高梁CRY2基因的克隆及其表达分析

从高粱(Sorghum bicolor L. var.R1ll)幼苗中提取总RNA,利用RT-PCR和cDNA的3'末端的快速扩增方法(3'RACE),第一次克隆了高粱隐花色素2基因(CRY2)的cDNA序列.该序列包括了一个完整的开放阅读框,编码大小为690个氨基酸残基的蛋白质,与水稻、番茄和拟南芥CRY2蛋白质的同源性分别为8 7%、5 7%和45.5%.高粱CRY2基因组DNA含有3个内含子和4个外显子.RT-PCR检测结果表明,高粱CRY2基因在根、茎和叶中都有转录.Western blotting结果显示CRY2蛋白在根、茎和叶中表达,并在黑暗中积累,蓝光下降解.高粱CRY2可能在蓝光诱导的幼苗去黄化反应中起作用.     

红树林植物木榄水通道基因的克隆和表达

根据植物水通道基因保守区设计简并引物,采用RT-PCR方法,从木榄树叶中分离出水通道基因的cDNA片段;3′RACE获得3′端cDNA序列;再经5′RACE获得5′端部分cDNA序列,命名为PIP2,GenBank登录号为EF126757。该基因全长843个碱基,编码281个氨基酸,具有典型的植物水通道基因结构。该基因编码的蛋白质与含羞草(PIP2;5)、欧洲葡萄(PIP)、拟南芥(PIP3)等水通道蛋白的同源性分别为90%、91%、88%。Northern杂交分析表明,该基因在木榄树不同器官中的表达差异明显:根部有较高的表达水平,茎部较弱,而在叶中只能检测到微弱的信号。     

甘草肌动蛋白基因GuActin2的克隆和表达分析

以乌拉尔甘草根为材料,从中提取总RNA,根据植物肌动蛋白的5’和3＇末端设计简并引物。采用RT-PCR技术和5’RACE试剂盒,从乌拉尔甘草根中克隆到一个肌动蛋白基因编码区全长cDNA序列（GenBank登录号GQ404511）,长度为1137bp。该基因编码一个由377个氨基酸残基组成的蛋白质。甘草GuActin2具有肌动蛋白（YVGDEAQs．KRG和WISKgEYDE）和肌动蛋白类似物（LLTEApLNPkaNR）的特征信号序列。Northern blot分析表明,GuActin2在甘草的根、茎、叶组织中都有表达,在根中,尤其在胚根中的表达强于茎和叶中的表达。该基因属于营养型亚类。     

黑木耳核糖体失活蛋白cDNA 3′-端的克隆与分析

为了克隆黑木耳核糖体失活蛋白cDNA 3′-端,根据随机测得的中间一段蛋白质序列设计简并引物,应用RT-PCR和3′-RACE反应方法,将得到的基因片段与质粒连接,并转化至大肠杆菌中,进行蓝白筛选,再通过琼脂糖凝胶电泳法和PCR法验证白斑,从而得到阳性重组质粒,最后克隆出该目的蛋白基因片段。结果表明,克隆出的cDNA 3′-端为330bp的开放阅读框(ORF),编码107个氨基酸和2个终止密码子。经试验证明和文献检索,克隆得到的cDNA 3′-端为一种新基因。     

簸箕柳AP3同源基因SsMADS的克隆与特性分析

用cDNA末端快速扩增(rapid-amplification of cDNA ends,RACE)方法从簸箕柳雄花序中克隆了一个AP3同源基因的cDNA,长826 bp,包括完整的编码区、5′-UTR和3′-UTR,并将其相应的基因命名为SsMADS。该基因由7个外显子和6个内含子组成,编码区长723 bp,编码241个氨基酸,其N-端具有典型的MADS保守结构域。序列分析表明,SsMADS编码的氨基酸序列与毛果杨(Populus trichocarp)AP3同源蛋白有95.7%相似性,与其他几种柳属植物的AP3同源蛋白相似性达96.1%～99.6%。实时定量RT-PCR表明,SsMADS在叶、茎和根中表达量极低,在花序中表达量较高,并且其表达量在花器官的早中期发育阶段逐步提高,说明该基因在簸箕柳花器官的发育中起作用。     

野生种马铃薯PHYA基因cDNA的克隆与表达分析

采用RT-PCR技术从野生种马铃薯（Solanum pinnatisectum Dun）中克隆到1个光敏色素基因PHYA,其cDNA全长为3466bp,含有一个3 372bp的完整开放阅读框,编码一条长1123个氨基酸的蛋白,分子量为125kDa,等电点为5．8-该基因编码的蛋白序列与栽培种马铃薯、番茄和烟草基因编码的氧基酸序列同源性分别为96％、94％和91％。半定量RT-PCR分析表明,PHYA在根、茎和芽中的表达量较高;光对PHYA表达的作用在地上部器官中表现为抑制,而在地下部器官中则促进。     

水稻花粉[肌动蛋白]抑制蛋白基因的克隆和表达分析

[肌动蛋白]抑制蛋白（profilin）是一种低分子量、与肌动蛋白结构的蛋白质。通过筛选水稻成熟花粉的cDNA文库,获得了两个全长cDNA片段,序列分析结果表明,两个cDNA片段长度分别为821bp和805bp;共同拥有一个由131个氨基酸组成的开放密码框、5′末端翻译区和一个带有poly(A)的3′区域。[肌动蛋白]抑制蛋白与玉米、C.dactylon、H.brasiliensis、P.pratense中的该蛋白质的同源性分别为89％、87％、83％、89％。Southern杂交分析显示,在基因组至少有两个基因存在。Northern杂交和RT-PCR结果显示它在花粉和花粉中特异表达。     

牡丹PoSAD基因克隆与表达分析

以凤丹牡丹(Paeonia ostii)叶片为试验材料,采用RACE和RT-PCR方法,克隆得到凤丹牡丹硬脂酰-ACP去饱和酶基因SAD的cDNA全长,命名为PoSAD(GenBank登录号为KY038819)。序列分析表明,该基因cDNA序列全长1 559bp,其中开放阅读框1 197bp,编码398个氨基酸,3′端非编码区长172bp,5′端非编码区长123bp。多序列比对结果表明,凤丹牡丹PoSAD氨基酸序列含有2个保守结构域。系统发育分析结果显示,凤丹牡丹与蓖麻处于同一分支,其亲缘关系最近。TMHMM和TargetP亚细胞定位分析得知,PoSAD蛋白无跨膜区域,可能定位于叶绿体中发挥功能。组织特异性结果分析表明,PoSAD基因在凤丹牡丹的根、茎、叶、花瓣、雌蕊、雄蕊、种子中均有表达,且在花瓣中表达量最高,雌蕊中次之,在根中的表达量最低;不同时期种子中,60d表达量最高,80d次之,10d中表达量最低。     

小地老虎隐花色素基因cryptochromel和cryptochrome2的克隆及mRNA表达分析

隐花色素（cryptochrome,cry）是一类广泛存在于生物体内的蓝光和近紫外光受体,介导生物对蓝光的一系列反应并能导引生物钟。本研究利用RT—PCR和RACE方法获得了小地老虎Agrotisypsiloncryl和cry2基因,分别命名为Aycryl和Aycry2。Aycryl基因（GenBankNo．JQ616846）读码框1587bp,编码528个氨基酸,预测分子量60．5ku,等电点6．68。Aycry2基因（GenBankNo．JQ616847）读码框2439bp,编码812个氨基酸,预测分子量92．1ku,等电点8．45。保守区分析表明：Aycryl和Aycry2均含有FAD结合位点的PHR区域和c末端保守区域。氨基酸序列比对分析表明,小地老虎的AyCRY1和AyCRY2分别与其它鳞翅目昆虫的CRY1和CRY2有很高的一致性,其中与棉铃虫HelicoverpaaFmigera的一致性最高,分别为89．5％和73．8％。NJ聚类分析结果表明昆虫含有两类CRY,即CRY1和CRY2,它们可分别以目为单位进行聚类,其中AyCRY1和AyCRY2分别与其它鳞翅目昆虫的CRY1和CRY2聚到一起。以室内饲养的小地老虎为材料,以3h为间隔检测了Aycry1和Aycry2的24h昼夜表达变化,结果表明这2个基因均表现出一定的昼夜节律性。Aycry1和Aycry2表达趋势白天高于晚上,表达峰值出现在ZT7（Zeitgebertime）。方差分析其昼夜波动差异不显著。     

番茄LeNHX1基因植物过量表达载体的构建及表达分析

从番茄幼苗中提取RNA,根据NCBI中番茄LeNHX1基因序列设计引物,通过RT-PCR获得了番茄LeNHX1基因的cDNA序列,包含一个1 605 bp的开放阅读框,编码534个氨基酸。将cDNA序列连接到植物过量表达载体PBI121上,对所获得的重组质粒进行双酶切鉴定,结果表明,植物过量表达载体PBI121-LeNHX1已构建成功。半定量RT-PCR结果表明LeNHX1基因在根、茎和叶中均表达,盐、低温和脱落酸的诱导能提高LeNHX1基因的表达量,推测番茄LeNHX1基因在逆境应答中可能起着重要作用。     

Method for Label-Free Quantitative Proteomics for <Emphasis Type="Italic">Sorghum bicolor</Emphasis> L. Moench

Sorghum (Sorghum bicolor L. Moench) is a rapidly emerging high biomass feedstock for bioethanol and lignocellulosic biomass production. The robust varietal germplasm of sorghum and its completed genome sequence provide the necessary genetic and molecular tools to study and engineer the biotic/abiotic stress tolerance. Traditional proteomics approaches for outlining the sorghum proteome have many limitations like, demand for high protein amounts, reproducibility and identification of only few differential proteins. In this study, we report a gel-free, quantitative proteomic method for in-depth coverage of the sorghum proteome. This novel method combining phenol extraction and methanol chloroform precipitation gives high total protein yields for both mature sorghum root and leaf tissues. We demonstrate successful application of this method in comparing proteomes of contrasting cultivars of sorghum, at two different phenological stages. Protein identification and relative quantification analyses were performed by a label-free liquid chromatography tandem mass spectrometry (LC/MS-MS) analyses. Several unique proteins were identified respectively from sorghum tissues, specifically 271 from leaf and 774 from root tissues, with 193 proteins common in both tissues. Using gene ontology analysis, the differential proteins identified were finely corroborated with their leaf/root tissue specific functions. This method of protein extraction and analysis would contribute substantially to generate in-depth differential protein data in sorghum as well as related species. It would also increase the repertoire of methods uniquely suited for gel-free plant proteomics that are increasingly being developed for studying abiotic and biotic stress responses.     

A study of the blue-light-dependent phosphorylation, degradation, and photobody formation of Arabidopsis CRY2

Arabidopsis cryptochrome 2 (CRY2) is a blue-light receptor mediating blue-light inhibition of hypocotyl elongation and photoperiodic promotion of floral initiation. CRY2 is a constitutive nuclear protein that undergoes blue-light-dependent phosphorylation, ubiquitination, photobody formation, and degradation in the nucleus, but the relationship between these blue-light-dependent events remains unclear. It has been proposed that CRY2 phosphorylation triggers a conformational change responsible for the subsequent ubiquitination and photobody formation, leading to CRY2 function and/or degradation. We tested this hypothesis by a structure-function study, using mutant CRY2-GFP fusion proteins expressed in transgenic Arabidopsis. We show that changes of lysine residues of the NLS (Nuclear Localization Signal) sequence of CRY2 to arginine residues partially impair the nuclear importation of the CRY2K541R and CRY2K554/5R mutant proteins, resulting in reduced phosphorylation, physiological activities, and degradation in response to blue light. In contrast to the wild-type CRY2 protein that forms photobodies exclusively in the nucleus, the CRY2K541R and CRY2K554/5R mutant proteins form protein bodies in both the nucleus and cytosol in response to blue light. These results suggest that photoexcited CRY2 molecules can aggregate to form photobody-like structure without the nucleus-dependent protein modifications or the association with the nuclear CRY2-interacting proteins. Taken together, the observation that CRY2 forms photobodies markedly faster than CRY2 phosphorylation in response to blue light, we hypothesize that the photoexcited cryptochromes form oligomers, preceding other biochemical changes of CRY2, to facilitate photobody formation, signal amplification, and propagation, as well as desensitization by degradation.     

QTL for nodal root angle in sorghum (<Emphasis Type="Italic">Sorghum bicolor</Emphasis> L. Moench) co-locate with QTL for traits associated with drought adaptation

Nodal root angle in sorghum influences vertical and horizontal root distribution in the soil profile and is thus relevant to drought adaptation. In this study, we report for the first time on the mapping of four QTL for nodal root angle (qRA) in sorghum, in addition to three QTL for root dry weight, two for shoot dry weight, and three for plant leaf area. Phenotyping was done at the six leaf stage for a mapping population (n = 141) developed by crossing two inbred sorghum lines with contrasting root angle. Nodal root angle QTL explained 58.2% of the phenotypic variance and were validated across a range of diverse inbred lines. Three of the four nodal root angle QTL showed homology to previously identified root angle QTL in rice and maize, whereas all four QTL co-located with previously identified QTL for stay-green in sorghum. A putative association between nodal root angle QTL and grain yield was identified through single marker analysis on field testing data from a subset of the mapping population grown in hybrid combination with three different tester lines. Furthermore, a putative association between nodal root angle QTL and stay-green was identified using data sets from selected sorghum nested association mapping populations segregating for root angle. The identification of nodal root angle QTL presents new opportunities for improving drought adaptation mechanisms via molecular breeding to manipulate a trait for which selection has previously been very difficult.     

Migration of the Fungal Protein Cryptogein within Tobacco Plants

Cryptogein (CRY), a protein secreted by Phytophthora cryptogea, causes necrosis on tobacco (Nicotiana tabacum) plants at the site of application (the stem or the roots) and also on distant leaves. Autoradiography of plantlets after root absorption of radioiodinated CRY demonstrated a rapid migration of the label to the leaf lamina via the veins. Using an anti-CRY antiserum, a CRY-related antigen was detected in the stem and leaves of CRY-treated plants at a distance from the site of application. This antigen had the same molecular weight as CRY and was detected in the leaves as early as 1 hour after stem treatment, i.e. long before necrosis was detectable. The antigen was also detected in plants inoculated with P. cryptogea. The distant location of the necrosis induced by the fungus or by CRY can be ascribed to the migration of this protein, which is toxic to tobacco cells. It is proposed that CRY, which also elicits defense reactions in tobacco, might contribute to the hypersensitive response of tobacco to P. cryptogea.     

Molecular Genetics of Cryptopleurine Resistance in Saccharomyces Cerevisiae: Expression of a Ribosomal Protein Gene Family

The Saccharomyces cerevisiae CRY1 gene encodes the 40S ribosomal subunit protein rp59 and confers sensitivity to the protein synthesis inhibitor cryptopleurine. A yeast strain containing the cry1-δ1::URA3 null allele is viable, cryptopleurine sensitive (Cry(S)), and expresses rp59 mRNA, suggesting that there is a second functional CRY gene. The CRY2 gene has been isolated from a yeast genomic library cloned in bacteriophage λ, using a CRY1 DNA probe. The DNA sequence of the CRY2 gene contains an open reading frame encoding ribosomal protein 59 that differs at five residues from rp59 encoded by the CRY1 gene. The CRY2 gene was mapped to the left arm of chromosome X, centromere-proximal to cdc6 and immediately adjacent to ribosomal protein genes RPS24A and RPL46. Ribosomal protein 59 is an essential protein; upon sporulation of a diploid doubly heterozygous for cry1-δ2::TRP1 cry2-δ1::LEU2 null alleles, no spore clones containing both null alleles were recovered. Several results indicate that CRY2 is expressed, but at lower levels than CRY1: (1) Introduction of CRY2 on high copy plasmids into Cry(R) yeast of genotype cry1 CRY2 confers a Cry(S) phenotype. Transformation of these Cry(R) yeast with CRY2 on a low copy CEN plasmid does not confer a Cry(S) phenotype. (2) Haploids containing the cry1-δ2::TRP1 null allele have a deficit of 40S ribosomal subunits, but cry2-δ1::LEU2 strains have wild-type amounts of 40S ribosomal subunits. (3) CRY2 mRNA is present at lower levels than CRY1 mRNA. (4) Higher levels of β-galactosidase are expressed from a CRY1-lacZ gene fusion than from a CRY2-lacZ gene fusion. Mutations that alter or eliminate the last amino acid of rp59 encoded by either CRY1 or CRY2 result in resistance to cryptopleurine. Because CRY2 (and cry2) is expressed at lower levels than CRY1 (and cry1), the Cry(R) phenotype of cry2 mutants is only expressed in strains containing a cry1-δ null allele.     

Characterization of photolyase/blue-light receptor homologs in mouse and human cells.

We isolated and characterized mouse photolyase-like genes, mCRY1 (mPHLL1) and mCRY2 (mPHLL2), which belong to the photolyase family including plant blue-light receptors. The mCRY1 and mCRY2 genes are located on chromosome 10C and 2E, respectively, and are expressed in all mouse organs examined. We raised antibodies specific against each gene product using its C-terminal sequence, which differs completely between the genes. Immunofluorescent staining of cultured mouse cells revealed that mCRY1 is localized in mitochondria whereas mCRY2 was found mainly in the nucleus. The subcellular distribution of CRY proteins was confirmed by immunoblot analysis of fractionated mouse liver cell extracts. Using green fluorescent protein fused peptides we showed that the C-terminal region of the mouse CRY2 protein contains a unique nuclear localization signal, which is absent in the CRY1 protein. The N-terminal region of CRY1 was shown to contain the mitochondrial transport signal. Recombinant as well as native CRY1 proteins from mouse and human cells showed a tight binding activity to DNA Sepharose, while CRY2 protein did not bind to DNA Sepharose at all under the same condition as CRY1. The different cellular localization and DNA binding properties of the mammalian photolyase homologs suggest that despite the similarity in the sequence the two proteins have distinct function(s).