- [Show abstract] [Hide abstract] ABSTRACT: UDP-glucosyltransferase enzymes (UGTs) are known to function in plant disease defense responses. In our previous RNA-seq analysis, TaUGT expression was found to be strongly up-regulated in Agrobacterium-infected wheat immature embryos, as compared to untreated controls. In this study, we isolated the TaUGT-B2, located on wheat 2BS chromosome, and characterized its function in tobacco, Arabidopsis, and wheat. Transient overexpression of TaUGT-B2 reduced the number of Agrobacterium cells attached to the surface of wheat cells, and hindered Agrobacterium-mediated GUS transient expression in wheat cells. Transiently silenced TaUGT-B2 expression resulted in an increased rate of shoot regeneration from Agrobacterium-infected wheat tissues. Therefore, we concluded that the overexpression of TaUGT-B2 impeded T-DNA transformation by reducing number of Agrobacterium cells attached to wheat tissues and lowering the regeneration efficiency of transgenic plantlets. In order to further investigate the function of TaUGT-B2, it was introduced into tobacco and Arabidopsis, and transgenic plants were then re-transformed with another vector carrying the GUS gene. The results of these experiments indicated that the expression of TaUGT-B2 in tobacco and Arabidopsis led to a reduction in the efficiency of Agrobacterium-mediated transformation. GUS transient expression was also impeded following overexpression of TaUGT-B2 in tobacco and Arabidopsis. Therefore, we conclude that TaUGT-B2 plays a negative role in Agrobacterium-mediated transformation of plants, especially in wheat.
- [Show abstract] [Hide abstract] ABSTRACT: Wheat is recalcitrant to genetic transformation. A potential solution is to manipulate the expression of some host proteins involved in T-DNA integration process. VirE2 interacting protein 2 (VIP2) plays an important role in T-DNA transport and integration. In this study, a TaVIP2 gene was cloned from common wheat. Southern blot and allele-specific polymerase chain reaction (AS-PCR) combined with an online chromosomal location software tool revealed that three TaVIP2 genes were located on wheat chromosomes 1AL, 1BL, and 1DL. These three homoeoallelic TaVIP2 genes all contained 13 exons and 12 introns, and their coding sequences were the same; there were a few single nucleotide polymorphisms (SNPs) among the three genes. The heterologous expression of the TaVIP2 gene in tobacco led to enhancement of the Agrobacterium-mediated transformation efficiency up to 2.5-fold. Transgenic tobacco plants expressing TaVIP2 showed enhanced resistance to powdery mildew. Further quantitative real-time PCR (qRT-PCR) revealed that overexpression of TaVIP2 in transgenic tobacco up-regulated the expression of an endogenous gene, NtPR-1, which likely contributed to powdery mildew resistance in transgenic tobacco. Our study indicates that the TaVIP2 gene may be highly useful in efforts to improve Agrobacterium-mediated transformation efficiency and to enhance powdery mildew resistance in wheat.
- [Show abstract] [Hide abstract] ABSTRACT: Agrobacterium-mediated plant transformation is an extremely complex and evolved process involving genetic determinants of both the bacteria and the host plant cells. However, the mechanism of the determinants remains obscure, especially in some cereal crops such as wheat, which is recalcitrant for Agrobacterium-mediated transformation. In this study, differentially expressed genes (DEGs) and differentially expressed proteins (DEPs) were analyzed in wheat callus cells co-cultured with Agrobacterium by using RNA sequencing (RNA-seq) and two-dimensional electrophoresis (2-DE) in conjunction with mass spectrometry (MS). A set of 4,889 DEGs and 90 DEPs were identified, respectively. Most of them are related to metabolism, chromatin assembly or disassembly and immune defense. After comparative analysis, 24 of the 90 DEPs were detected in RNA-seq and proteomics datasets simultaneously. In addition, real-time RT-PCR experiments were performed to check the differential expression of the 24 genes, and the results were consistent with the RNA-seq data. According to gene ontology (GO) analysis, we found that a big part of these differentially expressed genes were related to the process of stress or immunity response. Several putative determinants and candidate effectors responsive to Agrobacterium mediated transformation of wheat cells were discussed. We speculate that some of these genes are possibly related to Agrobacterium infection. Our results will help to understand the interaction between Agrobacterium and host cells, and may facilitate developing efficient transformation strategies in cereal crops.
- [Show abstract] [Hide abstract] ABSTRACT: Dwarfing germplasms are crucial to the development of wheat (Triticum aestivum L.) varieties with ideal plant height and combinations of agronomic traits. A novel dwarfing wheat line, AS34, was developed in this study by somatic variation. This new wheat germplasm was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, two-dimensional gel electrophoresis, genomic in situ hybridization, and genomic scanning with 44 simple sequence repeat (SSR) markers, and our analysis showed that AS34 was clearly derived from the somatic variation of Yumai66 (YM66), its tissue culture parent, but not from natural hybridization. Molecular tests using known primers specific to Rht8 and Rht-B1b genes indicated that AS34 and its parent contained Rht8 but not Rht-B1b in their genomes, which was not consistent with previous reports. Similar to YM66, AS34 was sensitive to gibberellic acid, and this characteristic might be induced only by Rht8 or by Rht8 and the new dwarfing genes simultaneously. Genetic analysis revealed that the new dwarfing trait in AS34 was controlled by multiple quantitative genes, and data from the F 2 population yielded no key quantitative trait locus. According to the results of genomic scanning for different SSR markers, some of the new dwarfing genes in AS34 might be located on chromosome 4B. The new dwarfing material had positive effects on the agronomic traits related to grain yield in F-2 populations, particularly the variation range in plant height. This dwarfing resource will be potentially used in wheat breeding for the development of new wheat varieties with available agronomic traits.
- [Show abstract] [Hide abstract] ABSTRACT: Key message: Seven kinds of transgenic tobacco plants transformed with combinations of three FBE genes were obtained. The transgenic plants transformed with Ta1-SST + Ta6-SFT genes appeared to have the highest fructan or soluble sugar content and the strongest salt tolerance. Fructan is thought to be one of the important regulators involved in plant tolerance to various abiotic stresses. In this study, wheat-derived genes, Ta1-SST, Ta6-SFT, and Ta1-FFT, encoding fructan biosynthesis enzymes (FBE) were isolated and cloned into vectors modified pBI121 or pZP211. Seven different combinations of the three target genes were transformed into tobacco plants through an Agrobacterium-mediated approach, and transgenic tobacco plants were identified by PCR, ELISA, and Southern blotting. Compared with tobacco plants transformed with other six combinations of the three target genes and with wild-type plants, the transgenic plants transformed with Ta1-SST + Ta6-SFT genes contained the highest fructan and soluble sugar content. All seven types of transgenic tobacco plants displayed a much higher level of tolerance to drought, low temperature, and high salinity compared with the wild type. Differences of drought and low temperature tolerance between the transgenic plants containing a single FBE gene and those harboring two or three FBE genes were not significant, but the salt tolerance level of the transgenic plants with different FBE gene combinations from high to low was: Ta1-SST + Ta6-SFT > Ta1-SST + Ta6-SFT + Ta1-FFT > Ta1-SST + Ta1-FFT > Ta1-SFT + Ta1-FFT > single FBE gene. These results indicated that the tolerances of the transgenic tobacco plants to various abiotic stresses were associated with the transformed target gene combinations and the contents of fructan and soluble sugar contained in the transgenic plants.
- [Show abstract] [Hide abstract] ABSTRACT: With 4 figures and 2 tablesAbstractThe bifunctional α-amylase/subtilisin inhibitor (BASI) encoded by the Isa gene on barley chromosome 2HL could inhibit wheat α-amylase activity, thereby reducing sprouting and improving the quality of wheat. Regenerated plants were derived from immature embryo culture of hybrids of common wheat varieties ‘Zhengmai 9023’, ‘CB037’ and ‘Zhongmai 16’ with the wheat–barley 2H alien substitution lines 2H(2A) and 2H(2B) after callus induction, subculture and differentiation. The presence of barley 2H chromatin was detected in regenerated plants (SC1) and their selfed progeny (SC2–5) using homoeologous group 2 SSR markers from barley and further identified in selected SC4 and SC5 lines using genomic in situ hybridization with barley genomic DNA as probe and CS genomic DNA for blocking. The Isa gene from the identified SC4 and SC5 lines was also amplified using Isa-specific primers. We identified wheat–barley 2H chromosome translocation lines carring the Isa gene.
- [Show abstract] [Hide abstract] ABSTRACT: Barley yellow dwarf virus (BYDV) may cause a serious disease affecting wheat worldwide. True resistance to BYDV is not naturally found in wheat. BYDV resistance genes are found in more than 10 wild relative species belonging to the genera of Thinopyrum, Agropyron, Elymus, Leymus, Roegneria, and Psathyrostachy. Through wide crosses combining with cell culture, use of ph mutants, or irradiation, 3 BYDV resistance genes in Th. intermedium, including Bdv2, Bdv3 and Bdv4, were introgressed into common wheat background. Various wheat-Th. intermedium addition and substitution, translocation lines with BYDV-resistance were developed and characterized, such as 7D-7Ai#1 (bearing Bdv2), 7B-7Ai#1, 7D-7E (bearing Bdv3), and 2D-2Ai-2 (bearing Bdv4) translocations. Three wheat varieties with BYDV resistance from Th. intermedium were developed and released in Australia and China, respectively. In addition, wheat-Agropyron cristatum translocation lines, wheat-Ag. pulcherrimum addition and substitution lines, and a wheat-Leymus multicaulis addition line (line24) with different resistance genes were developed. Cytological analysis, morphological markers, biochemical markers, and molecular markers associated with the alien chromatin carrying BYDV resistance genes were identified and applied to determine the presence of alien, chromosomes or segments, size of alien chromosome segments, and compositions of the alien chromosomes. Furthermore, some resistance-related genes, such as RGA, P450, HSP70, protein kinases, centrin, and transducin, were identified, which expressed specifically in the resistance translocation lines with Bdv2. These studies lay the foundations for developing resistant wheat cultivars and unraveling the resistance mechanism against BYDV.
- [Show abstract] [Hide abstract] ABSTRACT: The wheat line H960642 is a homozygous wheat-Thinopyrum intermedium translocation line with resistance to BYDV by genomicin situ hybridization (GISH) and RFLP analysis. The genomic DNA ofTh. intermedium was used as a probe, and common wheat genomic DNA as a blocking in GISH experiment. The results showed that the chromosome segments ofTh. intermedium were transferred to the distal end of a pair of wheat chromosomes. RFLP analysis indicated that the translocation line H960642 is a T7DS-7DL-7XL translocation by using 8 probes mapped on the homoeologous group 7 in wheat. The translocation breakpoint is located between Xpsr680 and Xpsr965 about 90–99 cM from the centromere. The RFLP markers psr680 and psr687 were closely linked with the BYDV resistance gene. The gene is located on the distal end of 7XL around Xpsr680 and Xpsr687.
Chinese Academy of Agricultural Sciences
Beijing, Beijing Shi, China
- Institute of Crop Breeding and Cultivation