抗盐酸克伦特罗单链抗体基因的克隆、序列分析及表达

提取分泌抗盐酸克伦特罗单克隆抗体杂交瘤细胞株的总RNA,通过RT-PCR技术,扩增VH和VL,用一段柔性肽链-(G4S)3将VH和VL连接成ScFv。测序后经NCBI Blast分析,所得ScFv具有重组功能性鼠抗体可变区基因的特征。将所得目的基因与pET-22b( )连接,转化E.coli BL21,用IPTG诱导表达,表达蛋白经SDS-PAGE分析,37℃培养5 h表达量较大,25℃诱导表达以可溶性为主。     

大豆品种间DNA限制性片段长度多态性（RFLP）的分析

用６２个大豆ＤＮＡ分子探针与５种限制性内切酶组合，对大豆基因组ＤＮＡ限制性片段长度多态性（ＲＦＬＰ）进行分析，结果表明，所选用的两对材料，其多态性ＲＦＬＰ标记的频率均高达６０％，可作为ＲＦＬＰ作图较理想的亲本，ＲＦＬＰ标记的多态性类型表现为共显性，少数为显性，其中一些标记揭示了２个或２个以上的独立分离的基因座位，不同限制性内切酶的揭示多态性的能力上差异不显著，在杂交片段长度上差异显著并都大于预期，     

Genetic Relationships among Prunus mume var. pendula Using AFLP Markers

Genetic relationships among Prunus mume var. pendula were studied by using AFLP markers. 18 accessions representing 14 cultivars ofPrunus murne var. pendula were selected from the germplasm collection at the Research Center of China Mci Flower. Seven Mse I-EcoR I AFLP primer combinations revealed 450 legible bands, and 269 of which were polymorphic markers. A similarity matrix was prepared using the simple matching coefficient of similarity and Nei‘s (72) distance coefficient. A UPGMA dendrogram demonstrated the genetic relationships of the cultivars. The information given by AFLP markers was basically consistent with the morphological classification and the evolutionary history of the morphotypes, and roughly supported the new revised classification system for Chinese Mci Cultivars. But there were still several exceptions: 1) the ‘Guhong Chuizhi‘ inserted between the ‘Tiaoxue Chuizhi‘ and the ‘Danfen Chuizhi‘; 2) the ‘Wufu Chuizhi‘ kept off the Pink Pendant Form, and the ‘Moshan Chuizhi‘ was removed from Viridiflora Pendant Form; 3) the ‘Danbi Chuizhi‘ and the ‘Shuangbi Chuizhi‘ of Viridiflora Pendant Form got together well but fell within the Pink Pendant Form.     

多角体蛋白基因核苷酸序列对HBsAg(PreS2+S)在家蚕中表达的作用

应用PCR突变的方法 ,在乙肝病毒表面抗原 (HBsAg)前S2序列的 5’端融合了BmNPV多角体蛋白基因5’端的 12个碱基 ,获得了融合乙肝表面抗原中蛋白基因 (HBMp) ;通过同源重组将其插入到BmNPV基因组多角体启动子后 ,构建了重组杆状病毒BmPAK HBMp。用重组病毒BmPAK HBMp和BmPAK HBM(带有非融合乙肝表面抗原中蛋白基因 )感染家蚕细胞及蛹 ,对两种病毒的表达产物用ELISA进行了跟踪检测 ,结果表明融合蛋白比非融合蛋白HBsAg活性提高了 6 0 %～ 80 % ,作为HBsAg构成部分的PreS2抗原性提高了 8～ 10倍 ;并且HBsAg和PreS2 Ag表达的时相曲线不同 ,PreS2 Ag的表达量比HBsAg早 1～ 2d达到最大值。ELISA检测和Westernblotting分析表明 ,多角体基因序列的存在更有利于中蛋白全基因 (PreS2 +S)的表达。     

Identification and breeding behaviour of a major deletion of chromosome 5D ofTriticum aestivum

Summary A study was undertaken to evaluate the breeding behaviour and to identify a spontaneously produced putative chromosomal deletion in the winter wheat (Triticum aestivum L. em. Thell.) cv Norstar. Male and female transmission studies of plants heterozygous for the deletion chromosome indicated 9.5% and 48.8% transmission through the pollen and the egg, respectively. Meiotic analyses of progeny from deletion heterozygotes indicated that the deletion chromosome was eliminated from half of the plants, which agreed with the male and female transmission frequencies. Testcrosses of the deletion chromosome with telocentrics and nullisomic-tetrasomic combinations suggested that the deletion involved the long arm of chromosome 5D. This was confirmed by restriction fragment length polymorphism (RFLP) analysis. Also, monosomic plants obtained in progeny of deletion heterozygotes were shown to be monosomic for 5D. Studies of plants homozygous for the deletion showed relatively normal pairing between the deletion chromosomes, and with the short arm (5DS), but not the long arm (5DL). Deletion homozygotes were self-sterile, and morphologically similar to plants nullisomic for 5D, but plants that also contained 5DL, or a homoeologous chromosome were self-fertile and had normal morphology. Studies of chromosome morphology indicated that the deletion chromosome was metacentric, and the length of the long arm was reduced by approximately 60%. RFLP studies showed that, in terms of genetic distance, 90% of the arm was missing.     

Integration of AFLP markers into a linkage map of sugar beet (Beta vulgaris L.)

The AFLP (amplified fragment length polymorphism) technique has been applied in establishing an extended linkage map of sugar beet. A total of 120 AFLPs were integrated into an existing linkage map based on RFLP markers. Four primer combinations yielded between 19 and 40 polymorphic bands in an F₂ population consisting of 94 plants. The AFLP loci were evenly distributed over the nine linkage groups, with the exception of linkage group V where the number of AFLPs was significantly low. The AFLPs were found to be reproducible even against the background of different combinations of Taq DNA polymerases and buffers. However, the quantity of higher molecular weight fragments (>400 bp) was reduced when using plant DNA of poor quality as a template. The results of these experiments are discussed, together with possible applications of AFLPs in sugar beet breeding.     

A molecular marker linked to the Prv1 gene that confers resistance to Papaya ringspot virus-type W in melon

Papaya ringspot virus‐type W (PRSV‐W) is the most prevalent and important viral pathogen of cucurbits in Brazil. It can be effectively controlled by the incorporation of genetic resistance into susceptible melon cultivars. The present study identified amplified fragment length polymorphic (AFLP) markers linked to the PRSV‐W resistance Prv¹ allele. The susceptible yellow‐fleshed melon‐breeding line AF426^prv1 and its nearly isogenic‐resistant line AF426^Prv1, which carries the Prv¹ allele resident in the Indian cantaloupe U.S. Plant Introduction (PI) 180280, were screened for AFLP marker polymorphisms. Of 30 251 AFLP loci, only three were polymorphic between the nearly isogenic lines. Segregation analyses for these three polymorphic markers and the Prv¹ allele using a BC₁ population of 197 plants indicated close linkage (0.5% recombination frequency) between marker EK190 (HindIII‐CGA and MseI‐GTG; 190 bp) and Prv¹. Thus, EK190 might be a useful marker in breeding programmes aiming to develop melon cultivars resistant to PRSV‐W. The other two markers are closely linked to each other, but distantly linked to Prv¹.     

Precision of genetic relationship estimates based on molecular markers

Genetic progress through selection is directly related to the amount of variability present in the population and the quality of genes contributed by the parents. Genetic relationships between lines were studied using DNA marker-based estimates of genetic similarity. A statistical methodology using the width of a confidence interval was developed to determine the number of probes to be surveyed and the precision in the estimation of genetic distance between pairs of cultivars. Precision was affected by type of genetic distance used, the number of cultivars, and amount of genetic diversity present in the studied group. The width of a (1-α)% confidence interval decreased as the number of RFLP fragments increased. Oat and wheat diversity studies were used to illustrate the methodology. This revised version was published online in July 2006 with corrections to the Cover Date.