均匀设计在南林95杨叶片遗传转化体系优化中的应用

为优化南林95杨叶片遗传转化体系,以南林95杨叶片为外植体,采用均匀设计U12(12×63),利用组织化学染色定位法,根据GUS基因的瞬时表达量,研究了农杆菌浸染时间、农杆菌菌液OD600值、共培养时间、乙酰丁香酮浓度等4个因素对遗传转化的影响。建立相关的线性回归方程,并优化了南林95杨叶片的农杆菌介导遗传转化体系,即农杆菌浸染时间35 min、农杆菌OD600值1.1、共培养时间4 d。     

尾赤桉DH201-2遗传转化体系建立的研究

以建立的尾赤桉(Eucalyptus urophylla × E. camaldulensis)DH201-2无性系高频再生体系为基础,利用GUS染色组织分析法研究了根癌农杆菌菌株、农杆菌预培养时间、浸染时间和共培养时间等因素对尾赤桉茎段和叶片外植体遗传转化的影响,探讨了适宜尾赤桉转化的卡那霉素和抑菌抗生素头孢塞污钠的使用浓度.结果表明,较优的转化条件是:采用农杆菌菌株GV3101,以茎段为受体材料,经过预培养6 d,在光密度OD600=0.5的菌液中浸泡30 min,然后转移到共培养培养基中共培养4 d,再转移到含90 mg/L卡那霉素和300 mg/L头孢噻肟钠的筛选培养基上,进行转化植株的再生,同时在共培养培养基和菌液中添加100 μM的乙酰丁香酮能够提高茎段外植体的转化效率.     

南方四季杨高效遗传转化体系的建立

以南方四季杨叶片作为受体材料,分别设置梯度实验,研究了Kan(卡那霉素)选择压以及遗传转化过程中影响转化效率的5个主要因素(预培养时间、浸染时间、共培养温度、共培养时间和乙酰丁香酮浓度),得出30mgL-1的Kan浓度作为南方四季杨遗传转化过程中的选择压。高效遗传转化体系为:叶片预培养3 d;OD600=0.4的菌液浸染10 min～15 min;浸染前菌液中加入AS(乙酰丁香酮)300 umol·L-1;22℃～25℃条件下暗处共培养3d。     

农杆菌介导的杜仲叶片愈伤组织遗传转化体系

【目的】优化杜仲叶片愈伤组织的再生体系,研究愈伤组织对抗生素和抑菌剂的敏感性,探索影响农杆菌介导杜仲愈伤组织遗传转化的最适转化因子水平,构建农杆菌介导的杜仲愈伤组织瞬时转化体系,使杜仲成年植株外植体为受体的遗传转化成为可能,为杜仲基因功能的研究与定向改良奠定基础。【方法】以杜仲成年植株叶片为材料诱导愈伤组织,通过添加不同浓度植物生长调节剂与大量元素的MS培养基进行不定芽的诱导与增殖,确定最适培养基。在此基础上,在培养基中添加不同浓度抗生素及抑菌剂,研究愈伤组织对其敏感性。以获得的叶片愈伤组织受体系统为基础,通过L_(16)(4~5)的正交试验,探索不同转化因子对农杆菌介导叶片愈伤组织遗传转化的瞬时转化效率的影响,建立最适瞬时转化体系。使用获得的瞬时转化体系对愈伤组织进行遗传转化操作,筛选抗性芽,对抗性芽进行GUS组织化学染色与PCR检验。【结果】再生体系优化的结果表明,3/4大量元素浓度的MS培养基能够促进杜仲愈伤组织不定芽的诱导及生长;愈伤组织诱导不定芽的最适培养基为3/4MS+0. 27μmol·L~(-1)NAA+4. 4μmol·L~(-1)6-BA,诱导率为83%±10. 0%;不定芽复壮的最适培养基为3/4MS+0. 054μmol·L~(-1)NAA+4. 4μmol·L~(-1)6-BA,平均伸长长度为(2. 47±1. 33) cm。抗生素与抑菌剂敏感性试验表明,遗传转化的选择培养基中,抑菌剂头孢霉素的最适浓度为200 mg·L~(-1),筛选用的抗生素卡那霉素最适浓度为70 mg·L~(-1)。转化因子的正交试验表明,最适的农杆菌介导杜仲叶片愈伤组织遗传转化的转化因子组合为:预培养5天、侵染10 min和共培养3天。使用最适瞬时转化体系对约200个愈伤组织进行遗传转化操作,共筛选获得3个抗卡那霉素的抗性芽; GUS组织化学染色显示GUS基因在抗性芽中得到了表达,PCR检测证明这些抗性芽中存在NPTⅡ基因。【结论】杜仲叶片愈伤组织诱导不定芽的最适培养基为3/4MS+0. 27μmol·L~(-1)NAA+4. 4μmol·L~(-1)6-BA,不定芽复壮的最适培养基为3/4MS+0. 054μmol·L~(-1)NAA+4. 4μmol·L~(-1)6-BA。农杆菌介导的杜仲叶片愈伤组织瞬时转化体系为:预培养5天、侵染10 min和共培养3天,筛选培养基为3/4MS+0. 054μmol·L~(-1)NAA+4. 4μmol·L~(-1)6-BA+200 mg·L~(-1)Cef+70 mg·L~(-1)Km。利用此体系共获得3个抗性芽,PCR分析和GUS组织化学染色都表明T-DNA已整合到抗性芽基因组中。     

柚木愈伤组织的LBA4404转化条件

对农杆菌LBA4404转化柚木愈伤组织的适宜条件进行了优化．结果表明;LBA4404转化柚木愈伤组织的适宜预培养时间为11～15d,菌液光密度为0．5～0．8,共培养时间为13d,共培养的pH为5．6～6．2;重新悬浮的作用明显,乙酰丁香酮的作用不明显;不同无性系对LBA4404侵染的敏感性不一样,选择适宜的无性系可显著提高转化效率．     

四倍体刺槐转甜菜碱醛脱氢酶基因的研究

试验以所建立的四倍体刺槐高频再生体系为基础,通过农杆菌介导法转化甜菜碱醛脱氢酶(BADH)基因,以GUS染色组织分析法为依据探讨了影响转化效率的各种因素,建立了优化转化体系,结果如下:20mg·L~(-1)乙酞丁香酮可显著提高转化效率;菌液浓度OD_(600)值0.3～0.7、预培养2d为宜;转化后暗培养对转化效率没有影响。并在以上研究基础上,成功地建立了高效、可重复的遗传转化体系,选择培养基上头孢霉素400mg·L~(-1)可有效地抑制农杆菌;卡那霉素50mg·L~(-1)时愈伤组织的白化死亡率达96.4％。经PCR检测,外源基因已成功的整合到植株的基因组DNA中,获得了15个转基因株系。     

沙田柚高效转化体系建立初报

以沙田柚(Citrus grandis(L)Osbeck cv．)上胚轴为对象,农杆菌为载体,进行了抗菌肽shivaA基因转入沙田柚影响因素研究。研究结果表明,沙田柚上胚轴卡那霉素选择浓度为70mg／L,抑菌用抗菌素采用羧苄青霉素效果好于其它种类抗菌素。共培养期间,50mg／L羧苄青霉素的加入可促进转化频率的提高。实生苗苗龄以20～25天为宜。预培养3天、农杆菌再悬浮液与共培养基中加入3％烟草叶片提取液、选择剂起始浓度降为50mg／L有利于转化频率的提高。     

农杆菌介导的楸树遗传转化体系

【目的】以楸树胚性愈伤组织为受体,建立有效的楸树遗传转化体系,为今后楸树性状的遗传改良奠定基础。【方法】通过农杆菌EHA105介导以胚性愈伤组织作为外植体进行遗传转化,通过正交试验获得最优的遗传转化条件,进而将外源基因转入到楸树基因组中。【结果】在1/2 MS培养基中添加不同梯度浓度的卡那霉素(Kana)进行选择压力筛选,在添加了60 mg·L~(-1)Kana的1/2 MS培养基中,楸树胚性愈伤组织的分化率为0.00%,存活率仅为5.71%,因此确定60 mg·L~(-1)为遗传转化的选择压。采用正交设计L18(37)进行农杆菌介导的楸树遗传转化试验,通过GUS化学组织染色统计瞬时表达率,正交试验直观分析和单因素方差分析结果表明:在预培养时间为2天,采用农杆菌菌株EHA105、菌液浓度OD600值为0.7、添加乙酰丁香酮(AS)浓度为300μmol·L~(-1)、侵染时间为10 min,共培养时间为5天的条件下,农杆菌介导的转化效率最高,且对转化效率影响最大的2个因素是乙酰丁香酮浓度和预培养时间。对浸染后的胚性愈伤组织进行8个月的筛选培养,共获得32个抗性组织团,对其中15个增殖较多的抗性愈伤组织进行PCR检测,表明86.67%的抗性组织团中有外源基因整合到楸树基因组中。内源激素水平会对植物体细胞胚分化产生影响,细胞分裂素(CTK)和脱落酸(ABA)促进体胚发生,生长素(IAA)和赤霉素(GA)对体胚发生有抑制作用。通过测定内源激素可知,转基因的抗性组织中内源CTK和ABA水平显著低于野生型的楸树胚性愈伤组织,而内源IAA和GA则显著高于野生型胚性愈伤组织,推测内源激素水平可能是转基因抗性组织体胚分化能力比较差的原因。【结论】建立了农杆菌介导的楸树胚性愈伤组织的遗传转化体系,对筛选获得的15个抗性愈伤组织进行PCR检测,其中13个抗性愈伤组织中有外源基因的整合。内源激素水平的变化可能是导致楸树转基因抗性愈伤组织难以分化的原因。     

黑林1号杨抗生素敏感性测定

以黑林1号杨叶片和茎段为材料,针对蜘蛛杀虫肽+Bt毒蛋白嵌合基因的遗传转化进行了抗生素筛选。结果表明,随2种抗生素浓度的增加,叶片分化率和不定芽率逐渐下降,卡那霉素和头孢噻肟钠临界浓度分别为15、600mg/L;在茎段生根筛选阶段,卡那霉素和头孢噻肟钠的适宜浓度分别为25、700mg/L。     

金银花发根诱导初探

研究了发根农杆菌的菌株类型、菌种浓度、浸染时间、预培养和共培养时间等对金银花发根诱导的影响。结果表明:农杆菌R1000培养OD600至0．6左右,稀释40倍浸染4 min时,诱导率最高,诱导率达58％。金银花外植体预培养5 d,与农杆菌共培养3 d,然后转入含500 mg／L羧苄青霉素的培养基杀菌培养是最适合的。     

Agrobacterium-mediated transformation of lombardy poplar (Populus nigra L. var.italica Koehne) using stem segments

Genetically transformed lombardy poplar (Populus nigra L. var.italica Koehne) plants were regenerated by co-cultivation of stem segments withAgrobacterium tumefaciens strain LBA4404 that harbored a binary vector (pBI121) which included genes for β-glucuronidase (GUS) and neomycin phosphotransferase. Successful transformation was confirmed by the ability of stem segments to produce calli in the presence of kanamycin, histochemical and fluorometric assays of GUS activity in plant tissues, and Southern blot analysis.     

苏云金杆菌杀虫晶体蛋白基因转化美洲黑杨的研究

本研究首先建立了美洲黑杨叶片外植体的再生系统，并利用Ｈｏｒｓｃｈ等人１９８５年发明的叶盘法，将分别带有嵌合基因ＮＰＴＩＩ和１．８ｋｂ或２．１ｋｂ Ｂｔ．基因的土壤杆菌ＬＢＡ４４０４与美洲黑杨叶片共培养。采用３０ｍｇ／ｌ和５０ｍｇ／ｌ两种浓度的卡那霉素筛选转子，约２８天左右的时间，有不定芽从外植体切口处形成。两种卡那霉素浓度下共形成２７５个伸长芽，存活了１８７株。这１８７株分别进入卡那霉素３０ｍｇ／     

杨树皮储藏蛋白基因启动子的克隆和功能研究

杨树树皮储藏蛋白BSP是类似种子储藏蛋白的氮素储藏物 ,冬季在韧皮部薄壁细胞中大量积累 ,是落叶树氮代谢中的重要成分。为了研究BSP基因启动子在转基因植物中的表达特性 ,探索其在植物基因工程研究中潜在的应用价值 ,我们用PCR方法从美洲黑杨基因组中DNA扩增得到了BSA启动子片段。与GUS基因融合构建中间载体后 ,转化烟草 ,获得了一批PCR检测为阳性的转化再生植株。经GUS组织化学检测 ,发现若干转基因烟草的茎和叶柄韧皮部以及叶脉都呈GUS染色阳性 ,初步证明杨树BSP基因启动子确有韧皮部表达特性 ,可介导GUS基因在转基因烟草韧皮部特异表达。     

Genetic transformation of loblolly pine using mature zygotic embryo explants by Agrobacterium tumefaciens

Introduction1Genetictransformationinconifershasthepotentialtoallowtheselectiveimprovementofindividualtraitsinelitecloneswhilestillmaintainingtheexistingcombinationofgenesresponsibleforthesuperiorphenotype(Charestetal.1991;Jamesetal.1996;Walteretal.1999).Atpresent,althoughconsiderableresearchefforthasbeendevotedtothegeneticengineeringofconiferspecies(Sederoffetal.1986;Bekkauoietal.1988,1990;Robertsonetal.1992;Bomminenietal.1993;Shinetal.1994;Klimaszewskaetal.1997),ithaslaggedbehindadvancesma…     

Transgenic poplar with enhanced growth by introduction of the ugt and acb genes

The transgenic poplar (Populus tremula L.) was obtained by transfer of the ugt and acb genes via triparental mating, which was employed to deliver large fragments of TDNA as a cluster. Freshly harvested seeds of local poplar were placed on MS agar medium and plantlets were obtained. After 1 year of subcultivation, plantlets were infected with a transconjugant of triparental mating with target ugt and acb genes into axillary buds. The transformed sprouts so obtained were cut and subcultivated on agar medium with an addition of 0.6 mg/l indole-3-butyric acid as an auxin source. The transformed sprouts showed GUS activity and resistance to gentamycin and kanamycin. The integrity of the target ugt and acb genes into poplar genome was demonstrated via PCR and Southern blot hybridisation. The transgenic poplar plants revealed a higher growth energy, corresponding to a higher content of IAA as opposed to control plants. Both transgenic and non-transformed plants were potted into soil for outdoor acclimatisation and subsequently transferred to earth in beds. Growing outside during 3 years, the transgenic poplar demonstrated a higher growth rate with fast bud and branch development.     

转抗菌肽基因毛白杨的培育及其对溃疡病的抗性

以毛白杨为转基因受体材料,利用根癌农杆菌介导法转化来源于益母草的阳离子抗菌肽基因LJAMP2。经卡那霉素筛选,共获得50株抗性植株。GUS组织染色和PCR检测显示有30株抗性植株呈阳性,初步证明外源目的基因已整合到毛白杨基因组中。RT-PCR证实抗菌肽基因LJAMP2在转基因植株中能大量表达。离体抗病性试验表明:转基因毛白杨细胞粗提液的抑菌能力明显强于非转基因植株。进一步将溃疡病菌接种在转基因和野生型毛白杨茎段上培养30天,转基因植株的病级指数均低于非转化植株。上述抗性试验结果表明:在毛白杨中超量表达益母草抗菌肽基因LJAMP2能显著提高其溃疡病抗病性。     

Factors influencing Agrobacterium-mediated embryogenic callus transformation of Valencia sweet orange (Citrus sinensis) containing the pTA29-barnase gene

Valencia sweet orange (Citrus sinensis (L.) Osbeck) calluses were used as explants to develop a new transformation system for citrus mediated by Agrobacterium tumefaciens. Factors affecting Agrobacterium-mediated transformation efficiency included mode of pre-cultivation, temperature of cocultivation and presence of acetosyringone (AS). The highest transformation efficiency was obtained with a 4-day pre-cultivation period in liquid medium. Transformation efficiency was higher when cocultivation was performed for 3 days at 19 degrees C than at 23 or 28 degrees C. Almost no resistant callus was obtained if the cocultivation medium lacked AS. The transformation procedure yielded transgenic Valencia plants containing the pTA29-barnase gene, as verified by PCR amplification and confirmed by Southern blotting. Because male sterility is a common factor leading to seedlessness in citrus cultivars with parthenocarpic characteristics, production of seedless citrus genotypes by Agrobacterium-mediated genetic transformation is a promising alternative to conventional breeding methods.     

Genetic transformation of loblolly pine using mature zygotic embryo expiants byAgrobacterium tumefaciens

Agrobacterium tumefaciens strain LBA 4404 carrying pBI121 plasmid was used to transform mature zygotic embryos of three genotypes (E-Hb, E-Ma, and E-Mc) of loblolly pine. The results demonstrated that the expression frequency of β-glucuronidase reporter gene (GUS) varied among genotypes after mature zygotic embryos were infected withAgrobacterium tumefaciens cultures. The highest frequency (27.8%) of GUS expressing embryos was obtained from genotype E-Mc with mean number of 21.9 blue GUS spots per embryo. Expression of β-glucuronidase reporter gene was observed on cotyledons, hypocotyls, and radicles of transformed mature zygotic embryos, as well as on organogenic callus and regenerated shoots derived from co-cultivated mature zygotic embryos. Nineteen regenerated transgenic plants were obtained from GUS expression and kanamycin resistant calli. The presence and integration of the GUS gene was confirmed by polymerase chain reaction (PCR) and Southern blot analysis. These results suggested that an efficientAgrobacterium tumefaciens-mediated transformation protocol for stable integration of foreign genes into loblolly pine has been developed and that this transformation system could be useful for the future studies on transferring economically important genes to loblolly pine.