野生罂粟COR、BBE基因片段融合及其RNAi载体构建

可待因酮还原酶(COR)与小檗碱桥酶(BBE)是吗啡合成代谢途径的关键酶,其活性大小直接影响着吗啡合成途径中生物碱的代谢合成。采用RT-PCR从罂粟幼叶克隆出COR和BBE基因全序列,同源性比较结果显示,它们与GenBank上已报道的COR和BBE基因高度同源。利用blast及分子生物学软件DNAStar对COR和BBE基因的cDNA序列同源性进行分析比较,分别从各基因中筛选和克隆了一段同源性极低、约400～500 bp的片段;并应用重叠PCR法将其拼接成744 bp的融合基因BC,以中间载体pHANNIBAL和植物表达载体pART27为基础,构建了以CaMV 35S启动子驱动的含有“正向BC融合片段- pdk内含子-反向BC融合片段”的ihRNAi植物表达载体,通过转化野生罂粟,初步研究了以COR和BBE基因为靶标的RNAi对内源吗啡合成的抑制效果,为进一步培育低吗啡高蒂巴因的罂粟种质提供了依据。     

二氢黄酮还原酶基因的克隆与转基因烟草的获得

以蒺藜苜蓿(Medicagotrunctulacv.5160)幼果总RNA为模板,采用RT-PCR技术克隆到二氢黄酮还原酶(DFR)基因的cDNA序列,所获得的cDNA序列全长1018 bp,具有完整的ORF,编码337个氨基酸。Blast分析表明,该片段与GenBank中注册的DFR基因同源性为99.80%。以植物表达载体pBI121为基础,构建了Ca MV35S启动子驱动的DFR基因植物表达载体pBIDFR;采用直接转化法将pBIDFR导入根癌农杆菌EHA105,用该菌株对普通烟草进行遗传转化获得6株转基因植株。     

决明查尔酮合成酶全长cDNA的克隆及其原核表达载体的构建

从决明子叶中提取查尔酮合成酶(Chalcone synthase)总RNA,经过RT-PCR获得查尔酮合成酶cDNA,纯化后与pMD18-T载体连接,转化大肠杆菌Top10,获得了查尔酮合成酶全长基因序列.序列测定表明,查尔酮合成酶全长核苷酸长度为1 173 bp,编码390个氨基酸.与GenBank中已发表序列EU430077进行比较,核苷酸同源性为100%.将该基因片段克隆到原核表达栽体pET-28a(+)中,构建重组质粒pET-28a(+)/Chalcone synthase,所获重组质粒经过双酶切、PCR和序列测定,证实含有目的片段,且连接、构建正确,为查尔酮合成酶的进一步表达奠定了基础.     

筛选代表小鼠植入前胚胎紧密化相关基因的EST

分别收集181及241枚昆明白小鼠8细胞早期胚胎及8细胞紧密化胚胎,采用SMART PCR方法直接合成胚胎双链cDNA.进而运用抑制消减杂交技术(SSH)对8细胞早期胚胎及8细胞紧密化胚胎的基因表达进行研究,并将所获得的差异表达产物按片段大小分段分离纯化后克隆入pUCm-T载体中,经PCR鉴定后挑选阳性克隆进行测序,筛选出27个代表8细胞早期胚胎和紧密化8细胞胚胎差别表达基因的cDNA片段;经与GenBank中收录的序列进行同源性匹配分析,证实其中17个eDNA片段为新的EST,提交GenBank后被接受并给予了新序列编号.这1 7个片段均可能为与紧密化密切相关的新基因的表达片段,为今后进一步克隆新的紧密化相关基因的全长cDNA及后续新基因的结构和功能研究打下基础.通过采用不同长度大小片段分别克隆的方法,可获得较长片段的EST,避免差异表达大片段的丢失.     

分蘖洋葱查尔酮异构酶基因的克隆及表达载体的构建

旨在得到分蘖洋葱查尔酮异构酶基因,探明该基因的功能。克隆查尔酮异构酶基因并构建了其超表达和干扰载体,以期得到用于研究查尔酮异构酶基因功能的表达载体。从分蘖洋葱叶片中克隆得到查尔酮异构酶基因cDNA全长743 bp,GenBank登录号为KJ489062。结果显示,该基因633 bp的开放读码框编码210个氨基酸的多肽序列。将PCR扩增克隆得到的分蘖洋葱查尔酮异构酶基因片段连接到干扰载体pCAMBIA1 301和超表达载体SOL2095中,成功构建了35S启动子控制的植物表达双元载体pCAMBIA1301-CHI和SOL2095-CHI。     

一种小鼠可溶性Fas cDNA的克隆与表达

为获得调节鼠Fas Fas配体系统诱导凋亡的作用 ,根据GenBank中小鼠Fas基因碱基序列 ,设计扩增可溶性Fas(solubleFas ,sFas)引物 ,用RT PCR方法从幼鼠胸腺组织中克隆出与人可溶性Fas类似的新的鼠sFas (FasC)cDNA序列 .该序列缺乏Fas基因的跨膜区片段 ,但不改变其阅读框 .采用定向克隆的方法将之插入pUC19中间载体 ,DNA序列测定证实该片段序列与预期序列完全一致 .利用亚克隆的方法将鼠FasC的cDNA片段克隆到真核表达载体pCA13上 ,构建出重组载体pCA13 FasC ,通过脂质体LF2 0 0 0转染至 2 93细胞 .RT PCR和Western印迹证实 ,鼠FasC在 2 93细胞获得高效表达 .凋亡诱导实验表明 ,鼠FasC的表达可阻断Fas诱导凋亡的作用 ,证实了所转染鼠FasC的生物活性 .     

柽柳翻译起始因子(eIF-5A)基因的克隆及原核表达

根据柽柳cDNA文库中获得的eIF-5A基因片段,用RACE技术克隆出其全长cDNA序列.cDNA长度为799 bp,编码159个氨基酸.将该cDNA序列克隆到原核表达载体pET28a中,获得重组质粒pET28a-eIF5A.不同浓度NaCl胁迫下大肠杆菌(Escherichia coli)BL21(pET28a-eIF5A)比E.coli BL21(pET28a)有明显的抗盐性,前者菌株存活率在1.0 mo1·L-1NaCl盐胁迫下是后者的9.3倍,据此认为E.coliBL21(pET28a-eIF5A)的耐盐性可能与eIF-5A基因的表达相关.该基因的GenBank登录号为AY587771(基因)、AAT01416(蛋白).     

小鼠canstatin C端片段基因的克隆及其在大肠杆菌中的表达

为了构建小鼠canstatinC端片段的原核表达载体并在大肠杆菌中表达。以小鼠肝脏组织总RNA为模板,通过RT-PCR扩增小鼠canstatinC端片段(mCan-C)基因,克隆到pMD18-T载体中并进行序列分析。将mCan-C基因定向克隆于原核表达载体pET30a(+)中,构建表达载体pET／mCan-C,转化大肠杆菌BL21(DE3),IPTG诱导表达。结果表明,小鼠canstatinC端片段的cDNA长度为399bp,含有1个终止密码,编码132个氨基酸,与已知的人canstatinC端片段氨基酸的同源性为61％。IPTG诱导mCan-C在大肠杆菌E．coliBL21中表达,表达量约占菌体总蛋白量的28％,重组蛋白主要以包涵体形式存在。首次克隆了小鼠canstatinC端片段的cDNA,IPTG诱导mCan-C在大肠杆菌E．coliBL21中高效表达。小鼠canstatinC端片段的cDNA序列已收入GenBank,接受号为:AY502947。     

木薯SBEI基因片段克隆及块根特异性反义表达载体的构建

为了抑制木薯支链淀粉的合成,提高直链淀粉含量,改变木薯淀粉结构,培育工业用燃料酒精型木薯品种,本研究利用RT-PCR方法,用木薯块根cDNA克隆获得支链淀粉合成的关键酶基因SBEI的部分片段,对其进行序列分析表明与GenBank序列高度同源,同源性为99.22%;并以pBI121为基础,构建了以块根特异性表达启动子Sporamin驱动的SBEI基因反义结构的植物表达载体.     

荠菜CRABS CLAW的克隆与拟南芥遗传转化

根据GenBank收录的CRC基因cDNA序列设计引物,以短角果荠菜(Capsella bursa-pastoris)为材料,通过RT-PCR扩增出与拟南芥CRC基因同源的全长cDNA,进行测序、比对及同源性分析.结果显示,克隆的该基因cDNA序列与报道的拟南芥CRC序列一致性达到93%,可以推断其为荠菜CRC基因的cDNA(cbCRC).以Ti质粒pWM101为载体,构建了由CaMV35S启动子调控的cbCRC基因植物表达载体pWM101-cbCRC, 采用根癌农杆菌滴注柱头法转化拟南芥,获得了转cbCRC基因的拟南芥植株.转基因拟南芥心皮果荚形态大小发生了一定的变化,说明荠菜cbCRC基因在拟南芥中的表达对拟南芥心皮形态和大小都产生了一定影响,但其并没有使拟南芥表现出荠菜短角果的形态.     

Isolation and characterization of cold-regulated transcriptional activator <Emphasis Type="Italic">LpCBF3</Emphasis> gene from perennial ryegrass (<Emphasis Type="Italic">Lolium perenne</Emphasis> L.)

In plants, low temperatures can activate the CBF cold response pathway playing a prominent role in cold acclimation by triggering a set of cold-related gene expressions. CBF homologous gene, designated as LpCBF3, from a cold-tolerant perennial ryegrass (Lolium perenne L.) accession was identified. It carries the sequences for nuclear localization signal (NLS), AP2 DNA-binding domains and an acidic activation present in most of the plant CBF proteins. Southern analysis indicated the presence of three homologs of LpCBF3 gene in perennial ryegrass genome, and only one amino acid variation in LpCBF3 protein between cold-tolerant and -sensitive perennial ryegrass accessions. In their putative promoter regions, some differential regions were found. Northern blotting and RT-PCR analysis found that LpCBF3 reached the highest expression after 1.5 h of cold treatment (4 degrees C). The COR homologous gene, a downstream gene of CBF, can be expressed in the plant stem of cold-tolerant perennial ryegrass accessions without cold treatment. Without cold treatment, the COR gene cannot be activated in cold-sensitive perennial ryegrass accessions. Cold treatment can prompt expression levels of COR homologous genes in both perennial ryegrass accessions. In transgenic Arabidopsis, the overexpression of LpCBF3 with the 35S promoter resulted in dwarf-like plants, later flowering and greater freezing tolerance.     

Characterization of Plasmids Encoding the Phytotoxin Coronatine in Pseudomonas syringae

Coronatine (COR) is a nonhost-specific phytotoxin that substantially contributes to the virulence of several pathovars (pvs.) of Pseudomonas syringae. The COR gene cluster in P. syringae is generally plasmid-encoded in pvs. atropurpurea, glycinea, morsprunorum, and tomato but chromosomally encoded in pv. maculicola. In the present study, we investigated whether the COR plasmids in four pathovars shared other traits including self-transmissibility, conserved oriV/par loci, and insertion sequences (ISs) known to reside on other plasmids in P. syringae. Three COR plasmids were shown to be self-transmissible, and all COR plasmids shared a related oriV/par region. Two COR plasmids hybridized to IS801, an IS element widely distributed in P. syringae. Further analysis of p4180A, a 90-kb COR plasmid in P. syringae pv. glycinea, indicated that multiple copies of IS801 were present on this plasmid, and all copies mapped outside the COR gene cluster. Sequence analysis of the region adjacent to the COR gene cluster in p4180A indicated the presence of additional IS elements including IS870, IS51, and IS1240. The IS elements borne on p4180A may have contributed to horizontal transfer of the COR gene cluster and the evolution of the COR biosynthetic pathway.     

An unusual Group 2 LEA gene family in citrus responsive to low temperature

Six cDNAs representing unique cold-induced sequences have been cloned from the hardy citrus relative Poncirus trifoliata. Among these, pBCORc115 and pBCORc119 were found to belong to the same gene family. Sequencing data indicated that pBCORc115 and pBCORc119 each contained an open reading frame, coding for a 19.8 kDa protein (COR19) and a smaller 11.4 kDa protein (COR11) respectively. Inspection of the deduced amino acid sequences revealed three large repeats in COR19, but only one was present in the COR11. Two elements: a Q-clustered tract and a K-rich motif were identified in each repeat. The K-rich motifs were similar to those of cotton D-11 and Group 2 LEA proteins. A Serine-cluster, a common feature in many Group 2 LEA-like proteins, was also found in these proteins, but it was in an unusual position at the carboxy-terminus. A bipartite motif of basic residues, similar to known nuclear targeting sequences, was also present in COR19 and COR11, suggesting that members of this protein family may have a nuclear targeting function. The expression of COR19 mRNA in response to cold acclimation, drought, flooding, and salinization was examined. COR19 expression in leaf tissue was induced in response to cold acclimation, but repressed during drought and flooding stress.     

Duplication and adaptive evolution of the COR15 genes within the highly cold-tolerant Draba lineage (Brassicaceae)

Plants have evolved diverse adaptive mechanisms that enable them to tolerate abiotic stresses, to varying degrees, and such stresses may have strongly influenced evolutionary changes at levels ranging from molecular to morphological. Previous studies on these phenomena have focused on the adaptive evolution of stress-related orthologous genes in specific lineages. However, heterogenetic evolution of the paralogous genes following duplication has only been examined in a very limited number of stress-response gene families. The COR15 gene encodes a low molecular weight protein that plays an important role in protecting plants from cold stresses. Although two different copies of this gene have been found in the model species, Arabidopsis thaliana, evolutionary patterns of this small gene family in plants have not been previously explored. In this study, we cloned COR15-like sequences and performed evolutionary analyses of these sequences (including those previously reported) in the highly cold-tolerant Draba lineage and related lineages of Brassicaceae. Our phylogenetic analyses indicate that all COR15-like sequences clustered into four clades that corresponded well to the morphological lineages. Gene conversions were found to have probably occurred before/during the divergence of Brassica and Draba lineage. However, repeated, independent duplications of this gene have occurred in different lineages of Brassicaceae. Further comparisons of all sequences suggest that there have been significant inter-lineage differences in evolutionary rates between the duplicated and original genes. We assessed the likelihood that the differences between two well-supported gene subfamilies that appear to have originated from a single duplication, COR15a and COR15b, within the Draba lineage have been driven by adaptive evolution. Comparisons of their non-synonymous/synonymous substitution ratios and rates of predicted amino acid changes indicate that these two gene groups are evolving under different selective pressures and may be functionally divergent. This functional divergence was confirmed by comparing site-specific shifts in evolution indexes of the two groups of predicted proteins. The evidence of differential selection and possible functional divergence suggests that the duplication may be of adaptive significance, with possible implications for the explosive diversification of the Draba lineage during the cooling Quaternary stages and the following worldwide colonization of arid alpine and artic regions.     

雷琼牛GH基因第五外显子遗传多样性及其分化

对16头雷琼牛GH基因第5外显子序列进行分析,发现了1个变异位点,定义了2种单倍型。引用巴州牦牛2个个体GH基因同源区序列并结合GenBank中牛属普通牛、其它瘤牛和牦牛3个种群与水牛1个远缘种GH基因同源区序列,分别采用邻接（NJ）法和最大简约（MP）法构建分子系统发育树,得到基本一致的拓扑结构,结果显示GH基因的分化早于雷琼牛（瘤牛）、其它瘤牛、普通牛、牦牛和水牛的分化,瘤牛物种内存在多型,同时证实了GH基因第5外显子区有着较高的突变率。     

大黄鱼16SrRNA和18SrRNA基因的测定与序列分析

利用多对引物,扩增并测定出大黄鱼16SrRNA基因和18SrRNA基因的部分序列,其长度分别为1202bp和1275bp,16SrRNA基因序列的GC含量为46．12％,18SrRNA基因的Gc含量为53．oo％。将大黄鱼16SrRNA基因序列与GenBank中15种硬骨鱼类的同源序列结合,同时将其18SrRNA基因序列与GenBank中9种脊索动物的同源序列相结合,运用软件获得各自序列间差异百分比,转换和颠换数值等信息。基于这两种基因序列,利用NJ法和BI法,分别构建16种硬骨鱼类和10种脊索动物的分子系统树。18SrRNA构建的系统树包括三大支,一支为哺乳类、鸟类和爬行类共6个物种,一支为两栖类的1个物种,另一支为2种硬骨鱼类。16SrRNA构建的系统树显示大黄鱼所在的石首鱼科与鲈科和盖刺鱼科亲缘关系较近。此外还讨论了这两个基因的序列特征。