DREB1B基因在转基因小麦后代的稳定表达

本研究对基因枪导入了pBAC128F/R质粒(含有玉米Adh1内含子1的CaMV35S启动子驱动的bar基因作为选择标记,以来自拟南芥菜逆境诱导表达类型--亲水蛋白rd29b基因的启动子驱动脱水应答转录因子DREBIB基因的逆境诱导表达类型质粒)的T4代转基因小麦进行了稳定表达研究.PCR、PCR-Southem和Southern杂交分析表明,外源转录因子DREBlB基因已稳定整合到转基因株系的基因组中.半定量RT-PCR结果表明,部分转基因株系的DREB1B基因的相对表达量有所增强.叶片除草剂抗性检测结果显示有8个转基因株系可抗到150 mg/L.叶片脯氨酸含量测定结果表明,有4个株系(编号为1,18,30和76)的脯氨酸含量提高幅度较大,同一株系,干旱与未干旱处理相比,脯氨酸含量提高了3～5倍.干旱处理后的转基因植株与非转基因植株相比,脯氨酸含量高2～3倍.在干旱条件下,T4代田间小区产量统计数据分析结果表明,有4个转基因株系(编号为30,51,70和76)的产量与非转基因植株相比有显著增加.研究表明,利用逆境诱导型rd29b基因的启动子来增强外源DREB1B基因的表达,能显著改良小麦的抗旱性.     

SDS-PAGE和PCR检测1B/1R易位系研究初报

采用18个冬小麦品种作为供试材料,分别通过SDS-PAGE检测黑麦Secalin蛋白和PCR检测黑麦1RS染色体或其片段,结果表明:SDS-PAGE检测不出不带有secalin蛋白的1B/1R小片段易位系,而PCR则能检测出,从而提高1B/1R易位系的检出率;SDS-PAGE和PCR结合是检测1B/1R易位系的一种快速经济的有效方法。     

过量表达拟南芥NPR1基因提高小麦纹枯病的抗性

拟南芥NPR1基因是植物重要的抗病调控因子,转基因过表达该基因可以赋予植物广谱抗性.为明确该基因在小麦抗纹枯病基因工程中的应用潜力,本研究构建了由玉米ubiquitin启动子驱动的拟南芥NPR1表达载体,采用基因枪介导法导入到小麦品种扬麦12号中,获得20株转基因植株.对14个外源基因纯合株系的半定量RT-PCR和测序分析结果显示,外源拟南芥NPR1基因已经导入转基因小麦并有不同水平的表达,部分转基因株系拟南芥NPR1基因发生重排；纹枯病抗性鉴定结果表明,转基因株系中拟南芥NPR1基因的正确表达可以减轻纹枯病菌引起的病症发展,提高转基因小麦的纹枯病抗性.     

对转蚕丝芯蛋白轻链基因棉花的分析

利用根癌农杆菌介导转化法,将棉纤维特异表达启动子GAE6-3A驱动的蚕丝芯蛋白轻链基因（FBN）转入陆地棉R15中。T₀代转基因再生株FNB基因PCR检测阳性率达70%。随机选取4个转基因株系T₁代材料的Southern杂交显示,3个为双拷贝插入、1个为单拷贝插入;Northern分析结果证实FBN基因在转基因棉纤维中表达。转基因后代的纯合选育主要以田间卡那霉素检测结合实验室内GUS组织化学检测进行,其中4个株系已获得了T₃代转基因材料,其他若干个转化子也获得T₁代和T₂代的不同材料。对6个转基因棉花株系后代纤维检测结果表明,蚕丝芯蛋白轻链基因对棉花纤维品质的影响主要体现于对纤维强度的改良。H18、H32、H34株系转基因棉花后代纤维强度较对照显著提高,其中H18纤维强度提高12.3%。     

过表达TaPK-R1基因增强了小麦对纹枯病的抗性和耐冻性

小麦纹枯病菌(Rhizoctonia cerealis)和倒春寒是小麦生产中重要的生物和非生物胁迫因子,本研究目的是利用转基因技术改良小麦的纹枯病抗性和耐冷性。利用构建的转基因载体pAHC25-MYC-TaPK-R1,通过基因枪介导法,将小麦AGC蛋白激酶基因TaPK-R1导入春小麦品种扬麦20,获得TaPK-R1过表达的转基因小麦。对T1至T4代植株进行PCR、RT-PCR、qRT-PCR、Western blot分析,筛选到3个转基因小麦株系,外源TaPK-R1基因能够在其植物体内遗传和超量转录,并翻译成蛋白质。利用致病力不同的R. cerealis菌株R0301和WK207,分别对3个转基因株系T1至T2代和T3至T4代进行纹枯病抗性鉴定,发现TaPK-R1过量表达提高了转基因小麦的纹枯病抗性,3个转基因株系各世代的纹枯病平均病级为1.16~2.11, 病情指数为23.20~42.10,而对照扬麦20的纹枯病病级为2.55~3.60,病情指数为51.00~72.00。用?9℃低温处理三叶一心期小麦幼苗24 h,对照扬麦20的存活率为17%,3个转基因小麦株系的存活率分别为52%、79%和96%。上述结果表明,TaPK-R1过量表达能显著增强小麦对纹枯病的抗性及对冻害的耐受性, 所获得的3个转基因小麦株系可作为潜在的抗源材料。     

利用1RS特异标记和染色体原位杂交技术鉴定小麦1BL·1RS易位系

以小麦-黑麦1BL·1RS易位系(Kavkaz、山农030-1)、1AL·1RS易位系(Amigo)、荆州黑麦、八倍体小黑麦劲松49、1R-7R二体异附加系以及普通小麦中国春、辉县红、铭贤169、Chancellor等为材料,对65个黑麦1RS特异标记进行鉴定,从中筛选出8个稳定的标记,即NOR-1、SECA2/SECA3、SCSS30.2、Sec1Gene、Sec1Pro、ω-Sec-P1/P2、ω-Sec-P3/P4和IB-267,可用于检测1AL·1RS易位系或1BL·1RS易位系;另外3个特异标记O-SEC5′-A/O-SEC3′-R、IAG95-1和SCM-9可用于区别1RS来源不同的1AL·1RS和1BL·1RS易位系。利用这11个标记和染色体原位杂交技术对40份山东省近年育成小麦品种(系)进行检测,发现潍麦8号、鲁麦14、济宁13、山农664、山农优麦3号和烟农25为1BL·1RS易位系,而且是1RS的整臂易位系,未检测到1AL·1RS易位系和其他易位类型。     

小麦-黑麦远缘杂交后代高分子麦谷蛋白亚基变异分析

采用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE) 方法, 分析了小麦-黑麦远缘杂交后代的66个株系的高分子量谷蛋白亚基(HMW-GS) 组成.结果表明:(1)在29个母本为MY 11的株系中,有6个株系的Glu-1等位基因相对于母本MY 11发生了变异,1 B/1 R易位系中Glu-1位点发生变异的频率为66.67%,非1 B/1 R易位系中Glu-1位点发生变异的频率为8.69%.在36个母本为A 42912株系中,3个株系的Glu-1等位基因发生变异且这3个株系皆为1 B/1 R易位系,1 B/1 R易位系中Glu-1位点发生变异的频率为60.0%,非1 B/1 R易位系Glu-1位点没有变异;(2)对于母本为MY 11的株系,Glu-1三个位点上等位基因的变异均只有2种,即Glu-A 1 a(86.20%)、Glu-A 1 c(13.79%)、Glu-B 1 b(89.65%)、Glu-B 1 c(10.34%)、Glu-D 1 a(3.45%)、Glu-D 1 d(96.55%).对于母本为A 42912的株系,仅在Glu-B 1位点发生变异,即Glu-B 1 b(91.66%)、Glu-B 1 c(8.33%);(3)供试株系共出现6种高分子谷蛋白亚基组合,即(1,7 8,5 10)、(Null,7 8,5 10)、(Null,7 9,5 10)、(Null,7 8,2 12)、(1,7 8,5 10)、(1,7 9,5 10),两种母本不同的株系类型均是母本型HMW-GS组合(1,7 8,5 10)占绝对优势.本文分析了1 B/1 R易位株系具有高频率HMW-GS变异的原因,并就这些材料在小麦优质育种中的利用策略作了探讨.     

利用CRISPR/CAS9基因编辑技术创制香型郑稻19新种质

有特殊香味的稻米深受我国消费者欢迎,是优质水稻的重要指标之一,本研究通过基因编辑创制香型优质水稻新种质。水稻中甜菜碱醛脱氢酶基因Badh2是控制香味的关键基因,以适于直播品种郑稻19为供体材料,利用CRISPR/CAS9技术定点突变水稻Badh2基因。获得T0代转基因阳性植株11株,其中9株子粒有香味,检测6个香型T1株系靶点序列,6个株系均发生突变,发现7种突变类型,5种是缺失类型,2种插入类型,其中3个株系（T1-2、T1-3和T1-7）出现双等位突变。6个T1代株系共测序22个单株,检测到纯合突变10株;T2代检测28株,其中纯合突变21株。以潮霉素抗性基因引物检测T1-2、T1-6和T1-7株系后代单株载体脱落情况,T1代3个株系未获得无载体纯合突变单株,T2代中获得8株无载体纯合突变单株,来源于T1-2株系有3株,来源于T1-7株系5株。T0代9株香型单株子粒2-AP含量1.259±0.072μg/g,T1代8个突变株系2-AP含量0.537±0.111μg/g,均显著高于对照郑稻19。考察中选T1-2、T1-7株系的农艺性状,与郑稻19无显著差异。这些无载体突变单株可作为香型种质在种质创新和育种中应用。     

转大豆Na~+/H~+逆向转运蛋白GmNHX1基因植株的获得

制约大豆生产的因素很多,土壤盐渍化就是其中之一。大量研究证明过表达NHX逆向转运蛋白可以提高植物的耐盐性。为获得耐盐性良好的转基因大豆材料,我们将大豆Na~+/H~+逆向转运蛋白(GmNHX1)基因构建到植物表达载体pCAMBIA3300上,应用农杆菌介导法将GmNHX1导入大豆品种黑农56和黑农59中,共获得18个转基因株系,并对T_1代转基因株系进行了PCR和实时荧光定量PCR检测。PCR结果表明转基因后代株系呈阳性的植株有4株;经Real-time PCR检测该4个PCR阳性转基因株系的G NHX1基因表达水平均高于对照株系;200 mmol/L NaCl溶液的盐试结果表明;对照植株的生长速度滞缓于4个转基因株系的生长速度。     

川麦42的1BS染色体臂对小麦主要农艺性状的遗传效应

川麦42的1BS染色体臂来源于人工合成小麦亲本Syn769。利用川麦42与含1BL/1RS易位系的四川小麦品种川农16构建的127个重组自交系(RIL, F₈),经3年4个环境的遗传评价,比较了川麦42的1BS和川农16的1RS染色体臂对小麦产量构成因子和产量的遗传效应。结果表明,RIL群体中川麦42的1BS染色体臂株系和川农16的1RS染色体臂株系在分蘖力、成穗率、全生育期、小穗数、收获指数和籽粒产量6个性状上存在显著差异; 1BS染色体臂有利于提高成穗率和收获指数,而1RS染色体臂有利于提高分蘖能力和增加小穗数,1BS株系的籽粒平均产量比1RS株系增加2.91%。鉴于1RS染色体臂上的抗条锈病基因丧失抗性,其携带的黑麦碱基因对加工品质有明显的负向作用,而川麦42的1BS染色体臂携带高抗条锈病基因YrCH42, 并对小麦籽粒产量有正向作用,因此建议在小麦遗传改良中利用川麦42的1BS替换1RS染色体臂。     

The Effect of Chromosome 1B/1R Translocation on the Yield Potential of Certain Spring Wheats (Triticum aestivum L.)

Nearly 50 percent of the 1988 advanced breeding lines of the CIMMYT bread wheat breeding program possess the 1B/1R homozygous translocation. Hence, a trial was conducted to estimate the effect of 1B/1R chromosome translocation on the yield potential of some of our high-yielding spring wheats, where non-limiting levels of fertility, moisture, preventive pest and disease programs were used. In conclusing the 1B/1R lines seemed to have increased their above-ground biomass yield, number of spikes per meter², 1000-grain weight and test weight. They also exhibited a slight advantage over the 1B homozygous lines on grain yield. The observed difference, however, was non-significant, as was the plant height difference observed among the two groups. Varietal comparisons indicated that the 1B/1R group headed later than the 1B group.     

SDS-PAGE of the high-molecular-weight subunits of wheat glutenin and the characterization of 1R (1B) substitution and 1BL/1RS translocation lines

Summary The high-molecular-weight subunits of glutenin from wheat 1R(1B) substitution and 1BL/1RS translocation lines were fractionated by SDS-PAGE. Two new subunits denoted R₁ and R₂ were characterized in 1R(1B) substitution, but not in 1BL/1RS translocation lines. R₁ and R₂ were proved to be rye proteins by 2d electrophoresis (NEPHGE x SDS-PAGE).In contrast to literature citations it was demonstrated that the cultivar Winnetou is a 1R(1B) substitution line and the cultivars Clement and Mildress both are 1BL/1RS translocation lines.     

The 1BL/1RS chromosome translocation effect on yield characteristics in a Triticum aestivum L. cross

Comparisons involving 28 random F₂-derived F₆ wheat (Triticum aestivum L.) lines from the cross, ‘Nacozari’/‘Seri 82’, suggested that advanced derivatives with the 1BL/1RS chromosome translocation possess superior agronomic performance in both full and reduced irrigation conditions when compared with 1B derivatives. This performance advantage was attributed to high grain yield, above-ground biomass at maturity, grains/spike, 1000-grain weight and test weight. The 1BL/1RS lines were shorter with delayed flowering and maturity. The superiority of the 1BL/1RS translocation group on grains/m² was expressed only under the full irrigation environment. Higher harvest index, longer spike-length and grain-filling period were detected only under reduced irrigation conditions. A significant grain yield relationship with test weight was detected only among the 1BL/1RS genotypes, indicating that they possess heavier and plumper grains than the 1B genotypes.     

The effect of chromosome 1AL/1RS translocation on agronomic performance of 85 F2-derived F6 lines from three Triticum aestivum L. crosses

Summary The effect of the 1AL/1RS chromosome translocation on grain yield and other agronomic characteristics of 85 random F₂-derived F₆ bulks from three 1AL 1RS × 1A bread wheat crosses was determined under optimum and reduced irrigation conditions at CIANO, Yaqui Valley, Sonora, Mexico, during the 1991–1992 and 1992–1993 crop production cycles. Harvest plots of 5.0 m² were arranged in an alpha lattice design with three replications. The 1AL/1RS translocation increased grain yield, above-ground biomass, spikes/m², and test weight under both irrigated and dryland conditions. Homozygous chromosome 1A lines, on the other hand, possessed longer spikes with more grains. The 1AL/1RS cultivars had an advantage in 1000-grain weight, which was detected only under optimum irrigation. The translocation lines showed later maturity and longer grainfilling period than the 1A genotypes under one irrigation treatment. A significant relationship between grain yield and test weight was detected only among the 1AL/1RS genotypes, indicating that they possess heavier and plumper grains than the 1A genotypes. These results encourage the continued use of the 1AL/1RS translocation in wheat improvement.     

小麦品质性状分子标记多重PCR体系的建立

小麦高分子量谷蛋白亚基组成、1B/1R易位、籽粒硬度、直链淀粉含量和穗发芽抗性等性状与加工品质密切相关，建立相关性状的多重PCR体系在小麦品质分子育种中具有重要意义。本研究利用现有品质性状基因的分子标记，建立了适合不同品质类型品种评价和分子聚合育种的3个多重PCR体系，并用已知基因的品种（系）进行验证。多重PCR体系Ⅰ包括ω-secalin（1B/1R）、Vp1B3和Pinb-D1b基因的分子标记检测，可用于一般的品质检测；体系Ⅱ包括ω-secalin、Ax2*、Bx17和Dx5 基因的检测，可望用于强筋小麦品种的选育；体系Ⅲ包括Wx-A1、Wx-B1和Wx-D1位点的检测，可用于淀粉品质或糯小麦的选育。每个体系内的引物之间不存在相互抑制作用和错配，检测品种（系）的结果可靠、重复性好，成本低。3个多重PCR体系用于小麦品质育种的亲本评价和杂交后代优质基因的聚合，将会提高优质专用小麦品种评价和选育的效率。     

Genetic mapping of sequence-specific PCR-based markers on the short arm of the 1BL.1RS wheat-rye translocation

Wheat cultivars carrying the 1BL.1RStranslocation were crossed with newly synthesised octoploid triticale lines involving four rye genotypes having ο-secalin banding patterns different from each other and from that of the 1BL.1RS translocation. Homologous recombination was expected between the short arm of the 1R chromosomes of the rye genotypes and the 1RS arm of the 1BL.1RSwheat/rye translocation. Seven sequence-specific PCR-based markers:Xiag95, RMS13, Bmac0213, GPI, Xpsr960, 5Sand SCM9, and ο-secalinproteins were used to detect recombination events in the BC₁F₂ generation. Segregation analysis demonstrated that a barley SSR marker (Bmac0213) locus was present on the 1RS chromosome arm. Of 834plants tested in four different BC₁F₂ populations, 246individuals were found to carry recombined1BL.1RS translocation chromosomes. Genetic linkage analysis was performed on the eight markers in the four different mapping populations. The physical positions of the markers are discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

转Rs-AFP2基因小麦的分子分析及其纹枯病抗性

Rs-AFP2属于r-硫堇类抗菌肽,主要通过形成离子通道直接破坏细胞来杀灭病原菌。本研究通过基因枪介导法结合对目标基因的分子检测,证明已将外源Rs-AFP2基因转入小麦推广品种扬麦12中。通过逐株抗纹枯病接种鉴定、PCR、PCR-Southern blot、Southern blot和 RT-PCR/荧光定量RT-PCR(Q-RT-PCR)分析,对转Rs-AFP2基因小麦T₁至T₄代植株跟踪检测。结果表明,Rs-AFP2在转基因小麦中能够稳定遗传,以单拷贝整合到小麦基因组中,遗传方式符合孟德尔遗传规律,并能在转录水平上表达。对转Rs-AFP2基因小麦的抗病性、主要农艺性状以及Rs-AFP2表达活性分析结果表明,与受体扬麦12相比,Rs-AFP2表达活性高的转基因小麦植株对纹枯病抗性有明显提高,其抗病性可以遗传,而主要农艺性状没有明显差异,证明可以利用Rs-AFP2基因和基因工程途径创制抗纹枯病小麦新种质。     

Inheritance and field performance of transgenic Korean Bt rice lines resistant to rice yellow stem borer

Transgenic Korean rice plants containing the cry1Ab gene were developed for resistance against yellow stem borer (Scirpophaga incertulas, YSB). More than 100 independent transgenic lines from three Korean varieties (P-I, P-II and P-III) were generated. The amount of Cry1Ab in transgenic T₀ plants was as high as 2.88% of total soluble proteins. These levels were sufficient to cause 100% mortality of YSB larvae. The majority of T₁ transgenic lines originated from the varieties P-I and P-II followed a Mendelian fashion of segregation. Deviation from the expected segregation ratio was observed in a small number of the transgenic lines of P-I and P-II origins. However, this deviation was primarily observed in the P-III originated lines. Segregation analysis of the T₁ generation indicated that 1–3 copies of the cry1Ab gene were integrated into the genome of the majority of the transgenic lines originating from varieties P-I and P-II. Stunted and semi-fertile mutants were observed in some transgenic lines. These aberrations were either independent or closely linked to the introduced cry1Ab gene loci in different transgenic lines. Reduction in GUS expression levels and loss of toxicity against YSB larvae were found in some transgenic lines. The transgenic T₃ and T₄ lines causing 100% mortality of third instar YSB larvae in the lab were completely protected in the field. Analysis of important yield components on nine selected transgenic lines indicated that stem length, panicle length, grain number per panicle, and seed setting rates were reduced in transgenic plants compared to those in non-transgenic parental rice lines. Number of panicles per cluster, however, was significantly higher in transgenic plants. The numerical value of the average yield was in general greater in the controls than in all the transgenic lines, indicating some ‘yield drag’. Since some selected lines were highly resistant to the YSB with good yielding potential, they offer effective potential for use in insect resistance management programs.     

反义trxs基因导入对不同品质类型小麦产量和品质性状的影响

以转反义trxs基因小麦TY18和TY34及其相应未转基因对照为材料,于2007-2009年通过大田试验系统研究了反义trxs基因导入对两种品质类型小麦籽粒产量性状和加工品质指标的影响。结果表明,4个转基因株系的穗粒数和产量较对照显著提高,反义trxs基因对小麦籽粒淀粉品质有一定的正效应。4个转基因株系的淀粉含量、支链淀粉含量、峰值黏度、低谷黏度、最终黏度均显著提高,直/支比降低。反义trxs基因对小麦籽粒蛋白质品质的影响因品种类型而异,其中弱筋小麦的总蛋白、清蛋白、球蛋白、谷蛋白含量显著减少,醇溶蛋白含量显著增加,面粉的形成时间和稳定时间降低。强筋小麦除清蛋白、醇溶蛋白、谷蛋白含量减少外,面团的粉质和拉伸参数变化不明显。上述结果说明,反义trxs基因导入有利于两种品质类型小麦籽粒淀粉品质的改善,并在一定程度上改善了弱筋小麦的烘焙品质。     

Genetic, agronomic and quality comparisons of two 1AL.1RS. wheat-rye chromosomal translocations

The 1AL.1RS wheat-rye chromosomal translocation originally found in ‘Amigo’ wheat possesses resistance genes for stem rust, powdery mildew and greenbug biotypes B and C, but also has a negative effect on wheat processing quality. Recently, a second 1AL.1RS translocation carrying Gb6, a gene conferring resistance to greenbug biotypes B, C, E, G and I, was identified in the wheat germplasm line ‘GRS1201′. Protein analytical methods, and the DNA polymerase chain reaction were used to identify markers capable of differentiating the 1RS chromosome arms derived from ‘Amigo’ and ‘GRS1201′. The secalin proteins encoded by genes on 1RS chromosome arms differed in ‘Amigo’ and ‘GRS1201′. A 70 kDa secalin was found in the ‘Amigo’1AL.1RS, but did not occur in the ‘GRS1201’1AL.1RS. Polymorphisms detected by PCR primers derived from a family of moderately repetitive rye DNA sequences also differentiated the two translocations. When ‘GRS1201’was mated with a non-1RS wheat, no recombinants between 1RS markers were observed. In crosses between 1RS and non-1RS parents, both DNA markers and secalins would be useful as selectable markers for 1RS-derived greenbug resistance. Recombination between 1RS markers did occur when 1RS from ‘Amigo’ and 1RS from ‘GRS1201’were combined, but in such intermatings, the molecular markers described herein could still be used to develop a population enriched in lines carrying Gb6. No differences in grain yield or grain and flour quality characteristics were observed when lines carrying 1RS from ‘Amigo’ were compared with lines with 1RS from ‘GRS1201′. Hence, differences in secalin composition did not result in differential quality effects. When compared with sister lines with 1AL.1AS derived from the wheat cultivar ‘Redland’, lines with ‘GRS1201’had equal grain yield, but produced flours with significantly shorter mix times, weaker doughs, and lower sodium dodecyl sulphate sedimentation volumes.