蓖麻蚕DNA导入家蚕引起遗传变异的研究-基因组DNA的RAPD检测

借助于精子介导,在家蚕受精的过程中将蓖麻蚕DNA转入家蚕卵内,从它们后代获得了新的变异品系。本文采用RAPD技术对这些品系基因组DNA进行了分析。结果表明,所用50种10mer随机引物中有49个检测出DNA的多态性,统计分析图谱中各类扩增带,其中变异品系与其相应受体的差异带占其总带数的26-37%,提示外源DNA导入受体后引起后代基因组的显著变异,并对这些变异的意义了讨论。 Abstract:With the aid of domesticated silkworm sperms,eri silkworm DNA was transferred into domesticated silkworm eggs during insemination,and variant strains were obtained from the progenies.Genomes of three new strains were analyzed using RAPD assay.Polymorphic fingerprints were obtained from 49 out of 50 primers.Different kinds of amplified bands in RAPD patterns were calculated and analyzed,the variant bands between variants and their recipients counted for 26~37% of the total bands of each variant.The results indicated that exogenous DNA introduced into recipients induced remarkable variation in progeny genomes.The significance of the variation was discussed.     

Molecular Genetic Variation in a Clonal Plant Population of Leymus chinensis （Trin.） Tzvel.

Randomly amplified polymorphic DNA （RAPD） analysis was used to investigate the genetic variation among populations, between populations, and within populations, relationships between genetic distance and geographic distance, and the molecular variation and population size. The effects of geographic and genetic distances, as well as of genetic differentiation and population size, on genetic variations of Leymus chinensis （Trin.） Tzvel. are discussed. The present study showed that there was significant RAPD variation between the Baicheng region population and the Daqing region population, with a molecular variance of 6.35% （P 〈 0.04）, and for differentiation among area populations of the Daqing region, with a molecular variance of 8.78% （P 〈 0.002）. A 21.06% RAPD variation among all 16 populations among two regions was found （P 〈 0.001）, as well as 72.59% variation within populations （P 〈 0.001）. Molecular variation within populations was significantly different among 16 populations.     

SSR based linkage and mapping analysis of C,a yellow cocoon gene in the silkworm,Bombyx moil

The yellow color of the cocoon of the silkworm Bombyx mori is controlled by three genes, Y （Yellow haemolymph）, 1 （Yellow inhibitor） and C （ Outer-layer yellow cocoon）, which are located on linkage groups 2, 9 and 12, respectively. Taking advantage of a lack of crossing over in females, reciprocal backcrossed F1 （BC1） progeny were used for linkage analysis and mapping of the C gene using silkworm strains C 108 and KY, which spin white and yellow cocoons, respectively. DNA was extracted from individual pupae and analyzed for simple sequence repeat （SSR） markers. The C gene was found to be linked to seven SSR markers. All the yellow cocoon individuals from a female heterozygous backcross （BC1F） showed a heterozygous profile for SSR markers on linkage group 12, whereas individuals with light yellow cocoons showed the homozygous profile of the strain C108. Using a reciprocal heterozygous male backcross （BC1M）, we constructed a linkage map of 36.4 cM with the C gene located at the distal end, and the closest SSR marker at a distance of 13.9 cM.     

Molecular characters and morphological genetics of CAL gene in Chinese cabbage

BepCAL,the homologous gene of CAL,was isolated from Chinese cabbage.Unlike BobCAL of cauliflower,BcpCAL did not hold the terninating mutation in the fifth exon.After crosses of cauliflower with Chinese cabbage,the resultant hybrids failed to form curd,which implicates the genetic complement of BcpCAL to the mutated BobCAL in the function of curd formation.One of CAL gene isolated from the hybrid apparently comes from the female parent(Chinese cabbage) even though there are a few of the bases substituted and deleted.The result offers the molecular and genetic evidences for the study of biological function of CAL in morphological genetics of curd.     

甘蓝型油菜Ogura 雄性不育×白菜的回交杂种后代与亲本之间蕾期基因表达差异比较研究*

以甘蓝型油菜（Brassica napus L, AACC,2n＝38）Ogura细胞质雄性不育材料为母本,以不同白菜（B campestris ssp. chinensis Makino, AA, 2n＝20） 自交系‘新选一号’和‘矮脚黄’为父本进行杂交,获得了杂种F1、BC1、BC2代。利用cDNA-AFLP技术对两种材料的不同回交世代BC1、BC2代与其亲本在蕾期的基因表达进行分析。结果表明,两种白菜回交世代与其亲本的基因表达有明显差异,在质和量上都存在差异。基因表达模式有5类共7种：（1）单亲沉默型（2种）,（2）单亲一致型（2种）,（3）双亲共沉默型,（4）杂种特异型,（5）表达一致型。随着回交世代的增加,回交杂种和亲本的基因表达在单亲沉默型、双亲共沉默型呈增加趋势。而在母本一致型、父本一致型、杂交种特异型、表达一致型呈下降的趋势。两种白菜在F1、BC1、BC2 3个世代与回交亲本花蕾间的基因差异表达有15种类型,其中以在轮回亲本、F1、BC1、BC2中共同出现表达的带的比例最高。Abstract: Crosses between female parent of Ogura male sterility Brassica napus L. and male parents of B. campestris ssp. chinensis Makino were made and F1, BC1 and BC2 generations produced. Gene expression of two Chinese cabbage backcross hybrid BC1, BC2 and their parents at bud stage was analyzed by means of cDNA-AFLP technique. The results indicated that the patterns of gene expression differ significantly between BC1 and BC2 generations and their parents. There were many patterns of gene expression, including gene overexpression and gene silencing. Five patterns (seven kinds) of gene expression were observed, which include: (1) bands occurring in only one parent (two kinds); (2) bands observed in hybrids and one parent (two kinds); (3) bands occurring in only parents (one kind); (4) bands visualized in only hybrids (one kind); (5) bands observed in parents and hybrids (one kind). In accompany with the addition of backcross, the increase trend in backcross hybrids and their parents were described in the aspects of differential gene expression, bands expressed only in one parent and bands expressed only in both parents. The declined trend in backcross hybrids and their parents were observed in the aspects of bands expressed in both hybrids and one parent (two kinds), bands visualized in only hybrids and bands observed in parents and hybrid. Fifteen patterns of gene expression were observed in F1、BC1、BC2 and backcross parents. The percent of bands expressed in F1、BC1、BC2 and backcross was highest.     

采用RAPD标记探讨瓣蕊唐松草(毛茛科唐松草属)的遗传多样性及其地理分布格局(英文)

Random amplified polymerphic DNA(RAPD)method was applied to assessg enetic variation and population structure of Thahctrum petalotdeum L(Ranunoulaceae),Two hundred and forty-six individuals from 11 populations of the species were investigated by RAPD profiles Twenty selected RAPD primers generated 125 bands．in which 120 were polymorphic Ther esults revealed a high level of genetic variation(ercentage of polymorphIc bands(PPB was 96％．Nei’s gene diversity(りwas 03502 and shannon’s information index(I) was 0．5199 at the species level) The differentiation among the populations was high(Gst=0．3511)in this species．Result of analyzing of molecularvariance(AMOVA)showedthat38．88％of genetic variance was found among the populations Positive correlation withr r=01945(P=00002)was found between genetic distance and geographic distance amongpo pulations Two populations distributed in the drainage basin of YanELz River affined genedcally and formed one clada and the rest nine populations formed the other clade in both unweighted pair-group method using arithmetic average(UPGMA)trees made by two different method different methods. It was yen/clear that these two populations were very special, andmust be closely related in history, despite the fact that they now share quite weak link to the restpopulations through gene communication.     

Loss of Genetic Diversity of Domesticated Panax notoginseng F H Chen as Evidenced by ITS Sequence and AFLP Polymorphism： A Comparative Study with P. stipuleanatus H T Tsai et K M Feng

In the present study, we evaluated the genetic diversity of Panax notoginseng F H Chen, a domesticated species, and P. stipuleanatus H T Tsai et K M Feng, an endangered wild species in southeastern Yunnan and adjacent areas in Vietnam, using sequences of the internal transcribed spacer (ITS) regions of nuclear ribosomal DNA and amplified fragment length polymorphism (AFLP) markers. Twenty-four accessions from three plantations of P. notoginseng and 51 samples from eight populations of P. stipuleanatus were assayed. A total of 694 bp of partial sequences of 18S, ITS 1, 5.8S, ITS2, and partial sequences of 26S were obtained. No sequence variation was detected within P. notoginseng and nine sites (1.30%) were variable in P. stipuleanatus. Two-thirds of the variable sites were found between Langqiao and other populations. In P. notoginseng, four pairs of AFLP primer combinations generated 312 bands, of which 240 (76.9%) were polymorphic and 60.15% of the polymorphisms were harbored within plantations. Approximately 41.0% and 66.9% of bands were polymorphic in population D7 and 5589, respectively. In P.stipuleanatus, the same four primer combinations produced 346 bands, of which 334 (96.5%) were polymorphic and approximately 62.14% of polymorphisms were maintained within populations. Considerable variations were observed. The percentage of polymorphic bands ranged from 50.2% to 84.9% and the average over populations was 70.9%. Cluster analysis did not show correlation of genetic differentiation with the distinctive leaf morphology of P. stipuleanatus (i.e. one form with bipinnatifid leaflets and the other with undivided leaflets). Because over 40% of genetic variations were maintained among populations and because of the very restricted distribution of P. stipuleanatus, all natural populations of this species should be conserved in situ. Considering that there are variations in P. notoginseng within and among plantations, we suggest establishing a genetic resource conservation garden or reintroducing P. notoginseng into its native habitats in southwestern China. Such reintroduction should be carefully executed after large-scale screening of genetic variation within the species.     

Molecular mapping of split rice spikelet mutantsrs-1 and analysis of its homeotic function in rice

srs-1, a new floral organ identity gene in rice, was mapped using RAPD and RFLP markers. Firstly, the cross was made between "ZhaiYeQing 8" (ZYQ8, indica) and split rice spikelet (SRS, japonica) mutant. The ratio of wild-type individuals and mutant plants in F2 population is 3:1, which indicates that the mutant characteristics are controlled by single recessive gene, srs-1. Consequently, BSA method was adopted and 520 random 10-mer primers were used to screen polymorphic bands between two bulks. Six primers could amplify polymorphic bands, of which S465 generates the most stable RAPD patterns. Then, S465 that cosegregates in F2 population has been converted into an RFLP marker successfully. Furthermore, srs-1 gene was mapped on chromosome 3 using DH mapping population. The effect of srs-1 gene results in the mutant of split rice spikelet. The mutant has longer and softer palea/lemma than those of wild-type, and two small palea/lemma-like organs between palea and lemma. In addition, there is a flower wit     

Genomic variation in the hybrids of white crucian carp and red crucian carp: evidence from ribosomal DNA

In this study, we conducted a cross of white crucian carp(♀)×red crucian carp(♂)(WR), and characterized the morphology, reproduction and genetics of the progeny. Different from parents, WR with the gray color showed the hybrid morphological traits of both parents. WR possessed normal gonads producing mature eggs or sperm, and exhibited high fertilization rate(90.2%) and high hatchery rate(80.5%), which contributed to produce and enlarge the population. WR with the same DNA content as parents was a diploid fish with 100 chromosomes(2n=100). Amplified ITS of 45 S r DNA, in WR the sequences consisting of 884 bp bases of the entire ITS-1 region, 5.8 S region, and entire ITS-2 region. The sequences showed high similarity between WR and its parents and leaned towards male inheritance. In WR, NTS of 5S r DNA consisted of three length types with total 654 bp bases. From sequence analysis of NTS, WR shared 94.2% and 95.1% similarities with their female and male parent, respectively. Sequence analysis of ITS and NTS revealed that there existed recombination and variation in the hybrid progeny, which was the genetic base for adaptation and speciation. In conclusion, we obtained WR from hybridization and it exhibited hybrid traits in morphology and variation in genetic composition showing essential difference with its parents. The obtainment of WR has important significance in fish genetic breeding.     

Changes in DNA base sequences in the mutant of Arabidopsis thaliana induced by low-energy N+ implantation

To reveal the mutation effect of low-energy ion implantation on Arabidopsis thaliana in vivo, T80II, a stable dwarf mutant, derived from the seeds irradiated by 30 keV N⁺ with the dose of 80×10¹⁵ ions/cm² was used for Random Amplified Polymorphic DNA (RAPD) and base sequence analysis. The results indicated that among total 397 RAPD bands observed, 52 bands in T80II were different from those of wild type showing a variation frequency 13.1%. In comparison with the sequences of A. thalianain GenBank, the RAPD fragments in T80II were changed greatly in base sequences with an average rate of one base change per 16.8 bases. The types of base changes included base transition, transversion, deletion and insertion. Among the 275 base changes detected, single base substitutions (97.09%) occurred more frequently than base deletions and insertions (2.91%). And the frequency of base transitions (66.55%) was higher than that of base transversions (30.55%). Adenine, thymine, guanine or cytosine could be replaced by any of other three bases in cloned DNA fragments in T80II. It seems that thymine was more sensitive to the irradiation than other bases. The flanking sequences of the base changes in RAPD fragments in T80II were analyzed and the mutational “hotspot” induced by low-energy ion implantation was discussed.     

An integrated database for the enhanced identification of silkworm gene resources

The National Academy of Agricultural Science (NAAS) has developed a web-based database to provide characterization information in silkworm. The silkworm database has four major function menus: variety searching, characterization viewing, general information and photo gallery. It provides 321 silkworm varieties characterization information for six different regions namely, Korean, Japanese, Chinese, European, Tropical and non-classified group. Additionally, the database provides 1,132 photo images regarding life cycle of various silkworm varieties. A specific characterization information table provides accession number, variety, strain and larval marking, blood color, cocoon color, cocoon shape, egg colors, remarks and image table provides photos which consist of shape and color in the different stages of larval, egg and cocoon stages. AVAILABILITY: The database is available for free at http://www.naas.go.kr/silkworm/english/     

一种简便的提取蚕蛹蛋白新方法

以蚕蛹为原料,采用一种简便的提取工艺提取蚕蛹蛋白质,结果表明：干蛹约占鲜蛹重的32．5％,干蛹中蛋白质含量为54．2％,成品中蚕蛹蛋白质含量为83．31％,其蚕蛹蛋白收率达干蛹的61．32％,是文献报道用减法提取收率的2．27倍,蚕蛹中蛋白质的回收率达94．33％。     

家蚕分子生物学研究进展

近年来,应用RAPD技术,通过构建近等基因系的方法,已筛选出家蚕性别、抗性等连锁的分子标记。日本已成功地构建了家蚕28个连锁群的RAPD分子连锁图,并有7个连锁群和经典的遗传图相对应．我国也公布了RAPD和AFLP分子连锁图,由于密度不够出现28个以上的连锁群;家蚕生物反应器研究和开发已近20年历史,表达了数百种外源基因,由于表达量不高及产物分离纯化难度和成本问题,至今未能进入产业化;家蚕转基因有过比较好的尝试,改进转基因技术提高外源基因的整合率是今后主攻方向。     

Expression of hIGF-I in the silk glands of transgenic silkworms and in transformed silkworm cells

To express human insulin-like growth factor-I (hIGF-I) in transformed Bombyx mori cultured cells and silk glands, the transgenic vector pigA3GFP-hIGF-ie-neo was constructed with a neomycin resistance gene driven by the baculovirus ie-1 promoter, and with the hIGF-I gene under the control of the silkworm sericin promoter Ser-1. The stably transformed BmN cells expressing hIGF-I were selected by using the antibiotic G418 at a final concentration of 700—800 μg/mL after the BmN cells were transfected with the piggyBac vector and the helper plasmid. The specific band of hIGF-I was detected in the transformed cells by Western blot. The expression level of hIGF-I, determined by ELISA, was about 7800 pg in 5×10⁵ cells. Analysis of the chromosomal insertion sites by inverse PCR showed that exogenous DNA could be inserted into the cell genome randomly or at TTAA target sequence specifically for piggyBac element transposition. The transgenic vector pigA3GFP-hIGF-ie-neo was transferred into the eggs using sperm-mediated gene transfer. Finally, two transgenic silkworms were obtained after screening for the neo and gfp genes and verified by PCR and dot hybridization. The expression level of hIGF-I determined by ELISA was about 2440 pg/g of silk gland of the transgenic silkworms of the G1 generation.     

Analysis of heterosis and recombination loss for fitness and productivity traits in different hybrids of mulberry silk moth Bombyx mori

Three different races of lepidopteron silk moth Bombyx mori were used in reciprocal and inter se crosses to determine heterosis effects at F₁ and recombination loss at the F₂ generation for three fitness traits (fecundity, larval duration, survival rate) and four productivity traits (larval weight, cocoon weight, shell weight, filament length). Eleven mating types were represented in the present study, including three pure breeds and a variety of F₁ and F₂ populations arising from regular and reciprocal crosses, respectively. Equations were derived to evaluate heterosis, maternal and overdominance effects for the above traits. Estimates of heterosis and overdominance effects revealed significant heterosis effects for all the traits, but overdominance was only seen for larval duration (favorable effect) and survival rate (unfavorable effect). Maternal effects were significant for the majority of the traits under study. The results revealed significant reduction for all the quantitative traits from F₁ to F₂, except for larval duration. The most obvious explanation for the reduction of fitness parameters and productive traits is the reduction in heterozygosity from F₁ to F₂ (it is expected that one half of the heterozygosity of F₁ is lost in F₂). For larval duration this explanation seems insufficient and breakdown of epistatic gene effects (i.e. recombination loss) has been suggested.     

Gene expression analysis in the larval silk gland of the eri silkworm Samia ricini

Insects produce silk for a range of purposes. In the Lepidoptera, silk is utilized as a material for cocoon production and serves to protect larvae from adverse environmental conditions or predators. Species in the Saturniidae family produce an especially wide variety of cocoons, for example, large, golden colored cocoons and those with many small holes. Although gene expression in the silk gland of the domestic silkworm (Bombyx mori L.) has been extensively studied, considerably fewer investigations have focused on members of the saturniid family. Here, we established expression sequence tags from the silk gland of the eri silkworm (Samia ricini), a saturniid species, and used these to analyze gene expression. Although we identified the fibroin heavy chain gene in the established library, genes for other major silk proteins, such as fibroin light chain and fibrohexamerin, were absent. This finding is consistent with previous reports that these latter proteins are lacking in saturniid silk. Recently, a series of fibrohexamerin‐like genes were identified in the Bombyx genome. We used this information to conduct a detailed analysis of the library established here. This analysis identified putative homologues of these genes. We also found several genes encoding small silk protein molecules that are also present in the silk of other Lepidoptera. Gene expression patterns were compared between eri and domestic silkworm, and both conserved and nonconserved expression patterns were identified for the tested genes. Such differential gene expression might be one of the major causes of the differences in silk properties between these species. We believe that our study can be of value as a basic catalogue for silk gland gene expression, which will yield to the further understanding of silk evolution.     

Ecdysteroids in Developing ovarian follicles of Hyalophora cecropia

Developing ovarian follicles of the silkmoth Hyalophora cecropia accumulate large amounts of ecdysteroids during oogenesis. As measured by an ecdysteroid radioimmunoassay (RIA), this accumulation begins near the end of vitellogenesis, just prior to nurse cell collapse, and continues through the beginning of chorion formation. Analysis of ovarian ecdysteroids by a combination of high-performance liquid chromatography and RIA demonstrates that the major proportion of these are present in a highly polar form, most likely as conjugates; ecdysone and 20-hydroxyecdysone were present as well, in much lower proportions. Light microscopic autoradiographs of photoactivated follicles after in vivo incubation with [³H]ecdysone indicate that within the oocyte ecdysteroids are associated with the yolk sphere membranes.     

应用频数分布面积法定位家蚕QTL

利用生态型差异较大的两个家蚕品种７８２和ｏｄ１００作正反杂交,得到Ｆ１。让Ｆ１雄蚕与叠双隐性标记的ｏｄ１００回 到回交子代。     

In vivo site-specific integration of transgene in silkworm via PhiC31 integrase-mediated cassette exchange

Current techniques for genetic engineering of the silkworm Bombyx mori genome utilize transposable elements, which result in positional effects and insertional mutagenesis through random insertion of exogenous DNA. New methods for introducing transgenes at specific positions are therefore needed to overcome the limitations of transposon-based strategies. Although site-specific recombination systems have proven powerful tools for genome manipulation in many organisms, their use has not yet been well established for the integration of transgenes in the silkworm. We describe a method for integrating target genes at pre-defined chromosomal sites in the silkworm via phiC31/att site-specific recombination system-mediated cassette exchange. Successful recombinase-mediated cassette exchange (RMCE) was observed in the two transgenic target strains with an estimated transformation efficiency of 3.84–7.01%. Our results suggest that RMCE events between chromosomal attP/attP target sites and incoming attB/attB sites were more frequent than those in the reciprocal direction. This is the first report of in vivo RMCE via phiC31 integrase in the silkworm, and thus represents a key step toward establishing genome manipulation technologies in silkworms and other lepidopteran species.