分子鉴定拟南芥 myb26／mail sterile35突变体中表达下调基因 At2 g47500

利用不可产生花粉的突变体作为研究花药和花粉发育的途径,以证实涉及花粉发育的基因及其在基因网络中的作用。研究结果表明,拟南芥中MYB26/MALE STERILE35（ MS35）雄性不育基因会调控药室内壁次生加厚发育,影响其后的花药开裂,并可上调木质素生物合成途径。而At2g47500基因是在芯片分析ms35/myb26突变体中涉及花粉发育表达下调的一个未被鉴定的基因。通过生物信息学分析发现,At2g47500基因是一个与微管发育有关的基因,在整个植株都表达,包括花发育阶段。利用拟南芥基因库筛选该基因中的T-DNA插入突变体,并通过PCR鉴定,得到插入位点在启动子和外显子的纯合突变体。通过表型分析和表达筛选发现,At2g47500突变并未对表现型产生影响,表明T-DNA插入位点在外显子的突变体基因并未在花药中表达;但转录表达分析认为,该基因在花粉里表达,并通过启动子：GUS表达结构得到进一步证实。35S启动子超量表达At2g47500,并未导致其在野生型植物中异位的过量表达;将其转入突变体中,基因表达量恢复,也未有表现差异。说明其对花粉发育的影响不明显。定量PCR分析微管发育其他相关基因,以及花发育中次生壁发育有关基因在突变体中的表达,At2g47500对花粉发育影响不明显。在突变体中,大部分基因也未因其缺失、表达受到明显影响。综上所述,该基因在花药发育中不受ms35/myb26的调控,发挥次要作用,受到主效基因的调控。     

一个新的水稻细条纹叶色突变体特性分析和基因克隆

叶色突变体作为一种易于观察的性状突变,是开展光合系统的结构和功能、叶绿素生物合成及其调控机制研究的理想材料。本研究以水稻T-DNA插入细条纹叶色突变体为研究材料,通过叶片的形态鉴定、石蜡切片、叶绿素和光合速率测定,分析突变体的表型和光合特性;采用TAIL-PCR技术,定位和克隆该突变基因。结果表明:突变体苗期幼叶上呈现间断的白色细条纹,而叶鞘、叶脉表现正常,三叶期后至分蘖期叶片逐渐转绿;突变体叶色对温度变化敏感;是国内外尚未报道的一种新型水稻细条纹叶色突变体。根据T-DNA插入标签,确定突变候选基因位于第7染色体上,并对候选基因进行了克隆已获得部分cDNA序列,暂命名为fs3(t)。     

利用CRISPR-Cas9基因编辑技术获得AtLCR67基因敲除突变体

拟南芥AtLCR67基因属于低分子量富含半胱氨酸(low-molecular-weight cysteine-rich,LCR)基因家族。在正常生长条件下,该基因在发育的种子中特异表达。为了研究该基因在发育或代谢过程中的可能作用,在没有合适T-DNA插入突变缺乏的情况下,我们利用CRISPR-Cas9定向敲除技术来创制该基因沉默的拟南芥突变体材料。通过构建AtLCR67基因的CRISPR-Cas9敲除质粒,借助农杆菌介导的浸花法转化野生型拟南芥Col-0,最终获得12株T0代转基因植株。对T0转基因植株的AtLCR67基因进行测序分析发现,在设计的靶位点处存在短片段缺失12个碱基和增加一个T碱基两种突变类型,进一步做RT-PCR检测分析发现突变体中AtLCR67基因完全不表达,说明成功获得拟南芥lcr67突变体。突变体材料的获得为进一步研究AtLCR67基因在发育或代谢方面的的功能奠定了良好基础。     

3种基因突变方法在微生物中的应用研究进展

为了解某个基因的功能,最常用的方法就是构建该基因缺失的突变体,通过测定突变体的某些性状来分析该基因的功能,因此,获得由基因型决定的表型突变体在生物学研究中极为重要。本研究主要介绍了3种构建基因突变体的方法：转座子插入突变、同源重组和定点突变技术,转座子插入方法多用于构建突变体库,同源重组多用于基因功能的研究,定点突变多用于蛋白质功能的研究。3种方法各自侧重点不同,将同源重组和定点突变技术结合起来是将来研究基因突变的一个方向。最后,论述了基因突变技术存在的缺陷和不足,提出该技术还亟待完善。     

研究找到调控水稻根系发生关键基因

<正>中科院遗传与发育所植物基因组学国家重点实验室储成才课题组,通过大规模筛选水稻T-DNA插入突变体库,获得了一个水稻根数目显著增加的突变体,从而找到调控水稻根系发生的关键基因。据了解,此突变体的表型由编码另一个细胞质型细胞分裂素氧化酶/脱氢酶基因OsCKX4被特异性激活而形成。OsCKX4受植物激素细胞分裂素和生长素     

拟南芥失绿突变体生理特性及其基因定位

通过筛选T-DNA插入突变体库得到一种叶色失绿拟南芥突变体yg (yellow green),为了研究该失绿突变体的性状变化及发生原因,对其表型、光合色素含量、叶绿体超微结构进行观察或测量,并通过染色体步移法定位了突变基因和T-DNA插入位点。研究发现,突变体yg整个生长期均呈浅黄绿色或黄色,植株瘦弱,生长迟缓,干鲜比、植株高度、基部抽薹数均低于野生型;其叶绿素a、叶绿素b、类胡萝卜素含量相对野生型分别减少了90.6%、89.6%、79.7%;透射电镜观察表明,该突变体叶绿体发育异常,类囊体排列松散,基粒片层断裂,淀粉粒较少,嗜锇颗粒数量显著增加;基因定位发现yg的CHLI1基因发生突变,T-DNA插入点位于5’UTR第24 bp后,导致该基因表达量显著降低。本研究明确了该突变体的失绿机制,进一步证实了CHLI1基因在叶绿素合成过程中的重要功能。     

T-DNA插入产生的水稻多分蘖突变的遗传分析

在筛选和鉴定水稻T-DNA(含Basta抗性基因Bar)插入纯合体的过程中，观察到一个多分蘖的突变体。其分离群体中多分蘖和正常分蘖两种植株类型的分离比率为3∶1，符合1对基因的显性遗传。Basta 抗性检测、PCR分子检测及Southern杂交证实，该突变体是由单一T-DNA插入引起的，多分蘖性状与T-DNA共分离。该突变材料可用于插入座位的     

大丽轮枝菌致病缺陷型T-DNA插入突变体的筛选与鉴定

【目的】从大丽轮枝菌T-DNA突变体库中筛选鉴定致病缺陷型突变体并分析致病相关基因。【方法】将落叶型大丽轮枝菌菌株Vd991的294个T-DNA插入突变体在寄主棉花上进行致病力测定。利用Southern杂交和hiTAIL-PCR(High-efficiency thermal asymmetric interlaced PCR)技术分别对筛选获得的9个致病力显著降低的突变体进行拷贝数和侧翼序列分析。【结果】与接种野生型Vd991的棉花相比,接种突变体的棉花的病情指数极显著降低。Southern杂交表明其中1个突变体中T-DNA为双拷贝插入,其余8个突变体均为单拷贝插入。生物学特性分析发现T-DNA的插入导致这些突变体在生长速率和产孢量等方面与野生型Vd991相比均有不同程度的降低。Blast比对分析明确了这些突变体中T-DNA的插入位置和在基因组上的分布,并从野生型菌株Vd991中成功克隆得到了这些可能影响大丽轮枝菌致病力的相关基因。【结论】筛选和鉴定T-DNA插入突变是在全基因组范围内快速获得致病相关基因信息的1种有效方法,极大的促进了大丽轮枝菌的致病分子机制等研究,为进一步培育抗病棉花品种奠定了基础。     

水稻光叶突变新基因的克隆和亚细胞定位

水稻叶片表皮毛突变体是研究单子叶植物细胞分化和水稻品种改良的重要材料。本研究对T-DNA标签技术获得的水稻光叶突变体(单隐性核基因控制,稳定遗传)进行田间观测和扫描电镜观察,以分析突变体叶片毛状体类型和分布特征;并采用DNA-Walking和RT-PCR方法定位并克隆该突变体基因,通过对该基因进行初步的生物信息学分析,构建绿色荧光蛋白融合载体,进行亚细胞定位。研究结果显示:突变体叶缘无毛,叶面毛状体缺失,缺失的毛状体类型主要为宏毛(即钩毛),但种子颖壳表面毛状体正常生长,是国内外尚未报道的一种新型的光叶突变体;对T-DNA标签插入序列进行相似性比对,确定插入位点,表明突变体候选基因在水稻第6号染色体上,通过基因克隆获得了c DNA序列,暂命名为GLL;生物信息学分析表明,GLL基因含有8个外显子,7个内含子,该基因的开放阅读框(ORF)有702 bp,编码233个氨基酸;对GLL蛋白结构域进行分析,发现该蛋白有一个PEX11保守结构域;系统进化关系表明,GLL与预测的水稻中PEX11-5同源性最高;亚细胞定位表明该基因在细胞核、细胞质和细胞膜中表达。本研究为解析水稻及单子叶植物中毛状体发育分子机制和进行水稻品种改良奠定了基础。     

灰葡萄孢致病力增强突变体BCt98的突变基因分析

为获得灰葡萄孢的致病相关基因并研究其基因功能,筛选灰葡萄孢的T-DNA插入突变体库,获得了一株致病力增强的突变体BCt98。利用PCR和Southern Blotting技术,对突变体BCt98进行鉴定。利用TAIL-PCR技术结合生物信息学方法,确定了突变体BCt98中T-DNA插入位点位于BC1G_07014.1基因的第3个外显子上。利用RT-PCR技术,确定了突变体BCt98的突变基因为BC1G_07014.1。突变体BCt98生长速度较快,菌落颜色较浅,菌丝较为致密,不产生分生孢子和菌核,且胞壁降解酶(PMG、PG和Cx)及毒素活性较野生型明显增强。表明BC1G_07014.1基因在灰葡萄孢生长、发育和致病力调控方面发挥重要作用,且参与调控病菌的胞壁降解酶活性和毒素活性。     

Location of a high-lysine gene and the DDT-resistance gene on barley chromosome 7

Summary The high-lysine gene in Risø mutant 1508 conditions an increased lysine content in the endosperm via a changed protein composition, a decreased seed size, and several other characters of the seed. The designation lys3a, lys3b, and lys3c, is proposed for the allelic high-lysine genes in three Risø mutants, nos 1508, 18, and 19. Linkage studies with translocations locate the lys3 locus in the centromere region of chromosome 7. A linkage study involving the loci lys3 and ddt (resistance to DDT) together with the marker loci fs (fragile stem), s (short rachilla hairs), and r (smooth awn) show that the order of the five loci on chromosome 7 from the long to the short chromosome arm is r, s, fs, lys3, ddt. The distance from locus r to locus ddt is about 100 centimorgans.     

Biological Activity and Quantification of Suspected Allelochemicals from Alfalfa Plant Parts

Autotoxicity restricts reseeding of alfalfa (Medicago sativa L.) after alfalfa until autotoxic chemical(s) breaks down or is dispersed into external environments. A series of aqueous extracts from leaves, stems, roots and seeds of alfalfa ‘Vernal’ were bioassayed against alfalfa seedlings of the same cultivar to determine their autotoxicity. The highest inhibition was found in the extracts from the leaves. Extracts at 40 g dry tissue l^?1 from alfalfa leaves were 15.4, 17.5 and 28.7 times more toxic to alfalfa root growth than were those from roots, stems and seeds, respectively. A high‐performance liquid chromatography (HPLC) analysis with nine standard compounds showed that the concentrations and compositions of allelopathic compounds depended on the plant parts. In leaf extracts that showed the most inhibitory effect on root growth, the highest amounts of allelochemicals were detected. Among nine phenolic compounds assayed for their phytotoxicity on root growth of alfalfa, coumarin, trans‐cinnamic acid and o‐coumaric acid at 10^?3 m were most inhibitory. The type and amount of causative allelochemicals found in alfalfa plant parts were highly correlated with the results of the bioassay, indicating that the autotoxic effects of alfalfa plant parts significantly differed.     

Simultaneous Determination of Four Phenolic Acids in Radix Salviae Miltiorrhizae Formula Granules by QAMS

[Objectives]This study aimed to establish a QAMS(quantitative analysis of multi-components by single-marker)method for simultaneous determination of four phenol...     

Changes in Seed Yield and Quality with Maturity in Onion (Allium cepa L., cv. 'Early Cream Gold')

Development of onion (Allium cepa L., cv. ‘Early Cream Gold’) seed under cool climate conditions in Tasmania, Australia occurred over a longer duration than previously reported, but similar patterns of change in yield components were recorded. In contrast to previous studies, umbel moisture content declined from 85 to 67 % over 57 days while seed moisture content decreased from 85 to 31 %. Seed yield continued to increase over the duration of crop development, with increasing seed weight compensating for seed loss resulting from capsule dehiscence in the later stages of maturation. Germination percentage was high and did not vary significantly from 53 to 77 days after full bloom (DAF), but mean germination time declined and uniformity of germination increased significantly over the same time period. The percentage abnormal seedlings declined with later harvest date, resulting in highest seed quality at 77 DAF. The results of this study suggest that the decision to harvest cool climate onion seed crops before capsule dehiscence will result in a loss of potential seed yield and quality.     

Water Extraction Process of Chinese Herbal Compound Man Gan Ning

[Objectives]To optimize the water extraction process of Chinese Herbal Compound Man Gan Ning and establish a method for its extraction and content determination...     

Present status and future strategy in breeding faba beans (Vicia Faba L.) for resistance to biotic and abiotic stresses

Progress is being made, mainly by ICARDA but also elsewhere, in breeding for resistance to Botrytis, AScochyta, Uromyces, and Orobanche; and some lines have resistance to more than one pathogen. The strategy is to extend multiple resistance but also to seek new and durable forms of resistance. Internationally coordinated programs are needed to maintain the momentum of this work.Tolerance of abiotic stresses leads to types suited to dry or cold environments rather than broad adaptability, but in this cross-pollinated species, the more hybrid vigor expressed by a cultivar, the more it is likely to tolerate various stresses.     

Pollen and pollination experiments. III. The viability of apple and pear pollen as affected by irradiation and storage

Summary Apple and pear pollen was irradiated with doses of 0, 50, 100, 250 and 500 krad (gamma rays) and stored at 4°C and 0–10% r.h. From the in-vitro germination percentages an average LD 50 dose of about 220 krad was estimated. For both irradiated and untreated pollen a close and corresponding lineair relationship existed between germination percentage and pollen tube growth.Irradiated pollen was much more sensitive to dry storage conditions than untreated pollen, resulting in less germination and more bursting. Apparently, irradiation caused the pollen cell membrane to lose its flexibility faster than normal. Rehydration of dry-stored, irradiated pollen in water-saturated air restored germination percentages up to their initial levels. The importance of this procedure in germination trials is stressed.     

Optimization of Extraction Technology and Determination of Content of Total Phenols of Leaves in Acanthopanax giraldii Harms

[Objectives] To determine the optimum extraction technology for total phenols of leaves in Acanthopanax giraldii Harms.[Methods]The single factor test and ortho...     

Cytoplasmic male sterility,resistance to gall mite and mildew,and season of leafing out in black currants

Summary Cytoplasmic male sterility (cms) is described in the F₁ hybrids Ribes × carrierei (R. glutinosum albidum × R. nigrum) and R. sanguineum × R. nigrum. In backcrosses to R. nigrum, progenies with R. glutinosum cytoplasm were either all male sterile, or segregated for full male fertility (F) and complete (S) and partial (I) male sterility. Ratios of F:I+S suggested that two linked genes controlled cms, F plants being dominant for one (Rf ₁) and recessive for the other (Rf ₂).Segregation for cms in relation to three linded genes, Ce (resistance to the gall mite, Cecidophyopsis ribes), Sph ₃(resistance to American gooseberry mildew, Sphaerotheca mors-uvae) and Lf ₁(one of two dominant additive genes controlling early season leafing out) indicated that Rf ₁and Rf ₂were in this linkage group. The gene order and approximate crossover values appeared to be: % MathType!MTEF!2!1!+-% feaafiart1ev1aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn% hiov2DGi1BTfMBaeXafv3ySLgzGmvETj2BSbqef0uAJj3BZ9Mz0bYu% H52CGmvzYLMzaerbd9wDYLwzYbItLDharqqr1ngBPrgifHhDYfgasa% acOqpw0xe9v8qqaqFD0xXdHaVhbbf9v8qqaqFr0xc9pk0xbba9q8Wq% Ffea0-yr0RYxir-Jbba9q8aq0-yq-He9q8qqQ8frFve9Fve9Ff0dme% aabaqaciGacaGaamqadaabaeaafaaakeaacaWGdbGaamyzamaamaaa% baGaaiiiaiaacccacaGGWaGaaiOlaiaacgdacaGG0aGaaiiiaiaacc% caaaGaaiiiaiaacccacaGGGaGaamOuaiaadAgaliaaigdakmaamaaa% baGaaiiiaiaacccacaGGGaGaaiiiaiaaccdacaGGUaGaaiOmaiaacs% dacaGGGaGaaiiiaiaacccacaGGGaGaaiiiaaaacaWGsbGaamOzaSGa% aGOmaOWaaWaaaeaacaGGGaGaaiiiaiaacccacaGGGaGaaiiiaiaacc% cacaGGGaGaaiiiaiaacccaaaGaamitaiaadAgaliaaigdakmaamaaa% baGaaiiiaiaacccacaGGGaGaaiiiaiaacccacaGGGaGaaiiiaiaacc% cacaGGGaGaaiiiaiaacccacaGGGaaaaiaadofacaWGWbGaamiAaSGa% aG4maaaa!6E4D!\[Ce\underline { 0.14 } Rf1\underline { 0.24 } Rf2\underline { } Lf1\underline { } Sph3\]. Crossover values of 0.36 for Ce-Lf ₁, and 0.15 for Lf ₁-Sph ₃were estimated from the relative mean differences in season of leafing out between seedlings dominant and recessive for Ce and Sph ₃.It is suggested that competitive disadvantage of lf ₁-carrying gametes and/or zygotes at low temperatures may be implicated in the almost invariable deficit of plants dominant for the closely linked mildew resistance allele Sph ₃. Poor performance of lf ₁- (and possibly lf ₂-) carrying gametes and young zygotes during periods of low temperature at flowering might also account for the liability of some late season cultivars and selections to premature fruit drop (running off).     

Screening techniques and sources of resistance to parasitic angiosperms

Parasitic angiosperms cause great losses in many important crops under different climatic conditions and soil types. The most widespread and important parasitic angiosperms belong to the genera Orobanche, Striga, and Cuscuta. The most important economical hosts belong to the Poaceae, Asteraceae, Solanaceae, Cucurbitaceae, and Fabaceae. Although some resistant cultivars have been identified in several crops, great gaps exist in our knowledge of the parasites and the genetic basis of the resistance, as well as the availability of in vitro screening techniques. Screening techniques are based on reactions of the host root or foliage. In vitro or greenhouse screening methods based on the reaction of root and/or foliar tissues are usually superior to field screenings and can be used with many species. To utilize them in plant breeding, it is necessary to demonstrate a strong correlation between in vitro and field data. The correlation should be calculated for every environment in which selection is practiced. Using biochemical analysis as a screening technique has had limited success. The reason seems to be the complex host-parasite interactions which lead to germination, rhizotropism, infection, and growth of the parasite. Germination results from chemicals produced by the host. Resistance is only available in a small group of crops. Resistance has been found in cultivated, primitive and wild forms, depending on the specific host-parasite system. An additional problem is the existence of pathotypes in the parasites. Inheritance of host resistance is usually polygenic and its transfer is slow and tedious. Molecular techniques have yet to be used to locate resistance to parasitic angiosperms. While intensifying the search for genes that control resistance to specific parasitic angiosperms, the best strategy to screen for resistance is to improve the already existing in vitro or greenhouse screening techniques.