与高等植物吸收(NO_3)~-/(NH_4)~+、(PO_4)~(3-)和K~+有关的膜转运蛋白编码基因的分子生物学研究现状

高等植物对土壤中营养元素的吸收是其一切生命活动过程的基础,尤其在营养元素缺乏的状态下,更与其抗营养饥饿等特性息息相关。兼于土壤中Ｎ、Ｐ、Ｋ元素缺乏的严重性与普遍性,以及Ｎ、Ｐ、Ｋ对高等植物生长和发育的重要性,有关高等植物吸收营养元素的膜转运蛋白编码基因的分子生物学研究已引起有关学者的高度重视。ＮＯ－３／ＮＨ＋４、ＰＯ３－４与Ｋ＋膜转运蛋白均有低亲和力和高亲和力系统（ＬｏｗＡｆｉｎｉｔｙＴｒａｎｓｐｏｒｔｅｒ＆ＨｉｇｈＡｆｉｎｉｔｙＴｒａｎｓｐｏｒｔｅｒ）。对ＰＯ４３－和Ｋ＋而言,低亲和力系统是组成性表达的系统,在正常营养状态下对根系吸收营养起重要作用。而高亲和力系统是受营养缺乏而诱导表达的系统,对于植物的抗逆性、耐营养饥饿至关重要。迄今为止,与之有关的基因的全长ｃＤＮＡ或全基因已在几种植物中被克隆。此外,对基因的表达特性亦有广泛研究。本文简要概述这三大营养元素的膜转运蛋白编码基因的分子生物学研究现状。     

Expression studies of plant genes differentially expressed in leaf and root tissues of tomato colonised by the arbuscular mycorrhizal fungus Glomus mosseae

Arbuscular mycorrhizal (AM) fungi are a multifaceted group of mutualistic symbionts that are common to terrestrial ecosystems. The interaction between AM fungi and plant roots is of environmental and agronomic importance. Understanding the molecular changes within the host plant upon AM fungal colonisation is a pre-requisite to a greater understanding of the mechanisms underlying the interaction. Differential mRNA display was conducted on leaf tissue of tomato plants colonised and non-colonised by the AM fungus Glomus mosseae and five putative differentially regulated cDNAs were identified. All cDNAs isolated shared high sequence similarity to known plant genes. Differential screening was initially used to establish whether the cDNAs were differentially expressed. Semi-quantitative RT-PCR was used to establish gene expression patterns for all five clones within leaf and root tissue of mycorrhizal and non-mycorrhizal colonised tomato plants. Differential regulation was observed for all five cDNAs. Down-regulation within the leaf tissue of mycorrhizal plants was observed for 4 out of the 5 cDNAs with an up-regulation observed only for one. Tissue specific regulation was observed for several cDNAs, with down-regulation observed in mycorrhizal leaf tissue and up-regulation observed within mycorrhizal root tissue as compared to non-mycorrhizal tissue.     

Regulation of sulfate uptake and expression of sulfate transporter genes in Brassica oleracea as affected by atmospheric H(2)S and pedospheric sulfate nutrition

Demand-driven signaling will contribute to regulation of sulfur acquisition and distribution within the plant. To investigate the regulatory mechanisms pedospheric sulfate and atmospheric H(2)S supply were manipulated in Brassica oleracea. Sulfate deprivation of B. oleracea seedlings induced a rapid increase of the sulfate uptake capacity by the roots, accompanied by an increased expression of genes encoding specific sulfate transporters in roots and other plant parts. More prolonged sulfate deprivation resulted in an altered shoot-root partitioning of biomass in favor of the root. B. oleracea was able to utilize atmospheric H(2)S as S-source; however, root proliferation and increased sulfate transporter expression occurred as in S-deficient plants. It was evident that in B. oleracea there was a poor shoot to root signaling for the regulation of sulfate uptake and expression of the sulfate transporters. cDNAs corresponding to 12 different sulfate transporter genes representing the complete gene family were isolated from Brassica napus and B. oleracea species. The sequence analysis classified the Brassica sulfate transporter genes into four different groups. The expression of the different sulfate transporters showed a complex pattern of tissue specificity and regulation by sulfur nutritional status. The sulfate transporter genes of Groups 1, 2, and 4 were induced or up-regulated under sulfate deprivation, although the expression of Group 3 sulfate transporters was not affected by the sulfate status. The significance of sulfate, thiols, and O-acetylserine as possible signal compounds in the regulation of the sulfate uptake and expression of the transporter genes is evaluated.     

Differential regulation of nramp and irt metal transporter genes in wild type and iron uptake mutants of tomato

Metal transporters regulated by iron can transport a variety of divalent metals, suggesting that iron regulation is important for specificity of iron transport. In plants, the iron-regulated broad-range metal transporter IRT1 is required for uptake of iron into the root epidermis. Functions of other iron-regulated plant metal transporters are not yet established. To deduce novel plant iron transport functions we studied the regulation of four tomato metal transporter genes belonging to the nramp and irt families with respect to environmental and genetic factors influencing iron uptake. We isolated Lenramp1 and Lenramp3 from tomato and demonstrate that these genes encode functional NRAMP metal transporters in yeast, where they were iron-regulated and localized mainly to intracellular vesicles. Lenramp1 and Leirt1 revealed both root-specific expression and up-regulation by iron deficiency, respectively, in contrast to Leirt2 and Lenramp3. Lenramp1 and Leirt1, but not Lenramp3 and Leirt2, were down-regulated in the roots of fer mutant plants deficient in a bHLH gene regulating iron uptake. In chloronerva mutant plants lacking the functional enzyme for synthesis of the plant-specific metal chelator nicotianamine Leirt1 and Lenramp1 were up-regulated despite sufficient iron supply independent of a functional fer gene. Lenramp1 was expressed in the vascular root parenchyma in a similar cellular pattern as the fer gene. However, the fer gene was not sufficient for inducing Lenramp1 and Leirt1 when ectopically expressed. Based on our results, we suggest a novel function for NRAMP1 in mobilizing iron in the vascular parenchyma upon iron deficiency in plants. We discuss fer/nicotianamine synthase-dependent and -independent regulatory pathways for metal transporter gene regulation.     

Two tomato non-symbiotic haemoglobin genes are differentially expressed in response to diverse changes in mineral nutrient status

The role of plant non‐symbiotic haemoglobins remains undefined, but recent findings suggest a possible role in plant nitrate nutrition. This study sought to characterize patterns of gene expression for two tomato (Lycopersicon esculentum L.) non‐symbiotic haemoglobin genes, and to examine how diverse changes in nutrient status influences the expression of these two genes. The deduced amino acid sequences of the genes examined, SOLly GLB1 and SOLly GLB2, are 55% identical. SOLly GLB1 mRNA is present in highest abundance in roots and older stems, whereas SOLly GLB2 mRNA abundance is highest in leaves, but detectable in other tissues including roots. The SOLly GLB1 mRNA levels increased rapidly in roots in response to each nutrient treatment examined in hydroponic culture, including the individual removal of phosphate, potassium and iron from the culture medium, as well as by the addition of nitrate. In contrast, the levels of SOLly GLB2 mRNA were not significantly altered in response to these same treatments. These results are the first report that non‐symbiotic haemoglobin gene expression can be influenced by a broad range of changes both in mineral macronutrient and micronutrient status, suggesting a previously unrecognized generic role for non‐symbiotic haemoglobins in processes associated with mineral nutrient nutrition.     

Symbiotic phosphate transport in arbuscular mycorrhizas

Arbuscular mycorrhizal fungi colonize the root systems of most land plants and modulate plant growth by enhancing the availability of nutrients, mainly phosphorus, for plant nutrition. Recently identified genes encoding mycorrhiza-specific plant phosphate transporters have enabled fundamental problems in arbuscular mycorrhizal symbiosis research to be addressed. Because phosphate transport is a key feature of this symbiosis, the study of phosphate transport mechanisms and their gene regulation will further our understanding of the intimate interaction between the two symbiotic partners.     

与芒果子叶切段不定根形成相关基因的cDNA片段的克隆

在研究芒果子叶横切所形成的远轴面和近轴面的不定根形成时发现,只在近轴面形成不定根.该研究利用抑制性扣除杂交(suppres sive subtractivehybridization,SSH)方法,以子叶切段远轴切面作为参照样品,近轴切面作为检测样品,构建正向差异cDNA文库,分离与芒果子叶切段不定根形成相关基因的cDNA片段.经VirtualNorthern杂交分析,获得6个阳性克隆.序列分析和同源性比较结果表明这些cDNA片段均为首次报告,它们分别与转运蛋白、转录调控因子及酶基因的DNA序列同源.     

Regulation of expression of two novel flower-specific genes from tomato (Solanum lycopersicum) by gibberellin.

Two flower-specific cDNAs have been isolated after differential screening of an anther cDNA library. This library was constructed 48 h after GA(3) treatment of buds of the GA-deficient gib-1 mutant of tomato. Northern blot analysis during flower development in tomato demonstrated that the expression of both genes is regulated by gibberellins (GAs). Application of GA(3) to developmentally arrested gib-1 flower buds induced new expression of tgas100 mRNA 48 h post-treatment, while an increased accumulation of tgas105 mRNA was found after 8 h. In situ analyses showed the spatial distribution of the expression of both genes within the tomato flower. One of the deduced polypeptides (TGAS105) displays similarities to cysteine-rich extensin-like proteins, while the other (TGAS100) shows significant homology with a stamen-specific gene of Antirrhinum majus. Based on the deduced protein sequences, the possible function of the encoded proteins is discussed.     

Microarray analysis of the nitrate response in Arabidopsis roots and shoots reveals over 1,000 rapidly responding genes and new linkages to glucose,trehalose-6-phosphate,iron, and sulfate metabolism

The genomic response to low levels of nitrate was studied in Arabidopsis using the Affymetrix ATH1 chip containing more than 22,500 probe sets. Arabidopsis plants were grown hydroponically in sterile liquid culture on ammonium as the sole source of nitrogen for 10 d, then treated with 250 microm nitrate for 20 min. The response to nitrate was much stronger in roots (1,176 genes showing increased or decreased mRNA levels) than in shoots (183 responding genes). In addition to known nitrate-responsive genes (e.g. those encoding nitrate transporters, nitrate reductase, nitrite reductase, ferredoxin reductase, and enzymes in the pentose phosphate pathway), genes encoding novel metabolic and potential regulatory proteins were found. These genes encode enzymes in glycolysis (glucose-6-phosphate isomerase and phosphoglycerate mutase), in trehalose-6-P metabolism (trehalose-6-P synthase and trehalose-6-P phosphatase), in iron transport/metabolism (nicotianamine synthase), and in sulfate uptake/reduction. In many cases, only a few select genes out of several in small gene families were induced by nitrate. These results show that the effect of nitrate on gene expression is substantial (affecting almost 10% of the genes with detectable mRNA levels) yet selective and affects many genes involved in carbon and nutrient metabolism.     

High-affinity nitrate transport in roots of Arabidopsis depends on expression of the NAR2-like gene AtNRT3.1

The NAR2 protein of Chlamydomonas reinhardtii has no known transport activity yet it is required for high-affinity nitrate uptake. Arabidopsis (Arabidopsis thaliana) possesses two genes, AtNRT3.1 and AtNRT3.2, that are similar to the C. reinhardtii NAR2 gene. AtNRT3.1 accounts for greater than 99% of NRT3 mRNA and is induced 6-fold by nitrate. AtNRT3.2 was expressed constitutively at a very low level and did not compensate for the loss of AtNRT3.1 in two Atnrt3.1 mutants. Nitrate uptake by roots and nitrate induction of gene expression were analyzed in two T-DNA mutants, Atnrt3.1-1 and Atnrt3.1-2, disrupted in the AtNRT3.1 promoter and coding regions, respectively, in 5-week-old plants. Nitrate induction of the nitrate transporter genes AtNRT1.1 and AtNRT2.1 was reduced in Atnrt3.1 mutant plants, and this reduced expression was correlated with reduced nitrate concentrations in the tissues. Constitutive high-affinity influx was reduced by 34% and 89%, respectively, in Atnrt3.1-1 and Atnrt3.1-2 mutant plants, while high-affinity nitrate-inducible influx was reduced by 92% and 96%, respectively, following induction with 1 mm KNO(3) after 7 d of nitrogen deprivation. By contrast, low-affinity influx appeared to be unaffected. Thus, the constitutive high-affinity influx and nitrate-inducible high-affinity influx (but not the low-affinity influx) of higher plant roots require a functional AtNRT3 (NAR2) gene.     

两个小麦磷转运蛋白基因的分离、功能鉴定和表达研究

磷是能量代谢、核酸以及许多生物膜合成的重要底物。在光合作用、呼吸作用等过程中发挥了重要作用。中国大多数小麦产区的土壤存在着缺磷的问题。磷饥饿给小麦生产造成了很大损失。培育耐低磷小麦是解决这一问题的一个重要途径。在磷饥饿的过程中,哪些基因的表达发生了变化．它们是如何变化的,弄清楚这些问题对于培育转基因耐低磷小麦具有重要的意义。磷转运蛋白基因在植物吸收磷的过程中发挥着重要作用。利用RT—PCR的方法,我们从普通小麦“小偃54”中分离了两个磷转运蛋白基因TaPT8和TaPHT2;1。通过与酵母突变体互补分析表明这两个基因都能够与磷吸收功能存在缺陷的酵母突变体实现功能互补,在低磷条件下有促进酵母突变体吸收磷的作用。进一步分析表明TaPT8属于Pht1家族。TaPHT2;1属于Pht2家族。运用RQRT—PCR的方法进行分析后发现TaPT8在根中表达,受磷饥饿的诱导;TaPHT2;1主要在绿色组织中表达,受磷饥饿的抑制,受光的诱导。TaPT8可能主要参与了小麦的根从土壤中吸收磷的过程。TaPHT2;1可能在磷从细胞质向叶绿体内转运的过程中发挥了重要作用。