Cloning And Characterization Of Regeneration Candidate Genes Relevant To Tissue Culture Response In Wheat

Somatic embryogenesis regeneration is an essential step for wheat cell engineering breeding and transgenic research. All along, in-depth investigation on the improvement of regeneration potential has been taken for different explants and genotypes from the physiological characters and culture process in wheat, and some useful results have been achieved. However, in comparison with other model plants, the researches on molecular level of the regeneration traits by molecular biology methods, isolation and characterization of candidate genes relevant to wheat regeneration in vitro, and their function analysis by transgenic approach and application in obtaining of marker-free transgenic wheat plants are relatively behindhand. This study introduced a novel culture method for efficiently improving regeneration potential of wheat immature embryos by summarizing results of our research group. Meanwhile, three types of regeneration-related candidate genes were isolated and characterized based on the results of mRNA differential display with results as the followings:1. 2, 4-D break treatment and not (labeled SD0 and SD2, respectively) were designed for the exploration on the improvement of regeneration potential of wheat immature embryo, using wheat genotype Zhong8601 as materials. The results showed that 8-d pre-culture on 2, 4-D 2.0mg/L contained medium followed by 12-d auxin break culture on the medium significantly increased the regeneration capacity of wheat immature embryos from 12.3% to 208.2%. The successful application to different wheat genotypes further confirmed that the developed method was less genotype-dependent. Meanwhile, the pretreatment effect of different temperatures and water stress on the regeneration of wheat immature embryos was significantly different.2. TaNiR coding for nitrite reductase in wheat was first successfully cloned based on homologous sequence alignment in this research. BiFC analysis confirmed the interaction between TaNiR and Fd (ferredoxin). Quantitative assay showed that the expression of wheat TaNiR was induced by KNO₃ and higher embryogenic callus (E) than in non-embryogenic callus (NE). TaNiR expression was also observed be induced by different temperature in pretreatment culture and SD0 culture regimen. Chromosome location of TaNiR showed a copy on wheat chromosomes 6A and 6B each and one copy was assigned to chromosome 6BL. Deletion analysis for functional elements investigation showed weak promotion of the tested four missing fragments of the regulatory elements. PNiR4 containing the first intron of TaNiR displayed a stronger promotion than the other three truncated fragments. Transgenic assay confirmed the overexpression of TaNiR seemed essential for the improvement of regeneration potential.3. By screening the results of mRNA differential display, CAT was determined and cloned using in silico technology. TaCAT3, a member in wheat CAT family, was also isolated. Sequence alignment confirmed the presence of three types of TaCAT3, according to the size and the presence or not of the second intron. One In/Del mutation in the promoter region of TaCAT2 was found with probably the same insertion mechanisms as the intron II of TaCAT3. Truncation analysis on TaCAT1 promoter showed weak promotion. Quantitative analysis confirmed the inductive role of SD0 treatment, pretreatments of 25℃and water stress in TaCATs expression and H₂O₂ accumulation in wheat calli. Preliminary functional analysis of T0 transformed wheat genotype KN199 with TaCAT1-RNAi cassette showed negative effect on regeneration efficiency.4. Three wheat SERKs genes were revealed in this study, of them, two were demonstrated to have full ORF (TaSERK1^N and TaSERK3^N). Sequence alignment and expression analysis showed that the sequences and advanced structures of TaSERK1^N and TaSERK2^N were very similar to each other. The expression level of TaSERKs^N was the highest in immature embryos and modestly high in E during the induction stage of wheat immature embryos. Pretreatments of 25℃and water stress and addition of AGP and BR can improve the regeneration potentials and induce the expression of TaSERKs^N. Prediction on TaSERKs^N chromosome mapping indicated that TaSERK1^N and TaSERK2^N might be positioned on chromosome 2 while TaSERK3^N might be located on chromosome 4 and/or long arms of chromosome 5.5. Based on wheat functional genomics study and the development of marker-free transgenic wheat plants for biosafety evaluation, vector pWMB006 was constructed for RNAi analysis for gene gun-mediated wheat transformation by using several intermedium expression vectors, pWMB014, a new binary vector with two T-DNA regions, pWMB025 construct using EPSPS as selective marker, and pWMB022 containing plant pigment regulatory genes Lc and C1. The construction of these expression vectors is of significant importance to wheat research on functional genomics and biosafty transgenic events.