农杆菌浸种处理对小麦种子萌发及幼苗生长发育的影响

以小麦品种(系)郑9023、中13和西农1376为主区,分别以农杆菌菌液浓度、农杆菌生长阶段和农杆菌侵染时间为副区,研究了农杆菌浸种处理后小麦种子萌发及幼苗生理生化特性变化.结果表明,小麦品种(系)分别与农杆菌菌液浓度、农杆菌生长阶段和侵染时间的互作效应不显著;随着农杆菌菌液浓度的增加,小麦种子发芽率、幼苗株高、鲜重、叶绿素含量呈下降趋势,MDA含量、白化苗率、卡那霉素抗性苗率则呈上升趋势,而POD活性则呈先升后降的趋势,小麦种子及幼苗受到的伤害逐渐增大,尤其是农杆菌菌液浓度超过1.5 OD后小麦幼苗受到的伤害更为明显;对数生长阶段的农杆菌活性最高,该阶段处理的小麦种子发芽率、幼苗株高、鲜重、叶绿素含量达到最低,而POD活性、MDA含量、白化苗则达到最大,小麦种子及幼苗受到的伤害也最为明显;随着农杆菌侵染时间的延长,小麦种子萌发及幼苗生长发育的各项指标表现出与农杆菌菌液浓度处理相同的变化趋势,尤其是侵染时间超过1.5 h时,小麦种子萌发及幼苗生长发育受到了显著抑制;各小麦不同品种(系)对农杆菌的反应存在一定基因型差异.研究发现,依据小麦种子萌发及幼苗生理生化特性影响和卡那霉素抗性苗率表现,浸种转化法中的农杆菌菌液浓度应控制在1.5 OD左右,生长阶段宜选用对数生长期,侵染时间以1.5 h较为适宜.     

超声波辅助农杆菌介导CP基因转化番小瓜(Carioca papaya L.)的有效方法

本研究探索了通过农杆菌介导,超声波辅助处理,转化番木瓜胚性愈伤组织,获得转基因植株的有效方法。分别将含有日本PLDMV 外壳蛋白基因(PTi-Epj-TL-PLDMV)和含有台湾PRSV 菌株、美国夏威夷PRSV 菌株、泰国PRSV 菌株及日本PLDMV 菌株的多元外壳蛋白基因编码序列(PTi-NP-YKT)插入双元载体质粒pGA482G,借助于农杆菌系LBA4404将双元载体上的外壳蛋白基因和新霉素磷酸转移酶基因(nptⅡ)转移到番木瓜品种Sunset 的胚性愈伤组织中,从而获得抗卡那霉素的转化再生植株。试验着重在转化方法上进行探索。结果表明,农杆菌过夜培养后,用高渗透压培养液(1/2 MS 6%蔗糖 1%葡萄糖,pH 5.7)调整至光密度OD_(600(?)m)=0.15-0.20,然后用该菌液感染材料30min,其间辅以超声波处理,可以大大提高转化效率。用15ml 无菌离心管装载胚性愈伤材料进行15s 的超声波处理,在80块被转化的胚性愈伤中获得21个CP 基因G 转化系(26.3%),而在对照处理64块胚性愈伤中仅获得1个转化系(1.6%);在经过15s 的超声波处理48块被转化的胚性愈伤中获得8个CP 基因B 转化系(16.7%),而在对照处理25块胚性愈伤中未出现转化系。上述操作方法用在两种CP 基因转化上均表现出相似的效果。试验还表明:120mg/L 是卡那霉素抗性筛选的最佳浓度。抗性筛选9个月后,在421块胚性愈伤组织中产生了42个抗卡那霉素的转化系。所获得的转基因植株分别用PCR 和Southern 印迹杂交进行了鉴定。     

超声波辅助农杆菌介导CP基因转化番木瓜(Carioca papaya L.)的有效方法

本研究探索了通过农杆菌介导,超声波辅助处理,转化番木瓜胚性愈伤组织,获得转基因植株的有效方法.分别将含有日本PLDMV外壳蛋白基因(PTi-Epj-TL-PLDMV)和含有台湾PRSV菌株、美国夏威夷PRSV菌株、泰国PRSV菌株及日本PLDMV菌株的多元外壳蛋白基因编码序列(PTi-NP-YKT)插入双元载体质粒pGA482G,借助于农杆菌系LBA4404将双元载体上的外壳蛋白基因和新霉素磷酸转移酶基因(nptⅡ)转移到番木瓜品种Sunset的胚性愈伤组织中,从而获得抗卡那霉素的转化再生植株.试验着重在转化方法上进行探索.结果表明,农杆菌过夜培养后,用高渗透压培养液(1/2 MS+6%蔗糖+1%葡萄糖,pH 5.7)调整至光密度OD600nm=0.15-0.20,然后用该菌液感染材料30min,其间辅以超声波处理,可以大大提高转化效率.用15m1无菌离心管装载胚性愈伤材料进行15s的超声波处理,在80块被转化的胚性愈伤中获得21个CP基因G转化系(26.3%),而在对照处理64块胚性愈伤中仅获得1个转化系(1.6%);在经过15s的超声波处理48块被转化的胚性愈伤中获得8个CP基因B转化系(16.7%),而在对照处理25块胚性愈伤中未出现转化系.上述操作方法用在两种CP基因转化上均表现出相似的效果.试验还表明120mg/L是卡那霉素抗性筛选的最佳浓度.抗性筛选9个月后,在421块胚性愈伤组织中产生了42个抗卡那霉素的转化系.所获得的转基因植株分别用PCR和Southern印迹杂交进行了鉴定.     

农杆菌介导的大麦Mlo反义基因转化小麦获得抗白粉病后代

从大麦‘斯特林’幼叶总RNA中分离Mlo基因cDNA完整编码区,反向连接到植物双元载体（pBI-121.2）35S启动子下游,通过农杆菌介导的苗端转化法获得两种小麦基因型（‘烟优2801’和‘烟优361’）的转基因小麦。T0代405株中有55株PCR检测阳性,平均转化率达到13.58%,T0和T1基因组DNA Southern杂交可以证明大麦Mlo基因片段已整合到小麦基因组中并可传递到后代。两种基因型的转基因小麦T0和T1植株在温室及大田中均表现出对白粉病抗性的提高。农杆菌介导的苗端转化法可以简单、快速、高效地获得转基因株系;排除体细胞变异对转基因植株的影响;克服基因型对农杆菌转化的限制,是小麦遗传转化的一种实用方法。     

番茄红素β-环化酶基因的玉米转化及 后代遗传分析

利用农杆菌介导法将番茄红素β-环化酶基因(Lycb)转入由玉米自交系天塔五号植株,分析基因在T0转化及后代的遗传情况,结果表明,在27株T0转基因植株中,PCR初步检测后8株呈阳性;将T1代转基因植株以株系为单位用200mg/L草铵膦抗性筛选后,收获抗性植株种子。T2代转基因植株进一步进行PCR、RT-PCR和田间草铵膦涂抹检测,结果表明,PCR、RT-PCR为阳性的6个株系植株均具有草铵膦抗性。选取6株阳性植株提取叶片总类胡萝卜素,经HPLC分析其β-胡萝卜素含量显著高于野生型,表明目的基因Lycb成功的转入玉米,并得到了稳定遗传。     

农杆菌共培养条件对小麦种子萌发和幼苗生理生化特性的影响

以‘郑9023’、‘中13’和‘西农1376’3个小麦品种(系)为主区,再分别以农杆菌共培养时间、共培养温度以及乙酰丁香酮(AS)浓度为副区,对农杆菌浸种处理后小麦种子萌发及幼苗生理生化特性进行了研究。结果表明,各小麦品种(系)与共培养时间、共培养温度以及AS浓度的互作效应不显著;随共培养时间的延长,小麦种子发芽率、幼苗株高、鲜重、叶绿素含量呈下降趋势,MDA含量、白化苗率、卡那霉素抗性苗率则呈上升趋势,而POD活性则呈先升后降的趋势,农杆菌对小麦种子及幼苗的伤害随共培养时间的延长而增大,且当共培养时间超过2 d时其伤害作用更为明显;共培养温度为25℃时,小麦种子发芽率、幼苗株高、鲜重、叶绿素含量达到或接近最低值,POD活性、MDA含量、白化苗率则达到最大值,此时农杆菌对小麦种子及幼苗不利影响最为明显;加入AS能促进农杆菌对小麦的侵染效果,并以150μmol/L AS的促进作用最强,对小麦种子萌发及幼苗生理生化指标的影响也最大;小麦不同品种(系)对农杆菌的反应存在一定基因型差异。依据共培养条件下小麦种子萌发和幼苗生理生化特性及卡那霉素抗性苗率综合分析认为,农杆菌浸种法转化小麦时较适宜的条件为:共培养时间应控制在2～3 d、共培养温度22～25℃、AS浓度为150μmol/L。     

超声波辅助农杆菌介导CP基因转化番木瓜（Carioca papaya L.）的有效方法

本研究探索了通过农杆菌介导,超声波辅助处理,转化番木瓜胚性愈伤组织,获得转基因植株的有效方法。分别将含有日本PLDMV外壳蛋白基因(PTi-Epj-TL-PLDMV)和含有台湾PRSV菌株、美国夏威夷PRSV菌株、泰国PRSV菌株及日本PLDMV菌株的多元外壳蛋白基因编码序列(PT—NP—YKT)插入双元栽体质粒pGA482G,借助于农杆菌系LBA4404将双元载体上的外壳蛋白基因和新霉素磷酸转移酶基因(nptⅡ)转移到番木瓜品种Sunset的胚性愈伤组织中,从而获得抗卡那霉素的转化再生植株。试验着重在转化方法上进行探索。结果表明,农杆菌过夜培养后,用高渗透压培养液(1／2MS 6％蔗糖 1％葡萄糖,pH5．7)调整至光密度OD600nm=15-0．20,然后用该菌液感染材料30min,其间辅以超声波处理,可以大大提高转化效率。用15ml无菌离心管装载胚性愈伤材料进行15s的超声波处理,在80块被转化的胚性愈伤中获得21个CP基因G转化系(26．3％),而在对照处理64块胚性愈伤中仅获得1个转化系(1．6％);在经过15s的超声波处理48块被转化的胚性愈伤中获得8个CP基因B转化系(16．7％),而在对照处理25块胚性愈伤中未出现转化系。上述操作方法用在两种CP基因转化上均表现出相似的效果。试验还表明：120mg／L是卡那霉素抗性筛选的最佳浓度。抗性筛选9个月后,在421块胚性愈伤组织中产生了42个抗卡那霉素的转化系。所获得的转基因植株分别用PCR和Southern印迹杂交进行了鉴定。     

真空渗入转化法中农杆菌在植株体内的分布和活力变化

以真空渗入处理后的白菜植株为材料,采用组织化学染色法和细菌平板培养的方法,研究了农杆菌在植株体内的分布特点及其活力变化。结果表明：不同器官中农杆菌的分布量不同,以花器官中分布最多,叶中次之,茎中最少;农杆菌存在于细胞间隙中,维管束及其周围分布较集中,在胚珠中大量分布。处理后植株体内,各器官中农杆菌的生活力及其数量都随时间延长而减少,但是花器官中的农杆菌存活量较大,处理15d后的花蕾中仍然有一定量(约10.3个CFU/g组织)具有活力的农杆菌存在。讨论了这些研究结果在揭示真空渗入转化法的转化过程和提高转化频率中的意义。     

提高农杆菌基因转化率方法的研究

通过甘蓝型油菜带柄子叶转化过程中转化受体是否进行预培处理、转化前菌液的对数浓度确定以及合适的再生筛选体系的摸索,研究了提高农杆菌基因转化效率的方法。试验结果表明：转化受体经过MD培养基3d的预培处理,用处于对数生长期OD600为0．6～0．8的菌液稀释至所需浸染浓度,浸染所需时间共培后,转入降低卡那霉素浓度的初筛分化培养基中,再经过后期较高浓度的卡那霉素继代筛选培养,大大提高了转化后卡那霉素抗性绿苗的得率。此研究不仅优化了油菜的转化体系和提高了油菜的转化效率,而且为农杆菌转化其它不同物种的受体材料提供了可指导的借鉴。     

农杆菌介导的半夏凝集素基因(Pinellia Ternate Agglutinin Gene,pta)对小麦的遗传转化及鉴定

以甘肃主要推广春小麦品种陇春22幼胚为转基因受体材料,建立了农杆菌介导的小麦遗传转化体系。以预培养4天的幼胚愈伤组织为受体,C58c1农杆菌菌株为供体,将含有半夏凝集素基因的重组质粒pBIpta转入了小麦,经G418 25 mg/L抗性筛选、PCR检测和荧光定量PCR检测共获得转基因植株3株,外源基因的插入拷贝数分别为2、1、3。同时对转基因小麦的T₁代植株进行了PCR检测和抗虫性分析,表明半夏凝集素基因在转基因植株的后代中得到了遗传并有一定的抗蚜虫作用。     

高效烟草遗传转化体系的建立及甜蛋白基因的导入

以烟草无菌茁叶片为外植体,通过根癌农杆菌LBA4404介导法,将Thamnatin基因导人烟草中,经梯度卡那霉素(Kana-mycin,Km)筛选,获得可在含75mg／L、100mg／L Km选择生根培养基上再生的抗性植株,其中部分Km抗性植株经PCR检测为阳性,转化率为31．3％,初步鉴定已成功地建立了烟草遗传转化系统,为进一步探讨甜蛋白在植物中的转化和表达情况奠定基础。     

Preliminary Study on Transformation of Wheat Apical Point

Apical points of young seedlings of wheat (Triticum aestivum) cultivar "Jing 411” and somatic calli of cultivar "FK8” were transformed with plasmids pBI121 and (or) pBIAH-A+ by using microprojectile bombardment. Histochemical assay of GUS activity showed positive reaction on some of the transformation processed apical points and calli. This demonstrated that foreign genes were introduced into the apical meristematic cells as well as the callus cells. The plantlets of cv. "Jing 411” survived after apical point transformation with pBIAH-A+ were transplanted into the field and the progenies were screened with kanamycin. 4 % of the screened seeds germinated into green seedlings with kanamycin resistance. Dot hybridization of total DNA from kanamycin resistant plants showed the existence of foreign DNA in some of the detected plants.     

Waxy基因的RNA沉默使转基因小麦种子中直链淀粉含量下降

通过RNAi策略转化小麦,以降低小麦种子中直链淀粉的含量。小麦中直链淀粉合成的关键酶是颗粒结合型淀粉合成酶（Granule—bound starch synthase l,GBSSI,即WAXY蛋白）,通过RT—PCR方法从小麦种子中分离出Waxy基因。Southern杂交分析表明,在基因组中存在3个Waxy基因。Northern杂交分析显示出在授粉后的小麦种子中检测到Waxy mRNA。利用RNA沉默策略,将Waxy编码区683bp的正向和反向片段以及150bp内含子,连接于表达载体pCAMBIA3300中玉米ubil启动子下游。以扬麦10号授粉后15d的幼胚为外植体,利用农杆菌介导的方法进行转化。通过PCR、RT-PCR和叶片离体褪绿实验鉴定出4株转基因植株。小麦胚乳I2-KI染色和直链淀粉含量测定表明这4株转基因植株直链淀粉含量明显下降。研究结果表明Waxy基因的RNA沉默使转基因小麦种子直链淀粉的含量下降。     

甲型肝炎病毒衣壳蛋白融合基因植物表达载体的构建 与遗传转化柑桔的研究

基因工程领域的研究进展使得植物体成为具有重要经济价值的药用蛋白的生产体系。以含甲型肝炎病毒结构基因cDNA的克隆载体pCDNAⅡA16为模板,用甲型肝炎病毒衣壳蛋白融合基因特异引物进行PCR扩增,得到全长2.2kb衣壳蛋白融合基因序列。经测序鉴定后正向克隆于植物表达载体pBI121中,衣壳蛋白融合基因位于pBI121质粒T-DNA左右边界区间内,处于CaMV35S启动子控制之下。经限制性内切酶分析和PCR鉴定后利用冻融法将重组质粒pBI121-A导入根癌农杆菌LBA4404。以锦橙 (Citrus. Sinensis Osbeck) 上胚轴为转化材料,通过根癌农杆菌介导法将衣壳蛋白融合基因转 化到植物基因组中。120株转化外植体经卡那霉素50 mg/L筛选,其中13株生长状况良好未出现白化现象的拟转化芽微嫁接到实生砧木继续培养。PCR分析证明,13株拟转化植株中有5株植物基因组中已导入甲型肝炎病毒衣壳蛋白融合基因,转化率为4.1%。此研究是对遗传转化柑桔表达外源蛋白的初步探讨,为进一步研究食用疫苗开辟了新途径。Abstract: The use of edible plants for the production and delivery of vaccine proteins could provide an economical alternative to fermentation systems. The construction of the plant expression vector pBI121-A was reported, which contained a fusion gene encoding hepatitis A capsid proteins. The gene was located between the left and right Ti border sequences under the control of CaMV35S promoter. The vector was identified via PCR and restriction enzyme analysis and was introduced into Agrobacterium tumerifacience LBA4404. The transgenic Citrus plants were produced by Agrobacterium-mediated transformation of epicotyl segments. 13 putatively transformed plants through the kanamycin selection were micrografted onto the seedlings. The presence and integration of the transgene had been verified by PCR analysis. The result showed that five transformants were integrated and the transformation efficiency was 4.1%.     

农杆菌介导抗储粮害虫转基因小麦(Triticum aestivum L.)的获得及分析

以本实验室选育的小麦优良品系的胚性愈伤组织为材料,采用农杆菌介导将抗虫基因豇豆胰蛋白酶抑制剂基因CpTI转入小麦培养细胞,经筛选获得抗卡那霉素的愈伤组织并再生植株。经PCR和实时PCR检测、PCR-Southern和Southernblot验证,确定了3株独立再生植株为含有CpTI的转基因植株。农杆菌菌浓度、侵染时间及转化处理方式对小麦转化率均有明显影响。3株转基因植株正常可育并结籽,形成转基因株系。外源基因在转基因植株T1代中的分离呈多样性,部分株系(转基因株系T-Ⅰ、T-Ⅲ)表现出孟德尔遗传规律。抗虫试验表明,3株转基因植株T2代籽粒对储粮害虫麦蛾具有一定的抗性,转基因株系T-Ⅰ、T-Ⅱ、T-Ⅲ及非转基因植株的T2代籽粒虫蛀率分别为19·8%、21·9%、32·9%和58·3%。转基因植株T1代群体农艺性状调查显示,3个株系具有良好的农艺性状,为小麦的遗传改良提供了新的种质抗虫材料。     

Early infection of scutellum tissue with Agrobacterium allows high-speed transformation of rice

Several approaches have recently been adopted to improve Agrobacterium-mediated transformation of rice, both to generate the large number of T-DNA insertion plants needed for functional analysis of the rice genome, and for production of rice with additional agronomical value. However, about 3 months of in vitro culture is still required for isolation of transgenic rice plants. Here, we report the competency of scutellum tissue from 1-day pre-cultured seeds for Agrobacterium-mediated transformation. Furthermore, early infection of rice seeds with Agrobacterium enhanced efficient selection of transformed calli. Using our system, we successfully regenerated transgenic rice plantlets within a month of the start of the aseptic culture of mature seeds. Our new system should reduce the somaclonal variation accompanying prolonged culture of rice cells in the dedifferentiated state and facilitate the molecular breeding of rice.     

Agrobacterium tumefaciens-mediated genetic transformation of mungbean (Vigna radiata L. Wilczek) — a recalcitrant grain legume

Agrobacterium-mediated transformation of Vigna radiata L. Wilczek has been achieved. Hypocotyl and primary leaves excised from 2-day-old in-vitro grown seedlings produced transgenic calli on B₅ basal medium supplemented with 5×10⁻⁶ M BAP, 2.5×10⁻⁶ M each of 2,4-D and NAA and 50 mg l⁻¹ kanamycin after co-cultivation with Agrobacterium tumefaciens strains, LBA4404 (pTOK233), EHA105 (pBin9GusInt) and C58C1 (pIG121Hm) all containing β-glucuronidase (gusA) and neomycin phosphotransferase II (nptII) marker genes. Transformed calli were found resistant to kanamycin up to 1000 mg^.l⁻¹. Gene expression of kanamycin resistance (nptII) and gusA in transformed calli was demonstrated by nptII assay and GUS histochemical analysis, respectively. Stable integration of T-DNA into the genome of transformed calli of mungbean was confirmed by Southern blot analysis. Transgenic calli could not regenerate shoots on B₅ or B₅ containing different cytokinins or auxins alone or in combination. However, for the first time, transformed green shoots showing strong GUS activity were regenerated directly from cotyledonary node explants cultured after co-cultivation with LBA4404 (pTOK233) on B₅ medium containing 6-benzylaminopurine (5×10⁻⁷ M) and 75 mg l⁻¹ kanamycin. The putative transformed shoots were rooted on B₅+indole-3-butyric acid (5×10⁻⁶ M) within 10–14 days and resulted plantlets subsequently developed flowers and pods with viable seeds in vitro after 20 days of root induction. The stamens, pollen grains and T₀ seeds showed GUS activity. Molecular analysis of putative transformed plants revealed the integration and expression of transgenes in T₀ plants and their seeds.     

Genetic transformation of Nicotiana africana Merxm. with plasmids containing lox recombination

An efficient genetic transformation method for african tobacco Nicotiana africana Merxm. has been established. African tobacco is a valuable source for cytoplasmic male sterility (CMS) and nuclear encoded resistance to potato virus Y (PVY). N. africana transgenic plants have been obtained using both Agrobacterium-mediated and direct transformation of leaf explants with gold particle bombardment using particle inflow gun. Plasmid vectors containing phosphinothricin resistance gene (bar gene) coding region without promoter and independent 35S promoter between lox sites (lox-bar-35S-lox) and nptII gene were used. Transgenic plants were selected according to growth capacity on the selective medium containing 50 mg/l kanamycin. PCR analyses of kanamycin-resistant plants confirmed the presence of nptII and bar genes in their genome. Agrobacterium-mediated transformation of root explants has proved to be the most efficient transformation method for N. africana.     

Evaluation of transgenic canola plants under field conditions.

Eleven independent transgenic canola (Brassica napus ssp. oleifera L. cv. Westar and Regent) lines were evaluated in the field. The plants carried a neomycin phosphotransferase (NPTII) gene for kanamycin resistance that was introduced via Agrobacterium-mediated transformation. NPTII enzyme assays, Southern blot by hybridizations and progeny analysis, confirmed the stable, heritable integration and expression of the introduced NPTII gene. A number of agronomic characteristics evaluated under field conditions, including maturity yield, and oil and protein content, were all statistically comparable between the transformed and nontransforemd platns. These results indicate that canola can be genetically engineered successfully, and that the Agrobacterium-based transformation system employed does not induced any adverse effects on the intrinsic agronomic and qualitative traits critical to the agricultural industry.