in root tissues of wheat under the water stress,a candidate gene TaSNACl,which closely related to the water-stress response and coding a putative transcription factor,was cloned via RT-PCR method and its expression patterns were characterized during the wheat dehydration.The main content of the study was summarized as follows:1.Immature embryos of several wheat varieties Yumai 18,Yumai 34,Yumai 49, Yumai 70 and Zhengmai 9023 were used to study on the genotype screening for genetic transformation via microprojectile bombardment.The results showed that the properties of proliferation,differentiation and regeneration of Zhengmai 9023 and Yumai 18 were better than other varieties;Subcultivation times affected the differentiation of the calli to some extent and subcultivate 2 times of induced calli could significantly enhance the differentiation rate;a proper plant hormone concentration,IAA(0.5mg/L)and CCC (1.5mg/L),in the differential medium can effectively promote the root differentiation and is very benefit to transplanting of regenerated plantlets.2.The test of different PPT concentrations(0-120 mg/L)showed that 100mg/L was the proper concentration for transgenic assay on wheat leaves of Yumai 18,Yumai 34. Under this concentration,transgenic leaves can survive and grow normally,while the no-transgenic leaves become wither and finally died after 5 days.3.Based on the optimized methods of genetic transformation,the wheat variety Yumai 18 was transformed with the fusion gene TPSP for for trehalose synthesis via microprojectile bombardment.1286 embryo generated calli were used as the target tissues,and 54 putative transgenic plantlets were obtained after the ppt resistant screening,differentiation and plant regeneration.After the PCR identification and the ppt resistant assay,5 transgenic plantlets(T₀)were obtained.4.Wheat cultivars,Yumai 18,Yumai 34,Yumai 49,Yumai 70 and Zhengmai 9023, were transformed with the fusion gene TPSP by the method of pollen-tube-pathway.In total,5848 flowers from the 5 cultivars were treated and 3726 seeds were obtained with a seed-set percentage of 63.7%.T₀ seeds were sowed in the field individually and 3526 T₁ plantlets were got with a seedling percentage of 94.63%.Tests of T₁ Yumai 34 offspring were carried out,including PPT resistence and PCR identification,and finally 4 plantlets were selected as candidate transgenic plantlets from 421 tested plantlets.5.A transcriptional factor gene TaSNACl,which strongly induced by the the water stress,was cloned by Reverse Transcription-Polymerase Chain Reaction(RT-PCR) from wheat root.The cloned gene including a sequence of 1169bp in length and a coding region 1029bp,the deduced amino acids sequence(342 aa)of this gene is about 38.14KD and the pI is 6.26.Anylysis showed that the conserved function domains from amino acid sequence belongs to NAC genoid and the homology of it to NAC genes from wheat(AY625682,DQ022842 and DQ022843)is up to 96 or so.The expression pattern of the cloned gene was characterized via RT-PCR and the results indicated that the TaSNACl gene was induced in roots during the water stress accumulation,while the expression of the gene was not detected in leaves.