甜橙液泡转化酶基因(CSVI)的分离及全序列分析

根据不同植物液泡转化酶基因序列的保守区设计合成引物，采用滤纸吸附噬菌体PCR法，首次从甜橙基因组文库筛选出含甜橙液泡转化酶基因的阳性X噬菌体，经过大量培养并提取其DNA，限制性内切酶消化后进行DIG southern印迹分析，将阳性条带回收、加工并克隆测序。序列分析表明，该基因全长4722bp，编码588个氨基酸，包括6个外显子和5个内含子。在GenBank进行Blast检索的结果表明，该基因编码的氨基酸序列与马铃薯、番茄和酸樱桃液泡转化酶基因编码的氨基酸序列同源性分别为68％、68％和62％。系统进化关系聚类分析结果表明，该基因属液泡转化酶基因类型。该基因已经在GenBank登录(登录号：AF433643)。该基因的获得为从分子水平研究柑桔果实发育过程中蔗糖积累和分解机制以及通过基因工程改良果实品质奠定了基础。     

中华大节竹耐盐基因Is SOS1的克隆与序列分析

通过RACE技术,从中华大节竹根中克隆出一个质膜型Na+/H+逆向转运蛋白基因,并命名为Is SOS1(基因登录号:KC113048)。Is SOS1序列分析结果显示,Is SOS1的全长为3 516 bp,其中开放读码框为3 105 bp,编码了1 035个氨基酸多肽。氨基酸同源性和聚类分析证实,Is SOS1氨基酸序列与拟南芥At SOS1和水稻Os SOS1的氨基酸序列同源性较高,分别为50. 3%和76. 63%,但是与液泡型的Na+/H+逆向转运蛋白氨基酸序列同源性较低。     

茶树2个MYB转录因子基因的克隆及表达分析

MYB类转录因子是一类包含一段保守的DNA结合结构域的基因家族,广泛地参与植物发育和植物次生代谢的调节。根据前期芯片杂交和文库筛选得到的2个MYB转录因子的部分序列,采用RT-PCR和RACE技术分离得到它们的全长基因:CsMYB1和CsMYB2,在GenBank的登录号分别为HQ660373和HQ660374。序列分析表明:CsMYB1基因全长1132bp,开放阅读框长879bp,编码292个氨基酸,推测的蛋白分子量约为32.9ku,理论等电点为8.13;CsMYB2基因全长1020bp,其中开放阅读框长675bp,编码224个氨基酸,推测的蛋白分子量约为25.4ku,理论等电点为9.05。2个基因编码的蛋白均具有明显的R2R3MYB结构域,且在R3结构域的下游都含有1个相对保守的C1(LIXXGIDPXTHR)基序。同源性分析表明:茶树CsMYB1和CsMYB2编码的氨基酸序列与其他植物的MYB类转录因子具有较高的相似性,其中CsMYB1编码的氨基酸序列与陆地棉MYB1的相似性为57%,CsMYB2编码的氨基酸序列与葡萄MYBC2的相似性为75%。利用荧光定量PCR技术检测2个转录因子基因在遮荫处理条件下的表达规律,及其在茶树不同组织中的表达特性,结果表明:CsMYB1和CsMYB2在不同组织中均有表达,但表达量具有明显区别,其中CsMYB2在叶片中的相对表达量是根中的100多倍;而遮荫处理能明显降低叶片中的花青素含量,并提高CsMYB1的表达,但对转录因子CsMYB2的影响不大。     

杉木CCoAOMT基因部分cDNA克隆与序列分析

利用CODEHOP设汁CCoAOMT基因的简并引物,通过RT-PCR的方法,从杉木茎段中克隆到1个长度为614 bp的PCR产物,产物经pMD18-T载体克隆转化至DH5α大肠杆菌中,序列通过同源性比对后显示该片段与其它植物的CCoAOMT基因具有较高的同源性.利用生物信息学方法分析发现,该序列在112～651 bp片段上有一个开放性阅读框,该序列编码的是一个酸性蛋白,亲水性较弱.疏水性较强;信号肽序列为第23位至第45位,二级结构以α-螺旋(40.62%)与不规则盘绕(45.31%)为主,没有发现β转角区域.     

我国木本植物致病性土壤杆菌的分子检测和比较鉴定

根据根癌土壤杆菌Ti质粒的保守序列设计了2对引物,对我国不同木本植物上分离鉴定的10株致病性根癌土壤杆菌进行PCR,致瘤性根癌土壤杆菌(Agrobacterium tumefaciens)扩增出427 bp和338 bp的特异性DNA片段,而发根土壤杆菌(A.rhizogenes)仅扩增出338 bp片段,放线土壤杆菌(A.radiobacter)、丁香假单胞菌(Pseudomonas syringae)和泡桐丛枝病植原体(phytoplasma)皆未有特异扩增带.用CYT/CYT'引物对也可鉴定T-DNA遗传转化瘤组织,而VirD2A/VirD2E可用于工程土壤杆菌株侵染能力鉴定.从北京通州杨树苗圃和河北廊坊桃园的病树冠瘿组织和土壤中经选择培养基分离菌株,然后PCR筛选出6个致病性根癌土壤杆菌菌株,而未能从施用过抗根癌生防制剂的北京玉渊潭公园樱花根癌病园中分离和鉴定出典型的致病根癌土壤杆菌株.将杨树根癌土壤杆菌菌株(CFCC1001)异戊烯基转移酶基因(ipt)片段克隆和序列测定结果显示与A.tumefaciens Ti15955菌株的ipt基因序列同源性为83.64%.用此片段制备的ipt cRNA基因探针进行斑点杂交和Northern blot,结果显示此探针与杨树根癌土壤杆菌以及玫瑰、樱花、海棠、桃树等木本植物上分离鉴定的致病土壤杆菌菌株所扩增出的427 bp片段都有明显杂交信号,但与引起泡桐丛枝病植原体染色质和染色质外DNA均未有明显杂交信号.     

杨树木质素合成酶hct基因的克隆及核苷酸序列分析

根据毛果杨全序列（AC185363.2）中唯一具有转移酶功能的保守区设计引物,以正在分化的2年生欧美杨107次生木质部总RNA为模板经RT-PCR扩增出hct基因片段,与pMD20-T载体连接,重组质粒经特异引物扩增、限制性内切酶酶切和测序鉴定。结果表明,扩增片段长度为709bp,包含一个708bp的开放阅读框,与毛果杨全序列及HCT2（EU603314）的同源性均为98%,编码的氨基酸序列与毛果杨HCT2氨基酸（ACC63883.1）的同源性为98%,断定我们克隆的cDNA为hct,属于转移酶超家族,GenBank登录号为FM202091,该重组质粒命名为pMD20-T-hct。     

麻疯树磷酸烯酮式丙酮酸羧化酶em>pepc基因全长cDNA克隆及序列分析

应用RT-PCR和RACE法从麻疯树总RNA中分离出pepc基因全长cDNA,长度为3 142 bp,阅读框2 898 bp,编码965个氨基酸,在GenBank中登录,序列号为EU069413。它的氨基酸序列与蓖麻、陆地棉、橙、大豆、花生、烟草、油菜、拟南芥的氨基酸同源性分别为94.94%、92.46%、90.60%、90.50%、90.50%、88.33%、84.61%、82.44%。该基因编码了pepc基因家族中的pepc1,蛋白属于C₃型PEPC。推测了pepc编码蛋白分子量为110.6 kD,并对其稳定性、二级结构、疏水性等特性进行了分析,最后确定了该蛋白的功能位点和结构域。     

杨树Na+/H+反向运输蛋白基因(PtNHX1、PtNHX6)的克隆和检测

Na /H 反向运输蛋白基因(NHX)是在细菌、植物和动物体内普遍存在的一类膜蛋白基因家族.迄今已在模式植物拟南芥中分离出AtNHX1、AtNHX2、AtNHX3、 AtNHX4、 AtNHX5和 AtNHX6共6个成员,并发现部分成员对盐胁迫有不同程度的响应.以AtNHX1和AtNHX6的cDNA核苷酸序列为信息探针, 基于可利用的杨树EST数据库和毛果杨全基因组测序结果,通过电子杂交辅助的克隆技术,从毛白杨形成层cDNA中分离得到长度分别为1 635 bp和1 709 bp的2个cDNA,其分别含有编码544个和526个氨基酸残基的完整开放阅读框. 由它们所推导的蛋白质序列与拟南芥、水稻、小麦和玉米的NHX1和NHX6基因的蛋白质序列高度同源, 同源性分别为79%、76%、69%和74%以及82%、82%、27%和26%, 故将其命名为PtNHX1和PtNHX6(GenBank 注册号分别为AY660749和AY832912).Southern 杂交分析表明, PtNHX1和PtNHX6均为低拷贝基因(或有一些高度同源的基因),在杨树基因组中以1 ～ 4个拷贝形式存在.组织特异性RT-PCR结果显示, PtNHX1和PtNHX6基因在杨树根、茎和叶片中均有表达,但其表达模式稍有不同:PtNHX1在根部、形成层、未成熟木质部、成熟叶和嫩叶中均高度表达,在韧皮部和成熟木质部中有少量表达,而PtNHX6在根部、成熟叶和嫩叶中表达丰度较高,在韧皮部、形成层、未成熟木质部表达丰度较低,在成熟木质部中表达丰度极低.NaCl诱导的杨树叶片差异表达RT-PCR检测结果初步表明,PtNHX1和PtNHX6基因均受盐诱导表达,当溶液中NaCl浓度在100～400 mmol·L-1内,随着盐浓度的提高,PtNHX1和PtNHX6基因的表达丰度逐渐增强,但超过400 mmol·L-1后,其表达丰度均有所降低,这与所测定的叶片ABA含量匹配.     

版纳龙竹CONSTANS同源基因的克隆与序列分析

以版纳龙竹基因组DNA为模板,采用前人基于水稻CO同源基因Hd1序列的保守区所设计的特异引物COS1和COA1,运用PCR方法扩增出一条1 520 bp的DNA片段,并克隆到pGEM-T载体。测序和序列分析结果显示:该片段含有1个590 bp的内含子,编码区930 bp共编码310个氨基酸;该基因被命名为DxCO1,其DNA序列在G enB ank中的注册号为GQ358925。在G enB ank中进行同源性检索的结果显示:其核苷酸序列与其它禾本科植物CO同源基因的氨基酸序列同源性高达81%~91%;推测的DxCO1蛋白质序列与其它种子植物CO同源基因蛋白质序列的系统发育分析结果显示:DxCO1与小麦Hd1-like等5个基因聚成了一个强烈支持的分支;另外,在该片段推测的蛋白质序列的氨基端含有一个类似锌指蛋白的B-box(Cx2Cx8Cx7Cx2Cx4Hx8H)结构域,羧基端含有一个CCT(CO,CO-like,TOC1)结构域。序列和结构的高度同源性表明:DxCO1是版纳龙竹的1个CO-like基因,可能对其开花调控有着重要作用。     

CODEHOP在线简并引物设计与杉木4CL基因片段克隆

利用CODEHOP设计4CL酶的简并引物,利用设计的3对简并引物进行RT—PCR,从杉木幼茎中克隆到2个长度分别为502bp和536bp的PCR产物,产物经pMD18-T载体克隆转化至DH5α大肠杆菌中,序列通过Blastx检索与Genbank进行同源性比对后,发现2片段都与其它植物的4-CL基因具有较高的氨基酸序列同源性。研究表明,该程序设计的简并引物可信性强,阳性率高。该基因的成功克隆将为研究杉木木质素的合成提供科学依据。     

Cloning and Sequence Analysis of a Glucose-6-Phosphate Dehydrogenase Gene PsG6PDH from Freezing-tolerant Populus suaveolens

A 1 207 bp cDNA fragment (PsG6PDH) was amplified by RT-PCR from cold-induced total RNA of the freez- ing-tolerant P. Suaveolens, using primers based on the highly conserved region of published plant glucose-6-phosphate dehydro- genase (G6PDH) genes. The sequence analysis showed that PsG6PDH coding region had 1 101 bp and encoded 367 predicted amino acid residues. Moreover, the nucleotide sequence of PsG6PDH showed 83%, 82%, 79%, 79% and 78% identity, and the derived amino acid sequence shared 44.2%, 44.7%, 42.0%, 40.5% and 43.9% identity with those of the Solanum tuberosum, Nicotiana ta- bacum, Triticum aestivum, Oryza sativa and Arabidopsis thaliana, respectively. The results show that PsG6PDH is a new member of G6PDH gene family and belongs to the cytosolic G6PDH gene. This is the first report on cloning of the G6PDH gene from woody plants.     

Cloning and sequence analysis of a glucose-6-phosphate dehydrogenase gene <Emphasis Type="Italic">PsG6PDH</Emphasis> from freezing-tolerant <Emphasis Type="Italic">Populus suaveolens</Emphasis>

A 1207 bp cDNA fragment (PsG6PDH) was amplified by RT-PCR from cold-induced total RNA of the freezing-tolerant P. Suaveolens, using primers based on the highly conserved region of published plant glucose-6-phosphate dehydrogenase (G6PDH) genes. The sequence analysis showed that PsG6PDH coding region had 1 101 bp and encoded 367 predicted amino acid residues. Moreover, the nucleotide sequence of PsG6PDH showed 83%, 82%, 79%, 79% and 78% identity, and the derived amino acid sequence shared 44.2%, 44.7%, 42.0%, 40.5% and 43.9% identity with those of the Solanum tuberosum, Nicotiana tabacum, Triticum aestivum, Oryza sativa and Arabidopsis thaliana, respectively. The results show that PsG6PDH is a new member of G6PDH gene family and belongs to the cytosolic G6PDH gene. This is the first report on cloning of the G6PDH gene from woody plants.     

Spatial and temporal expression profiling of cell-wall invertase genes during early development in hybrid poplar

Cell-wall invertase genes are spatially and temporally regulated in several plant species, including Daucus carota L., Lycopersicon esculentum L. and Solanum tuberosum L. However, few studies of cell-wall invertase genes of trees have been conducted, despite the importance of trees as a source of lignocellulosic biopolymers. We identified three putative cell-wall invertase genes in hybrid poplar (Populus alba L. x grandidentata Michx.) that showed higher homology to each other than to cell-wall invertases of other dicotyledonous species, with two of the genes (PaxgINV2 and PaxgINV3) appearing as a genomic tandem repeat. These genes are more similar to each other than to tandemly repeated cell-wall invertases of other plants, perhaps indicating parallel evolution of a duplication event with cell-wall invertases in dicotyledons. Spatial and temporal expression analyses throughout a complete annual cycle indicated that PaxgINV1 and PaxgINV2 are highly regulated in vegetative tissues during three distinct growth phases: early growth, dormancy and post-dormancy. Expression of the third gene (PaxgINV3) appears to be tightly regulated and may represent a floral-specific cell-wall invertase. Of the two genes expressed in vegetative tissues, PaxgINV1 appears to be exclusively involved in processes related to dormancy, whereas PaxgINV2 appears to encode an enzyme involved in phloem unloading and in providing actively growing tissues, such as developing xylem, with the energy and carbon skeletons necessary for respiration and cell wall biosynthesis.     

油桐尺蛾核型多角体病毒广西株全基因组序列

油桐尺蛾核型多角体病毒广西株基因组序列由Sanger测序的方法得到。拼接后获得了121 268 bp序列,GC含量为36.76%。以长度不小于50 aa的序列认定为功能基因,共预测到131个开放阅读框,通过Blast比对,其中87个能注释到功能基因。对基因组序列进重复序列分析,共发现SSR位点174个,同源重复序列1个。在SSR位点中,包含6种长度的重复基序,且以富含AT的重复基序为主。同源重复序列两端为一59 bp片段或其一部分组成的重复序列,两端分别重复12次和7次,中间则为一段与重复片段无关的236 bp片段。与武汉株相比,广西株病毒在37个核心基因中有4个存在蛋白序列长度上的差异,而在其他非核心基因中有6个基因仅存在于一个病毒株中,另有13个基因存在蛋白序列长度上的差异。     

叶绿体全基因组序列确定钻天柳在杨柳科中的系统发育位置

目的 为解决濒危物种钻天柳在杨柳科中分类学位置的争议。 方法 本研究通过二代测序技术,从头测序、拼接得到钻天柳叶绿体基因组全序列,并与已发表的9种杨属植物和4种柳属植物的叶绿体基因组全序列进行比较,采用最大似然法、最大简约法和贝叶斯推断法分析了这些物种的系统发育关系。 结果 研究发现：钻天柳总基因组为155 661 bp,由长度为84 536 bp的长单拷贝（LSC）区域和16 215 bp的短单拷贝（SSC）区域,以及一对分隔开它们的27 455 bp的反向重复序列（IRS）组成。钻天柳叶绿体基因组总GC含量为36.68%,共有113个不同的基因,包括79个蛋白质编码基因,30个tRNA基因和4个rRNA基因,其中,有20个基因分布于反向重复区;在所有基因中,有14个基因包含1个内含子,3个基因（rps12、clpP、ycf3）内含有2个内含子;系统发育分析以100%的支持率将钻天柳与柳属黄花柳亚属的2个物种聚为一支,杨属的所有物种聚为另一支。 结论 本研究首次组装并注释了钻天柳叶绿体基因组全序列,并明确支持钻天柳并入柳属,而非单独成属,这将为钻天柳甚至杨柳科的系统进化研究提供重要参考。     

慈竹β-tubulin基因片段的克隆及序列分析

利用Trizol一步法提取慈竹幼笋总RNA,运用RT-PCR方法首次在慈竹中克隆到3条β-tubulin基因部分序列,命名为Na-βtub1、Naβ-tub2、Naβ-tub3,长度分别为958bp、958bp和959bp,分别编码318、318和319个氨基酸。核酸和氨基酸的序列比对分析结果表明,慈竹中3条β-tubulin基因核酸和推测的氨基酸序列之间相似性分别为92.46%和98.22%,与禾本科植物水稻、玉米、小麦的β-tubulin基因核酸和氨基酸序列相似性分别在89%和95%以上。     

Discrimination of Bursaphelenchus xylophilus and Bursaphelencus mucronatus by PCR-RFLP technique

A polymerase chain reaction-restriction fragment length polymorphism analysis was used to discriminate isolates of Bursaphelenchus xylophilus and B. mucronatus. The amplifications of B. xylophilus isolates yielded one fragment of approximately 890 bp and that of B. mucronatus was about 930 bp. Digestion of amplified products of each nematode isolate with five restriction endonucleases revealed the following results: 1) Dra I digestion of the internal transcribed spacer (ITS) products of B. xylophilus populations yielded two fragments of 510 and 380 bp. Dra I could not digest the ITS products of B. mucronatus populations; 2) Sal I could not digest the ITS products of all B. xylophilus populations, but it could digest those of B. mucronatus populations into two fragments, which were 720 and 220 bp; 3) digested products of four B. xylophilus populations by Msp I yielded two fragments of 530 and 360 bp, except GZ02, which could not be digested. B. mucronatus populations yielded three fragments: 340, 290, and 180 bp; 4) all populations of B. xylophilus and B. mucronatus could not be digested by Apa I; 5) digestion of the ITS products of B. xylophilus and B. mucronatus yielded two fragments of 520 and 370 bp, and 530 and 400 bp respectively. The restriction endonucleases Dra I and Sal I could be used to identify B. xylophilus and B. mucronatus. Because the results of digestion of B. xylophilus and B. mucronatus were markedly different, they were very easy to be identified and applied; Msp I and Xho I were not suitable for identification of B. xylophilus and B. mucronatus and Apa I could not identify and distinguish between B. xylophilus and B. mucronatus. __________ Translated from Journal of Nanjing Forestry University, 2005, 30(4): 5–9 [译自: 南京林业大学学报]