【作者】 黄双龙；

【导师】 赖钟雄；

【作者基本信息】 福建农林大学 ， 园林植物与观赏园艺， 2008， 硕士

【摘要】 本研究以中国水仙（Narcissus tazetta var.chinensis）和根癌农杆菌菌株LBA4404为试验材料,首次对根癌农杆菌介导的ACS反义基因转化中国水仙进行研究。主要内容包括:①选择中国水仙高频离体再生途径;②建立并优化再生小鳞茎和愈伤组织两种中国水仙转基因受体系统:③采用根癌农杆菌介导法进行ACS反义基因转化中国水仙并优化转化体系;④筛选抗性小鳞茎并实现其植株再生;⑤对转ACS反义基因的再生小鳞茎（切块）和愈伤组织进行GUS瞬时表达检测;⑥对再生植株叶片进行GUS稳定表达检测和gus基因的PCR检测。主要研究结果如下:1、中国水仙高频离体再生途径的选择。试验以中国水仙鳞茎盘切块、子房切片以及花药等为外植体,对中国水仙离体再生途径进行研究,初步筛选出适合的外植体及其高频的离体再生途径。中国水仙鳞茎在0.1%升汞+3-5滴吐温的试验条件下消毒效果良好,在相对优化的培养基MS+5.0 mg·L^-1BA+0.1 mg·L^-12,4-D条件下,不同外植体直接诱导小鳞茎再生频率高低依次是鳞茎盘切块、子房切片和花药;在相对优化的培养基MS+2.0 mg·L^-12,4-D+0.1 mg·L^-1BA条件下,不同外植体愈伤组织诱导频率由高至低依次是鳞茎盘切块、花药和子房切片;而在相对优化的培养基MS+5.0 mg·L^-1BA+0.1 mg·L^-12,4-D条件下,不同外植体来源的愈伤组织诱导小鳞茎再生频率也存在显著差异,其中鳞茎盘切块来源的再生频率最高,而子房切片和花药来源的则依次之。2、中国水仙转基因受体系统的建立及优化。根据中国水仙高频离体再生途径的选择结果并综合考虑再生频率和浸染效果,试验建立再生小鳞茎（切块）以及愈伤组织作为根癌农杆菌介导ACS反义基因转化中国水仙的受体系统。100 mg·L^-1和110 mg·L^-1卡那霉素浓度分别适合于小鳞茎切块以及愈伤组织进行转化操作的筛选抗生素浓度,而头孢霉素浓度范围在0-300 mg·L^-1时,小鳞茎切块、愈伤组织小鳞茎分化率均稳定高于80.00%。在中国水仙遗传转化过程中,9-10月、11-12月以及次年1-3月这3个接种时期均适于转化操作;较优生长调节剂组合为中浓度(5.0 mg·L^-1)BA附加低浓度(0.1 mg·L^-1)2,4-D,适合于小鳞茎切块受体系统的高频率稳定再生,而影响愈伤组织受体系统小鳞茎再生的主次因子分别是KT、2,4-D以及BA,其中附加高浓度的KT(5.0 mg·L^-1)、中高浓度(2.0 mg·L^-1)的BA以及低浓度(0.1 mg·L^-1)的2,4-D的组合效果最佳,其小鳞茎分化率比优化前提高了近6.00%,且其有效繁殖系数仍保持在12.00以上;试验选择的小鳞茎切块受体系统的继代周期为30 d,愈伤组织受体系统的继代周期为15 d;1/2小鳞茎（外缘有微纵切）微切处理较适合小鳞茎切块受体系统以用于浸染转化操作,而愈伤组织受体系统经细胞学观察研究分析具有较强的分化和再生能力,遗传稳定性良好。3、农杆菌介导的ACS反义基因转化中国水仙。在前述研究基础上,试验对农杆菌介导的中国水仙两种受体系统的遗传转化新策略进行研究,相关较适技术参数显示,相同之处表现在稀释液均为1/2 MS液体培养基,农杆菌活化物均为添加一定浓度的葡萄糖,共培养时间均为4 d,共培养温度均为25℃以及共培养培养基pH值均为5.8等;不同之处则表现在预培养时间前者为6 d而后者为10 d,前处理方式前者为局部针刺而后者为局部针刺+风干30 min,农杆菌重悬液浓度OD₆₀₀值前者为0.5而后者为0.3,重悬液中添加的蔗糖浓度前者为100 g·L^-1而后者为80 g·L^-1,浸染液前者为重悬液而后者为重悬液+石英砂以及浸染时间前者为20 min而后者为15 min等。浸染后的两种受体系统在除菌时均采用含有300 mg·L^-1的头孢霉素的无菌水来除菌。4、抗性小鳞茎的筛选、植株再生及检测。小鳞茎切块和愈伤组织筛选过程中,间歇筛选方式的抗性小鳞茎分化率较高,而逐步加压、直接筛选方式则依次之。而小鳞茎切块和愈伤组织分化的抗性小鳞茎GUS稳定表达检测中,直接筛选方式抗性小鳞茎阳性率较高,而逐步加压、间歇筛选方式则依次之。总体而言,愈伤组织受体系统的抗性小鳞茎分化率和GUS稳定表达率均高于小鳞茎切块受体系统。经筛选共获得6个转ACS反义基因的中国水仙抗性小鳞茎系。对6个抗性小鳞茎系叶片的gus基因的PCR检测表明,在其中的1个抗性小鳞茎系,gus基因已经导入并整合进中国水仙基因组中。经鉴定后的中国水仙转基因植株经炼苗后已移栽成活。更多还原

【Abstract】 In this experiment,Chinese narcissus（Narcissus tazetta var.chinensis）as well as Agrobacterium tumefaciens strain LBA4404 were used as materials for first studies on the introduction of antisense ACS gene to Narcissus tazetta vat.chinensis mediated by Agrobacterium tumefaciens,which mainly included the selection of high-frequency in vitro regeneration approaches,the establishment and optimization of two classes of transgenic receptor system,both regenerated bulblets and calli,and the introduction of antisense ACS gene into Chinese narcissus mediated by Agrobacterium tumefaciens and the optimization of that transformation system,and resistant bulblets screening and plant regeneration,transient expression of GUS of gene-transformed slivers of regenerated bulblets and calli,and stable expression of GUS of regenerated leaves from gene-transformed slivers of regenerated bulblets and calli,and the gus gene PCR assay of leaves from transformed bulbs.The main results were as follows:1.The selection of high-frequency in vitro regeneration approaches of Narcissus tazetta var. chinensis.The slivers of bulbs,slices of ovaries as well as anthers were used as explants to carry out studies on the in vitro regeneration of Narcissus tazetta var.chinensis and choose the suitable explants as well as their high-frequency regeneration approaches.The preferable sterilization way for the bulbs was 0.1%mercuric chloride added with 3-5 drops of Tween-20.The ordinal effect of direct bulblet regeneration was slivers of bulb,slices of ovary and anthers on the all and the relative optimized medium of MS added with 0.1 mg·L^-12,4-D and 5.0 mg·L^-1BA,the ordinal effect of callus induction was slivers of bulbs,anthers and slices of ovaries on the all and the relative optimized medium MS added with 2.0 mg·L^-12,4-D and 0.1 mg·L^-1BA,and the ordinal effect of bulblet regeneration from calli was slivers of bulbs,slices of ovaries and anthers on the all and the relative optimized medium of MS added with 0.1 mg·L^-12,4-D and 5.0 mg·L^-1BA.2.The establishment and optimization of transgenic receptor systems for Narcissus tazetta var.chinensis.Integrating the former screening results with regeneration frequency as well as infection effects,both regenerated bulblets and calli were established as transgenic receptor systems.100 mg·L^-1and 110 mg·L^-1kanamycin concentrations were respectively suitable for transgenic operations on slivers of regenerated bulblets and calli,both whose bulblet differentiation rates were stably higher than 80.00%where the cepha1ycin concentration range was 0-300 mg·L^-1.There were three inoculation suitable phases for transgenic operations,namely September-October,November-December and January-March in the next year.The optimized growth regulator combinations for stably high-frequency regeneration of system slivers of regenerated bulblets was 5.0 mg·L^-1BA added with 0.1 mg·L^-12,4-D,while the effect - ranking factors on calli was KT afterward 2,4-D and BA,of which the bulblet differentiation rate was evaluated almost 6.00%presented in the combination of 5.0 mg·L^-1KT and 2.0 mg·L^-1BA, added with 0.1 mg·L^-12,4-D,where the efficient propagation coefficient is more than 12.00.The subculture period for system slivers of regenerated bulblets was 30 d and that of system calli was 15 d.1/2 bulblet（with slight-cut in the exterior edge）treatment was relatively suitable for the receptor system of slivers of regenerated bulblets used in transgenic operations,and the receptor system of calli was provided with strong ability of differentiation and regeneration,showing favorable genetic stability by cytology observation and analysis.3.The introduction of antisense ACS gene into Chinese narcissus mediated by Agrobacterium tumefaciens.Based on the above results,new transgenic strategies of both regenerated bulblets and calli were performed.The technical parameters of transgenic operations on two systems showed that the sameness was:1/2 MS liquid medium for diluents,a certain concentration of glucose for the activation of Agrobacterium tumefaciens,time,temperature,and pH values of coculture were respectively 4 d,25℃and 5.8,etc.On the other hand,the differences represented in preculture time（the former 6 d while the latter 10 d）,pretreatment modes（the former partly acupunctured while the latter air-dried for 30 min and meanwhile partly acupunctured）, suspension concentration OD₆₀₀values（the former 0.5 while the latter 0.3）,the sucrose concentrations in suspension(the former 100 g·L^-1while the latter 80 g·L^-1),and components of infection liquid（the former suspension while the latter suspension added with some quartz）, Agrobacterium inoculation time（the former 20 min while the latter 15 min）,etc.The asepsis water with 300 mg·L^-1cephamycin was used in removing Agrobacterium turnefaciens.4.The resistant bulblets screening,plant regeneration as well as assays.During the screening course,the ordinal differentiation rate of resistant bulblets in different screening modes was intermittent mode,afterward gradual-adding mode and direct mode.While the ordinal stable expression rate of GUS in resistant bulblets from both slivers of regenerated bulblets and calli presented to direct mode,afterward gradual-adding mode and intermittent mode.As a whole,both differentiation rate and GUS stable expression rate of resistant bulblets in the receptor system of calli were higher than that in regenerated bulblets.After screening a total of six antisense ACS gene-transformed resistant bulblet lines were obtained.Moreover,the gus gene PCR assay of leaves from transformed bulblets showed that one of the lines appeared clearly visible strip,which might indicate gene transformed and conformed into the genome.Furthermore,the identified transgenic plants of Narcissus tazetta var.chinensis survived after and seedling training and transplant.更多还原