ACC合酶cDNA克隆及其对美洲黑杨体内乙烯产生的反义抑制

利用ＲＴＰＣＲ技术克隆了大豆ＡＣＣ合酶基因ＧＭＣＡＣＣＳＩ编码区１５ｋｂ的ｃＤＮＡ片段,经酶切图谱分析和序列分析鉴定后,反向插入到２元载体ｐＢｉｎ４３８中,构建了表达ＡＣＣ合酶反义ＲＮＡ的植物表达载体,转化农杆菌后用该农杆菌侵染美洲黑杨叶片,在含卡那霉素的ＭＳ培养基上选择转化子和植株再生,通过ＰＣＲ检测从抗卡那植株中选到１５株转基因植株,Ｓｏｕｔｈｅｒｎ杂交分析初步确证了外源基因是以单拷贝插入到杨树基因组ＤＮＡ中;对杨树幼苗乙烯释放的测定结果表明转基因杨树幼苗的乙烯释放量为对照的２２％。     

ACC氧化酶cDNA克隆及其对美洲黑杨体内乙烯产生的反义抑制

利用ＲＴ－ＣＲ技术克降了番茄ＡＣＣ氧化酶基因ＬＥＥＴＨＹＢＲ编码区０．９ｋｂ的ｃＤＮＡ片段,经酶切图谱分析笔序列分析鉴定后,反向插入到植物表达载体ｐＢｉｎ４３８中,构建了表达ＡＣＣ氧化酶反义和ＲＮＡ的二元载体。     

转Bt基因和蛋白酶抑制剂基因杨树抗虫性的研究

虫害是杨树造林生产中所面临的一大难题,通过农杆菌介导,将经过人工改造的苏云金杆菌杀虫结晶蛋白基因转化杨树,得到杨树再生植株,然后,再将蛋白酶抑制剂基因（ＰＩ）导入已含Ｂｔ基因的转基因杨树,经ＰＣＲ检测和Ｓｏｕｔｈｅｒｎ杂交分析证明,最终获得既含有Ｂｔ基因又含有蛋白酶抑制基因的转基因杨树植株。利用这种杨种叶片饲同喂舞毒蛾幼虫的杀虫试验结果表明,转基因杨树具有明显的杀虫活性,同时表明含有Ｂｔ基因和蛋白     

抗虫转基因欧美杨的培育

以携带３５Ｓ－Ω－Ｂ．ｔ－Ｎｏｓ嵌合基因的双元载体农杆菌ｐＢ４８．２１４和ｐＢ４８２．１５（Ｂ．ｔ基因片断长度分别为２．１ｋｂ和１．８ｋｂ），转化欧美杨叶片和茎段外植体，共获得２２５株转化再生植株。以舞毒蛾（ＬｙｍａｎｔｒｉａｄｉｓｐａｒＬｉｎｎａｅｕｓ）幼虫进行生物测定后，获得杀虫率分别为５５—８０％的抗虫转基因植株共３株（Ｒ－１３５，Ｒ－１２３和Ｒ－１４０），经组培苗和苗圃移栽植株的ＰＣＲ分析，苗圃移栽植株ＰＣＲ产物杂交和Ｓｏｕｔｈｅｒｎｂｌｏｔ分析表明，Ｂ．ｔ毒蛋白基因已整合到上述抗虫植株的细胞ＤＮＡ中，并表达出杀虫活性。     

杨树细菌溃疡病的研究概况

杨树细菌溃疡病的研究概况刘晓光，高克祥（河北林学院林学系保定０７１０００）关键词杨树，细菌溃疡，研究进展中图分类号Ｓ７６３．１３ＲＥＶＩＥＷＯＮＴＨＥＢＡＣＴＥＲＩＡＬＣＡＮＫＥＲＯＦＰＯＰＬＡＲＬｉｕＸｉａｏｇｕａｎｇＧａｏＫｅｘｉａｎｇ（Ｆｏｒｅ...     

桦木次生林经营效果分析及对策

桦木次生林经营效果分析及对策姚庆玉（崇礼县国营和平林场０７６３５０）关键词桦木次生林，经营效果，对策中图分类号Ｓ７９２．１５ＡＮＡＬＹＳＩＳＯＦＭＡＮＡＧＩＮＧＥＦＦＥＣＴＡＮＤＣＯＵＮＴＥＲＭＥＡＳＵＲＥＳＦＯＲＳＥＣＯＮＤＡＲＹＢＩＲＣＨＦＯＲＥ...     

雄性毛白杨离体叶片再生及抗虫基因转化

用雄性毛白杨（Ｐｏｐｕｌｕｓｔｏｍｅｎｔｏｓａ）试管植株无菌叶片作外植体，筛选出诱导不定芽分化、增殖和生根培养基。在组培室的光照条件下培养３７ｄ，平均每片叶产生２５个不定芽。已建立雄性毛白杨最佳转化和再生系统。用含有完全改造的Ｂｔ抗虫基因表达载体ｐＢ４８．７和部分改造的Ｂｔ抗虫基因表达载体ｐＢ４８．６转化雄性毛白杨，经在含有卡那霉素的培养基上选择和ＰＣＲ检测，已获得初步确定的转基因植株。     

生物技术林业应用研究工作进展

生物技术林业应用研究工作进展郑均宝（河北林学院基础部保定０７１０００）关键词木本植物，茎尖培养，转基因细胞系，胚培养，离体根培养，玻璃化中图分类号：Ｓ７２２．３７ＡＤＶＡＮＣＥＳＯＦＢＩＯＴＥＣＨＮＩＱＵＥＡＰＰＬＩＣＡＴＩＯＮＩＮＦＯＲＥＳＴＲＥＳ...     

84K杨PagMSBP1/2a基因对木质素合成的影响

【目的】膜类固醇结合蛋白(MSBP)是一类定位在细胞质膜上的类固醇受体蛋白,通过响应激素调节信号、参与类固醇代谢影响植物生长发育过程。研究杨树MSBP对生长发育过程的影响,尤其是对木材形成过程的作用,为杨树木材品质改良提供支撑。【方法】以拟南芥MSBP1蛋白序列在毛果杨、银腺杨84K基因组中进行同源序列比对,获取杨树MSBP的核苷酸和氨基酸序列,构建系统进化树并绘制基因结构图谱;利用qPCR分析杨树MSBP基因在84K杨顶芽、根、茎、叶中的相对表达量;在AspWood表达谱数据中分析同源基因在木材形成中的表达模式;构建35 S∷GFP-PagMSBP1/2a植物表达载体,通过农杆菌介导瞬时转化烟草叶片,激光共聚焦显微镜观察其亚细胞定位情况;构建35 S∷PagMSBP1/2a植物表达载体,利用农杆菌介导的叶盘法转化84K杨,通过震荡切片观察转基因和对照植株茎段木质部差异;采用乙酰溴法和间苯三酚染色法,分析转基因和对照植株的木质素含量以及木质素在杨树茎段维管组织中的分布。【结果】同源比对鉴定出杨树7个MSBP成员,根据进化关系和基因结构对杨树基因进行命名,其中与AtMSBP1、AtMSB...     

论文献课教学内容和实习教学

论文献课教学内容和实习教学刘景会，王晓苏（河北林学院图书馆，保定０７１００１）关键词文献检索课；教学内容；实习教学中图分类号Ｇ４２３ＡＤＩＳＣＵＳＳＩＯＮＡＢＯＵＴＴＨＥＴＥＡＣＨＩＮＧＣＯＮＴＥＮＴＡＮＤＰＲＡＣＴＩＣＥＯＦＬＩＴＥＲＡＴＵＲＥＣＯ...     

香石竹ACC氧化酶重复基因植物表达载体的构建及转基因植株的获得

香石竹ACC氧化酶基因从核NDA中克隆之后,将其首先构建成正义及反义的单拷贝植物表达载体,在此基础上又同向插入一个ACO基因片段,从而获得ACO的正义重复基因和反义重复基因植物表达载体,所得载体已通过酶切分析和PCR鉴定。将该表达载体导入根癌农杆菌LBA4404菌株,并转化香石竹品种Master、爱卡迪幼叶,经PCR检测和Southem杂交鉴定,获得了8株转化植珠。     

Molecular Cloning of a Cellulose Synthase Gene PtoCesA1 from Populus tomentosa and Its Genetic Transformation in Tobacco

A 3 125 bp cellulose synthase gene, PtoCesA1, which has a 98% identity to PtrCesA1 from Populus tremuloides, was cloned from cDNA prepared from secondary xylem of P tomentosa. Four anti-expression vectors with different fragments of PtoCesAl, named as pBIPF, pBICC1, pBIPR and pBIBR, were constructed. Some traits of transformed tobacco of pBICC1, pBIPR and pBIBR differed from wild types, such as small leaves, ＂dwarf＂ phenotype and thinner xylem and fiber cell walls than wild plants consistent with a loss of cellulose. It indicated that the growth of transgenic tobacco was restrained by the expression of anti-PtoCesA1. Transgenic tobacco was obtained and the contents of cellulose and lignin were analyzed as well as the width and length of fiber cells, and xylem thickness for both transgenic and control plants. Transformed tobacco showed a different phenotype from control plants and it implied that PtoCesA1 was essential for the cellulose biosynthesis in poplar stems.     

抗虫转基因杨树的培育及其抗虫性初步研究

以杨树优良新品种欧美杨107(Populus×euramericanacl."74/76")为试材,通过根癌农杆菌(Agrobacteriumtumefaciens)介导法,将Bt毒蛋白基因导入107杨的叶片及茎段外植体,经潮霉素抗性筛选,获得了转基因再生植株。提取转基因植株的总DNA,经PCR扩增,部分植株呈阳性反应,证明目的基因已经整合到杨树基因组中。以转基因杨树叶片饲喂天幕毛虫幼虫的杀虫试验结果表明,转基因杨树表现出一定的杀虫活性。     

RNA干涉培育低木质素杨树

采用改良的CTAB法提取南林95杨的基因组DNA,经PCR扩增得到肉桂酰辅酶A还原酶CCR基因的第4个外显子部分序列,通过中间载体pUCCRNAi,构建含正反向干涉片段的pBI121表达载体,导入农杆菌LBA4404。利用叶盘法侵染南林95杨,获得3株转基因植株,经分子鉴定证实干涉片段已导入南林95杨。测定Klason木质素及综纤维素含量的结果显示:转基因植株Klason木质素含量与对照相比平均降低了9.86%,综纤维素含量与对照相比平均增加了3.17%,纤维长宽比明显增加,均表明转基因植株更有利于造纸。     

杨树木质素合成酶hct基因的克隆及核苷酸序列分析

根据毛果杨全序列（AC185363.2）中唯一具有转移酶功能的保守区设计引物,以正在分化的2年生欧美杨107次生木质部总RNA为模板经RT-PCR扩增出hct基因片段,与pMD20-T载体连接,重组质粒经特异引物扩增、限制性内切酶酶切和测序鉴定。结果表明,扩增片段长度为709bp,包含一个708bp的开放阅读框,与毛果杨全序列及HCT2（EU603314）的同源性均为98%,编码的氨基酸序列与毛果杨HCT2氨基酸（ACC63883.1）的同源性为98%,断定我们克隆的cDNA为hct,属于转移酶超家族,GenBank登录号为FM202091,该重组质粒命名为pMD20-T-hct。     

Constitutive Expression of Sense & Antisense PtAP3, an AP3 Homologue Gene of Populus tomentosa, Affects Growth and Flowering Time in Transgenic Tobacco

To analyze the function of PtAP3, an APETALA3 (AP3) homologue gene isolated from Populus tomentosa Carr., the full length sequence (1 797 bp) and a fragment (870 bp) of PtAP3 were fused to a CaMV 35S promoter of pBI121 to generate the sense and antisense constructs of PtAP3. These constructs were transformed into tobacco by Agrobacterium infection of leaf disks and selection on kanamycin medium. Some sense and antisense transgenic tobacco plants were obtained by PCR and Southern blot analysis. Great phenotypic differences in transgenic tobacco plants were observed. Almost all of sense PtAP3 to transgenic tobaccos showed a higher growth rate than those of antisense transformants and a few developed pregnancy earlier than wild type seedlings and antisense transformants under the same conditions.     

Constitutive Expression of Sense & Antisense PtAP3, an AP3 Homologue Gene of Populus tomentosa, Affects Growth and Flowering Time in Transgenic To-bacco

To analyze the function of PtAP3, an APETALA3 (AP3) homologue gene isolated from Populus tomentosa Carr., the full length sequence (1 797 bp) and a fragment (870 bp) of PtAP3 were fused to a CaMV 35S promoter of pBI121 to generate the sense and antisense constructs of PtAP3. These constructs were transformed into tobacco by Agrobacterium infection of leaf disks and selection on kanamycin medium. Some sense and antisense transgenic tobacco plants were obtained by PCR and Southern blot analysis. Great phenotypic differences in transgenic tobacco plants were observed. Almost all of sense PtAP3 to transgenic tobaccos showed a higher growth rate than those of antisense transformants and a few developed pregnancy earlier than wild type seedlings and antisense transformants under the same conditions.     

Antisense expression of a caffeic acidO-methyltransferase ofStylosanthes humilis in transgenic poplar: Effect of expression onO-methyltransferase activity and lignin composition

Poplar (Populus tremula) was transformed with a construct carrying an antisense caffeic acidO-methyltransferase (COMT) cDNA (pOMT8) from a tropical pasture legume,Stylosanthes humilis. pOMT8 shows 83% overall homology to the corresponding COMT gene (pPCLA) of poplar. Of the 200 putatively-transformed plants regenerated on selective media after co-cultivation of poplar stem explants withAgrobacterium tumefaciens harbouring a CaMV 35S-antisensepOMT8 construct, a subset of 20 plants were randomly chosen for further analysis. PCR and Southern blot analysis demonstrated the stable integration of T-DNA into the genome of these plants. Antisense expression ofpOMT8 resulted in reductions in total COMT activity in the majority of the transgenic plants with the lowest total COMT activities (61–70% of untransformed control plants) being observed in four transgenic plants. The composition of lignin in transgenic plants was also changed, as detected by reductions in the content of syringyl units using infrared spectroscopy. However, no changes were found in the amount of insoluble lignin in transgenic plants as compared to untransformed control plants. These results indicate the potential of thepOMT8 gene to partially suppress COMT activity and modify the composition of lignin in transgenic poplar. This work was partly supported by General Management of Turkish Pulp and Paper Mills.     

杨树微管功能研究植物表达载体构建及验证

[目的]分别构建84K杨的微管蛋白TUA5和TUB16与红色荧光蛋白mCherry融合的植物过表达载体,瞬时表达验证载体在植物体内表达后的荧光信号,为研究杨树微管功能奠定基础。[方法]以毛果杨微管蛋白TUA5和TUB16的基因序列为模板,设计84K杨的皿5和7TZB76基因的引物,提取野生型84K杨的RNA并反转录成cDNA,同源克隆得到84KTUA5和84KTUB16基因,分别连接在pCAMBIA 1300载体mCh-ep荧光标签的N,端和C端,转化到大肠杆菌TOP10感受态细胞中,通过菌落PCR和测序鉴定获得阳性单克隆,并通过电击法将重组质粒转化到农杆菌GV3101中,瞬时转化烟草后进行荧光观察。[结果]克隆得到了84KTUA5和84KTUB16基因,成功与pCAMBIA 1300-mCherry载体连接,烟草瞬时表达荧光观察结果显示:仅目的基因与mCherry标签C,端相连的融合蛋白能够成功表达,且荧光明显。[结论]成功构建了84K杨皿5和TUB16基因与pCAMBIA 1300-mCherry的融合表达载体,为进一步研究杨树微管功能提供了背景材料。