万寿菊论文：万寿菊属植物染色体核型分析及万寿菊psy基因遗传转化体系影响因素的研究

万寿菊论文：万寿菊属植物染色体核型分析及万寿菊psy
基因遗传转化体系影响因素的研究
【中文摘要】本研究以八个万寿菊属（Tagetes L.）品种为材料,进行染色体核型分析,从细胞学水平为万寿菊遗传育种提供科学依据。

以万寿菊（Tagetes erecta L.）N-091、N-092、N-093和N-094为材料,根据gus基因瞬时表达情况,筛选易转化基因型及外植体种类,在此基础上对根癌农杆菌介导的万寿菊psy基因遗传转化体系的影响因素进行研究,为实现psy基因过量表达,获得高叶黄素含量的
万寿菊转基因品种奠定基础。

本研究具体结果如下:（1）对八个万寿菊属品种进行染色体核型分析。

结果如下:观赏万寿菊丰收、泰山、奇迹、完美黄、完美橙黄、印卡橙黄和印卡黄的核型公式分别为
2n=2x=24=6sm+18m, 2n=2x=24=14sm+10m, 2n=2x=24=24m ,
2n=2x=4sm+20m, 2n=2x=24m,2n=2x=2sm+22m,2n=2x=24m;染色体长度组成分别为
2n=4L+6M2+10M1+4S,2n=6L+4M2+10M1+4S,2n=4L+8M2+8M1+4S,2n=6L+ 6M2+6M1+6S,2n=2x=4sm+20m,2n=4L+8M2+8M1+4S,2n=4L+4M2+12M1+2S 。

核型类型分别为1B, 2B, 1B, 1B, 1B, 1B, 1B。

孔雀草金门的核型公式为2n=4x=48=4sm+44m;染色体长度组成为
2n=12L+8M2+16M1+12S;核型类型为1B。

（2）以万寿菊N-091、N-092、N-093和N-094的叶片及带腋芽茎段为实验材料,各品种gus基因瞬时表达阳性率结果为:N-094（86.6%）>N-093（73.3%）>N-091
（43.3%）>N-092（40%）,易转化的基因型为N-094。

（3）以N-094
的叶片和带腋芽茎段为外植体,再生诱导率结果为带腋芽茎段
（93.3%）>叶片（50%）,表明带腋芽茎段可作万寿菊再生体系的外植体。

其诱导培养基为:MS + 6-BA 0.1 mg/L+ NAA 0.1 mg/L+植物凝胶6 g/L。

（4）以N-094带腋芽茎段为外植体,研究psy基因遗传转化体系各影响因素,结果为:预培养1 d,农杆菌浓度OD600=0.8,1/2 MS悬浮,共培养时培养基添加AS浓度为100μmol/L,侵染采用80 W、10 min 的超声波针刺法处理,黑暗共培养2 d,选择培养基中抗生素Kan浓度为80 mg/L,Cef400 mg/L。

利用上述体系,本研究侵染外植体485个,获得4个抗性株系,经过PCR检测,有两株呈阳性,阳性植株获得率为0.41%。

【英文摘要】Karyotypes of eight genus Tagetes L. accessions were studied, and he result could provide the scientific basis for genetic breeding in the cytology level. Based on the marigold （Tagetes erecta L.） N-091, the N-092, N-093, the N-094 for the materials, according to transient expression of gus gene, the suitable kind of variety and explant types for conversion have been chosen, and the factors in the Agrobacterium-mediated genetic transformation system have been studied, This will provide basic information for new marigold which with higher lutein content by making psy gene overexpressied. The detailed study results are as follows.
（1） The karyotype formula of T. erecta L. Harvest , Taishan , Marval , Perfection Yellow , Perfection Orange , Inca Orange , Inca Yellow was like:
2n=2x=6sm+18m,2n=2x=14sm+10m,2n=2x=24m,2n=2x=4sm+20m,2n=2x= 24m, 2n=2x=2sm+22m,2n=2x=24m; the length type was
2n=4L+6M2+10M1+4S,
2n=6L+4M2+10M1+4S,2n=4L+8M2+8M1+4S,2n=6L+6M2+6M1+6S,2n=2x=4 sm+20m,2n=4L+8M2+8M1+4S,2n=4L+4M2+12M1+2S;The KA type was of 1B, 2B, 1B, 1B, 1B, 1B, 1B. The karyotype formula of T.patula L. Golden Gate was 2n=4x=48=4sm+44m; the length type was
2n=12L+8M2+16M1+12S; The KA type was of 1B.（2） Based on the marigold N-091, the N-092, N-093, the N-094 leaves and stems with axillary buds for experimental material. Results of gus gene transient expression: N-094 （86.6%） > N-093 （73.3%） > N-091 （43.3%） > N-092 （40%）. N-094 was the variety which was most easily converted.（3） For N-094, based on leaves and stems with axillary buds as the explants, the results of regeneration induction rate: stems with axillary buds （93.3%） > leaves （50%）. Stems with axillary buds was available to be the explant in the regeneration system of marigold.The inductive medium for stems with axillary buds: MS + 6-BA 0.1 mg/L + NAA 0.1 mg/L + 6 g/L plant gel.（4） According
to transient expression of gus gene, the optimal transformation conditions were obtained: take the stems with axillary buds as explants, precultured for 1d, agrobacterium tumefacien infection with OD600=0.8, suspension cultured in1/2 MS0 medium, As 100μmol/L in 1/2 MS0 medium and cocultivation medium, needle puncturing method was more useful during invasion, and co-cultivation for 2 days. In this way a total of 485 explant has been infected, and four resistant plant were got. After the PCR detection, two resistance plants were positive.The rate of positive plant for PCR detection was 0.41%.
【关键词】万寿菊叶黄素遗传转化体系 psy 染色体核型分析【英文关键词】Tagetes erecta L lutein genetic transformation system psy karyotype
【目录】万寿菊属植物染色体核型分析及万寿菊psy基因遗传转化体系影响因素的研究摘要3-5ABSTRACT5-6第一章绪论
11-24 1.1 万寿菊概述11-13 1.1.1 形态及生长习性
11 1.1.2 功能11-12 1.1.3 栽培及育种12-13 1.2 植物染
色体核型分析研究进展13-17 1.2.1 方法与标准13-16 1.2.2 万寿菊核型分析现状及存在的问题16-17 1.3 万寿菊的遗传转化
概况17-23 1.3.1 万寿菊的组培现状17-18 1.3.2 根癌农杆菌介导的遗传转化方法与机理18-19 1.3.3 遗传转过过程中的影响
因素19-20 1.3.4 万寿菊遗传转化研究现状20-21 1.3.5 PSY
基因的研究现状21-23 1.4 本研究的目的和意义23-24第二章万寿菊属植物染色体核型分析24-38 2.1 材料24-25 2.2 方法25-28 2.2.1 万寿菊属植物根尖染色体压片技术25-26 2.2.2 染色体核型分析标准26 2.2.3 Photoshop 核型分析方法
26-28 2.3 结果与分析28-35 2.3.1 万寿菊染色体核型分析结果28-29 2.3.2 孔雀草染色体核型分析结果29-35 2.4 讨论
35-38 2.4.1 万寿菊属不同种间染色体核型差异35 2.4.2 同种不同品种间染色体核型差异35-36 2.4.3 万寿菊属植物遗传进化关系36-38第三章万寿菊psy 基因遗传转化体系影响因素的研究38-57 3.1 材料38-40 3.1.1 植物材料38-39 3.1.2
菌株和载体39 3.1.3 实验试剂39-40 3.1.4 培养基的制备
40 3.2 方法40-44 3.2.1 菌液制备40-41 3.2.2 GUS 检测方法41 3.2.3 易转化基因型的筛选41 3.2.4 外植体种类及再生培养基配方的筛选41-42 3.2.5 遗传转化过程影响因素水平的确立42-43 3.2.6 转基因植株的PCR 检测43-44 3.3 结果与
分析44-50 3.3.1 易转化基因型的筛选44-45 3.3.2 外植体种类及再生培养基配方的筛选45-46 3.3.3 带腋芽茎段遗传转化体系影响因素水平的确定46-49 3.3.4 转基因植株的PCR 检测
49 3.3.5 侵染外植体与对照外植体的生长表型差异49-50 3.4 讨论50-57参考文献57-64致谢64-65攻读学位期间发表
的学术论文65-68附件68
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