苹果多酚氧化酶双链RNA干扰(RNAi)研究

以成熟苹果果实的RNA为模板,经RT-PCR扩增并克隆苹果多酚氧化酶(APPO)长度为710bp的反义、正义基因片段.以副球菌中类胡罗卜素合成有关的(crtW+crtY)融合基因片段YYT为间隔区,将APPO反义基因片段、YYT和APPO正义基因片段串联,构成全长为2446bp的DNA并插入到植物双元载体pYPX145中,构成可表达苹果多酚氧化酶双链RNA的植物双元载体pYF7704.以根癌农杆菌介导的叶盘转化法转化苹果栽培品种红富士,通过50mg/L卡那霉素筛选和GUS检测,获得了转基因苹果抗性芽.荧光定量RT-PCR检测结果显示,转基因苹果抗性芽内多酚氧化酶基因的干扰效果达91.69%以上,研究结果证实多酚氧化酶双链RNA干扰在转基因苹果上是可行的.     

苹果α-法尼烯合成酶基因双链RNAi载体的构建

RNA干扰属于转录后后基因基因沉默机制(PTGS),是一种新型的基因沉默手段,用于基因表达和功能的研究。以成熟苹果的果皮RNA为模板,经RT-PCR扩增并克隆α-Farnesene合成酶基因(AFS),设计特异引物从AFS基因中扩增515bp和396bp的反义和正义基因片段,将正义和反义基因片段串联,把构成的长为911bp的DNA插到PBI121表达载体中,构建成可表达α-Farnesene合成酶双链RNA载体PBI-AFS。     

鸭梨多酚氧化酶基因反义表达载体的构建及农杆菌介导的遗传转化

根据鸭梨多酚氧化酶基因序列设计引物,PCR扩增该基因3′端450bp的片段,并将该片段反向插入真核表达载体pB I121的CaMV 35S启动子和NOS终止子之间,首次构建了鸭梨PPO基因的反义表达载体;其后,在农杆菌EHA105的介导下,成功实现了PPO反义基因对鸭梨组培苗的遗传转化。经Northern杂交和酶活检测证实,转基因鸭梨植株体内的多酚氧化酶基因转录和翻译水平均得到明显抑制,从而为耐褐化梨新品种的培育奠定了基础。     

ACC氧化酶和ACC合成酶反义RNA融合基因导入番茄和乙烯合成的抑制

从番茄品种强力米寿的总DNA中克隆番茄果实特异启动子2A11,以番茄成熟果实的RNA为模板,进行RT-PCR扩增,克隆番茄全长的ACC氧化酶基因和ACC合成酶基因片段。完成两个基因的克隆和测序后,将888bp的番茄ACC氧化酶基因和943bp的ACC合成酶基因片段串联,构成全长1837bp的融合基因。将该融合基因以反义的方向插入植物双元载体pYPX145中番茄果实表达特异启动子下游,获得ACC氧化酶基因和ACC合成酶基因融合的植物双元载体pOSACC。该载体外源基因表达单元的两端含两个烟草SAR序列,利于转基因的稳定遗传。以番茄栽培品种合作903子叶和下胚轴为外植体,利用根癌农杆菌进行基因转化,通过200mg/L卡那霉素选择和GUS检测,获得了105株番茄GUS阳性植株,转基因番茄果实在当代表现明显耐贮特点。经过4代的耐贮和果实农艺性状的综合选择,获得了两个表现良好的株系DR-1和DR-2,两株系果实乙烯释放量显著下降,是未转基因材料的9.5%,番茄的贮存期在50天以上。     

ACC氧化酶和ACC合成酶反义RNA融合基因导入番茄和乙烯合成的抑制

从番茄品种强力米寿的总DNA中克隆番茄果实特异启动子2A11,以番茄成熟果实的RNA为模板,进行RT-PCR扩增,克隆番茄全长的ACC氧化酶基因和ACC合成酶基因片段。完成两个基因的克隆和测序后,将888bp的番茄ACC氧化酶基因和943bp的ACC合成酶基因片段串联,构成全长1837bp的融合基因。将该融合基因以反义的方向插入植物双元载体pYPX145中番茄果实表达特异启动子下游,获得ACC氧化酶基因和ACC合成酶基因融合的植物双元载体pOSACC。该载体外源基因表达单元的两端含两个烟草SAR序列,利于转基因的稳定遗传。以番茄栽培品种合作903子叶和下胚轴为外植体,利用根癌农杆菌进行基因转化,通过200mg／L卡那霉素选择和GUS检测,获得了105株番茄GUS阳性植株,转基因番茄果实在当代表现明显耐贮特点。经过4代的耐贮和果实农艺性状的综合选择,获得了两个表现良好的株系DR-1和DR-2,两株系果实乙烯释放量显著下降,是未转基因材料的9．5％,番茄的贮存期在50天以上。     

草酸氧化酶基因转化烟草的研究

为研究草酸氧化酶基因(OxO)对植物抗病的作用,将含有CaMV 35s启动子的植物表达载体以根癌农杆菌(Agrobacterium tumefaciens)介导的叶盘方法,转化了烟草97131。具有卡那霉素抗性的再生植株经PCR检测,得到了与阳性对照一致的470bp的片段,进一步对PCR产物测序表明OxO基因已整合进烟草基因组中。在对草酸的耐受性实验中,转基因烟草对草酸的抗性明显高于未转化烟草。     

35S驱动Pib基因启动区和编码区的转基因初步分析

将包含Pib基因启动区及下游完整编码区的9.9 kb DNA片段克隆到双元载体pPZP2Ha3(+)中, 构建了35S驱动的正义表达载体pNAR701(20.3 kb); 同时将Pib基因编码区6 986~9 392 bp之间的DNA片段, 克隆到双元载体pPZP2Ha3(-)中, 构建了35S驱动的反义表达载体pNAR703(12.8 kb); 用农杆菌介导法转入中感稻瘟病水稻品种R109中。PCR、Southern blot鉴定以及转基因T0代种子的潮霉素抗性鉴定证明, 目的基因已经整合到R109基因组中, 并能在后代稳定遗传。Northern blot分析表明含有启动区及下游完整编码的Pib基因片段在35S驱动下能够在转基因后代中表达。对T1代苗期转基因植株和分蘖期离体叶片进行抗稻瘟病初步分析, 结果显示pNAR701转基因植株对稻瘟病生理小种ZD1和ZG1的抗性较对照增强, 而转反义片段的pNAR703转基因植株对稻瘟病的抗性较对照减弱。     

外源RNA干涉基因在烟草中的转化及表达

依据RNA干涉机制,以TMV复制酶基因为靶标基因,针对TMV 5个株系复制酶基因间高度同源序列设计引物,经RT-PCR反应获得靶序列,构建靶序列反向重复结构的RNA干涉双元载体.用根癌农杆菌介导将外源基因转化至烟草品种K326基因组中,培育RNA干涉转基因烟草.人工接种病毒验证转基因烟草中外源基因在植物抗病毒能力方面的表达效果,实时荧光定量PCR分析转基因烟草抗病毒能力.结果表明,实验培育的RNA干涉转基因烟草67%对TMV呈现高度抗性;荧光定量PCR分析显示,对TMV具高度抗性的转基因烟草中病毒复制酶基因转录产物mRNA存在很大程度的降解,证实了RNA干涉技术在培育抗病毒烟草品种中的效果.     

利用转hpRNA基因水稻抗水稻矮缩病毒

具有发夹结构的双链RNA(hairpin RNA,hpRNA)能高效诱导转录后基因沉默的发生.以水稻(Oryza sativaL.)矮缩病毒(RDV)基因组中第八片段编码区128～754 bp的序列为臂构建hpRNA,并克隆到植物表达载体pROK-2上.通过农杆菌介导的方法转化水稻"中花11".Southern blot分析表明,共获得12株阳性转化体.用带有RDV的叶蝉(Nephotettix cincticeps)接种Tl代转hpRNA水稻,结果表明转基因水稻对RDV具有高抗性或表现为症状延迟.而相同序列的有义链的转基因水稻和空载体的转基因水稻表现为典型的RDV侵染症状.HpRNA在转基因水稻中对RDV高抗性发挥重要作用.     

砂梨脂氧合酶cDNA片段克隆与RNAi载体构建

以砂梨‘若光’的成熟果实为材料,根据脂氧合酶氨基酸保守区设计1对简并引物,采用RT-PCR克隆到1段长827 bp的序列,经Blast比对和DNAstar软件聚类分析,结果表明由该序列推导出的氨基酸序列含有脂氧合酶氨基酸保守结构域,与马铃薯脂氧合酶基因的氨基酸序列相似性达到77.5%,判断其为砂梨脂氧合酶基因片段,命名为LOX1,并将序列登录到GenBank,登录号为EF215448。根据RNAi载体的构建原则,选择LOX1一开放阅读框设计携带酶切位点的特异引物,通过PCR扩增正、反向基因片段,再与YYT间隔区串连,插入植物表达载体pYF7713中的相应位置,成功地构建了干扰LOX基因表达的RNAi的植物双元表达载体pYL028,为深入研究该基因在砂梨果实成熟过程中的功能及耐贮藏基因工程育种奠定了基础。     

Investigation of Agrobacterium-mediated transformation of apple using green fluorescent protein: high transient expression and low stable transformation suggest that factors other than T-DNA transfer are rate-limiting

To investigate early events of Agrobacterium-mediated transformation of apple cultivars, a synthetic green fluorescent protein gene (SGFP) was used as a highly sensitive, vital reporter gene. Leaf explants from four apple cultivars (Delicious, Golden Delicious, Royal Gala and Greensleeves) were infected with Agrobacterium EHA101 harboring plasmid pDM96.0501. Fluorescence microscopy indicated that SGFP expression was first detected 48 h after infection and quantitative analysis revealed a high T-DNA transfer rate. Plant cells with stably incorporated T-DNA exhibited cell division and developed transgenic calli, followed by formation of transgenic shoots at low frequencies. The detection of SGFP expression with an epifluorescence stereomicroscope confirmed the effectiveness of SGFP as a reporter gene for detection of very early transformation events and for screening of putative transformants. The efficiency of the transformation and regeneration process decreased ca. 10000-fold from Agrobacterium infection to transgenic shoot regeneration, suggesting that factors other than Agrobacterium interaction and T-DNA transfer are rate-limiting steps in Agrobacterium-mediated transformation of apple.     

Different effects on ACC oxidase gene silencing triggered by RNA interference in transgenic tomato

RNA interference (RNAi) is a potent trigger for specific gene silencing of expression in a number of organisms and is an efficient way of shutting down gene expression. 1-Aminocyclopropane-1-carboxylate (ACC) oxidase catalyzes the oxidation of ACC to ethylene, a plant growth regulator that plays an important role in the tomato ripening process. In this research, to produce double-stranded (ds)RNA of tomato ACC oxidase, we linked the sense and antisense configurations of DNA fragments with 1,002-bp or 7-nt artificially synthesized fragments, respectively, and then placed these under the control of a modified cauliflower mosaic virus 35S promoter. The dsRNA expression unit was successfully introduced into tomato cultivar Hezuo 906 by Agrobacterium tumefaciens-mediated transformation. Molecular analysis of 183 transgenic plants revealed that the dsRNA unit was integrated into the tomato genome. With respect to the construct with the 1,002-bp linker, the severity of phenotypes indicated that 72.3% of the transformed plants had non-RNA interference, about 18.1% had semi-RNA interference, and only 9.6% had full-RNA interference. However when the construct with the 7-nt linker was used for transformation, the results were 13.0%, 18.0%, and 69.0%, respectively, indicating that the short linker was more efficient in RNAi of transgenic tomato plants. When we applied this fast way of shutting down the ACC oxidase gene, transgenic tomato plants were produced that had fruit which released traces of ethylene and had a prolonged shelf life of more than 120 days. The RNA and protein analyses indicated that there was non-RNA interference, semi-RNA interference and full-RNA interference of ACC oxidase in the transgenic tomato plants.     

Changes in the activities of catalase, peroxidase, and polyphenol oxidase in apple buds during bud break induced by thidiazuron

The breaking of dormancy in apple buds (Malus domestica Borkh cv. York Imperial) by thidiazuron (N-phenyl-N-1,2,3,-thidiazol-5-ylurea) was investigated in relation to catalase, peroxidase, and polyphenol oxidase activities and their isoenzyme patterns. The activity and number of isoenzymic components of catalase increased progressively during bud break, then decreased after buds started to grow. Peroxidase activity was highest during dormancy and declined during bud swell, increased at bud break, and decreased after bud expansion. Several isoperoxidases were observed in gel electrophoresis. Similar patterns were found at different growth stages of apple buds except for one peroxidase isoenzyme, P3, which disappeared 12 days after thidiazuron treatment. There was an inverse relationship between the activities of polyphenol oxidase and peroxidase during the development of apple buds. Apple buds have a very similar polyphenol oxidase isoenzyme pattern throughout bud development. However, the appearance and disappearance of minor isoenzymes were also observed. Phloridzin, rutin, p-coumaric, epicatechin, naringin, chlorogenic acid, and catechol were found in apple buds. Among them, phloridzin, rutin, and p-coumaric were the dominant phenolic compounds. Dormant buds contained a high amount of phenolic substances which decreased after bud break (4 days after thidiazuron treatment) then increased until the start of bud expansion. Phenolic compounds are found to be potent modifiers of catalase, peroxidase, and polyphenol oxidase activity, as both inhibitors and stimulators in apple buds.     

Immunological and molecular comparison of polyphenol oxidase in Rosaceae fruit trees.

An antibody raised against apple polyphenol oxidase (PPO) cross-reacted with PPOs from Japanese pear (Pyrus pyrifolia), pear (Pyrus communis), peach (Prunus persica), Chinese quince (Pseudocydonia sinensis) and Japanese loquat (Eriobotrya japonica). Core fragments (681 bp) of the corresponding PPO genes were amplified and characterized. The deduced protein sequences showed identities of 85.3 to 97.5%. Chlorogenic acid oxidase activity of these PPOs showed higher activities when assayed at pH 4 than at pH 6. These results indicate that PPOs in Rosaceae plants are structurally and enzymatically similar.     

Changes in the activities of catalase,peroxidase, and polyphenol oxidase in apple buds during bud break induced by thidiazuron

The breaking of dormancy in apple buds (Malus domestica Borkh cv. York Imperial) by thidiazuron (N-phenyl-N′-1,2,3,-thidiazol-5-ylurea) was investigated in relation to catalase, peroxidase, and polyphenol oxidase activities and their isoenzyme patterns. The activity and number of isoenzymic components of catalase increased progressively during bud break, then decreased after buds started to grow. Peroxidase activity was highest during dormancy and declined during bud swell, increased at bud break, and decreased after bud expansion. Several isoperoxidases were observed in gel electrophoresis. Similar patterns were found at different growth stages of apple buds except for one peroxidase isoenzyme, P3, which disappeared 12 days after thidiazuron treatment. There was an inverse relationship between the activities of polyphenol oxidase and peroxidase during the development of apple buds. Apple buds have a very similar polyphenol oxidase isoenzyme pattern throughout bud development. However, the appearance and disappearance of minor isoenzymes were also observed. Phloridzin, rutin, p-coumaric, epicatechin, naringin, chlorogenic acid, and catechol were found in apple buds. Among them, phloridzin, rutin, and p-coumaric were the dominant phenolic compounds. Dormant buds contained a high amount of phenolic substances which decreased after bud break (4 days after thidiazuron treatment) then increased until the start of bud expansion. Phenolic compounds are found to be potent modifiers of catalase, peroxidase, and polyphenol oxidase activity, as both inhibitors and stimulators in apple buds.     

A transgenic apple callus showing reduced polyphenol oxidase activity and lower browning potential

Polyphenol oxidase (PPO) is responsible for enzymatic browning of apples. Apples lacking PPO activity might be useful not only for the food industry but also for studies of the metabolism of polyphenols and the function of PPO. Transgenic apple calli were prepared by using Agrobacterium tumefaciens carrying the kanamycin (KM) resistant gene and antisense PPO gene. Four KM-resistant callus lines were obtained from 356 leaf explants. Among these transgenic calli, three calli grew on the medium containing KM at the same rate as non-transgenic callus on the medium without KM. One callus line had an antisense PPO gene, in which the amount and activity of PPO were reduced to half the amount and activity in non-transgenic callus. The browning potential of this line, which was estimated by adding chlorogenic acid, was also half the browning potential of non-transgenic callus.