长穗偃麦草DNA导入小麦后代变异系醇溶蛋白的研究

应用花粉管通道技术,将抗逆性强的长穗偃麦草总ＤＮＡ导入普通小麦甘麦８号,后代中出现了广泛的变异,并筛选出两个高产、分蘖力强、抗条锈病的新品系。以这两个品系为材料,以供体和受体作为对照,研究了外源ＤＮＡ导入后籽粒醇溶蛋白的变化,发现醇溶蛋白的增加、缺失和电泳迁移率的变化,新增加的组分与供体长穗偃麦草某些组分相对应。由此推测外源ＤＮＡ导入受体后有可能某些ＤＮＡ片段插入受体基因组从而导致受体基因表达改变或基因突变。     

耐盐辣椒分子育种

利用自花授粉后形成的花粉管通道将海岸耐盐植物红树总ＤＮＡ导入辣椒,其后代的耐盐性明显增强,在海滩上试种。用海水直接浇灌,约５５％的转化株能开花,结果,而对照株全部死亡。进行蛋白质ＳＤＳ－ＰＳＧＥ电泳和ＲＡＰＤ分析,分别发现一条１７．５ＫＤ蛋白质和一条１．１Ｋｂ ＤＮＡ的特异性谱带,表明通过化粉管道导入外源ＤＮＡ是可行的,其后代植株耐盐能力提高与基因组变异有关。     

外源DNA导入春麦引起蛋白质性状变异的研究

应用外源DNA导入春麦改良主裁品种的某些性状,结果表明:利用花粉管通道进行外源DNA直接导入是改良春麦品种的有效技术.导入D_2代在表现型及基因型均发生变异,变异具有随机性,不受亲缘关系的影响,获得一批优良性状的变异株(系),特别是高蛋白质含量的变异株系,比原品种蛋白质含量提高2—4%.     

高效的植物DNA提取方法

利用液氮研磨植物幼芽,以苯酚─氯仿─异戊醇─核糖核酸酶法提取了大豆、菜豆、玉米、高梁等几种农作物的总ＤＮＡ。所提取的ＤＮＡ经岛津ＵＶ－２６５紫外分光光度计检测及０．６％的琼脂糖凝胶电泳,结果证明：该方法所提取的植物总ＤＮＡ纯度高,片段长度整齐,约５０ｋｂ左右,符合外源ＤＮＡ导入作物的要求,并且ＤＮＡ收率很高。     

大豆DNA直接导入茄子引起变异

本文报道了利用花粉管通道技术,将大豆DNA直接导入茄子,引起茄子部分表型及蛋白质、赖氨酸含量的变异,并且这种变异可以遗传。结果表明,利用此项技术,在实现作物科间的遗传物质转移,在一定条件下是可能的。 自从周光宇先生提出了利用花粉管通道技术来直接导入外源DNA并实现了棉花抗病育种以来,这项技术被愈来愈多的科学工作者所采用,使我国农业分子育种首先进入了应用阶段。这一技术的采用也引起一些科学工作者在探索植物除种间以外的,如在属间甚至科间来实现遗传物质转移可能性的极大兴趣。 1989年我们利用花粉管通道技术进行大豆种间的DNA的导入的同时,进行了大豆DNA直接导入茄科的推广品种龙茄一号茄子中,旨在探索利用花粉管通道技术来实现超远缘物种间遗传物质转移的可行性,进而使高蛋白植物大豆的优良特性在其它作物上加以利用。     

耐盐辣椒分子育种

利用自花授粉后形成的花粉管通道将海岸耐盐植物红树总 Ｄ Ｎ Ａ 导入辣椒,其后代的耐盐性明显增强,在海滩上试种,用海水直接浇灌,约５５％ 的转化株能开花、结果,而对照株全部死亡。进行蛋白质 Ｓ Ｄ Ｓ Ｐ Ａ Ｇ Ｅ电泳和 Ｒ Ａ Ｐ Ｄ 分析,分别发现一条１７５ Ｋ Ｄ蛋白质和一条１１ Ｋｂ Ｄ Ｎ Ａ 的特异性谱带,表明通过花粉管通道导入外源 Ｄ Ｎ Ａ 是可行的,其后代植株耐盐能力提高与基因组变异有关。     

几丁质酶基因导入西瓜植株及其抗病性鉴定研究

利用西瓜有性生殖过程 ,将携带有外源几丁质酶基因的质粒DNA涂抹在授粉后的柱头上 ,使其沿花粉管通道进入到生殖细胞 ,得到转化处理种子。转化T1代植株经除草剂Basta筛选和PCR扩增获得转化植株。对T3 代株系进行田间自然发病和人工接种鉴定获得 3个抗枯萎病的株系。结果表明 :几丁质酶对镰刀菌引起的西瓜枯萎病有一定的抑制作用 ,利用花粉管通道法直接导入西瓜活体植株的技术是可行的。     

刺梨花粉管萌发的荧光显微观察

花粉管作为连接柱头与胚囊的唯一结构,是外源DNA导入的通道.因此,必须首先形成花粉管通道,才能进行外源DNA的导入.本研究利用醋酸洋红染色对刺梨雌蕊、雄蕊进行显微观察,并在人工授粉后的0.5h、1h、3h、6h、10h、12h、14h、16h、18h、20h、24h和30h不同时期取其雌蕊,用苯胺蓝染色后进行荧光显微观察.研究结果初步探明了刺梨人工授粉的最佳时期在花蕾露红期;其花粉管通道从人工授粉后10 h左右开始萌发,到18 h左右完成萌发,花粉管萌发完成后的2~9 h是利用花粉管通道法介导刺梨遗传转化的最佳时期.本研究结果将为花粉管通道法介导刺梨遗传转化技术体系的建立提供基础资料.     

导入外源DNA小麦变异类型的光合特性研究(简报)

花粉管通道法是我国学者周光宇等在调查远缘杂交时提出的一种外源基因导入方法。本实验系用花粉管通道技术将C_4植物高粱总DNA(2D)导入C_3作物小麦甘麦8号(G8)和陇春13号(L13),结果在其后代中出现了广泛变异,并从中选育到了丰产、抗逆性强的小麦新品系89144(G8×2D)和89122(L13×2D)。连续几年的大田试验结果表明,其株形、抗锈性及经济产量显著优于其小麦亲本品种。为了进一步阐明这两个变异系光合性状优于其亲本     

经大豆DNA溶液处理的水稻后代种子的粗蛋白的氨基酸含量分？…

以大豆为外源ＤＮＡ供体,用浸种及幼苗期浇灌法和花粉管通道法直接将大豆总ＤＮＡ导入受体水稻,经常规栽培获得水稻后代种子。采用微量凯氏定氮法和氨基酸自动分析仪进行水稻后代种子糙米的粗蛋白含量和氨基酸含量的测定。结果显示：三组经大豆ＤＮＡ溶液处理获得的水稻后代种子糙米粗蛋白平均含量分别为１６．４２％、１６．８０％和１９．８７％,与对照组相比有明显提高,统计分析都达到极显的差异（Ｐ＜０．０１）;在氨基酸     

HSP_(70)基因表达对高粱育性的影响(英文)

高粱细胞质雄性不育系３１９７Ａ（３Ａ）在常温条件下是不育的（Ｆｉｇｓ．１１＆２）,经热激（４５℃）诱导不同程度地恢复了育性（Ｆｉｇｓ．１３＆４）,为研究其不育机理提供了线索。热激２ｈ后,３Ａ中即可产生一类线粒体热激蛋白（ＨＳＰｓ）。其中,分子量为７０ｋＤ的ＨＳＰ７０含量最高,也最为稳定。不过,３Ａ中ＨＳＰｓ的稳定性弱于保持系３１９７Ｂ（３Ｂ）（Ｆｉｇ．２,Ｐａｎｅｌｓ１～４）。放线菌素Ｄ抑制ＨＳＰｓ的合成,而氯霉素无此作用（Ｆｉｇ．２,Ｐａｎｅｌｓ５＆６）,表明：ＨＳＰｓ是由核基因编码、在细胞质中合成、再跨膜转运到线粒体中的。３Ａ幼穗经热激后,线粒体的总蛋白量猛增了２．７倍（Ｆｉｇ．３）,达到３Ｂ的水平,育性亦变为可育的。Ｆｉｇ．４表明：ＨＳＰ７０反义链ｃＤＮＡ（Ｒ１）能进入到３Ｂ花药细胞中,并与靶ＲＮＡ（ＨＳＣ７０ｍＲＮＡ）结合,而对照、正义链ｃＤＮＡ（Ｄ）链无此反应。由此、再增加一个通用保守序列的反义链ｃＤＮＡ（Ｒ２）、共两个探针（Ｒ１、Ｒ２）,可以检测到：３Ａ在常温下没有能力合成ＨＳＣ７０ｍＲＮＡ（Ｆｉｇ．５）,而在热激条件下,转变为有能力（Ｆｉｇ．６）。启示：３Ａ在热激条件下由不育转变为可育     

Genetic Variation in Sorghum for the Inhibition of Maize Pollen Tube Growth

Aniline blue fluorescence was used to study the growth of maizepollen tubes in the stigmas of 13 diverse sorghum accessions.In 12, only short maize pollen tubes were formed, but in thesingle exception (Sorghum nervosum Nr481) maize pollen tubesgrew at least as far as the base of the style. The S. bicolorgenotypes S9B and CMS (a cytoplasmic male sterile line) werehybridized with Nr481, and analysis of maize pollen tube growthin F₁ plants, and BC₁ plants using Nr481 as the recurrent parent,suggested that differences in inhibition of pollen tube growthwere due to variation at a single locus, which we propose todesignate lap (Inhibition of alien pollen tubes). AccessionNr481 appears to be homozygous for a recessive allele permittingmaize pollen tube growth. Attempts were made to produce sorghumx maize hybrids using Nr481 and CMS derivatives which were knownto allow maize pollen tube growth to the base of the style.A putative hybrid endosperm was obtained in one Nr481 x Seneca60 maize cross, but this was not repeatable and no hybrid plantswere produced. A fundamental problem may be the large size ofthe maize pollen tube, which could have difficulty growing throughthe sorghum ovary and in entering the micropyle. Sorghum bicolor spp. bicolor (L.) Moench, Zea mays L, sorghum, maize, pollen tube growth, hybridization barriers     

Harnessing Genetic Variation in Leaf Angle to Increase Productivity of Sorghum bicolor

The efficiency with which a plant intercepts solar radiation is determined primarily by its architecture. Understanding the genetic regulation of plant architecture and how changes in architecture affect performance can be used to improve plant productivity. Leaf inclination angle, the angle at which a leaf emerges with respect to the stem, is a feature of plant architecture that influences how a plant canopy intercepts solar radiation. Here we identify extensive genetic variation for leaf inclination angle in the crop plant Sorghum bicolor, a C4 grass species used for the production of grain, forage, and bioenergy. Multiple genetic loci that regulate leaf inclination angle were identified in recombinant inbred line populations of grain and bioenergy sorghum. Alleles of sorghum dwarf-3, a gene encoding a P-glycoprotein involved in polar auxin transport, are shown to change leaf inclination angle by up to 34° (0.59 rad). The impact of heritable variation in leaf inclination angle on light interception in sorghum canopies was assessed using functional-structural plant models and field experiments. Smaller leaf inclination angles caused solar radiation to penetrate deeper into the canopy, and the resulting redistribution of light is predicted to increase the biomass yield potential of bioenergy sorghum by at least 3%. These results show that sorghum leaf angle is a heritable trait regulated by multiple loci and that genetic variation in leaf angle can be used to modify plant architecture to improve sorghum crop performance.     

Comparative Genome Mapping of Sorghum and Maize

Linkage relationships were determined among 85 maize low copy number nuclear DNA probes and seven isozyme loci in an F2 population derived from a cross of Sorghum bicolor ssp. bicolor x S. bicolor ssp. arundinaceum. Thirteen linkage groups were defined, three more than the 10 chromosomes of sorghum. Use of maize DNA probes to produce the sorghum linkage map allowed us to make several inferences concerning processes involved in the evolutionary divergence of the maize and sorghum genomes. The results show that many linkage groups are conserved between these two genomes and that the amount of recombination in these conserved linkage groups is roughly equivalent in maize and sorghum. Estimates of the proportions of duplicated loci suggest that a larger proportion of the loci are duplicated in the maize genome than in the sorghum genome. This result concurs with a prior estimate that the nuclear DNA content of maize is three to four times greater than that of sorghum. The pattern of conserved linkages between maize and sorghum is such that most sorghum linkage groups are composed of loci that map to two maize chromosomes. This pattern is consistent with the hypothesized ancient polyploid origin of maize and sorghum. There are nine cases in which locus order within shared linkage groups is inverted in sorghum relative to maize. These may have arisen from either inversions or intrachromosomal translocations. We found no evidence for large interchromosomal translocations. Overall, the data suggest that the primary processes involved in divergence of the maize and sorghum genomes were duplications (either by polyploidy or segmental duplication) and inversions or intrachromosomal translocations.     

Evaluation of Public Sweet Sorghum A-Lines for Use in Hybrid Production

A fundamental need for commercialization of sweet sorghum [Sorghum bicolor (L.) Moench] as a bioenergy crop is an adequate seed supply, which will require development of hybrid varieties using dwarf seed-parent lines. A set of six public sweet sorghum A-lines (Dwarf Kansas Sourless, KS9, N36, N38, N39, and N4692) were crossed with a set of six public sweet sorghum cultivars (Brawley, Kansas Collier, Dale, Sugar Drip, Waconia, and Wray). Grain, fiber, and sugar yields were determined, and conversion formulas were applied to estimate ethanol yields. Hybrids were grown in fields at Ithaca, NE, USA, in 1983–1984 fertilized with 112 kg ha^?1 N. In terms of yield components and overall ethanol yields, one A-line, N38, was inferior. Average total ethanol yields from hybrids made on the other A-lines were not significantly different, suggesting that any of those five A-lines could be useful seed-parents. With the exception of grain yield, cultivars used as pollen parents were among the highest-performing entries for all traits. For all traits directly contributing to total ethanol yield (grain yield, juice yield, % soluble solids, sugar yield, fiber yield), hybrids were also among the highest-performing entries. Results of this study demonstrate that hybrid sweet sorghum with performance criteria equivalent to existing sweet sorghum cultivars can be produced on the sweet sorghum seed-parent lines A-Dwarf Kansas Sourless, A-KS9, A-N36, A-N39, and A-N4692. Identification of specific seed-parent × pollen parent lines with characteristics best suited for particular growing regions and end-user needs will be critical for commercial hybrid development.     

RFLP-based assay of Sorghum bicolor (L.) Moench genetic diversity

Sixty-two single-copy sorghum DNA clones were used to compare restriction fragment patterns of 53 sorghum accessions from Africa, Asia and the United States. Included were accessions from five morphological races of the cultivated subspecies bicolor, and four races of the wild subspecies verticilliflorum. From two to twelve alleles were detected with each probe. There was greater nuclear diversity in the wild subspecies (255 alleles in ten accessions) than in the domestic accessions (236 alleles in 37 accessions). Overall, 204 of the 340 alleles (60%) that were detected occurred in both subspecies. Phylogenetic analysis using parsimony separated the subspecies into separate clusters, with one group of intermediate accessions. Though exceptions were common, especially for the race bicolor, accessions classified as the same morphological race tended to group together on the basis of RFLP similarities. Selection for traits such as forage quality may have led to accessions genetically more similar to other races being classified as bicolors, which have a loose, small-grained panicle similar to wild races. Population statistics, calculated using four nuclear and four cytoplasmic probes that detect two alleles each, revealed a low but significant amount of heterozygosity, and showed little differentiation in alleles in the wild and cultivated subspecies. Outcrossing with foreign pollen appears to have been more important than migration via seed dispersal as a mechanism for gene flow between the wild and domestic accessions included in this study.     

Exine-Detached Pollen of Nicotiana tabacum as an Electroporation Target for Gene Transfer

In developing alternative systems for plant transformation the authors investigated the use of male gametophyte as the foreign gene receptor. However, delivery of foreign DNA into pollen is difficult because of the existence of a thick exine, therefore a new experimental system was developed using exine-detached pollen (EDP) of Nicotiana tabacum as an electroporation target which was also compared with germinating pollen (GP) and pollen grains (P). A transient GUS expression assay was conducted to analyze the effects of different electroporation conditions and promoter activity. The pollen-specific promoter Zml3 from Zea mays mediated high level of GUS gene expression but CaMV 35S only had very low activity in both EDP and GP. The optimal field strength for gene transfer was obtained at 750 V/cm for EDP and 1250 V/cm for GP when the time constant of pulse was 13 ms. The GUS activity in EDP had a 5-fold increase as compared with GP and P respectively. The level of GUS gene expression was slightly increased when adding 10 % PEG into the electroporation buffer. This result indicates that pollen deprived of exine responds much better to foreign gene transfer than the previously used intact pollen grains and may be a better vector to introduce, via pollen tube, genes into the egg cell and offsprings.     

A functional study of stylar hydroxyproline-rich glycoproteins during pollen tube growth

Class III pistil-specific extensin-like proteins (PELPIII) are chimeric hydroxyproline-rich glycoproteins with properties of both extensins and arabinogalactan proteins. The abundance and specific localization of PELPIII in the intercellular matrix (IM) of tobacco (Nicotiana tabacum) stylar transmitting tissue, and translocation of PELPIII from the IM into the pollen tube wall after pollination, presume the biological function of these glycoproteins to be related to plant reproduction. Here we show that in in vitro assays the translocation of PELPIII is specifically directed to the callose inner wall of the pollen tubes, indicating that protein transfer is not dependent on the physiological conditions of the transmitting tract. We designed a set of experiments to elucidate the biological function of PELPIII in the stylar IM. To study the function of the specific interaction between PELPIII proteins and the pollen tube wall, one of the PELPIII proteins (MG15) was ectopically expressed in pollen tubes and targeted to the tube wall. We also generated transgenic tobacco plants in which PELPIII proteins were silenced. In vitro bioassays were performed to test the influence of purified PELPIII on pollen tube growth, as compared to tobacco transmitting tissue-specific proteins (TTS) that were previously shown to stimulate pollen tube growth. The various tests described for activity of PELPIII proteins all gave consistent and mutually affirmative results: the biological function of PELPIII proteins is not directly related to pollen tube growth. These data show that similar stylar glycoproteins may act very differently on pollen tubes.     

Direct pollination of Zea mays ovules in vitro with Z. mays,Z. mexicana and Sorghum bicolor pollen

Summary Sorghum (Sorghum bicolor) pollen tubes penetrated and grew in corn (Zea mays) styles. The limited length of the sorghum pollen tubes (3–5 mm) and the absence of stigmatic hairs on the basal (5–10 mm) section of the corn styles prevented effective pollination in vivo and in vitro. Normal fertilisation occurred after in vitro pollination of exposed corn ovules with either corn or teosinte (Zea mexicana) pollen. Six per cent of corn ovules pollinated directly with sorghum pollen responded by rapid, massive growth of nucellar tissue.     

烟草脱外壁花粉的电激基因转移

以 β-葡糖苷酸酶 GUS 基因作为报告基因 ,通过瞬间表达的检测 ,比较了烟草 Nicotianatabacum L . 脱外壁花粉、未萌发与萌发花粉的电激导入效果 ,探讨了不同电激条件及启动子对外源基因瞬间表达的影响 .结果表明 :当脉冲时间常数为 13 ms时 ,导致脱外壁花粉和萌发花粉生活力下降约50 %的电场强度分别为 750 V/ cm和 12 50 V/ cm,在此条件下电激 ,二者的导入效果最好 .脱外壁花粉的GUS基因表达水平约为萌发花粉的 5倍、花粉粒的 30倍 .玉米花粉特异启动子 Zm13- 2 60 能启动 GUS基因在脱外壁花粉和萌发花粉中高效表达 ,而 Ca MV 35S的启动活性很低