Association of NOS1AP variants and depression phenotypes in schizophrenia

Background The nitric oxide synthase 1 adaptor protein gene (NOS1AP) has previously been recognised as a schizophrenia susceptibility gene due to its role in glutamate neurotransmission. The gene is believed to inhibit nitric oxide (NO) production activated by the N-methyl-d-aspartate (NMDA) receptor and reduced NO levels have been observed in schizophrenia patients. However, association studies investigating NOS1AP and schizophrenia have produced inconsistent results, most likely because schizophrenia is a clinically heterogeneous disorder. This study aims to investigate the association between NOS1AP variants and defined depression phenotypes of schizophrenia. Methods Nine NOS1AP SNPs, rs1415259, rs1415263, rs1858232, rs386231, rs4531275, rs4656355, rs4657178, rs6683968 and rs6704393 were genotyped in 235 schizophrenia subjects screened for various phenotypes of depression. Result One NOS1AP SNP (rs1858232) was associated with the broad diagnosis of schizophrenia and eight SNPs were associated with depression related phenotypes within schizophrenia. The rs1415259 SNP showed strong association with sleep dysregulation phenotypes of depression. Conclusion Results suggest that NOS1AP variants are associated with various forms of depression in schizophrenia and are more prevalent in males. Limitation Schizophrenia is a clinically heterogeneous disease that can vary greatly between different ethnic and geographic populations so our observations should be viewed with caution until they are independently replicated, particularly in larger patient cohorts.

Phylogenetic analysis of betanodavirus isolates from Australian finfish

In Australia, disease caused by betanodavirus has been reported in an increasing number of cultured finfish since the first report of mortalities in 1990. Partial coat protein gene sequences from the T2 or T4 regions of 8 betanodaviruses from barramundi Lates calcarifer, sleepy cod Oxyeleotris lineolata, striped trumpeter Latris lineata, barramundi cod Cromileptes altivelis, Australian bass Macquaria novemaculata and gold-spotted rockcod Epinephelus coioides from several Australian states were determined. Analysis of the 606 bp nucleotide sequences of the T2 region of 4 isolates demonstrated the close relationship with isolates from the red-spotted grouper nervous necrosis virus (RGNNV) genotype and the Cluster Ia subtype. Comparison of a smaller 289 bp sequence from the T4 region identified 2 distinct groupings of the Australian isolates within the RGNNV genotype. Isolates from barramundi from the Northern Territory, barramundi, sleepy cod, barramundi cod and gold-spotted rockcod from Queensland, and striped trumpeter from Tasmania shared a 96.2 to 99.7%, nucleotide identity with each other. These isolates were most similar to the RGNNV genotype Cluster Ia. Isolates from Australian bass from New South Wales and from barramundi from South Australia shared a 98.6% sequence identity with each other. However, these isolates only shared an 85.8 to 87.9%, identity with the other Australian isolates and representative RGNNV isolates. The closest nucleotide identity to sequences reported in the literature for the New South Wales and South Australian isolates was to an Australian barramundi isolate (Ba94Aus) from 1994. These 2 Australian isolates formed a new subtype within the RGNNV genotype, which is designated as Cluster Ic.

A quantitative-trait genome-wide association study of alcoholism risk in the community: findings and implications

BACKGROUND Given moderately strong genetic contributions to variation in alcoholism and heaviness of drinking (50% to 60% heritability) with high correlation of genetic influences, we have conducted a quantitative trait genome-wide association study (GWAS) for phenotypes related to alcohol use and dependence. METHODS Diagnostic interview and blood/buccal samples were obtained from sibships ascertained through the Australian Twin Registry. Genome-wide single nucleotide polymorphism (SNP) genotyping was performed with 8754 individuals (2062 alcohol-dependent cases) selected for informativeness for alcohol use disorder and associated quantitative traits. Family-based association tests were performed for alcohol dependence, dependence factor score, and heaviness of drinking factor score, with confirmatory case-population control comparisons using an unassessed population control series of 3393 Australians with genome-wide SNP data. RESULTS No findings reached genome-wide significance (p = 8.4 x 10(-8) for this study), with lowest p value for primary phenotypes of 1.2 x 10(-7). Convergent findings for quantitative consumption and diagnostic and quantitative dependence measures suggest possible roles for a transmembrane protein gene (TMEM108) and for ANKS1A. The major finding, however, was small effect sizes estimated for individual SNPs, suggesting that hundreds of genetic variants make modest contributions (1/4% of variance or less) to alcohol dependence risk. CONCLUSIONS We conclude that: - 1) meta-analyses of consumption data may contribute usefully to gene discovery; - 2) translation of human alcoholism GWAS results to drug discovery or clinically useful prediction of risk will be challenging, and; - 3) through accumulation across studies, GWAS data may become valuable for improved genetic risk differentiation in research in biological psychiatry (e.g., prospective high-risk or resilience studies).

Digestion capasity, nutrient digestibilities and physico-chemical conditions in the intestine influenced by the age of growing turkeys

Six experiments have been conducted to examine digestibility and feeding value of domestic Finnish fibre-rich cereals (barley and oats as compared to maize and wheat) and protein sources (rapeseed meal and cake, peas, faba beans, lupin seeds) for growing turkeys and to investigate effects of age of the birds (from 3 to 12 weeks of age) on digestion process and estimated nutrient digestibility and energy values. Besides, an objective of the study was to test applications of digestibility research methodology for turkeys. Total tract digestibility and apparent metabolizable energy (AME) was assayed in experimental cages using excreta collection, and a slaughter method was applied to sample small intestinal digesta for determination of apparent ileal crude protein digestibility (AICPD), jejuno-duodenal digesta viscosity and caecal volatile fatty acid (VFA) concentration. Digesta viscosity decreased and caecal VFA production increased with age of growing turkeys. Digesta retention times in the small intestine were generally longer in the older birds than in the younger ones. Crude fat digestibility and AME increased with age of growing turkeys, especially with viscous diets. AICPD seemed to decrease with age in most cases. Supplementation with β-gucanase-xylanase decreased viscosity, improved crude fat digestibility and metabolizable energy value and increased VFA production especially in barley-fed turkeys and especially in the young birds. Poor protein digestibility and low energy value of rapeseed meal and rapeseed cake decreased their feeding value for turkeys. In addition, a typical goitrogenic effect of rapeseed feeding was detected. Use of legume seeds as feed for growing turkeys is limited mostly by the low energy value in lupin seeds and the low ileal protein and amino acid digestibility in faba beans. Digestibility of fibre-rich protein sources was not improved with age of the turkeys. Euthanizing the turkeys for AICPD determination by carbon dioxide and bleeding led to lower digestibility values than mechanical stunning and cervical dislocation, suggesting inferiority of carbon dioxide stunning in experimental use. Comparison of AICPD and AME results obtained using different markers showed that considerable differences may occur, especially on total tract level, when acid-insoluble ash gave considerably lower AME values than titanium dioxide and chromic oxide.

A single point mutation disrupts the capsid assembly in Sesbania Mosaic Virus resulting in a stable isolated dimer

Protein-protein interactions play a Crucial role in Virus assembly and stability. With the view of disrupting capsid assembly and capturing smaller oligomers, interfacial residue mutations were carried Out in the coat protein gene of Sesbania Mosaic Virus, a T=3 ss (+) RNA plant virus. A single point mutation of a Trp 170 present at the five-fold interface of the virus to a charged residue (Glu or Lys) arrested assembly of virus like particles and resulted in stable Soluble dimers of the capsid Protein. The X-ray crystal structure of one of the isolated dimer mutants - rCP Delta N65W170K was determined to a resolution of 2.65 angstrom. Detailed analysis of the dimeric mutant protein structure revealed that a number of Structural changes take place, especially in the loop and interfacial regions during the course of assembly. The isolated chiller was ``more relaxed'' than the dimer found in the T=3 or T=1 capsids. The isolated dimer does not bind Ca2+ ion and consequently four C-terminal residues are disordered. The FG loop, which interacts with RNA in the Virus, has different conformations in the isolated dimer and the intact Virus Suggesting its flexible nature and the conformational changes that accompany assembly. The isolated choler mutant was much less stable when compared to the assembled capsids, suggesting the importance of inter-subunit interactions and Ca2+ mediated interactions in the stability of the capsids. With this study, SeMV becomes the first icosahedral virus for which X-ray crystal Structures of T=3, T=1 capsids as well as a smaller oligomer of the capsid protein have been determined.

ORP2 is a sterol receptor that regulates cellular lipid metabolism

ORP2 is a member of mammalian oxysterol binding protein (OSBP)-related protein/gene family (ORPs), which is found in almost every eukaryotic organism. ORPs have been suggested to participate in the regulation of cellular lipid metabolism, vesicle trafficking and cellular signaling. ORP2 is a cytosolic protein that is ubiquitously expressed and most abundant in the brain. In previous studies employing stable cell lines with constitutive ORP2 overexpression ORP2 was shown to affect cellular cholesterol metabolism. The aim of this study was to characterize the properties and function of ORP2 further. ORP2 ligands were searched for among sterols and phosphoinositides using purified ORP2 and in vitro binding assays. As expected, ORP2 bound several oxysterols and cholesterol, the highest affinity ligand being 22(R)hydroxycholesterol. In addition, affinity for anionic membrane phospholipids, phosphoinositides was observed, which may assist in the membrane targeting of ORP2. Intracellular localization of ORP2 was also investigated. ORP2 was observed on the surface of cytoplasmic lipid droplets, which are storage organelles for neutral lipids. Lipid droplet targeting of ORP2 was inhibited when 22(R)hydroxycholesterol was added to the cells or when the N-terminal FFAT-motif of ORP2 was mutated, suggesting that oxysterols and the N-terminus of ORP2 regulate the localization and the function of ORP2. The role of ORP2 in cellular lipid metabolism was studied using HeLa cell lines that can be induced to overexpress ORP2. Overexpression of ORP2 was shown to enhance cholesterol efflux from the cells resulting in a decreased amount of cellular free cholesterol. ORP2 overexpressing cells responded to the loss of cholesterol by upregulating cholesterol synthesis and uptake. Intriguingly, also cholesterol esterification was increased in ORP2 overexpressing cells. These results may be explained by the ability of ORP2 to bind and thus transport cholesterol, which most likely leads to changes in cholesterol metabolism when ORP2 is overexpressed. ORP2 function was further investigated by silencing the endogenous ORP2 expression with short interfering RNAs (siRNA) in A431 cells. Silencing of ORP2 led to a delayed break-down of triglycerides under lipolytic conditions and an increased amount of cholesteryl esters in the presence of excess triglycerides. Together these results suggest that ORP2 is a sterol-regulated protein that functions on the surface of cytoplasmic lipid droplets to regulate the metabolism of triglycerides and cholesteryl esters. Although the exact mode of ORP2 action still remains unclear, this study serves as a good basis to investigate the molecular mechanisms and possible cell type specific functions of ORP2.

Mode of action of antitumour antibiotics-III: Modulation of permeability of nuclear membrane in the presence of the antibiotics

The binding characteristics of the antibiotics to nuclei and their effect on the permeability of nuclear membrane with respect to histones and ribonucleic acids have been investigated. The binding constant for chromomycin A3 was found to be 1.4 × 104M?1 and number of binding sites was equal to 3.48 ± 1.08 × 1012 molecules/nuclei. The antibiotic chromomycin A3 enhanced the uptake of lysine-rich histone, actinomycin D decreased the uptake and ethidium bromide had no effect. Chromomycin A3 also enhanced the release of acid insoluble fraction containing RNA from the nuclei, actinomycin D and ethidium bromide inhibited the release of acid insoluble fraction containing RNA. The relevance of this finding to the role of nuclear envelope in understanding the mechanism of action of the antibiotic has been discussed.

GmcA Is a Putative Glucose-Methanol-Choline Oxidoreductase Required for the Induction of Asexual Development in Aspergillus nidulans

18 p.

Phosphorylation of the carboxy-terminal domain of histone H1: effects on secondary structure and DNA condensation

Linker histone H1 plays an important role in chromatin folding. Phosphorylation by cyclin-dependent kinases is the main post-translational modification of histone H1. We studied the effects of phosphorylation on the secondary structure of the DNA-bound H1 carboxy-terminal domain (CTD), which contains most of the phosphorylation sites of the molecule. The effects of phosphorylation on the secondary structure of the DNA-bound CTD were site-specific and depended on the number of phosphate groups. Full phosphorylation significantly increased the proportion of -structure and decreased that of -helix. Partial phosphorylation increased the amount of undefined structure and decreased that of -helix without a significant increase in -structure. Phosphorylation had a moderate effect on the affinity of the CTD for the DNA, which was proportional to the number of phosphate groups. Partial phosphorylation drastically reduced the aggregation of DNA fragments by the CTD, but full phosphorylation restored to a large extent the aggregation capacity of the unphosphorylated domain. These results support the involvement of H1 hyperphosphorylation in metaphase chromatin condensation and of H1 partial phosphorylation in interphase chromatin relaxation. More generally, our results suggest that the effects of phosphorylation are mediated by specific structural changes and are not simply a consequence of the net charge.

野大豆贮藏蛋白的基因的克隆及早期发育的基因表达的研究

豆科植物是动物获取植物蛋白的主要来源之一。豆类种子储藏蛋白作为一种多基因家族的产物专一性地积累于种子的发育过程。植物遗传工程需要深入地了解基因的特异性表达及调控。种子储藏蛋白基因为这类研究提供了一个理想的研究系统。在我国生长着大量的野生豆科资源，许多优良性状被期望应用于将来的植物遗传工程研究．本文选择了一种富含种子储藏蛋白高达50%的野生大豆品种Glycine soja 79-34作为材料进行了以下的研究： 1、大豆7s储藏蛋白基因同源片段的克隆。 以原生质体作为初始材料游离大片段染色体DNA，原生质体经低渗处理而破裂，方法简便，得到的DNA分子量大且重复率高。大片段染色体DNA经EcoRI酶切后，用32P标记的大豆a'-亚基基因作为探针进行分子杂交，得同源片段的杂交带。其中分子量大约为6.3kb的同源片段被克隆到Puc9质粒中，通过x-gal/IPTG培养基及原位杂交初步筛选出5个克隆。进一步的Sourthern分子杂交确认了一个克隆包含7s储藏蛋白a'-亚基基因的同源片段。其详细的序列分析尚需进一步进行。 2、种子储藏蛋白基因在体细胞胚胎发生中的表达。 以授粉20天左右的幼胚作为外植体，在含有2mg/l 2,4,5-T的B5液体培养基中直接诱导愈伤组织，一个月内得到了均一的野生大豆悬浮培养体系。悬浮培养细胞转至含有0.5mg／l萘乙酸，O.1mg／l 2,4-D，0.5mg／l BA，0.5mg/l玉米素的B5液体培养基中，得到了处于不同发育阶段的体细胞胚。分别从体细胞胚及悬浮培养细胞中提取总RNA，以种子储藏蛋白基因为探针，RNA斑点杂交首先证明种子储藏蛋白基因在体细胞胚中转录水平上的表达，而在末分化的悬浮培养细胞中则检测不到．从处于不同发育时期（球形胚，心形胚，鱼雷形胚及带有分化子叶的胚）的体细胞中分别提取盐溶蛋白，用大豆储藏蛋白的兔抗血清做Western blotting分析，发现最早在球形胚中即能检测到种子储藏蛋白的积累，大大早于合子胚发育过程中种子储藏蛋白积累的时期．随着体细胞的发育，储藏蛋白的积累略有增加，但总体水平上不如合子胚发育中的明显。本研究从几个方面探讨了种子储藏蛋白基因在体细胞胚及合子胚发育过程中表达的相似性及差异，并对其产生差异的原因作出了推测。 3、果蝇同源转化基因在植物基因组中的检测及表达的探讨。 果蝇同源转化基因(Homeotic gene)被认为是早期胚胎发育过程中形态发生的开关基因。在动物界中已有80多个同源序列从不同进化水平的动物中分离出来，是一个非常保守而与发育密切相关的基因。本研究以果蝇同源转化基因为探针，分别在野生大豆及土豆的基因组中都检测到了该基因同源序列的存在。用野生大豆染色体DNA的多种内切酶片段做分子杂交分析发现至少有一个拷贝的基因同源性非常高。进一步关于该基因表达的研究发现，在未成熟胚及浸泡过夜的种子中都能检测到该基因的表达，但在休眠种子，萌发5天以上的种子及成熟叶片中未能检测到。该基因的表达只局限于早期发育，因而推测其功能有与动物界中的一般性。

细胞分裂素的测定及其转基因植物的基因表达和调控的研究

细胞分裂素是一类重要的植物激素，它参与调节许多植物的生命活动过程。本文从几个方面研究了细胞分裂素的作用。 在细胞分裂素的活性测定中，通过改进尾穗蔸苋红素合成法建立了一种简便、准确的生物试法，同时还建立了根据物理化学和免疫学原理而测定细胞分裂素的HPLC和ELISA方法，使得细胞分裂素的定量更加准确。经过对上述三种方法的相互验证实验表明，同时采用二种方法可以保证细胞分裂素分析的准确和可靠。 细胞分裂素可以促进黄瓜子叶的扩张。利用离体黄瓜子叶，分析BA诱发其扩张与子叶内源细胞分裂素之间的关系，实验证明，BA能促进玉米素及其核苷的迅速积累，进而诱发子叶的扩张。上述结果还表明，黄瓜子叶可能具有合成细胞分裂素的能力。 荸荠球茎是一种贮藏器官，但实验测定发现其中含有细胞分裂素的生理活性形式——异戊烯基腺嘌呤核苷(iPA)，而且合成它的前体腺嘌呤的含量也十分丰富，考虑到球茎与种子的类似之处，推测它可能做为合成细胞分裂素的一个源，而且其合成途径可能有别于植物其它组织。 农杆菌中的异戊烯基转移酶(ipt)基因是负责细胞分裂素生物合成的关键基因。将ipt基因克隆后对其启动子进行了改造，分别构建了如下三种基因：(1) ipt启动子+ipt编码区和3，区(ipt)，(2)磷酸核酮糖羧化酶小亚基启动子SSU 301+ipt编码区和3，区(SSU -ipt)，(3)豌豆种子特异性启动子viciln+ipt编码区和3，区(vic-ipt)。上述三种基因经农杆菌介导转化烟草，获得了16株再生植株，经Southern杂交证明其中15株的基因组上含有正常整合的ipt基因。Northern杂交表明有13株转基因烟草中的ipt基因能转录出大小正常的ipt mRNA并促进了细胞分裂素的生物合成。 实验表明，转基因烟草中ipt基因的表达受到多种因素的调控。首先启动子决定了ipt基因的表达模式，SSU -ipt基因的表达受光的诱导，黑暗中这种基因的转录完全停止，而vic-ipt基因的表达是种子特异性的，它不在烟草营养生长器官如根、茎、叶和愈伤组织中表达。第二，生长素能降低ipt基因的表达活性。第三，在整体植物的根中，存在某些反式因子，能够控制ipt基因的过量表达，这其中可能涉及到细胞内的蛋白因子、基因的甲基化作用及细胞分裂素的反馈调节等。 vic-ipt基因在烟草种子中的特异性表达导致种子内形成了一个细胞分裂素合成的源(source)。对种子中营养物质积累的研究表明，ipt基因的表达促进了种子干物质的积累，其中作用最明显的是增加种子内蛋白质的合成。转入vic-ipt基因后的烟草种子其萌发率没有显著变化，但幼苗的生长速率明显加快，这表明细胞分裂素能调节植株的生长。 通过Northern杂交检测转基因烟草中基因表达的调控，实验证明，ipt基因的表达明显抑制一组植物病理相关蛋白(PR)基因的转录活性，这组基因编码：几丁质酶，β-1，3一葡萄糖苷酶，伸展蛋白和渗调蛋白。对这些调控作用的生理学意义还有待进一步探索。 上述结果表明，在高等植物中，除了传统上认为根是合成细胞分裂素的部位之外，其它组织和器官也具有合成细胞分裂素的能力，其中合成能力最强的是一些离体组织和贮藏器官。农杆菌中的细胞分裂素生物合成基因(ipt)能够在高等植物的基因组中正常的整合和表达，并受到植物体内生理、发育等多种因素的调控，而与整体植物的正常生理过程协调一致。ipt基因的表达还能够调节植物体的生长和发育，包括种子发育时营养物质的积累、幼苗的生长和某些相关基因的表达。对上述问题的深入研究,必将促进细胞分裂素及其相关生理学和发育学研究的进展。

Hybridization between two serranids, the coney (Cephalopholis fulva) and the creole-fish (Paranthias furcifer), at Bermuda

Intergeneric hybridization between the epinepheline serranids Cephalopholis fulva and Paranthias furcifer in waters off Bermuda was investigated by using morphological and molecular characters. Putative hybrids, as well as members of each presumed parent species, were analyzed for 44 morphological characters and screened for genetic variation at 16 nuclear allozyme loci, two nuclear (n)DNA loci, and three mitochondrial (mt)DNA gene regions. Four of 16 allozyme loci, creatine kinase (CK-B*), fumarase (FH*), isocitrate dehydrogenase (ICDH-S*), and lactate dehydrogenase (LDH-B*), were unique in C. fulva and P. furcifer. Restriction fragments of two nuclear DNA intron regions, an actin gene intron and the second intron in the S7 ribosomal protein gene, also exhibited consistent differences between the two presumed parent species. Restriction fragments of three mtDNA regions—ND4, ATPase 6, and 12S/16S ribosomal RNA—were analyzed to identify maternal parentage of putative hybrids. Both morphological data and nuclear genetic data were found to be consistent with the hypothesis that the putative hybrids were the result of interbreeding between C. fulva and P. furcifer. Mean values of 38 morphological characters were different between presumed parent species, and putative hybrids were intermediate to presumed parent species for 33 of these characters. A principal component analysis of the morphological and meristic data was also consistent with hybridization between C. fulva and P. furcifer. Thirteen of 15 putative hybrids were heterozygous at all diagnostic nuclear loci, consistent with F1 hybrids. Two putative hybrids were identified as post-F1 hybrids based on homozygosity at one nuclear locus each. Mitochondrial DNA analysis showed that the maternal parent of all putative hybrid individuals was C. fulva. A survey of nuclear and mitochondrial loci of 57 C. fulva and 37 P. furcifer from Bermuda revealed no evidence of introgression between the parent species mediated by hybridization.

水稻(Oryza sativa L. ssp japonica)幼苗根质膜蛋白质组分析

根质膜具有重要的生物学功能，它参与了根响应脱落酸（ABA）的一系列活动。尽管已经有很多有关ABA影响根的生长和发育的报道，但是在蛋白质组水平上研究参与ABA信号转导及相关活动的质膜蛋白质的报道还未见到。我们期望利用蛋白质组学技术平台研究外源ABA胁迫下水稻根质膜与ABA功能相关的蛋白质组的变化。 本论文通过双向电泳（2DE）结合质谱（MALDI-TOF MS 和 MALDI-TOF/TOF MS）分析的方法鉴定了102个质膜相关蛋白质。这些蛋白质功能涉及到跨膜运输（16.2%）、胁迫反应（14.3%）、物质运输（4.8%）、细胞骨架动态变化（5.7%）、细胞壁重建（3.8%）、碳代谢和能量循环（13.3%）、蛋白质代谢（14.3%）、信号转导（18.1%）和其他功能的蛋白质（4.8%），以及未知功能的蛋白质（2.9%）。其中大约30％的蛋白质以同工型的形式存在。在这些鉴定结果中，有10个斑点（代表10种蛋白质）已被报道为质膜特异的蛋白质;68个蛋白质斑点（代表58种蛋白质）是质膜相关蛋白质。其余54个蛋白质斑点（代表42种蛋白质）是首次在水稻根的质膜囊泡中被鉴定出来。 在ABA处理条件下，我们在2DE胶上发现了15个响应ABA调节的蛋白质斑点。9个上调的蛋白质斑点分别代表以下9种蛋白质：vacuolar proton-ATPase A subunit, vacuolar ATPase B subunit、patatin、 Salt-stress root protein RS1、谷氨酰氨合成酶（Glutamine synthetase，GS）、OSR40c1、H+-exporting ATPase （vacuolar ATPase E subunit）、甘油醛-3-磷酸脱氢酶I型（glyceraldehyde-3- phosphate dehydrogenase, type I，GADPH）和醛缩酶C-1（aldolase C-1）。6个下调的蛋白质斑点分别代表4种蛋白质：endosperm lumenal binding protein、remorin protein、富含脯氨酸蛋白质（glycine-rich protein，GRP）和蔗糖合成酶（sucrose synthase, SuSy）。其中，OSR40c1和endosperm lumenal binding protein与蛋白质合成相关，从它们与ABA的关系中可以看出，ABA可能抑制了细胞的蛋白质合成。而vacuolar proton-ATPase A subunit、vacuolar ATPase B subunit和 H+-exporting ATPase参与了细胞质pH的调控，ABA致使了细胞质pH的上升。甘油醛-3-磷酸脱氢酶I型、醛缩酶C-1和蔗糖合酶参与了细胞壁的生长发育，ABA的作用可能导致了细胞壁生长发育的延迟。ABA促使Patatin上升，其作用可能与质膜膜脂的降解有关。而ABA的刺激也使谷氨酰氨合成酶的表达显著上升，谷氨酰氨合成酶可以去除细胞内有害的游离NH+4。同时还有未知功能的富含脯氨酸蛋白质（glycine-rich protein，GRP）同样受到ABA的诱导，但具体的功能及其与ABA的关系还要进一步的实验证据。

复苏植物牛耳草Boea hygrometrica干旱诱导基因的筛选和功能分析

干旱对植物的影响和植物对干旱的反应是十分复杂的，涉及到植物的各种生理活动，由胁迫强度及时间、植物本身的遗传特性、发育阶段和生理状况以及其他环境因子共同决定。现代分子生物学和生物技术的发展深化了对植物逆境反应的研究，对植物抗旱分子反应的研究成为这个领域的热点，也引发了抗逆基因资源的争夺战。 以拟南芥等植物为实验材料的研究对深入了解植物对干旱胁迫感知和反应机制提供了重要信息, 但对植物抗旱机制的了解仍然十分匮乏。其中， 限制人们对抗旱机制深入了解的一个重要因素就是植物抗旱机制的复杂性和多样性，这种抗旱机制的复杂性决定了不是所有的机制都可通过拟南芥等植物来加以揭示。因此利用特殊生境植物来研究相关基因的表达对揭示植物对环境适应机制有着极为重要的价值。 我们实验室以复苏被子植物旋蒴苣苔（Boea hygrometrica (Bunge) R Br.，牛耳草）为实验材料，开展了多方面的工作，以期从复苏植物的角度加深人们对抗旱机制的了解。目前我们实验室已成功地建立了利用cDNA微阵列技术研究牛耳草基因表达谱的体系，并比较了4562个cDNA克隆在干旱前后的表达差异, 发现434个cDNA在干旱条件下表达水平增加一倍以上。 本工作是在上述工作的基础上，对这些表达差异的cDNA克隆并测序，利用Northern blot进一步验证这些基因。序列分析表明，这434个cDNA片段实际上代表着42个基因。根据序列同源性分析表明其中36个克隆与已知功能的基因具有同源性，它们分别是细胞壁相关基因、LEA基因和糖类、抗氧化酶类的编码基因等。另外，4个克隆未能找到同源序列，这可能意味着它们是一些新基因；2个克隆虽找到同源基因但功能未知。36个克隆中有3个编码的是细胞壁相关基因，它们在干旱早期就被诱导，而编码LEA蛋白的基因在干旱中期或后期大量诱导，这说明牛耳草耐旱反应的启动是程序化的，随干旱时间的延长和程度的加强，一步步地启动相应的基因来发挥作用，多方面地对植物细胞进行保护和修复。 本实验室的前期工作表明牛耳草脱水复水过程中细胞超微结构分发生了明显变化，其中细胞壁脱水时发生折叠复水时恢复原状。鉴于细胞壁如此显著的变化及其重要作用，我们以两个细胞壁相关的基因BhGRP1和BhGLP1为对象，对其表达的时间空间特点和对不同胁迫信号的应答、编码产物的理化性质、过量表达或抑制表达的转基因植物的表现型及转基因植物对不同逆境胁迫的抗/感性状等方面的进行研究，综合分析其在耐旱反应中可能参与的代谢途径或信号途径，以期为揭示牛耳草耐旱复苏机制提供有力的佐证。 我们利用Northern blot和半定量RT-PCR对两基因进行了表达模式分析，发现BhGRP1在干旱早期被诱导，干旱后期其转录本水平下降。而BhGLP1在早期诱导后一直保持高的表达。两者在不同胁迫、激素等处理下都有不同的响应。经PSORT分析两基因编码的蛋白都具有N-端信号肽，意味着两蛋白定位于胞外基质。构建BhGRP1-GFP和BhGLP1-GFP融合蛋白进行亚细胞定位分析，质壁分离后BhGRP1-GFP的信号仅保留在细胞壁，而BhGLP1-GFP则在胞壁胞膜上都存在。过量表达BhGRP1后发现它能赋予植物更强的耐旱复苏能力及机械强度，而抑制GLP表达的植株的抗旱性明显弱于野生型，表明BhGRP1和BhGLP1与牛耳草的耐旱复苏有密切的关系。