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。     

浅黄根须腹菌磷酸盐转运蛋白基因异源表达分析

为探究外生菌根真菌对油松磷吸收作用的分子机理,以油松优良乡土外生菌根真菌——浅黄根须腹菌Rhizopogon luteolus的磷酸盐转运蛋白基因（RlPT）为对象,在缺陷酵母MB192中进行了异源表达研究。结果显示,该cDNA编码的蛋白质能够互补高亲和力磷酸盐转运蛋白pho84的功能;由不同pH条件下生长试验可知,该蛋白是一个与质子相偶联的运输蛋白;RlPT测算的K_m值为57.90μmol/L磷酸盐;通过酸性磷酸酶的活性检测,进一步验证该基因是具有高亲和力磷酸盐转运蛋白功能的基因。激光共聚焦显微观察表明,该蛋白在低磷条件下多定位于酵母细胞膜上发挥其功能。     

水稻磷酸盐转运蛋白基因的克隆、表达及功能分析

以水稻叶片为材料, 设计一对特异引物, 获得了编码磷酸盐转运蛋白基因OsPT6:1. 聚类和氨基酸保守位点分析指出该基因可能为水稻高亲和力磷酸盐转运蛋白编码基因. 原位杂交与RT-PCR表达结果确定此基因在根与叶片中均表达, 尤以低磷诱导下叶片的叶肉细胞表达量最高. 同源重组表明该基因的表达可以提高毕氏酵母对磷素的吸收效率, 同时其基因的导入可以使高亲和力磷酸盐转运蛋白缺失的酵母突变体的磷素吸收功能得以恢复. 以上结果表明, OsPT6:1为水稻高亲和力磷酸盐转运蛋白的编码基因.     

油茶Pht1;2的克隆及生物信息学分析

目的:克隆和分析油茶高亲和磷转运蛋白基因,为研究其结构和功能打下基础。方法:以油茶品种‘华硕’为试材,通过RT-PCR和RACE的方法克隆出油茶磷酸转运子Pht1基因家族一个成员的全长cDNA序列,命名为CoPht1;2(GenBank登录号:JX412956.1),通过生物信息学技术对其序列的理化性质、结构与功能进行分析和预测。结果:CoPht1;2 CDS长度为1 590bp,编码530个氨基酸,与其它物种的Pht1氨基酸序列具有较高的相似性,其中与杨柳科毛果杨的Pht1相似性最高,达到77.5%;该基因所编码蛋白质的分子量为58.02 kDa,理论等电点pI为8.97,二级结构主要由α-螺旋、β-折叠和不规则卷曲构成,包含12个明显的跨膜螺旋拓扑结构。结论:预测显示该蛋白是一个疏水跨膜蛋白,具有磷转运蛋白的主要特征,初步判定其与油茶磷吸收有关,其功能有待进一步验证。     

植物磷营养高效的分子生物学研究进展

挖掘利用植物自身的磷高效营养遗传资源是农业可持续发展的关键.磷高效营养性状涉及根形态、根分泌物、膜与体内磷转运以及菌根等许多方面,表现为数量遗传性状及受多基因控制.近年来,许多高亲和磷转运子基因已被克隆,磷向地上部转运和磷吸收负反馈调节的控制基因也被发现,对于根系分泌有机酸和酸性磷酸酶的基因的控制也有了一定的了解,但目前对于根毛、排根、根构型以及菌根的营养学意义性状的分子生物学研究进展缓慢.     

植物磷营养高效的分子生物学研究进展

挖掘利用植物自身的磷高效营养遗传资源是农业可持续发展的关键。磷高效营养性状涉及根形态、根分泌物、膜与体内磷转运以及菌根等许多方面,表现为数量遗传性状及受多基因控制。近年来,许多高亲和磷转运子基因已被克隆, 磷向地上部转运和磷吸收负反馈调节的控制基因也被发现, 对于根系分泌有机酸和酸性磷酸酶的基因的控制也有了一定的了解, 但目前对于根毛、排根、根构型以及菌根的营养学意义性状的分子生物学研究进展缓慢。     

磷高效基因型小麦对缺磷胁迫的根际适应性反应

采用奶箱分隔栽培试验法,进行了磷高效与磷低效小麦基因型根际土壤ＰＨ与有效磷变化的研究,结果表明：小麦根际土壤ＰＨ和有效磷含量皆明显低于外围土壤,表现出明显的根际效应特征;磷高效基因型小麦的根际ＰＨ和有效磷含量明显低于磷低效基因型,ＰＨ变异范围和磷素亏缺区也表现明显较大。为了进一步验证磷高效小麦基因型这的这一根际特征,同时进行了施用水溶性,枸溶性磷肥的试验研究,结果表明,以水溶性磷肥对根际ＰＨ和有效     

OsPT8过量表达提高转基因烟草的耐低磷能力

高亲和磷转运蛋白负责植物在低磷条件下吸收和转运磷酸盐,对植物的生长发育至关重要。将水稻中关键的高亲和磷转运蛋白基因OsPT8(A high affinity phosphate transporter gene OsPht1;8,以下简称OsPT8)通过农杆菌介导的方法转入烟草云烟87,以转基因烟草和野生型(云烟87)为材料,设置正常供磷(1 mmol/L Pi)和低磷(0.1 mmol/L Pi)两个处理的沙培试验,检测烟株地上部和地下部的生物量、全磷及有效磷的含量,分析烟草高亲和磷转运蛋白家族基因(NtPT1和NtPT2)的表达差异。结果显示,低磷条件下,OsPT8过量表达转基因株系生物量均显著高于野生型;在正常供磷和低磷条件下,OsPT8过量表达烟草株系全磷含量和有效磷含量均显著高于野生型,这表明高亲和磷转运蛋白基因OsPT8可以提高转基因烟草的耐低磷能力。RT-PCR和Q-PCR结果显示,转基因株系显著提高了烟草高亲和磷转运蛋白基因NtPT1和NtPT2的表达量,表明OsPT8对烟草磷吸收和转运的影响是通过OsPT8基因和烟草NtPT1、NtPT2基因等一个复杂的过程起作用的。     

大豆磷、硫转运蛋白研究进展

磷、硫转运蛋白是大豆（Glycine max（L.）Merr.）体内磷、硫转运的重要载体,参与调节磷和硫酸盐的吸收与转运,对提高大豆的磷、硫利用效率至关重要。大豆磷转运蛋白可划分为Pht1、Pht2、Pht3、Pho1和Pho2 5大家族,目前对Pht1的研究最为深入。大豆14个Pht1家族可分为3个亚家族,他们对磷吸收和转运具有重要作用。大豆硫转运蛋白基因GmSULTR1;2b可在大豆根中特异性表达并被低硫胁迫诱导。本文基于大豆磷、硫的营养吸收、转运与利用过程中的相关性,对Pht1家族以及GmSULTR1;2b基因在大豆中的研究进展进行了综述,并对近年来大豆磷、硫转运蛋白的研究进展及未来的研究方向进行了展望。     

不同基因型小麦磷素营养阈值的研究

利用温室盆栽试验,对不同磷效率小麦基因型进行了磷素营养阈值的研究。结果表明,磷低效基因对缺磷反应敏感,在不施或施磷量较低时,籽粒产量及生物量均较低,随着施磷量增加,产量缓慢增加,表现出对磷肥效应较迟钝的钝的现象,而磷高效小麦基因型在不同施磷肥或施磷量较低条件下的产量与生物量相对较高,表现出明显的耐低磷特性及对土壤难溶性磷的高效活化、吸收能力。且随产丰施磷量增加,磷高效基因型的产量急剧增加,达到一定     

Two cDNAs from potato are able to complement a phosphate uptake-deficient yeast mutant: identification of phosphate transporters from higher plants.

Acquisition as well as translocation of phosphate are essential processes for plant growth. In many plants, phosphate uptake by roots and distribution within the plant are presumed to occur via a phosphate/proton cotransport mechanism. Here, we describe the isolation of two cDNAs, StPT1 and StPT2, from potato (Solanum tuberosum) that show homology to the phosphate/proton cotransporter PHO84 from the yeast Saccharomyces cerevisiae. The predicted products of both cDNAs share 35% identity with the PHO84 sequence. The deduced structure of the encoded proteins revealed 12 membrane-spanning domains with a central hydrophilic region. The molecular mass was calculated to be 59 kD for the StPT1 protein and 58 kD for the StPT2 protein. When expressed in a PHO84-deficient yeast strain, MB192, both cDNAs complemented the mutant. Uptake of radioactive orthophosphate by the yeast mutant expressing either StPT1 or StPT2 was dependent on pH and reduced in the presence of uncouplers of oxidative phosphorylation, such as 2,4-dinitrophenol or carbonyl cyanide m-chlorophenylhydrazone. The K(m) for Pi uptake of the StPT1 and StPT2 proteins was determined to be 280 and 130 microM, respectively. StPT1 is expressed in roots, tubers, and source leaves as well as in floral organs. Deprivation of nitrogen, phosphorus, potassium, and sulfur changed spatial expression as well as the expression level of StPT1. StPT2 expression was detected mainly in root organs when plants were deprived of Pi and to a lesser extent under sulfur deprivation conditions. No expression was found under optimized nutrition conditions or when other macronutrients were lacking.     

Isolation and characterization of a sodium-dependent phosphate transporter gene in Dunaliella viridis

A sodium-dependent phosphate transporter gene, DvSPT1, was isolated from a cDNA library using a probe derived from a subtracted cDNA library of Dunaliella viridis. Sequencing analyses revealed a cDNA sequence of 2649 bp long and encoded an open-reading frame consisting of 672 amino acids. The deduced amino acid sequence of DvSPT1 exhibited 31.2% identity to that of TcPHO from Tetraselmis chui. Hydrophobicity and secondary structure prediction revealed 11 conserved transmembrane domains similar to those found in PHO89 from Saccharomyces cerevisiae and PHO4 from Neurospora crassa. Northern blot analysis indicated that the DvSPT1 expression was induced upon NaCl hyperosmotic stress or phosphate depletion. Functional characterization in yeast Na+ export pump mutant G19 suggested that DvSPT1 encoded a Na+ transporter protein. The gene sequence of GDvSPT1 (7922 bp) was isolated from a genomic library of D. viridis. Southern blot analysis indicated that there exist at least two homologous genes in D. viridis.     

Further Readings in Geomicrobiology

We screened 4908 non-essential gene deletion mutant yeast strains for uranium sensitivity and low accumulation by growth in agar medium containing uranium. All mutant strains grew successfully on agar media containing 0 or 0.2 mM uranium for one week at 30^o C. Thirteen strains with single gene deletions showed reduced growth in the agar medium containing 0.5 mM uranium and were identified as uranium-sensitive mutant strains. The phosphate transporter genes of PHO86, PHO84, PHO2, and PHO87 were among the deleted genes in the uranium-sensitive mutant strains, suggesting that genes concerned with phosphate transport contribute to uranium tolerance. Seventeen single-deletion strains showed lower uranium accumulation than the wild-type after exposure to agar medium containing 0.5 mM uranium, and were identified as mutant strains with low uranium accumulation. Among the deleted genes in these strains were cell membrane proteins, phospholipid-binding proteins, and cell wall proteins, suggesting that cell surface proteins contribute to uranium accumulation.     

OsCYCP1;1, a PHO80 homologous protein,negatively regulates phosphate starvation signaling in the roots of rice (Oryza sativa L.)

Phosphorus is one of the most essential and limiting nutrients in all living organisms, thus the organisms have evolved complicated and precise regulatory mechanisms for phosphorus acquisition, storage and homeostasis. In the budding yeast, Saccharomyces cerevisiae, the modification of PHO4 by the PHO80 and PHO85 complex is a core regulation system. However, the existence and possible functions in phosphate signaling of the homologs of the PHO80 and PHO85 components in plants has yet to be determined. Here we describe the identification of a family of seven PHO80 homologous genes in rice named OsCYCPs. Among these, the OsCYCP1;1 gene was able to partially rescue the pho80 mutant strain of yeast. The OsCYCP1;1 protein was predominantly localized in the nucleus, and was ubiquitously expressed throughout the whole plant and during the entire growth period of rice. Consistent with the negative role of PHO80 in phosphate signaling in yeast, OsCYCP1;1 expression was reduced by phosphate starvation in the roots. This reduction was dependent on PHR2, the central regulator of phosphate signaling in rice. Overexpression and suppression of the expression of OsCYCP1;1 influenced the phosphate starvation signaling response. The inducible expression of phosphate starvation inducible and phosphate transporter genes was suppressed in the OsCYCP1;1 overexpression lines and was relatively enhanced in the OsCYCP1;1 RNAi plants by phosphate starvation. Together, these results demonstrate the role of PHO80 homologs in the phosphate starvation signaling pathway in rice.     

Phosphate transport and sensing in Saccharomyces cerevisiae.

Cellular metabolism depends on the appropriate concentration of intracellular inorganic phosphate; however, little is known about how phosphate concentrations are sensed. The similarity of Pho84p, a high-affinity phosphate transporter in Saccharomyces cerevisiae, to the glucose sensors Snf3p and Rgt2p has led to the hypothesis that Pho84p is an inorganic phosphate sensor. Furthermore, pho84Delta strains have defects in phosphate signaling; they constitutively express PHO5, a phosphate starvation-inducible gene. We began these studies to determine the role of phosphate transporters in signaling phosphate starvation. Previous experiments demonstrated a defect in phosphate uptake in phosphate-starved pho84Delta cells; however, the pho84Delta strain expresses PHO5 constitutively when grown in phosphate-replete media. We determined that pho84Delta cells have a significant defect in phosphate uptake even when grown in high phosphate media. Overexpression of unrelated phosphate transporters or a glycerophosphoinositol transporter in the pho84Delta strain suppresses the PHO5 constitutive phenotype. These data suggest that PHO84 is not required for sensing phosphate. We further characterized putative phosphate transporters, identifying two new phosphate transporters, PHO90 and PHO91. A synthetic lethal phenotype was observed when five phosphate transporters were inactivated, and the contribution of each transporter to uptake in high phosphate conditions was determined. Finally, a PHO84-dependent compensation response was identified; the abundance of Pho84p at the plasma membrane increases in cells that are defective in other phosphate transporters.     

Breeding of wastewater treatment yeasts that accumulate high concentrations of phosphorus

Inorganic phosphate is an essential nutrient. In general, microorganisms take up phosphorus when the extracellular phosphorus concentration is low, but not when it is high. In Saccharomyces cerevisiae, the major phosphate transporters, such as Pho84p, and acid phosphatases (APases), such as Pho5p, are regulated in parallel by the phosphate signal transduction pathway (PHO pathway). We found that PHO mutants expressing PHO84 and PHO5, even under high-P conditions, could take up phosphorus at twice the rate of the wild-type strain. The regulatory pathway for phosphorus accumulation in two wastewater treatment yeasts, Hansenula fabianii J640 and Hansenula anomala J224-1, was found to be similar to that in S. cerevisiae. We screened for mutants of these yeasts that constitutively expressed APase. Such mutants formed blue colonies on high phosphorus concentration agar plates containing 5-bromo-4-chloro-3-indolylphosphate (X-phosphate). We found four mutants of H. fabianii J640 and one mutant of H. anomala J224-1 that accumulated from 2.2 to 3.5 times more phosphorus than the parent strains. The growth rates and abilities to remove dissolved total nitrogen and dissolved organic carbon of the mutants were similar to those of the parent strains. In addition, the mutants removed 95% of dissolved total phosphorus from shochu wastewater, while the parent strain removed only 50%.