荔枝采后果皮褐变过程中差异表达基因的SSH分析

 以‘妃子笑’荔枝果实为材料,采用抑制差减杂交（SSH）与cDNA 微阵列技术相结合,研 究了采后荔枝果皮褐变过程中的基因差异表达。分别以采后0 h 与32 h 的果皮总RNA 为驱动组与检测组, 构建了正向与反向SSH 文库,分别获得了282 个与76 个阳性克隆。通过cDNA 微阵列杂交筛选获得了在 采后32 h 果皮中上调表达克隆17 个,下调表达克隆49 个,分别代表了在采后32 h 果皮中上调表达基因 16 个和下调表达基因17 个,其中有较多的热击蛋白基因、糖代谢相关基因、细胞壁代谢相关基因等。 RT-PCR 检测基因表达结果与cDNA 微阵列杂交结果一致。     

家蚕黄茧近等基因系SSH文库的构建及部分EST序列分析

以家蚕黄茧品系KY(YYCC)和白茧品系C108(+Y+Y+C+C)组配黄茧(C)近等基因系。挑取回交11代的黄茧(C)近等基因系群体中的黄血个体的黄色和浅黄色中部丝腺作为实验材料,构建家蚕黄茧近等基因系抑制消减杂交(SSH)cDNA文库。通过对SSHcDNA文库中随机挑选的104个阳性克隆测序和聚类拼接,得到15个假定基因(contig)、25个已知功能基因(unigenes)和5个未知功能基因(unknown)。获得的表达序列标签(EST)经Blast比对和同源性分析,初步发现这些基因参与家蚕体内能量代谢、基因转录、信号传导、物质转运、细胞生长分裂、细胞结构形成、蛋白质合成与降解、核酸及蛋白质加工、机体防御等诸多生物学过程。分析结果可作为深入研究家蚕茧着色分子机制的基础信息。     

孔石莼在干出胁迫下上调表达基因消减cDNA文库的构建及分析

利用抑制消减杂交技术构建了潮间带绿藻孔石莼(Ulva pertusa)在干出胁迫状态下上调表达基因的消减cDNA文库.获得的阳性克隆经测序得到150条上调表达的EST片段,其中21条为冗余序列(RS),137条非冗余序列(NRS).片段中最长833 bp,最短106 bp,平均长度364 bp.参与比对的EST片段按照...     

链状亚历山大藻抑制消减杂交文库的构建及分析

为了筛选链状亚历山大藻特异表达的发光相关基因,应用抑制消减杂交技术,构建了链状亚历山大藻特异表达的cDNA消减文库,共获得500个克隆,通过基因PCR初步筛选表明插入片段的大小主要集中在250～1000 bp,随机挑选10个阳性克隆进行双向测序和序列比对分析,有2个克隆基因片段与已知基因序列高度同源,分别为荧光素结合蛋白和羧化酶/加氧酶,另有8个克隆基因片段在GenBank中未查找到相应的同源基因,可能为未知新基因序列.这些基因的获得为进一步研究链状亚历山大藻特异表达基因,阐明其发光机理及建立特异甲藻的新型检测方法提供重要依据.     

落叶松体细胞胚原胚团阶段差异表达基因文库的构建与分析

以日本落叶松×华北落叶松杂种无性系胚性细胞系Y35继代培养阶段胚性愈伤组织的cDNA为实验组,体细胞胚成熟阶段培养物的cDNA为对照组,利用抑制性消减杂交技术(SSH)构建了原胚团阶段差异表达基因文库.随机选取800个阳性克隆进行测序,共获得400个UniESTs,将其分为19类,功能分析结果表明这些UniESTs参与代谢、转录、信号转导、转运、细胞生长分裂、细胞结构、细胞命运、蛋白质合成与降解、核酸及蛋白质加工、发育、防御等诸多生物学过程.这些差异表达ESTs的获得为研究植物体细胞胚胎发生的分子机理奠定了基础.     

乙烯利诱导橡胶树胶乳cDNA消减文库的构建

为解析乙烯利刺激巴西橡胶树橡胶增产的分子机制,应用抑制性消减杂交技术(suppression subtractive hybridization,SSH)成功构建了乙烯利刺激条件下橡胶树胶乳与未处理橡胶树胶乳差异表达的cDNA消减文库。经蓝、白斑筛选,共得到了118个阳性克隆。菌落PCR分析结果表明,92%左右的阳性克隆含有大小200～500bp的插入片段。随机选取25个克隆进行了测序,并用基因检索工具(Genbank)对获得的EST进行了Blastx分析,结果表明,5个EST属于无任何序列线索的未知序列,6个属于未知功能的推测蛋白基因,2个属于未知功能的蛋白基因,3个与已知功能基因具有较高的同源性。应用半定量PCR技术,对编号203片段进行的表达分析结果表明,该片段为乙烯利诱导特异表达基因,在乙烯利处理后24h范围内随处理时间的增加而表达量增强,叶片中没有检测到该基因的表达。     

Temporal segregation in spawning between native Yellowstone cutthroat trout and introduced rainbow trout

Hybridisation with introduced taxa poses a threat to native fish populations. Mechanisms of reproductive isolation can limit or prevent hybridisation between closely related species. Understanding how these mechanisms interact between the same species across geographically distinct occurrences of secondary contact, and how regional factors influence them, can inform our understanding of hybridisation as a threat and management actions to mitigate this threat. We used data collected on adult fish migration timing and approximate emergence timing of subsequent juvenile fish paired with genomic data to assess whether temporal isolation in the timing of spawning exists between Yellowstone cutthroat trout, rainbow trout and hybrids in the North Fork Shoshone River drainage in northwest Wyoming. We found evidence that Yellowstone cutthroat trout spawn, on average, two to four weeks later than rainbow trout and hybrids and two environmental covariates related to water temperature and discharge were associated with differences in spawning migration timing. Despite statistical support for Yellowstone cutthroat trout spawning later, disproportionately high numbers of rainbow trout and hybrids, paired with extended spawning seasons, lead to substantial overlap between all genotypes. Our results provide further evidence of temporal segregation in the timing of spawning as a mechanism of reproductive isolation between closely related species, but substantial spawning overlap suggests temporal segregation alone will not be enough to curtail hybridisation in conservation populations.