采用土壤农杆菌感染和粒子轰击法对爱字棉和珂字棉抗真菌构建基因的遗传转育

采用土壤农杆菌感染和粒子轰击法对爱字棉和珂字棉抗真菌构建基因的遗传转育试验采用几个抗真菌构建基因进行遗传转育以抑制黄曲霉菌的生长。用ＧＵＳ和ＮＰＴⅡ标志基因对珂字棉和爱字棉（陆地棉）品种就土壤农杆菌和粒子轰击两种不同的遗传转育方法进行最优化比较。试验...

小黑麦×普通小麦及其选育小麦遗传材料的研究

对小黑麦与普通小麦的可杂交性、小黑麦×普通小麦杂种Ｆ１、Ｆ２自交结实性及性状分离表现进行了分析。利用小黑麦×普通小麦杂交方式育成了几个特殊材料，并对特殊材料进行细胞遗传学和同工酶分析，发现含有黑麦血缘，现正在用于小麦育种实践中。

雌激素对载脂蛋白AⅠ基因启动子不同区域转录活性的影响

目的:探讨雌激素对载脂蛋白(Apo)AⅠ基因启动子不同区域转录调节活性的影响。方法:利用含荧光素酶报告基因的表达载体pGL2构建携带ApoAⅠ基因启动子不同区域片段的重组质粒和同时携带ApoCⅢ/AⅣ基因片段的重组质粒。阳离子脂质体法将重组质粒与pRL-null内参质粒共转染HepG2细胞,加入10μmol/L雌激素刺激24h检测细胞荧光素酶报告基因的荧光强度,以反映ApoAⅠ的转录水平。结果:pGL2/-256AⅠ,pGL2/-2500AⅠ,pGL2/-256AⅠCⅢAⅣ及pGL2/-2500AⅠCⅢAⅣ转染后雌激素组荧光素酶相对活性明显高于对照组(P<0.05);而pGL2/-41AⅠ及pGL2/-41AⅠCⅢAⅣ质粒转染后雌激素组与对照组荧光素酶活性差异无统计学意义(P>0.05)。结论:ApoAⅠ启动子-256^-41区域可能包含与雌激素作用相关的反应元件,受雌激素刺激后转录活性增强。

三种转染试剂介导pGPU6/GFP/Neo真核表达载体转染鸡胚成纤维细胞的比较研究

目的:采用不同转染试剂介导pGPU6/GFP/Neo质粒转染鸡胚成纤维细胞UMNSAH/DF-1,以获得最优化的方法。方法:分别采用不同剂量(1.25μl、2.0μl、2.5μl)的Lipofectamin2000、Gbfectene-Elite及HilyMax转染试剂介导1μg质粒进行转染,观察其转染效率,测定其转染细胞存活率。结果:HilyMax组的转染效率为(86.85%±2.32%),较Lipofectamin2000组(48.33%±3.24%)、Gbfectene-Elite组(37.35%±5.41%)高(F=18.882,P<0.05);其中,HilyMax 2.5μl组的转染效率最高为(90.53%±1.15%)。Lipofectamin2000组的细胞存活率(65.76%±5.78%)明显低于HilyMax组(89.54%±0.86%)、Gbfectene-Elite组(82.45%±3.56%)(F=90.676,P<0.05)。结论:HilyMax对于鸡胚成纤维细胞具有最好的转染效率和最低的细胞毒性,推荐使用2.5μl HilyMax:1μg质粒进行鸡胚成纤维细胞的转染。

外源类甜蛋白基因在马铃薯中的表达

将“津引薯 8号”马铃薯薄片和含有CaMV35S启动子控制的类甜蛋白 (TLP)基因与紧密连锁的Ubiquitin启动子控制的bar标记基因的农杆菌双元表达载体进行共培养。切下马铃薯薄片表面产生的幼芽 ,用含有除草剂bialaphos的MS培养基进行初步筛选。当获得的生根抗性芽生长成株后用TLP蛋白抗体和TLP基因特异引物对成株进行间接ELISA检测和PCR检测。两种检测结果一致率为 90 % ,推断以bar基因为筛选标记。

Maize Transformation of cry1Ac3 Gene and Insect Resistance of Their Transgenic Plants

The success for genetic transformation of maize (Zea mays L.) is highly related to genotype of target material. A few model varieties can be induced into type Ⅱ callus, which can be easily transformed with high regeneration frequency. However, most of cultivars could be only induced into type Ⅰ callus, which is difficult to be transformed with low regeneration. Thus, studying on the conditions of induction and transformation for type Ⅰ callus will show great importance for improving elite of maize directly with genetic engineering. Bacillus thuringiensis toxin protein (cry1Ac3) gene was successfully delivered into type Ⅰ calli of two elite inbred lines of maize, 340 and E28, via particle bombardment in this work. Fertile transgenic corn plants were obtained through phosphinothricin (PPT) or hygromycin B (HygB) selection, and the results of PCR, Southern blot assay and ELISA showed that foreign genes had been integrated into maize genome and expressed. In the meantime, strong resistance of some transgenic plants to corn borer was showed through bioassay. In addition, the comparison of selective effect between PPT and HygB showed that PPT, as a selective agent, was better than HygB for the growth and regeneration of resistant calli.

The Factors of Genetic Transformation for Indica Rice Kasalath

Objective The aim was to explore conditions of genetic transformation for Indica rice Kasalath and laid a foundation for further study on molecular biology. Method With callus of Kasalath as transformation receptor, Agrobacterium tumefaciens-mediated method was used to conduct genetic transformation. The genetic transformation system was optimized from several aspects, including co-culture mode, co-culture time and the affertreatment method of co-culture. Result The results showed that two days is the best co-culture time for genetic transformation, the acquisition rate of resistant callus was up to 84.1%, and transformation rate was up to 73%. Whether callus contact to the culture medium directly or indirectly has no significant effect on transformation. [ Conclusion] Genetic transformation successfully transferred exogenous gene OsMAPk2 into the rice genome.

Genetic Transformation of Aloe barbadensis Miller by Agrobacterium tumefaciens

Despite the importance of aloe in cosmetic and pharmaceutical industries, improvement of aloe （Aloe barbadensis Miller） by genetic engineering was seldom reported previously. In this study, regeneration and transformation conditions, including explant selection and surface sterilization, use of different Agrobacterium strains, and co-culture processing, are optimized. The use of 20.0% sodium hypochloride （25 rain） for sterilization was less detrimental to the health of explant than 0.1% mercuric chloride （10 min）. Regeneration frequency from stems was much higher than that from leaves or sheaths. Explants were infected by Agrobacterium （30 rain） in liquid co-cultural medium, and this was followed by three days co-culture on sterile filter papers with light for 10 h per day at 24℃. Histochemical data demonstrated that the transient expression of GUS gene in the stem explants of aloe infected with Agrobacterium strains EHAI05 and C58CI was 80.0% and 30.0%, respectively, suggesting the higher sensitivity of the explants to EHAI05 than to C58C1. Infected tissues were selected using G418 （10.0-25.0 mg/L） to generate transformants. Sixty-seven G418 resistant plantlets were generated from the infected explants. Southern blotting, PCR, and ELISA analyses indicated that the alien gene were successfully transferred into aloe and was expressed in the transgenic plants. This newly established transformation system could be used for the genetic improvement of aloe.

Genetic Transformation in Triticeae Crops

Wheat, triticale, tritordeum, barley, oat and rye are the most important crops in human consumptions and industry in the world. Transformation technology supplies a new source of improving Triticeae crops. In the past decade, transformation of wheat crops has considerably progressed. Many transgenic plants of Triticeae crops with various genes were produced via nricroprojectile bombardment, Agrobacterium-mediated transformation, PEG-uptake DNA technique, electroporation, microinjection, injection inflorescence and silicone carbide. Integration and expression of transgenes, inheritance and variation of transgenic plants have been studied. Technical improvements of genetic transformation for wheat crops will be extensively useful in commerce and benefit significantly to human being in the world.

Studies on Genetic Transformation of NPR1 Gene into Maize by Microprojectile Bombardment

[Objective] This study aimed to explore the conditions of transformation of maize by microprojectile bombardment. [Method] Immature embryo-derived callus of maize inbred line 7239 was used as explants to study the effects of shoot distance, helium pressure, vacuum and bombardment frequency on the transformation efficien- cy in the particle bombardment system of maize. [Result] Considering the transfor- mation efficiency, particle bombardment with 100 μg/P of golden particles, at a shoot distance of 9 cm from the target cells, under helium pressure of 1 350 psi and vac- uum 25 inHg, and bombarding twice could achieve relatively ideal results. After se- lection on media supplemented with different concentration of hygromycin, some re- generated plants were obtained. The results of PCR and Southern blotting analysis demonstrated that the NPR1 gene had been integrated into the genome of trans- genic maize plants, with an average transformation efficiency of 1.76%. [Conclusion] The study laid the foundation for the cultivation and breeding of excellent resistant varieties of maize.

Establishment of Green Cotton Regeneration System

[Objective]The aimed to optimize transformation system for Agrobacterium-mediated green cotton shoot tip and provide reference for genetic engineering-assisted breeding of color cotton. [Method]With shoot tip of natural green cotton as receptor and by using Agrobacterium-mediated transformation method,the optimum conditions of regeneration system were obtained. [Result]Combining with vacuum infiltration,3-old-day shoot tips were infected for 10 minutes in Abarobacterium suspension with OD600 value about 0.5 followed by co-culture for 24 h on the medium containing MS＋l mg/L KT,pH value of 5.8. The regeneration culture was conducted in MS medium containing l mg/L KT and increasingly 30,50 and 70 mg/L Kan. The highest transformation frequency was 34.6%,and the positive resistant plantlets of green cotton 9804 were obtained. [Conclusion]Green cotton is more beneficial than white cotton to conduct genetic transformation.

Construction of Exogenous Expression Vector of Trichoderma reesei

[Objective] The study was to construct a exogenous expression vector for Trichoderma reesei.[Method] Using CBHI promoter and terminator of T.reesei strain 40359,we constructed an expression vector of T.reesei strain 40359 for expressing Hpt gene and got six strains capable of growing on basic medium containing 175 mg/L of hygromycin B,further conducted hygromycin resistance test.[Results] In comparison with the original strain（wild type）,hygromycin resistance the six engineered strains was increased by 75%;the hygromycin resistance could inherit stably.[Conclusion] Our results laid basis for biological study on T.reesei at molecular and genetically engineering levels.

Establishment of a New Method for Genetic Transformation of Dunaliella salina

The exogenous gene was integrated into Dunaliella salina successfully by using LiAc/PEG mediating method for the first time. According to the results of histochemical staining, transgenic D. salina was blue, showing that the exogenous GUS gene was successfully expressed in the cells of D. salina. Meanwhile, the effects of growth state of D. salina, plasmid concentration and temperature on its transformation efficiency were studied, and the transformation conditions were optimized. The results show that the optimum conditions for the genetic transformation of D. salina are shown as follows： D. salina was in the early logarithmic phase; plasmid DNA concentration was 600 μg/ml; temperature was 29 ℃, and transformation efficiency was up to 74.8‰ under the best conditions. According to the results of PCR amplification and PCR-Southern hybridization, the target gene had been integrated into genome and was hereditary.

A Genetic Transformation System for Rosa multiflora Thunb. var. cathayensis Rehd. et Wils through Callus Induction

An efficient genetic transformation system is a preparation for Rosa multi-flora Thunb. var. cathayensis Rehd. et Wils to diversify its flower color through ge-netic engineering. We firstly optimized the explants and culture conditions on callus induction, hormone concentrations and dark period of culture time on bud differentia-tions in particular, with sterilized seedlings to establish the regeneration system of R. multiflora. It showed that callus induction frequency reached 100% after the ex-plants being cultured in dark for 21 d when MS was chosen to be the initial culture medium. The bud differentiation rate was 48% after cal i being cultured under dark for 8 d on MS medium supplemented with TDZ （1.5 mg/L） and NAA （0.05 mg/L）. The cal i was used as the explants that were infected with Agrobacterium tumefa-ciens harboring a DFR-RNAi construct. The transformation rate reached as high as 50%. The establishment of a highly efficient rose gene transformation system out-lined in this report is prerequisite for genetic improvement in rose flower colors.

Inheritance and Sequence Homology Analysis of the Maize DNA Introgressed into the Wheat Doubled Haploid Plant Through Wheat×Maize Cross

A maize (Zea mays L.) genome_specific repeated DNA sequence (clone MR64) has been transferred into one DH line of wheat through wheat (Triticum persicum Vav. ex Zhuk.) and maize cross. In the present study by RFLP analysis the authors proved that this DNA sequence could stably transmit into DH3 plants, the next generation derived from DH2 self_crossing. A similarity search in all DNA databases using BLASTN program showed that the DNA sequence of MR64 had as high as 93% identity to PREM_2 and 79% to Opie_2 in nucleotides. Both PREM_2 and Opie_2 are known as retrotransposons in maize genome, suggesting that MR64 likely is the partial sequence of a maize retrotransposon. Therefore, the results indicate that some retrotransposon might involve the DNA introgression from maize to wheat genome through wide fertilization. Stable inheritance of this maize genome_specific retrotransposon_like DNA in the wheat genome opens up the possibility of using retrotransposon as a new tool for gene tagging, function analysis, and insertional mutagenesis in wheat genome.

Optimization of Genetic Transformation System of Tobacco K326 Mediated by Agrobacterium

[Objective]The aim was to optimize genetic transformation system in tobacco K326 mediated by Agrobacterium.[Method]The leaf of tobacco aseptic seedling was taken as explants to study the optimization of Agrobacterium-mediated genetic transformation system.[Result] The highest transformation efficiency was obtained when the explants were pre-cultured in the medium of MS ＋ 2 mg/L 6-BA ＋ 0.2 mg/L IAA for 2 d,and then infected with Agrobacterium GV3101（OD600 =0.6） for 5 min.The PCR detection proved that npt II gene had been integrated into the regenerated tobacco plants.[Conclusion]A highly efficient genetic transformation system of tobacco leaf mediated by Agrobacterium was established.

Genetic Polymorphism of Wheat by IRAP Analysis Based on Retrotransposon Wis2-1 A

[Objective] To analyze genetic polymorphism of different species of wheat. [Method] The DNA of young seedlings from 21 species of wheat was isolated,and their genetic polymorphism was analyzed by inter-retrotransposon amplified polymorphism （IRAP） using a molecule marker technology based on wheat retrotransposon Wis2-1 A. [Result] As shown by clustering map of the electrophoresis results,19 species of wheat assembled as cluster with different genetic distance. Most of the wheat species were distinguished. The genetic polymorphism among different species of wheat could be evaluated by this method objectively. [Conclusion] The analysis of IRAP based on wheat retrotransposon Wis2-1A could give a basis for breeding of wheat.

Cloning and Gentic Transformation of NbDAD1 Gene from Nicotiana benthamiana

[Objective] The aim was to clone NbDAD1 gene from Nicotiana benthami- ana and study its genetic transformation. [Method] NbDAD1 gene was isolated from N. benthamiana by using RT-PCR technology and over-expression vectors were con- structed to obtain NbDADl-overexpression resistant plants and NbDADl-overexpres- sion resistant plants carrying HA tag. [Result] The 351 bp long NbDAD1 gene was cloned from N. benthamiana; recombinant plasmids pCAMBIA1301-NbDAD1 and pCAMBIA1301-NbDAD1HAtag were constructed successfully; 50T0-generation N. ben- thamiana Hyg-resistant transgenic lines of three genotypes were obtained, including 23 positive transgenic plants. [Conclusion] This study laid the foundation for investi- gating the specific functions of NbDAD1 gene in N. benthamiana and exploring the possible functional mechanism of DAD1 protein in programmed cell death of plants.

Cloning and Genetic Transformation of OsOle1 Gene in Rice

[Objective] The cloning and transformation of rice OsOle1 gene were conducted in the research.[Method] OsOle1 gene was cloned by RT-PCR.The amplified OsOle1 was then ligased to pCAMBIA 1300 to construct GUS overexpression vector.Then the Agrobacterium-mediated method was used in rice callus transformation.[Result] The full-length of OsOle1 gene was 498 bp and it encoded 189 amino acids.The over-expression vector Ub：：OsOe1-GUS was prepared and transgenic plants were successfully obtained.[Conclusion] The transgenic lines laid the foundation for the function research of OsOle1.

The Inheritance and Expression of cry1A Gene in Transgenic Maize

We investigated the inheritance and expression of cry1A gene in transgenic maize ( Zea mays L.) by Southern blotting analysis and enzyme_linked immunosorbent assays (ELISA). The results showed that cry1A had been transmitted to progeny of transgenic maize as a single gene. Contents of cry1A insecticidal protein were significantly different among transgenic maize lines and various tissues of the same transgenic lines. High expression of cry1A protein occurred in green tissues, such as leaf and husk leaf, and low expression occurred in pith, tassel, ear pith, pollen and silk. The results also showed that the contents of cry1A insecticidal protein in leaves of transgenic maize increased with the advance of development and there was no significant difference in cry1A expression level among various generations of transgenic maize.