青枯菌M5菌株特异基因组消减文库的构建

为筛选青枯菌M5菌株特有的序列,利用SSH技术对青枯菌生理小种5号菌株M5（tester）和生理小种1号菌株GMI1000（driver）基因组进行了比较。分别提取二者基因组DNA,RsaⅠ酶切,将酶切后M5菌株DNA分为两份,分别与接头1和接头2R连接,然后进行两轮杂交和两轮PCR扩增,并检测连接效率与消减效率。将获得的消减PCR产物,即差异基因/差异DNA片段,与pGEM-T载体连接,转化E.coli DH5α感受态细胞,建立消减文库。随机挑取96个白色克隆,经菌液PCR法测定,80个为预期阳性,含有预期的插入片段。首次构建了青枯菌M5菌株特异基因的抑制消减文库,为进一步筛选、克隆青枯菌M5菌株特异核苷酸片段奠定了基础。     

番茄NP24基因超表达载体的构建

根据NCBI上提供的NP24基因(Gen Bank登录号为543979)的序列,设计全长引物,扩增编码区c DNA,通过克隆至中间载体p MD18-T,将类甜蛋白基因NP24插入植物表达载体PBI121,构建超表达载体PBI121-NP24,采用Ca Cl2冻融法将其转入农杆菌EHA105菌株中。酶切鉴定表明,目的基因已经正确地插入到PBI121载体中,超表达载体构建成功。菌液PCR鉴定,超表达载体已转入农杆菌中。     

植物表达载体pGMAR-BADH的构建

将甜菜碱醛脱氢酶基因BADH和植物表达载体pGMAR通过内切酶BamHI和KpnI双酶切,T4连接酶进行连接反应。重组质粒在大肠杆菌菌株DH5α内扩增,提取并纯化质粒。经鉴定,基因BADH已被完整、正确的插入到pGMAR载体中,并成功将BADH插入到载体pGMAR的两个MAR序列之间。     

苦瓜MAP30基因的克隆与载体构建

首先提取苦瓜的基因组DNA,然后根据Genbank中已公开发表的MAP30序列,设计一对特异引物MAP301,MAP302,并增添特异定位于内质网的小肽Kozak序列。采用PCR技术,从苦瓜的基因组DNA中扩增出一分子量为800bp左右的片段,回收克隆测序,结果表明,克隆获得片段与已公开发表的MAP30序列同源性达100%,将阳性克隆进行酶切,获得的目标片段与用同样酶切所获得的植物表达载体pEV1和pEV2大片段连接,酶切鉴定重组子构建成特异表达于果实的载体。     

黄瓜棒孢叶斑病菌的分子鉴定及rDNA-ITS序列分析

为了分析不同地区黄瓜棒孢叶斑病菌的种内分化,能够准确地鉴定该病菌,以服务于黄瓜抗病育种,对2007-2014年采集的黄瓜棒孢叶斑病菌多主棒孢经单孢纯化获得34个菌株。利用公开发表的特异性引物CCF/CCR对其进行了PCR分子鉴定,结果表明:34个菌株均扩增得到了预期272 bp的特异片段,说明均为多主棒孢菌。应用真菌核糖体rDNA区通用引物ITS1和ITS4分别扩增各个菌株的r DNA内转录间隔区序列,经测序并比对分析,34个菌株均扩增得到了559 bp的特异片段,其中32个从黄瓜上分离的多主棒孢病菌菌株碱基序列完全一致,2个从番茄上分离的菌株碱基序列完全一致,二者的差异在于2个SNP位点T-C、G-A的突变,二者相似度为99.64%。说明,多主棒孢菌属种间的ITS区序列高度保守,利用ITS序列分析方法可以为多主棒孢菌的分类鉴定及系统发育研究提供依据,可以区分出黄瓜和番茄寄主上的多主棒孢菌。     

Y 系山定子 AP1同源基因的克隆及序列分析

以Y系山定子为试材研究AP1基因,试图为缩短果树童期提供理论依据。根据已登录的苹果AP1基因保守序列设计引物,以Y系山定子的基因组DNA为模板进行PCR扩增,获得1010 bp的基因片段;PCR产物纯化后,将其连接到pMD18-T载体上,连接产物转化大肠杆菌DH5α菌株感受态细胞,通过蓝白斑筛选、质粒长度和酶切鉴定,获得重组质粒并测序。测序结果表明,该序列有2个外显子、1个内含子及2个内含子的一部分,编码区共编码81个氨基酸;对氨基酸序列保守性分析发现,第13～81个氨基酸为MADS-box基因家族特有的K区域;其氨基酸序列在GenBank中同源性检索表明,Y系山定子与其他植物AP1同源基因的氨基酸序列同源性为67．9％～100％。研究结果为揭示Y系山定子早花现象的分子机制奠定了基础,对果树育种具有重要的理论价值和实际意义。     

拟南芥菜花药绒毡层启动子的克隆和序列分析

以拟南芥菜(Arabid　op　sis thaliana)基因组DNA为模板 , 通过PCR扩增得到绒毡层特异 表达基因A9的启动子片段, 克隆到pUC18载体上。 序列分析表明, 该启动子大小为36 0 bp , RNA聚合酶识别序列TATA box, 花药特异表达和增强序列TGTGG、 TGTGA两个Motifs皆完整 , 与已报道的序列比较仅有3个核苷酸发生改变, 同源性为99.     

草莓乙烯受体FaEtr2基因的克隆及其反义表达载体的构建

为了构建草莓乙烯受体FaEtr2基因的反义表达载体,在已报道的FaEtr2基因序列的基础上,设计特异引物,克隆草莓乙烯受体FaEtr2基因部分特异序列,将该片段反向插入植物表达载体pBI121的CaMV 35S启动子和NOS终止子之间,构建了反义表达载体pBI121Etr2。通过双酶切鉴定后,导入农杆菌EHA105中,酶切和PCR鉴定表明质粒已导入到农杆菌中。本研究为后期该反义基因转化草莓品种以改良草莓果实耐贮运性打下基础。     

陆地棉L-D1等位基因特异性分子标记的开发及应用

【目的】L-D1基因调控陆地棉叶形。本研究设计特异分子标记精准鉴定L-D1等位基因,为其在陆地棉冠层结构改良中的应用提供支撑。【方法】利用具有鲁棉研28号遗传背景的L-D1等位基因近等基因系,分析不同等位基因组合对叶形的影响。根据4个等位基因的启动子和编码区的多态性设计特异分子标记,并对不同叶形中的等位基因进行检测。【结果】L-D1基因从3叶期开始调控叶片叶裂的形成,4～8叶期叶裂不断加深,从9叶期开始基本稳定;L-D1等位基因不同组合可形成相似叶形,仅从形态上难以准确辨别。克隆获得L-D1位点4个等位基因起始密码子前约4 kb的启动子片段,发现24个SNPs(Single nucleotide polymorphism,单核苷酸多态性)、1个133 bp及1个14 bp的插入缺失。根据等位基因启动子及编码区的SNP和缺失插入开发了1个Indel分子标记InDel＿8和2个衍生酶切扩增多态性序列标记dCAPS＿192、dCAPS＿519,分别为l2、L2o、L2s的特异分子标记。【结论】根据陆地棉中控制叶形的L-D1等位基因间的差异开发了3个特异性分子标记,可用来鉴定不同的L-D1等位基因。     

无花果ACC合成酶基因克隆及序列分析

为进一步利用基因工程手段调控无花果乙烯的合成,以无花果果肉为材料提取基因组DNA,根据已报道的无花果ACC合成酶基因序列设计引物,采用PCR技术扩增得到一条约600 bp的特异片段,将该片段克隆到pGM-Teasy vector上经PCR、酶切和测序鉴定。序列分析结果表明,基因全长590 bp,编码196个氨基酸,该序列与GenBank上已登陆的Masui Dauphine-ACS1的cDNA序列同源性达99%,氨基酸同源性达98%。结果表明,成功克隆到了无花果ACS基因片段。     

茶树菇复合群系统类群及交配分析

［目的］茶树菇复合群是一类具有重要经济价值的菌类,其菌株资源多样,命名混乱。为了给育种工作提供参考依据,［方法］本研究利用线粒体小亚基V4-6-9区序列和交配,对四个代表菌株进行分子和交配特征分析,［结果］结果表明：(1)线粒体小亚基序列作为类群区分的有效标记,有效区分杨柳田头菇类群、茶树菇/茶薪菇类群和杨树菇类群;(2)交配特征显示杨柳田头菇为独立生物学种,而其它类群属同一生物学种,建议使用茶树菇这一名称。［结论］研究结果可以为茶树菇资源鉴定及育种研究提供参考 。     

1株野生茶树菇的鉴定及其母种培养条件研究

为更好地开发利用茶树菇,同时丰富国内茶树菇种质资源,以采自贵州的1 株野生茶树菇NC1 菌株为材料,对其进行内转录间隔区ITS(internal transcribed spacer)鉴定和母种培养条件研究,包括对菌丝生长的最适碳源、氮源、C/N 比、最适温度、最适pH 等。结果表明：该菌株为柱状田头菇(Agrocybe aegerita),其菌丝生长的最适碳源为玉米淀粉,其次为葡萄糖或果糖,最适氮源为酵母粉,其次为蛋白胨,最适C/N 比为30/1~40/1,最适温度为29℃,最适pH 5.0~6.0。研究结果可为野生茶树菇NC1 菌株进一步驯化栽培和开发利用提供参考。     

不同物种RAB27A基因完整编码区生物信息学分析

应用比较基因组学和生物信息学方法比较分析了人、黑猩猩、恒河猴、家鼠、挪威鼠、狗、猪、牛、马、短尾负鼠、鸡和非洲爪蛙RAB27A基因完全编码区(cds),并对该基因的遗传多样性及分子系统发育进行了分析。结果表明,来自12个物种的52条基因序列检测到239个多态位点,共生成17种单倍型。RAB27A基因物种内保守性较强,但在物种间核苷酸多样性和氨基酸多样性相对丰富。     

Bph15在非洲栽培稻、药用野生稻和宽叶野生稻中的BAC-FISH比较物理定位

用覆盖抗褐飞虱基因Bph15的两个BAC克隆,即20M14 (27 kb)和64O9 (36 kb)作为探针,对非洲栽培稻、药用野生稻和宽叶野生稻体细胞染色体进行荧光原位杂交(FISH)。两个BAC克隆均被定位于非洲栽培稻和药用野生稻第4染色体的短臂上,杂交信号的百分距离分别为37.03±4.11和81.22±3.62,相应的信号检出率为41.18%和38.22%。在宽叶野生稻中,有两对同源染色体同时检出信号,分别定位于染色体的短臂和着丝粒区,信号距着丝粒平均百分距离分别为87.78±5.23和0,信号检出率为52.58%。由此推知,这两个BAC克隆在非洲栽培稻和药用野生稻的第4染色体分布同线且同区,并且在宽叶野生稻的DD基因组也存在Bph15基因的同源序列。在未封阻的情况下,BAC克隆在非洲栽培稻的多条染色体上有杂交信号,表明它和栽培稻C_0t-1 DNA在一定程度上具有同源性。上述结果初步显示Bph15在3个稻种染色体中的相对位置。文章讨论了Bph15在3个种间的关系,为有效分离和利用Bph15基因提供了有益的依据,对不同基因组及二倍体和四倍体中功能基因可能进化机制的分析提供了线索。     

Strategies to develop polymorphic markers for <Emphasis Type="Italic">Coffea arabica</Emphasis> L.

This study reports the implementation of three strategies for the development of genetic markers and their evaluation in both progenitors of an F2 population used for the construction of a genetic map of Coffea arabica. The strategies were Cleaved Amplified Polymorphic Sequences (CAPS), Single Strand Conformational Polymorphism (SSCP), and sequence analysis predicted Single Nucleotide Polymorphism (SNP). The methodologies were developed from different sequence sources: For CAPS, we used 25 COS sequences derived from Hedyotis spp. and 29 COSII sequences derived from Solanaceae and Rubiaceae species; for SSCP, we used 111 coffee EST sequences, 50 COSII sequences, and 10 C. arabica BAC end sequences. A low polymorphism was identified with the CAPS and SSCP methodologies. A total of 61 SNPs were identified in silico from 5,371 ESTs of coffee and from amplified, cloned, and sequenced COSII markers. Sixteen of these SNPs were validated with Luminex technology and 2 of them were polymorphic in C. arabica genotypes. This study highlights the difficulties of finding polymorphism in the species C. arabica where SNP identification seems to be the best strategy to search for polymorphic markers for this low diversity plant.     

植物特异DNA序列应用研究进展

植物特异DNA序列是指在某些植物属、种、基因组或染色体上单独具有的特异存在的DNA序列。几乎所有的高等生物基因组中都有一些物种或基因组特异(有)的DNA序列。研究这些特定序列,对研究物种间在进化过程中的关系及现有物种间的亲缘关系、特异性状表达等方面有着重要的意义。本文简述了植物特异DNA序列的种类,总结了利用特异DNA序列作为与某一性状基因连锁的分子标记的应用情况,以及特异DNA序列在鉴定外源染色体的来源、某一染色体组或基因组、某一物种或属植物等方面的应用情况,提出了存在的问题与应用前景。     

Analysis of puroindoline a and b sequences from Triticum aestivum cv. `Penawawa' and related diploid taxa

Flanking sequences of the puroindoline a(pinA) and puroindoline b (pinB) genes from Triticum monococcum, T. urartu, Aegilops speltoides and A. tauschii were obtained by inverse PCR. Two accessions from each of these related diploid taxa were sequenced, and the sequences compared to those of the soft hexaploid wheat cultivar `Penawawa'. As expected from the origin of the D-genome in hexaploid wheat, the highest sequence identity was observed for the A. tauschii sequences, whereas the sequences for A.speltoides were the most divergent. The promoter sequences were further analyzed for putative regulatory elements, and the possible significance of several highly conserved sequence motifs is discussed. Western blots of Triton X-114 extracted proteins separated by SDS-PAGE confirmed the accumulation of pinA and pinB gene products in the endosperm of all the germplasm lines studied. The deduced amino acid sequences for the related diploid taxa do not imply any major structural changes as compared to the wild-type T. aestivum puroindolines, and is therefore in agreement with the soft endosperm texture of these species. This revised version was published online in August 2006 with corrections to the Cover Date.     

SCAR and CAPS markers flanking the Brassica oleracea L. Pp523 downy mildew resistance locus demarcate a genomic region syntenic to the top arm end of Arabidopsis thaliana L. chromosome 1

We recently mapped the Pp523 locus that includes a single, dominant gene conferring resistance to downy mildew expressed in adult plants to a 75.1 cm long linkage group on a genetic linkage map of Brassica oleracea L. More recently, we identified a new AFLP marker 2.8 cm downstream from the resistance gene. The five DNA markers within an 8.5 cm region encompassing the Pp523 gene were cloned and sequenced. Three of these markers were transformed into SCARs (sequence characterised amplified regions), however, two among them were monomorphic and were analysed as CAPS (cleaved amplified polymorphic sequence) markers among the mapping population. Searched against genomic databases, the five B. oleracea DNA-marker sequences matched Arabidopsis thaliana L. gene sequences that delimit a conserved syntenic region in the top arm end of chromosome 1 of this last species. Considering the close genetic relatedness between both species, the information on this specific genomic region in A. thaliana is particularly useful for the construction of a fine-scale map of the corresponding genomic region in B. oleracea. The identified SCAR and CAPS markers can be used for marker assisted selection (MAS) in breeding programs aimed at the introgression of the Pp523 resistance locus, allowing the reliable indirect identification of plants harbouring the resistance gene with a margin of error of approximately six in ten-thousand selected plants.     

A rapid and reliable PCR-restriction fragment length polymorphism (RFLP) marker for the identification of Amaranthus cruentus species

A rapid and reliable PCR-restriction fragment length polymorphism (RFLP) marker was developed to identify the Amaranthus cruentus species by comparing sequences of the starch branching enzyme (SBE) locus among the three cultivated grain amaranths. We determined the partial SBE genomic sequence in 72 accessions collected from diverse locations around the world by direct sequence analysis. Then, we aligned the gene sequences and searched for restriction enzyme cleavage sites specific to each species for use in the PCR-RFLP analysis. The result indicated that MseI would recognize the sequence 5′-T/TAA-3′ in intron 11 from A. cruentus SBE. A restriction analysis of the amplified 278-bp portion of the SBE gene using the MseI restriction enzyme resulted in species-specific RFLP patterns among A. cruentus, Amaranthus caudatus and Amaranthus hypochondriacus. Two different bands, 174-bp and 104-bp, were generated in A. cruentus, while A. caudatus and A. hypochondriacus remained undigested (278-bp). Thus, we propose that the PCR-RFLP analysis of the amaranth SBE gene provides a sensitive, rapid, simple and useful technique for identifying the A. cruentus species among the cultivated grain amaranths.     

二倍体野生棉与四倍体栽培棉基于GISH的遗传关系分析

[目的]研究二倍体野生棉与四倍体栽培棉间的遗传亲缘关系,进一步探索各棉种间的起源与进化.[方法]以5个二倍体基因组的代表种B1(异常棉)、C1(斯特提棉)、E2(索马里棉)、F1(长萼棉)以及G1(比克氏棉)的基因组DNA(gDNA)为探针,以2个四倍体栽培种(陆地棉中棉所16、海岛棉新海7号)有丝分裂中期染色体为靶DNA,进行了基因组原位杂交(Genomic in situ hybridization,GISH)分析.[结果]以B1、E2和F1gDNA为探针时,杂交信号主要分布在2个栽培种较长的13对A亚组染色体上;各产生3对较强的GISH-NOR信号,其中1对分布在较长的A亚组上,2对分布在较短的D亚组上,其GISH-NOR信号强度与分布情况与以D基因组棉种为探针时相似.说明二倍体B、E、F基因组与四倍体棉A亚基因组具有较高的同源性,亲缘关系更近.这一点与它们的地理分布情况相符;而它们基因组中的45S rDNA重复序列与二倍体D基因组的45SrDNA重复序列同源性较高.C1和G1中以gDNA为探针时,杂交信号分布在2个栽培种全部26对染色体上,无法区分开A或D亚组染色体,都有3对较强的GISH-NOR信号.这一现象与D基因组拟似棉(D6) gDNA为探针的GISH相似,表明二倍体C和G基因组与四倍体棉的A和D亚基因组均具有较高的同源性,或者C和G基因组同时含有A基因组(或其他非洲棉基因组)和D基因组成分,进一步证实了其基因组成分的杂合性;而它们基因组中的45S rDNA重复序列同属D基因组类型.[结论]这些发现可为棉花杂交育种和棉属起源与演化研究提供有用信息.