反义trxs基因对转基因小麦种子内源trxh基因表达的影响

以转反义硫氧还蛋白基因(anti-trxs)株系01TY70-1-17-5及其对照小麦品种‘豫麦70’为试验材料,以小麦中稳定表达的肌动蛋白基因actin为内标,用半定量反转录聚合酶链式反应(semi-QRT-PCR)方法,对转基因株系及其对照种子中trxh基因时空表达情况进行了检测。检测结果表明,花后15~30d转基因株系trxh基因转录量平均比对照下降了20.1%,花后25d显著低于对照(P<0.05);胚乳trxh基因转录量最低,平均比对照低19.4%;种子吸涨24h时间内,转基因株系trxh基因转录量较对照均略低,但差异不显著。表明,外源trxs基因的导入直接干扰了内源基因的表达。     

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.     

转反义trxs基因对小麦种子萌发过程中淀粉水解酶的影响

检测转反义trxs基因小麦种子萌发过程中胚乳内α-淀粉酶、β-淀粉酶和支链淀粉酶活性变化的结果表明,在种子萌发4d内,转基因小麦3种酶的活性态酶活性均低于野生型小麦,而3种酶的抑制态酶活性则均高于野生型小麦,转基因小麦淀粉酶的抑制程度高于野生型小麦,这可能是转基因小麦酶活性降低的原因之一。     

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).     

转反义硫氧还蛋白基因小麦萌发种子中蛋白质的变化

硫氧还蛋白h（thioredoxin h,Trx h）是一类广泛存在于生物体内的多功能活性蛋白,分子量约为12kD,它通过还原靶蛋白中的二硫键参与酶活性调节、抗胁迫、信号传导等许多重要的生命活动。硫氧还蛋白h能促进谷物类种子萌发过程,主要表现在以下2个方面：（1）在籽粒萌发期间,硫氧还蛋白可通过还原储存蛋白的分子内二硫键使其更易于被降解;（2）硫氧还蛋白也可以直接地通过将酶还原或者间接地通过使酶抑制蛋白失活而激活酶。源于Phalaris coerulescens的trxs基因（thioredoxin s,trxs）与小麦硫氧还蛋白h基因（thioredoxin h,trx h）同属于硫氧还蛋白基因家族,它们的cDNA有94％的同源性,表达产物也有相似的生物功能。我们采用基因枪法将反义trxs基因导入小麦,获得了可稳定遗传的小麦,并检测出转基因种子中硫氧还蛋白h表达量、水溶蛋白和醇溶蛋白的还原状态以及α-淀粉酶活性均低于对照小麦;另外,通过模拟降雨抗穗发芽试验证实转基因株系具有很强的抗穗发芽能力。以转反义trxs基因抗穗发芽小麦为材料,检测反义trxs基因小麦籽粒萌发过程中蛋白质的变化,探讨转反义trxs基因小麦的抗穗发芽机理。研究表明反义trxs基因能够减缓KCl可溶性蛋白中Chloroform-methanol（CM）蛋白向代谢类蛋白的转化进程,在萌发初期降低籽粒代谢类蛋白的含量,使籽粒代谢速度下降,而CM蛋白主要包含一些分子量小于20kD的蛋白质。在籽粒成熟过程中,硫氧还蛋白能够阻止麦谷蛋白亚基形成谷蛋白聚合体的过程,在转基因小麦中麦谷蛋白更易于形成大分子量的谷蛋白大聚合体,使得转基因小麦中的谷蛋白在萌发初期更难于被水解,因此转基因小麦籽粒会因谷蛋白难于降解而萌发较慢。另外,反义trxs基因减慢了麦胚中10kD蛋白的降解过程。     

转反义Trx s基因小麦灌浆期间籽粒和成熟后种子萌发期间的过氧化物酶和过氧化氢酶活性变化

检测转反义Trx s基因小麦灌浆期间籽粒和萌发期种子中过氧化物酶(POD)和过氧化氢酶(CAT)活性及其同工酶变化的结果表明,在籽粒成熟过程中,转基因小麦籽粒中POD和CAT活性均低于非转基因小麦:在小麦种子萌发过程中,转基因小麦种子中的POD和CAT活性也均低于非转基因小麦.     

种子特异表达异源DGAT1基因提高大豆种子含油量和营养品质

为提高大豆Glycine max种子含油量和营养品质,文中以二酰甘油酰基转移酶1(Diacylglycerol acyltransferase 1,DGAT1)基因为遗传修饰靶标。将来自高油植物斑鸠菊Vernonia galamensis L.编码DGAT1酶蛋白的c DNA克隆Vg DGAT1A在大豆种子特异超表达。连续选择获得高代(T7)Vg DGAT1A转基因大豆株系。转基因株系表型鉴定显示,在大豆种子发育中期(30–45 DAF),Vg DGAT1A高表达,相应地DGAT酶活性是非转基因野生型和空载体转化对照的7.8倍。转基因成熟种子含油量比对照提高了5.1%,淀粉含量比对照减少2%–3%,蛋白质含量与对照无显著差异。此外,转基因大豆种子百粒重(14.5 g)和种子萌发率(95.6%)与对照亦无明显差异。种子油脂脂肪酸成分分析显示,转基因大豆种子油中抗氧化的油酸(C18:1Δ9)含量比对照提高8.2%,相应地易氧化的亚油酸(C18:2Δ9,12)和亚麻酸(C18:3Δ9,12,15)分别减少6%和2%。这些数据表明,种子特异超表达外源Vg DGAT1A基因,打破了大豆种子含油量和蛋白质含量的负连锁,显著提高种子含油量且未导致蛋白含量降低。转基因大豆种子重量和萌发率亦未显负效应,而且种子油脂抗氧化性和营养品质得以改善。研究表明应用这一高酶活性Vg DGAT1A的基因工程是提高种子含油量和改善油脂品质的一条有效途径。     

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。     

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)。     

Fractional Analyses of Sterols in Soybean Harvested in Japan

Debranching enzyme activity in rice seeds increased during the early stage of ripening and then decreased, and increased again during germination. The inactive enzyme accumulated rapidly in ripening seeds from the 20th day after flowering.

Radioactive amino acids were readily incorporated into the active debranching enzyme after their absorption into immature rice seeds. Subsequently, the radioactivity increased in the inactive enzyme, accompanying a decrease in the active enzyme. We concluded that the debranching enzyme in rice seeds is synthesized during ripening in active form and that it accumulates in inactive form, which can be reactivated during germination.     

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.     

Investigating seed dormancy in cotton (Gossypium hirsutum L.): understanding the physiological changes in embryo during after‐ripening and germination

• The dormancy of seeds of upland cotton can be broken during dry after‐ripening, but the mechanism of its dormancy release remains unclear.
• Freshly harvested cotton seeds were subjected to after‐ripening for 180 days. Cotton seeds from different days of after‐ripening (DAR) were sampled for dynamic physiological determination and germination tests. The intact seeds and isolated embryos were germinated to assess effects of the seed coat on embryo germination. Content of H₂O₂ and phytohormones and activities of antioxidant enzymes and glucose‐6‐phosphate dehydrogenase were measured during after‐ripening and germination.
• Germination of intact seeds increased from 7% upon harvest to 96% at 30 DAR, while embryo germination improved from an initial rate of 82% to 100% after 14 DAR. Based on T₅₀ (time when 50% of seeds germinate) and germination index, the intact seed and isolated embryo needed 30 and 21 DAR, respectively, to acquire relatively stable germination. The content of H₂O₂ increased during after‐ripening and continued to increase within the first few hours of imbibition, along with a decrease in abscisic acid (ABA) content. A noticeable increase was observed in gibberellic acid content during germination when ABA content decreased to a lower level. Coat removal treatment accelerated embryo absorption of water, which further improved the accumulation of H₂O₂ and changed peroxidase content during germination.
• For cotton seed, the alleviation of coat‐imposed dormancy required 30 days of after‐ripening, accompanied by rapid dormancy release (within 21 DAR) in naked embryos. H₂O₂ acted as a core link between the response to environmental changes and induction of other physiological changes for breaking seed dormancy.

     

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.     

不同发育时期黄皮种子脱水敏感性的研究

自花后４６ｄ到８８ｄ果实成熟．黄皮种子的发芽率由０升至１００％．而活力指数逐渐上升,到花后７４ｄ达到最大值,之后略有下降．每粒种子的呼吸强度在花后４６－６７ｄ持续增加,此后则渐渐减弱,但湿藏２ｄ后又回升．黄皮种子的发育明显超前于果实．花后７４ｄ时．每粒种子的干重已接近最大值,这时种子活力最大．而果实的鲜重虽然已接近最大值．但其干重却只有成熟时的７３％。花后４６－５３ｄ的种子,其发芽率小于１００％,轻微脱水能提高种子的发芽率及活力指数,花后６０ｄ至果实成熟．种子发芽率均为１００％．这时任何程度的脱水都会引起活力指数的下降,但不同发育时期的黄皮种子耐脱水力有差别．其中以花后６７ｄ的耐脱水力最强．花后８８ｄ果实成熟时种子的耐脱水力最弱。     

Enzymic mechanism of starch breakdown in germinating rice seeds I. An analytical study

Time-sequence analyses of carbohydrate breakdown in germinating rice seeds shows that a rapid breakdown of starch reserve in endosperm starts after about 4 days of germination. Although the major soluble carbohydrate in the dry seed is sucrose, a marked increase in the production of glucose and maltooligosaccharides accompanies the breakdown of starch. Maltotriose was found to constitute the greatest portion of the oligosaccharides throughout the germination stage. α-Amylase activities were found to parallel the pattern of starch breakdown. Assays for phosphorylase activity showed that this enzyme may account for much smaller amounts of starch breakdown per grain, as compared to the amounts hydrolyzed by α-amylase. There was a transient decline in the content of sucrose in the initial 4 days of seed germination, followed by the gradual increase in later germination stages. During the entire germination stage, sucrose synthetase activity was not detected in the endosperm, although appreciable enzyme activity was present in the growing shoot tissues as well as in the frozen rice seeds harvested at the mid-milky stage. We propose the predominant formation of glucose from starch reserves in the endosperm by the action of α-amylase and accompanying hydrolytic enzyme(s) and that this sugar is eventually mobilized to the growing tissues, shoots or roots.     

铜尾矿对5种豆科植物种子萌发和幼苗生长影响的初步研究

通过水培实验和土培实验,研究了铜陵铜尾矿对5种豆科植物种子萌发和幼苗生长的影响。结果表明,五种豆科植物种子在尾矿浸提液中均能萌发,但萌发率比对照组低,而且浸提液对种子萌发产生一定的阻滞作用,酸化尾矿浸提液对种子萌发初期的吸胀吸水有促进作用。尾矿与土壤5种不同改良方式的基质试验也发现铜尾矿对种子萌发着阻滞作用,其中大豆、赤豆、赤小豆在70d内未发现死苗现象。