泡桐体外植株再生及反义LFY基因遗传转化研究
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摘要
泡桐(Paulownia spp．)是我国重要的一个速生用材和园林绿化树种。近年来，科技工作者对其优良品种选育进行了大量的研究，然而在利用细胞工程和基因工程培育新品种方面的研究较少。本文重点研究了泡桐体外器官发生植株再生系统和体细胞胚胎发生及植株再生系统。建立了泡桐体外植株高效再生系统，并在此基础上进行了泡桐农杆菌介导转反义LFY基因的遗传转化研究，首次将有意的目的基因转入泡桐，探索并验证了利用生物技术培育泡桐新品种的可行性。
通过对毛泡桐(Pauliwnia tomentosa)、南方泡桐(P．australis)、白花泡桐(P．fortunei)、兰考泡桐(P．elongata)和豫杂一号泡桐(P．tomentosa×P．fortune)叶片愈伤组织诱导及植株再生的研究。结果表明，毛泡桐、南方泡桐、白花泡桐、兰考泡桐和豫杂一号泡桐叶片愈伤组织诱导在MS、1／2MS、WPM、B_5、N_6和PC基本培养基中，MS为最适宜的基本培养基。毛泡桐叶片愈伤组织诱导的最适培养基为MS+0.5NAA+4BA、南方泡桐为MS+0.3NAA+2BA、白花泡桐为MS+0.5NAA+4BA、兰考泡桐为MS+0.3NAA+6BA和豫杂一号为MS+0.3NAA+8BA。毛泡桐叶片愈伤组织芽诱导的最适培养基为MS+0.3NAA+12BA、南方泡桐为MS+0.3NAA+12BA、白花泡桐为MS+0.5NAA+12BA、兰考泡桐为MS+0.5NAA+12BA和豫杂一号泡桐为MS+0.7NAA+12BA。毛泡桐、南方泡桐、白花泡桐、兰考泡桐和豫杂一号泡桐叶片愈伤组织根诱导的最适培养基分别为1／2MS十0.1NAA、1／2MS+0.1NAA、1／2MS、1／2MS+0.5NAA和1／2MS+0.3NAA。
兰考泡桐、白花泡桐和毛泡桐无菌苗叶片和茎段体细胞胚胎发生及植株再生的试验结果表明：胚性愈伤组织诱导的最适培养基分别为：兰考泡桐叶片为MS+0.3NAA+8BA，茎段为MS+0.3NAA+11BA；白花泡桐叶片为MS+0.3NAA+14BA，茎段为MS+0.3NAA+8BA；毛泡桐叶片和茎段均为MS+0.3NAA+17BA。3种泡桐不同基因型、不同外植体种类在体细胞胚胎发育方面虽有差异，但整体上又有相似的趋势。体细胞胚胎发生经历胚性细胞、早期原胚、球形胚、心形胚和鱼雷胚等阶段，最终达到成熟子叶胚，历程与合子胚相似。在试验条件下，泡桐体细胞胚胎发生过程中，其胚性愈伤组织诱导、各阶段体细胞胚胎发生、体细胞胚胎成熟及体外植株再生均在同一培养条件下出现。
从农杆菌介导反义LFY基因兰考泡桐苗的研究结果可以看出：兰考泡桐叶片DNA用CTAB-Ⅰ、CTAB-Ⅱ、SDS-Ⅰ和SDS-Ⅱ4种方法提取，提取的泡桐叶片DNA得率约为3.5-5.2μg／10mg，A_(260)／A_(280)约为1.7-1.8，A_(260)／A_(230)在2.0以上，分子量在23kb以上，能够被EcoRⅠHindⅢ酶切和进行RAPD分析。综合考虑得率、纯度和质量等因素，以SDS-Ⅰ法为最佳方案。兰考泡桐叶片农杆菌介导转反义LFY基因获得较高转化率的最适参数
为:Klll选择压为30mgL一，，脱菌剂头抱霉素浓度为250mgL一’，预培养时间4d的叶片，
农杆菌0D60。值二0.45侵染时间10min抗性愈伤组织得率较高。愈伤组织经培养得到的再
生植株叶片经GUS和PCR扩增检测，可以初步证明己经将外源反义LFY基因转化到兰
考泡桐中。
Paulownia (Paulownia spp.) is one of the important fast-growing trees in timber production and garden planting in China. Some scientists have been doing much works on selection of good cultivar and new variety breeding recent years. But little progress was made in application of gene engineering and cell engineering for variety improved. The emphases were put on the system development of in vitro organogenesis and somatic embryogenesis and Agrobacterium mediated antisense LFY transformation of Paulownia in this thesis, which laid a solid foundation for its new cultivars breeding in applying biotechnological methods.
The results indicated that MS medium was optimum for inducing calli from 5 species of Paulownia plant leave. Callus induction media of leaves and their bud induction ones of Pauliwnia tomentosa, P. australis, P. fortunei, P. elongata and P. tomentosa xP. fortune were MS+0.5NAA+4BA, MS+0.3NAA+2BA, MS+0.5 NAA+4BA, MS+0.3NAA+6BA and MS+0.5NAA+8BA, MS+0.3NAA +12BA MS+0.3NAA+12BA, MS+0.5NAA+12BA, MS+0.5NAA+12BA and MS+0.7NAA + 12BA respectively; And that 1/2MS+0.1NAA, 1/2MS+0.1NAA, 1/2MS, l/2MS+0.5NAA and 1/2MS+0.5NAA were the shoot rooting media.
The best media for embryogenic callus induction from young stem segments and leaves of P. tomentosa, P. elongata and P. fortunei were MS+0.3NAA+17BA, MS+0.3NAA+11BA, MS+0.3NAA+8BA and MS+0.3NAA+17BA, MS+0.3NAA+ 8BA, MS+0.3NAA+14BA respectively. Somatic embryogenesis undergoes 5 phases as following, pre-embryo, globular embryo, heart-shaped embryo, torpedo-shaped embryo and cotyledonary embryo. The course is just similar to zygotic embryogenesis. The induction of embryogenic callus, its embryogenesis and even the plant regeneration might be carried out on the same media.
DNA extracted from P. elongata leaves with four different methods (CTAB-I, CTAB-II, SDS-I and SDS-II ) belonged to CTAB and SDS categories, which were tested by ultraviolet spectrophotometer analysis, agarose gel electrophoresis, restriction enzyme digestion and RAPD reaction in the thesis, were all suitable for digestion with two restriction enzymes and RAPD analysis. Besides, the DNA production rate are 3.5-5.2ug per 10 mg of fresh leaves, A260/A280
=1.7-1.8, A260/A230>2.0, the molecular weights above 23 Kb. But SDS-I was the best method as comprehensive consideration.
Plantlet regenerated from P. elongata leaves infected with agrobacterium mediated antisense LFY gene and confirmed by GUS and PCR amplification showed that the transformation was successful and the optimum parameters in transformation procedures were as follows: 30mg.L-1 Km selection pressure concentration, 250 mg. L-1 Cef concentration, 4 d preculture and 0.35 agrobacterium concentration (OD600).
通过对毛泡桐(Pauliwnia tomentosa)、南方泡桐(P．australis)、白花泡桐(P．fortunei)、兰考泡桐(P．elongata)和豫杂一号泡桐(P．tomentosa×P．fortune)叶片愈伤组织诱导及植株再生的研究。结果表明，毛泡桐、南方泡桐、白花泡桐、兰考泡桐和豫杂一号泡桐叶片愈伤组织诱导在MS、1／2MS、WPM、B_5、N_6和PC基本培养基中，MS为最适宜的基本培养基。毛泡桐叶片愈伤组织诱导的最适培养基为MS+0.5NAA+4BA、南方泡桐为MS+0.3NAA+2BA、白花泡桐为MS+0.5NAA+4BA、兰考泡桐为MS+0.3NAA+6BA和豫杂一号为MS+0.3NAA+8BA。毛泡桐叶片愈伤组织芽诱导的最适培养基为MS+0.3NAA+12BA、南方泡桐为MS+0.3NAA+12BA、白花泡桐为MS+0.5NAA+12BA、兰考泡桐为MS+0.5NAA+12BA和豫杂一号泡桐为MS+0.7NAA+12BA。毛泡桐、南方泡桐、白花泡桐、兰考泡桐和豫杂一号泡桐叶片愈伤组织根诱导的最适培养基分别为1／2MS十0.1NAA、1／2MS+0.1NAA、1／2MS、1／2MS+0.5NAA和1／2MS+0.3NAA。
兰考泡桐、白花泡桐和毛泡桐无菌苗叶片和茎段体细胞胚胎发生及植株再生的试验结果表明：胚性愈伤组织诱导的最适培养基分别为：兰考泡桐叶片为MS+0.3NAA+8BA，茎段为MS+0.3NAA+11BA；白花泡桐叶片为MS+0.3NAA+14BA，茎段为MS+0.3NAA+8BA；毛泡桐叶片和茎段均为MS+0.3NAA+17BA。3种泡桐不同基因型、不同外植体种类在体细胞胚胎发育方面虽有差异，但整体上又有相似的趋势。体细胞胚胎发生经历胚性细胞、早期原胚、球形胚、心形胚和鱼雷胚等阶段，最终达到成熟子叶胚，历程与合子胚相似。在试验条件下，泡桐体细胞胚胎发生过程中，其胚性愈伤组织诱导、各阶段体细胞胚胎发生、体细胞胚胎成熟及体外植株再生均在同一培养条件下出现。
从农杆菌介导反义LFY基因兰考泡桐苗的研究结果可以看出：兰考泡桐叶片DNA用CTAB-Ⅰ、CTAB-Ⅱ、SDS-Ⅰ和SDS-Ⅱ4种方法提取，提取的泡桐叶片DNA得率约为3.5-5.2μg／10mg，A_(260)／A_(280)约为1.7-1.8，A_(260)／A_(230)在2.0以上，分子量在23kb以上，能够被EcoRⅠHindⅢ酶切和进行RAPD分析。综合考虑得率、纯度和质量等因素，以SDS-Ⅰ法为最佳方案。兰考泡桐叶片农杆菌介导转反义LFY基因获得较高转化率的最适参数
为:Klll选择压为30mgL一，，脱菌剂头抱霉素浓度为250mgL一’，预培养时间4d的叶片，
农杆菌0D60。值二0.45侵染时间10min抗性愈伤组织得率较高。愈伤组织经培养得到的再
生植株叶片经GUS和PCR扩增检测，可以初步证明己经将外源反义LFY基因转化到兰
考泡桐中。
Paulownia (Paulownia spp.) is one of the important fast-growing trees in timber production and garden planting in China. Some scientists have been doing much works on selection of good cultivar and new variety breeding recent years. But little progress was made in application of gene engineering and cell engineering for variety improved. The emphases were put on the system development of in vitro organogenesis and somatic embryogenesis and Agrobacterium mediated antisense LFY transformation of Paulownia in this thesis, which laid a solid foundation for its new cultivars breeding in applying biotechnological methods.
The results indicated that MS medium was optimum for inducing calli from 5 species of Paulownia plant leave. Callus induction media of leaves and their bud induction ones of Pauliwnia tomentosa, P. australis, P. fortunei, P. elongata and P. tomentosa xP. fortune were MS+0.5NAA+4BA, MS+0.3NAA+2BA, MS+0.5 NAA+4BA, MS+0.3NAA+6BA and MS+0.5NAA+8BA, MS+0.3NAA +12BA MS+0.3NAA+12BA, MS+0.5NAA+12BA, MS+0.5NAA+12BA and MS+0.7NAA + 12BA respectively; And that 1/2MS+0.1NAA, 1/2MS+0.1NAA, 1/2MS, l/2MS+0.5NAA and 1/2MS+0.5NAA were the shoot rooting media.
The best media for embryogenic callus induction from young stem segments and leaves of P. tomentosa, P. elongata and P. fortunei were MS+0.3NAA+17BA, MS+0.3NAA+11BA, MS+0.3NAA+8BA and MS+0.3NAA+17BA, MS+0.3NAA+ 8BA, MS+0.3NAA+14BA respectively. Somatic embryogenesis undergoes 5 phases as following, pre-embryo, globular embryo, heart-shaped embryo, torpedo-shaped embryo and cotyledonary embryo. The course is just similar to zygotic embryogenesis. The induction of embryogenic callus, its embryogenesis and even the plant regeneration might be carried out on the same media.
DNA extracted from P. elongata leaves with four different methods (CTAB-I, CTAB-II, SDS-I and SDS-II ) belonged to CTAB and SDS categories, which were tested by ultraviolet spectrophotometer analysis, agarose gel electrophoresis, restriction enzyme digestion and RAPD reaction in the thesis, were all suitable for digestion with two restriction enzymes and RAPD analysis. Besides, the DNA production rate are 3.5-5.2ug per 10 mg of fresh leaves, A260/A280
=1.7-1.8, A260/A230>2.0, the molecular weights above 23 Kb. But SDS-I was the best method as comprehensive consideration.
Plantlet regenerated from P. elongata leaves infected with agrobacterium mediated antisense LFY gene and confirmed by GUS and PCR amplification showed that the transformation was successful and the optimum parameters in transformation procedures were as follows: 30mg.L-1 Km selection pressure concentration, 250 mg. L-1 Cef concentration, 4 d preculture and 0.35 agrobacterium concentration (OD600).
引文
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