Thermostability Improvement of a Streptomyces Xylanase by Introducing Proline and Glutamic Acid Residues

Protein engineering is commonly used to improve the robustness of enzymes for activity and stability at high temperatures. In this study, we identified four residues expected to affect the thermostability of Streptomyces sp. strain S9 xylanase XynAS9 through multiple-sequence analysis (MSA) and molecular dynamic simulations (MDS). Site-directed mutagenesis was employed to construct five mutants by replacing these residues with proline or glutamic acid (V81P, G82E, V81P/G82E, D185P/S186E, and V81P/G82E/D185P/S186E), and the mutant and wild-type enzymes were expressed in Pichia pastoris. Compared to the wild-type XynAS9, all five mutant enzymes showed improved thermal properties. The activity and stability assays, including circular dichroism and differential scanning calorimetry, showed that the mutations at positions 81 and 82 increased the thermal performance more than the mutations at positions 185 and 186. The mutants with combined substitutions (V81P/G82E and V81P/G82E/D185P/S186E) showed the most pronounced shifts in temperature optima, about 17°C upward, and their half-lives for thermal inactivation at 70°C and melting temperatures were increased by >9 times and approximately 7.0°C, respectively. The mutation combination of V81P and G82E in adjacent positions more than doubled the effect of single mutations. Both mutation regions were at the end of long secondary-structure elements and probably rigidified the local structure. MDS indicated that a long loop region after positions 81 and 82 located at the end of the inner β-barrel was prone to unfold. The rigidified main chain and filling of a groove by the mutations on the bottom of the active site canyon may stabilize the mutants and thus improve their thermostability.     

Genetic regulation of developmental phases in winter wheat

The orderly development of winter wheat through its life cycle can be marked at three stages: stem elongation, heading date, and physiological maturity. The duration of a developmental phase between two stages is important in yield component generation. In this study the three developmental stages were characterized and 350 markers were mapped in a population of recombinant inbred lines (RILs) generated from a cross between two winter wheat cultivars (‘Jagger’ and ‘2174’). Three major QTLs were found to control variation in developmental process, and each of them was tightly associated with a known flowering gene, VRN-A1 on chromosome 5A, PPD-D1 on chromosome 2D, and VRN-D3 on chromosome 7D. The average contribution of the gene marker for each QTL to the total phenotypic variation (R ²) was evaluated over 3 years. The effect of VRN-A1 ranged from 21.5% at stem elongation to 17.4% at physiological maturity. The effect of PPD-D1 was minor (6.7%) at stem elongation but increased to 29.7% at heading and 20.1% at physiological maturity. The effect of VRN-D3 was not detected at stem elongation but increased to 14.6% at heading and to 20.5% at physiological maturity. Hence, the VRN-A1 locus, the PPD-D1 locus, and the VRN-D3 locus had greatest impact on development at stem elongation, heading date, and physiological maturity, respectively. Whereas the Jagger VRN-A1 and VRN-D3 alleles accelerated development, the Jagger PPD-D1 allele delayed the developmental process due to its sensitivity to photoperiod. Our findings suggest that through the appropriate combination of alleles at these three loci one would be able to regulate the various developmental phases to accommodate different agricultural needs.     

Cloning of <Emphasis Type="Italic">TaTPP-6AL1</Emphasis> associated with grain weight in bread wheat and development of functional marker

Trehalose 6-phosphate phosphatase (TPP) dephosphorylates trehalose 6-phosphate to trehalose, an important growth regulator, and is involved in starch accumulation and grain yield. In this study, wheat TPP homologs were isolated from chromosomes 6AL, 6BL, and 6DL, designated as TaTPP-6AL1, TaTPP-6BL1, and TaTPP-6DL1, respectively. Sequence alignment showed a single-nucleotide polymorphism (SNP) at TaTPP-6AL1 locus between cultivars with contrasting thousand grain weight (TGW), forming alleles TaTPP-6AL1a and TaTPP-6AL1b, respectively. A cleaved amplified polymorphic sequence (CAPS) marker, TaTPP-6AL1-CAPS, was developed to differentiate the two alleles. TaTPP-6AL1 was mapped within the interval of IWB65749 and IWB60449 in a recombinant inbred line (RIL) population derived from Zhou8425B/Chinese Spring using the wheat 90K SNP assay. A QTL for TGW identified in the interval explained 12.1–19.1% of the phenotypic variance across five environments. Association analysis on 141 Chinese wheat cultivars also indicated a significant correlation of TaTPP-6AL1 with TGW. In conclusion, TaTPP-6AL1 and its functional marker are valuable to improve grain yield in wheat breeding.     

1971-2010年中国大陆潜在蒸散变化的年代际转折及其成因

潜在蒸散时间演变的年代际转折研究有助于全面认识潜在蒸散对气候变化的响应。基于修正的FAO56 Penman-Monteith公式和中国580个台站逐日气象观测资料,利用气候变化趋势转折判别模型分析了1971—2010年中国潜在蒸散变化的年代际转折特征,并探讨转折前、后的变化趋势及其主导因素。结果表明:1971—2010年中国年平均潜在蒸散在20世纪90年代初期由显著下降(-2.46 mm/a)转变为显著上升(1.57 mm/a),这与影响潜在蒸散变化的4个气象因子趋势的年代际转折密切相关。90年代之前,全国风速和日照时数普遍下降引起的负贡献超过气温上升引起的正贡献,导致潜在蒸散显著下降;90年代之后,全国大部分地区的增暖加剧和干旱化使得气温和相对湿度的正贡献明显增大,超过由于风速和日照时数下降趋势减缓甚至转折而减小的负贡献,导致潜在蒸散显著上升。潜在蒸散趋势转折现象在全国80%以上的站点普遍存在,且转折前、后主导因子的空间分布格局存在差异。90年代之前,风速和日照时数分别是北方和南方多数站点的主导因子;90年代之后,以气温和相对湿度为主导因子的站点明显增多,尤其是在西北地区、青藏高原和东南沿海部分地区。     

珠三角城市绿地CO2通量的季节特征

城市绿地是城市碳循环的重要组成部分,利用长期定位观测资料估算珠三角典型城市绿地的CO2通量,可以为应对气候变化、评价区域碳源汇提供参考。应用2009、2010年,东莞市植物园内的涡度相关法CO2通量定位观测资料,分析了净生态系统交换量(NEE)的年变化及其与气象要素的关系,结果表明:(1)年平均NEE总量为-104.2 gC.m-.2a-1,表明城市绿地生态系统具有固碳能力。(2)NEE随光温条件变化呈现明显的季节动态,12至3月表现为碳源,其他月份表现为碳汇。(3)根据白天NEE与光合有效辐射(PAR)逐月拟合Michaelis-Menten方程,得到年平均表观初始光能利用率(α)为(0.00134±0.00035)mgCO.2μmol-1光子,年平均光饱和生态系统生产力(Pmax)为(1.006±0.283)mgCO.2m-.2s-1。(4)利用夜间呼吸(Reco)与5 cm土壤温度(Ts)拟合指数方程,得到年平均Reco总量为1378.1 gC.m-.2a-1。(5)NEE与PAR、气温(Ta)和饱和水压差(VPD)的相关性分析显示,NEE与PAR偏相关系数的绝对值大于Ta和VPD,表明PAR对NEE的影响最大。     

Molecular differentiation of null alleles at <Emphasis Type="Italic">ZCCT</Emphasis>-<Emphasis Type="Italic">1</Emphasis> genes on the A,B, and D genomes of hexaploid wheat

A dominant allele of the vernalization gene Vrn-2 is the wild type conferring winter growth habit, whereas a recessive vrn-2 allele confers spring growth habit. The recessive vrn-2 allele is mutated due to the deletion of the complete gene (a null form) or alternation of a key amino acid in the VRN-2 protein (a nonfunctional form) in diploid wheat or tetraploid wheat. VRN-2 is also denoted ZCCT due to the presence of a zinc finger and a CCT domain in its protein. There are two paralogous ZCCT genes at the VRN-2 locus in diploid Triticum monococcum and three paralogous ZCCT genes on each of the A and B genomes in tetraploid wheat, but little is known about the allelic variation in VRN-2 in hexaploid wheat. In the study reported here, we performed a one-shot PCR to simultaneously amplify the promoter regions of the three ZCCT-1 genes from hexaploid wheat, including the 302-bp fragment from ZCCT-A1, the 294-bp fragment from ZCCT-B1, and the 320-bp fragment from ZCCT-D1. Each amplicon could be differentiated by electrophoresis in an acrylamide/bisacrylamide gel. This PCR marker for different lengths of the three ZCCT-1 genes was used to search for null alleles in hexaploid wheat. A null allele was found in each of ZCCT-A1, ZCCT-B1, and ZCCT-D1 among 74 cultivars and genetic stocks of U.S. hexaploid wheat. Among 54 Chinese wheat cultivars, breeding lines, and landraces, we identified three accessions carrying a single null allele at ZCCT-A1, three accessions carrying a null allele at ZCCT-B1, and one accession carrying a double null allele at both ZCCT-A1 and ZCCT-D1. The potential application of these natural ZCCT-1 mutant materials in wheat breeding programs and studies on the genetics of wheat is discussed.     

Genome-Wide Association of Stem Water Soluble Carbohydrates in Bread Wheat

Water soluble carbohydrates (WSC) in stems play an important role in buffering grain yield in wheat against biotic and abiotic stresses; however, knowledge of genes controlling WSC is very limited. We conducted a genome-wide association study (GWAS) using a high-density 90K SNP array to better understand the genetic basis underlying WSC, and to explore marker-based breeding approaches. WSC was evaluated in an association panel comprising 166 Chinese bread wheat cultivars planted in four environments. Fifty two marker-trait associations (MTAs) distributed across 23 loci were identified for phenotypic best linear unbiased estimates (BLUEs), and 11 MTAs were identified in two or more environments. Liner regression showed a clear dependence of WSC BLUE scores on numbers of favorable (increasing WSC content) and unfavorable alleles (decreasing WSC), indicating that genotypes with higher numbers of favorable or lower numbers of unfavorable alleles had higher WSC content. In silico analysis of flanking sequences of trait-associated SNPs revealed eight candidate genes related to WSC content grouped into two categories based on the type of encoding proteins, namely, defense response proteins and proteins triggered by environmental stresses. The identified SNPs and candidate genes related to WSC provide opportunities for breeding higher WSC wheat cultivars.