人朊蛋白基因的克隆与原核表达

以人血液中提取的总DNA为模板,克隆朊蛋白(prion protein,PrP)基因Prnp户的开放阅读框(ORF),与pMD -18T载体连接后转入E．coli JM109,用Blast软件比较重组质粒序列,结果表明获得的人Prnp的ORF与Genbank登录的相应核苷酸序列最高同源性为99．6％,推导的氨基酸序列同源性为99．8％,将编码人成熟PrP的基因定向克隆到 pET-30a(+)表达载体,将重组质粒pET-homPrP转化E．coli BL21(DE3),在37℃下用1．0 mmol／L IPTG诱导,重组融合蛋白获得高效表达,SDS-PAGE显示表达产物以包涵体形式存在,占菌体总蛋白的20％～25％．用Ni-NTA 树脂亲和层析纯化了表达产物．Western-blotting分析表明,融合蛋白被抗PrP的单克隆抗体特异识别．因此,在大肠杆菌中表达的人成熟PrP融合蛋白可以作为制备PrP抗体的免疫原．     

23个杨树无性系引种试验研究

通过对白城４１＃等２３个杨树无性系进行育苗、造林随机区组试验、差异性分析结果指出,各无性系之间苗期的高、径生长量都具有显著差异,但造林后第４ａ差异不显著（α〈０．０５）。系统聚类分析指出,２３个无性系可以分为６类：Ⅰ白城小黑等５个;Ⅱ桓仁等３个;Ⅲ金县等４个;Ⅳ箭杆杨;Ⅴ昌图等７个;Ⅵ健康杨等３个,在此基础上综合评价筛选出适合科尔沁沙地栽植的无性系为健杨、法库１＃、兴城、白林２＃、白城４１＃、鞍杂     

鸡白介素-6(ChIL-6)基因的克隆与序列分析

分别用鸡传染性法氏囊病病毒感染和用ConA免疫刺激健康鸡，无菌取处理鸡的脾脏，匀浆后用Tr-izol抽提细胞总RAN，应用随机引物反转录获得cDNA，设计引物，经PCR扩增获得鸡白介素-6（ChIL-6)基因，应用pGEM-T载体克隆该基因，经测序，表明其与Gene Bank中公布的唯一的一个ChIL-6基因为同源基因。     

杨树基因工程研究进展

运用现代分子生物学技术进行林木遗传改良研究具有重要的现实意义。介绍了杨树基因工程方面的研究进展，综述了杨树遗传图谱构建、基因克隆及转基因应用研究方面的状况，并对杨树基因工程研究存在的问题和前景进行了讨论。总结了杨树基因工程研究的规律，以促进林木基因工程研究的发展。     

杉木采穗圃营建技术的研究

从1980年起,先后在湖南省零陵地区林科所和江永县高泽源林场营建杉木采穗圃7.5亩。实践表明:杉木采穗圃繁殖系数大、产量高,且技术容易掌握,便于推广,是加速杉木造林良种化的有效途径。同时说明,科学地选择建圃材料,采用浅栽、控制冠幅、弯干和截顶,以及加强对圃地的经营管理,是实现建圃目标,提高穗条产量和质量的关键措施。     

木麻黄抗逆无性系苗期生长比较试验

采用通过筛选的2个木麻黄抗逆无性系与当地生长较好的惠2无性系进行水培育苗试验,结果表明:(1)不同无性系间苗高和地径差异不大,方差分析均未达显著水平。从遗传参数来看,3个无性系间苗高和地径的遗传力均为中等,分别为0.486和0.557,方差分量也不大,分别为66.06%、69.27%,苗高的遗传变异系数较小,其值为14.23%,而地径的遗传变异系数较大,其值为18.22%,利用苗高和地径指标对木麻黄抗逆品系和对照进行比较,不能取得较理想的结果;(2)不同无性系在侧根数、主根长、总干质量/总鲜质量等指标上均未达显著水平,且变异系数不大,仅为25.26%、13.70%、9.28%;但在地下部分干质量/地上部分干质量指标上达极显著水平,且变异系数较大,达46.42%。从苗木根系性状和生物量的遗传变异分析,木麻黄抗逆品系和对照没有什么差异。     

杜鹃红山茶CaAPX基因的克隆、表达及功能分析

[目的]利用分子生物学技术探讨杜鹃红山茶CaAPX基因的表达模式及在模式植物烟草中所起的抗寒、耐热作用,为今后山茶花抗逆育种奠定基础。[方法]根据山茶花同源序列设计特异性引物,利用同源克隆和3’,5’-RACE技术,从杜鹃红山茶嫩叶组织中克隆出抗坏血酸过氧化物酶(APX),命名为CaAPX,基因全长1 097 bp,开放阅读框753 bp,编码250个氨基酸。实时荧光定量PCR对杜鹃红山茶7种组织和温度胁迫下该基因的表达模式进行分析。[结果]表明:CaAPX基因在杜鹃红山茶7种组织中均得到表达,但表达水平不一,表达量由高到低依次为:未成熟果实嫩叶花苞叶芽种胚花瓣花芽,其中,未成熟果实中的表达量是其它组织的2.6811.44倍;温度胁迫处理8 h后该基因呈上调表达,CaAPX基因表达量分别是0 h的3.49和2.67倍。CaAPX基因转化烟草分析表明,过量表达CaAPX基因后,APX活性提高了2.58 4.09倍,抗坏血酸(As A)含量提高了2.673.56倍,并且转基因烟草植株的抗寒、耐热能力也获得提高。[结论]通过过量表达CaAPX基因能够提高烟草植株的抗寒、耐热性,为山茶花抗逆育种提供了科学依据。     

Quantitative Estimation of Genetic Variability in Morphological Characteristics of Eastern Cottonwood (Populus deltoides Bartr.) Clones

The study was carried out to analyze the genetic variability for different growth parameters in poplar clones at the age of 2 and 3 yr in the nursery. Forty-nine exotic and indigenous clones of poplar were evaluated for eight morphological traits. Clones were planted in randomized block design (RBD) with three replications with four clones in a block in each treatment. Observations for different characters were recorded on six selected competitive clones per genotype. Results showed a high interclonal variability for most parameters. Statistically significant differences among clones indicated that the majority of study characters are controlled by genetic factors, specific to each clone. Highly significant genotypic difference supported by wide range of variation of mean and range values were observed for the characters under study. Significant and positive correlation was observed between diameter at breast height (DBH) and plant height. Leaf lamina length showed positive and significant correlation with petiole length, total leaf length, leaf width, and L/B ratio. Total leaf length showed positive and significant correlation with leaf width and L/B ratio. High estimates of heritability (in a broad sense) were observed for almost all characters in the study. High genetic advance expressed as percent of mean was recorded for petiole length (60.90), followed by plant height (60.78) and collar diameter (44.19) at 2 yr. At the age of 3 yr, genetic advance was found maximum for petiole length (60.05), followed by collar diameter (47.62) and plant height (45.29). The efforts for selecting new clones and their field-testing must continue under a long-term improvement plan so that the best clones can be recommended for plantations and hybridization programs.     

Cloning and sequence analysis of nine novel MYB genes in Taxodiaceae plants

Myeloblastosis(MYB) is one of the largest transcribed factor families in plants. To gain an overall picture of the evolution of MYB genes in relict plants, we cloned nine novel MYB genes in Taxodiaceae plants(Taxodium distichum, Taxodium ascendens, Cryptomeria japonica var. Sinensis, Cryptomeria japonica cv. Araucarioides, Cryptomer Japonica, Metasequoia glyptostroboides, Cunninghamia lanceolata, Taiwania cryptomerioides and Glyptostrobus pensilis). The deduced amino acid sequences for MYBs showed that the nine MYB proteins contained two DNA binding domains. The first domain is from amino acid position 29 to 78, wherein three tryptophanes at 33, 53 and 73 were separated by 19 amino acids, respectively. The second domain is from amino acid position 82 to 127, wherein three tryptophanes at 86, 105 and 124 were separated by 18 amino acids, respectively, whereas the first tryptophane at amino acid position 86 is replaced by a phenylalanine. The characterization of these conserved domains at nine MYBs indicated that they all belong to the R2R3-MYB group. The secondary structure analysis showed that α-helix and β-turn are the major motifs of the predicted secondary structure of MYBs. The three dimensional model of each MYB protein showed that the structure is like clip, making it more flexible and mobile. The similarities between the nine MYB proteins in Taxodiaceae were calculated. The highest identical value of 99% is between CjsMYB, CjMYB and CjaMYB, whereas the lowest value of 82% is between TaMYB and ClMYB. According to the phylogenetic tree, the distances between different genera were relatively large whereas those within genera were relatively small. As expected, accessions of the same genus formed a subgroup before being grouped with other genera.     

马铃薯纺锤块茎类病毒山西分离物侵染性克隆的构建

马铃薯纺锤块茎类病毒(Potato spindle tuber viriod,PSTVd)侵染马铃薯会严重影响薯块的产量和品质。根据PSTVd的同源序列设计引物,以山西省马铃薯感病块茎中提取的总RNA为模板,经RT-PCR方法扩增出全长cDNA片段,将其克隆到质粒pBS-T载体上,进行序列分析。结果表明,该类病毒全长359 nt,能通过分子内序列互补形成致密的二级结构。以该序列作为种子序列进行Blast搜索,该序列与PSTVd荷兰分离物(GenBank登陆号:AY372400.1)的序列一致性为100%。将该序列与东北株系、河北株系及已公布的弱株系(GenBank登陆号:M14814.1)、强株系(GenBank登陆号:U23058.1)、中间株系(GenBank登陆号:AY937179.1)比对,有12个核苷酸具有多态性,其中有5个发生在致病区。将线性化的质粒pBS-PSTVd及其PSTVd的体外转录产物接种番茄矮红宝的无毒苗,20 d后接种株轻微显症,RT-PCR检测均呈阳性。将RT-PCR产物测序,其结果与接种的PSTVd原序列完全一致,证实构建的PSTVd山西分离物的克隆具有侵染性。