Effects of Cor15a-IPT Gene Expression on Leaf Senescence in Transgenic Petunia x hybrida and Dendranthema x grandiflorum.

To prevent leaf senescence of young transplants or excised shoots during storage under dark and cold conditions, the cytokinin biosynthetic gene isopentenyl transferase (ipt) was placed under the control of a cold-inducible promoter cor15a from Arabidopsis thaliana and introduced into Petunia x hybrida 'Marco Polo Odyssey' and Dendranthema x grandiflorum (chrysanthemum) 'Iridon'. Transgenic cor15a-ipt petunia and chrysanthemum plants and excised leaves remained green and healthy during prolonged dark storage (4 weeks at 25 degrees C) after an initial exposure to a brief cold-induction period (4 degrees C for 72 h). However, cor15a-ipt chrysanthemum plants and excised leaves that were not exposed to a cold-induction period, senesced under the same dark storage conditions. Regardless of cold-induction treatment, leaves and plants of non-transformed plants senesced under prolonged dark storage. Analysis of ipt expression indicated a marked increase in gene expression in intact transgenic plants as well as in isolated transgenic leaves exposed to a short cold-induction treatment prior to dark storage. These changes correlated with elevated concentrations of cytokinins in transgenic leaves after cold treatment. Cor15a-ipt transgenic plants showed a normal phenotype when grown at 25 degrees C.

sporamin A ipt嵌合质粒的构建及其对马铃薯的转化

细胞分裂素在植物的生长、发育过程中起着重要的作用，来源于农杆菌Ti质粒上基因4区域的ipt基因特异地编码控制细胞分裂素生物合成中的关键酶－－异戊烯基转移酶。 启动子是一类非常重要的基因表达调控元件，在植物的生长、发育过程中控制着基因的时空及顺序表达。来源于甘薯的Sporamin有两种类型（A和B），它是甘薯块根中的主要贮藏蛋白，并且特异地在块根中表达。我们将sporamin A启动子和ipt基因嵌合构成双元载体pBz213，使之进入农杆菌，然后再转化马铃薯，已经得到了具有卡那霉素抗性的小植株，进一步的检测工作正在进行中。在马铃薯块茎中可望异戊烯基转移酶特异地合成，并且能够增加细胞分裂素的水平。

种子特异启动子调控ipt基因表达对烟草种子发育的影响

细胞分裂素（cytokinin，CTK）是五大类植物激素之一，它参与了植物许多生理过程与代谢的调控，主要有促进细胞的分裂和扩大，诱导芽、根和叶绿体的分化，促进种子与果实的发育，解除顶端优势，延缓叶片衰老及增强植物胁迫抗性，调节叶绿体发育基因、营养代谢基因及其它功能基因的表达，调控营养物质的运输和分配等。其调节的植物生理过程也受到其他不同因素的影响。细胞分裂素也是参与植物信号途径间相互作用的一类重要激素。 早期有关细胞分裂素生理作用的研究是基于外源激素的施用来进行的。由于通过外源施用细胞分裂素，其在植物体内的吸收，转运及代谢过程的复杂性和未知性，使得实验研究的因果关系难以确定。随着分子生物学的发展和植物转基因技术的日趋成熟，采用基因工程的方法来研究和探讨细胞分裂素对植物生长发育的调节作用及作用机理是近年来研究的热点，同时也为应用植物激素进行遗传育种提供了广阔的前景。 近年来，越来越多的真核生物启动子的分离克隆，促进了细胞分裂素基因工程的发展。利用具有组织特异性、发育特异性的启动子调控ipt基因，可使ipt基因在植物的特定组织或某一发育阶段进行表达。从而可根据不同的研究目的调控植物转化体中细胞分裂素合成的部位、时间和表达水平。尽管应用一些组织特异启动子融合ipt基因进行了一些细胞分裂素有关生理作用的研究，但是，有关细胞分裂素在胚和种子发育过程中的细胞学方面的研究还很少。 为研究ipt基因在种子发育过程中的作用，我们用大豆种子特异启动子-lectin融合ipt基因转化烟草，获得再生烟草植株。从生理学和细胞学上分析了ipt基因在lectin启动子的控制下的基因表达对种子生长发育的影响。发现在转基因烟草中，lectin-ipt基因的表达促进了种子胚及胚乳的细胞分裂，促使种子胚的生长加快，种子胚的增大为物质的贮存提供了条件，使营养物质更多的向种子运输，主要是可溶性蛋白质含量增加。由此进一步提高了转基因烟草种子干重的增加，种子的萌发与幼苗生长的加快，幼苗鲜重增加。

roIC基因和MT基因特性及其表达的研究

植物种子萌发、开花结实和衰老等一系列生长发育过程，都受到植物激素的影响。细胞分裂素作为重要的生长调节物质，对其传统生物化学和生理学特性的研究已积累子大量资料。随着分子生物学的发展，对植物激素的研究又进一步从单纯的生物学描述阶段深入到分子水平研究的阶段。尤其是近年来对来自病原微生物植物激素相关基因的研究，为揭示细胞分裂素的作用机理和细胸分裂素的水平调节机制的阐明开辟了新的途径。 根瘤农杆菌T-DNA上ipt、iaaM和iaaH基因和发根农杆菌的rol基因表达产物与植物激素的代谢有关。rolC基因是位于发根农杆菌T-DNA区的12号开放读框，编码细胞分裂素-β-葡萄糖苷酶，水解结合态细胞分裂素为自由态细胞分裂素。ipt基因编码异戊烯基转移酶，是细胞分裂素合成过程中的关键酶。 本文用PCR方法从发根农杆菌（Agrobacterium rhizogenes）1601质粒中扩增 rolC基因，并构建CaMV 35S启动子驱动下的双元表达载体。以农杆菌介导的叶盘法，分别对野生型烟草（Nicotiana tabacum L. cv. W38）和已转入异戊烯基转移酶基因（ipt）的3F1和3F2烟草进行转达化。Southern blot和Northern Dot Blot分析表明，rolC基因已导入烟草植株，并具有转录活性。转基因烟草的形态特征与细胞分裂素过量表达的植株表现出的特征一致。 用ELISA方法测定转基因烟草植株中激素的含量，结果显示，单独转rolC基因烟草和同时转入rolC和ipt两个基因的烟草，细胞分裂素的水平有不同程度的提高。转基因烟草表现多芽、节间缩短、叶色深绿等现象。同时，转基因烟草内部发生生理变化，如总自由氨基酸、脯氨酸在正常情况下较对照减少，气孔延迟关闭。在干旱胁迫下，转基因烟草随水势的降低、总自由氨基酸和脯氨酸的变化与对照不同。转基因烟草在开始干旱阶段较对照的总自由氨基酸和脯氨酸含量低，随着干旱胁迫的加深，植物中自由氨基酸的含量增加，但转基因植物自由氨基酸的含量高峰值出现时间较对照推迟。干旱胁迫48小时后，恢复给水，转基因植物较对照易恢复正常生长状态，表明转细胞分裂素基因植物抗旱能力增强。另外，叶片总蛋白SDS-PAGE电泳分析表明，转基因植物蛋白质含量高于对照，某些蛋白组分所占比例也明显提高。 综上所述，转rolC和ipt基因烟草的形态和生理变化，是细胞分裂素过量表达引起植物体内激素失衡的结果。

Controlled Cytokinin Production in Transgenic Tobacco Using a Copper-Inducible Promoter

The cytokinin group of plant hormones regulates aspects of plant growth and development, including the release of lateral buds from apical dominance and the delay of senescence. In this work the native promoter of a cytokinin synthase gene (ipt) was removed and replaced with a Cu-controllable promoter. Tobacco (Nicotiana tabacum L. cv tabacum) transformed with this Cu-inducible ipt gene (Cu-ipt) was morphologically identical to controls under noninductive conditions in almost all lines produced. However, three lines grew in an altered state, which is indicative of cytokinin overproduction and was confirmed by a full cytokinin analysis of one of these lines. The in vitro treatment of morphologically normal Cu-ipt transformants with Cu2+ resulted in delayed leaf senescence and an increase in cytokinin concentration in the one line analyzed. In vivo, inductive conditions resulted in a significant release of lateral buds from apical dominance. The morphological changes seen during these experiments may reflect the spatial aspect of control exerted by this gene expression system, namely expression from the root tissue only. These results confirmed that endogenous cytokinin concentrations in tobacco transformants can be temporally and spatially controlled by the induction of ipt gene expression through the Cu-controllable gene-expression system.

Axillary bud outgrowth: sending a message

Mutants that branch profusely in the presence of a growing shoot tip have highlighted the role of graft-transmissible signals that are produced in roots and stem. Orthologous genes in Arabidopsis, pea and petunia are involved in the transmission of a novel long-distance message. These genes show varying degrees of regulation by auxin and an auxin-independent feedback system, and encode enzymes that might act on carotenoid-like substrates. Axillary bud outgrowth is under homeostatic control, involving developmental stages or checkpoints. Perturbation of the long-range messaging and auxin depletion does not guarantee that bud outgrowth will ensue at a particular node.

Enhanced Cold Tolerance in Transgenic Tobacco Expressing a Chloroplast Omega-3 Fatty Acid Desaturase Gene under the Control of a Cold-inducible Promoter

A new cold-inducible genetic construct was cloned using a chloroplast-specific omega-3-fatty acid desaturase gene (FAD7) under the control of a cold-inducible promoter (cor15a) from Arabidopsis thaliana. RT-PCR confirmed a marked increase in FAD7 expression, in young Nicotiana tabacum (cv. Havana) plants harboring cor15a-FAD7, after a short-term exposure to cold. When young, cold-induced tobacco seedlings were exposed to low-temperature (0.5, 2 or 3.5 degrees C) for up to 44 days, survival within independent cor15a-FAD7 transgenic lines (40.2-96%) was far superior to the wild type (6.7-10.2%). In addition, the major trienoic fatty acid species remained stable in cold-induced cor15a-FAD7 N. tabacum plants under prolonged cold storage while the levels of hexadecatrienoic acid (16:3) and octadecatrienoic acid (18:3) declined in wild type plants under the same conditions (79 and 20.7% respectively). Electron microscopy showed that chloroplast membrane ultrastructure in cor15a-FAD7 transgenic plants was unaffected by prolonged exposure to cold temperatures. In contrast, wild type plants experienced a loss of granal stacking and disorganization of the thylakoid membrane under the same conditions. Changes in membrane integrity coincided with a precipitous decline in leaf chlorophyll concentration and low survival rates in wild type plants. Cold-induced double transgenic N. alata (cv. Domino Mix) plants, harboring both the cor15a-FAD7 cold-tolerance gene and a cor15a-IPT dark-tolerance gene, exhibited dramatically higher survival rates (89-90%) than wild type plants (2%) under prolonged cold storage under dark conditions (2 degrees C for 50 days).

No evidence for association of ataxia-telangiectasia mutated gene T2119C and C3161G amino acid substitution variants with risk of breast cancer

Background There is evidence that certain mutations in the double-strand break repair pathway ataxia-telangiectasia mutated gene act in a dominant-negative manner to increase the risk of breast cancer. There are also some reports to suggest that the amino acid substitution variants T2119C Ser707Pro and C3161G Pro1054Arg may be associated with breast cancer risk. We investigate the breast cancer risk associated with these two nonconservative amino acid substitution variants using a large Australian population-based case–control study. Methods The polymorphisms were genotyped in more than 1300 cases and 600 controls using 5' exonuclease assays. Case–control analyses and genotype distributions were compared by logistic regression. Results The 2119C variant was rare, occurring at frequencies of 1.4 and 1.3% in cases and controls, respectively (P = 0.8). There was no difference in genotype distribution between cases and controls (P = 0.8), and the TC genotype was not associated with increased risk of breast cancer (adjusted odds ratio = 1.08, 95% confidence interval = 0.59–1.97, P = 0.8). Similarly, the 3161G variant was no more common in cases than in controls (2.9% versus 2.2%, P = 0.2), there was no difference in genotype distribution between cases and controls (P = 0.1), and the CG genotype was not associated with an increased risk of breast cancer (adjusted odds ratio = 1.30, 95% confidence interval = 0.85–1.98, P = 0.2). This lack of evidence for an association persisted within groups defined by the family history of breast cancer or by age. Conclusion The 2119C and 3161G amino acid substitution variants are not associated with moderate or high risks of breast cancer in Australian women.

The Expanded Human Kallikrein (KLK) gene family : genomic organisation, tissue specific expression and potential functions

The tissue kallikreins are serine proteases encoded by highly conserved multigene families. The rodent kallikrein (KLK) families are particularly large, consisting of 13 26 genes clustered in one chromosomal locus. It has been recently recognised that the human KLK gene family is of a similar size (15 genes) with the identification of another 12 related genes (KLK4-KLK15) within and adjacent to the original human KLK locus (KLK1-3) on chromosome 19q13.4. The structural organisation and size of these new genes is similar to that of other KLK genes except for additional exons encoding 5 or 3 untranslated regions. Moreover, many of these genes have multiple mRNA transcripts, a trait not observed with rodent genes. Unlike all other kallikreins, the KLK4-KLK15 encoded proteases are less related (25–44%) and do not contain a conventional kallikrein loop. Clusters of genes exhibit high prostatic (KLK2-4, KLK15) or pancreatic (KLK6-13) expression, suggesting evolutionary conservation of elements conferring tissue specificity. These genes are also expressed, to varying degrees, in a wider range of tissues suggesting a functional involvement of these newer human kallikrein proteases in a diverse range of physiological processes.

Membrane associated transporter protein gene (SLC45A2) and the genetic basis of normal human pigmentation variation

This work is concerned with the genetic basis of normal human pigmentation variation. Specifically, the role of polymorphisms within the solute carrier family 45 member 2 (SLC45A2 or membrane associated transporter protein; MATP) gene were investigated with respect to variation in hair, skin and eye colour ― both between and within populations. SLC45A2 is an important regulator of melanin production and mutations in the gene underly the most recently identified form of oculocutaneous albinism. There is evidence to suggest that non-synonymous polymorphisms in SLC45A2 are associated with normal pigmentation variation between populations. Therefore, the underlying hypothesis of this thesis is that polymorphisms in SLC45A2 will alter the function or regulation of the protein, thereby altering the important role it plays in melanogenesis and providing a mechanism for normal pigmentation variation. In order to investigate the role that SLC45A2 polymorphisms play in human pigmentation variation, a DNA database was established which collected pigmentation phenotypic information and blood samples of more than 700 individuals. This database was used as the foundation for two association studies outlined in this thesis, the first of which involved genotyping two previously-described non-synonymous polymorphisms, p.Glu272Lys and p.Phe374Leu, in four different population groups. For both polymorphisms, allele frequencies were significantly different between population groups and the 272Lys and 374Leu alleles were strongly associated with black hair, brown eyes and olive skin colour in Caucasians. This was the first report to show that SLC45A2 polymorphisms were associated with normal human intra-population pigmentation variation. The second association study involved genotyping several SLC45A2 promoter polymorphisms to determine if they also played a role in pigmentation variation. Firstly, the transcription start site (TSS), and hence putative proximal promoter region, was identified using 5' RNA ligase mediated rapid amplification of cDNA ends (RLM-RACE). Two alternate TSSs were identified and the putative promoter region was screened for novel polymorphisms using denaturing high performance liquid chromatography (dHPLC). A novel duplication (c.–1176_–1174dupAAT) was identified along with other previously described single nucleotide polymorphisms (c.–1721C>G and c.–1169G>A). Strong linkage disequilibrium ensured that all three polymorphisms were associated with skin colour such that the –1721G, +dup and –1169A alleles were associated with olive skin in Caucasians. No linkage disequilibrium was observed between the promoter and coding region polymorphisms, suggesting independent effects. The association analyses were complemented with functional data, showing that the –1721G, +dup and –1169A alleles significantly decreased SLC45A2 transcriptional activity. Based on in silico bioinformatic analysis that showed these alleles remove a microphthalmia-associated transcription factor (MITF) binding site, and that MITF is a known regulator of SLC45A2 (Baxter and Pavan, 2002; Du and Fisher, 2002), it was postulated that SLC45A2 promoter polymorphisms could contribute to the regulation of pigmentation by altering MITF binding affinity. Further characterisation of the SLC45A2 promoter was carried out using luciferase reporter assays to determine the transcriptional activity of different regions of the promoter. Five constructs were designed of increasing length and their promoter activity evaluated. Constitutive promoter activity was observed within the first ~200 bp and promoter activity increased as the construct size increased. The functional impact of the –1721G, +dup and –1169A alleles, which removed a MITF consensus binding site, were assessed using electrophoretic mobility shift assays (EMSA) and expression analysis of genotyped melanoblast and melanocyte cell lines. EMSA results confirmed that the promoter polymorphisms affected DNA-protein binding. Interestingly, however, the protein/s involved were not MITF, or at least MITF was not the protein directly binding to the DNA. In an effort to more thoroughly characterise the functional consequences of SLC45A2 promoter polymorphisms, the mRNA expression levels of SLC45A2 and MITF were determined in melanocyte/melanoblast cell lines. Based on SLC45A2’s role in processing and trafficking TYRP1 from the trans-Golgi network to stage 2 melanosmes, the mRNA expression of TYRP1 was also investigated. Expression results suggested a coordinated expression of pigmentation genes. This thesis has substantially contributed to the field of pigmentation by showing that SLC45A2 polymorphisms not only show allele frequency differences between population groups, but also contribute to normal pigmentation variation within a Caucasian population. In addition, promoter polymorphisms have been shown to have functional consequences for SLC45A2 transcription and the expression of other pigmentation genes. Combined, the data presented in this work supports the notion that SLC45A2 is an important contributor to normal pigmentation variation and should be the target of further research to elucidate its role in determining pigmentation phenotypes. Understanding SLC45A2’s function may lead to the development of therapeutic interventions for oculocutaneous albinism and other disorders of pigmentation. It may also help in our understanding of skin cancer susceptibility and evolutionary adaptation to different UV environments, and contribute to the forensic application of pigmentation phenotype prediction.

The proximal first exon architecture of the murine ghrelin gene is highly similar to its human orthologue

BACKGROUND: The murine ghrelin gene (Ghrl), originally sequenced from stomach tissue, contains five exons and a single transcription start site in a short, 19 bp first exon (exon 0). We recently isolated several novel first exons of the human ghrelin gene and found evidence of a complex transcriptional repertoire. In this report, we examined the 5' exons of the murine ghrelin orthologue in a range of tissues using 5' RACE. -----FINDINGS: 5' RACE revealed two transcription start sites (TSSs) in exon 0 and four TSSs in intron 0, which correspond to 5' extensions of exon 1. Using quantitative, real-time RT-PCR (qRT-PCR), we demonstrated that extended exon 1 containing Ghrl transcripts are largely confined to the spleen, adrenal gland, stomach, and skin. -----CONCLUSION: We demonstrate that multiple transcription start sites are present in exon 0 and an extended exon 1 of the murine ghrelin gene, similar to the proximal first exon organisation of its human orthologue. The identification of several transcription start sites in intron 0 of mouse ghrelin (resulting in an extension of exon 1) raises the possibility that developmental-, cell- and tissue-specific Ghrl mRNA species are created by employing alternative promoters and further studies of the murine ghrelin gene are warranted.