白菜小孢子超低温保存及复苏后胚状体诱导

随着白菜游离小孢子培养技术的不断优化，通过诱导小孢子胚状体获得大量DH系在白菜育种等科研领域得到了广泛应用。但小孢子供体植株受生长季节、生长期等影响，无法长期持续获取高生活力白菜小孢子，成为目前阻碍白菜细胞工程相关研究顺利推进的重要问题之一。本试验利用超低温技术对10份白菜材料小孢子开展冻存复苏研究，结果表明：利用改良B5液体培养基作为冷冻保护剂，白菜小孢子生活力明显高于传统冷冻保护剂二甲基亚砜处理的白菜小孢子；小孢子-80 ℃(冰箱)和-196 ℃(液氮)冻存180 d后生活力仍保持在85%以上，且两个温度处理间差异不显著，实际应用中可选择-80 ℃冰箱冻存替代传统-196 ℃液氮冻存；利用0.5 mL冻存管保存的白菜小孢子最适复苏条件为37 ℃恒温水浴40 s，在-80 ℃冰箱冻存90 d后复苏的小孢子平均生活力达87.9%。复苏冻存30、60、90 d后易出胚材料的白菜小孢子诱导获得了胚状体及再生植株；复苏冻存180 d的白菜小孢子在高温热激诱导条件下仍可感应胁迫，表现为体积膨大，且具胚胎发生能力的小孢子能够进行细胞分裂。本试验为国内首次开展的白菜小孢子超低温冻存研究，建立了白菜...     

金太阳杏花粉超低温保存方法研究

为探讨杏花粉种质保存的方法,进而为花粉培养、遗传转化和杂交育种提供材料,以杏品种金太阳的新鲜成熟花粉为试材,采用干燥法和玻璃化法2种超低温保存技术,对花粉干燥时间、解冻方式和贮藏时间等影响保存后花粉萌发率的有关因素进行研究。结果表明,干燥时间对金太阳杏花粉超低温保存后的萌发率有显著影响,其中干燥法宜在4℃下硅胶干燥8h、玻璃化法宜在(20±1)℃下以玻璃化液PVS2(体积分数15%二甲基亚枫+15%乙二醇+30%甘油+0.4mol/L蔗糖)处理60min;干燥法保存后分别以4℃2h、20℃30min和40℃70s等3种方式化冻,花粉萌发率无明显差别,而对玻璃化法则以40℃70s化冻后的花粉萌发率最高;以2种方法分别保存1、10、30、60d后花粉萌发率无显著变化。     

软枣猕猴桃休眠芽超低温保存技术研究

【目的】探讨软枣猕猴桃种质资源的长期保存技术体系。【方法】以软枣猕猴桃‘魁绿’休眠芽为试材,研究了预培养液种类、预培养时间、装载时间、玻璃化保护液、恢复培养基等因素对玻璃化法超低温保存后休眠芽成活率的影响。【结果】休眠芽在0.3 mol·L~(-1)蔗糖+1 mol·L~(-1)甘油的预培养液中震荡培养2 d,常温下用装载液(2 mol·L~(-1)甘油+0.4mol·L~(-1)蔗糖+MS)处理20 min,0℃条件下用PVS2(30%甘油+15%乙二醇+15%二甲基亚砜+0.4 mol·L~(-1)蔗糖+MS)脱水120 min,换新鲜PVS2后迅速投入液氮中冻存24 h以上。取出后立即用38℃水浴解冻2 min,用含1.2 mol·L~(-1)蔗糖的MS卸载液洗涤30 min,无菌滤纸吸干后接种到MS+2 mg·L~(-1)6-BA+0.02 mg·L~(-1)NAA恢复培养基上,休眠芽成活率达86.30%。用流式细胞仪鉴定再生植株倍性,没有发现明显变化。【结论】建立了高效的软枣猕猴桃‘魁绿’休眠芽玻璃化超低温保存体系。     

不同保存方式对扁桃花粉保存效果的影响

采用干燥法和玻璃化法2种方法,对新疆莎车县扁桃品种晚丰18号花粉进行了超低温保存研究。结果表明:晚丰18号花粉最佳干燥方式为4℃硅胶干燥,适宜干燥时间为6⁸ h,干燥6h和8 h的花粉含水量分别为53.74%、52.74%,超低温保存后花粉萌发率为61.22%、63.77%,相对保存率为91.68%、97.13%;在室温(20±1)℃下以玻璃化液PVS2(30%甘油+15%乙二醇+15%二甲基亚砜+0.4 mol/L蔗糖)处理208 h,干燥6h和8 h的花粉含水量分别为53.74%、52.74%,超低温保存后花粉萌发率为61.22%、63.77%,相对保存率为91.68%、97.13%;在室温(20±1)℃下以玻璃化液PVS2(30%甘油+15%乙二醇+15%二甲基亚砜+0.4 mol/L蔗糖)处理20³0 min后,超低温保存的花粉萌发率最高,为40.88%30 min后,超低温保存的花粉萌发率最高,为40.88%⁴1.53%;不同解冻方式间花粉萌发率差异极显著,以35℃解冻70 s的萌发效果最好;干燥法保存效果较玻璃化法保存好,超低温保存时间对花粉萌发率无显著影响。     

应用茎尖滴冻法超低温保存香蕉资源研究(英文)

采用正交试验探讨了滴冻法中装载液处理时间、玻璃化溶液处理时间和过渡培养时间的最优组合;另外还研究了不同冰冻保护剂、茎尖大小、恢复培养基中大量元素浓度对滴冻法保存效果的影响。通过试验筛选出了最佳的滴冻法保存方案:剥取含有1~2个叶原基的茎尖,长约1.5~2.5 mm,先在室温下用装载液预处理20~120 min,再用玻璃化溶液PVS2于0℃下处理30~50 min,接着将PVS2溶液滴于铝箔上,每个液滴中放一个茎尖,迅速投入液氮。保存1 h后,将铝箔取出投入卸载液(Suc.1.2 mol.L-1+MS)中快速解冻。15 min后取出茎尖,用滤纸吸去水分,转入过渡培养基上(Suc 0.3 mol.L-1+MS)暗培养24 h。再转到恢复培养基上,暗培养1周后转移到光下常规培养。采用滴冻法成功保存了分属于5个基因组型的15份香蕉资源,每种基因组型的平均成活率为:AAA 78.0%,AAB 57.8%,ABB72.1%,AA 43.3%,野生蕉42.2%。     

香蕉种质超低温保存技术研究进展

对于香蕉这种营养繁殖的植物,超低温保存是长期有效地保存其种质的一种有效方法。主要综述了香蕉胚性悬浮细胞系、合子胚和茎尖分生组织的超低温保存技术方面近十几年来的研究进展。总结了影响香蕉超低温保存效果的几个主要因素,主要包括材料、预处理、装载处理以及冰冻保护剂。并分析了当前应用在香蕉分生组织上的4种超低温保存方法的优缺点,分别为:分生组织团简单冻存法、分生组织团玻璃化法、单个分生组织玻璃化法、滴冻玻璃化法,认为滴冻玻璃化法效果最好。     

草菇基因文库的构建(英文)

应用基因重细技术,我们建立丁一个草菇总DNA的部分基因文库。通过限制性内切酶Sau 3AI部分酶切纯化的草菇总DNA,得到小于4kb的DNA片段。这些DNA片段插入到经Bam HI酶切及碱性磷酶酯酶处理的pUC18质粒,转化感受态细胞DH5α。在此研究中。我们比较了两种转化方法(氯化钙转化法和电转化法)的转化效率,电转化法的转化效率高于氯化钙转化法数千倍,达到2×10(2)CFU/μg DNA。在此实验中DNA重组率是64%,这个草菇总DNA的部分基因文库由8000个克隆组成,平均每个克隆含有1.5kb的草菇DNA。此基因文库含有15%的草菇总DNA。可作为分子标记用于DNA指纹图谱分析及作为遗传学标记用于RFLPs的遗传图谱分析。     

小西葫芦黄花叶病毒外壳蛋白基因植物表达载体的构建

小西葫芦黄花叶病毒(Zucchiniyellowmosaicvirus,ZYMV)是危害中国葫芦科作物主要病毒之一。试验以该病毒中国分离物外壳蛋白基因的克隆载体pZCP-87(含ZYMV的CP基因)为材料,经SalⅠ和BamHⅠ双酶切,从胶上回收目的基因,与经过同两种酶酶切的植物表达载体pBIN438连接,转化感受态的大肠杆菌细胞,提取质粒,经PCR和SalⅠ/BamHⅠ双酶切验证,已将该病毒外壳蛋白基因克隆到植物表达载体上(重组质粒命名为pBZCP5),采用冻融法完成了对pBZCP5质粒的农杆菌转化。该工作是通过转基因获得抗ZYMV病毒研究的基础。     

生物磷肥对普通白菜生长、品质及土壤养分的影响

采用盆栽试验方法,研究解磷细菌肥对普通白菜生长、品质及土壤理化性质的影响。结果表明:施用解磷细菌肥后土壤细菌数量极显著升高,解磷细菌数量随解磷细菌肥施用量的增加而极显著增加,每盆施用45g解磷细菌肥处理的土壤解磷细菌可达2.05×106cfu·g-1;同时土壤p H值下降。随着解磷细菌肥施用量的增加,普通白菜地上部磷积累量极显著增加,分别比其相应对照增加121.06%、226.43%和302.09%;地上部鲜质量呈显著增高趋势,分别比其相应对照增加40.19%、52.71%和33.56%;根冠比显著降低,其中每盆施用45g解磷细菌肥处理的根冠比最低,为0.013。每盆施用45g解磷细菌肥处理的普通白菜叶片VC、可溶性糖、可溶性蛋白含量及游离氨基酸含量分别比其相应对照增加29.97%、39.94%、30.86%、17.36%,但硝态氮含量降低21.83%。综上,以麸皮为主要载体制作的解磷细菌肥可以极显著提高普通白菜对土壤难溶性磷素的利用,并能显著提高普通白菜的产量和品质。     

冻结终温和贮藏温度对梨枣果实的影响

以梨枣为试材,研究了冷冻、冻结终温和冻藏温度对果肉细胞相对活力、果肉细胞膜透性和果实品质的影响.试验结果表明:梨枣冷冻后,果肉细胞活力下降了85.0%.冻藏8个月后,贮藏在-35℃中的枣果果肉细胞相对活力比贮藏在-22℃中的高27.4%.同时,果肉细胞相对活力和果肉细胞膜透性之间呈极显著负相关(r=-0.9023).冻结终温和贮藏温度对果肉细胞膜透性有显著的影响.梨枣冷冻后,果肉硬度极显著下降,而在贮藏过程中,没有明显变化.梨枣在冻藏的后4个月,抗坏血酸含量显著下降,下降幅度达37.1%～40.2%;可溶性固形物含量在贮藏前4个月显著下降,后4个月又显著上升;可滴定酸含量没有受到冷冻、冻结终温和贮藏温度的显著影响.     

Cloning and expression of mouse canstatin cDNA in E.coli

AIM: To clone and express mouse canstatin (m canstatin) cDNA and provide a basis for the further research on its anti-angiogenic activity and potential application for cancer therapy. METHODS: Total RNA was extracted from mouse liver tissue by Trizol Reagent, and mouse canstatin cDNA was amplified by RT- PCR, then cloned into vector pMD18-T for sequencing. pET30a(+)-m canstatin recombinant plasmid was constructed and expressed in E.coli BL21 with induction of IPTG. RESULTS: Mouse canstatin cDNA is 684 bp coding 227 amino acids. The sequences of both cDNA and amino acid share high homology with human canstatin, with cDNA identity at 89% and amino acids identity at 96% to human canstatin. In the present study, pET30a(+)-m canstatin recombinant plasmid was expressed in E.coli BL21. CONCLUSION: Mouse canstatin cDNA has been cloned for the first time. Constructed pET30a(+)-m canstatin recombinant plasmid is highly expressed in E.coli BL21.     

Expression of recombinant human tumor suppressor NDPK-A in E. coli

AIM: To construct E. coli expression plasmid of recombinant human NDPK-A with a 6×His tag, optimize the expression condition and identify the activity of the product. METHODS: nm23-H1 was subcloned from plasmid pBVNMH1 to pQE40 which contain 6×His purification tag. The expression condition was modulated in grades to get the optimal expression. We purified protein with the Ni⁺-NTA affinity chromatography column, identified the immunogenicity of the product with Western blot, and measured the kinases activity with HPLC. In addition, angiogenesis inhibition activity of rhNDPK was identified by CAM. RESULTS: The sequence of nm23-H1 subclone in pQE40 was exactly correct. The expression rate of rhNDPK-A was 49.6%. Purified rhNDPK-A specially recognized the antiserum of NDPK-A. It also inhibited angiogenesis. CONCLUSION: PQE-nm23H1 containing 6×His can express target protein at high level. This purification method is simple than other methods, and the product has the same activity as natural human NDPK-A.     

Expression features of human decorin in E.coli DH5α

AIM: To study the expression features of human decorin in E. coli DH5α.METHODS: The pGEX-4T-1-decorin fusion clone was expressed in E. coli DH5α. Positively expressed clone was selected by SDS-PAGE. The optimized inducing time by 1 mmol/L IPTG was determined. The solubility of GST-decorin fusion protein was analyzed by ultrasonic crush method, and its quality was examined by Western blotting. RESULTS: With the induction of 1 mmol/L isopropy-β-D-thiogalactoside (IPTG), the fusion protein was expressed in E. coli DH5α. The optimized inducing time by 1 mmol/L IPTG was 4 hours. Most of fusion protein existed in the form of inclusion body. The expressed protein was GST fusion protein.CONCLUSION: It is suggested that the fusion protein of GST-decorin may be expressed in E.coli DH5α in a large amount in the form of inclusion body.     

High efficient expression of human endostatin in E.coli and its antiangiogensis activity

AIM: To examine the expression of human endostatin in E.coli, produce its fusion protein antibody and observe its biological activity. METHODS: Endostatin gene was amplified by polymerase chain reaction,recombined with plasmid vector pGEX-2T and induced expression with IPTG.The protein activity was tested by endothelial cell proliferation inhibitory assay.Inclusion body crudely purified was used to generate polyclonal antibody to detect its expression at mouse's liver and kidney etc. RESULTS: The protein expressed was 20kD after digestion by thrombin,it appeared the anti-angiogenesis activity and Western blotting indicated the expression of endostatin in liver and kidney of mouse. CONCLUSION: The successful expression of human endostatin and the preparation of polycolonal antibody indicated its potential application in anti-angiogenesis therapy and diagnosis tumors.     

阔叶猕猴桃果实GalDH cDNA 克隆及其在大肠杆菌中的表达

 以猕猴桃属植物中果实维生素C含量最高的阔叶猕猴桃(Actinidia latifolia Merr. ) 果实为试 材, 经RT2PCR扩增获得约110 kb的L - 半乳糖脱氢酶cDNA片段。生物信息学分析表明, 该cDNA片段长为997 bp, 最大开放阅读框为960 bp, 可编码319 个氨基酸残基, 命名为A lGalDH, GenBank登录号为EU525846, 核苷酸序列及其推导的氨基酸序列与已知其它植物GalDH核苷酸、氨基酸序列间的同源性分别在76%和70%以上, 且具有醛酮还原酶的保守结构域。构建了其原核表达载体pET-A lGalDH并转化大肠杆菌BL21, 经0.1 mmol·L ^{- 1} IPTG诱导, 获得具有较高活性的表达融合蛋白6 ×His-AlGalDH。经Ni-His亲和磁珠分离纯化, 获得单一的融合目的蛋白条带, 测定其酶活为120 pmol·min^{- 1} ·mg^{- 1}。     

新疆红肉苹果PGIP基因的克隆及原核表达

【目的】研究克隆新疆红肉苹果[Malus sieversii f.neidzwetzkyana(Dieck)Langenf]PGIP基因并进行原核表达,探讨其抗病机制。【方法】根据Genbank中已经发表的‘金冠’苹果PGIP保守区域设计1对特异引物,以新疆红肉苹果叶片总RNA为模板,T/A克隆后进行序列测定,并对该序列进行分析。随后将该蛋白成熟肽cDNA片段连接到原核表达载体pET30a(+)中,构建融合表达质粒,转化到E.coli BL21(DE3)中进行表达。【结果】序列分析表明,新疆红肉苹果PGIP基因cDNA编码区全长993 bp,编码330个氨基酸残基,命名为MsPgip,GenBank登录号为JQ001783。MsPgip分子质量为36.6 kD,等电点为7.05,有6个潜在的N-糖基化位点,信号肽为N端24个氨基酸残基。该蛋白质还具有2个连续的24个氨基酸残基大小的LRR基序(LSQLKNLTFLDLSFNNLTGAIPSSLSQ LPNLNALHLDRN-KLTGHIPIS)。与已克隆的‘澳洲青苹’、‘金冠’、‘富士’苹果PGIP氨基酸序列同源性均高达99%。原核表达产物经SDS-PAGE分析表明,表达蛋白的分子质量与预期一致。【结论】克隆了新疆红肉苹果PGIP基因,并可在大肠杆菌中表达。     

Expression of phenylalanine ammonia-lyase cDNA from rice in E. coli BL21DE3

AIM: To study the expression and its kinetics of rice phenylalanine ammonia-lyase gene encoding into E. coli as the basis of treatment for phenylketouria. METHODS: The phenylalanine ammonia-lyase-1-cDNA(rPAL-1-cDNA) from rice was recombined into E. coli high expression vector pET-28c and transformed into E. coli host strain BL21DE3. Engineering bacteria was then inducted by isopropyl-β-D-thiogalactoside (IPTG) for 1, 3, 5, 7 hours, in order to obtain high level expression. RESULTS: After induction, the expression level of fusion protein was 21.40%, 30.60%, 35.40%, 35.43% respectively. The fusion protein exhibited a band of 78.6 kD on SDS-PAGE analysis, but was not found in controls.The target protein was mainly existed in the form of inclusion body. CONCLUSION:Rice PAL gene expressing E. coli was established by gentic engineering technique.     

Synthesis of multiple copies of neurotrophic peptide and their high level expressions in E.coli

AIM: To construct and express containing multiple tandem copies of a peptide (neurotrophic peptide,NP),which was designed according to the NP sequence of prosaposin.METHODS: DNA sequence of peptide NP was synthesized by the preferred codons of E.coli.Two copies of NP fragments were produced by PCR and inserted into pUC18 vector.The fragments from pUC18-2NP by EcoT 14I were ligated into tandem multi-NP fragments through self-ligation,and subcloned into pET-EcoT vector,which has a unique EcoT14I cloning site allowing unidirectional insertion of a desired sequence.Multi-NP clones were screened by PCR-array.RESULTS: The constructs with different repeats of NP were obtained and expressed as fused-proteins at high level in E.coli BL21 (DE3).In order to get monomer peptide,each copy of peptide was interspersed by a unique site where the fused-protein could be cleaved by cyanogens bromide.CONCLUSION: Peptide NP could be highly expressed in E.coli.This work builds a solid foundation for further study on its bioactivity.