共转化CBF4和bar基因蒙农杂种冰草植株的分子检测

在获得转CBF4和bar基因蒙农杂种冰草植株的基础上,应用PCR和Southern杂交技术检测外源基因在转基因蒙农杂种冰草植株中的整合及拷贝数.确定外源基因CBF4和bar基因已经整合到冰草基因组中,并且是以多拷贝的整合方式插入受体细胞的染色体上.说明外源基因对蒙农杂种冰草成功地进行了转化,可以作为后期转基因冰草植株研究及培育转基因冰草新品种的材料.     

表达雪花莲外源凝集素基因的油菜转基因植株的获得

利用农杆菌素LBA4404（pCAMBIA3300RG)转化优良甘蓝型油菜恢复系W723的下胚轴节段.pCAM-BIA3300RG含有Rssl启动子引导的雪花莲外源凝集素基因（gna）和CaMV-35S启动子引导的除草剂抗性基因（bar）。经过两轮除草剂（2.5mg/L bialaphos）筛选（两周/轮）,除草剂抗性再生芽被传入生根培养基中生根,对根系旺盛生长的植株中所含gna基因进行PCR分析,PCR分析证实了这些植株确为转基因植株,利用Western印迹法对随机选择的5株含gna基因的转基因植株的分析发现,其中4株表达了gna基因。目前正对这些表达gna基因的转基因植株进行后代遗传分离分析。     

转抗菌肽B基因水稻(Oryza sativa L.)植株的再生

通过研究除草剂Basta对水稻品种台安 1号愈伤组织生长的影响 ,确定了能抑制愈伤组织生长的最低剂量 ;在此基础上将抗菌肽B基因 (cecropinB)和bar基因导入了台安 1号基因组。除草剂抗性鉴定结果表明 :转基因植株表现出对Basta较强的抗性 .     

利用RNAi沉默技术对水稻OsHAD1基因的功能分析

生物信息学分析推测了水稻OsHAD1基因属于HAD(haloacid dehalogenase)超级家族水解酶.为研究其功能,本实验设计构建了OsHAD1-RNAi载体,通过农杆菌介导的方法转化水稻,成功获得了转基因植株.结果表明,转基因植株与野生型(日本晴)相比,转基因植株从T0代到T2代结实率均有20%～50%的降低;实时荧光定量PCR分析显示,T2代独立转基因植株中OsHAD1基因的表达量均有50%以上的下调;花粉染色结果显示,转基因植株花粉染色异常,败育率高,平均败育率达71.19%;石蜡切片结果显示,转基因植株花粉形状不规则,在花粉囊中排列松散,并且植株表现出明显的雄性不育.因此,推测OsHAD1基因参与水稻的生殖发育.     

甘蓝型油菜BnBRI1基因过量表达载体和RNA干扰载体的构建及遗传转化

构建了含甘蓝型油菜BnBRI1基因过量表达载体pFGC5941-BnBRI1和RNA干扰载体pFGC5941-BnBRI1-Ri,以根癌农杆菌介导的方法转化甘蓝型油菜"W679"下胚轴外植体,经PCR鉴定,获得18株转基因阳性植株,其中1株为BnBRI1基因过量表达转基因植株,17株为BnBRI1基因RNA干扰转基因植株.经RT-PCR鉴定显示,在过表达转基因植株中BnBRI1基因表达量比野生型甘蓝型油菜高,而在RNA干扰转基因植株中表达量比野生型低,RNA干扰BnBRI1基因的表达导致了植株的矮化,矮化植株将可用于油菜株型的遗传改良,以培育适于机械化生产的中矮秆油菜新品种.     

用基因枪法转bar基因以培育抗除草剂的直播稻

用基因枪法将 p CB1 质粒 (含 bar基因和 cecropin B基因 )导入到三种有应用前景的直播稻品种中 ,受体材料为来源于水稻未成熟胚的胚性愈伤组织和来源于成熟胚的悬浮细胞系 .当代转化植株显示出很强的对除草剂 Basta的抗性 ,Southern-blot分析显示 :两个外源基因共整合到转化植株基因组中 ;子一代植株中仍含有外源基因 ;来源于同一块愈伤组织的转化植株的整合模式可能是相似的 ,也可能完全不同 .     

松树根特异性启动子驱动的转CMO/BADH基因水稻的耐盐性研究

运用松树根特异性启动子PmPgPR10驱动把CMO/BADH双价基因转入水稻.对转基因植株根和叶的CMO酶、BADH酶活性及其它生理生化指标进行了测定.结果表明:CMO/BADH双价基因能在根部特异性表达.文章还对转基因提高植株耐盐性的机理进行了探讨.     

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.     

转蔗糖: 蔗糖-1-果糖基转移酶基因提高烟草的耐旱性

蔗糖: 蔗糖-1-果糖基转移酶（sucrose: sucrose 1-fructosyltransferase, 1-SST）以蔗糖为底物催化生成蔗果三糖等低聚合度的果聚糖.将从莴苣中克隆的1-SST基因重组到pCAMBIA1300-als中，构建了在CaMV 35S启动子调控下的植物表达载体，利用农杆菌介导的叶盘转化法将1-SST基因导入烟草中，PCR和Southern杂交检测表明获得了转基因植株，RT-PCR结果表明该基因在烟草中正常表达. 对T₀代转基因烟草进行的耐旱性分析结果表明，干旱胁迫6d的转基因植株丙二醛含量和电解质渗漏率显著低于未转基因对照，叶片相对含水量下降速度也明显比对照慢. 对转基因植株叶片糖分分析表明，转基因烟草植株积累果聚糖，并在干旱胁迫后含量明显增加，而未转基因对照植株不积累果聚糖. 在14%PEG溶液中未转基因烟草种子的萌发率仅为转基因烟草种子的一半；在附加200mmol/L甘露醇的培养基中未转基因烟草种子根的生长明显受到抑制，而转基因烟草根的生长发育正常. 以上研究结果表明，转1-SST基因烟草植株耐旱性的提高可能与该基因的表达有关.     

HAK基因对玉米的遗传转化及其耐盐性研究

摘要：用农杆菌介导法将高亲和性钾离子转运体基因（HAK）和Bar基因转入5个优良玉米自交系7922、P138、265、238和271中,并对影响其遗传转化效率因素进行了优化．经PCR和RT-PCR检测证实获得阳性植株．除草剂涂抹实验证明Bar基因已经整合进玉米基因组．用不同浓度的盐溶液处理转基因植株和对照植株,发现转基因植株叶片中的K＋含量、脯氨酸和叶绿素含量均高于未转化植株,而Na＋含量低于未转化植株,表明HAK基因已经整合进玉米基因组,并通过过量表达提高了植株的耐盐性．     

Transgene directionally integrated into C-genome of Brassica napus

Transgenic Brassica napus has been widely planted in Canada, the United States, and some other countries. In China, although the policy for genetically modified foods has not yet opened, genetically modified rape- seed oil as raw material for biodiesel of…     

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     

Expression of the mouse metallothionein mutant ββ-cDNA in the lettuces (Lactuca sativa L.)

Mouse metallothionein (MT) domain mutant ββ-cDNA gene has been inserted into the plant expression vector pGPTVd35S which has herbicide (PPT)-resistance gene bar as a selective marker. The chimeric gene was introduced into the lettuce (Lactuca sativa L. cv Salinas 88) by Agrobacterium-mediated transformation. PCR and Southern blot analysis of some putative transformants indicated that the introduced ββ-cDNA was integrated into lettuce genome and inheritted by sexual reproduction. The expression of ββ gene in lettuce plants was demonstrated by Northern and Western blot analysis. Meanwhile, the zinc content of the transformed lettuce plants is up to 400 μg/g dry weight, remarkably higher than the control plants.     

多基因双T-DNA植物表达载体的构建及拟南芥转化

多基因植物表达载体用于植物遗传转化是培育具有多种优良品质作物的有效策略. 双T-DNA系统是实现筛选完成后选择标记基因删除的一种简便可行的方式. 为培育高度抗逆或去除标记基因的农作物，构建了多基因双T-DNA植物表达载体2T-bbgdD，其中含有一个抗除草剂基因bar, 3个抗逆相关基因(DREB1A, Na+依赖性Pi转运体基因（d5）, betA)和一个报告基因gfp. 利用农杆菌介导法将该载体转入拟南芥，获得了多基因共转化及去除标记基因的转基因拟南芥. 可将此植物表达载体进一步用于作物的遗传转化.     

bar基因表达框和完整质粒转化对水稻农艺性状的变异效应比较

通过分析转基因植株的株高、分蘖数、结实率、千粒重四个主要农艺性状的变异,比较基因枪法转化的基因表达框(仅含启动子、基因开放阅读框和终止子序列)和完整质粒两种基因载体形式对水稻农艺性状的变异效应.结果表明,与非转基因亲本相比,转基因植株的千粒重和分蘖数与对照无显著差异;株高变异也不大,17个转基因株系中,仅基因表达框转化的2个株系XFb-36和XFb-63株高变矮;而结实率变异最大,有9个转基因株系结实率显著降低,变异率达50%以上.总体上看,基因表达框和完整质粒两种基因载体形式对水稻农艺性状的变异效应差异不明显,基因枪转化对水稻农艺性状的变异效应主要表现为降低植株的育性,转基因水稻植株的农艺性状变异主要来源于转基因过程中组织培养引起的无性系变异.     

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.     

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.     

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.