cDNA(EST)文库的高通量生物信息学分析体系的构建与应用

应用生物信息学方法,构建了一套针对cDNA或EST文库的高通量、自动化分析体系,CLASP(cDNA Library Analysis SystemPrimary)。CLASP基于Linux操作系统,主要由Perl程序构成。它以cDNA文库(ESTs)序列为分析对象,具有自动查找序列同源基因并进行染色体定位(包括细胞遗传学定位和SIS定位)、EST自动延伸等功能;并对不同来源序列进行聚类分析。应用该体系对3对肺癌相关抑制性消减杂交(SSH)cDNA文库进行了分析。结果在所有3对文库的2083条EST中有1492条找到了同源基因,其中1365条得到染色体定位。对所余591条未知基因的EST进行了电子延伸,其中有214条EST得到不同程度的延伸。对上述cDNA文库中已知基因的EST以及电子延伸后的EST再分别进行聚类分析,而后综合两个聚类分析的结果,由此可发现不同文库间的共同与差异表达基因,可用于特定性状相关的基因功能预测。     

高山植物黄管秦艽cDNA文库构建与表达序列标签（EST）分析

黄管秦艽（Gentiana officinalis）是一种重要的藏药高山植物,本研究构建了该物种开花期的eDNA文库。经检测达到中等cDNA文库水平,文库滴度为1．2×10^7pfu／ml,重组率95．9％,插入片段平均长度大于500bp。对343个随机挑选的重组克隆进行部分测序,获得的ESTs经编辑后共有181条有效序列。经生物信息学方法分析181条表达序列标签（EST）代表144个单克隆序列,其中55个与已鉴定的基因同源,35个序列与未鉴定的EST匹配,54个未找到同源序列;后两者共有89个EST序列未发现功能相似的蛋白。对已鉴定的EST进行功能分析发现,相关基因主要编码以下蛋白：与蛋白表达相关的占35％;光合作用相关的占笠％;新陈代谢相关的占18％;抗性相关的占11％;质膜运输和细胞分裂相关的分别占5％;染色体变化和细胞信号转导的分别占2％。根据有效EST序列设计引物,通过RT-PCR进一验证了所得EST的准确性。这些研究结果为将来研究黄管秦艽的功能基因以及该物种与相关物种的群体遗传学、进化生物学等方面提供了基础。     

杨树基因组中染色体基因丰度分化及EST序列对基因覆盖度的检测(英文)

高等植物基因组中,大部分序列为非表达序列,基因序列所占的比例很小,了解基因在基因组中的分布是研究基因组结构的一个重要方面。在美国能源部资助下,一个毛果杨无性系的基因组测序已经完成并对公众发布。杨树全基因组序列的完成,为我们了解林木基因组中基因的分布提供了一个特例。在本文中,我们利用泊松分析对杨树基因组中基因在各个染色体上的密度进行了检测,结果表明杨树基因组中各条染色体的基因含量存在显著差异。杨树全基因组测序项目揭示现代杨树基因组起源于一次古全基因组复制事件(称为杨柳科基因组复制),所以杨树基因组不同染色体间存在很大的同源复制片段。但是我们的研究显示,杨树基因组中大多数高度同源的染色体上基因的密度与染色体间的同源性没有明显关系,这说明杨柳科全基因组复制事件后,各个高度同源染色体上的基因发生了流失,且基因流失的速率是不一样的。同时本文还对近九万条毛果杨EST序列进行了比对分析,结果显示这些EST序列覆盖的基因仅占杨树基因组中基因总数的16.8%左右。EST测序虽然是发现基因的一个重要手段,但小规模EST测序对基因的覆盖度很低,所以小规模EST测序的应用价值是有限的。     

三叶木通转录组分析及三萜皂苷生物合成途径关键酶基因的发掘

为了解三叶木通(Akebia trifoliata(Thunb.)Koidz.)的三萜皂苷合成途径及其关键酶,本研究对其花、叶、根、茎进行转录组测序,组装获得了57.25 Gb数据,含140 859个unigenes,序列平均长度为1350 bp。KEGG代谢通路富集结果显示,517个unigenes参与三萜皂苷合成相关的3条代谢途径,其中415个unigenes编码三萜皂苷生物合成途径的19个关键酶。对三萜皂苷生物合成过程中的关键酶角鲨烯环氧酶(SE)进行序列分析和同源建模,发现其具有保守的底物结合结构域。将三叶木通茎与花、叶、根的基因表达水平进行比较,发现茎与根相比较其上调基因数目最多,其中295个差异表达基因(DEGs)与三萜皂苷生物合成途径相关。     

XerCD/dif位点特异性重组

大约10%~15%的大肠杆菌在染色体复制过程中会形成染色体二聚体。大肠杆菌染色体编码的重组酶XerC和XerD作用于染色体复制终点区的dif序列,以同源重组的方式将染色体二聚体解离为单体,使细菌得以正常复制分裂。编码霍乱毒素的噬菌体CTXΦ以位点特异的方式整合入霍乱弧菌染色体,但其基因组中不含有任何重组酶基因,其整合过程需要细菌染色体编码的XerC和XerD重组酶,且整合位点与大肠杆菌dif序列相似。XerCD重组酶基因和dif位点在细菌染色体广泛存在,表明其可能是染色体二聚体解离,噬菌体及其他外源基因成分整合入染色体过程中一种广泛存在的途径。文章对XerCD/dif位点特异性重组在细菌染色体二聚体解离、外源基因整合的研究进展进行综述。     

葡萄果实(E)-β-丁香烯合酶基因的克隆及其表达分析

利用生物信息学方法,通过电子克隆获得葡萄(Vitis vinifera Linn.) (E)-β-丁香烯合酶基因的cDNA序列;以从葡萄品种‘德引84-1’(‘Deyin 84-1’)果肉中提取的mRNA为cDNA模板,利用特异PCR技术克隆得到1个全长1 880 bp的基因,被命名为Vv-ECar(GenBank登录号JF808010),该基因序列包括开放阅读框1 674 bp、3’非翻译编码区209 bp和poly+ (A) 28 bp,可编码557个氨基酸.比对结果显示:葡萄Vv-ECar基因的核苷酸序列与葡萄VvGwECar2基因的同源性达93％,二者编码的氨基酸序列同源性达90.8％,均含有植物萜类合酶家族共有的保守域DDXXD;葡萄Vv-ECar与茶[Camellia sinensis (Linn.)0.Kuntze]和杨(Populus balsamifera subsp.trichocarpa×P.deltoids)的萜类合酶相关基因同源性均在73％以上;分子进化树的分析结果也显示葡萄Vv-ECar基因编码的氨基酸序列与其他植物的同源序列具有高度保守性.半定量RT-PCR和荧光定量PCR分析结果显示:在葡萄果实发育的不同阶段均有Vv-ECar基因的表达,但其相对表达量随果实的发育呈先低后高的趋势,其中在幼果期相对表达量最高.     

高山植物黄管秦艽cDNA 文库构建与表达序列标签(EST) 分析

黄管秦艽( Gentiana officinalis) 是一种重要的藏药高山植物, 本研究构建了该物种开花期的cDNA 文库。经检测达到中等cDNA 文库水平, 文库滴度为1 . 2×107 pfu􊄯ml , 重组率95.9% , 插入片段平均长度大于500 bp。对343 个随机挑选的重组克隆进行部分测序, 获得的ESTs 经编辑后共有181 条有效序列。经生物信息学方法分析181 条表达序列标签(EST) 代表144 个单克隆序列, 其中55 个与已鉴定的基因同源, 35 个序列与未鉴定的EST 匹配, 54 个未找到同源序列; 后两者共有89 个EST 序列未发现功能相似的蛋白。对已鉴定的EST进行功能分析发现, 相关基因主要编码以下蛋白: 与蛋白表达相关的占35%; 光合作用相关的占22%; 新陈代谢相关的占18%; 抗性相关的占11%; 质膜运输和细胞分裂相关的分别占5% ; 染色体变化和细胞信号转导的分别占2%。根据有效EST 序列设计引物, 通过RT-PCR 进一验证了所得EST 的准确性。这些研究结果为将来研究黄管秦艽的功能基因以及该物种与相关物种的群体遗传学、进化生物学等方面提供了基础。     

基于油桐种子3个不同发育时期转录组的油脂合成代谢途径分析

油桐是我国重要的木本油料植物, 过去对油桐的研究主要集中于栽培和常规育种, 与油桐种仁油脂合成相关的分子机理研究还未见报道。文章采用RNA-seq技术对油桐种子油脂合成的3个不同时期的转录组进行比较, 获得了大量差异表达的Unigene序列。在此基础上, 通过GO分类和Pathway富集性分析将这些差异表达Unigene归类于128个代谢途径, 其中包含与油脂合成相关的脂肪酸生物合成和甘油磷脂代谢途径。桐酸经脂肪酸生物合成途径合成后通过甘油磷脂代谢途径以桐油的形式贮存。将这两个代谢途径的Unigene序列在KEGG数据库中进行比对, 获得了一些关键酶的同源蛋白质。文章通过对编码这些同源蛋白质的基因在油桐种子油脂合成期的表达模式进行分析, 以期为油桐油脂合成, 尤其是桐酸合成机理的解析提供理论参考, 并为油桐的遗传改良提供潜在的基因资源, 从而提高油桐的单位面积产量。     

谷子弯孢菌海藻糖酶基因(ClTRE)及其启动子的克隆和序列分析

海藻糖代谢调控真菌生长、发育及致病性,为了进一步研究海藻糖代谢在谷子弯孢病菌中的功能,对海藻糖酶基因及其启动子进行了克隆.根据植物病原真菌海藻糖酶基因的保守序列设计简并引物,扩增得到海藻糖酶基因同源序列.通过RACE技术,首次在谷子弯孢病菌中克隆得到了海藻糖酶基因(ClTRE)全长cDNA序列.序列分析表明,该基因最大开放阅读框为2037 bp,编码679个氨基酸;二级结构预测表明,该蛋白含约40.48％的α螺旋,12.99％的延伸串,6.5％的β转角,40.03％的不规则卷曲;蛋白保守结构域分析发现其含有海藻糖酶特有的保守位点,与其他植物病原真菌中海藻糖酶基因有51％-86％同源性;利用SignalP3.0软件预测谷子弯孢海藻糖酶蛋白具有信号肽.通过染色体步移技术克隆得到其上游启动子序列,利用TFSEARCH软件分析含有多个与逆境胁迫相关的顺式作用元件,初步推测该基因与逆境胁迫相关.谷子弯孢病菌ClTRE基因及其启动子的克隆,为进一步研究该基因在病菌致病中作用以及以该基因为靶位点的化学防控奠定基础.     

表达序列标签数据库搜索鉴定小鼠UBAP1基因及其数字化表达分析

UBAP1(ubiquitin associated protein 1)基因是最近克隆的一个定位于人类染色体9p21-22鼻咽癌杂合性丢失高频区的泛肽相关蛋白家族新成员.为了深入研究UBAP1基因的功能,利用计算机对表达序列标签(expressed sequence tag, EST)、UniGene等数据库进行综合搜索分析,结合cDNA克隆测序的方法, 成功地获得了UBAP1基因在小鼠中的同源基因.小鼠UBAP1基因cDNA全长为2 676 bp,编码一个由441个氨基酸组成的蛋白质,在其蛋白质C端只有一个泛肽相关功能域（UBA domain）.与人UBAP1基因相比,两者编码的氨基酸序列有89%相同.基于EST的数字化表达分析显示UBAP1基因在小鼠正常组织中广泛高表达.     

A genetic map of candidate genes and QTLs involved in tomato fruit size and composition

In order to screen for putative candidate genes linked to tomato fruit weight and to sugar or acid content, genes and QTLs involved in fruit size and composition were mapped. Genes were selected among EST clones in the TIGR tomato EST database (http://www.tigr.org/tdb/tgi/lgi/) or corresponded to genes preferentially expressed in the early stages of fruit development. These clones were located on the tomato map using a population of introgression lines (ILs) having one segment of Lycopersicon pennellii (LA716) in a L. esculentum (M82) background. The 75 ILs allowed the genome to be segmented into 107 bins. Sixty-three genes involved in carbon metabolism revealed 79 loci. They represented enzymes involved in the Calvin cycle, glycolysis, the TCA cycle, sugar and starch metabolism, transport, and a few other functions. In addition, seven cell-cycle-specific genes mapped into nine loci. Fourteen genes, primarily expressed during the cell division stage, and 23 genes primarily expressed during the cell expansion stage, revealed 24 and 26 loci, respectively. The fruit weight, sugars, and organic acids content of each IL was measured and several QTLs controlling these traits were mapped. Comparison between map location of QTLs and candidate gene loci indicated a few candidate genes that may influence the variation of sugar or acid contents. Furthermore, the gene/QTL locations could be compared with the loci mapped in other tomato populations.     

Proanthocyanidin synthesis and expression of genes encoding leucoanthocyanidin reductase and anthocyanidin reductase in developing grape berries and grapevine leaves

Proanthocyanidins (PAs), also called condensed tannins, can protect plants against herbivores and are important quality components of many fruits. Two enzymes, leucoanthocyanidin reductase (LAR) and anthocyanidin reductase (ANR), can produce the flavan-3-ol monomers required for formation of PA polymers. We isolated and functionally characterized genes encoding both enzymes from grapevine (Vitis vinifera L. cv Shiraz). ANR was encoded by a single gene, but we found two highly related genes encoding LAR. We measured PA content and expression of genes encoding ANR, LAR, and leucoanthocyanidin dioxygenase in grape berries during development and in grapevine leaves, which accumulated PA throughout leaf expansion. Grape flowers had high levels of PA, and accumulation continued in skin and seeds from fruit set until the onset of ripening. VvANR was expressed throughout early flower and berry development, with expression increasing after fertilization. It was expressed in berry skin and seeds until the onset of ripening, and in expanding leaves. The genes encoding LAR were expressed in developing fruit, particularly in seeds, but had low expression in leaves. The two LAR genes had different patterns of expression in skin and seeds. During grape ripening, PA levels decreased in both skin and seeds, and expression of genes encoding ANR and LAR were no longer detected. The results indicate that PA accumulation occurs early in grape development and is completed when ripening starts. Both ANR and LAR contribute to PA synthesis in fruit, and the tissue and temporal-specific regulation of the genes encoding ANR and LAR determines PA accumulation and composition during grape berry development.     

Abscisic acid,sucrose, and auxin coordinately regulate berry ripening process of the Fujiminori grape

The aim of this study was to examine the effect of abscisic acid (ABA), sucrose, and auxin on grape fruit development and to assess the mechanism of these three factors on the grape fruit ripening process. Different concentrations of ABA, sucrose, and auxin were used to treat the grape fruit, and the ripening-related indices, such as physiological and molecular level parameters, were analyzed. The activity of BG protein activity was analyzed during the fruit development. Sucrose, ABA, and auxin influenced the grape fruit sugar accumulation in different ways, as well as the volatile compounds, anthocyanin content, and fruit firmness. ABA and sucrose induced, but auxin blocked, the ripening-related gene expression levels, such as softening genes PE, PG, PL, and CELL, anthocyanin genes DFR, CHI, F3H, GST, CHS, and UFGT, and aroma genes Ecar, QR, and EGS. ABA, sucrose, and glucose induced the fruit dry weight accumulation, and auxin mainly enhanced fruit dry weight through seed weight accumulation. In the early development of grape, starch was the main energy storage; in the later, it was glucose and fructose. Sucrose metabolism pathway-related gene expression levels were significant for glucose and fructose accumulation. BG protein activity was important in the regulation of grape ABA content levels. ABA plays a core role in the grape fruit development; sucrose functions in fruit development through two pathways: one was ABA dependent, the other ABA independent. Auxin blocked ABA accumulation to regulate the fruit development process.