八棱海棠转PuNHA基因的遗传转化研究

以八棱海棠茎尖分生组织为外植体,经农杆菌介导将PuNHA基因导入八棱海棠,在MS+BA 4 mg/L+NAA 0.2 mg/L+除草剂4 mg/L+羧苄青霉素500 mg/L的培养基中筛选培养,转化率8.7%。结果表明:经PCR、Southern Blot和Northern Blot分析得出,PuNHA基因已经整合到八棱海棠基因组内,并且可以转录为mRNA,获得了转基因植株;同时将转基因植株进行盐胁迫处理,耐盐能力有显著提高,由原来的耐盐量2‰提高到了3‰,其耐盐能力提高了50%。     

超声波辅助农杆菌介导八棱海棠转rolC基因

应用超声波辅助农杆菌介导法,对八棱海棠进行rolC基因转化,以期提高转化效率并获得转基因植株。利用gus基因瞬间表达的方法研究了超声波处理时间、处理时期和农杆菌悬浮液中乙酰丁香酮(As)浓度对rolC基因转化率的影响。结果表明,叶盘在D600nm为0．6且含有75 mg／L As的农杆菌悬浮液中侵染2 min后,用功率为100 W的超声波处理30 s,再浸泡2．5 min,然后放到再生培养基上共培养3 d,能获得最佳的gus基因瞬间表达率。最佳处理条件下转化683枚八棱海棠叶片,共得到138个抗性愈伤组织和15株抗性苗,转化率为2．2％。GUS染色、PCR及Southern blotting检测结果显示,有12个八棱海棠株系的基因组中整合了完整的外源rolC基因。     

珠美海棠叶片离体培养与植株再生

将珠美海棠试管苗叶片接种于附加NAA0.3 mg·L-1和不同体积分数的6-BA(1.0～6.0 mg·L-1)的MS培养基中进行离体培养,结果表明,6-BA为1.0～3.0 mg·L-1的处理芽再生率为6.7%～31.8%,而根的再生率为26.7%～28.3%.6-BA为4.5和6.0 mg·L-1处理芽再生率分别高...     

珠美海棠MzWRKY基因家族盐胁迫应答模式研究

对苹果属植物现有的58个WRKY 的EST序列进行分析,得到了37条uniESTs。珠美海棠（Malus zumi Mats）是耐盐的优良苹果砧木,能在土壤全盐含量为0.6%的盐碱地上存活。根据已知的37条uniESTs序列,通过半定量RT-PCR研究了珠美海棠中MzWRKY基因家族的盐应答模式。28个MzWRKY基因能检测到RT-PCR的产物,其中21个基因受盐诱导,1个受盐抑制。根据MzWRKY基因盐应答模式可将这些基因分为两组,基因表达模式的差异说明MzWRKY在珠美海棠的盐应答中角色的多样性。     

农杆菌介导三价融合基因Rirol转化八棱海棠的研究

研究了以口蹄疫病毒FMDV 2A序列融合多基因在八棱海棠遗传转化上的应用,将DREB,IRT1和rolC融合三价基因(Rirol)通过农杆菌介导法转化八棱海棠,获得了一批nptⅡ抗性植株,经报告基因检测、PCR和Southern杂交分析证明融合基因成功导入到八棱海棠中。对转基因八棱海棠相关性状分析表明,转基因八棱海棠比对照具有较强的耐盐性,在表型上,表现节间缩短,分枝性强,侧根发达,证明外源融合基因在转基因植株中得到表达。试验初步证明了利用FMDV 2A序列进行多基因转化八棱海棠的可行性,为苹果等果树多基因转化提供了新的思路和途径。     

提高农杆菌介导番茄遗传转化效率的研究

利用农杆菌介导法对番茄进行遗传转化,比较了不同的农杆菌种类、农杆菌侵染时间、外植体种类及番茄品种对农杆菌侵染效率的影响.转化后得到的再生植株,利用PCR、Southern杂交检测GUS基因是否整和进入基因组.结果表明:农杆菌介导法成功介导了GUS基因导入番茄.农杆菌EHA105侵染番茄的效率明显高于LBA4404,侵染时间3 min较好.子叶外植体的转化明显优于下胚轴,黄色串珠樱桃番茄的遗传转化效率高于中蔬5号;优化的转化条件可以使番茄获得较高的遗传转化效率.     

根癌农杆菌介导的矮牵牛遗传转化体系研究

研究建立了根癌农杆菌介导的矮牵牛遗传转化体系.结果表明最佳的不定芽诱导分化和继代培养基为:MS 6-BA1.5 mg/L NAA0.2 mg/L;选择培养基中30 mg/L卡那霉素和300 mg/L的头孢霉素是适宜的抗生素使用浓度;侵染过程中0.05 MPa负压处理5 min和共培养过程中添加20 mg/L的乙酰丁香酮均可提高转化效率.卡那霉素抗性植株经2次选择继代培养后,PCR检测全部为阳性.对部分PCR阳性植株进行PGR-Southern杂交,证实外源基因已整合进入矮牵牛基因组中.     

农杆菌介导的BnCS基因对黄瓜遗传转化研究

以黄瓜为受体,用农杆菌介导的方法,将同源克隆的抗冷基因BnCS转入黄瓜,建立农杆菌介导的遗传转化体系,获得转化的抗性植株;对抗性植株和后代进行PCR鉴定,证明BnCS基因已经转入黄瓜.对T0种子和T1植株进行耐冷性鉴定.结果表明:后代耐冷性有所增强;试验获得的耐冷材料已应用于育种中.     

湖北海棠二价融合表达载体MhT2AC的构建及转化烟草耐盐性分析

 利用口蹄疫病毒2A序列将湖北海棠MhTGA2转录因子和几丁质酶基因（Mhchitl）两个抗病相关基因融合在同一个开放阅读框下,构建二价融合植物双元表达载体,命名为MhT2AC。将该载体转化烟草,获得了8个转基因株系,MhTGA2基因和Mhchitl基因在烟草中共表达,并且转基因株系中NtSOD、NtAPX和NtPPO等抗逆基因的表达量均加强;转基因烟草T₁代植株抗盐性增强。     

农杆菌介导ODREB2B基因转化马铃薯的研究

以马铃薯品种黄麻子和中薯3号脱毒微型薯为外植体,利用根癌农杆菌介导法,将来源于诸葛菜的抗逆相关转录因子ODREB2B基因导入马铃薯,并对遗传转化的影响因素进行优化。获得抗性植株黄麻子33株、中薯3号27株,经PCR-Southern杂交分析,两品种分别有16株和14株呈阳性,证明ODREB2B基因已整合到马铃薯基因组中。     

盐胁迫对珠美海棠和山定子膜保护酶系统的影响

实验以耐盐种珠美海棠(MaluszumiMats)和盐敏感种山定子[Malusbaccata(L.)Borkh]为试材,比较研究了在盐胁迫下叶片质膜透性、叶绿素和丙二醛(MDA)含量、膜保护酶超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、过氧化物酶(POD)活性及根质膜透性的变化,为探索苹果属植物耐盐机制提供素材。结果表明,盐胁迫对根质膜透性没有明显的影响;但导致叶片质膜透性增加,盐敏感种山定子的质膜透性高于耐盐的珠美海棠。随盐胁迫加重,叶绿素a、b含量降低,叶绿素a/b比值增加,类胡萝卜素变化不明显。MDA含量随盐胁迫而升高,但珠美海棠低于山定子。山定子叶片中SOD、POD、CAT活性随盐胁迫加重而降低,而珠美海棠SOD活性在盐胁迫下保持稳定,POD、CAT活性增高。由此可见珠美海棠在盐胁迫条件下有活性较高的膜保护酶系统。     

苹果属植物变叶海棠遗传多样性形成机理研究进展

过去20余年国内外学者对苹果属植物的起源、分类、区系地理、种内和种间遗传变异等开展了广泛的研究。然而,有关苹果属植物遗传多样性形成机理迄今未见任何直接报道。作者从变叶海棠的遗传多样性及其起源,物种的居群分化及其近缘种的形成几方面重点介绍过去15a西南大学在研究变叶海棠的遗传多样性和居群分化等方面所取得的进展。我们的研究工作为进一步探索苹果属植物遗传多样性形成机理奠定了基础,并为变叶海棠珍贵基因资源的保护和利用提供了理论依据。     

植物基因转化方法的研究概况

综述了目前用来进行植物基因转化的各种方法及其优缺点 ,以期为植物转基因方法的选择提供借鉴     

根癌农杆菌介导的西瓜遗传转化研究

利用带有内含子的GUS基因的瞬时表达,研究影响根癌农杆菌介导的西瓜遗传转化的若干因素。结果表 明:西瓜子叶块外植体对潮霉素较为敏感,15 mg/L是适宜的筛选浓度,可明显抑制非转化组织的生长;脱菌过程中 采用500 mg/L的头孢霉素,对外植体生长影响较小;农杆菌菌株EHA105对西瓜子叶块的侵染能力较强;预培养有 利于转化;共培养3-4 d有利于提高转化频率并避免了农杆菌的过度生长;OD600值为0.3的菌液侵染10min效果最 佳;共培养培养基中添加乙酰丁香酮可提高转化频率。     

葡萄组培苗耐盐性研究

对中国野生葡萄4个种的6个株系、2个种间杂种、5个鲜食葡萄品种、1个酿酒葡萄品种和5个砧木品种通过组织培养获得试管苗,以不同浓度的NaCl作为盐胁迫因子,分别加入生根培养基中进行耐盐筛选。结果表明,在盐胁迫的50d内,试管苗的侧芽萌发倍数、增殖系数、生根率、生根数目均随着盐浓度的升高而降低,试管苗的受害指数、胁迫敏感指数随着盐浓度的升高而增大。在0.6%NaCl胁迫下,随着盐胁迫时间的延长,受害率和受害指数呈上升趋势。将供试的19个材料耐盐性分为4个类型:耐盐葡萄类型,包括酿酒葡萄EndFine、燕山葡萄燕山-1、1-1-6(88-110)、莫里莎无核和克瑞森无核;较耐盐葡萄类型,包括红地球、1-1-8(88-110)、户太8号、山葡萄通化-3和秋葡萄平利-7;盐较敏感葡萄类型,包括京秀、101-14MG、3309C和蘡奠葡萄泰山-1、安林-3;盐敏感葡萄类型,包括SO4、1103P、110R和山葡萄华县-47。     

果树中基因枪法遗传转化的研究进展

目前,应用于果树遗传转化的方法主要有农杆菌介导法和DNA直接导入法。介绍了DNA直接导入法中基因枪转化技术的原理、类型和基因枪的轰击参数、受体材料、基因型、轰击前后的培养条件等对其遗传转化频率的影响,并系统地概述了基因枪法在选择标记基因、报告基因、改良果树性状相关基因、启动子及多基因遗传转化中的应用研究进展,同时对果树基因枪转化研究中存在的问题及应用前景进行了讨论。     

The effects of NaCl pre-treatments on salt tolerance of melons grown under long-term salinity

The response of melon (Cucumis melo) plants to long-term salinity was investigated to determine the availability of the NaCl pre-treatments (seed priming + seedling conditioning) as an interesting strategy for increasing the salt tolerance. Seeds of melon cultivars “Hasanbey” and “Kirkagac” were primed with 18 dS m⁻¹ NaCl solution for 3 days at 20 °C. During emergence and seedling growth, non-primed seeds were irrigated with local irrigation water (EC: 0.3 dS m⁻¹) whereas primed groups were treated with 9.0 dS m⁻¹ saline solution for 35 days. Seedlings derived from pre-treated (P) and non-pre-treated (NP) groups were transplanted to 8 l pots. After transplanting, salinity treatments were started with the first irrigation. The salinity treatments consisted of five levels (control, 4.5, 9.0, 13.5 and 18.0 dS m⁻¹) of irrigation solution for a period of 90 days. NaCl pre-treatments diminished the inhibiting effect of salinity on growth of melon plants. However, competence for salt adaptation varied with cultivar and the level of salinity. The physiological response of the P plants was also maintained in the long-term. Stomatal conductance and relative chlorophyll content of P plants tended to be higher than those of the NP ones. In addition, NaCl pre-treatments enhanced K and Ca concentrations of leaves and stems, and prevented toxic effects of salinity because less Na accumulated in stems. These results suggest that the use of NaCl pre-treatments could be a useful strategy to increase the salt tolerance of melon plants in the long-term and also to permit the establishment of melon crop by direct sowing in a saline medium.     

Study on genetic instability of nm23-H1 gene and expression of hMLH1,hMSH2 in sporadic gallbladder carcinoma

AIM: To examine the MSI and LOH of locus D17S396 and their influence on the expression of nm23-H1 in gallbladder tumors,and to examine the protein expression of hMLH1/hMSH2,which may provide experimental evidence for the tumor occurrence and metastasis.METHODS: Techniques such as DNA extraction,CR-SSCP,ordinary silver stain were used to study MSI and LOH of locus D17S396.Envision IHC was used to assess the expression of nm23-H1 and hMLH1/hMSH2.RESULTS: ① The frequency of heredity instability of gallbladder carcinoma was 42.55%.The frequency of LOH in liver and lymph node metastasis cases and in stage Nevin IV and V was significantly higher than that without metastasis and stage I,II and III.However,the frequency of MSI showed contrary correlation with some clinicopathologic characteristics.② The expression of nm23-H1 was 46.81%.The case with lymph node metastasis and Nevin stage IV and V showed significantly lower expression than that without lymph node metastasis and stage I,II and III.③ The expressions of hMLH1 and hMSH2 were 51.06% and 42.55% respectively.hMLH1 in lymph node and liver metastasis cases and in stage Nevin IV and V were significantly lower than that without metastasis and in stage I,II and III.④ Positive frequency of hMLH1 in MSI positive group was higher than that in MSI negative group.The positive frequency of nm23-H1 and hMSH2 protein in LOH positive group was lower than that in negative group.CONCLUSION: The heredity instability of nm23-H1 gene may be implicated pathogenesis and progression of gallbladder carcinoma.Both MSI and LOH of nm23-H1 control the development of gallbladder carcinoma independently in different paths.Abnormal expression of hMLH1/hMSH2 may be a molecule marker in early stage of gallbladder carcinoma.     

阔叶猕猴桃遗传转化技术参数的优化

为了提高阔叶猕猴桃的遗传转化效率,以阔叶猕猴桃叶柄为试材,通过根癌农杆菌介导法进行了遗传转化技术参数的研究。结果表明,叶柄预培养3～4d、用农杆菌悬浮液(D600nm值0.5)感染10min、共培养48h、共培养时在培养基中加入200μmol/L乙酰丁香酮处理可以获得较高的gus基因表达率。在供试的1200个叶柄中获得了49个抗性芽,转化频率为4.1%。对转基因抗性材料进行PCR检测和gus基因组织化学染色,证实了外源基因已整合到阔叶猕猴桃的基因组中,并得到稳定表达。     

Lentivirus mediated CCN1 gene on growth and migration of rat bone marrow mesenchymal stem cells

AIM：To investigate the role of cysteine-rich 61 (Cyr61/CNN1) in proliferation and migration of bone marrow mesenchymal stem cells (BMSCs). METHODS：The lentiviral vector carrying CCN1 (Lenti-GFP-CCN1) was constructed and then transfected into the rat BMSCs. The cells were divided into non-transfection group, transfection group (transfected with Lenti-GFP-CCN1) and negative control group (Lenti-GFP). The fluorescence intensity of the transfected BMSCs was observed under inverted fluorescence microscope. The effects of CCN1 on the proliferation and migration of BMSCs were detected by MTT assay and scratch wound healing assay. RESULTS：The proliferation of BMSCs transfected with Lenti-GFP CCN1 had no significant difference compared with negative control group and control group. The width/thickness ratio of migrated BMSCs in wound healing was significantly higher in Lenti-GFP-CCN1 group than that in negative control group and control group (P<0.05). CONCLUSION：Exogenous CCN1 promotes the migration of BMSCs.