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作者：熊盛华南热带农业大学

学位级别：硕士

The straw mushroom, Volvariella volvacea, is one of the most important cultivated edible mushrooms, representing a valuable source both in economy and in nutrition. But the straw mushroom has comparably lower individual yield and less substrate utilization efficiency. It is also unable to stand freeze preservation. Thus breeding novel strains (highly resistant and high- yield strain) is one important problem in straw mushroom production and research.It is designed in this study to establish an efficient transformation system of straw mushroom with plasmid pCAMBIA1301 and recombinant plasmid pCTH823 by particle bombardment and Agrobacterium-mediated DNA entry. So that it can contribute to the genetic modification of straw mushroom varieties and can be helpful in the study of its functional genome.The main content of the study as follows:Optimization of parameters used in particle bombardment by visualization of the growth of putative transformants on selective medium.Establishment of transformation conditions applied in Agrobacteriwn-mediated DNA entry.Visualization of the expression of GUS gene in putative transformants using histochemical assay. The results show that GUS gene is integrated into the genome of straw mushroom.The results of Cropping trial and GUS enzyme activity assay indicated that straw mushroom transformants can develop into normal fruiting body and GUS gene can be expressed in this kind of fruiting body.The resistant strains of Straw mushroom obtained in the screening after bombardment were detected by Southern dot blotting. Positive signals were visualized after hybridization for 16 hours, which shows that GUS gene was integrated into the genome of straw mushroom from the level of gene.Straw mushroom transformed by /Igro&actermm-mediated DNA entry was assayed by PCR. 2 strains gave positive signals, which proves that transgenic straw mushroom was obtained.

关键词： Straw mushioom(Volvariella volvacea) genetic transformation GUS gene Particle bombardment Agrobacterium-mediated DNA entry THP gene

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Expression Pattern of the Foreign Gene Regulated by the Tomato rbcS3A Promoter in Transgenic Tomato

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Agricultural Science & Technology 2013年第4期14卷 559-563页

作者：徐晓霞金烨杨万年华中师范大学生命科学学院湖北武汉430079

[Objective] This study aimed to reveal the expression pattern of foreign genes regulated by tomato rbcS3A promoter in transgenic tomato. [Method] Rubisco small subunit promoter rbcS3A was cloned by PCR, fused to the upstream of Gus coding region in a binary vector, and transformed into tomato plants mediated by Agrobacterium. Histochemical staining on PCR positive plants was performed to ana- lyze the expression pattern of the foreign gene regulated by the tomato rbcS3A pro- moter in transgenic tomato. [Result] A total of 15 positive plants were obtained, ac- counting for 33.3%. Histochemical staining showed that the expression level of Gus fusion gene was highest in mature leaf, lower in reproductive organs such as fruit, and not detected in seed. [Conclusion] More positive seedlings were obtained using the modified tissue culture method. Under the control of tomato rbcS3A promoter, exogenous gene highly expressed in transgenic plant leaves, but did not express in seeds and tomato pulp.

关键词： Tomato rbcS3A promoter Tomato genetic transformation Gus staining

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Construction of Exogenous Expression Vector of Trichoderma reesei

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Agricultural Science & Technology 2011年第2期12卷 308-312页

作者：张晓烜王傲雪东北农业大学成栋学院黑龙江哈尔滨150030 东北农业大学生命科学学院黑龙江哈尔滨150030

[Objective] The study was to construct a exogenous expression vector for Trichoderma reesei.[Method] Using CBHI promoter and terminator of *** strain 40359,we constructed an expression vector of *** 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 *** at molecular and genetically engineering levels.

关键词： Trichoderma reesei Vector genetic transformation

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Advances in Transgenic Techniques in Apple

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Agricultural Science & Technology 2009年第4期10卷 43-46页

作者： ZHOU Rui-jin DU Guo-qiang SHI Xiao-xin College of Landscape Architecture Henan Institute of Science and Technology Xinxiang 453003 College of Horticulture Agricultural University of Hebei Baoding 071001

Main progress of transgenic techniques in apple made in recent years was summarized, and related research achievements in various aspects like transformation method, regenerating ability of explants, strain type and infection conditions of Agrobacterium were reviewed. On this basis, the current bottlenecks in transgenic techniques in apple were put forward.

关键词： Apple genetic transformation Agrobacterium Regeneration of leaf

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Establishment of Green Cotton Regeneration System

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Agricultural Science & Technology 2010年第1期11卷 8-11页

作者：侍福梅王超聊城大学生命科学学院山东聊城252059

[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.

关键词： Green cotton Shoot tip genetic transformation Agrobacterium

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Cloning and Gentic transformation of NbDAD1 Gene from Nicotiana benthamiana

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Agricultural Science & Technology 2012年第5期13卷 938-941,944页

作者：徐玲玲陈崇崇王梨嬛潘永娟杨莉浙江师范大学化学与生命科学学院浙江金华321004

[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.

关键词： NbDAD1 gene genetic transformation Nicotiana benthamiana PCD

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通过基因工程创造对非生物胁迫抗性的梨新种质(英文)

通过基因工程创造对非生物胁迫抗性的梨新种质(英文)

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作者：文晓鹏庞晓明 Narumi Matsuda Masayuki Kita Hiromichi Inoue Chikako Honda Takaya Moriguchi Guizhou University Guiyang Guizhou 550025China National Institute of Fruit Tree Science Tsukuba Ibaraki 305-8605Japan Key Laboratory of genetics and Breeding in Forest Trees and Ornamental Plants Ministry of EducationBeijing Forestry University

An apple spermidine synthase(SPDS) gene(MdSPDS1)was verified to encode a func-tional protein by the complementation of the spe3 yeast mutant,which lacks the SPDS *** justify our hypothesis that apple SPDS is involved in abiotic stress responses and to obtain transgenic fruit trees tolerant to abiotic stresses as well,MdSPDS1-over-expressing transgenic European pear(Pyrus communis L. ’Ballad’) plants were created by Agrobacterium-mediated transformation.A total of 21 transgenic lines showing various spermidine(Spd) titers and MdSPDS1 expression levels were *** lines were exposed to salt(150 mM NaCl),osmosis(300 mM mannitol),and heavy metal(500 mM CuSO) stresses for evaluating their stress *** line no.32,which was revealed to have the highest Spd accumulation and expression level of MdSPDS1,showed the strongest tolerance to these *** growth increments,electrolyte leakage(EL),and values of thiobarbituric acid reactive substances(TBARS) were monitored,line no.32 showed the lowest growth inhibition and the least increase in EL or TBARS under stress *** titers in wild-type and transgenic lines showed diverse changes upon stresses, and these changes were not consistent with the changes in MdSPDS1 ***- over,there were no differences in the sodium concentration in the shoots between the wild type and line no.32,whereas the copper concentration was higher in the wild type than in line *** the mechanism(s) underlying the involve-ment of polyamines in stress responses is not known,these results suggest that the over-expression of the SPDS gene substantially increased the toler-ance to multiple stresses by altering the polyamine titers in ***,MdSPDS1-over-expressing transgenic pear plants could be used to improve desert land and/or to repair polluted environments.

关键词： Abiotic stress tolerance European pear(Pyrus communis L.’ Ballad’) genetic transformation Polyamine Spermidine synthase

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