顺乌头酸酶基因在杀雄剂SQ-1诱导小麦雄性不育花药中的表达分析

采用RT-PCR技术,克隆了小麦胞质顺乌头酸酶基因(cACO)部分cDNA序列.该cDNA序列长1368bp,编码456个氨基酸,GenBank登录号为GU475062.半定量RT-PCR结果表明,在生理型不育和可育花药发育的单核早期至三核期,cACO基因的表达水平均表现为先升后降;在生理型不育花药发育的单核晚期cACO基因表达水平与同期可育花药相比显著升高,到二核期和三核期明显降低,ACO酶活性变化表现出相同趋势.这反映出在小麦生理型不育系中,cACO基因在花药败育关键期异常表达可能影响了花药发育过程中正常的能量供应和物质代谢,导致花粉发育能量不足和所需物质匮乏,从而导致了非遗传型花药败育现象.     

基因芯片分析杀雄剂SQ-1诱导小麦生理型雄性不育的基因差异表达

为揭示小麦生理型雄性不育的分子机理,更好地为小麦杂种优势利用提供理论依据和技术支撑,本研究以SQ-1诱导的西农558生理型雄性不育的花药为试验材料,以未经SQ-1处理的西农558的花药为对照,利用基因芯片技术对两者的基因表达差异进行了分析,并对部分基因运用半定量PCR技术进行了验证.结果表明,在55 052个转录本中, 两材料间差异表达的转录本有2 052条, 其中1 294个基因表达上调,758个基因表达下调.功能分类表明这些基因主要参与了毒性物质响应、逆境响应、多糖代谢及信号转导等重要生命过程.为验证芯片数据的可信性, 利用cDNA半定量 PCR 法对11个差异表达显著的基因(Ta.116, Ta.5629, TaAffx.122333, Ta.30726, Ta.13682, Ta.4057, Ta.4101, Ta.4139, Ta.11957, Ta.25934, Ta.27552) 进行验证.结果证明,无论是上调表达的还是下调表达的基因,其表达模式都与基因芯片的检测结果的一致.这些基因可作为育性相关候选基因开展下一步研究.     

Localization and organization of tRNA genes on the mitochondrial genomes of fertile and male sterile lines of maize

Summary Maize mitochondrial (mt) tRNA genes were localized on the mt master circles of two fertile lines (WF9-N and B37-N) and of one cytoplasmic male sterile line (B37-cmsT) of maize. The three genomes contain 16 tRNA genes with 14 different anticodons which correspond to 13 amino acids. Out of these 16 tRNA genes, 6 show a high degree of homology with the corresponding chloroplast (cp) tRNA genes and were shown to originate from cp DNA insertions and to be expressed in the mitochondria. The organization of the mt tRNA genes in both fertile lines is similar. The same genes are found, in the same environment, as judged from the restriction maps, in fertile and male sterile lines that have the same nuclear background, but the relative organization of the mt tRNA genes on the master circle is completely different.     

Use of Chlorella as a test system for understanding the mode of action of a chemical hybridising agent

Studies were made on Chlorella zofingiensis to elucidate the mode of action of a chemical hybridising agent (gametocide) for small grain cereals, azetidine-3-carboxylic acid (A3C). The compound reduced the rate of cell division, but undivided cells continued to grow in size up to 26 times the volume of the largest cells in the control medium. The total biomass of cultures was not affected. Electron microscopy revealed that cells treated with A3C contained no autospores. When samples were returned to control medium, however, division took place rapidly. The results led to the conclusion that A3C blocks cell division prior to the formation of partition membranes and walls. A hypothesis was erected to explain its effects on pollen and was subsequently tested. The test system described is suitable for screening other potential gametocides and assessing their modes of action.     

Metabolism of indole-3-acetic acid and indole-3-methanol in wheat leaf segments

Indole-3-methanol is a product of indole-3-acetic acid metabolism in wheat leaves ( Triticum compactum Host., cv. Little Club). It leads either to the production of the corresponding aldehyde and carboxylic acid, to the production of a polar glucoside which releases indole-3-methanol on β-glucosidase treatment, or to an unidentified apolar product on mild alkaline hydrolysis in aqueous methanol. With reference to a published pathway of indole-3-acetic acid degradation, the results provide evidence for a prominent role of indole-3-methanol and also for the occurrence of co-oxidation processes in wheat leaves involving indole-3-acetic acid and phenolic cosubstrates.     

Tight linkage between a nuclear male-sterile locus and an enzyme marker in tomato

Summary The linkage relationship of a nuclear male sterile locus, ms-10, was tested with two enzyme marker loci known to be on the same chromosome (long arm of chromosome 2). The results indicate the gene order is Est-1 — 18 cM — Prx-2 — 1.5 cM — ms-10. The linkage intensity of ms-10 and Prx-2 (1.5 cM) suggests that Prx-2 might provide a selectable marker for male-sterility. In accordance with this idea, the ms-10 allele was placed in cis with a rare-allele of Prx-2 (Prx-2 ¹). Selection on the basis of the codominant Prx-2 ¹ allele should allow for more rapid and efficient transfer of the recessive male sterile allele into an array of genetic backgrounds, thus promoting its use in hybrid seed production.     

CRISPR/Cas9-mediated disruption of TaNP1 genes results in complete male sterility in bread wheat

Male sterile genes and mutants are valuable resources in hybrid seed production for monoclinous crops.High genetic redundancy due to allohexaploidy makes it difficult to obtain the nuclear recessive male sterile mutants through spontaneous mutation or chemical or physical mutagenesis methods in wheat.The emerging effective genome editing tool,CRISPR/Cas9 system,makes it possible to achieve simultaneous mutagenesis in multiple homoeoalleles.To improve the genome modification efficiency of the CRISPR/Cas9 system in wheat,we compared four different RNA polymerase(Pol) Ⅲ promoters(TaU3 p,TaU6 p,OsU3 p,and OsU6 p) and three types of sgRNA scaffold in the protoplast system.We show that the TaU3 promoter-driven optimized sgRNA scaffold was most effective.The optimized CRISPR/Cas9 system was used to edit three TaNP1 homoeoalleles,whose orthologs,OsNP1 in rice and ZmIPE1 in maize,encode a putative glucose-methanol-choline oxidoreductase and are required for male sterility.Triple homozygous mutations in TaNP1 genes result in complete male sterility.We further demonstrated that anyone wild-type copy of the three TaNP1 genes is sufficient for maintenance of male fertility.Taken together,this study provides an optimized CRISPR/Cas9 vector for wheat genome editing and a complete male sterile mutant for development of a commercially viable hybrid wheat seed production system.     

Single gene restoration of cytoplasmic male sterility in wheat and its implications in the breeding of restorer lines

Summary Cultivars of T. aestivum crossed onto two lines with male sterility induced by the cytoplasm of T. timopheevi gave a high level of restoration in the F₁ generation. The ratio of fertile to sterile plants segregating in the F₂ generation was consistent with that expected for a single dominant restorer gene. The possible association between this gene and mildew resistance or some other desirable character derived from CI 12633, a common ancestor of the cultivars used, is discussed.     

Changes in the level of MACC during cold hardening and dehardening in winter wheat

Changes in the level of 1-(malonylamino)-cyclopropane-1-carboxylic acid (MACC) were determined in 6 winter wheat cultivars during cold hardening at 4°C. The cultivars differed by one degree of frost resistance within the range of degree II to VII of the COMECON scale. The time-course of changes in MACC level showed a similar pattern in all 6 cultivars; i.e. increase till day 6, no changes for the next 10 days, and then a steady decrease till the end of the hardening period. There was little difference between the final and the initial levels. The increase of MACC level, expressed as per cent of the original level, was not directly correlated with either the degree of frost resistance of the actual percentage of survival. In some cultivars. mean errors exceeded the difference in MACC accumulation between cultivars closest on the resistance scale.
The fate of MACC during the second half of hardening and after transfer of plants to 25°C was studied in cultivars Bezostaya and San Pastore. During the second half of the hardening period the level of MACC decreased in the leaves of both cultivars, but increased significantly in the roots. Within two days of transfer of the hardened plants to 25°C, the MACC level in leaves increased again, while that in the roots decreased. This finding, together with the preliminary evidence of very low MACC metabolism, strongly suggest that MACC accumulates in roots during the hardening period and when transferred to 25°C, it moves from roots to leaves.     

胡萝卜育种研究(综述)

胡萝卜是高度异花授粉作物,由于存在近交衰退,因而很难通过系内授粉培育出综合性状优良的品种。随着雄性不育系的发现,进而培育出整齐一致的一代杂种。本文综述国内外胡萝卜育种与科研现状,提出胡萝卜育种的主要目标,着重介绍雄性不育在胡萝卜育种中的应用,为加快胡萝卜育种研究提供思路。     

Azetidine-2-carboxylic acid in the food chain

Azetidine-2-carboxylic acid (Aze) 1 is a non-protein amino acid present in sugar beets and in table beets (Beta vulgaris). It is readily misincorporated into proteins in place of proline 2 in many species, including humans, and causes numerous toxic effects as well as congenital malformations. Its role in the pathogenesis of disease in humans has remained unexplored. Sugar beet agriculture, especially in the Northern Hemisphere, has become widespread during the past 150 years, and now accounts for nearly 30% of the world’s supply of sucrose. Sugar beet byproducts are also used as a dietary supplement for livestock. Therefore, this study was undertaken as an initial survey to identify Aze-containing links in the food chain. Herein, we report the presence of Aze 1 in three sugar beet byproducts that are fed to farm animals: sugar beet molasses, shredded sugar beet pulp, and pelleted sugar beet pulp.     

Ethylene-dependent action of gibberellin in seed germination of Amaranthus caudatus

The role of gibberellins in the germination of seeds of Amaranthus caudatus L. was examined. Tetcyclacis (BAS 106), an inhibitor of gibberellin biosynthesis, inhibited germination of the seeds. The inhibition caused by BAS 106 was antagonised by gibberellin A₄₊₇ (GA₄₊₇). Ethephon (2-chloroethylphosphonic acid) and 1-aminocyclopropane-1-carboxylic acid (ACC) could replace GA₄₊₇. Ethephon and ACC counteracted also the side effects of BAS 106 that are not reversible by GA₄₊₇. The rate of seed germination was not increased by gibberellin in the presence of aminoethoxyvinylglycine (AVG). AVG increased the effect of BAS 106. GA₄₊₇ could not reverse the effect of BAS 106 when AVG was simultaneously applied. The results indicate that the biosynthesis of endogenous gibberellins may be required for germination of A. caudatus seeds and that main physiological effects of GA₄₊₇ on seed germination may depend on ethylene biosynthesis.     

杀雄剂SQ-1诱导小麦雄性不育花粉粒差异蛋白质组学研究

采用固相pH梯度/SDS-PAGE双向凝胶电泳对经杀雄剂SQ-1处理和未处理的小麦(Triticum aestivum)成熟期花粉总蛋白质进行了分离, 银染显色, 获得了分辨率和重复性较好的双向电泳图谱. 通过PDQuest 2DE图像软件的分析, 在等电点4～7之间可识别350个以上较为清晰的蛋白质点, 其中差异表达明显的蛋白质点数为21个. 将11个差异点采用基质辅助激光解析电离飞行时间质谱进行了肽质量指纹图谱分析, 采用Mascot软件在Swiss-prot数据库查询, 鉴定出了7个蛋白质, 它们分别是液泡转化酶、动力蛋白轻链TCTEX-1、锰超氧化物歧化酶、果糖-1,6-二磷酸醛缩酶、抗坏血酸过氧化物酶、凝集素蛋白激酶和一种未知功能的蛋白. 对已知蛋白的功能进行分析, 推测杀雄剂SQ-1诱导小麦雄性不育可能与能量代谢失衡、淀粉合成受抑制、活性氧积累、细胞凋亡以及花器官发育调节基因作用失控等有关.     

Stereoselectivity and structural determinants in molecular recognition by the ACC transport system in isolated maize mesophyll vacuoles

The ethylene precursor, 1-aminocyclopropane-1-carboxylic acid (ACC), is actively transported across the tonoplast of plant cells, impacting cellular compartmentation of ACC and ethylene biosynthesis. In the present study, the effects of ACC and amino acid analogs on ACC uptake into isolated maize (Zea mays L. cv. Golden Cross Bantam) mesophyll vacuoles were investigated to identify the stereospecific and structural features that are important in molecular recognition by the ACC transport system. Of the four stereoisomers of l-amino-2-ethylcyclopropane-l-carboxylic acid (AEC), (1S, 2R)-(–)-AEC having a configuration corresponding to an L-amino acid was the preferred substrate for the ACC transport system, competitively inhibiting ACC transport with a Ki of 18 μM. Of 11 neutral amino acid stereoisomers, L-isomers were stronger inhibitors of ACC transport than corresponding D-isomers. Neutral L-amino acids with nonpolar side chains generally were more inhibitory than those with polar side chains, whereas several cationic and anionic L-amino acids were ineffective antagonists of ACC transport. These observations suggest that the ACC transport system is stereospecific for relatively nonpolar, neutral L-amino acids. This conclusion was supported by the observation that group additions, substitutions, or deletions at the carboxyl. α-amino and the Pro- (R) methylene or hydrogen moieties (analogous to D-amino acids) of ACC and other neutral amino acids and analogs essentially eliminated transport inhibition. In contrast, L-amino acid analogs with variable substitutions at the distal end of the molecule remained antagonists. The relative activity of analogs was influenced by the length and degree of unsaturation of the side chain and by the location of side chain branching. Increasing the ring size of ACC analogs reduced antagonism whereas incorporating the α-amino group into the ring structure as an L-amino acid increased antagonism. The kinetics of L-methoxyvinylglycine, L-methionine. p-nitro-L-phenylalanine and 1-aminocyclobutane-l-carboxylic acid were competitive with Ki values of 3, 13, 16 and 19 μM, respectively. These results indicate that the ACC transport system can be classifie as a neutral L-amino acid carrier having a relatively high affinity for ACC and other nonpolar amino acids. The results also suggest that the carrier interacts with the carboxyl, α-amino and Pro-(R) groups and with other less restricted side chain substituents of substrate amino acids.     

强优势杂交小麦产量结构模式的研究

利用1999-2001年选用的9个性状差异较大的品种（系）,按完全双列杂交（正交）组配成36个杂交组合,用一种新的分组法-Nair单性状水平分组法将杂交组合的各性状进行了分组,按产量分组结果对产量与各性状间的关系进行了研究,结果表明：随产量水平的升高单位面积穗数对产量的作用逐渐弱化,穗粒数对产量的作用逐渐增强,单位面积穗数与穗粒数间的相互作用构成限制产量的主要因素,千粒重对产量的作用较稳定;千粒重与单位面积穗数的负相关性较弱、与穗粒数无明显的相关性;在7500-9000kg/hm^2水平下,产量结构模式为：504.0-607.5万穗/hm^2,33.6-41.3粒/穗,35.7-42.3g/千粒重;在9000-10500kg/hm^2水平下,产量结构模式为：525-645万穗/hm^2,36-50粒/穗,37-45g/千粒重。     

Genetic control of male fertility in Arabidopsis thaliana: structural analyses of postmeiotic developmental mutants

Seven new male-sterile mutants (ms7–ms13) of Arabidopsis thaliana (L.) Heynh. (ecotype columbia) are described that show a postmeiotic defect of microspore development. In ms9 mutants, microspores recently released from the tetrad appear irregular in shape and are often without exines. The earliest evidence of abnormality in ms12 mutants is degeneration of microspores that lack normal exine sculpturing, suggesting that the MS12 product is important in the formation of pollen exine. Teratomes (abnormally enlarged microsporocytes) are also occasionally present and each has a poorly developed exine. In ms7 mutant plants, the tapetal cytoplasm disintegrates at the late vacuolate microspore stage, apparently causing the degeneration of microspores and pollen grains. With ms8 mutants, the exine of the microspores appears similar to that of the wild type. However, intine development appears impaired and pollen grains rupture prior to maturity. In ms11 mutants, the first detectable abnormality appears at the mid to late vacuolate stage. The absence of fluorescence in the microspores and tapetal cells after staining with 4′,6-diamidino-2-phenylindole (DAPI) and the occasional presence of teratomes indicate degradation of DNA. Viable pollen from ms10 mutant plants is dehisced from anthers but appears to have surface abnormalities affecting interaction with the stigma. Pollen only germinates in high-humidity conditions or during in-vitro germination experiments. Mutant plants also have bright-green stems, suggesting that ms10 belongs to the eceriferum (cer) class of mutants. However, ms10 and cer6 are non-allelic. The ms13 mutant has a similar phenotype to ms10, suggesting is also an eceriferum mutation. Each of these seven mutants had a greater number of flowers than congenic male-fertile plants. The non-allelic nature of these mutants and their different developmental end-points indicate that seven different genes important for the later stages of pollen development have been identified. Received: 14 August 1997 / Accepted: 7 October 1997     

免疫测定光敏核不育水稻中光敏色素Ⅰ含量

光敏核不育水稻（农垦５８Ｓ）是中国水稻研究中的一个重要发现,在水稻杂交育种上有巨大潜力。研究表明：农垦５８Ｓ的雄性不育性受光周期调控,光敏色素是育性转变过程中光周期反应的光受体,然而,鉴于高等植物中存在至少两种不同的光敏色素分子,并且它们同时存在于水稻中,人们对调节农垦５８Ｓ雄性不育过程的光敏色素分子种类及作用方式仍然不清楚。本文采用酶联免疫吸附测定法（ＥＬＩＳＡ）,比较了不同光周期下农垦５８Ｓ和对照品种农垦５８叶片（光周期感受器官）中光敏色素Ｉ（ｐｈｙＡ）的含量。从农垦５８Ｓ的二次枝梗原基分化期开始,进行１０天的光周期处理。一组为短日照（ＳＤ）,另一组为长日照（ＬＤ）。在第１０个暗期结束前,于暗绿光下收获每株水稻的最上部两片新展叶,立即保存在液氮中。样品在研钵中磨成液氮粉,然后加入提取液（含５０ｍｍｏｌ／ＬＴｒｉｓ－ＨＣＩ和０．２ｍｏｌ／Ｌ巯基乙醇,ｐＨ８．５）匀浆。粗提液经０．５％聚乙烯亚胺（ＰＥＩ）沉淀后,上清液供ＥＬＩＳＡ分析。以燕麦ｐｈｙＡ的多克隆抗体、单克隆抗体分别作ＥＬＩＳＡ的一抗和检测抗体。采用ＥＬＩＳＡ可以专一性地检测水稻ｐｈｙＡ（Ｔａｂｌｅ１）。几次独立的实验说明,光周期处理对ｐｈｙＡ含