转基因番木瓜的抗病性及分子鉴定

对T1代转番木瓜环斑病毒(PRV)复制酶(RP)突变体基因的两个番木瓜株系,进行了抗病性和分子生物学分析。结果表明,转基因番木瓜对PRV抗性达到高抗或免疫,目的基因RP遗传至转基因后代并在RNA水平表达,PCR可检测到CaMV35S启动子序列、标记基因NPTII。 Abstract:Virus resistance in field and molecular biological characterizations of the transgenes were analyzed for two lines of T1 generation of transgenic papaya with the replicase mutant gene from papaya ringspot virus (PRV).The transgenic plants showed highly resistant or immune against PRV.Results indicated that the transgenes inherited to and expressed at RNA level in the progenies.     

花粉管通道法介导PRSV-CP基因dsRNA转化番木瓜

以番木瓜‘蔬罗Ⅰ号’植株为受体材料,采用花粉管通道技术将番木瓜环斑病毒外壳蛋白(PRSV-CP)基因3′-端同源序列dsRNA转入番木瓜子房中。用PCR方法对T0代种子进行了分子检测,获得53株转基因番木瓜,转化率达到8.9%;对获得的T0代转基因番木瓜进行了田间抗病性鉴定,结果表明,转基因番木瓜植株对番木瓜环斑病毒(PRSV)表现为不同程度的抗性,可以推迟发病。     

转柽柳晚期胚胎富集蛋白基因烟草T1代的耐盐性评价

目的:验证转柽柳晚期胚胎富集(LEA)蛋白基因烟草T1代的耐盐性。方法:采用盐胁迫方式,对转柽柳LEA蛋白基因烟草T1代的6个株系及非转基因对照烟草T1代进行不同浓度NaCl胁迫处理,分析了NaCl胁迫下转基因烟草的生长量、根系的发育及盐害程度。结果:各转基因烟草T1代组培苗在150mmol/L的NaCl培养基上根系生长良好,平均增重(鲜重)是非转基因对照的7.72倍,平均高生长是非转基因对照的3.51倍,盐害指数低于或等于50%;而非转基因对照烟草T1代组培苗生长缓慢,根系几乎不能生长发育,盐害指数达65%。结论:柽柳LEA蛋白基因的导入提高了T1代烟草的耐盐性。     

转不可翻译PVY^N CP基因烟草的抗病性分析

我们曾报道表达不可翻译PVY^N CP基因的转基因烟草抗病性是由RNA介导的,其抗病性类似于转录后的基因沉默(PTGS)。本研究以这类不同抗性的T0代转基因烟草植株为材料,对自交后的T1代转基因植株的遗传和抗病性进行了分析,并选取部分T1代抗病株系自交留种。对T2代RNA介导抗病性转基因植株进行了分子分析和一系列抗病性研究。结果表明,含1—2个转基因拷贝的T0代感病植株,在T1代中的Km抗性分离符合单位点插入的3：1的遗传规律;含3个或3个以上转基因拷贝的T0代中抗或高抗植株,在T1代中的Km抗性分离符合多位点插入的15：1或63：1的遗传规律。大多数T1、T2代转基因植株的抗病性与转基因拷贝数成正相关,转基因在T1、T2代植株中能够转录表达,且转基因植株之间转基因mRNA在细胞质中的积累水平与转基因植株的抗病性成负相关。转基因植株的抗病性能够在T1、T2代中遗传,且T2代转基因植株的抗病性具有以下特征：1)既抗病毒粒体又抗病毒RNA的侵染,且这种抗病性不受接种物剂量的影响;2)抗病谱较窄,只对PVY的某些株系具有高度抗病性;3)与传毒方式无关,既抗摩擦接种又抗带毒蚜虫接种;4)与植株的发育阶段没有关系。     

转反义蜡质基因‘湘晴’及其杂交稻米的直链淀粉含量研究

利用根癌农杆菌介导将反义蜡质基因(anti-Waxy)导入三系恢复系湘晴水稻获得转基因植株.从T1代外源基因呈单拷贝整合的转基因湘晴水稻中选取3个稻米直链淀粉含量降低较明显的单株继续种植得到纯合后代.以纯合转基因植株及湘晴水稻(对照)为恢复系分别与寒丰不育系杂交,获得4组杂交稻后代(F2).3个T3代纯合转基因湘晴水稻糙米(T4)直链淀粉含量分别为14.42%、13.96%和14.72%,对照为16.04%;3组由转基因湘晴水稻为父本杂交制种后代(F2)糙米直链淀粉含量分别为14.53%、13.77%和14.64%,对照杂交稻(F2)为16.22%.研究表明,导入湘晴水稻中的反义蜡质基因不仅能够降低湘晴稻米直链淀粉含量,还能够进一步抑制杂交后代的稻米直链淀粉合成.     

转基因玉米株系纯合系选育及其农艺性状表现

目的：以携带水稻矮缩病毒（RDV）运动蛋白缺陷型（MP-）基因的转基因玉米种子T0为试验材料,通过分子分析、抗病鉴定及农艺性状筛选得到转基因纯合株系。方法：首先对转化种子进行潮霉素抗性筛选,其后对各代转基因材料进行PCR检测、农艺性状调查和抗病鉴定。结果：从645粒T0转化种子得到抗潮霉素植株246株,即T1转基因植株。T1、T2、T3、T4代材料的PCR检测阳性率分别为56.9%、83.9%、94.6%和99.8%,证明RDVMP-基因已被导入玉米自交系中,且目的基因可以稳定遗传到转基因植株及其后代。田间人工接种抗病鉴定结果表明,经过连续筛选,转基因后代植株（系）的抗病性不断提高,T1、T2、T3和T4代中的高抗病材料分别占总材料数的8.9%、31.5%、70.7%和100%;所选纯合系连续2年的发病率均为0,抗病性比相应对照株系提高4级。农艺性状调查结果表明,转基因株系的株高比对照株系高15.0～33.4cm,穗位高提高13.4～20.2cm,;穗长增加2.0～3.8cm,穗粒数增加10.2～22.8粒。结论：根据分子检测、田间抗病鉴定及农艺性状鉴定结果,选育到96C0502、96C0507和96C0513等抗矮花叶病转基因玉米纯合株系。     

转基因抗衰老番茄F2代果实品质研究

为了研究转基因抗衰老番茄F2代果实贮藏后品质,本研究以丽春番茄为对照,转反义NR、F1'、F2和F3基因番茄为试材,分析了采后3~20d果实总糖、总酸、Vc的含量变化,结果为:转基因番茄除F2基因型总糖含量显著低于对照外,其余均无差异;总酸含量在贮藏20d后除F3与对照无差异外,均高于对照;各个基因型番茄在贮藏期Vc含量均高于对照41%~72%。综合分析结果显示F3即转反义ACS-CTR双价番茄果实贮藏后品质最好,F2(CTR)最差,NR、F1'(ACS)与对照相同。     

土壤水分对富士苹果果实外观品质的影响

为探究不同果园土壤水分处理对富士苹果果实品质的影响,并重点探究外观品质的差异,筛选出最适宜生产中采用的控水方法,以‘烟富3号’富士苹果品种作为材料,按照土壤相对含水量设置两组水分处理,一组为LT1(55%~65%)、LT2(65%~75%)和LT3(75%~85%),另一组为LT4(前期55%~65%,后期45%~55%)、LT5(前期65%~75%,后期55%~65%)和LT6(前期75%~85%,后期65%~75%),以生理落果为节点,划分处理前期和后期,以普通果园水分管理作为对照处理(LCK),分别测定土壤含水量以及相应果实可溶性固形物含量,计算果实裂纹指数、着色指数和光洁指数,并用扫描电镜观测果皮超微结构。结果表明:(1)LT1、LT4、LT5处理的果实可溶性固形物含量比LCK分别显著提高13.4%、15.2%、13.1%。(2)与LCK相比,LT2、LT3、LT5、LT6处理果实的裂纹指数分别降低了49.8%、19.8%、49.8%、30.1%,LT1、LT2、LT5处理果实的着色指数分别提高了3.4%、14.0%、17.4%,LT1~LT6处理果实的光洁指数分别提高了50.4%、150.4%、125.6%、45.2%、175.9%、100.8%。(3)果皮显微结构观察表明,控制处理前、后期土壤水分不稳定会导致果皮角质层的严重开裂,LT5处理避免了这一情况的发生,而健康果实的果皮角质层厚度大于裂果的果皮。(4)健康果实的水势(-1.22 MPa)明显高于裂果的水势(-1.44 MPa),且果实水势与裂纹发生呈负相关关系。研究认为,适当的土壤水分胁迫(65%~75%)不影响果实食用品质,同时可显著降低果实表面裂纹的发生率,提高果面光洁度;在果实发育后期,适当的轻度控水处理会促进果实的糖积累并减少裂纹的产生;果实裂纹的发生与果皮角质层厚度和果实水势相关,厚度越大、水势越高,裂纹发生越少。土壤相对含水量LT2与LT5处理的苹果果实兼具良好的食用品质与外观品质。     

番木瓜

番木瓜原产子热带美洲,目前广泛分布在世界各热带地区。其学名为Carica papa-ya。主要俗名有:Pawpaw Papaw (英国),Mamao (巴西),Lechoso (危内瑞拉),Fruta bomba (古巴,当地人认为papaya是误称)。番木瓜通常用作一种早餐食品、冷餐水果和各种餐后甜点。果实含糖约10%,富含维生素A,还有一些维生素C。     

转基因番木瓜PCR 快速检测体系的优化

随着公众对转基因食品安全性的关注, 转基因番木瓜的快速检测技术成为关键问题。优化了番木瓜总DNA的提取方法, 选用木瓜蛋白酶基因(Papain)作为内源参照基因, 建立了转基因番木瓜GM YK 和华农一号的双重PCR定性检测方法。通过对广州市超市和批发市场222 个样品的检验, 并与SN/T 2653-2010 行业标准对比, 证明该方法具有污染少、速度快、成本低的优点。     

Analysis on virus resistance and fruit quality for T4 generation of transgenic papaya

Molecular biological characterization,fruit characters,and nutrients were analyzed for T4 generation of transgenic papaya.All transgenic papaya plants with the mutated replicase (RP) gene from papaya ringspot virus (PRSV) showed high resistance or immunity against PRSV in the field.The RP transgene can be steadily inherited to,and expressed at RNA level,the progenies.The growth characteristics of transgenic papaya were much better than nontransgenic papaya in the field.The non-transgenic papaya seedlings began to show typical symptoms caused by PRSV after being inoculated with PRSV.They died quickly and never grew to produce fruit.The adult trees developed yellow leaves and produced smaller fruits and were doomed to a slow death after some time,while most oftransgenic papaya plants (about 91.8%) did not show any symptoms caused by PRSV,and produced more,bigger,and high quality fruits.Compared with non-transgenic plants,the fresh fruit length of T4 generation of transgenic papaya increased 2.6%-5%,and the diameter decreased 0.6%-1.5%.The flesh thickness of fresh fruit increased 12%-15%,which made it fitter for eating.Although the fresh fruit quality changed,there was no significant difference between transgenic and non-transgenic papaya.The quality characteristics of dry fruit including the contents of water,lipid,N,protein,reduced sugar,vitamin A,vitamin C,and carotene in the T4 generation of transgenic papaya were all the same as their non-transgenic parents.This means that transgenic plants and non-transgenic plants are substantially equivalent,and the transgene has no effect on dry fruit quality.In this study,we found that vitamin A and vitamin C in red-fleshed papaya were 1.4-1.8 and 1.78-2.07 times more than the yellow-fleshed ones,respectively,while N and protein were only 84.2%-92.1% and 82.1%-98.9% of the yellow-fleshed ones.     

Analysis on virus resistance and fruit quality for T4 generation of transgenic papaya

Molecular biological characterization, fruit characters, and nutrients were analyzed for T4 generation of transgenic papaya. All transgenic papaya plants with the mutated replicase (RP) gene from papaya ringspot virus (PRSV) showed high resistance or immunity against PRSV in the field. The RP transgene can be steadily inherited to, and expressed at RNA level, the progenies. The growth characteristics of transgenic papaya were much better than non-transgenic papaya in the field. The non-transgenic papaya seedlings began to show typical symptoms caused by PRSV after being inoculated with PRSV. They died quickly and never grew to produce fruit. The adult trees developed yellow leaves and produced smaller fruits and were doomed to a slow death after some time, while most of transgenic papaya plants (about 91.8%) did not show any symptoms caused by PRSV, and produced more, bigger, and high quality fruits. Compared with non-transgenic plants, the fresh fruit length of T4 generation of transgenic papaya increased 2.6%–5%, and the diameter decreased 0.6%–1.5%. The flesh thickness of fresh fruit increased 12%–15%, which made it fitter for eating. Although the fresh fruit quality changed, there was no significant difference between transgenic and non-transgenic papaya. The quality characteristics of dry fruit including the contents of water, lipid, N, protein, reduced sugar, vitamin A, vitamin C, and carotene in the T4 generation of transgenic papaya were all the same as their non-transgenic parents. This means that transgenic plants and non-transgenic plants are substantially equivalent, and the transgene has no effect on dry fruit quality. In this study, we found that vitamin A and vitamin C in red-fleshed papaya were 1.4–1.8 and 1.78–2.07 times more than the yellow-fleshed ones, respectively, while N and protein were only 84.2%–92.1% and 82.1%–98.9% of the yellow-fleshed ones. Translated from Acta Ecologica Sinica, 2005, 25(12): 3301–3306 [译自: 生态学报]     

Field Released Transgenic Papaya Affects Microbial Communities and Enzyme Activities in Soil

Soil properties, microbial communities, and enzyme activities were studied in soil planted with transgenic or nontransgenic papaya under field conditions. The transgenic papaya contained a replicase (RP) mutant gene of the papaya ringspot virus (PRSV), which conferred resistance to the virus, the neomycin phosphotransferase II (NPT II) marker gene, which conferred Km resistance, and a cauliflower mosaic virus 35S promoter (CaMV 35S). There were significant differences (P < 0.05) in the total number of colony forming units (CFUs) of bacteria, actinomycetes, and fungi between soils planted with RP-transgenic and nontransgenic plants; total CFUs of bacteria, actinomycetes, and fungi in soil planted with transgenic papaya were significantly higher by 0.43, 0.80, and 0.46 times, respectively. Significantly higher (P < 0.05) CFUs of bacteria, actinomycetes, and fungi resistant to kanamycin (Km) were present in soils planted with the transgenic papaya than in those planted with nontransgenic papaya. Resistance quotients (CFU in the presence of a chemical relative to that without) of Km-resistant bacteria, actinomycetes and fungi were higher in soil planted with transgenic papaya, and the resistance quotients of Km-resistant bacteria, actinomycetes, and fungi in soils planted with transgenic papaya increased statistically significantly (P<0.05) from 1.5 to 2.5, from 1.2 to 2.6, and from 0.9 to 2.8 times, respectively. Soils planted with transgenic papaya had significantly higher enzyme activities of arylsulfatases (+5.4 times), alkaline phosphatases (+0.5 time), invertase (+0.5 time) and phosphodiesterases (+0.2 time), but lower enzyme activities of proteases (−2.1 times), polyphenol oxidases (−1.4 times), urease (−0.2 time) than the soils planted with nontransgenic papaya. Our results suggest that transgenic papaya could alter chemical properties, enzyme activities, and microbial communities in soil.     

Cloning of the papaya ringspot virus (PRSV) replicase gene and generation of PRSV-resistant papayas through the introduction of the PRSV replicase gene

Papaya ringspot virus (PRSV) can cause a destructive disease in papaya (Carica papaya L.). Based on observations that viral replicase (RP) gene confers resistance to virus in other plants, we designed a pair of primers and cloned the RP gene from PRSV by RT-PCR. The 3'-truncated and 5'-extended RP gene fragment was then oriented under the control of the CaMV35 S promoter and nos termination sequence in the mini Ti plasmid vector pRok to construct a plant expression vector, designated pRPTW. Papaya (C. papaya L.) cv. Tai-nong-2 embryogenic calli were transformed by Agrobacterium tumefaciens LBA4404 harboring the pRPTW vector. After selection on 100 mg/ml kanamycin, 20 putative transgenic papayas were regenerated and confirmed by PCR-Southern blot and Southern blot analyses. PRSV inoculation tests showed that the RP gene conferred resistance to PRSV in transgenic papayas and those offspring carrying the RP gene. The consistency of the presence of the RP gene and PRSV resistance indicates that replicase-mediated resistance against PRSV was attained in papaya. Possible mechanisms include RNA-mediated resistance and protein-mediated resistance, as well as others, although further studies are required.     

High-efficiency vitrification protocols for cryopreservation of in vitro grown shoot tips of transgenic papaya lines

In vitro grown shoot tips of transgenic papaya lines (Carica papaya L.) were successfully cryopreserved by vitrification. Shoot tips were excised from stock shoots that were preconditioned in vitro for 45–50-day-old and placed on hormone-free MS medium with 0.09 M sucrose. After loading for 60 min with a mixture of 2 M glycerol and 0.4 M sucrose at 25°C, shoot tips were dehydrated with a highly concentrated vitrification solution (PVS2) for 80 min at 0°C and plunged directly into liquid nitrogen. The regeneration rate was approximately 90% after 2 months post-thawing. Successfully vitrified and warmed shoot tips of three non-transgenic varieties and 13 transgenic lines resumed growth within 2 months and developed shoots in the absence of intermediate callus formation. Dehydration with PVS2 was important for the cryopreservation of transgenic papaya lines. This vitrification procedure for cryopreservation appears to be promising as a routine method for cryopreserving shoot tips of transgenic papaya line germplasm.     

Comparative effects of dietary administered transgenic and conventional papaya on selected intestinal parameters in rat models

Transformation of agricultural crops with novel genes has significantly advanced disease-resistance breeding, including virus resistance through the expression of virus sequences. In this study, the effects of long-term, repeated exposure to transgenic papayas carrying the coat protein gene of Papaya ringspot virus and conventional non-transgenic papaya on the histology and selected biochemical parameters of the intestinal tract were compared. For 3 months, male and female Wistar rats received diets containing transgenic or non-transgenic papaya at twice the equivalent of the average daily consumption of fresh papayas. Gross and macroscopic appearance of intestinal tissues, as well as stomach tissues, was comparable (P < 0.05) as were total intestinal bacterial counts and activities of β-glucuronidase. Activities of disaccharidases were not affected, neither were those of amylase (P < 0.05). Although significant differences were noted in the activity of Ca²⁺ and Na⁺/K⁺ ATPase brush border enzymes, no morphological alteration in the integrity of the intestinal mucosa was found. Overall, negligible effects on feed intake, body weight, and fecal output were observed (P < 0.05). Taken together, long-term exposure to diets formulated with transgenic papaya did not result in biologically important unintended effects.     

Transgenic papaya plants from Agrobacterium-mediated transformation of somatic embryos

Summary Transgenic papaya (Carica papaya L.) plants were regenerated from embryogenic cultures that were cocultivated with a disarmed C58 strain of Agrobacterium tumefaciens containing one of the following binary cosmid vectors: pGA482GG or pGA482GG/cpPRV-4. The T-DNA region of both binary vectors includes the chimeric genes for neomycin phosphotransferase II (NPTII) and ß-glucuronidase (GUS). In addition, the plant expressible coat protein (cp) gene of papaya ringspot virus (PRV) is flanked by the NPTII and GUS genes in pGA482GG/cpPRV-4. Putative transformed embryogenic papaya tissues were obtained by selection on 150 g·ml^–1 kanamycin. Four putative transgenic plant lines were obtained from the cp gene^– vector and two from the cp gene⁺ vector. GUS and NPTII expression were detected in leaves of all putative transformed plants tested, while PRV coat protein expression was detected in leaves of the PRV cp gene⁺ plant. The transformed status of these papaya plants was analyzed using both polymerase chain reaction amplification and genomic blot hybridization of the NPTII and PRV cp genes. Integration of these genes into the papaya genome was demonstrated by genomic blot hybridizations. Thus, like numerous other dicotyledonous plant species, papayas can be transformed with A. tumefaciens and regenerated into phenotypically normal-appearing plants that express foreign genes.Journal Series no. 3757 of the Hawaii Institute of Tropical Agriculture and Human Resources     

Cloning and Sequencing of Coat Protein Gene of Papaya Ringspot Virus and Sequence Comparison of Strains of PRV-P and PRV-YS

Papaya rinspot virus (PRV) was isolated and purified from infected papaya (Carica papaya L. )leaves in Hainan Island. The first strand of cDNA was synthesized with Olig(dT)as a primer. The coat protein gene of PRV was obtained by PCR techniques with primers synthesized accoding to the DNA sequence of PRV-P. Full length of cDNA clone encoding coat protein of PRV was sequenced and analysed. The result shows that the strain of PRV we isolated contains 881 nucleotides with 287 amino acids. Sequences among several strains of PRV were compared and it indicates an over 90% homology in DNA sequence of PRV-G the strain we isolated with PRV-P and PRV-YS. Highly convered sequence was located in carboxyl end and interestingly highly variable region was in N-terminal.     

棉田棉铃虫寄生峰对常规棉及转Bt棉品种的趋性反应

连续4年采用人工定期释放棉铃虫卵、幼虫和定期回收法,研究了不同棉花品种与棉铃虫卵期、幼虫期寄生蜂种群数量变化的关系．通过对卵、幼虫寄生率反正弦转换后进行方差分析表明,无论是在棉铃虫卵期还是在其幼虫期,转基因棉田中棉铃虫卵和幼虫的被寄生率始终显著低于其亲本对照棉．运用“Y”型嗅觉仪测定转基因棉及其亲本对照棉对棉铃虫幼虫寄生蜂——中红侧沟茧蜂的选择性行为反应,用成对数据进行方差分析,并进行了“T”测验．结果表明,转基因棉对棉铃虫寄生蜂有较强的忌避反应,且取食转基因棉的棉铃虫幼虫与被害转基因棉两者的共同组合与单独被害棉之间对寄生蜂的忌避效应基本一致．