玉米茎节再生体系建立及AGPL基因遗传转化的研究

以玉米优良自交系郑58的子粒为材料,切取无菌苗的第1个茎节,上下各保留0.5 cm,接种于诱导培养基(1/2MS+0.3 mg/L NAA)培养,直接获得再生苗。腺苷二磷酸葡萄糖焦磷酸化酶(AGPase)是玉米淀粉合成过程中的一个限速酶,AGPase酶大亚基(AGPL)是酶的调节中心,在玉米胞质中过表达能够提高玉米淀粉含量。利用农杆菌介导法将玉米AGPL基因导入郑58的茎节体系,转化后的茎节在含有3mg/L双丙氨膦的1/2 MS培养基上进行筛选培养,对筛选后的抗性苗进行PCR和PCR-Southern blot检测,获得了4株转基因植株。     

Effects of Baking and Boiling on the Nutritional and Antioxidant Properties of Sweet Potato [<Emphasis Type="Italic">Ipomoea batatas</Emphasis> (L.) Lam.] Cultivars

The effects of baking and boiling on the nutritional and antioxidant properties of three sweet potato cultivars (Beniazuma, Koganesengan, Kotobuki) cultivated in Turkey were investigated. The samples were analyzed for proximate composition, total phenolic content, ascorbic acid, β-carotene, antiradical activity, and free sugars. The dry matter, protein, and starch contents of the sweet potatoes were significantly changed by the treatments while the ash and crude fiber contents did not differ as significantly. The β-carotene contents of baked and boiled sweet potatoes were lower than those of fresh sweet potatoes; however, the total phenolic and ascorbic acid contents of the baked and boiled sweet potatoes were higher than those of the fresh samples. Generally, the antiradical activity of the sweet potatoes increased with the treatments. Sucrose, glucose, and fructose were quantified as free sugars in all fresh sweet potatoes; however, maltose was determined in the treated samples. In terms of the analyzed parameters, there were no explicit differences among the sweet potato cultivars.     

Evaluation of performance of sorghum varieties grown in Tokyo for sugar accumulation and its correlation with vacuolar invertase genes SbInv1 and SbInv2

ABSTRACT

The sugar-accumulating potential of global and local sweet and grain sorghum varieties were tested under the local conditions. The basis for this study was the dependency of sugar accumulation on temperature and photoperiod. Thus, the efficacy of cultivars as a bioenergy source would need to be determined based on their performance under the local environmental conditions. A strong correlation of sucrose content with brix was observed, enabling large-scale screening of varieties for high sucrose content. The morphological characteristics inherent in sweet sorghum, such as tall stems, greater number of leaves and a longer vegetative period, were found to correlate with the total stem sugar content. Assessment of sugars along the stem revealed maximum sugar accumulation in the upper intermediate to upper internodes in most of the varieties tested. The maximum theoretical ethanol yield (MTEY), a function of brix and juice yield, was determined as a better indicator of testing the performance of a variety as a potential source of bioethanol, mainly due to a negative correlation of stem juiciness and sucrose content in the varieties tested. Further, the relative expression of vacuolar invertase genes, SbINV1 and SbINV2, was studied, and a strong negative correlation of SbINV2 to stem sucrose content was observed. This reveals a possibility of involvement of vacuolar invertase gene, SbINV2, in sugar accumulation in sweet sorghum stems, and as a key candidate for molecular breeding studies for higher stem sugar content.     

玉米AGPase小亚基基因的扩张分析

采用生物信息学方法分析AGPase家族基因的系统发育关系和基因结构特征，基于玉米B73参考基因组(AGPv4)新鉴定玉米AGPase小亚基编码基因，同时根据序列比对结果重新注释基因组中发生扩张的AGPS片段基因。结果表明，新鉴定并重新注释的ZmAGPS2-3、ZmAGPS2-4、ZmAGPS2-5基因包含1个序列高度相似的外显子，由祖先基因ZmAGPS2-2的多个外显子(无内含子)序列拼接形成。其编码蛋白的保守结构域存在不同程度的缺失，且这3个基因在染色体上的位置无共线性关系，也不相邻。因此推测，ZmAGPS2-3、ZmAGPS2-4和ZmAGPS2-5基因起源方式为反转录转座复制，在演化过程中丢失了部分序列后成为功能缺失的假基因。     

Overexpression of a Maize SPS Gene Improves Yield Characters of Potato under Field Conditions

Abstract

We analyzed the yield characters of field-grown transgenic potato plants (Solanum tuberosum) carrying a maize gene for sucrose-phosphate synthase (SPS), the key enzyme in sucrose synthesis. The SPS activity in the leaves of transgenic plants (line Ag1203) was 2 times that of the control (cv. May Queen). There was no difference in the photosynthetic CO₂ uptake rates between Ag1203 and May Queen plants, and the leaf starch content of Ag1203 was lower. These observations indicate that the introduction of a foreign SPS gene improved the supply of photosynthate from source (leaves) to sink (tubers). Additionally, leaf senescence of the transgenic potato plants was delayed relative to that of May Queen. The average tuber weight and total yield of Ag1203 plants were at least 20% higher, and the tuber sucrose content, which is related to eating quality, was also higher. Increased translocation of photosynthate and longer period of photosynthetic activity in the leaves may have increased the yield of Ag1203. These results suggest that introduction of the SPS gene improved the yield characters and quality of potato tubers under field conditions.     

小麦淀粉合成关键酶基因和相关蛋白表达对不同施磷量的响应

为深入研究磷素在小麦淀粉产量和品质形成过程中的作用,以新疆冬小麦主栽品种新冬20号为试验材料,研究0 kg·hm^-2（CK）、105 kg·hm^-2（LP）和210 kg·hm^-2（HP）3种施磷（P₂O₅）量对小麦籽粒灌浆特性、淀粉合成关键酶(AGPase和GBSS)活性和基因表达、胚乳可溶性蛋白和淀粉粒结合蛋白含量及谱带特征、胚乳微观特性等的影响。结果表明,籽粒灌浆后期磷肥对粒重的促进作用逐渐增强;低磷（LP）条件下, agp1、 agp2、 gbss1、 gbss2基因表达量显著提高;3个处理下AGPase活性的变化趋势基本一致;籽粒灌浆中期（14～21 d）,低磷处理的GBSS活性显著高于CK;不同磷处理对淀粉粒结合蛋白和胚乳可溶性蛋白含量的影响显著,但对蛋白谱带特征的影响较弱。扫描电镜结果显示,不同磷处理的胚乳淀粉粒形态未发生明显变化;与CK和高磷（HP）处理相比,低磷处理下籽粒胚乳横断面蛋白基质较少,淀粉粒与蛋白质结合较疏松。说明常规施磷（低磷）条件下,淀粉合成关键酶基因（agp和gbss）相对表达量提高或酶活性变化可影响籽粒淀粉合成及胚乳中蛋白与淀粉粒的嵌合度,并最终造成粒重的增加和淀粉特性的变化。     

HARDY基因的克隆、原核表达及其植物表达载体的构建

为了给转HARDY(HRD)基因小麦研究奠定基础,利用PCR技术从拟南芥中克隆HRD基因,进行生物信息学分析,预测其编码的蛋白质结构与功能,构建原核表达载体pET28a(+)-HRD,转化大肠杆菌BL21(DE3),IPTG诱导蛋白的表达,同时构建pBin-HRD植物表达载体。测序分析结果表明,克隆的HRD与NCBI发布的拟南芥核苷酸序列的同源性为99%,其开放阅读框长555bp,可编码184个氨基酸,具有许多重要功能位点;成功构建了原核表达载体pET28a(+)-HRD和植物表达载体pBin-HRD,诱导表达出大小约为23.3kD的蛋白,与理论值相近。     

Development of Bt transgenic potatoes for effective control of potato tuber moth by using cry1Ab gene regulated by GBSS promoter

Transgenic of Indian potato cultivar Kufri Badshah expressing synthetic, modified cry1Ab gene were developed against potato tuber moth (Phthorimaea opercullela Z.) a destructive pest. The cry1Ab gene was in spatial and temporal expression under the control of tuber-specific GBSSi promoter. The transformation vector pBinCG1 was developed harbouring transgene expression cassette comprising cry1Ab gene under the control of potato GBSSi promoter, castor bean catalase intron (5′UTR) and OCS termination signals. Selected kanamycin-resistant putative transformed potato lines were evaluated by positive PCR amplification of 713 bp, 1206 bp and 700 bp with npt II, GBSSi promoter and cry1Ab gene specific primers, respectively. Gene integration in transformed potato plants was elucidated through Southern hybridization and in planta transgene expression analysis. Transgene expression was highest in stolons, followed by tubers matured leaves and young leaves. Insect bioassay of stored transgenic tubers resulted in significant retardation and mortality in neonate tuber moth larvae. RT-PCR and northern hybridization confirmed stable expression in stored tubers. These transgenic lines; thus can represent an effective resistance management strategy which can significantly reduce the selection pressure on target and non-target insect pests to a threshold level.     

Inactivation of trypsin inhibitors in sweet potato and taro tubers during processing

In order to understand the extent of elimination of trypsininhibitors during processing of sweet potato (Ipomoea batatas) andtaro (Colocasia esculenta) tubers, a detailed study was conductedusing tubers processed by oven drying, cooking, and microwavebaking. Between 80 and 90% trypsin inhibitor (TI) activity wasretained in sweet potato chips up to 2h at 70 ^°C. Among thefour cultivars of sweet potatoes, RS-III-2 trypsin inhibitors were more heatlabile. Heating at 100 ^°C led to rapid inactivation of TI of sweetpotatoes. Varietal differences in thermal stability were more pronouncedfor the trypsin inhibitors of taro than sweet potatoes. Taro inhibitors werealso more rapidly inactivated than sweet potato TI. Between 17 and31% TI activity was retained in cooked tuber pieces of sweet potatoes,while only 3–10% were retained in taro cultivars. Very effectiveinactivation of trypsin inhibitors of sweet potatoes and taro could beobtained through microwave baking. Flour prepared from taro was devoidof TI activity, while 5–12% TI activity was retained in the flour preparedfrom sweet potatoes. The study clearly established that among the fourtechniques used, microwave baking and flour preparation were the best methods to eliminate TI from sweet potatoes and taro.     

玉米ZmCOL3pro217启动子的克隆及功能分析

ZmCOL3是玉米开花期光周期调控网络中一个重要的功能基因,研究发现自然群体中该基因的启动子存在遗传变异,推测这些变异可能参与玉米开花调控。研究从热带血缘玉米材料CIMBL119中克隆了ZmCOL3启动子,命名为ZmCOL3pro217。序列比对发现,该启动子序列与温带血缘玉米B73序列存在1个217 bp大片段置换。生物信息学分析表明,ZmCOL3pro217含有分生组织特异性、光响应、胁迫响应等多个顺势作用元件,预测ZmCOL3pro217可能具有组织特异性,并通过响应光和胁迫反应参与玉米开花调控。进一步构建ZmCOL3pro217驱动gus报告基因表达载体,转化玉米,实时荧光定量PCR、酶联免疫吸附剂测定、GUS组织化学染色等研究结果发现,转基因植株中ZmCOL3pro217驱动gus基因在根和叶器官中高表达,而在茎、花丝和子粒中几乎不表达,证实ZmCOL3pro217是1个新的组织特异性表达启动子。     

耐盐碱转基因玉米的获得及其抗性分析

依据Gen Bank数据库中发表的拟南芥DREB基因序列进行优化,人工合成DREB基因序列,构建植物表达载体,通过农杆菌介导法将DREB基因转入玉米自交系Hi II中,获得转DREB基因玉米材料,经过分子检测获得4个阳性转化事件。在不同浓度水平的Na Cl溶液(40、80、120 mmol/L)和Na_2CO_3溶液(10、20、30 mmol/L)胁迫下,研究其耐盐碱性。结果表明,DREB基因已经整合到玉米基因组中,转DREB基因玉米的耐盐碱性获得显著提高,80 mmol/L的Na Cl溶液和20 mmol/L Na_2CO_3溶液可以作为转DREB基因玉米的耐盐碱分析条件。     

小麦隐性核不育保持系表达载体的构建及拟南芥遗传转化

利用小麦杂种优势能够创制稳定的雄性不育系。相比于细胞质雄性不育系和光温敏雄性不育系,细胞核雄性不育具有育性稳定、易恢复、不受环境影响等特点。小麦隐性核不育基因 ms1的克隆以及杂交制种技术(hybrid seed production technology, SPT)的开发,为雄性不育的保持和杂种优势利用奠定了基础。本研究将小麦花粉育性恢复基因 Ms1、花粉致死基因 ZmAA1、红色荧光蛋白基因 DsRed2及其特异性启动子和终止子连接到表达载体pGEII上,转化野生型拟南芥,得到6株转基因拟南芥。通过荧光显微镜观察拟南芥种子,发现野生型拟南芥种子表面平滑,不能发出红色荧光;而转基因种子表面呈现颗粒状,能够发出红色荧光。这说明表达载体构建成功,并能够在拟南芥中成功表达。这为创制人工小麦隐性核不育系奠定了理论和材料基础。     

Transgenic potato plants expressing oxalate oxidase have increased resistance to oomycete and bacterial pathogens

Summary Potato (cv. Bintje) was transformed with a gene encoding an oxalate oxidase from wheat under the control of the CaMV35S promoter. Transgenic potato plants produced high constitutive levels of H₂O₂ as visualized by 4-chloro-l-naphtol staining. The resistance of these plants was tested againstPhytophthora infestans. An increased level of resistance to the disease was marked by a reduced number of lesions as well as by a decreased number of sporangia formed per lesion. In addition, oxalate oxidase overexpressing plants also exhibited improved resistance toStreptomyces reticuliscabiei, the causal agent of netted scab. Increased expression of oxalate oxidase had no effect on the interaction withErwinia carotovora. These experiments show that overexpression of oxalate oxidase represents a potentially interesting approach for protection of potato to pathogens.     

Suppression of the invasive plant mile-a-minute (Mikania micrantha) by local crop sweet potato (Ipomoea batatas) by means of higher growth rate and competition for soil nutrients

Background

There are a variety of ways of increasing crop diversity to increase agricultural sustainability and in turn having a positive influence on nearby natural ecosystems. Competitive crops may provide potent management tools against invasive plants. To elucidate the competitive mechanisms between a sweet potato crop (Ipomoea batatas) and an invasive plant, mile-a-minute (Mikania micrantha), field experiments were carried out in Longchuan County of Yunnan Province, Southwest China, utilizing a de Wit replacement series. The trial incorporated seven ratios of sweet potato and mile-a-minute plants in 25 m² plots.

Results

In monoculture, the total biomass, biomass of adventitious root, leafstalk length, and leaf area of sweet potato were all higher than those of mile-a-minute, and in mixed culture the plant height, branch, leaf, stem node, adventitious root, flowering and biomass of mile-a-minute were suppressed significantly (P < 0.05). The relative yield (RY) of mile-a-minute and sweet potato was less than 1.0 in mixed culture, indicating that intraspecific competition was less than interspecific competition. The competitive balance index of sweet potato demonstrated a higher competitive ability than mile-a-minute. Except pH, other soil nutrient contents of initial soil (CK) were significantly higher than those of seven treatments. The concentrations of soil organic matter, total N, total K, available N, available P, available K, exchange Ca, exchange Mg, available Mn, and available B were significantly greater (P < 0.05) in mile-a-minute monoculture soil than in sweet potato monoculture soil, and were reduced by the competition of sweet potato in the mixture.

Conclusions

Evidently sweet potato has a competitive advantage in terms of plant growth characteristics and greater absorption of soil nutrients. Thus, planting sweet potato is a promising technique for reducing infestations of mile-a-minute, providing weed management benefits and economic returns from harvest of sweet potatoes. This study also shows the potential value of replacement control methods which may apply to other crop-weed systems or invaded natural ecosystems.     

Sorghum extrusion process combined with biofortified sweet potato contributed for high iron bioavailability in Wistar rats

This study aimed to evaluate the effect of sorghum and sweet potato on the bioavailability of iron, gene expression of proteins involved in iron metabolism and the plasma antioxidant capacity in animals fed with whole sorghum grains processed by dry heat or extrusion, combined or not with sweet potato flour with high content of carotenoids. Five experimental groups were tested (n = 7): dry heat sorghum flour (DS); extruded sorghum flour (ES); whole sorghum flour + sweet potato flour (DS + SP); extruded sorghum flour + sweet potato flour (ES + SP) and positive control (FS). The evaluations included: hemoglobin gain, hemoglobin regeneration efficiency, gene expression of divalente metal transporter 1 (DMT-1), duodenal citochroma B (DcytB), ferroportin, hephaestin, transferrin and ferritin and total plasma antioxidant capacity (TAC). The ES + SP group showed higher (p < 0.05) expression of DcytB, ferroportin and hephaestin when compared to the control group. The DS group showed high (p < 0.05) expression of DMT-1 and the ES showed high mRNA expression of transferrin and ferritin. The changes in the sorghum physicochemical properties from extrusion process reduced the iron and phytate content, and increased the gene expression of proteins involved in iron metabolism, improving iron bioavailability. The combination of sweet potato and sorghum flour (dry or extruded) improved the iron capture and total antioxidant capacity, probably due to the presence of β-carotene and antioxidant compounds.     

Resistance levels and fitness of glufosinate-resistant transgenic sweet potato in field experiments

Transgenic herbicide-resistant sweet potato plants [Ipomoea batatas (L.) Lam.] produced through a biolistic transformation were used in this study. The objectives of this research were to (a) quantify resistance levels of transgenic sweet potato expressing the bar gene to glutamine synthetase (GS)-inhibitors, glufosinate and methionine sulfoximine, protoporphyrinogen oxidase (PPO)-inhibiting herbicide, oxyfluorfen and photosynthesis-inhibiting herbicide, paraquat; (b) compare the relative chlorophyll content, quantum yield, growth, and tuberous root yield between transgenic and wild-type sweet potato; (c) compare the response of transgenic and wild-type sweet potato plants to chilling and high temperature, and (d) compare amino acids, minerals and sugar contents of transgenic and wild-type sweet potato tubers. The transgenic sweet potato lines were 20-82-fold more resistant to glufosinate than the wild-type. The representative transgenic line 7171 also showed resistance to methionine sulfoximine, but was not resistant to oxyfluorfen and paraquat, which have different target sites. The stem length, number of leaves, petiole length, shoot fresh weight, and storage roots of transgenic lines were lower than those of the wild-type in field experiments. Reduced growth and root yield were not due to reduced quantum yield and relative chlorophyll contents. Glufosinate increased the ammonium accumulation in leaves of wild-type plants, but had minimal or no effect on leaves of transgenic line 7171. There were differences in mineral contents, free sugars, total amino acids, and free amino acids in the petiole and storage roots between wild-type and line 7171. There was no difference in drought tolerance between wild-type and line 7171, but the transgenic line was more tolerant to chilling temperature than wild-type plants. Follow-up research is required to elucidate the mechanisms underlying the growth and yield differences, different nutritional values, and differential response to low temperature between wild-type and transgenic sweet potato.     

冬小麦miR398前体的克隆及其在低温条件下对靶基因CSD1表达的调控

miR398是受逆境胁迫负调控的miRNA,其靶基因CSD编码超氧化物歧化酶(SOD),使植物抵御活性氧(ROS)的毒害。为进一步了解低温胁迫下miR398的调控机制,从东农冬麦1号中克隆小麦miR398前体,构建过表达载体并转化拟南芥,用Real-time PCR检测T0代植株中小麦miR398及其靶基因CSD1在低温胁迫下的表达量。结果表明,随着低温胁迫时间的延长,miR398表达下调、CSD1基因表达上调,认为小麦miR398能响应低温胁迫、负调控CSD1基因表达,间接提高了拟南芥的抗寒性。     

不同氮素形态配比对菜用甘薯产量和品质的影响

菜用甘薯存在产量低、品质差以及在种植过程中由于不合理施肥造成生态环境恶化等问题,因此,针对以上问题开展本研究。氮肥作为农业肥料投入的主要部分,是影响菜用甘薯产量和品质的重要因素,通过设置不同氮素形态配比处理,根据菜用甘薯茎尖产量和品质相关指标的变化,揭示菜用甘薯产量和品质对氮素形态配比的响应机制,为提升菜用甘薯产量和品质的同时减轻因不合理施肥造成环境污染提供理论依据。本研究为大田试验,供试品种为生产上主栽的2个菜用甘薯品种‘福薯18’（F18）和‘鄂薯10号’（E10）,采用两因素裂区试验设计,设5个氮素形态配比处理为：（1）NH₄⁺-N∶NO₃^--N∶CONH₂-N =1∶1∶1 (N₁);（2）NH₄⁺-N∶NO₃^--N∶CONH₂-N = 1∶0∶2 (N₂);（3）NH₄⁺-N∶NO₃^--N∶CONH₂-N = 2∶0∶1 (N₃);（4）NH₄⁺-N∶NO₃^--N∶CONH₂-N = 1∶2∶0 (N₄);（5）NH₄⁺-N∶NO₃^--N∶CONH₂-N = 2∶1∶0 (N₅)。研究结果表明,N₄和N₅处理均可显著提高菜用甘薯的茎尖产量、总酚含量、总黄酮含量、可溶性糖含量、苯丙氨酸解氨酶(PAL)酶活性和IbPAL基因表达量;同一时期不同处理间差异显著,随着生育期的不断推进,各项指标的变化呈先升后降的趋势,其中以N₄处理效果更为显著,除可溶性糖含量在N₃处理下最低外,其他各项指标均在N₂处理下最低,由相关性分析证实,总酚、总黄酮和可溶性含量及PAL酶活性与IbPAL基因表达量呈显著正相关,而可溶性糖含量与基因表达量呈负相关,表明可溶性糖含量的变化与IbPAL基因表达无相关性;由氮素形态配比处理可知,相对于N₂和N₃处理,N₄和N₅处理为植株提供了更多的NO₃^--N,由此推断NO₃^--N在菜用甘薯生长发育过程中起到更为重要的促进作用,而CONH₂-N在本研究中对甘薯产量和品质的促进作用并不突出。而N₄处理即NH₄⁺-N∶NO₃^--N∶CONH₂-N = 1∶2∶0的配肥方案是促进菜用甘薯产量和品质提升的最佳配施组合。本研究结果可为合理配肥以获得理想的作物产量和品质提供理论依据,为在其他作物上开展相关研究提供参考。     

玉米逆境胁迫响应基因ZmbZIP15的克隆与抗旱功能分析

从转录组数据中筛选到27个参与干旱-复水胁迫响应的bZIP基因,构建共表达网络图。结果发现,ZmbZIP15处于核心节点位置,该基因位于第5号染色体,编码176个氨基酸,包含高度保守的bZIP结构域,属于亲水性蛋白。蛋白进化树分析发现,该蛋白与芒草、高粱的亲缘关系最近,与大麦、小麦的亲缘关系最远。ZmbZIP15基因ATG上游2 K启动子的顺式元件分析,发现含有多个参与调控脱落酸、低温和干旱的结合元件。实时荧光定量PCR (qRT-PCR)结果显示,ZmbZIP15是组成型表达基因,在雌穗高表达,幼茎的表达量最低。干旱、高温、盐、氮胁迫处理下,该基因的表达量显著上调,说明ZmbZIP15基因积极参与并调控非生物胁迫途径。过表达ZmbZIP15转基因拟南芥抗旱性检测分析,发现干旱胁迫处理下过表达ZmbZIP15基因能够提高拟南芥幼苗的抗旱性。亚细胞定位显示,该基因编码的蛋白定位于细胞核。     

小麦TaDHN2基因及其启动子的克隆与功能研究

为给深入研究Ta DHN2基因在小麦抗旱机制中的作用机理奠定基础,并为进一步丰富小麦DHN基因研究内容提供参考,本研究通过筛选石麦15基因组BAC文库和BAC克隆测序方法克隆了Ta DHN2基因及其启动子,并对Ta DHN2基因序列特征、表达模式和启动子功能等进行了分析和探讨。结果表明,Ta DHN2基因含有1个88 bp的内含子,开放读码框长为696 bp,编码1个含有231个氨基酸的脱水素蛋白。Ta DHN2蛋白具有Y-segment、S-segment和K-segment结构域,属于YSK2类型脱水素蛋白。此外,该蛋白含有明显的核定位信号序列S-segment和基序RRKK。Ta DHN2基因受渗透胁迫诱导表达,在根和叶中表达模式类似,叶中表达量显著高于根中。Ta DHN2基因启动子序列长为2 025 bp,预测含有9个脱水响应顺式元件。在转基因拟南芥中,Ta DHN2基因启动子能够启动GUS基因表达,并在渗透胁迫下诱导GUS基因上调表达。以上结果说明,Ta DHN2基因为脱水响应基因,其启动子为渗透胁迫强诱导启动子。