Authors:L.J. Wu, Y. Shang, T. Liu, W.J. Chen, B.L. Liu, L.Q. Zhang, D.C. Liu, B. Zhang, and H.G. Zhang
In this study, the cDNA of homocysteine S-methyltransferase was isolated from Aegilops tauschii Coss., with the gene accordingly designated as AetHMT1. Similar to other methyltransferases, AetHMT1 contains a GGCCR consensus sequence for a possible zinc-binding motif near the C-terminal and a conserved cysteine residue upstream of the zinc-binding motif. Analysis of AetHMT1 uncovered no obvious chloroplast or mitochondrial targeting sequences. We functionally expressed AetHMT1 in Escherichia coli and confirmed its biological activity, as evidenced by a positive HMT enzyme activity of 164.516 ± 17.378 nmol min−1 mg−1 protein when catalyzing the transformation of L-homocysteine. Compared with the bacterium containing the empty vector, E. coli harboring the recombinant AetHMT1 plasmid showed much higher tolerance to selenate and selenite. AetHMT1 transcript amounts in different organs were increased by Na2SeO4 treatment, with roots accumulating higher amounts than stems, old leaves and new leaves. We have therefore successfully isolated HMT1 from Ae. tauschii and characterized the biochemical and physiological functions of the corresponding protein.
Two lines, L-19-613 and L-19-626, were produced from the common wheat cultivar Longmai 19 (L-19) by six consecutive backcrosses using biochemical marker-assisted selection. L-19 (Glu-D1a, Glu-A3c/Gli-A1?; Gli-A1? is a gene coding for unnamed gliadin) and L-19-613 (Glu-D1d, Glu-A3c/Gli-A1?) formed a set of near-isogenic lines (NILs) for HMW-GS, while L-19-613 and L-19-626 (Glu-D1d, Glu-A3e/Gli-A1m) constituted another set of NILs for the LMW-GS/gliadins. The three L-19 NILs were grown in the wheat breeding nursery in 2007 and 2008. The field experiments were designed using the three-column contrast arrangement method with four replicates. The three lines were ranked as follows for measurements of gluten strength, which was determined by the gluten index, Zeleny sedimentation, the stability and breakdown time of the farinogram, the maximum resistance and area of the extensogram, and the P andWvalues of the alveogram: L-19-613 > L-19-626 > L-19. The parameters listed above were significantly different between lines at the 0.05 or 0.01 level. The Glu-D1 and Glu-A3/Gli-A1 loci had additive effects on the gluten index, Zeleny sedimentation, stability, breakdown time, maximum resistance, area, P and W values. Although genetic variation at the Glu-A3/Gli-A1 locus had a great influence on wheat quality, the genetic difference between Glu-D1d and Glu-D1a at the Glu-D1 locus was much larger than that of Glu-A3c/Gli-A1? and Glu-A3e/Gli-A1m at the Glu-A3/Gli-A1 locus. Glu-D1d had negative effects on the extensibility and the L value compared with Glu-D1a. In contrast, Glu-A3c/Gli-A1? had a positive effect on these traits compared with Glu-A3e/Gli-A1m.
Authors:J.Q. Xu, L. Wang, B.L. Liu, T.F. Xia, D.C. Liu, X. Chang, T.W. Zhang, H.G. Zhang, and Y.H. Shen
As one of the world’s earliest domesticated crops, barley is a model species for the study of evolution and domestication. Domestication is an evolutionary process whereby a population adapts, through selection; to new environments created by human cultivation. We describe the genome-scanning of molecular diversity to assess the evolution of barley in the Tibetan Plateau. We used 667 Diversity Arrays Technology (DArT) markers to genotype 185 barley landraces and wild barley accessions from the Tibetan Plateau. Genetic diversity in wild barley was greater than in landraces at both genome and chromosome levels, except for chromosome 3H. Landraces and wild barley accessions were clearly differentiated genetically, but a limited degree of introgression was still evident. Significant differences in diversity between barley subspecies at the chromosome level were observed for genes known to be related to physiological and phenotypical traits, disease resistance, abiotic stress tolerance, malting quality and agronomic traits. Selection on the genome of six-rowed naked barley has shown clear multiple targets related to both its specific end-use and the extreme environment in Tibet. Our data provide a platform to identify the genes and genetic mechanisms that underlie phenotypic changes, and provide lists of candidate domestication genes for modified breeding strategies.
Authors:H. Zhang, X. Yu, H. Sun, Y. Liu, Z. Nan, C. Lu, M. Ru, G. Mai, Y. Wang, and Z. Li
The power vs. time curves of the promoter bacteria of a nutrient drug were determined by using a 2277 Thermal Activity Monitor (Sweden). A new experimental model of bacterial growth were established. The growth rate constant, heat output and optimum concentration of specific promoter bacterial of nutrient drug were calculated.
Iron deficiency is the most common nutritional disorder, affecting over 30% of the world’s human population. The primary method used to alleviate this problem is nutrient biofortification of crops so as to improve the iron content and its availability in food sources. The over-expression of ferritin is an effective method to increase iron concentration in transgenic crops. For the research reported herein, sickle alfalfa (Medicago falcata L.) ferritin was transformed into wheat driven by the seed-storage protein glutelin GluB-1 gene promoter. The integration of ferritin into the wheat was assessed by PCR, RT-PCR and Western blotting. The concentration of certain minerals in the transgenic wheat grain was determined by inductively coupled plasma-atomic emission spectrometry, the results showed that grain Fe and Zn concentration of transgenic wheat increased by 73% and 44% compared to nontransformed wheat, respectively. However, grain Cu and Cd concentration of transgenic wheat grain decreased significantly in comparison with non-transformed wheat. The results suggest that the over-expression of sickle alfalfa ferritin, controlled by the seed-storage protein glutelin GluB-1 gene promoter, increases the grain Fe and Zn concentration, but also affects the homeostasis of other minerals in transgenic wheat grain.
Premature termination codons (PTCs) are an important reason for the silence of highmolecular- weight glutenin subunits in Triticum species. Although the Glu-A1y gene is generally silent in common wheat, we here isolated an expressed Glu-A1y gene containing a PTC, named 1Ay8.3, from Triticum monococcum ssp. monococcum (AmAm, 2n = 2x = 14). Despite the presence of a PTC (TAG) at base pair positions 1879–1881 in the C-terminal coding region, this did not obviously affect 1Ay8.3 expression in seeds. This was demonstrated by the fact that when the PTC TAG of 1Ay8.3 was mutated to the CAG codon, the mutant in Escherichia coli bacterial cells expressed the same subunit as in the seeds. However, in E. coli, 1Ay8.3 containing the PTC expressed a truncated protein with faster electrophoretic mobility than that in seeds, suggesting that PTC translation termination suppression probably occurs in vivo (seeds) but not in vitro (E. coli). This may represent one of only a few reports on the PTC termination suppression phenomenon in genes.
Authors:N. Zhang, R.Q. Pan, J.J. Liu, X.L. Zhang, Q.N. Su, F. Cui, C.H. Zhao, L.Q. Song, J. Ji, and J.M. Li
Plants with deficiency in Gibberellins (GAs) biosynthesis pathway are sensitive to exogenous GA3, while those with deficiency in GAs signaling pathway are insensitive to exogenous GA3. Thus, exogenous GA3 test is often used to verify whether the reduced height (Rht) gene is involved in GAs biosynthesis or signaling pathway. In the present study, we identified the genetic factors responsive to exogenous GA3 at the seedling stage of common wheat and analyzed the response of the plant height related quantitative trait loci (QTL) to GA3 to understand the GAs pathways the Rht participated in. Recombinant inbred lines derived from a cross between KN9204 and J411 with different response to exogenous GA3 were used to screen QTL for the sensitivity of coleoptile length (SCL) and the sensitivity of seedling plant height (SSPH) to exogenous GA3. Two additive QTL and two pairs of epistatic QTL for SCL were identified, meanwhile, two additive QTL and three pairs of epistatic QTL for SSPH were detected. For the adult plant height (PH) investigated in two environments, six additive QTL were identified. Three QTL qScl-4B, qSsph-4B and qPh-4B were mapped in one cluster near the functional marker Rht-B1b. When PH were conditional on SSPH, the absolute additive effect value of qPh-4B and qPh-6B were reduced, suggesting that the Rhts in both two QTL were insensitive to exogenous GA3, while the additive effect values of qPh-2B, qPh-3A, qPh-3D and qPh-5A were not significantly changed, indicating that the Rhts in these QTL were sensitive to exogenous GA3, or they were not expressed at the seedling stage.
Authors:L. Wei, S.G. Bai, X.J. Hou, J.M. Li, B. Zhang, W.J. Chen, D.C. Liu, B.L. Liu, and H.G. Zhang
Among 20 awnless Tibetan wheat cultivars analyzed by SDS-PAGE, the migration rate of an HMW-GS in XM001584 and XM001593, named 1BX23*. was shown to be slightly faster than 1Bx6. and slower than Bx7. Its nucleotide sequence was isolated based on homology clones. In a phylogenetic tree of 1Bx genes, 1Bx23* was apparently clustered with 1Bx23. Compared with 1Bx23. eight single nucleotide replacements caused four single amino acid replacements in 1Bx23*. The deletion of “G” at base pair 1463 and insertion of “A” at 1509 bps induced a 42-nucleotide frame shift. “GQRQQAGQWQRPGQ” was replaced by “DKGNRQDNGNDRDK”. The new segment cannot be found in other HMW-GSs, and it is very similar to a segment found in collagen. Moreover, an 18-nucleotide deletion made 1Bx23* six amino acids shorter than 1Bx23. The cultivar XM001593 had 28 chromosomes, which signifies that it was tetraploid wheat, and that the new HMW-GS 1Bx23* cannot be used directly for breeding in common wheat.
Authors:Y.Q. Wang, X.J. Hou, B. Zhang, W.J. Chen, D.C. Liu, B.L. Liu, and H.G. Zhang
Red coleoptile is an easily observed agronomic trait of wheat and has been extensively studied. However, the molecular mechanism of this trait has not yet been revealed. In this study, the MYB gene TaMYB-D1 was isolated from the wheat cultivar ‘Gy115’, which possesses red coleoptiles. This gene resided at the short arm of the homoelogous group 7 chromosomes. TaMYB-D1 was the only gene expressed in the coleoptiles of ‘Gy115’ and was not expressed in ‘Opata’ and ‘CS’, which have uncoloured coleoptiles. Phylogenetic analysis placed TaMYB-D1 very close to ZmC1 and other MYB proteins regulating anthocyanin biosynthesis. The encoded protein of TaMYB-D1 had an integrated DNA binding domain of 102 amino acids and a transcription domain with 42 amino acids, similar to the structure of ZmC1. Transient expression analysis in onion epidermal cells showed that TaMYB-D1 was located at the plant nucleus, which suggested its role as a transcription factor. The expression of TaMYB-D1 was accompanied with the expression of TaDFR and anthocyanin biosynthesis in the development of the coleoptile of ‘Gy115’. Transient expression analysis showed that only TaMYB-D1 induced a few ‘Opata’ coleoptile cells to synthesize anthocyanin in light, and the gene also induced a colour change to red in many cells with the help of ZmR. All of these results suggested TaMYB-D1 as the candidate gene for the red coleoptile trait of ‘Gy115’.
Authors:W.J. Chi, Z.Y. Wang, J.M. Liu, C. Zhang, Y.H. Wu, and Y.J. Bai
Nitrogen (N) is an important nutrient for plant growth and yield production, and rice grown in paddy soil mainly uses ammonium (NH4+) as its N source. Previous studies have shown that N status is tightly connected to plant defense; however, the roles of NH4+ uptake and assimilation in rice sheath blight disease response have not been studied previously. Here, we analyzed the effects of different N sources on plant defense against Rhizoctonia solani. The results indicated that rice plants grown in N-free conditions had higher resistance to sheath blight than those grown under N conditions. In greater detail, rice plants cultured with glutamine as the sole N source were more susceptible to sheath blight disease compared to the groups using NH4+ and nitrate (NO3–) as sole N sources. N deficiency severely inhibited plant growth; therefore, ammonium transporter 1;2 overexpressors (AMT1;2 OXs) were generated to test their growth and defense ability under low N conditions. AMT1;2 OXs increased N use efficiency and exhibited less susceptible symptoms to R. solani and highly induced the expression of PBZ1 compared to the wild-type controls upon infection of R. solani. Furthermore, the glutamine synthetase 1;1 (GS1;1) mutant (gs1;1) was more susceptible to R. solani infection than the wild-type control, and the genetic combination of AMT1;2 OX and gs1;1 revealed that AMT1;2 OX was less susceptible to R. solani and required GS1;1 activity. In addition, cellular NH4+ content was higher in AMT1;2 OX and gs1;1 plants, indicating that NH4+ was not directly controlling plant defense. In conclusion, the present study showed that the activation of NH4+ uptake and assimilation were required for rice resistance against sheath blight disease.