抗纹枯病、根腐病的转SN1基因小麦的获得与鉴定

SN1是源于马铃薯的一种抗菌肽, 可以抑制多种植物病原菌的生长。小麦纹枯病(主要病原菌为禾谷丝核菌Rhizoctonia cerealis)和根腐病(主要病原菌为平脐蠕孢菌Bipolaris sorokiniana)是小麦的主要土传真菌病害。本研究利用基因工程技术构建了SN1基因的单子叶植物表达载体pA25-SN1, 它受玉米泛素(ubiquitin)启动子的控制;采用基因枪法将pA25-SN1转化小麦推广品种扬麦18幼胚愈伤组织4 000块, 获得203株再生植株, 通过PCR检测出阳性植株55株, 转化率为1.38%。对转SN1基因小麦T₀~T₂代植株, 进行外源基因的PCR、Southern blot、RT-PCR、荧光定量RT-PCR(Q-RT-PCR)分析和小麦纹枯病菌与根腐病菌接种及其抗病性鉴定。结果表明, 转入的SN1基因已经整合到转基因小麦的基因组中, 能够在转基因小麦中遗传、转录与表达。SN1基因的表达提高了转基因植株对小麦纹枯病和根腐病的抗性, 其抗病性可以遗传。     

用基因枪法获得转异天南星基因aha抗蚜虫小麦

凝集素是一类具有特异糖结合活性的蛋白,对蚜虫等害虫有很强的抗杀作用。利用异天南星凝集素基因aha(Arisaema heterophyllum agglutinin)以及水稻Rubisco小亚基启动子,构建了aha基因植物绿色组织特异性表达载体,并采用基因枪转化方法, 与携带bar基因的pAHC20载体共转化到小麦品种科农199中。经过愈伤诱导、再生和筛选以及PCR鉴定,获得aha转基因植株42株,平均转化效率为2.41%。对转aha基因植株后代PCR鉴定表明,T₁代转基因植株的分离比例基本符合孟德尔遗传规律。利用室内多目标综合判别法评定抗蚜虫特性,8个T₁代转基因株系中有高抗材料1份,低抗材料3份,占参试比例44.4%。本研究为获得抗蚜虫转基因小麦新材料奠定了基础。     

转双价基因(Cry1 Ac+CpTI)抗虫棉(ZGK 9 71 2)对小地老虎抗虫性研究

室内测定了转 Cry1 Ac基因抗虫棉和转双价基因 (Cry1 Ac+Cp TI)抗虫棉对小地老虎的存活、生长发育、繁殖及营养效应的影响。结果表明 ,两种转基因抗虫棉对小地老虎幼虫均有一定的抗性 ,转 Cry1 Ac基因抗虫棉对低龄幼虫的抗性好于转双价基因抗虫棉 ,而转双价基因抗虫棉对高龄幼虫的抗性好于转 Cry1 Ac基因抗虫棉 ,但两种转基因抗虫棉对小地老虎幼虫的控制作用均不理想。两种转基因抗虫棉对小地老虎幼虫的生长发育、繁殖和营养效应均有较大的影响。表现为发育历期延长 ,虫重和蛹重减轻 ,羽化率、化蛹率、产卵量和卵孵化率降低 ;相对生长率、相对取食量、近似消化率、食物转化率和利用率明显降低 ,而相对代谢速率明显增加。转双价基因抗虫棉的影响更为明显     

中国水仙凝集素基因在大花烟草中的定量表达及抗蚜性分析

为了确定中国水仙凝集素基因NTL1的抗蚜功能及培育具有抗蚜功能的大花烟草,利用qPCR方法,研究了中国水仙凝集素基因(NTL1)在转基因大花烟草4个株系中的相对表达情况;利用自然感蚜、活体接种和离体接种方法研究了不同转基因株系的抗蚜能力。结果表明,NTL1在不同株系中的相对表达量差异显著,从高到低依次为3,4,1,2;抗蚜虫试验结果显示:转基因烟草具有一定抗蚜性,但不同烟草株系的抗蚜能力有所不同;活体转基因烟草单株与离体烟草叶片的平均蚜虫密度抑制率分别为8.22%~76.61%,4.62%~65.65%;转基因大花烟草可加速蚜虫的死亡。对比NTL1在不同转基因大花烟草株系中的相对表达量及抗蚜效果,发现NTL1在不同株系中的相对表达量与植株抗蚜性具有高度一致性。     

转抗虫基因三系优良保持系的获得

以质粒pBUSCK-HPT作抗虫基因供体,优良籼型保持系D62B为受体,采用基因枪转化法,获得了转抗虫基因sck的植株。将转基因保持系与不育系D62A回交,获得转抗虫基因不育系。分子证据表明,外源基因能稳定转移到不育系中。蛋白活性测定显示,转基因在不育系中得到表达。本文还讨论了转基因技术在水稻育种中的作用。     

转甜菜碱醛脱氢酶(BADH)基因小麦的耐盐耐旱性

采用室内模拟盐、旱胁迫，结合田间实际测定的方法，对基因枪法获得的转BADH基因小麦多个株系的不同世代材料进行了耐盐、耐旱性鉴定。结果表明，在旱、盐胁迫条件下，转BADH基因小麦在种子萌发、幼苗生长、根系发育以及质膜保护等方面均比受体品种（对照）具有明显的优势；在田间生长条件下的转基因株系，其叶片蒸腾强度比对照明显降低，而离体叶片失水速率与对照没有明显差别。     

抗根腐病的转GmPGIP3基因小麦扬麦18的获得与鉴定

GmPGIP3是大豆的一种多聚半乳糖醛酸酶抑制蛋白, 能够特异性地抑制部分病原真菌内切多聚半乳糖醛酸活性, 从而减弱病原菌对植株的侵害。利用基因重组技术构建了GmPGIP3基因的单子叶植物表达载体pA25-GmPGIP3, 通过基因枪介导法将pA25-GmPGIP3转入小麦品种扬麦18中。对转GmPGIP3基因扬麦18的T₀至T₂代植株进行PCR、Southern杂交、半定量RT-PCR和荧光定量Q-RT-PCR分析, 并对根腐病进行抗性鉴定。结果表明, GmPGIP3已转入扬麦18, 并在转基因小麦中遗传、转录和表达;比受体材料相比, 5个GmPGIP3过表达的转基因小麦株系对根腐病的抗性有明显提高。     

36份小麦种质资源抗麦长管蚜遗传多样性的鉴定与分析

为筛选抗麦长管蚜优异的小麦种质资源,以36份不同抗蚜水平的小麦种质资源为材料,调查记录不同时期供试材料.的蚜虫数量,并利用3对位于7D染色体的特异性简单重复序列(Simple Sequence Repeats,SSR)标记检测材料,采用蚜量比值、Shannon's信息指数及聚类分析的方法进行分析,在分子水平上探讨小麦抗麦长管蚜种质资源的遗传多样性.分子检测结果表明,引物Xgwm44、Xwmc182和Xwmc121扩增条带数平均为6.7条,其中多态性条带数分别为5条、5条和2条;平均位点的有效等位基因数范围为0.119 5～1.080 0,Shannon's信息指数范围0.000 0～1.329 7.聚类分析结果表明,36份小麦种质资源按照阚值λ=2.06可分为4大类群,其中发现抗性品种矮抗58与其它品种具有较远的亲缘关系,可作为最新的抗蚜材料.     

转AcAMP-sn基因抗全蚀病小麦新种质的创制与鉴定

抗菌肽是一类具有广谱抗菌性的小分子多肽,在植物防卫反应中起着重要作用。本研究人工合成了洋葱抗菌肽基因AcAMP-sn,利用基因重组技术构建了该基因的单子叶植物表达载体pAHC25::AcAMP-sn,使AcAMP-sn基因的表达受玉米Ubiqutin启动子控制。采用基因枪介导法,将AcAMP-sn基因导入小麦品种扬麦18,利用PCR、半定量RT-PCR及荧光实时定量RT-PCR检测、分析转基因小麦T0~T4代目的基因及其表达量,并对转基因植株进行全蚀病的抗性鉴定。PCR检测结果表明,导入的AcAMP-sn基因能够在转基因小麦中遗传。与受体扬麦18相比,在5个转基因小麦T4代株系中,AcAMP-sn基因的表达量及全蚀病抗性均显著提高,且全蚀病菌的相对含量明显下降,说明AcAMP-sn基因的过表达可以增强转基因小麦对全蚀病的抗性。     

转基因抗虫小麦中sgna基因的遗传分析及抗虫性鉴定

利用基因枪介导将抗蚜虫的sgna基因导入了长江中下游地区小麦栽培品种扬麦158，得到了6棵转基因植株。PCR和RT-PCR检测分别证实了sgna基因的整合和表达。通过分析转基因植株的T1代和T2代，发现sgna基因的遗传并不符合孟德尔的遗传规律。抗蚜虫鉴定试验证明含有sgna基因的植株对蚜虫有明显的抗性，转基因植株可以显著降低蚜虫     

A Monosomic Analysis of Russian Wheat Aphid Resistance in the Common Wheat PI 294994

Chromosome 7D of PI 294994 was indicated as carrying a single dominant gene for resistance to the Russian wheat aphid. The symbol Dn5 is proposed to designate the gene.     

Intergeneric Transfer (Rye to Wheat) of a Gene(s) for Russian Wheat Aphid Resistance

An octoploid triticale was derived from the F, of a Russian wheat aphid-resistant rye, ‘Turkey 77’, and ‘Chinese Spring’ wheat. The alloploid was crossed to common wheat, and to ‘Imperial’ rye/‘Chinese Spring’ disomic addition lines. F₂, progeny from these crosses were tested for Russian wheat aphid resistance and C-banded. A resistance gene(s) was found to be associated with chromosome arm IRS of the ‘Turkey 77’ rye genome. A monotelosomic IRS (‘Turkey 77’) addition plant was then crossed with the wheat cultivar ‘Gamtoos’, which has the 1BL.1RS ‘Veery’ translocation. Unlike the IRS segment in ‘Gamtoos’, the ‘Turkey 77’-derived 1 RS telosome did not express the rust resistance genes Sr31 and Ar26, which could then be used as markers. From the F, a monotelosomic 1 RS addition plant that was also heterozygous for the 1BL. 1 RS translocation was selected and testerossed with an aphid-susceptible common wheat, ‘Inia 66’ Meiotic pairing between the rye arms resulted in the recovery of five euploid Russian-wheat-aphid-resistant plants. One recombinant also retained Sr31 and Lr26 and was selfed to produce translocation homozygotes.     

中国水仙八氢番茄红素脱氢酶基因(PDS)的克隆及表达分析

八氢番茄红素脱氢酶（PDS）是影响类胡萝卜素合成的一个重要限速酶,在植物花色发育过程中起着重要作用。采用RT-PCR技术从中国水仙（Narcissustazetta var.chinensis）幼嫩花蕾中分离到一个新的水仙花色发育相关的PDS基因,命名为NTPDS1（GenBank登记号：EU138883）。该基因长1719bp,具有一个1713bp的完整开放读码框（ORF）,编码570个氨基酸。序列分析表明,NTPDS1编码的氨基酸序列与其它植物的PDS蛋白有很高的相似性。系统进化树分析显示,NTPDS1与喇叭水仙亲缘关系最近。利用半定量PCR技术进行组织表达模式分析发现,该基因在中国水仙的花、叶片和根中均有表达,但以花器官中表达量最高;在花瓣和副冠中的表达量高于雄蕊和雌蕊。     

小麦Mlo反义基因的转化及转基因植株的白粉病抗性分析

采用基因枪法将小麦反义Mlo基因导入扬麦158和济麦20的幼胚愈伤组织中,在含除草剂的分化培养基上经两轮筛选,获得抗性再生植株。PCR检测、PCR-Southern杂交、基因组DNA斑点杂交和除草剂BASTA抗性分析结果证实已获得转基因扬麦158和济麦20阳性植株,荧光定量表达分析亦证明Mlo基因发生沉默。对T₀和T₁代转基因植株的白粉病抗性鉴定表明,有6个转基因株系高抗白粉病。对T₁代转基因小麦接种白粉菌后孢子发育的显微观察结果显示,Mlo反义基因的导入明显加快了乳突的形成和维持时间,有效抑制了吸器的发育,因而使转Mlo反义基因材料表现抗病性。     

小麦抗病基因同源序列(RGAs)的克隆与分析

RGA(抗性基因同源序列)法是克隆植物抗性基因的一种经济有效的方法,成为近年来的研究热点。本实验综合分析了拟南芥,西红柿,水稻,烟草等植物已克隆的抗性基因,并以这些抗性基因的NBS(核酸结合位点),LRR(富含亮氨酸重复),STK(丝氨酸/苏氨酸激酶)保守结构域设计并合成了几十对RGA引物,对小麦抗条锈病材料进行PCR扩增,获得以Xal-NBS为引物的R88RGA片段,经克隆和序列比对分析,发现该片段与逆境条件下植物抗病信号传导相关,与蛋白激酶同源性达到96%。此项研究对抗病机理的研究和基因的发掘有重要的指导意义。     

小麦抗条锈病基因YrZH84的RGAP标记及其应用

利用RGAP标记及基于标记基因型和表型选择的分离群体分组分析法,对小麦抗条锈病骨干亲本周8425B和感病品种中国春及其F₂分离群体进行分析,获得了与抗条锈基因YrZH84紧密连锁的RGAP标记Xrga-1,连锁距离为0.8 cM。其RGA片段长度为343 bp,BLAST分析结果表明,该RGA序列与已克隆的大麦抗秆锈病基因Rpg1核苷酸序列同源性为93%,与大麦抗白粉病基因Mla家族的核苷酸序列同源性为92%。用标记Xrga-1对黄淮麦区58个小麦品种(系)进行了检测,结合系谱分析和抗病性鉴定结果,发现周8425B的衍生品种周麦11、周麦17、周麦20、周麦22、矮抗58、04中36、源育3号、05中37、宛抗18、豫展10号携带抗条锈病基因YrZH84。这些研究结果对小麦抗条锈病分子育种和YrZH84基因克隆有重要作用。     

The Transfer of Resistance to the Russian Wheat Aphid from Triticum monococcum L. to Common Wheat

Two Triticum monococcum accessions were found to be highly resistant to the Russian wheat aphid. An attempt was made to transfer the resistance to common wheat through direct hybridization or by using bridge species. In each of the four crossing strategies tried, a gradual loss of resistance occurred as hybrids with higher ploidy levels were obtained. It appeared that the level of resistance observed was directly proportional to the ratio of the Triticum monococcum genome relative to other genomes. This would indicate suppression of the resistance gene(s) by the added genomes or dilution of its product(s) by those of homoeoloci. The degree of protection afforded by the gene(s) at the hexaploid level may prove to be small. Plants suspected to be homozygous for the resistance gene(s) were identified; however, further backcrossing to common wheat will be required to improve their agronomic types and meiotic regularities.     

Inheritance and allelism for resistance to Russian Wheat Aphid in an Iranian Spring Wheat

Russian wheat aphid (RWA), Diuraphis noxia (Mordvilko), is an important pest of wheat (Triticum aestivum L.) in the United States of America. Developing adapted wheat cultivars with genetic resistance to RWA is an effective control strategy. Genetic studies were conducted to determine the mode of inheritance of gene(s) conferring resistance to RWA in an Iranian landrace wheat line, G 5864. For the inheritance study, G 5864 was crossed with the susceptible wheats ‘Yecora Rojo’ and ND 2375. Seedlings of F1, reciprocal F1, F2, BC1 to the susceptible parent (BCS), and BC1 to the resistant parent (BCR) were screened for RWA reaction. Several phenotypic segregation ratios were tested in the F2 populations for goodness of fit; the 9:3:3:1 ratio (resistant: rolled leaves: stunted plants: susceptible) was an acceptable fit in all cases. Thus, resistance in G 5864 seemed to be controlled by two independent dominant genes with additive gene effects. The allelic relationships of gene(s) in this line with genes in other resistant lines, PI 137739 (Dn1), PI 262660 (Dn2), PI 372129 (Dn4), PI 294994 (Dn5), and PI 243781 (Dn6), were also studied. Segregation patterns observed in G 5864 × resistant (R × R) F2 populations were inconclusive. However, no susceptible plants were observed in these F2 populations. If previous reports concerning the number of resistance genes present in the other resistant lines are correct, then given the high manifestation of resistance observed in G 5864, and given the absence of susceptible plants in the R × R F2 populations, it is indicated that RWA resistance in G 5864 is either controlled by different alleles at the same loci as the other resistance genes, or that G 5864 shares a resistance gene with each of the other resistant lines. This revised version was published online in July 2006 with corrections to the Cover Date.