沙地水稻各农艺性状与产量关系的回归分析

采用通辽地区沙地水稻生产上常用的品种为试验材料,对其产量与主要农艺性状进行了相关及多元回归分析,结果表明：水稻单穴产量与结实率和千粒重呈显著正相关,与空粒数呈显著负相关,与其它性状呈不显著负相关;通过逐步回归分析建立了数学模型,通径分析结果表明,穗长、实粒数、千粒重对产量具有正向效应,而生育期、株高、分蘖数却对水稻的单穴产量具有负向效应。在沙地衬膜条件下,应选择穗长、生育期适中、千粒重大、分蘖数较少、实粒数多、株高适中的品种易获得高产。     

水稻OsNAP基因的功能分析及其在育种价值方面的研究

NAC转录因子是植物特异的转录因子.本研究通过从水稻日本晴(Oryza sativa L.)的基因组中克隆到一个与拟南芥衰老相关的基因AtNAP(Atlg69490)的同源基因OsNAP.OsNAP的ORF为1 179bp,推测其编码蛋白含有392个氨基酸,等电点为8.55,分子质量为42.195ku.为进一步研究OsNAP的功能,利用显性抑制元件SRDX构建其pCAMBIA1304-35S:OsNAP-SRDX表达载体,通过农杆菌浸染方法将其转化水稻中花11品种.T1代转基因植株通过潮霉素、RealtimePCR、和Western blot鉴定,筛选出4个OsNAP高表达的阳性植株.在种子成熟期,观察到T1代转基因4号植株较野生型相比有明显的延缓衰老的表型,其他农艺性状如单株分蘖数、单株总粒数、单穗饱和穗粒数、千粒重、结实率等都优于野生型.实验证明抑制OsNAP基因的表达能够延缓水稻叶片衰老并可以提高产量,具有潜在的育种价值.     

水稻OsDDB2基因过量表达载体的构建及其转化

利用农杆菌介导法将构建好的水稻OsDDB2过表达载体导入水稻,共获得植株4株,经PCR检测确定4株皆为转基因植株.田间性状分析发现转基因植株的结实率(平均6.05%)远远低于野生型植株的结实率(平均87.4%).相应地,花粉粒I_2-KI染色结果显示转基因植株的花粉数远少于野生型植株的花粉数.这些初步实验结果为进一步研究水稻DDB2基因的功能奠定了基础.     

水稻OsDTH10基因CRISPR/Cas9编辑突变体的创建及功能分析

为了探究OsDTH10基因在水稻生长发育及开花中的作用,利用CRISPR/Cas9基因编辑技术对OsDTH10基因进行定向编辑.在OsDTH10基因的第4个外显子处设计sgRNA的靶位点,通过PCR扩增与Os U6SK空载体连接形成OsU6SK-sg RNA融合载体,再将获得的sg RNA和Cas9连接到植物双元载体pCAMBI1300中,得到重组载体pCAMBI1300-CRISPR/Cas9-OsDTH10.利用农杆菌介导的植物转化法得到转基因植株,对其进行鉴定并测序.本研究共获得3个株系8株转基因植株,测序结果显示,2号株系的4株转基因植株由于发生了单碱基的插入,基因测序出现了套峰.田间表型观察发现,2号株系的4株T0代转基因植株抽穗期早于空载体对照,获得的T1代转基因植株抽穗期比对照的提早8 d.这些结果表明,重组载体pCAMBI1300-CRISPR/Cas9-OsDTH10成功实现了对水稻OsDTH10基因的定向编辑.     

利用基因枪法钭多个抗虫基因导入超级杂交水稻培矮—64S的研究

应用基因枪法对超级杂交水稻培矮－64S进行了转化,质粒载体pKC－3串联了三个抗虫基因,将其转化成熟胚诱导的愈伤组织,共获得33株转基因植株,分别对不同的基因进行PCR分析,结果表明,三个抗虫基因均已整合到水稻基因组。     

AGP转基因粳稻产量性状在低世代中的遗传与变异

云南粳稻合系３８、合系４２经ＡＧＰ基因导入后,株高,有效穗数,实粒数,千粒重等产量性状的群体变异频率在Ｔ１和Ｔ２代中显著增高,由于水稻自花授粉的特性,导入目的基因的两条同源染色体配对的频率也较高,因此在代世代中已经出现了综合性状较好的株系。     

拟南芥AtPLC4基因RNAi载体的构建及遗传转化

利用RNAi技术,构建了AtPLC4基因的RNAi双元载体,并进行了拟南芥的遗传转化,通过抗性筛选及PCR鉴定,获得20株遗传转化株系.进一步通过RT-PCR检测,得到3株AtPLC4基因表达降低的转基因植株,为运用反向遗传学的方法深入研究AtPLC基因家族的生物学功能提供基础.     

基于Gene-Deletor系统的安全复合性状转基因烟草研究

本研究利用农杆菌介导法将带有水稻花粉特异启动子籼稻花粉过敏原基因(OSIPA)启动子驱动的Gene-Deletor外源基因清除系统,水稻Os GA3ox2(D18)启动子驱动水稻Os GA2ox1基因,玉米Ubiquitin启动子驱动BAR::GUS融合基因为筛选标记基因以及水稻Actin1启动子驱动驱动抗虫Cry1Ab基因复合性状植物表达载体p GM626-D18-Os GA2ox1遗传转化三星烟草。通过GUS组织化学染色及PCR分子鉴定,获得了43株转基因烟草植株。结果表明,水稻OsGA2ox1基因在烟草中表达能降低转基因烟草植株高度,但不影响植株生殖生长。转基因烟草对烟草斜纹夜蛾幼虫具有抗性,可抑制烟草斜纹夜蛾幼虫的取食与发育。以50 mg/L的除草剂Basta处理野生型和转基因烟草离体叶片,发现BAR基因提高了烟草抗除草剂的能力。对12株转基因烟草T0代花粉外源基因清除效率进行研究,发现部分烟草株系外源基因清除可达到100%,其他未完全清除外源基因株系的清除效率介于42.84%~99.97%之间。     

通过遗传转化获得含多基因的水稻转基因纯合植株

利用基因枪法将含有4个不同基因的3个质粒共转化由粳稻品种鄂宜105号和鄂晚5号种子胚诱导的愈伤组织（5－10d龄）。从轰击的986块愈合组织中共再生出169株独立的转基因水稻植株（转化率为17％）。PCR／Southern blot分析显示70％以上的转基因植株含有所有4个基因。GUS组织化学分析、Western blot和或RT－PCR分析表明所有4个基因的共表达率为70％。未观察到任何质粒在整合中存在优势,转基因拷贝数也与基因表达量无关。遗传分析证实外源基因在后代植株中大多以孟德尔方式遗传。从其R1代为3：1孟德尔方式遗传的后代R2代植株中,鉴定含有3个或4个不同基因的转基因纯合植株系。PCR／Southern blot分析证实了这些转基因纯合植株系。这些系的植株具有相似的外源基因表达量。我们证实通过基因枪介导的共转化,结合常规育种方法筛选可以获得含多基因的转基因水稻纯合植株。这项技术为利用基因同时改良作用多个性状提供了一种途径。     

转1Dx5基因小麦突变株系的研究

以转1Dx5基因小麦3个高分子量麦谷蛋白突变株系B73-6-1-1、B73-6-1-2和B73-6-1-3及转基因受体L88-6为材料,对其主要农艺性状、低分子量麦谷蛋白表达情况及染色体核型进行研究.结果表明:在田间栽培条件下,纯合稳定转基因突变株系之间及其与转基因受体之间主要农艺性状存在差异,其中株高和千粒重存在显著差异(P=0.05);同时,SDS-PAGE结果显示,在小麦低分子量麦谷蛋白区域,突变株系之间无明显差异,而突变株系与转基因受体之间部分区域表达量存在明显差异并产生一个突变条带;通过核型分析比较,得出突变株系之间及其与转基因受体之间,染色体无论是相对长度还是臂比值都没有显著差异.     

Transformation of GbSGT1 gene into banana by an Agrobacterium-mediated approach

SGT1 is a homologue of the yeast ubiquitin ligase-associated protein. It controls some protein degradation and activates defense pathway in plants. Cotton GbSGT1 gene (Gossypium barbadense) has been isolated and characterized in previous work. In this study, the plant expression vector pBSGT1 with bar gene as a selection agent was constructed and transgenic banana was obtained via Agrobacterium-mediated transformation with the assistance of particle bombardment and screened with PCR and Basta spreading on banana plant leaves. Estimating of transgenic banana plants for resistance to Panama wilt is in progress.     

Transformation of GbSGT1 gene into banana by an Agrobacterium-mediated approach

SGT1 is a homologue of the yeast ubiquitin ligase-associated protein. It controls some protein degradation and activates defense pathway in plants. Cotton GbSGT1 gene (Gossypium barbadense) has been isolated and characterized in previous work. In this study, the plant expression vector pBSGT1 with bar gene as a selection agent was constructed and transgenic banana was obtained via Agrobacterium-mediated transformation with the assistance of particle bombardment and screened with PCR and Basta spreading on banana plant leaves. Estimating of transgenic banana plants for resistance to Panama wilt is in progress.     

抗除草剂旱稻转基因植株的获得

以旱稻丹粳旱５－５５、６－２４、６－３７的悬浮细胞及未成熟胚为受体材料，采用基因枪法将含ＢＡＲ基因的ｐＤＭ３０２质粒ＤＮＡ导入旱稻细胞中，经ＰＰＴ筛选获得抗性愈伤组织，筛选出的愈伤组织在分化培养基上再生出完整的旱稻转基因植株。Ｓｏｕｔｈｅｒｎ分子杂交分析表明，外源ＢＡＲ基因已整合到水稻基因组内，抗除草剂试验结果表明，转化植株对０．０１％Ｂａｓｔａ（有效成分为ＰＰＴ）有一定程度的抗性，说明外源ＢＡＲ     

New technology to examine and improve the purity of hybrid rice with herbicide resistant gene

The herbicide resistant bar gene has been widely used as selectable marker genes in the study on plant genetic transformation. Owing to the integration of the gene into rice genome, transgenic rice was resistant to the herbicide Basta. Therefore, selection of transgenic plant md genetic analysis became easier. In the studies, bar gene was introduced as a genetic marker gene into ria restore lie variety of two-line or three-line. Combined with conventional breeding method, the good herbicide resistant individual plant was derived as the new restorc line for hybrid combination. After sprayed with Basta at seeding phasc. the real hybrid rice that hnd herbicide resistance could grow normally, while the fnke hybrid rice and weeds, having no bar gene, were killed by herbicide. The method described above helped rapidly to examine and improve the purity of hybrid rice.     

几种水稻籼型恢复系和不育系离体培养和遗传转化的研究

对二系杂交水稻亲本籼型恢复系 2 88,198,112 5和籼型不育系株 1- s进行了离体培养和遗传转化的研究 ,建立了这些籼稻新品系的胚性愈伤组织高频诱导与再生系统及其基因枪转化系统 .它们的胚性愈伤组织诱导率分别可达 87.4 % ,90 .6% ,78.9%和 88.0 % ;其分化率分别可达 60 .0 % ,4 5 .8% ,4 6.9%和 60 .6% ;其基因枪转化率分别可达 16.69% ,2 .65 % ,2 .0 4 %和 10 .78% ,并获得了一批可育的水稻转基因植株     

Overexpression of glutamine synthetases confers transgenic rice herbicide resistance

Glutamine synthetase (GS, E.C.6.3.1.2) is a key enzyme involved in the assimilation of inorganic nitrogen in higher plants and gram-negative microorganisms. GS is the targeting enzyme of a herbicide phosphinothricin (PPT) or Basta. In order to generate PPT-resistant transgenic rice via overexpression of GS, we constructed a plant expression vector p2GS harboring two different isoenzymes GS1 and GS2 cDNAs under the control of constitutive promoters of rice Act1 and maize Ubiquitin(Ubi) genes. The p2GS was introduced into rice genome by Agrobacterium-mediated transformation and confirmed by PCR and Southern blot hybridization. GS-transgene expression was first detected by Northern blot analyses. Results from Basta test indicated that GS-transgenic plants can tolerate as high as 0.3% Basta solution. In addition, our results also demonstrated that GS overexpression conferred transformed rice calli PPT resistance. Thus, GS cassette can serve as a selective marker gene instead of bar cassette for selection of PPT transformants.     

重要籼型杂交稻亲本“93-11”稻米食味品质的改良

以目前两系杂交稻大量应用的籼型水稻"93-11"和实验室已转化只含双份反义蜡质基因无抗性基因和报告基因纯合水稻"B3"为亲本,通过杂交和多次与"93-11"水稻回交以及分子标记辅助选择反义蜡质基因,将双份反义蜡质基因渗入到"93-11"水稻品种中,获得两种改良类型"93-11"水稻新材料.主要农艺性状考察、拷种以及糙米重量和体积分析显示,除了千粒重、糙米重量和体积,两种改良后水稻的株高、有效穗、每穗实粒数和结实率都与"93-11"对照水稻差异不显著(P0.05).两种改良后水稻蜡质糙米的直链淀粉含量(5.39%和5.01%)均比"93-11"对照(14.06%)极显著下降(P0.01);同时糙米胶稠度分别为93 mm和101 mm,与"93-11"对照(48 mm)比较也达到极显著差异水平(P0.01).研究为今后改良以"93-11"配组的两系杂交水稻食味品质奠定了重要基础.     

Transformation of japonica rice with RHL gene and salt tolerance of the transgenic rice plant

Overexpression of the yeast HAL2 gene increases salt tolerance of yeast and plant. Rice HAL2-like (RHL) gene was introduced into a japonica rice cultivar HJ19 with Agrobacterium tumefaciens-mediated transformation. Transgenic plants in R0 generation were selected on the principle of GUS-positive, RHL gene PCR-positive and normal growth. Hygromycin-resistant plants of some transgenic lines in R1 generation increased salt tolerance during the seedling and booting stage, being less damaged in the cytomembrane and stronger in leaf tissue viability under salt stress during booting period. Southern analysis of transgenic lines tolerant to salt in R1 generation showed that the RHL gene expression cassette had been successfully integrated into rice genome. Moreover, gene engineering breeding methodology and really salt-tolerant rice cultivar were discussed.     

水稻籼粳中间型RIL系主要农艺性状杂种优势分析

分析了遗传背景相同的籼粳中间型重组自交系（RIL）9个品系与5个籼、偏籼型光温敏核不育系杂交的F1主要农艺性状的杂种优势．结果表明：9个农艺性状均表现为正向超亲优势,以单株产量、穗实粒数、株高和穗总粒数表现的优势较强,有效穗数、结实率和千粒重表现的优势较弱;F1单株产量仅与父本穗长呈显著负相关,而与杂种有效穗数、穗实粒数、结实率和千粒重呈极显著和显著正相关;F1单株产量的超亲优势与父本单株产量、有效穗数、结实率均呈极显著负相关,与父本抽穗天数和千粒重却呈显著负相关,而与杂种的抽穗天数、有效穗数、穗实粒数、结实率和千粒重优势呈极显著正相关．亚种间杂交稻超高产的获得关键在于要协调好产量目标、亲本产量构成因素和其杂种优势三者的关系．