玉米自交系响应花粒期高温胁迫差异表达基因的分析

为了探明玉米重要自交系花粒期高温胁迫下转录组基因的表达差异,发掘玉米响应高温胁迫的关键基因和蛋白质,明确高温胁迫引起玉米减产的机理,从而利用分子生物学技术手段提高玉米耐高温胁迫性。首先,利用Ampha~(TM)Z30花粉活性分析仪检测高温胁迫和正常生长条件下玉米自交系昌7-2、郑58的花粉活性。然后收集花粉提取总RNA,利用Illumina Hiseq2000~(TM)高通量测序技术分别对昌7-2、郑58花粒期高温胁迫材料和正常生长条件下的对照材料进行全转录组测序,序列结果分析后获得差异显著表达基因。通过差异基因维恩图(VENN图)重叠区域分析、GO功能分类和富集统计、KEGG pathway通路分类和富集分析以及转录因子分析,获得玉米花粒期高温胁迫相关的重要差异表达基因和蛋白质。花粉活性检测结果显示,高温胁迫后和正常生长条件下,昌7-2花粉活性分别为24. 37%,42. 89%,郑58花粉活性分别为35. 57%,64. 83%。高温胁迫后在昌7-2花粉转录组中共检测到4 176个显著差异表达基因,郑58花粉转录组中共检测到5 487个显著差异表达基因。KEGG pathway通路分类和富集分析结果显示,高温胁迫后郑58的花粉转录组中有2 062个差异表达基因富集到399个相关通路上,昌7-2的花粉转录组中有1 943个差异表达基因富集到352个相关通路上。转录因子分析结果表明,共获得55个转录因子家族,其中,有45个转录因子包含10个以上差异表达基因。研究表明,通过对玉米自交系花粒期高温胁迫后花粉转录组测序获得了大量的差异显著表达基因信息,昌7-2、郑58对高温胁迫响应机制存在明显差异,热激蛋白(Hsps)、热激转录因子(Hsfs)、生长素(IAA)基因和磷脂酰基醇-4,5-二磷酸基因家族(PIP2)在响应玉米花粒期高温胁迫过程中起着重要作用。     

病毒复制酶基因介导玉米抗矮花叶病的研究

本研究将病毒复制酶基因(NIa、NIb)通过花粉介导法,转入玉米优良自交系中.对所转化的玉米后代进行了PCR检测,并对检测呈阳性的植株于次年播种于试验田,对转化的植株连续2代(T1和T2代)利用人工摩擦接种矮花叶病毒的方法进行田间抗病试验.将连续2代抗病的植株通过PCR以及PCR-Southern Blot检测结果证明:病毒复制酶基因已成功导入玉米自交系中,结合田间抗性鉴定,获得了对玉米矮花叶病抗病性增强且农艺性状良好的玉米株系.     

转基因抗丝黑穗病玉米的遗传、表达及选育研究

本研究以几丁质酶基因导入玉米自交系海92-1所获得的转基因植株的种子作为试验材料,对其To代种子进行了潮霉素抗性筛选试验,结果表明T1代的分离比分别为3:1和15:1(表1).这证实大多数转基因植株中几丁质酶基因是以单拷贝、单位点整合到植株的基因组中,个别转基因植株中是以单拷贝、多位点整合到转基因植株的基因组中的.农艺性状调查分析,转基因株系的株高和穗位高都高于对照植株,单株结穗数基本不变,而转基因植株的穗长值和穗粒数值均比对照植株大,穗长增加4～7 cm,穗粒数增加1%～7%.对T1,T2,T3代株系的PCR检测结果说明目的基因在转基因植株中可稳定遗传.田间抗病鉴定结果显示,转基因株系的发病率比当代对照株系明显减少2～4级,选育得到的纯合系06006和06012的发病率为0,表现出高抗病性.     

11个玉米自交系穗部性状的配合力分析

选用国内11个玉米自交系,采用GriffingⅣ双列杂交设计组配出55个杂交组合,对穗粒重、穗长、穗粗等8个穗部性状一般配合力和特殊配合力进行分析,结果表明:穗行数、行粒数、轴粗、秃尖等性状主要由基因的加性效应起作用,穗粒重主要是受非加性基因效应的作用;郑58×5003/412、昌7-2×综31、B73×昌7-2等10个组合的单穗粒重和千粒重的特殊配合力较高;昌7-2、P138、B73、242/B84具有较高单穗粒重性状GCA效应优势;昌7-2和郑58自交系秃尖性状上一般配合力为负数;5003/412、P138、综31具有穗长和行粒数性状GCA效应优势。     

转基因水稻中抗虫基因的遗传分析

通过PCR检测及正、反交试验对转双价抗虫基因水稻材料的8个T1代株系中抗虫基因的遗传特性进行了分析,结果表明：6个株系中抗虫基因的分离符合3:1的孟德尔单基因分离规律;1个株系中阳性和阴性植株之比接近于15:1,该株系中抗虫基因可能以双拷贝插入到基因组中的两个非连锁位点;1个株系中阳性和阴性植株之比极显著偏离3:1(接近于1:1),进一步分析表明该偏分离现象可能与转基因花粉竞争能力下降有关。     

电子束辐照玉米自交系M_3的变异分析

为探讨电子束辐照对玉米自交系诱变效果,本研究分别利用剂量为200、300、400 Gy电子束对2份玉米自交系‘18-599’、‘K169’种子进行了辐照诱变,分别获得诱变株系(M_3)32份、21份,并对诱变株系和对照株系当代主要农艺性状进行了调查与分析。结果表明:2份玉米自交系诱变M_3株系株高与对照存在显著性差异;而穗位高与对照有差异显著的株系较少;主要雌穗性状诱变集中体现在穗长和穗行数,‘18-599’M_3株系的穗长和穗行数与对照存在显著性差异;主要雄穗性状诱变效果因材料不同有差异;电子束辐照对油份和淀粉含量的改变效果较好,而2份自交系诱变M_3株系与对照的蛋白质含量均值均未达到显著水平。此研究结果表明,电子束辐照应用于玉米诱变育种可以引起较丰富的遗传变异,为利用电子束开展玉米自交系诱变育种以及玉米种质改良提供了依据。     

The Study on the Transformation and Selection of Insect-resistant Cotton Harboring Double-gene

采用花粉管通道法，将人工合成的ＧＦＭＣｒｙＩＡ和修饰的ＣｐＴＩ杀虫基因导入棉花优良品种（系）中棉所２３、中棉所２７、中９４０９和中９０系。经田间选择和室内生物鉴定，已获得了转基因抗虫植株。依据生物鉴定结果，对所获得的抗虫材料进行了ＰＣＲ分析，结果证明Ｂｔ和ＣｐＴＩ基因均整合于相应的抗虫材料染色体基因组中，通过自交选育，双价抗虫基因能够传递至ＺＧＫ９７０８抗虫材料Ｒ３代。抗虫鉴定表明，双价抗虫基因能够遗传，后代抗虫性稳定；在获得的Ｒ１代植株中，不同转化体对棉铃虫的抗性存在着差异。     

转AtHDG11基因玉米的获得及抗旱性鉴定

培育抗除草剂和抗旱的转基因玉米种质,通过超声波辅助农杆菌介导花粉非组培法将拟南芥抗旱基因At HDG11导入玉米自交系昌7-2中,获得转基因植株。通过逐代除草剂筛选、分子检测对获得的转基因植株进行检测,获得5株转基因株系。在此基础上,以非转基因自交系昌7-2为对照,对5个转基因株系进行抗旱性鉴测;对苗期和十叶期玉米植株进行干旱胁迫处理,测定转基因植株生理活性物质和光合参数。结果表明,在干旱胁迫条件下,转基因玉米叶片的Pro含量高于非转基因玉米,MDA含量低于非转基因玉米;不论在正常生长条件下还是干旱条件下,转基因株系的光合速率和水分利用率都明显高于对照植株;而转基因株系的蒸腾速率和气孔导度则明显低于对照植株。综合室内与田间的抗旱检测结果表明,超声波辅助农杆菌介导花粉的非组培转基因方法在玉米上是可行的。将拟南芥HDG11基因导入玉米,同样可以有效地提高玉米的抗旱性,为玉米的抗旱育种提供新的种质资源和新的转基因方法。     

N+离子束注入玉米自交系变异的生物学效应与分子标记分析

采用不同能量和剂量的N+离子束,对郑58、昌7-2和K12玉米自交系的干种子进行处理,结果表明,发芽率、发芽势和农艺性状变异都有很大差异,筛选出的N+离子束注入玉米自交系最佳诱变能量30 KV和剂量7×1016N +/cm2.同时利用10对SSR引物对玉米自交系进行扩增,共检测到39个等位基因,每对引物可检测等位基因2...     

转水稻OsSIK1基因玉米植株的获得及抗旱性分析

类受体激酶基因Os SIK1具有通过激活抗氧化系统,增强水稻对于干旱和盐胁迫抗性的作用。为了丰富可利用的作物抗旱基因,获得具有较高抗旱水平的玉米新种质,通过超声波辅助花粉介导法,将水稻类受体激酶基因Os SIK1导入玉米自交系郑58中,并对转化株进行卡那霉素筛选及T1、T2、T3的PCR及Southern Blotting杂交等分子检测,获得转化植株并在T3获得转基因纯合株系。对T3转基因玉米和非转基因玉米对照以16.1%的PEG模拟水分胁迫进行抗旱性分析。结果表明,与对照相比,在水分胁迫处理下,转基因玉米株系叶片相对含水量提高了7.4%~19.8%,叶绿素含量提高了11.3%~106.9%,SOD活性上升45.8%~93.4%,而转基因玉米叶片的相对电导率下降了35.4%~58.1%,MDA含量下降了25.7%~50.4%,说明转Os SIK1基因玉米植株抗旱性得到提高,其中,5个转化株系与对照在抗旱性方面有显著差异,且生长状况明显优于对照。综上所述,研究最终获得5个转Os SIK1基因玉米株系,并证明导入水稻Os SIK1基因可以提高玉米植株的抗旱性。     

Breeding of transgenic rice restorer line for multiple resistance against bacterial blight, striped stem borer and herbicide

In this paper, we described the breeding of transgenic rice restorer line for multiple resistance against bacterial blight, striped stem borer (SSB) and herbicide by conventional crossing of two transgenic parental lines transformed independently with different genes. Two stable transgenic rice lines used as donor parents were developed, one was Zhongguo91 which contained cry1Ab gene (for insect resistance) and bar gene (for tolerance of herbicide), and the other was Yujing6 which contained Xa21 gene (resistance to bacterial blight). The elite restorer line Hui773 was used as recipient and crossed with the two stable transgenic rice lines. Then five successive backcrosses were made using Hui773 as recurrent parent. Two rice elite restorers, T773-1 expressing cry1Ab and bar genes and T773-2 expressing Xa21 gene, were obtained, which were confirmed by PCR analysis and testing selectable marker genes in the hybrid progenies. The cross was made between T773-1 and T773-2 to select stable restorer line carrying Xa21, cry1Ab and bar genes. Finally, we obtained transgenic restorer line T773 with good agronomic traits and obvious multiple resistance to Xanthomonas oryzae pv. oryzae, striped stem borer (Chilo suppressalis) and herbicide. The hybrid F₁ generation produced from the cross between transgenic restorer line T773 and a corresponding male sterile line Zaohua2A maintained obvious resistance to rice bacterial blight, rice leaffolder and striped stem borer, and showed significant heterosis. Our results indicate that it is feasible to develop transgenic hybrid rice cultivar through breeding transgenic restorer lines.     

连续回交对消除农杆菌介导转化引起水稻体细胞变异的影响

农杆菌介导的转化引起许多体细胞变异, 影响了转基因植物的农艺性状。因此, 转基因作物的培育需要大量的T₀代再生植株。在本研究中, 我们将转基因水稻株系与原始品种连续回交, 然后评价其回交后代的表现, 消除体细胞变异, 恢复转基因亲本的农艺性状。回交的供体亲本是3个转基因水稻株系, 分别带有来自于苏云金芽胞杆菌(Bt)的抗虫基因。与原始品种连续回交至BC₃F₁代, 每代BC_nF₁单株再自交两代, 同时对各个世代进行抗虫性选择。通过发芽试验获得转基因纯合的BC_nF₃株系, 在室内抗性试验中, 所有的BC_nF₃纯合株系都能杀死100%的幼虫。在田间试验中, 这些株系的单株产量明显高于供体亲本, 大部分农艺性状与原品种没有显著的差异。这些结果说明连续回交能够在很大程度上恢复转基因水稻株系的农艺性状, 从而减少转基因育种过程中所需的工作量。     

不同供体及不同回交次数对玉米自交系R08的改良效应

以R08为轮回亲本,18个优良自交系为供体亲本,不同回交和自交次数,选育出遗传背景与R08相近、但相互之间又存在一定差异的BC₁F₃和BC₂F₂各18个R08改良系。通过抗病性鉴定、配合力及SSR分子标记分析,探讨不同供体及不同回交次数对R08的改良效果。结果表明,36个改良系中,29个抗或高抗大斑病,大部分改良系的多数产量性状一般配合力(GCA)与R08相比并无下降或有所提高;相同供体不同回交次数选系的比较显示,对大斑病抗性的改良,回交1次自交2次(BC₁F₃)优于回交2次自交1次(BC₂F₂),且改良后代选系多数产量性状GCA大体相当;相同回交次数不同供体选系的比较表明,供体对回交后代的影响较大,供体不同回交后代选系大斑病抗性及多数产量性状GCA存在较大的差异;SSR分子标记研究结果在一定程度上揭示相同供体不同回交次数所创造的遗传变异无明显差异,相同回交次数不同供体选系在分子水平上存在较大差异;供体昌7-2和川321对改良R08的大斑病抗性和产量性状GCA作用较大,属优良供体亲本;w4-1和w10-1属回交改良优良选系。因此,利用回交法改良玉米自交系,在选准供体亲本的基础上,回交1次后,在自交过程中加强目标性状的鉴定选择及配合力测定,可提高回交改良的育种效率。     

Insect-mediated seed-set evaluation of 21 soybean lines segregating for male sterility at 10 different loci

The cowpea trypsin inhibitor gene (CpTI) and neomycin phosphotransferase gene (nptII) were introduced into the embryonic callus cells of immature embryos of wheat elite line Shannong 995604 using Agrobacterium-mediated gene transfer. Independent plantlets were regenerated from kanamycin-resistant calli. PCR and real time PCR analysis, PCR-Southern and Southern blot hybridization indicated that there were three independently-dervied transgenic plants viz. transformed-I, II and III (T-I, T-II and T-III). The segregation of CpTI in the transgenic wheat progenies of T-Iand T-III were consistent with Mendelian inheritance. Resistance to the storage insect pest of wheat viz. the grain moth (Sitotroga cerealella Olivier) was improved significantly in seeds of the three transgenic wheat T₂ lines obtained from T₁ PCR-positive plants. The frequency of moth-eaten seed from T-I, T-IIand T-III was reduced 66.76%, 62.48% and 43.59% respectively. The investigation of agronomic traits of the three transgenic wheat T₁ PCR-positive plants revealed that the three transgenic lines had excellent agronomic traits. They provide good germplasm resource for wheat genetic improvement.     

Development of drought‐tolerant breeding lines derived from Helianthus annuus × H. argophyllus interspecific crosses

Helianthus argophyllus is a wild species known as “silver sunflower”, which possesses several traits, including morphological traits that increase drought tolerance. Therefore, introgressions between chosen cultivated lines and two H. argophyllus accessions were made, and segregating generations were established. Important agronomic traits including single heading, high pollen fertility, silver canopy (indicating more cuticular wax), and content of cuticular waxes and oil were selected over six segregating generations. The resulting F₆ lines showed introgression of water saving traits, as they had lower excise leaf water loss, with comparable yield to standard checks. However, these F₆ lines were late maturing and showed poor flowering synchronization between the cultivated and introgressed lines. Introgressed line “D‐22” was particularly promising as a breeding line, with superior agronomic and drought resistance traits. This line had the potential to be used as an inbred parental line for introgression of drought resistance traits into elite sunflower germplasm. Combining ability analysis of the introgressed lines further showed their potential for heterosis breeding or to be used as parental lines in breeding programme.     

转Cry1Ab基因水稻种质杂交后代的标记辅助选择及抗虫性评价

以转Cry1Mb基因的水稻株系TS．9(后命名为克螟稻)为种质,与优良亲本杂交,在分离世代,通过与目标基因紧密连锁的潮霉素抗性作为筛选标记,结合田间农艺性状选择,获得目标基因位点纯合的新品系。Cry1Mb蛋白含量检测结果表明大多数新品系携带的靶基因在经杂交重组、自交纯合后保留了原亲本高效表达的特性。抗虫鉴定结果表明,对二化螟(Chilo suppressalis)、稻纵卷叶螟等(Cnapha locrocis medinalis)鳞翅目害虫表现高抗性能,同时,在株高、生育期、产量构成因子等重要农艺性状上也有显著改良。从而,对转基因水稻种质的育种利用途径和选择方法作了有益探索。     

Construction of RNAi Vector of Cotton (Gossypium hirsutum L.) FAD2-1 Gene and Acquisition of New High Oleic Acid Germplasm

[Objective] Here, the aim was to create a new cotton (Gossypium hirsutum L.) germplasm with high oleic acid and low linoleic acid contents without affecting the fiber yield and quality. [Method] The fatty acid desaturation 2-1 (FAD2-1) gene catalyzes the formation of polyunsaturated linoleic acid from monounsaturated fatty acid in cotton. The protein’s properties, structure and functions were analyzed using the information analysis method. A conserved 381-bp fragment of GhFAD2-1 was cloned to construct an RNA interference vector, which was transformed into cotton by Agrobacterium-mediated hypocotyl infection. The fatty acid compositions and contents of T₂―T₄ transgenic plants were determined using gas chromatography-mass spectrometry. The T₄ transgenic lines were used to investigate the copy number, target gene expression, agronomic traits, and fiber quality. [Results] The RNA interference vector of GhFAD2-1 was successfully constructed and transformed into cotton. The target gene’s expression level was significantly lower in transgenic plants than in controls. The transgenic plants had high oleic acid and low linoleic acid contents, which could be stably inherited by the offspring. The oleic acid contents of transgenic cotton seeds increased by 224.1%, and the linoleic acid content decreased by 237.5%, compared with the control seeds. There were no significant differences in the agronomic traits and fiber quality of the transgenic lines compared with the control. [Conclusion] These results verified the function of GhFAD2-1 in cotton and provide a basis for breeding cotton varieties with high oleic acid contents.     

大豆花粉管通道技术转化雪花莲凝集素(GNA)基因

采用花粉管通道技术，用雪花莲凝集素基因（Galanthus nivalis agglutinin，GNA）转化吉林省主推品种吉林20号、吉林30号、吉林45号品种大豆。通过接蚜鉴定和PCR鉴定，从所获得的种子苗中筛选出转基因植株。对转基因植株的后代进行分子生物学鉴定：（1）PCR分析，转基因植株97TGR1和97TGR2的T2代表现阳性，第5代表现阳性纯合；97TGR1、97TGR2和98FD1～98FD20的T3代Western blotting检测结果证明，GNA基因在蛋白质水平有表达，最高表达量占总可溶性蛋白的0．7％；97TGR1、98TGR2和99JI45 TGR2的Southern blotting检测结果显示，GNA基因已插入大豆基因组；（2）遗传学分析，97TRG1的T2代呈孟德尔3：1分离，97TGR2的T3代出现种皮颜色不规则分离。经过抗蚜性鉴定和连续的筛选，获得抗性纯系；（3）抗蚜性鉴定，转基因株的T1、T2世代转基因植株可抑制蚜虫繁殖量50％～90％；（4）品系鉴定，转基因大豆的抗蚜性达到农学标准抗（R）和高抗（HR）水平；大面积环境释放试验自然感蚜鉴定，转基因系蚜虫发生的高峰比对照延迟，高峰期过后群体蚜量的下降速度也比对照快。本研究认为，大豆花粉管通道技术可以利用于大豆的转基因研究和应用中，GNA基因在改良大豆的抗蚜性上是可取的。     

Breeding of cabbage (Brassica oleracea L. var. capitata) with fusarium wilt resistance based on microspore culture and marker-assisted selection

Cabbage Fusarium wilt (CFW), caused by Fusarium oxysporum f. sp. conglutinans, is one of the most devastating diseases of cabbage worldwide. In recent years, CFW has been spreading across northern Chinad. Thus, development of resistant cultivars is necessary to control CFW. Here we report microspore culture from a cross of two elite cabbage inbred lines, 96–100 (highly resistant to CFW) and 01–20 (highly susceptible to CFW), in order to obtain doubled haploids (DHs) to cultivate cabbage materials for resistance breeding. A total of 196 DHs were obtained in 2011 and 2012 and three elite DH lines were selected from them through agronomic trait evaluation as well as marker-assisted selection. These three elite DH lines were crossed with elite cabbage inbred lines to create hybrids, which were then evaluated for their resistance and agronomic traits. Three excellent hybrids were selected. The use of microspore culture and marker-assisted selection greatly shortened the time required to obtain these excellent hybrids. These resistant varieties may become an effective control of CFW in diseased areas.