Bt水稻杂交育种中转基因的遗传分析

利用PCR、GUS染色和Western印迹杂交技术检测了Bt水稻杂交后代群体,发现在394株GUS阳性株中,共有392株表达Bt蛋白,协同表达株率达99.49%。由此表明,在杂交后代中报告基因Gus和目的基因crylAb紧密连锁遗传与表达。本试验还发现,在BC1、BC1F2和粳粳交F2群体中转基因呈单基因显性遗传,而在籼粳交F2群体中偏离3:1分离。 Abstract:Improved histochemical staining for GUS activity,PCR and Western blotting were used to detect the population of Bt rice crossed to conventional rice varieties.A total of 392 plants expressing Bt toxin protein were found in 394 GUS positive plants.The result demonstrated that cry1Ab gene closely inherited and expressed with reporter gene gus.Therefore,it is possible to develop GUS-assisted-selection to preliminarily identify the Bt gene and study the inheritance of transgenes in (back)cross breeding.Mendelian segragation of reporter gene Gus was observed in F2,BC1 and BC1F2 progenies.Thus indicated that transgenes inherited as a single dominant gene in the progenies of Bt rice crossed to conventional rice varieties.     

An in vitro whole plant selection system: paraquat tolerant mutants in the fern Ceratopteris

Summary A whole plant selection system using the haploid gametophyte generation of the fern Ceratopteris richardii has been developed to select for mutations that confer resistance or tolerance to various selection pressures. The expression of the mutations can be analyzed and characterized in both the haploid gametophyte and diploid sporophyte generations. Genetic analyses are facilitated by the fern's rapid life cycle and the ease of manipulating the gametophyte generation. Selection for tolerance to the herbicide paraquat has yielded two mutants which have an increased tolerance to the herbicide in both the gametophyte and sporophyte generations. Both mutants exhibit single nuclear gene inheritance patterns and appear to be closely linked or allelic.     

三个矮杆小麦的矮生性遗传研究

通过与事国春杂交,利用杂交后代Ｆ２和回交后代ＢＣ１Ｐ１及ＢＣ２Ｐ２,研究了三个小麦新矮杆品系和矮生性遗传特性。结果表明,０００４的矮生性受一对部分显性矮杆基因控制,５７４６和７５３９－各受两对部分显性矮杆基因控制。     

Inheritance and Expression of Copies of Transgenes 1Dx5 and 1Ax1 in Elite Wheat （Triticum aestivum L.） Varieties Transferred from Transgenic Wheat through Conventional Crossing

To study the inheritance and expression of multiple copies of transgenes from transgenic wheat lines, three crosses between transgenic wheat lines B72-8-11b and B102-1-2 and Chinese elite wheat varieties Chuan89-107 and Email 8 were carried out. Chuan89-107×B72-8-11b, Chuan89-107×B102-1-2 and Email 8×B72-8-11b, and F_1 plants were selfed or backcrossed to obtain different generation populations. Protein analysis in grains of F_1 and F_2 and backcross progenies of BC_1F_1, BC_1F_2, BC_1F_3, BC_2F_1, BC_2F_2 and BC_2F_3 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the transgenes lDx5 and lAx1 were expressed and segregated in the target wheat according to Mendelian laws. A range of lDx5 expression levels were observed in the progenies of Chuan89-107×B72-8-11b and Emai 18×B72-8-11b, but the expression levels of lAx1 in progenies of Chuan89-107×B102-1-2 rarely changed. It suggested that the two foreign genes had different mechanisms of expression in the cross progeny, even though they were produced in the same way and the foreign lDx5 gene of 5-10 copies had the more complicated expression mechanism than the lAx1 gene of 4-5 copies.     

Evaluation of BC2 progenies derived from 3x-2x and 3x-4x crosses of Lilium hybrids: a GISH analysis

An allotriploid (ALA, 2n=3 x=36) BC(1) plant was obtained by backcrossing a diploid F(1) interspecific hybrid (LA, 2n=2 x=24), derived from a Lilium longiflorum (L genome) and an Asiatic hybrid (A genome), to the latter parent. This allotriploid was backcrossed to a diploid Asiatic hybrid (2n=2 x=24) and to an allotetraploid (LLAA, 2n=4 x=48) LA hybrid. A total of 25 plants of these crosses were examined for ploidy level, and 12 individuals were analyzed for their genome constitution through genomic in situ hybridization (GISH). In most cases the progenies from the triploid-diploid (3 x-2 x) crosses consisted of aneuploids. Further more, there was evidence for the formation of near-haploid (x=12+2) to triploid (3 x=36) gametes in the allotriploid BC(1) plant. The progenies of triploid-tetraploid (3 x-4 x) cross also consisted of mostly aneuploids but in this case the triploid female parent had contributed predominantly near-triploid (2n) gametes for the origin of BC(2) progenies. The different ploidy levels observed between 3 x-2 x and 3 x-4 x crosses are possibly caused by preferential fertilization or survival resulting in a different ratio of chromosome numbers between the embryo and endosperm. Though Lilium has a tetrasporic, eight-nucleate type of embryo sac formation (Fritillaria type), the observed difference between the progeny types in 3 x-2 x and 3 x-4 x crosses is comparable to that of observed in monosporic eight nucleate types (Polygonum type) that predominate in most genera of Angiosperms. An important feature of the genome constitution of the progenies was that the homoeologous recombinant chromosomes were transmitted intact from BC(1) to BC(2) progenies in variable numbers. In addition, there was evidence for the occurrence of new homoeologous recombinations in the triploid BC(1). Of the two euploid BC(2) plants one had originated through the parthenogenetic development of a 2n egg and the other had originated through indeterminate meiotic restitution (IMR).     

Overexpression of osmotin gene confers tolerance to salt and drought stresses in transgenic tomato (Solanum lycopersicum L.)

Abiotic stresses, especially salinity and drought, are major limiting factors for plant growth and crop productivity. In an attempt to develop salt and drought tolerant tomato, a DNA cassette containing tobacco osmotin gene driven by a cauliflower mosaic virus 35S promoter was transferred to tomato (Solanum lycopersicum) via Agrobacterium-mediated transformation. Putative T0 transgenic plants were screened by PCR analysis. The selected transformants were evaluated for salt and drought stress tolerance by physiological analysis at T1 and T2 generations. Integration of the osmotin gene in transgenic T1 plants was verified by Southern blot hybridization. Transgenic expression of the osmotin gene was verified by RT-PCR and northern blotting in T1 plants. T1 progenies from both transformed and untransformed plants were tested for salt and drought tolerance by subjecting them to different levels of NaCl stress and by withholding water supply, respectively. Results from different physiological tests demonstrated enhanced tolerance to salt and drought stresses in transgenic plants harboring the osmotin gene as compared to the wild-type plants. The transgenic lines showed significantly higher relative water content, chlorophyll content, proline content, and leaf expansion than the wild-type plants under stress conditions. The present investigation clearly shows that overexpression of osmotin gene enhances salt and drought stress tolerance in transgenic tomato plants.     

Selection of a mutation conferring high NaCl tolerance to gametophytes of Ceratopteris

Summary Spores from a weakly salt tolerant strain of Ceratopteris richardii containing the mutation stl1 were irradiated and sown on nutrient medium supplemented with 200 mM NaCl. A single highly salt tolerant gametophyte was recovered and selfed to generate a homozygous sporophyte. Spores from this strain, 1023, were used to document the sexual transmission of the trait and to monitor the inheritance of tolerance in crosses to both the wild type and to the parental salt tolerant strain. Genetic analysis showed the 1023 strain to possess both the original stl1 mutation and an additional semi-dominant nuclear mutation, stl2, that individually conferred a high level of tolerance to gametophytes. In combination, both mutations had additive effects. Tolerance was also evident in sporophytes, but at a lower level than in gametophytes.     

Screening of pollen grains vis-à-vis whole plants of oilseed brassicas for tolerance to salt

Pollen grains and whole plants of 11 cultivars of oilseed brassicas (B. juncea,B. campestris,B. carinata) were screened for salt tolerance. Whereas pollen germination percentage in sitting drop cultures served as a reliable index of pollen tolerance to NaCl, pollen-tube growth did not. Seed yield in plants of the same 11 cultivars raised in artificially salinized soils also proved to be a good index of whole plant tolerance to soil salinity. A close correspondence between pollen (gametophyte) and whole plant (sporophyte) responses to salinity was discovered. Our studies show that tolerance to salt is yet another trait expressed in both the sporophyte and gametophyte.On study leave from the National Bureau of Plant Genetic Resources, New Delhi     

Identification of alien chromosomes through GISH and RFLP analysis and the potential for establishing potato lines with monosomic additions of tomato chromosomes.

To increase the potential for establishing a complete series of tomato chromosome addition-sbstitution lines in a potato background, six new BC1 progeny were produced. All of them originated from crosses between three different hexaploid potato (+) tomato fusion hybrids. Three different somatic hybrids, viz., C31-17-5, C31-17-24, and C31-17-51, were used as female parents, and four different tetraploids, viz., Katahdin, Frieslander, 6704-1, and AM66.42 were used as male parents. A characterisation of the genomes of the three fusion hybrids and the six BC1 progenies (6739, 2001, 2002, 2003, 2004, and 2005) through genomic in situ hybridization and restriction fragment length polymorphism (RFLP) analysis indicated that there was preferential tomato chromosome elimination in the fusion hybrids. Similar analyses of the six BC1 progeny indicated that a variable number of the alien tomato chromosomes (6-11) were present in individual plants. RFLP analysis using chromosome specific DNA probes indicated that BC1 progenies had retained all 12 tomato chromosomes, albeit in different individual plants. This means that the available BC1 progenies have the potential for establishing a complete series of tomato chromosome addition-substitution lines in a potato background.     

Apomixis and sexuality in Paspalum simplex: characterization of the mode of reproduction in segregating progenies by different methods

Segregating progenies of crosses between sexual and apomictic genotypes of Paspalum simplex were analysed for the formation of meiotic versus aposporous embryo sacs, zygotic versus parthenogenetic embryos, and autonomous versus pseudogamous endosperms by using cytoembryological and flow cytometric analyses. Reduced and unreduced 8-nucleated embryo sacs were the final product of female gametophyte development in sexual and aposporous genotypes, respectively. An incomplete penetrance of parthenogenesis was detected in aposporous genotypes. The relative DNA content of endosperm nuclei revealed the normal 2:1 maternal to paternal ratio in sexuals and a 4:1 ratio in apomicts, indicating insensitivity of the apomictic genotypes to endosperm imprinting. Apospory, parthenogenesis and pseudogamy are located on a relatively large linkage group and are inherited together with previously developed molecular markers as a single genetic unit in segregating progenies.     

雌雄异株地钱(Marchantia polymorpha)对镉胁迫生理响应的性别差异

为探究雌雄地钱在镉胁迫下性别响应差异,该文采用室内培养法,对野外采集的地钱雌雄配子体进行CdCl₂(100 mg·L^-1)溶液胁迫处理,定期取样观测其生理指标并在雌雄间进行比较。同时,利用隶属函数法计算雌雄地钱所有指标的综合评价值,评估雌雄地钱耐镉能力差异。结果表明:雌雄地钱在镉胁迫后,各个指标的变化趋势基本一致,超氧化物歧化酶(SOD)、谷胱甘肽(GSH)、丙二醛(MDA)均表现为显著上升,过氧化物酶(POD)呈现为先下降后上升,脯氨酸(Pro)、可溶性蛋白均表现为先上升后下降,叶绿素a(Chla)、叶绿素b(Chlb)均表现为显著下降; 雌性地钱叶绿素a/叶绿素b(Chla/Chlb)显著上升,雄性地钱仅在胁迫8 d显著上升,其余天数下无显著变化; 综合评价值显示耐镉能力为雄性地钱>雌性地钱。该研究结果为深入探讨雌雄异株植物对胁迫的性别响应差异提供了参考。     

小麦农家品种大籽糙抗条锈性的遗传分析

以抗条锈病的农家品种大籽糙作父本、感病品种铭贤169作母本杂交获得F1代杂交种,F1代植株自交获得F2代种子,F1代植株与铭贤169回交获得 BC1代种子。在人工控制条件下,用我国小麦条锈菌优势小种条中28号和条中32号,分别对F1、F2、BC1代及其亲本的幼苗进行人工接种,研究了它们的抗性表现和杂交后代中抗条锈性的分离情况。结果表明,大籽糙对条中32号小种的抗性由一对隐性基因控制;对条中28号小种的抗性由一对显性基因和一对隐性基因的互补作用控制。 Abstract:Dazicao,a native wheat variety with stripe rust resistance from Henan,China,was crossed with susceptible cultivar Mingxian 169 as the female parent.The F1 progeny was selfed to produce F2 progeny and backcrossed with Mingxian 169 to produce BC1 progeny.In air-conditioned greenhouse,seedlings of the F1,F2,BC1 progenies and their parents were inoculated with the prevalent races CY28 and CY32 of Puccinia striiformis respectively.The phenotypes of the F1,F2 and BC1 plants were analyzed for resistance to the two races.The results indicated that the resistance in the Dazicao to race CY32 was controlled by one recessive gene,and the resistance to race CY28 by complementary action of one dominant gene and one recessive gene.     

Polymorphism and inheritance of seed storage protein in sunflower

The data on polymorphism and inheritance of the seed storage protein helianthinin are presented. The results of hybrid analysis indicate that in the annual sunflower Helianthus annuus, helianthinin synthesis is controlled by at least three loci: HelA, HelB, HelB, and HelC. Codominant alleles controlling different electrophoretic variants of polypeptides were identified at each of the loci. The HelA locus was inherited independently of HelB and HelC in a series of dihybrid crosses. The frequencies of recombination between loci HelB and HelC estimated in F2 and BC of two crossing combinations were respectively 21.8 and 19.0%. Segregation of the Hel-C-controlled variants in the progenies from the crosses of cultured sunflower with annual wild species and forms corresponded to that theoretically expected for Mendelian inheritance. The maternal type of helianthinin inheritance was observed in the progenies from the crosses of inbred H. annuus lines with perennial diploid and polyploid Helianthus species. Altered expression of the HelC locus was detected in some hybrid combinations. These alterations appeared in early (F1, F2) hybrid generations and were similar in different hybrid combinations. They did not depend on the perennial paternal species being more influenced by the maternal genotype and by the mode of obtaining hybrids (in an embryo culture or in the field). These results are explained by "genomic shock" generated by hybridization of genetically incompatible species.     

烟草耐盐愈伤组织变异体对盐渍的适应性

用组织培养及逐步增加,NaCl浓度的方法,筛选出烟草耐盐愈伤组织变异体。能耐盐(2％ NaCl)的愈伤组织在2％ NaCl中继代29次,再移入无盐培养基中培养11和20代后不能保持提高的耐盐性,分别退化到只耐1.5％与1.0％ NaCl的水平。耐2％ NaCl愈伤组织产生的再生植株自交后代,其萌发种子、幼苗及成长植株均未能表现出耐盐性,说明用选择胁迫方法所筛选出的耐盐细胞系,其耐盐性的提高属于生理适应性。     

甘蔗野生种割手密远缘杂交后代品质性状的遗传研究

利用甘蔗品种Co419与野生种割手密云南75-1-2远缘杂交,ROC25与远缘杂交后代云野02-356进行回交,分别获得F1和BC1群体;利用R软件,分析了2个群体全部真实性后代品质性状的遗传表现.结果表明,杂交后代品质性状广义遗传力高,正态分布特性明显,品质性状间显著正相关;F1含糖量和纤维含量高于商业亲本,但甘蔗蔗糖分、蔗汁糖分、蔗汁锤度和简纯度等性状劣于商业亲本;BC1除含糖量高于双亲外,其他性状介于双亲之间,但主要性状均优于F1,后代性状恢复快.     

B-chromosomes in Iseilema laxum Hack. II. Genetics

Transmission of B-chromosomes to S₁ progenies and progenies of crosses involving parents with different numbers of B-chromosomes in Iseilema laxum Hack. is reported. The mean number of B's remained the same in S₁ progenies. In the progenies of the crosses, the mean number of B's increased and the range of their number in the offspring also is wider when they are transmitted through the male parent. The inheritance of B's is more or less normal on the female side. The phenomena of nondisjunction and precocious division of B's are more frequent in the male parent. Apart from nondisjunction and precocious divisions in the PMC's, there seems to be some post-meiotic mechanism responsible for the wide range and increase in the mean number of B's in crosses where the male parents contain B-chromosomes.     

Incomplete paternal inheritance of chloroplast DNA recognized in Chamaecyparis obtusa using an intraspecific polymorphism of the trnD-trnY intergenic spacer region

Using a fluorescence-based PCR-SSCP (single-strand conformation polymorphism), we verified imperfectibility in the paternal inheritance of chloroplast DNA (cpDNA) in Chamaecyparis obtusa (Cupressaceae) controlled crosses. An intraspecific sequence polymorphism of the intergenic spacer region between the trnD and trnY genes was utilized as a molecular marker. Of 361 progenies, in which the cpDNA haplotypes of their female and male parents were different, 352 (97.5%) possessed the same haplotypes as their male parents, and nine (2.5%) the same haplotypes as their female parents. The parentage of the nine progenies with female parental types was diagnosed using DNA fingerprinting based on fluorescence-based RAPD profiles. Their parentage showed convincing evidence of the low frequency of maternal inheritance. Moreover, heteroplasmy was observed in the open-pollinated seeds collected in a seed orchard. The confirmation of maternal plastid transmission in the full-sib families and the observation of heteroplasmy in seeds reveal that the paternal inheritance of cpDNA is not an exclusive phenomenon and that the mode of its inheritance is biparental in C. obtusa. Received: 15 April 2000 / Accepted: 13 July 2000     

Expression of a Novel Antiporter Gene from Brassica napus Resulted in Enhanced Salt Tolerance in Transgenic Tobacco Plants

Tobacco leaf discs were transformed with a plasmid pBIBnNHX1, containing the selectable marker neomycin phosphotransferase gene (nptII) and Na⁺/H⁺ vacuolar antiporter gene from Brassica napus (BnNHX1), via Agrobacterium tumefaciens-mediated transformation. Thirty-two independent transgenic plants were regenerated. Polymerase chain reaction (PCR) and Southern blot analyses confirmed that the BnNHX1 gene had integrated into plant genome and Northern blot analysis revealed the transgene expression at various levels in transgenic plants. Transgenic plants expressing BnNHX1 had enhanced salt tolerance and could grow and produce seeds normally in the presence of 200 mM NaCl. Analysis for the T₁ progenies derived from seven independent transgenic primary transformants expressing BnNHX1 showed that the transgenes in most tested independent T₁ lines were inherited at Mendelian 3:1 segregation ratios. Transgenic T₁ progenies could express _BnNHX1 and had salt tolerance at levels comparable to their T₀ parental lines. This study implicates that the BnNHX1 gene represents a promising candidate in the development of crops for enhanced salt tolerance by genetic engineering.