反义trxs基因导入对小麦籽粒脱支酶活性的影响

以转反义硫氧还蛋白基因株系01TY34-73-9及其对照品种‘豫麦34’为材料,运用PCR检测和酶活性测定的方法,对转基因株系遗传稳定性以及转基因与对照种子中脱支酶活性进行测定。结果显示:(1)外源基因已经稳定遗传至后代;(2)转基因种子在不同成熟时期和不同萌发过程中的脱支酶活性与对照相比均有不同程度的降低平均降低10.3%,但仅花后25 d到收获后5 d脱支酶活性显著低于对照,其中最低值出现在花后30 d,平均比对照下降了12.0%;(3)在花后30 d和后熟5 d萌发过程中,转基因种子脱支酶活性始终低于对照,平均下降6.2%和22.2%。表明反义trxs基因的导入干扰了小麦trxh基因的表达,使trxh转录量减少,小麦籽粒中脱支酶的活性受抑。     

外源脱水应答转录因子DREB基因在转基因小麦中的诱导型表达与抗干旱生理效果研究

以冬小麦品种8901、5-98、99-92和104等品种的幼穗和幼胚为材料,用基因枪转化含逆境诱导转录因子DREB和bar基因的质粒pBAC128F（7024bp）。经筛选与植株再生,共获得70多个转基因小麦植株及其后代株系。转基因株系经PCR分析和RNA点杂交检测,结果表明外源转录因子DREB基因已稳定整合到转基因植株及其后代株系中,并且在部分后代株系中获得了表达。叶片脯氨酸含量测定表明,有16个转基因株系的脯氨酸含量与非转基因对照相比,增加相当显著,其中10个株系的脯氨酸含量在1100μg/g以上,比对照提高了2倍多。室内抗旱模拟实验表明,转基因株系停止浇水15d后,叶片仍然表现绿色,而对照叶片则失绿、枯干;复水10d后,转基因株系恢复活力,对照则死亡。研究表明,利用逆境诱导型启动子（rd29B）来增强外源DREB基因的表达,能显著改良小麦的抗旱性。     

转反义trxs基因小麦籽粒后熟过程中水解酶活性的变化

以小麦品种'生抗1号'(对照)及其转反义trxs基因纯合株系为材料,测定了它们在种子后熟过程中硫氧还蛋白h(Trxh)、淀粉酶和蛋白酶活性以及呼吸速率等生理指标,以探讨转反义trxs基因小麦籽粒在后熟过程中的生理生化变化规律,为提高小麦耐储性探索有效途径.结果显示,在小麦后熟过程中,转基因小麦籽粒的Trxh活性、淀粉酶活性、蛋白水解酶活性和种子呼吸速率均比对照明显下降;后熟0～30 d,转基因小麦种子的Trxh活性、α-淀粉酶、β-淀粉酶、脱支淀粉酶、蛋白酶活性和种子呼吸速率平均比对照分别降低16.07%、31.64%、6.44%、21.63%、27.67%和52.90%.研究表明,反义trxs基因导入干扰或部分抑制了小麦内源同源基因的表达,致使小麦种子Trxh活性、呼吸速率和水解酶活性降低,从而延缓了籽粒储藏物质的转化速率,提高了小麦种子的耐贮藏性.     

以胆碱脱氢酶基因对小黑杨花粉植株的遗传转化

以小黑杨（Populus simoniixP．nigra）花粉植株叶片为外植体,用根癌农杆菌介导法将胆碱脱氢酶基因（betA）导入其中,将获得的4株卡那霉素抗性转基因株系进行PCR检测,结果均为阳性。用荧光定量PCR对转基因株系的betA基因转录结果检测表明,4个转基因株系均已表达外源基因,但表达量有差异。对获得的4株转基因株系及对照进行NaCl胁迫处理,当NaCl浓度为0．55％时,非转基因小黑杨花粉植株生根率为0,转基因株系生根率为80％～100％;在NaCl为0．70％～0.80％时,则转基因株系生根率也为0。4个转基因株系的甜菜碱含量显著高于未转基因对照,说明抛融基因的导入提高了转基因株系的耐盐性。     

脱水应答转录因子CBF1的克隆与转基因小麦的分子检测

根据已发表的小麦(T.aestivum)转录因子CBF1基因序列(GenBank Accession No.AF376136),设计引物从小麦品种‘京花1号’叶片中克隆出该基因,用拟南芥RD29B基因为启动子构建含CBF1基因的逆境诱导表达载体pBAC127F(6 967 bp),以‘99-92’、‘5-98’、‘104’和‘轮选987’等冬小麦品种(系)的幼穗和幼胚为材料,基因枪转化该表达载体。经筛选与植株再生,共获得14株转基因植株及其后代株系。这14个株系经PCR分析和点杂交检测,最终确认了5-98-40、5-98-41这2个株系为转基因株系,结果表明拟南芥RD29B启动子调控下的转录因子CBF1基因已稳定整合到转基因植株中。     

转TPSP融合基因小麦的耐旱相关特性

在获得转TPSP基因小麦纯合株系的基础上,对3个转基因株系的耐旱相关生理特性进行了分析。脯氨酸含量测定显示,干旱胁迫过程中小麦叶片中脯氨酸含量逐渐增加,且3个转基因株系叶片中脯氨酸的积累速度和积累量均显著高于非转基因对照;叶绿素荧光参数测定显示,3个转基因株系的Fv/Fm值在胁迫过程中均略高于非转基因对照,转基因株系4-4-4的Fv/Fo值显著高于非转基因对照,表明转基因株系在水分胁迫条件下光合系统II（PSII）的光合效率有所增强;转基因小麦耐旱性鉴定显示：模拟干旱胁迫100h时对照小麦叶片几乎全部萎蔫,而3个转基因株系均表现出较强的耐旱性;复水24h后转基因株系4-9-1、4-4-4和30-1-2的叶片黄化率分别为25.2%、23.3%和27.6%,显著低于非转基因对照（48.8%）。上述研究结果表明转TPSP基因小麦具有较强的耐旱能力,为转基因材料进一步应用于小麦抗旱育种提供了依据。     

花生AhFAD2基因抑制表达的转基因后代分析

FAD2(Δ~(12) fatty acid desaturase,Δ~(12)FAD或FAD2)是催化油酸在脂肪酸碳链Δ~(12)位脱氢生成亚油酸的关键酶。在花生中,FAD2酶活性下降或失活可提高籽粒中油酸的相对含量,改善花生籽粒及制品的品质和氧化稳定性。通过将种子特异性表达Lectin启动子和Ca MV35S启动子驱动的倒位重复Ah FAD2基因RNAi干扰结构转入花生,获得了以丰花1号(FH1)和花育23(HY23)为受体、携带上述2种转化结构、稳定遗传的花生转基因纯合体株系,转基因花生主要农艺性状与非转基因对照基本一致。实时荧光定量分析发现,各转基因株系发育种子中Ah FAD2基因的转录水平普遍下调。气相色谱法进一步测定了部分转基因后代株系种子的脂肪酸含量及组成,籽粒中油酸含量分别平均提高了15.09%(HY23为受体)、36.40%(FH1为受体),相应地,亚油酸含量平均下降了16.19%、29.81%,油亚比平均增加了38.02%、98.10%。各转基因株系的油酸含量显著提高;且在以FH1为受体的转基因株系后代籽粒以及种子特异性启动子驱动的转化结构中,RNAi的抑制效果更明显。通过RNAi技术抑制花生FAD2基因的表达,可以有效提高花生籽粒油酸含量。该技术体系可以为花生品质育种提供借鉴。     

转反义trxs基因小麦株系01TY18遗传分析及抗穗发芽特性

以豫麦18为受体导入反义trxs基因获得的株系01TY18(T0)为材料,运用PCR、实时荧光RT-PCR以及离体整穗发芽的方法,对反义trxs基因在转基因小麦中的遗传稳定性、基因表达和抗穗发芽特性进行了研究.结果表明,8个T1代转基因株系中有6个株系目的基因检测呈阳性,且在以后的世代中能够稳定遗传并呈典型的孟德尔单基因3∶1分离规律;反义trxs基因在6个转基因株系中能够正常表达且表现出显著的抗穗发芽特性.与非转基因对照相比,转基因株系穗粒发芽率和穗发芽度平均分别降低62%(P<0.01)和50.8%(P<0.01).     

同步抑制FAD2与FatB基因提高烟草种子油酸组分含量的研究

该研究以烟草品系NC89的无菌苗叶片为受体材料,采用前期构建的能同步抑制种子中FAD2(Δ12-油酸去饱和酶基因)与FatB(酰基转移酶基因)表达的RNAi载体,通过农杆菌介导转化获得了转基因烟草植株,分析转基因植株种子中的脂肪酸组分。结果显示:与对照相比,转基因植株种子中FAD2和FatB基因的表达水平分别降低了23%和11%;转基因植株种子的脂肪酸组分中,饱和脂肪酸棕榈酸和硬脂酸平均含量分别为8.02%和4.45%,多不饱和脂肪酸亚油酸平均含量为76.82%,较对照分别降低了2.91%、9.92%和3.47%;而转基因植株种子中单不饱和脂肪酸油酸含量高达7.48%,比对照提高46.38%。研究表明,同步抑制FAD2和FatB基因的表达能够显著提高烟草种子中油酸组分的含量,为进一步改良油料作物品质奠定了基础。     

蔗糖合酶基因AtSUS3干涉后对拟南芥角果发育的影响

蔗糖合酶(SuSy)是植物蔗糖代谢关键酶之一,该研究利用反向遗传学手段,采用RNAi技术抑制拟南芥中AtSUS3基因的表达,测定纯系转基因植株的抽苔率,并对酶活性、糖含量等指标以及糖代谢相关基因的表达进行了检测,探讨SuSy在植物发育中的作用。结果显示:(1)转基因拟南芥的抽苔平均早于野生型植株2～3d,且优先3～4d完成抽苔。(2)开花后生长天数对角果蔗糖和葡萄糖含量有显著影响,而对果糖含量影响不显著;开花后5d时,野生型株系的葡萄糖含量显著高于转基因株系SUS3-2,至15d时,两种转基因株系葡萄糖含量均显著低于野生型株系。(3)开花后生长天数对SuSy、SPS、INV的活性均有显著影响,随开花时间延长,野生型株系SuSy活性显著低于转基因株系,而SPS和INV则相反。(4)AtSUS3基因沉默对其他糖代谢基因有不同程度的影响,开花后5d时,转基因植株的角果中AtCesA1、AtCesA7和AtCINV1的表达量较野生型都有所增加;开花后15d时,转基因植株的角果中AtCesA1、AtCesA7的表达量较野生型高,而AtCINV、AtCwINV的表达量比野生型低。研究表明,拟南芥AtSUS3基因沉默后,在正常生长条件下未造成植株发育异常,同时还可能通过同源家族中其他SuSy的表达水平增加,促进了该酶及糖代谢相关基因整体水平的增加,有助于角果成熟。     

不同发育时期小麦种子活力的变化及其对环境温度的响应

以济麦22和山农23号为试验材料,利用标准发芽试验法对不同年份小麦种子发育过程中的种子活力变化进行研究,分析环境温度对不同发育时期小麦种子活力变化的影响,为早期小麦种子的利用及高活力种子的生产提供参考依据.结果表明: 伴随着小麦种子发育,鲜种子在花后26 d左右出现发芽能力,之后其发芽率整体呈上升趋势;干种子发芽势、发芽率和发芽指数在花后5～8 d迅速升高,之后保持相对稳定,活力指数主要受到幼苗单株干质量的影响而持续升高,一般在完熟前4～6 d达到最大值;不同发育时期小麦干种子的田间种植及其后代种子的活力测定表明,济麦22花后17 d以后的干种子田间出苗较好,并可成穗结实,其后代种子的发芽率和活力指数在不同样品间无显著差异.环境温度对不同发育时期小麦种子活力变化的影响显著,小麦花后日平均温度均值、日最高气温均值以及日最低气温均值均高,且花后日温差均值大的年份,种子发育时间短、百粒重及种子活力达到最大值的时间较早;反之,发育时间较长、百粒重及种子活力达到最大值的时间较晚,但完熟期积温高,种子活力较高.     

A Microarray-Based Comparative Analysis of Gene Expression Profiles During Grain Development in Transgenic and Wild Type Wheat

Global, comparative gene expression analysis is potentially a very powerful tool in the safety assessment of transgenic plants since it allows for the detection of differences in gene expression patterns between a transgenic line and the mother variety. In the present study, we compared the gene expression profile in developing seeds of wild type wheat and wheat transformed for endosperm-specific expression of an Aspergillus fumigatus phytase. High-level expression of the phytase gene was ensured by codon modification towards the prevalent codon usage of wheat genes and by using the wheat 1DX5HMW glutenin promoter for driving transgene expression. A 9K wheat unigene cDNA microarray was produced from cDNA libraries prepared mainly from developing wheat seed. The arrays were hybridised to flourescently labelled cDNA prepared from developing seeds of the transgenic wheat line and the mother variety, Bobwhite, at three developmental stages. Comparisons and statistical analyses of the gene expression profiles of the transgenic line vs. that of the mother line revealed only slight differences at the three developmental stages. In the few cases where differential expression was indicated by the statistical analysis it was primarily genes that were strongly expressed over a shorter interval of seed development such as genes encoding storage proteins. Accordingly, we interpret these differences in gene expression levels to result from minor asynchrony in seed development between the transgenic line and the mother line. In support of this, real time PCR validation of results from selected genes at the late developmental stage could not confirm differential expression of these genes. We conclude that the expression of the codon-modified A.␣fumigatus phytase gene in the wheat seed had no significant effects on the overall gene expression patterns in the developing seed.     

转反义trxs基因小麦株系00T89分子鉴定及抗穗发芽特性研究

以皖麦48为受体导入反义trxs基因已获得00T89第4代(T4)转基因株系,对其进行反义基因的PCR鉴定、相对定量RT-PCR基因表达检测以及抗穗发芽特性研究。结果表明,18个T4代转基因株系中,13个株系目的基因检测呈阳性;成熟期籽粒萌发过程中,8个株系转录水平上mRNA丰度极显著降低(P<0·01),mRNA丰度与穗发芽指标呈显著和极显著的相关性(r=0·7181)。其中6个株系在开花后30d至成熟后10d表现出明显的抗穗发芽特性。与非转基因对照相比,平均穗开始发芽时间推迟2·7d(P<0·01),穗粒发芽率和穗发芽度分别降低35·5%(P<0·01)和47·5%(P<0·01),成熟后25d这些株系又逐渐恢复发芽特性,无显著差异(P>0·05)。     

Constitutive Expression of Soybean Ferritin cDNA Intransgenic Wheat and Rice Results in Increased Iron Levels in Vegetative Tissues but not in Seeds

We used particle bombardment to produce transgenic wheat and rice plants expressing recombinant soybean ferritin, a protein that can store large amounts of iron. The cDNA sequence was isolated from soybean by RT-PCR and expressed using the constitutive maize ubiquitin-1 promoter. The presence of ferritin mRNA and protein was confirmed in the vegetative tissues and seeds of transgenic wheat and rice plants by northern and western blot analysis, respectively. The levels of ferritin mRNA were similar in the vegetative tissues of both species, but ferritin protein levels were higher in rice. Both ferritin mRNA and protein levels were lower in wheat and rice seeds. ICAP spectrometry showed that iron levels increased only in vegetative tissues of transgenic plants, and not in the seeds. These data indicate that recombinant ferritin expression under the control of the maize ubiquitin promoter significantly increases iron levels invegetative tissues, but that the levels of recombinant ferritin in seeds are not sufficient to increase iron levels significantly over those in the seeds of non-transgenic plants.     

Analysis of Expression of a Phenazine Biosynthesis Locus of Pseudomonas aureofaciens PGS12 on Seeds with a Mutant Carrying a Phenazine Biosynthesis Locus-Ice Nucleation Reporter Gene Fusion

A derivative of Pseudomonas aureofaciens PGS12 expressing a promoterless ice nucleation gene under the control of a phenazine biosynthesis locus was used to study the expression of a phenazine antibiotic locus (Phz) during bacterial seed colonization. Seeds of various plants were inoculated with wild-type PGS12 and a PGS12 ice nucleation-active phz:inaZ marker exchange derivative and planted in soil, and the expression of the reporter gene was monitored at different intervals for 48 h during seed germination. phz gene expression was first detected 12 h after planting, and the expression increased during the next 36-h period. Significant differences in expression of bacterial populations on different seeds were measured at 48 h. The highest expression level was recorded for wheat seeds (one ice nucleus per 4,000 cells), and the lowest expression level was recorded for cotton seeds (one ice nucleus per 12,000,000 cells). These values indicate that a small proportion of bacteria in a seed population expressed phenazine biosynthesis. Reporter gene expression levels and populations on individual seeds in a sample were lognormally distributed. There was greater variability in reporter gene expression than in population size among individual seeds in a sample. Expression on sugar beet and radish seeds was not affected by different inoculum levels or soil matric potentials of -10 and -40 J/kg; only small differences in expression on wheat and sugar beet seeds were detected when the seeds were planted in various soils. It is suggested that the nutrient level in seed exudates is the primary reason for the differences observed among seeds. The lognormal distribution of phenazine expression on seeds and the timing and difference in expression of phenazine biosynthesis on seeds have implications for the potential efficacy of biocontrol microorganisms against plant pathogens.     

RNAi mediated silencing of lipoxygenase gene to maintain rice grain quality and viability during storage

Lipoxygenase (LOX) is a common enzyme which catalyzes lipid peroxidation of seeds and consequently enhances seed quality deterioration and decreases seed viability. During seed storage, peroxidation of unsaturated fatty acids occur due to enhancement of LOX activity which directly leads to reduction in seed vigour and deterioration of grain nutritional quality. This study was undertaken to overcome these problem during rice seed storage by attenuating LOX activity using RNAi technology. To improve seed storage stability, we down regulated LOX gene activity by using a functional fragment of the LOX gene under the control of both constitutive (CaMV35S) and aleurone-specific (Oleosin-18) promoter separately. To understand the storage stability, RNAi–LOX seeds and non-transgenic control seeds were subjected to accelerated aging at 45 °C and 85 % relative humidity for 14 days. Our studies demonstrate that down regulation of LOX activity reduces the seed quality deterioration under storage condition. In addition GC–MS analysis revealed that reduction of fatty acid level in non-transgenic seeds during storage was higher when compared with that of transgenic rice seeds. Furthermore, the transgenic rice seeds with reduced LOX activity exhibited enhanced seed germination efficiency after storage than that of non-transgenic rice seeds. This study will have direct impact on nutritional stability of quality rice grains.