紫外线照射对梁平柚果皮基因表达的影响

 采用SSH技术以紫外照射的梁平柚[Citrus grandis（L.）Osbeck]果皮作为实验方（tester）,以未被照射的正常果皮作为驱动方（driver）,构建了一个梁平柚果皮紫外诱导基因的正向差减文库。经菌液PCR检测后随机挑取200个阳性克隆测序,获得168条表达序列标签（ESTs）。比对这168条ESTs,发现有分属于57个基因的114条ESTs与已知基因高度同源,54条ESTs同源性较低或没有同源性。功能分析发现,这些ESTs主要参与抗逆防御、生长发育、细胞凋亡、转录与翻译、细胞分化、信号传导、能量代谢、糖类及氨基酸代谢以及次生代谢等。     

利用抑制差减杂交技术分离茄子单性结实相关ESTs

以茄子（Solanum melongena）单性结实品系D-10和非单性结实品系03-2的子房cDNA分别为试验组（Tester）和驱动组（Driver）,利用抑制差减杂交（SSH）技术构建了正反两个SSH-cDNA文库,分别包含472和124个克隆。通过测序、序列拼接后,共获得384个Unique ESTs。将其与非冗余蛋白数据库进行BLASTx比对,在E值小于等于1e-10条件下,有257个ESTs能找到相匹配的蛋白质,其中121个ESTs与非冗余蛋白质数据库中已知功能的蛋白质具有高度的相似性。功能注释结果表明,具生物过程、分子功能和细胞组分的EST分别为107、102和119条。推测与单性结实相关的EST有5条：2条ESTs属于MADS-box转录因子家族,1条与生长素增长蛋白同源,1条属于P450还原酶系,1条属于MAPKK家族;另外136个ESTs代表了未知功能基因。     

辣椒细胞质雄性不育系和保持系SSH-cDNA文库的构建

以辣椒细胞质雄性不育系A1、A5为驱动方(driver),以其相应的保持系B1、B5为测验方(tester),利用抑制差减杂交技术构建了1个辣椒细胞质雄性不育保持系表达基因的正向差减cDNA文库。从差减文库中筛选到189个阳性单克隆,经高密度点阵膜杂交筛选和测序分析,共获得了42条表达序列标签(ESTs)。BLAST比对分析后,得到了35条有比对结果的序列,其中包括26条已知功能基因序列和9条未知功能基因序列,已知基因功能多与基础物质代谢、胁迫应答、信号转导等方面有关;有7条ESTs没有比对结果,可能代表一些新基因。     

‘岩溪晚芦’椪柑果皮与果肉差减cDNA文库的构建及初步分析

 以‘岩溪晚芦’(Citrus reticulata Blanco)成熟果皮和果肉为材料,采用抑制性差减杂交技术,成功构建了果皮（tester）与果肉（driver）的差减cDNA文库。结果显示：cDNA克隆外源插入片段大小介于100～500 bp之间。成功对411个克隆进行了测序,测序结果经与NCBI基因库中序列进行比较,377个EST 找到了同源序列,它们分属于126个基因,涉及到与色素合成、能量代谢、初级代谢、次生代谢、抗逆防御、蛋白代谢、信号转导、香味形成、果皮软化（衰老）、精油合成、抗氧化代谢等代谢途径和生理生化过程。还有一些基因涉及到三价铁螯合物、反转录转座子gag蛋白、查尔酮合成、硼离子转运、儿茶酚氧位甲基转移酶等功能。功能已知且没有重复的EST共有63个,占总数的49.2%。有5个基因共62个克隆虽在Blast搜索时找到了较高的同源性序列,但功能目前尚未明确,有待进一步深入分析。另有34个基因未搜索到同源序列,可能为新发现的基因。     

人工授粉对永嘉早香柚果实发育与贮藏品质的影响

永嘉早香柚是浙江温州地方名柚,具单性结实能力,可在无授粉品种混植时结无籽果,也可经人工授粉处理形成有籽果。研究表明,未授粉果实具有无籽优势,但糖酸指标则逊于授粉果。正常采收期(花后165d)未授粉果实果肉可溶性糖含量比授粉果低4.72mg/gFW,而果汁pH低0.07。75d贮藏期间果肉可溶性糖含量无明显变化,而果汁pH趋于上升,授粉果果汁pH上升0.29而未授粉果仅上升0.19。授粉果实在45d贮藏期间未发生明显枯水,而且不溶性膳食纤维含量下降38.10%,而未授粉果实贮藏期间易发生枯水,贮藏45d后枯水指数达0.5,不溶性膳食纤维含量上升至2.33mg/gFW,为同期授粉果实的2.56倍。未授粉果实枯水发生前期汁囊组织出汁率无明显下降而严重枯水(贮藏45d)时下降至64.08%(贮藏前为75.70%)。人工授粉显著改善了永嘉早香柚果实品质。     

资阳香橙根cDNA全长文库的构建及EST分析

以资阳香橙根RNA为材料,采用SMART技术构建了cDNA全长文库,并对其质量进行鉴定.结果显示,该文库的库容量为4.8×105pfu/mL,重组率为93%,文库插入片段大小主要集中在0.5～1.0kb.从文库中随机挑取450个克隆进行5′端单向测序,去除载体序列及低质量的序列后,共获得有效长度大于150 bp的高质量ESTs(expressed sequence tags)序列438条,利用BioEdit软件对有效ESTs序列进行片段重叠群分析和拼接后,获得251个独立基因(Unigenes),其中包括87个片段重叠群(Contigs)和164个独立的ESTs(Singlets);通过与NCBI的非冗余核酸数据库进行tBLASTx比对,初步确定已知功能基因146个,未知功能基因91个,另有14个unigene未与数据库中序列匹配.该研究为进一步分离克隆资阳香橙根部功能基因奠定了基础.     

胡柚果实采后枯水的研究

胡柚果实贮藏前期（６２天），果皮组织中的超氧物歧化酶（ＳＯＤ）活性趋缓慢下降，过氧化物酶（ＰＯＤ）活性迅速上升，随后ＳＯＤ活性持续增加，ＰＯＤ活性略有下降并维持较高水平，果肉组织中的ＳＯＤ活性则呈相反变化，前期活性增加，于第６２天达最高，之后持续下降，但ＰＯＤ活性很低，且变化无规律。随着果皮中ＳＯＤ活性的增加和果肉中ＳＯＤ活性的下降，果实开始出现枯水（贮藏１２０～１３０天），并日趋严重。贮藏过程中果皮和果肉的可溶性糖含量均呈减少趋势，但前者变化幅度明显小于后者。１００ｍｇ／ｋｇＧＡ３处理可减缓贮藏过程果皮组织中的ＳＯＤ和ＰＯＤ活性变化、推迟果肉组织中ＳＯＤ活性高峰的出现、提高果肉中可溶性糖含量，降低果皮中的可溶性糖水平，显著地减轻果实枯水的发生。     

利用SSH技术分离甘蓝抗黑腐病相关基因的研究

采用抑制差减杂交(SSH)技术,构建甘蓝抗病品种A21经黑腐病菌诱导6、8、12、24h后的混合SSH文库。经反向Northern筛选,共获得87条高质量ESTs。通过NCBI中BLAST数据库比对分析发现,83个ESTs与已知蛋白或基因具有不同程度的同源性。     

黄龙病诱导下椪柑SSH文库的构建与分析

以实生苗和平椪柑(Citrus reticulate Blanco)为材料,采用抑制性差减杂交技术,分别以感染黄龙病与未感染黄龙病的椪柑叶片为检测方(tester)和驱动方(driver),成功构建了黄龙病诱导的差减cDNA文库。挑选了100个阳性克隆并成功测序得到71条EST,经NCBI基因库同源性比对,有41条非冗余高质量EST序列找到了同源序列,另有10条非冗余未搜索到同源序列。同源序列的基因涉及抗逆防御、运输、能量代谢、光合作用、蛋白代谢、信号转导、抗氧化等代谢途径和生理生化过程。值得注意的是文库中有由病原引起的韧皮部相关的凝集素蛋白的前体积累。挑选了2条进行Q-PCR定量分析,结果表明感病1周表达量增强不大,2周后tester的表达量明显高于driver,说明材料感病早期这些基因表达增强。     

珠美海棠MzWRKY基因家族盐胁迫应答模式研究

对苹果属植物现有的58个WRKY 的EST序列进行分析,得到了37条uniESTs。珠美海棠（Malus zumi Mats）是耐盐的优良苹果砧木,能在土壤全盐含量为0.6%的盐碱地上存活。根据已知的37条uniESTs序列,通过半定量RT-PCR研究了珠美海棠中MzWRKY基因家族的盐应答模式。28个MzWRKY基因能检测到RT-PCR的产物,其中21个基因受盐诱导,1个受盐抑制。根据MzWRKY基因盐应答模式可将这些基因分为两组,基因表达模式的差异说明MzWRKY在珠美海棠的盐应答中角色的多样性。     

扩增核糖体DNA限制性分析(ARDRA)研究刺芹侧耳细菌病原菌

为分析刺芹侧耳(Pleurotus eryngii)工厂化栽培中发黄腐烂子实体的细菌组成,构建16SrDNA文库,利用限制性内切酶MspI和RsaI分别对随机克隆进行酶切筛选,并测定代表性克隆16SrDNA序列,BLAST分析结果表明刺芹侧耳子实体病状组织中存有两类菌群,分别为假单胞菌和芽孢杆菌属。同时,对刺芹侧耳子实体病状组织中的细菌进行分离培养、致病性测定、16SrDNA鉴定及BLAST分析,病原细菌与恶臭假单胞菌(Pseudomonas putida)的同源性较高,其作为引起刺芹侧耳工厂化栽培细菌性病害的病原菌在国内外尚属首次报道。     

Improvement of mishqui (Solanum muricatum) earliness by selection and ethephon applications

The long period of time required for fruit ripening is a main drawback to the adoption of mishqui (Solanum muricatum) as a new crop. Variation in the fruit ripening period was studied in 18 mishqui clones in response to ethephon sprays (0, 500 or 2000 mg l⁻¹). Significant differences in the length of the fruit ripening period were detected between clones, ethephon doses and their interactions. Some clones did not respond to ethephon sprays, while in others the ripening period was shortened by more than 60%. In general, the effect of ethephon was greater in the clones with a longer ripening period. Furthermore, differences between clones of up to 20 days were found in the fruit growth period. Lengths of both periods (fruit growth and ripening) were not correlated, indicating that independent selection can be performed for both traits. The effects of ethephon on fruit quality characters were not significant in the majority of clones, although in five clones ethephon produced a skin degreening. Fruits from these clones had a higher firmness and lower soluble solid content (SSC) after ethephon treatment. On the other hand, ethephon sprays did not affect either the postharvest behaviour or the sensitivity to bruising. The results obtained here point to the existence of genotypic variation in the fruit ripening physiology of this species and they give relevant information for the improvement of mishqui earliness.     

大白菜细菌人工染色体文库的构建及鉴定

 以我国优良的大白菜自交系‘85-1’为材料,利用pIndigoBAC-5为载体,通过对高分子量DNA的制备、大片段DNA的选择、连接转化条件等几个方面的优化,构建了大白菜细菌人工染色体文库。该文库由57 600个克隆组成,平均大小为98.4 kb,空载率为1.5%;覆盖大白菜基因组10.3倍;挑取6个克隆培养5 d后,经HindⅢ完全酶切检测,其指纹图谱稳定一致。大白菜细菌人工染色体文库的构建为重要功能基因的克隆和定位及比较基因组研究奠定了基础。     

A deep-coverage BAC library for Ponkan mandarin and its further use in screening ethylene regulated genes

A deep-coverage bacterial artificial chromosome (BAC) library of Yanxiwanlu Ponkan, a late maturing cultivar, was constructed and evaluated. Results showed that the library contained a total of 61,000 clones. Restriction analysis indicated that the DNA insert sizes ranged from 30 to 170 kb with an average of ∼82 kb. No empty clone was found. Therefore, the library should cover ∼13.6-fold of the citrus genome. Continuous sub-cultivation of 5 randomly chosen clones for up to 100 generations showed no detectable change in their restriction profiles. To demonstrate the application of the library in discovery of genes, 7 ethylene-regulated genes were screened by PCR amplification from hierarchically pooled library clones with gene-specific primers. Five positive clones, each representing a different gene, were thus identified from 3840 clones. Sequencing of the clones showed that all of them matched the expected genes, indicating that the library was highly representative.     

荔枝采后果皮褐变过程中差异表达基因的SSH分析

 以‘妃子笑’荔枝果实为材料,采用抑制差减杂交（SSH）与cDNA 微阵列技术相结合,研 究了采后荔枝果皮褐变过程中的基因差异表达。分别以采后0 h 与32 h 的果皮总RNA 为驱动组与检测组, 构建了正向与反向SSH 文库,分别获得了282 个与76 个阳性克隆。通过cDNA 微阵列杂交筛选获得了在 采后32 h 果皮中上调表达克隆17 个,下调表达克隆49 个,分别代表了在采后32 h 果皮中上调表达基因 16 个和下调表达基因17 个,其中有较多的热击蛋白基因、糖代谢相关基因、细胞壁代谢相关基因等。 RT-PCR 检测基因表达结果与cDNA 微阵列杂交结果一致。     

辽西地区引种野杏数量性状研究

以辽西地区26个野杏无性系为试材,采用变异性分析、方差分析以及主成分分析等方法,研究了其25个数量性状的变异特点,以期为野杏无性系的遗传多样性研究和良种选育提供重要参考依据。结果表明:19个数量性状呈正态分布;25个野杏无性系数量性状变异系数均值23%,其中22个数量性状的变异系数在10%以上,小枝长度变异系数最大,达64%,单果质量、单核质量、单仁质量的变异系数分别为24%、21%和18%,表明野杏无性系数量性状变异程度丰富,选择潜力较大。野杏无性系果实性状重复力普遍偏高,核仁主要性状重复力次之,树体主要数量性状重复力较二者偏低,均达到极显著水平;果核仁性状的重复力除单果质量、单核质量和仁厚分别为0.634、0.756和0.624外,其它指标皆在0.909以上,其中仁长重复力最高,达0.973。主成分分析结果表明,前6个主成分累积贡献率达83.50%,能够反映野杏无性系数量性状的大部分信息;其中第1主成分主要反映野杏果核仁等经济性状。     

Züchtungsinitiative Niederelbe

In the year 2002 the breeding initiative Lower Elbe (Züchtungsinitiative Niederelbe, ZIN) was founded with the aim to establish a private financed breeding program of apple cultivars in North Germany. Members of the breeding initiative are fruit farms, the fruit co-operative Marktgemeinschaft Altes Land and some fruit retailers. Breeding work happens in close cooperation with the nursery firm Carolus (Belgium) and the institutes of fruit growing and nursery of the University of Applied Sciences Osnabrück. In the year 2005 selection work in selection step I started. From 2010 12.000 to 14.000 clones will be tested each year. In 2008 the first 24 clones of the selection step II were planted on two locations. Yearly 25 to 50 clones will follow in this selection step. Scientific research projects flank the breeding work.     

Construction of phage random eight-peptide library

AIM: To construct random eight-peptide library for the study on atherosclerosis and restenosis. METHODS and RESULTS: Random oligodeoxynucleotides encoded eight peptides were synthesized and amplified by polymerise chain reaction( PCR).The product was cloned into phage surface display vector fUSE5 in Sfi I site and electroporated into competent MC1061. The library was identified through PCR, hybridization, DNA sequencing and affinity biopanning of streptavidin. Because the upstream primer is complementary to part vector clone site sequences and part exogenous gene sequences, and the other one complementary to pⅢ gene of vector, thus only clones inserted exogenous gene could be amplified easily. Additionally we used the probe oligodeoxynucleotide complementary to vec for clone site sequences to identify clones which were not inserted exogeneous genes. Furthermore, two hybridizing positive clones were sequenced. Their sequences are consistent with two oligodeoxynucleotide probe sequences. As a result, 2.1×10⁸ special clones were obtained. Affinity biopanning proved that the libraries could be amplified steadily.CONCLUSION: The eight-peptide library is reliable.     

Identification of differentially expressed genes during flower opening by suppression subtractive hybridization and cDNA microarray analysis in Eustoma grandiflorum

The forward and reverse suppression subtractive cDNA libraries were constructed in petals of Eustoma grandiflorum at bud stage (stage 1) and anthesis (stage 7). Approximately 1000 clones were isolated from stage 1- (S1) and stage 7-specific (S7) libraries. The clones were sequenced and assembled, which yielded 98 contigs and 444 singletons. BLAST search was conducted on these assembled sequences. Generally, probes isolated from the S7 library exhibited higher expression at stage 7 by microarray analysis, as did those of the S1 library at stage 1. A clone set from the S7 library contained genes from later steps of anthocyanin biosynthesis pathway, terpene synthases, GAST (gibberellic acid-stimulated) family proteins, xyloglucan endotransglucosylase/hydrolase, glycosidases, and stress- and senescence-related proteins. In contrast, the S1 library contained genes associated with flavonol biosynthesis, phenylpropanoid metabolism, terpenoid metabolism, and floral organ development. Gene expression profiling for flavonoid biosynthesis was in accordance with preferential accumulation of flavonols at bud stages and anthocyanins at anthesis.