外源基因1Dx5在转基因小麦后代中的表达

采用PCR和SDS-PAGE技术,分析研究了转基因小麦后代外源品质基因1Dx5的遗传分离及表达规律,结果显示:转基因小麦后代的遗传分离完全符合孟得尔遗传分离规律,转基因植株都达到了纯合状态,1Dx5基因在转基因植株中已经稳定整合并稳定遗传,并发现了超表达的差异表达现象,在转基因植株中没有发现基因沉默现象.     

两个杂交组合中转基因小麦外源1Dx5基因的遗传

利用杂交组合川89-107×B72-8-11b和绵阳26×B72-8-11b的亲本、F1、F2代,研究转基因小麦B72-8-11b中外源品质基因1Dx5表达的遗传.结果表明:外源1Dx5基因在F1代中呈现显性,在F2代中呈现3(有):1(无)的分离,有功能拷贝整合在1个位点,遵从孟德尔遗传模式,这对杂交育种选择策略的制订具有指导意义.     

杂交小麦早代HMW-GS基因Dx5的PCR检测

以小麦杂交后代为试验材料，从分蘖前叶片中提取DNA进行PCR扩增，探索利用特意引物PCR技术，鉴定小麦杂种后代HMW-GS基因Dx5的可能性。结果表明，杂种后代单株携带Dx5基因显带，不携带Dx5基因不显带。此法简便、快速、准确、科学，可检测优质面包烘烤品质基因为小麦品质早代选育提供了理论依据。     

花粉管通道法转基因技术在甜瓜品种河套蜜瓜上的应用

为建立甜瓜(Cucumis melo L.)简便易行转化频率高的转基因技术,以甜瓜品种河套蜜瓜为受体材料,以含gus基因的植物双元表达载体pPZP221为外源基因供体,进行了花粉管通道法转基因研究.自交授粉后分别于1、2、3、4、5、6、7、8、9、10 h,切去柱头上端1/3部分,立即滴加质粒DNA溶液,果实成熟后收获转化种子.对T0代植株进行PCR检测,结果表明不同时间处理所得到的T0代植株均具有较高的转化频率,其中授粉后7 h滴加DNA溶液所获得的转化率为28.3%.对一部分PCR阳性的T0代植株基因组DNA进行Southern杂交分析,结果表明所有样品均出现特异性杂交条带,证明外源基因已整合到受体植物基因组中.     

多重PCR法快速鉴定转基因小麦植株及后代

根据转基因植物中常用的报告基因uidA、选择基因bar的序列,设计合成两对不同的引物,建立了在转基因小麦材料分子确证中,应用一次PCR反应同时扩增检测两种或多种外源基因的多重PCR方法．通过对质粒pAHC25以及30个转基因小麦样品进行PCR扩增分析,比较多重PCR的检测结果与单基因的PCR扩增结果,发现两者结果完全一致．     

转基因小鼠乳腺表达人乳过氧化物酶的初步研究

从人基因组PAC文库中筛选出人乳过氧化物酶（hLPO）基因,利用长片段PCR的方法获得hLPO基因5’端约3kb片段,通过酶切方法获得hLPO基因3’端约27kb片段,将这两部分拼接并克隆到乳腺特异性表达载体pBC1上,构建以山羊β-casein启动区指导的hLPO的转基因表达载体pBC1-hLPO。利用显微注射的方法获得28只FO代小鼠,经PCR检测和Southerm杂交分析证实,有5只小鼠（4♂,1♀）为整合hLPO基因的转基因阳性小鼠,整合率为17.86％,整合拷贝数在1至5之间。利用SDS-PAGE凝胶电泳和Western blot印迹分析FO、F1代共3只雌性转基因小鼠乳样,结果表明hLPO重组蛋白的特异条带不明显。     

表达千穗谷Ah-AMP基因的转基因烟草抗病性研究

通过PCR从苋属植物千穗谷(Amarathus hypochondriacus)的总DNA中扩增出苋菜抗菌肽(Ah-AMP)的核基因片段,序列分析结果表明该基因长261bp,编码一个由86个氨基酸组成的Ah-AMP前体多肽.在构建Ah-AMP基因的植物表达载体pBinAH916后,通过根癌土壤杆菌介导方法转化了烟草.转化再生植株和T 1 代转基因烟草的PCR和Southern blot分析表明,Ah-AMP基因已整合到烟草的染色体中,并为单拷贝整合.T 1 代转基因烟草的Northern blot分析结果表明,Ah-AMP基因在转基因烟草中至少在mRNA水平上能正常表达.对T 0 代转基因烟草进行烟草青枯病的抗病性试验,筛选出两株抗性较强的植株.对T 1 代抗青枯病的统计分析发现,其抗病性比起始品种SR1分别提高了2.24和1.62个级别;其病情指数比起始品种降低49.6%和37.3%.对黑胫病也表现一定的抗性,主要表现在推迟发病时间,减缓发病速度上.这些结果表明Ah-AMP基因在植物抗病基因工程研究中可能是一个有潜在应用价值的基因.     

绵羊角蛋白关联蛋白KAP6-1基因5′端调控区的分子克隆及测序结果比较

根据绵羊毛囊角蛋白关联蛋白(KAP 6-1)基因已知DNA序列设计合成了两个特异性引物,以绵羊基因组DNA为模板,PCR扩增出1 042 bp的特异性片段,连接到pM D 19T载体中获得该片段克隆.经过快速提质粒法筛选、限制性内切酶分析表明该克隆包含所需的目的片段.DNA测序结果也证明该克隆片段与原基因5′端调控区序列相比具有很高的一致性.研究结果为今后制备转基因克隆动物,在毛囊细胞中特异性表达绒毛生长调控基因奠定了基础.     

人工合成启动子特异启动IGF-1真核载体的构建与绵羊成纤维细胞转染

构建了肌肉特异表达IGF-1载体,并转染绵羊成纤维细胞获得了稳定整合外源基因可用于细胞核移植的转基因细胞.通过人工合成骨骼肌特异启动子SP片段,与羊IGF-1相连,最终连接到骨架载体pCDsRed2中构建成骨骼肌特异表达IGF-的真核表达载体pSPICDS.使用脂质体法转染绵羊成纤维细胞,通过红色荧光与G418双重选择,获得了转基因细胞.经PCR检测证实得到的细胞克隆外源基因稳定整合,可以作为供体细胞用于体细胞核移植.     

优质早籼不育系HD9802S不育基因tms5的遗传分析及应用研究

tms5是从温敏核不育系安农S-1中鉴定到的不育基因.为验证tms5也是控制不育系HD9802S育性的关键基因,将R287中野生型TMS5基因通过农杆菌介导的转基因方法导入HD9802S中,通过转基因T0代植株育性观察及T1代单株的共分离检测,互补实验从遗传上证明不育系HD9802S不育基因即是tms5.同时本研究通过基因编辑的方法敲除R287中TMS5基因,转基因T0代单株在高温下表现为不育,说明选择合适的受体材料,通过基因编辑技术敲除TMS5基因可以创建新的不育系.     

Transgene inheritance and quality improvement by expressing novel HMW glutenin subunit （HMW-GS）genes in winter wheat

The expression vector pBPC30, which carries the high molecular weight glutenin subunit (HMW-GS) 1Dx5 and 1Dy10 genes, was transferred into hexaploid winter wheat cv. Jinghua No. 1, Jing411 and Jingdong No. 6 explants of immature embryos and immature inflorescence by particle bombardment. A large number of resistant transgenic plants were obtained under the selection of herbicide bialaphos or phosphinothricin (PPT). Confirmed transgenic plants of To generation showed successful integration of HMW-GS genes and bar gene into the wheat genome. T1 generation of transgenic plants can resist 20--150 mg/L PPT.Protein analysis of T2 seed by SDS-PAGE showed that HMW-GS 1Dx5 and 1DylO genes were well expressed in offspring seed of transgenic lines by co-expression with or substitution of endogenous 1Dx2 or 1DylO. In one transgenic line, TG3-74, a new protein band between endogenous protein subunits 7 and 8 (marked as 8*) of glutenin appeared,but endogenous subunit 8 (encoded by 1By8 gene) was absent. Analysis of gluten rheological quality on seed proteins of 102 T3 plants showed that the sedimentation value of 5 transgenic lines (44.2149.0 mL) was remarkably improved,59.6%---64.3% higher than that of wild type Jinghua No. 1 and Jingdong No. 6, similar to bread wheat Cheyenne (48.0 mL). Analysis of dough rheological properties of transgenic lines showed that the dough stable time of 5 transgenic lines range from 16 to 30 min, whereas the dough stable time of wild type was only between 3--7 min. Our research suggests that introducing novel HMW-GS genes into wheat is an efficient way to improve its bread-making quality.     

Genetic expression in progeny of transgenic plants obtained by using pollen-tube pathway (or delivery) method and approach to the transformation mechanism

The origin and the recent progress of pollen_tube pathway method (PTPM) is described. The results of transforming wheat during 1990 to 1994 are reported briefly. They are: (ⅰ) Transformation frequencies were 3%-6%, even reached 13.5%; (ⅱ) the genetic expression of four continuous generations obtained from single individuals of transgenic plants by self_fertilization was investigated. Plants with higher expression of foreign gene were found in every generation (from T1 to T4), but a phenomenon of gene silence, in which the genotype contained foreign gene but did not express, was also observed. In addition, from the angle of fertilization biology the transformation mechanism of PTPM (or PTDM) was analyzed and discussed. It was considered that the essential principle of this method was just like that of protoplast method.     

小麦与高冰草体细胞杂种F5代麦谷蛋白及SDS沉降值分析

通过对普通小麦与高冰草体细胞杂种F5代 175个株系的麦谷蛋白及SDS沉降值分析 ,发现杂种F5代有 10种不同的麦谷蛋白组成 ,并出现了 1Ax1,1Ax2 ,1Bx13,1By16 ,1By8,1Dx5等 6种不存在于亲本小麦中的高分子量麦谷蛋白亚基及 1Bx13 1By16 ,1Bx7 1By8,1Dx5 1Dy12等 3种新亚基组合 ,部分杂种中出现了高冰草的麦谷蛋白特征谱带 ;杂种株系的SDS沉降值表现出较大的差异 ,其中一些杂种的SDS沉降值明显高于亲本普通小麦 .结果表明 ,通过体细胞杂交有可能将野生禾草的优良性状引入普通小麦 ,创造出多种不同组成及组合的新种质 ,为小麦品质育种开辟新途径     

Identification of a novel HMW glutenin subunit and comparison of its amino acid sequence with those of homologous subunits

Aegilops tauschii is the donor of the D genome of common wheat (Triticum aestivum). Genetic variation of HMW glutenin subunits encoded by the Glu-1Dt locus of Ae. tauschii has been found to be higher than that specified by the Glu-1D locus in common wheat. In the present note, we report the identification of a novel HMW glutenin subunit, Dy13t, from Ae. tauschii. The newly identified subunit possessed an electrophoretic mobility that was faster than that of the Dy12 subunit of common wheat. The complete ORF of encoding the Dy13t subunit contained 624 codons (excluding the stop codons). The amino acid sequence deduced from the Dy13t gene ORF was the shortest among those of the previously reported subunits derived by the D genome. A further comparison of Dy13t amino acid sequence with those of the subunits characterized from the A, B, D, R genomes of Triticeae showed that the smaller size of the Dy13t subunit was associated with a reduction in the size of its repetitive domain.     

Generation of transgenic wheat resistant to wheat yellow mosaic virus and identification of gene silence induced by virus infection

The plasmid containing the promoter Act1, the coat protein (cp) gene of wheat yellow mosaic virus (WYMV) and the selectable bar gene, was delivered via particle bombardment, directly into immature embryos of a wheat cultivars. PCR and PCR-RFLP were employed to screen the existence of the cp gene in T0 and T1 generations. Seeds from the positive T1 plants were sowed in fields heavily contaminated with WYMV to detect their resistance. In field trial of virus infection, one of the transgenic wheat lines, P8-T2, exhibited highly disease-resistance. Western blot and RT-PCR analysis showed that the expression level of cp gene in the resistant transgenic line was reduced greatly compared to those susceptible to WYMV infection. This provided evidence to presume that the resistance obtained by the transgenic wheat line was stimulated by the mechanism of the virus induced gene silencing.     

茄青枯假单胞菌T2015致病相关基因突变体的构建

分离自花生植株的茄青枯假单胞菌(Ralstonia solanacearum)T2015的Tn5-lacZ插入突变体T2135是极性突变体.采用现代分子生物学实验技术,首先构建了同一操纵子中ORF2的表达质粒pGX6191,并进行生物学验证,然后采用三亲本结合技术导入极性突变体T2135,得到茄青枯假单胞菌T2015中与致病相关基因ORF3的突变体T2135/R.     

猪繁殖和呼吸综合征病毒福建毒株ORF5的序列分析及原核表达

根据猪繁殖和呼吸综合征病毒(PRRSV)已发表的核苷酸序列,设计了一对特异性引物(p1/p2),用RT-PCR扩增PRRSV福建毒株FJ-1的全长糖基化囊膜蛋白基因(ORF5),将扩增产物连接到pUCm-T载体并转化大肠杆菌DH10B,筛选阳性重组质粒进行序列测定与分析.再设计另一条引物(p3)与p2从重组载体pUCm-T-ORF5中扩增出缺失了N端30个氨基酸残基的tGP5的编码序列tORF5,克隆入表达载体pGEX-4T-3后在大肠杆菌中表达.结果表明,FJ-1毒株ORF5基因为603 bp,编码200个氨基酸,与北美型参考毒株VR-2332、CH-1a及欧洲型参考毒株LV的氨基酸同源性分别为87%、89%和53%,推定福建毒株FJ-1属于北美型毒株.原核表达产物经过SDS-PAGE及Western Blot分析,证实为GP5与谷胱甘肽转移酶(GST)的融合蛋白.分子量为41 kDa.表达量约占菌体蛋白的15%,主要以包涵体形式存在.     

小麦液泡质子焦磷酸酶基因EST的克隆与分析

通过RT-PCR的方法在矮苏3小麦的总cDNA中克隆到一个与大麦液泡质子焦磷酸酶基因(VP)高度同源的EST。以该序列为基础,利用生物信息学的方法构建了一个编码小麦VP蛋白的全长EST重叠群,长2764bp,其包含一个长2355bp的完整开放读码框(ORF),编码785个氨基酸多肽。通过Southern杂交,将该VP基因定位在小麦染色体的第7同源群上。     

Expression of lysine-rich protein gene and analysis of lysine content in transgenic wheat

Expression vector pBPC102, which carries winged bean lysine-rich protein (wblrp) gene and dihydropicolinate synthase (DHDPS) gene, was transferred into hexaploid winter wheat cv. Jinghua No.l, Jing411, You899 and Yangnongl5 explants of immature inflorescence and immature embryos by particle bombardment. More than 100 transgenic plants were obtained under the selection of s-(2-aminoethyl)-L-cysteine (AEC). Confirmed transgenic plants of To and TI generation by PCR and PCR-Southern blotting analyses showed successful integration of wblrp gene into wheat genome. Analysis of transgenic plant lines of T2 by Northern dot-blotting showed good expression of wblrp gene in offspring seed. The content of free lysine in leaves, contents of bound lysine and total proteins in seeds of T2 transgenie wheat lines were determined and analyzed. Among 34 tested transgenic lines, levels of free lysine content in leaves of 9 transgenic lines are 2~3times higher than un-trans-formed wild-type cultivars. Among 17 analyzed transgenic lines, bound lysine content of 4 transgenic lines is more than 10% higher than that of wild-type cultivars. Our research suggests that introducing wblrp gene into wheat is an effective way to improve its nutrition quality.