木糖异构酶基因xylA的克隆及其表达载体的构建

木糖异构酶基因xylA是一种正向选择标记基因,在植物基因工程中使用该标记可以获得安全的转基因植物.构建了以xylA基因为选择标记的植物表达载体.从大肠杆菌Top10中扩增出xylA基因,插入到质粒pCAMBIA2301的Xho Ⅰ位点,通过酶切和PCR检测插入片段的正确性,得到载体pCAMBIA2301-xylA,将pBI121载体上的‘35S-GUS-Nos'表达框插入到pCAMBIA2301-xylA的EcoR Ⅰ和Hind Ⅲ位点.得到中间载体pCAMBIA2301-xylA-GUS,用Sac Ⅰ和Sma Ⅰ切下克隆载体上的CBF1基因替代pCAMBIA2301-xylA-GUS中的GUS片段,用电转化法将获得的表达载体转化到农杆菌中,为将来获得安全的转基因抗寒植株奠定基础.     

两种质粒中gus基因在植物细胞和根癌农杆菌中的表达特性研究

以含有基因转化操作过程中常用的两种质粒载体pBI121和pCAMBIA2301的根癌农杆菌EHA105为材料,分别转化甜瓜子叶,应用组织化学方法检测了甜瓜子叶和子叶培养后的愈伤组织及根癌农杆菌菌液的瞬时转化效果,研究了两种不同的质粒载体上所含的gus基因在根癌农杆菌中和植物细胞中的表达特性.结果表明,不同质粒载体上所含的gus基因的表达特性不同,质粒载体pBI121上所含的gus基因既能在植物细胞中能表达,也能在根癌农杆菌细胞中表达,而质粒载体pCAMBIA2301上所含的gus基因能在植物细胞中表达,但是不能在根癌农杆菌细胞中表达.     

利用根癌农杆菌介导转化大豆成熟种子胚尖获得转基因植株

利用根癌土壤农杆菌EHA105/pCAMBIA2301对来自大豆成熟种子的胚尖外植体进行遗传转化,并对农杆菌侵染时间长短以及乙酰丁香酮(AS)浓度等影响转化频率的条件进行了探讨.发现浸染时间以20 h为佳,乙酰丁香酮最佳浓度为200 umo1/L,并探讨了恢复培养的重要性.分别从3个大豆品种合丰35、合丰39、东农42得到了转基因植株,GUS染色及Southern杂交结果证明外源基因整合到大豆基因组中,获得转基因大豆的频率达6.4%～12.1%.     

番木瓜β-Gal基因RNAi双T-DNA植物表达载体的构建及其遗传转化的初步研究

克隆了番木瓜(Carica papaya L.)果肉的细胞壁水解关键酶β-半乳糖苷酶(β-GAL)基因保守区,将其反向重复插入载体pKANNIBAL,构建RNAi中间表达载体pKAN/RG,将其上的发夹结构取代经改造的载体pCAMBIA 1300上hpt II基因,构建中间表达载体p1300~-/MFRG,分离单T-DNA区段,与载体pCAMBIA 2301构建RNAi双T-DNA植物表达载体p2301/TTRG.酶切分析和PCR检测表明,p2301/TTRG已被成功导入农杆菌EHA 105.通过遗传转化,初步获得了GUS染色呈阳性且具Kan抗性的番木瓜胚性愈伤组织.     

金针菇G蛋白偶联受体基因的CRISPR/Cas9基因组编辑载体构建及转化研究

通过氨基酸同源比对（Blast P）以及金针菇冷诱导前后菌丝阶段和原基阶段的转录组数据分析,获得了金针菇中的两个假定G蛋白偶联受体基因Fvgpcr1、Fvgpcr2。对获得的金针菇假定G蛋白偶联受体基因Fvgpcr1、Fvgpcr2构建了基因组编辑（CRISPR/Cas9）的pCAMBIA0390-hph-Fvcas9-Fvgpcr1- sgRNA1/sgRNA2、pCAMBIA0390-hph-Fvcas9-Fvgpcr2-sgRNA1/sgRNA2等4个表达载体。通过农杆菌介导（ATMT）将表达载体pCAMBIA0390-hph-Fvcas9-Fvgpcr-sgRNA转化金针菇菌丝体,采用潮霉素和头孢毒素低浓度初筛和高浓度复筛,经两段筛选获得金针菇拟转化子。经对拟转化子进行PCR鉴定、RT-qPCR检测和Western杂交验证,结果显示表达载体pCAMBIA0390-hph-Fvcas9-Fvgpcr-sgRNA成功整合进金针菇基因组中,FvCas9蛋白正常表达,但未得到Fvgpcr基因敲除突变体。本研究利用农杆菌介导转化法在金针菇中构建了CRISPR/Cas9敲除体系,对后续目标基因的敲除有着重要意义。     

农杆菌介导的水稻双载体共转化法中部分影响因素的研究

以T-DNA区分别只含有潮霉素选择标记基因(HPT)和GUS报告基因的双元载体pCAMBIA1300和pCAMBIA0301用于农杆菌介导的水稻共转化试验。根据经农杆菌浸染并共培养3d后水稻愈伤组织中的GUS瞬间表达情况及其稳定共转化率,测定了不同农杆菌菌株搭配及其不同浓度配比对共转化效率的影响。结果表明,在两种农杆菌菌液浓度比为1:1的情况下,农杆菌EHA105/pCAMBIA1300与EHA105/pCAMBIA0301组合共转化水稻的效率要高于其他菌株的组合;在以农杆菌EHA105/pCAMBIA1300与EHA105/pCAMBIA0301进行共转化时,两种菌液浓度比为1:2时共转化效率最高。     

意大利生菜转化体系建立及EV71抗原基因表达初探

以意大利生菜(Lactuca sativa L.var.ramosa Hort.)为试材,农杆菌LBA4404叶盘法转化子叶,通过植物组织培养技术,筛选获得再生体系最佳选择压为Kan 50 mg/L,最佳抑菌剂为Carb 400 mg/L,生菜子叶抗性愈伤组织诱导的最佳植物生长调节剂条件为6-BA 0.4 mg/L+NAA 0.15 mg/L或6-BA 0.3 mg/L+NAA 0.2 mg/L,其中在6-BA 0.3 mg/L+NAA 0.2 mg/L的培养基上诱生的抗性愈伤组织后续生芽效果较好。根据GenBank序列,优化设计合成适合植物表达的肠道病毒71型(Human enterovirus 71,EV71)C4亚型P1和3CD基因,运用基因重组技术,同时克隆入植物表达载体pCAMBIA2301,构建共表达植物表达载体pCAMBIA2301-P1-3CD。以农杆菌介导的叶盘法转化意大利生菜子叶,对叶片PCR阳性的抗性株进行蛋白质表达检测,Western-blot实验表明,两株抗性株叶片中表达出具有生物学活性的EV71抗原蛋白质,证明了利用生菜表达EV71抗原的可行性,为利用植物生物反应器生产抗EV71口服疫苗提供了理论和实验依据。     

蔗糖转运蛋白基因VvSUC12和VvSUC27在葡萄胚性和非胚性愈伤组织中的差异表达

本研究通过对霞多丽葡萄花前约10d的花丝进行组培诱导,获得胚性和非胚性两种愈伤组织,分别进行继代、组织结构观察和体细胞胚的诱导验证。为研究两种愈伤组织对培养基中主要碳源蔗糖的利用特点,根据GenBank中的定位于细胞质膜的葡萄蔗糖转运蛋白基因VvSUC12和VvSUC27的序列,设计了这两种蔗糖转运蛋白的PCR引物。以RNAplant试剂法,提取胚性愈伤组织和非胚性愈伤组织的RNA,进行半定量RT-PCR。研究表明,31个循环半定量RT-PCR结果中VvSUC12在胚性愈伤和非胚性愈伤中均有表达,且在非胚性愈伤组织中的表达水平稍高于胚性愈伤组织,表达差异未达到显著水平,VvSUC27的表达水平明显低于VvSUC12,且只在胚性愈伤组织中表达。提高至35个循环的半定量RT-PCR结果显示VvSUC27基因在非胚性愈伤组织中微弱表达,而在胚性愈伤组织中的表达强度较31个循环有所增加,且高于非胚性愈伤。     

根癌农杆菌介导的水稻高效转化系统的建立

比较了影响根癌农杆菌转化水稻的各种因素后,建立了农杆菌介导的水稻高效转基因实验体系。按该体系,水稻品种中花11号预培养4d的幼胚经农杆菌EHA105／pCAMBIA1301感染后,具有GUS基因瞬间表达的幼胚比例在50％以上,最高可达90％;按产生潮霉素抗性愈伤和转基因植株的比例计算,转化率分别达到87．6％和64．6％。转基因植株总DNA的Southern杂交分析表明T－DNA上的外源基因已整合进了水稻基因组,且在大多数转基因植株中表现为单拷贝插入;遗传分析证明T1代的表型分离符合孟德尔法则。此转化系统的建立为高效地将有用的外源DNA导入水稻植株奠定了基础。     

高温加速老化过程中甜菜种子线粒体小分子量热激蛋白22的变化

甜菜（Beta vulgaris L.cv.Loke）种子的人工老化（30℃,100％RH）导致种子的干重、生活力和活力逐渐丧失,线粒体的呼吸速率、细胞色素c氧化酶和苹果酸脱氢酶的性下降。线粒体蛋白质随着种子老化而显著变化,小分子量热休克蛋白（LMW HSP）22从老化的0d到30d增加,然后从30d到90d下降,可以认为甜菜种子人工老化过程中线粒体活性和LMW HSP22的含量变化与种子活力密切相关。     

Efficient embryogenic suspension culturing and rapid transformation of a range of elite genotypes of sweet potato (Ipomoea batatas [L.] Lam.)

Efficient Agrobacterium tumefaciens-mediated transformation was developed using embryogenic suspension cell cultures of elite sweet potato (Ipomoea batatas [L.] Lam.) cultivars, including Ayamurasaki, Sushu2, Sushu9, Sushu11, Wanshu1, Xushu18 and Xushu22. Embryogenic suspension cultures were established in LCP medium using embryogenic calli induced from apical or axillary buds on an induction medium containing 2 mg l⁻¹ 2,4-D. Suspension cultures were co-cultivated with A. tumefaciens strain LBA4404 harboring the binary plasmid pCAMBIA1301 with the hpt gene as a selectable marker and an intron-interrupted uidA gene as a visible marker. Several key steps of the sweet potato transformation system have been investigated and optimized, including the appropriate antibiotics and their concentrations for suppressing Agrobacterium growth and the optimal doses of hygromycin for transformant selection. A total of 485 putative transgenic plant lines were produced from the transformed calli via somatic embryogenesis and germination to plants under 10 mg l⁻¹ hygromycin and 200 mg l⁻¹ cefotaxime. PCR, GUS and Southern blot analyses of the regenerated plants showed that 92.35% of them were transgenic. The number of T-DNA insertions varied from one to three in most transgenic plant lines. Plants showed 100% survival when 308 transgenics were transferred to soil in the greenhouse and then to the field. Most of them were morphologically normal, with the production of storage roots after 3 months of cultivation in the greenhouse or fields. The development of such a robust transformation method suitable to a range of sweet potato genotypes not only provides a routine tool for genetic improvement via transgenesis but also allows us to conduct a functional verification of endogenous genes in sweet potato.     

Beet leafhopper (Hemiptera: Cicadellidae) settling behavior, survival, and reproduction on selected host plants

Experiments were conducted to determine the settling behavior, survival, and reproduction of the beet leafhopper, Circulifer tenellus (Baker), when maintained on selected host plants. This leafhopper was recently identified in the Columbia Basin of Washington and Oregon as the probable vector of the beet leafhopper-transmitted virescence agent phytoplasma, causal agent of several vegetable crop diseases, including potato purple top. Plants selected for study were sugar beet, Beta vulgaris L.; radish, Raphanus sativus L.; dry bean, Phaseolus vulgaris L.; potato, Solanum tuberosum L.; carrot, Daucus carota L.; and tomato, Lycopersicon esculentum Mill. Leafhopper adults were confined on caged plants, and settling behavior was observed during a 72-h period and survival was monitored for 40 d. Also, oviposition and nymphal production were investigated by maintaining leafhoppers for approximately 90 d on each of the selected plants. Sixty to 100% of leafhoppers settled on all studied plants during the first 5 h, but settling on bean and tomato declined sharply thereafter. Leafhopper mortality was very high on bean and tomato, with 95 and 65% of the leafhoppers, respectively, dying in about a week. In contrast, 77, 90, and 95% of leafhoppers maintained on potato, sugar beet, and radish, respectively, survived until the end of the 40-d experimental period. Beet leafhopper oviposition and nymphal production and development only occurred on sugar beet, radish, and potato; reproduction was lower on potato.     

人工合成根特异启动子SRSP的功能分析

根负责吸收水分和养分,是重要的植物组织,但易受生物及非生物胁迫,影响作物的生长发育和产量。设计合成根特异启动子,可为与胁迫相关的抗性基因在作物根部的功能研究及高效表达提供候选启动子。文中将4拷贝的根特异性顺式作用元件(OSE1ROOTNODULE、OSE2ROOTNODULE、SP8BFIBSP8AIB和ROOTMOTIFAPOX1)以串联排列方式设计合成了一个根特异性模块(pro-SRS),并与来自CaMV35S启动子的最小启动子融合,合成一个人工合成启动子SRSP。通过替换CaMV35S启动子将SRSP启动子克隆到pCAMBIA2300.1中以驱动GUS表达。将携带SRSP启动子的构建体通过农杆菌介导的方法转化到烟草中。GUS组织化学染色分析和实时PCR (RT-PCR)分析显示SRSP启动子在转基因烟草中赋予根特异性表达。说明顺式作用元件重复排列可实现启动子预期功能,本研究为理性设计植物组织特异启动子奠定了理论基础。     

Beet leafhopper (Hemiptera: Cicadellidae) transmits the Columbia Basin potato purple top phytoplasma to potatoes, beets, and weeds

Experiments were conducted to determine whether the beet leafhopper, Circulifer tenellus (Baker) (Hemiptera: Cicadellidae), transmits the purple top phytoplasma to potato, Solanum tuberosum L.; beets, Beta vulgaris L.; and selected weed hosts. The beet leafhopper-transmitted virescence agent (BLTVA) phytoplasma was identified as the causal agent of the potato purple top disease outbreaks that recently occurred in the Columbia Basin of Washington and Oregon. The phytoplasma previously was found to be associated almost exclusively with the beet leafhopper, suggesting that this insect is the probable vector of BLTVA in this important potato-growing region. Eight potato cultivars, including 'Russet Burbank', 'Ranger Russet', 'Shepody', 'Umatilla Russet', 'Atlantic', 'FL-1879', 'FL-1867', and 'FL-1833', were exposed for a week to BLTVA-infected beet leafhoppers. After exposure, the plants were maintained outdoors in large cages and then tested for BLTVA by using polymerase chain reaction after 6 to 7 wk. The leafhoppers transmitted BLTVA to seven of the eight exposed potato cultivars. Sixty-four percent of the exposed plants tested positive for the phytoplasma. In addition, 81% of the BLTVA-infected potato plants developed distinct potato purple top disease symptoms. Beet leafhoppers also transmitted BLTVA to beets and several weeds, including groundsel, Senecio vulgaris L.; shepherd's purse, Capsella bursa-pastoris (L.) Medik); kochia, Kochia scoparia (L.) Schrad; and Russian thistle, Salsola kali L. This is the first report of transmission of BLTVA to potatoes, beets, and the above-mentioned four weed species. Results of the current study prove that the beet leafhopper is a vector of the potato purple top disease.     

Sporamin-mediated resistance to beet cyst nematodes (Heterodera schachtii Schm.) is dependent on trypsin inhibitory activity in sugar beet (Beta vulgaris L.) hairy roots

Sporamin, a sweet potato tuberous storage protein, is a Kunitz-type trypsin inhibitor. Its capability of conferring insect-resistance on transgenic tobacco and cauliflower has been confirmed. To test its potential as an anti-feedant for the beet cyst nematode (Heterodera schachtii Schm.), the sporamin gene SpTI-1 was introduced into sugar beet (Beta vulgaris L.) by Agrobacterium rhizogenes-mediated transformation. Twelve different hairy root clones expressing sporamin were selected for studying nematode development. Of these, 8 hairy root clones were found to show significant efficiency in inhibiting the growth and development of the female nematodes whereas 4 root clones did not show any inhibitory effects even though the SpTI-1 gene was regularly expressed in all of the tested hairy roots as revealed by northern and western analyses. Inhibition of nematode development correlated with trypsin inhibitor activity but not with the amount of sporamin expressed in hairy roots. These data demonstrate that the trypsin inhibitor activity is the critical factor for inhibiting growth and development of cyst nematodes in sugar beet hairy roots expressing the sporamin gene. Hence, the sweet potato sporamin can be used as a new and effective anti-feedant for controlling cyst nematodes offering an alternative strategy for establishing nematode resistance in crops.     

甘薯MADS-box转录因子IbMADS11-Like的克隆及胁迫响应分析

MADS-box蛋白在植物生长发育及抗逆等过程中均发挥重要功能。本实验室根据甘薯近缘野生种I.trifida基因组序列,在甘薯栽培种徐薯22（Ipomoea batatas（L.） Xu22）中克隆到一个STMADS11亚家族MADS-box基因,命名为IbMADS11-Like。实时定量RT-PCR分析表明,IbMADS11-Like基因在甘薯根中大量表达,并且随着块根的形成和膨大表达量逐渐降低,表明该基因可能参与了甘薯块根的发育过程。胁迫处理分析表明,IbMADS11-Like基因的表达受干旱、盐和高温的诱导,而低温则抑制其表达。此外,IbMADS11-Like基因对ABA、IAA、ZT、BR、ACC、JA及GA等激素的处理也有不同程度的响应,暗示IbMADS11-Like基因可能参与了甘薯生长发育及胁迫的调控过程。这些结果为进一步分析IbMADS11-Like基因在甘薯块根发育和胁迫响应中的功能奠定了基础。