植物抗冻基因工程研究进展

提高作物的抗冻性对于提高作物产量有着非常重要的意义。目前,对冷诱导基因、CBF、ICE、抗冻蛋白基因、脂肪酸去饱和酶基因、脯氨酸基因、SOD基因与抗冻的关系进行了广泛的研究,研究表明对这些抗冻相关基因进行转基因,可以提高植物的抗冻性。而一些抗冻基因应用到作物上,也可以提高作物的抗冻性。     

宏基因组学应用于耐盐酶类及耐盐基因研究的进展

耐盐酶在高盐浓度下仍具备催化活性和稳定性,在高盐食品和海产品加工、洗涤及其它高盐环境生物技术领域被广泛应用;耐盐基因在高盐条件下可以使微生物维持正常功能,获取并研究不同环境中的耐盐基因对揭示微生物的耐盐机制,以及实现其在高盐环境中的定向应用具有的重要意义。宏基因组学避开纯培养技术探知微生物的多样性及其功能,为我们提供了一种发现新基因、开发新的微生物活性物质和研究微生物群落结构及其功能的新技术。文中结合本课题组的研究工作,综述了利用宏基因组学获取耐盐酶类及耐盐基因的策略,同时着重介绍利用宏基因组学从海洋、土壤、胃肠道等环境中获取耐盐酶类及耐盐基因的研究。     

Developing salt tolerant plants in a new century: a molecular biology approach

Soil salinity is a major abiotic stress in plant agriculture strongly, influencing plant productivity world-wide. Classical breeding for salt tolerance in crop plants has been attempted to improve field performance without success. Therefore, an alternative strategy is to generate salt tolerant plants through genetic engineering. Several species and experimental approaches have been used in order to identify those genes that are important for salt tolerance. Due to high level of salt tolerance, halophytes are good candidates to identify salt tolerance genes. However, other species such as yeast and glycophytes have also been employed. Three approaches are commonly used to identify genes important for salt tolerance. The first approach is to identify genes involved in processes known to be critical for salt tolerance (osmolyte synthesis, ion homeostasis, etc.). The second approach is to identify genes whose expression is regulated by salt stress. This is relatively simply and applicable to any plant species. Genetic amenability of some species allows the third approach, which consists in the identification of salt tolerance determinants based on functionality. At the moment, there is a large number of reports in the literature claiming that plants with increased salt tolerance have been obtained. The main problem is that different plant species, stage of development, organs, promoters and salt conditions used it is difficult to compare the degree of salt tolerance conferred by different genes. In this review, we discuss progress made towards understanding the molecular elements involved in salt stress responses that have been used in transgenic approaches to improve salt tolerance.     

Nickel tolerance and copper - nickel co - tolerance in Mimulus guttatus from copper mine and serpentine habitats

Previous work on M. guttatus suggested that nickel tolerance in copper mine populations may also be given by the genes for copper tolerance. It has been shown that copper tolerance in M. guttatus is controlled by a single major gene, plus a number of minor genes (or modifiers) which elevate copper tolerance. Crosses between nickel tolerant individuals from three families and non - tolerants showed that nickel tolerance in M. guttatus is heritable. In order to study the effects of the major copper tolerance gene on copper - nickel co - tolerance in M. guttatus, homozygous copper tolerant and non - tolerant lines were screened against nickel. Significant differences occur between these lines for copper, but were not found when analysed for nickel, indicating that copper - nickel tolerance is not governed by the major gene for copper tolerance. To test whether the minor genes for copper have a pleiotropic effect on nickel tolerance, five selection lines derived from three copper mines (Copperopolis, Penn and Quail) in Calaveras county, California, which vary in degree of tolerance to copper, by the presence or absence of minor copper genes, were also screened against nickel. Two out of three of the lines from Copperopolis showed elevated tolerance to nickel, but two further selection lines derived from Penn and Quail copper mines gave no indication of increased nickel tolerance. These results suggest that the minor genes for copper do not give tolerance to nickel. This was confirmed by the screening of modifier lines, in which modifiers for differing degrees of copper tolerance were inserted into a non - tolerant background. Genotypes possessing fewer copper modifiers yielded higher nickel tolerance than those genotypes which have a greater number of modifiers. Thus nickel tolerance in this species is heritable and under the control of different genes to those producing copper tolerance.     

Rapid identification of potential drought tolerance genes from Solanum tuberosum by using a yeast functional screening method

Identification of major stress tolerance genes of a crop plant is important for the rapid development of its stress-tolerant cultivar. Here, we used a yeast functional screen method to identify potential drought-tolerance genes from a potato plant. A cDNA expression library was constructed from hyperosmotic stressed potato plants. The yeast transformants expressing different cDNAs were selected for their ability to survive in hyperosmotic stress conditions. The relative tolerances of the selected yeast transformants to multiple abiotic stresses were also studied. Specific potato cDNAs expressed in the tolerant yeast transformants were identified. Sixty-nine genes were found capable of enhancing hyperosmotic stress tolerance of yeast. Based on the relative tolerance data generated, 12 genes were selected, which could be most effective in imparting higher drought tolerance to potato with better survival in salt and high-temperature stresses. Orthologues of few genes identified here are previously known to increase osmotic stress tolerance of yeast and plants; however, specific studies are needed to confirm their role in the osmotic stress tolerance of potato.     

Genomic library screens for genes involved in n-butanol tolerance in Escherichia coli

Background

n-Butanol is a promising emerging biofuel, and recent metabolic engineering efforts have demonstrated the use of several microbial hosts for its production. However, most organisms have very low tolerance to n-butanol (up to 2% (v/v)), limiting the economic viability of this biofuel. The rational engineering of more robust n-butanol production hosts relies upon understanding the mechanisms involved in tolerance. However, the existing knowledge of genes involved in n-butanol tolerance is limited. The goal of this study is therefore to identify E. coli genes that are involved in n-butanol tolerance.

Methodology/Principal Findings

Using a genomic library enrichment strategy, we identified approximately 270 genes that were enriched or depleted in n-butanol challenge. The effects of these candidate genes on n-butanol tolerance were experimentally determined using overexpression or deletion libraries. Among the 55 enriched genes tested, 11 were experimentally shown to confer enhanced tolerance to n-butanol when overexpressed compared to the wild-type. Among the 84 depleted genes tested, three conferred increased n-butanol resistance when deleted. The overexpressed genes that conferred the largest increase in n-butanol tolerance were related to iron transport and metabolism, entC and feoA, which increased the n-butanol tolerance by 32.8±4.0% and 49.1±3.3%, respectively. The deleted gene that resulted in the largest increase in resistance to n-butanol was astE, which enhanced n-butanol tolerance by 48.7±6.3%.

Conclusions/Significance

We identified and experimentally verified 14 genes that decreased the inhibitory effect of n-butanol tolerance on E. coli. From the data, we were able to expand the current knowledge on the genes involved in n-butanol tolerance; the results suggest that an increased iron transport and metabolism and decreased acid resistance may enhance n-butanol tolerance. The genes and mechanisms identified in this study will be helpful in the rational engineering of more robust biofuel producers.     

大鼠正加速度高耐力相关基因的分离

 为从基因水平上揭示正加速度 (+Gz)高耐力产生机理及寻找 +Gz高耐力相关功能性蛋白 ,利用抑制消减杂交技术分离 +Gz高耐力相关基因 .雄性SD大鼠在离心机上处理后 ,选取耐受终点在高、低两个极端的动物 ,立即取全脑 ,分离mRNA .以高耐力者为Tester ,低耐力者为Driver,利用抑制消减杂交技术进行 +Gz耐力处于高、低两个极端动物脑组织间基因表达差异显示 ,获得 +Gz高耐力大鼠脑组织相关cDNA .以高、低耐力大鼠脑组织mRNA来源的cDNA为探针 ,对获得的cDNA克隆进行斑点杂交 .分别以杂交筛选出的阳性克隆为探针 ,对高、低耐力大鼠脑组织总RNA进行Northern杂交分析 .两次杂交结果均选择高耐力组杂交信号是低耐力组 3倍以上的cDNA克隆 .经过斑点杂交筛选 ,从大鼠脑组织中获得了 6 7个在 +Gz高耐力大鼠脑组织中上调表达的cDNA克隆 .Northern杂交分析发现 ,钙离子 钙调蛋白依赖性蛋白激酶Ⅱβ亚基 (Camk2b)和一未知基因在 +Gz高耐力大鼠脑组织中的表达量增加 .结果提示 ,+Gz耐力处于高、低两个极端的大鼠脑组织基因表达有明显差异 ,这些差异表达的基因很可能与 +Gz高耐力的产生有关 ,且钙离子 钙调蛋白依赖性蛋白激酶Ⅱβ亚基和一未知基因是初步获得的与 +Gz高耐力的产生特异相关的基因     

大豆耐低磷相关基因的定位与克隆

大豆是食用油和植物蛋白的主要来源,土壤有效磷含量低是限制当前大豆生产的重要因素之一。鉴定优异耐低磷种质资源、快速发掘利用优异耐低磷基因、通过分子育种培育磷高效品种、改善大豆应对低磷胁迫的能力,是解决土壤有效磷含量低这一问题的有效途径。近年来,国内外开展了一些大豆磷效率相关基因的定位与克隆研究,但由于QTL连锁作图的精度较低,难以准确地分离候选基因,而大豆基因组的复杂性及相关功能基因分子机制的不明确阻碍了人们对大豆耐低磷特性本质的认识。文章综述了大豆耐低磷相关基因的定位、克隆及功能验证等方面的研究进展,分析了大豆耐低磷相关基因研究中存在的问题,以期为快速有效地分离目的基因、验证基因功能、解析大豆磷高效分子机制提供参考。     

Plant improvement for tolerance to aluminum in acid soils – a review

Development of acid soils that limit crop production is an increasing problem worldwide. Many factors contribute to phytotoxicity of these soils, however, in acid soils with a high mineral content, aluminum (Al) is the major cause of toxicity. The target of Al toxicity is the root tip, in which Al exposure causes inhibition of cell elongation and cell division, leading to root stunting accompanied by reduced water and nutrient uptake. Natural variation for Al tolerance has been identified in many crop species and in some crops tolerance to Al has been introduced into productive, well-adapted varieties. Aluminum tolerance appears to be a complex multigenic trait. Selection methodology remains a limiting factor in variety development as all methods have particular drawbacks. Molecular markers have been associated with Al tolerance genes or quantitative trait loci in Arabidopsis and in several crops, which should facilitate development of additional tolerant varieties. A variety of genes have been identified that are induced or repressed upon Al exposure. Most induced genes characterized so far are not specific to Al exposure but are also induced by other stress conditions. Ectopic over-expression of some of these genes has resulted in enhanced Al tolerance. Additionally, expression of genes involved in organic acid synthesis has resulted in enhanced production of organic acids and an associated increase in Al tolerance. This review summarizes the three main approaches that have been taken to develop crops with Al tolerance: recurrent selection and breeding, development of Al tolerant somaclonal variants and ectopic expression of transgenes to reduce Al uptake or limit damage to cells by Al.     

产酱香地衣芽孢杆菌CGMCC 3963耐受特征及基于转录组学的耐受机制分析

【目的】地衣芽孢杆菌是茅台酒高温大曲中能产酱香风味物质的主要微生物,对酱香型白酒的酿造具有重要价值。而酱香型白酒的酿造环境具有高渗、高温、酸性、高乙醇胁迫等特征,研究产酱香地衣芽孢杆菌在环境胁迫下的耐受特征有利于认识酱香型白酒的酿造特征。【方法】以一株产酱香地衣芽孢杆菌(Bacillus licheniformis CGMCC 3963)为研究对象,测定其耐渗、耐酸、耐乙醇特征,并从比较转录组学角度系统分析B.licheniformis CGMCC 3963的耐受机制。【结果】B.licheniformis CGMCC 3963在15%的KCl、15%的Na Cl、p H 4.0的酸性环境或6%乙醇浓度下的生长情况明显优于不产酱香的模式菌株B.licheniformis ATCC 14580。转录组比较分析显示B.licheniformis CGMCC 3963中一系列与耐受相关的基因表达有差异。【结论】来源于酿造环境的B.licheniformis CGMCC 3963耐受能力强于B.licheniformis ATCC 14580,一系列与耐受相关的基因表达有差异。编码脯氨酸和甜菜碱等溶质转运、离子外排、钾离子通道蛋白等基因的差异表达,使得高渗胁迫下B.licheniformis CGMCC 3963生长明显优于B.licheniformis ATCC 14580;编码II类热休克蛋白、乙醇脱氢酶、氧化应激、p H动态平衡等相关基因的差异表达,在提高菌株耐受酸性环境能力上起了重要作用;II类及III类热休克基因的高表达对B.licheniformis CGMCC 3963耐乙醇能力起了重要作用。