影响小麦遗传转化的因素及山菠菜BADH 基因导入研究

本研究在前人研究工作的基础上，以小麦转化系统的建立和完善为前提，将BADH基因导入小麦，获得外源BADH基因表达的小麦转基因植株。 （1）小麦不同基因型、不同外植体和不同器官对PPT或bialaphos选择的反应不同，两种试剂对小麦转化体的选择具有同样效果。轰击后的受体材料经过2-3天的恢复生长且植株分化时不用PPT选择可以提高转化效率。冀885-443和石90-4185两个品种对PPT敏感程度适中，具有较强的植株再生能力，得到的转基因植株数和转基因频率均较高。 （2）用pAHC25质粒转化冀885-443等小麦品种取得成功，获得转基因植株12株，平均转基因频率为0.4%。Southern杂交结果表明bar基因已经整合到小麦基因组中。根据研究结果认为，过快过高地提高PPT浓度是造成转基因频率低的主要原因。 （3）采用基因枪法成功地将山菠菜BADH基因（pABH9）导入到冀885-443等品种中。PCR检测和Southern杂交分析证实获得26株转基因植株，不同品种转化频率介于0.3-2.7%，外源BADH基因在转基因植株的叶片内表达。在胁迫条件下有15株转基因植株的BADH酶活力单位明显超过亲本;有6株的相对电导率显著比亲本低，说明这些植株在胁迫条件下细胞受到损伤比亲本低。 （4）采用花粉管通道法向小麦转化pAHC25，筛选出62株抗PPT，转化频率为3.97%。采用农杆菌介导转化冀885-443的成熟胚和幼胚愈伤组织，在转化愈伤组织中观察到gus基因的表达，也得到抗G418的愈伤组织，但没能得到再生植株。

BADH基因转化番茄提高耐盐性的研究

甜菜碱是植物在盐、干旱或其它胁迫下在细胞中迅速积累的一种相容性有机小分子化合物，它在细胞中的积累与植物抗盐性的提高密切相关。甜菜碱醛脱氢酶（BADH）催化甜菜碱醛转化为甜菜碱。我们将来源于耐盐植物山菠菜（Atriplx hortensis L.）的BADH基因通过农杆菌介导法导入‘百丽春’番茄(Lycopersicon esculentum L. ‘Bailichun’)中，并获得15株转化植株，PCR、Southern和Northern检测表明，其中的6株有外源BADH基因的整合，5株中BADH基因能够正常表达，但不同植株间BADH基因的表达水平和BADH酶活力有较大差异。对叶片电导率的测定表明，转基因植株比野生型的耐盐性有较大提高。T1代分析表明，检测的两个转基因株系后代遵循孟德尔分离规律，90mmol/L NaCl胁迫下种子发芽率提高了2~4倍，幼苗的苗高、根长和须根数三个指标均明显优于对照。部分T1代植株在水培条件下能够耐受180mmol/L NaCl胁迫。 植物耐盐的另一机理就是利用液泡膜上存在的转运蛋白将细胞内的有毒离子区域化。我们将已转入编码转运蛋白基因AtNHX1的番茄品种‘Moneymaker’（L. esculentum‘Moneymaker’）株系X1OEA1通过农杆菌介导法转入山菠菜BADH基因，以期获得转双基因耐盐番茄。目前已获得转基因植株，PCR结果证明部分抗性幼苗中已整合了BADH基因，其它各项分子检测正在进行中。

SeNHX1和BADH基因提高植物耐盐性的研究

土壤的盐碱化问题已经严重影响到世界范围内许多重要作物的生产。培育耐盐作物是解决这一问题的最有效途经。利用耐盐相关基因的转化可以在不改变或很少改变植物其它性状的情况下提高植物的耐盐性，因此基因工程方法对于改良植物耐盐性及其机理的研究具有重要的意义。目前植物耐盐基因工程从调控渗透调节物质和盐离子区隔化两个方面开展了较多的研究。已经获得一些耐盐性提高的转基因植物。 本研究拟用耐盐性较强植物山菠菜中的甜菜碱合成关键基因BADH和盐生植物盐角草的液泡膜Na+/H+ anitiporter基因SeNHX1对模式植物烟草进行转化，以确定其各自在耐盐性方面所起的作用。同时，现有的研究表明植物的耐盐性是多基因控制的复杂性状，因此拟把SeNHX1和BADH 这两个涉及不同耐盐机理的基因构建到同一个植物表达载体上，以比较单基因转化和双基因转化在提高植物耐盐性方面的优劣。除此之外，并对已经转入BADH基因番茄的耐盐性和遗传稳定性分析进行了研究。 转BADH基因番茄已经稳定遗传到T4世代。通过对5个转BADH基因番茄株系在T0世代、T3世代和T4世代的分析，表明除了株系T4-3由T0世代的3个拷贝变为1个拷贝外，其余各株系拷贝数均没有发生变化。外源基因编码的酶活性和最终催化产物甜菜碱在盐分胁迫下都能较容易的检测到，说明外源基因在番茄基因组中的遗传是稳定的，没有发生丢失。在连续2个世代的耐盐性鉴定中，各转基因株系的耐盐性较为一致，均比野生型有了较大的提高。其中株系T4-5连续2年表现出了较低的减产率，株系T4-8也在连续的2年中表现出了最高的单株产量。盐分胁迫下转BADH基因各个株系比野生型有较高的K+和Ca2+含量，较低的Na+含量，转基因株系较野生型有较低的脐腐病果率。 通过SeNHX1、BADH单独转化以及构建双价载体共转化的方法获得了3种类型的转基因烟草。Southern和Northern 检测结果表明，外源基因已经整合到烟草基因组中，并得到了正确的表达。转BADH基因烟草在盐分胁迫下能检测到明显的BADH酶活性和甜菜碱含量。转基因烟草T0代对盐分胁迫、氧化胁迫的抗性均较野生型对照有较大的提高。转基因株系在200 mM NaCl胁迫下较野生型有较高的光合速度。百草枯处理过的野生型叶盘比转基因株系积累了更多的丙二醛，表明野生型受到了更大的氧化胁迫。 已经获得3种转不同基因烟草的T1代，且T1代具有较强的耐渗透胁迫能力。转基因烟草的T0种子均能在含100 mg/L 卡钠霉素培养基上发芽和正常生长，其中部分种子能够在含200 mM NaCl 培养基上发芽并能较好的生长，而野生型根本不能发芽。从200 mM甘露醇胁迫1周后，又转移到营养液中的生长1周的情况来看，转基因烟草能较快的恢复正常的生长，有新的叶子和根长出，而野生型却不能，同时转基因株系比野生型具有更大的单株鲜重。 转BADH基因番茄在遗传上是稳定的，并且其耐盐性有了较大的提高。双基因转化烟草的抗盐性要好于单基因转化，但SeNHX1基因转化要好于BADH基因转化。说明SeNHX1基因在提高植物耐盐性方面要比BADH基因有更强的功能，同时，也表明多基因转化在植物的耐盐改良方面可能是一个更为有效的方法。

抗盐、耐海水蔬菜的生物技术培育与海水无土栽培应用

本研究是以植物起源于海洋的系统进化理论和植物细胞的全能性理论为依据的。 对芹菜(Apium graveolensL．)、油菜(B. rapa, chinese group)、叶用甜菜(Beta vulgaris(L.)Koch, Cicla group)、甘蓝(B. oleraceae, acephala group)、豆瓣菜(A'asturtiumofficinale R．Br*.）、番杏（Tetragonla expansa Ait.)、菠菜(Spinacia oleracea　L．)等蔬菜种类进行大规模种质资源筛选和鉴定， 从芹菜、油菜、叶用甜菜等植物中筛选出20多种能够耐受l%NaCI或1/3海水盐度的蔬菜品系。在耐盐蔬菜品种资源筛选的基础上，为了证明用生物技术提高盐敏感蔬菜耐盐性的可行性，本研究以植物体外培养细胞体系为操作平台，对盐敏感的蔬菜一一豆瓣菜进行了生物技术改造。一方面，筛选豆瓣菜的耐盐细胞变异体并使得耐盐细胞再生植株，获得了耐1/3海水的豆瓣菜变异体;另一方面，通过将盐生植物山菠菜(Atriplex hortensisL)的耐盐相关基因，甜菜碱醛脱氢酶(BADH)基因转入豆瓣菜，使得BADH基因在豆瓣菜中过量表达和积累甜菜碱，提高了豆瓣菜的渗透调节能力，从而提高了豆瓣菜的耐盐性。同时，本研究还将所获得的多种抗盐、耐海水蔬菜材料以海水无土栽培的方式进行生产和应用， 取得了很好的效果。 本文的结果证明了在陆地淡水栽培的蔬菜和野生蔬菜资源中，存在着部分耐盐性较强的蔬菜种质;通过生物技术改造能够提高盐敏感蔬菜的耐盐性，并获得抗盐、耐海水的蔬菜新品系。对这些抗盐、耐海水蔬菜材料进行1/3海水无土栽培应用的成功结果表明，某些陆地蔬菜具有重新适应海洋生境的潜能。

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.

Stem cell regulatory gene expression in human adult dental pulp and periodontal ligament cells undergoing odontogenic/osteogenic differentiation

Introduction During development and regeneration, odontogenesis and osteogenesis are initiated by a cascade of signals driven by several master regulatory genes. Methods In this study, we investigated the differential expression of 84 stem cell–related genes in dental pulp cells (DPCs) and periodontal ligament cells (PDLCs) undergoing odontogenic/osteogenic differentiation. Results Our results showed that, although there was considerable overlap, certain genes had more differential expression in PDLCs than in DPCs. CCND2, DLL1, and MME were the major upregulated genes in both PDLCs and DPCs, whereas KRT15 was the only gene significantly downregulated in PDLCs and DPCs in both odontogenic and osteogenic differentiation. Interestingly, a large number of regulatory genes in odontogenic and osteogenic differentiation interact or crosstalk via Notch, Wnt, transforming growth factor β (TGF-β)/bone morphogenic protein (BMP), and cadherin signaling pathways, such as the regulation of APC, DLL1, CCND2, BMP2, and CDH1. Using a rat dental pulp and periodontal defect model, the expression and distribution of both BMP2 and CDH1 have been verified for their spatial localization in dental pulp and periodontal tissue regeneration. Conclusions This study has generated an overview of stem cell–related gene expression in DPCs and PDLCs during odontogenic/osteogenic differentiation and revealed that these genes may interact through the Notch, Wnt, TGF-β/BMP, and cadherin signalling pathways to play a crucial role in determining the fate of dental derived cell and dental tissue regeneration. These findings provided a new insight into the molecular mechanisms of the dental tissue mineralization and regeneration