摘要
矮化和盆栽的木本花卉植物在园林绿化、室内装潢等方面有着重要作用,现有的木本花卉主要以乔木和小乔木为多。在控制木本花卉高生长的因素中,赤霉素合成酶(GA20-oxidase)通过反馈调节赤霉素含量,影响植物的高生长,是最重要的内源影响因子之一。本研究利用反义RNA及RNA干扰技术,在烟草上对GA20-oxidase基因的表达进行抑制,为最终实现木本花卉植物矮化性状的基因工程遗传改良奠定基础。主要研究结果如下:
1.从cDNA中克隆烟草NtGA20ox基因,构建了以NPTⅡ为标记基因的反义载体pBIantiW和RNA干扰载体pBIRNAiW;从载体pGEX-4T-2中克隆PttGA20ox基因,构建了以抗除草剂bar基因为标记基因的反义载体pBIbarantiG和RNA干扰载体pBIbarRNAiG。将四个载体转化到根癌农杆菌菌株LBA4404中。
2.建立并优化普通烟草W38及转基因烟草GW(转化超量表达PttGA20ox基因)的再生和遗传转化体系。继代培养基:MS+6-BA 1.0 mg/L+NAA 0.1 mg/L+琼脂5.5g/L;生根培养基:1/2 MS+NAA 0.1 mg/L+琼脂5.5 g/L;分化培养基:MS+6-BA 2.0 mg/L+NAA 0.1 mg/L+琼脂4.8 g/L。
3.建立最优遗传转化程序。通过选择剂敏感性试验,确立了叶片不定芽诱导的选择剂浓度为:Kan 75 mg/L,Glu 0.5 mg/L。茎段增殖和生根的选择剂浓度为Kan150 mg/L,Glu 1.0 mg/L。结合Cef对农杆菌的抑菌效果,在遗传转化和再生、扩繁、生根时都选择250 mg/L为最适抑制浓度。对预培养时间、菌液侵染时间、菌液浓度、共培养时间、延迟选择时间、AS浓度等影响转化的因素进行研究,结果表明,预培养时间、延迟选择时间、AS浓度对转化的影响不大;当菌液浓度OD_(600)=0.6时,10～15 min为比较合适的菌液侵染时间,此时抗性芽的转化效率最高;共培养最适宜时间为2 d。
4.对四种转化苗烟草AW(转反义NtGA20ox基因)、烟草RW(转反向重复NtGA20ox基因)、烟草AG(转反义PttGA20ox基因)、烟草RG(转反向重复PttGA20ox基因)进行叶片的再分化培养检测和分子生物学鉴定,证明目的片段已经插入到转基因植株基因组DNA中。
5.对转基因植株进行移栽和表型性状的统计与观测。转基因植株AW、GW与对照W38相比,明显矮化,茎段增粗,节间距变短,叶片变大,生根时间推迟且根数量相对稀少。转基因植株AG、RG与对照GW相比也呈现矮化状态,节间距明显缩短,茎段粗细变化不明显,叶片细长与对照相似,根稀少,生根时间推迟。
6.对转基因植株进行细胞学比较。显微观察显示,转基因植株在细胞层面上与对照植株有显著差异,生长初期叶片细胞更为致密,茎段细胞长度小于对照,实施RNA干扰的细胞甚至成为椭圆形,细胞长度远远小于对照。
7.对转基因植株进行内源激素含量的测定,并对转化苗进行外源GA喷施实验。内源激素测定结果表明,转化苗体内ABA、ZR的含量与对照相比没有显著差异,而其体内的IAA、GA_3、GA_4含量明显低于对照。外源GA喷施实验表明,转基因造成的GA含量降低是可逆转的,外源GA的喷施,可以提高植株体内GA的含量,使转基因植株的矮化状态得到恢复。
Dwarfed and bonsai's woody flowering plants are important in landscaping and upholstery,and most of them are arbors by now.GA20-oxidase,which is one of the most important endogenesis factors for controlling high growth of woody flowering plants,can influence high growth of plants by adjusting the content of GA.In this work,antisense RNA and RNA interference were used to suppress expression of the GA20-oxidase gene in tobacco.This study could be a foundation work for dwarfism of woody flowering plants.The main research work and the results are as follows:
1.The NtGA20ox gene was cloned from Nicotiana tabacum cDNA,and pBIantiW plasmid of antisense RNA and pBIRNAiW plasmid of RNA interference were constructed with marker gene NPTII;The PttGA20ox gene was cloned from pGEX-4T-2 plasmid,pBIbarantiG plasmid of antisense RNA and pBIbarRNAiG plasmid of RNA interference were constructed with marker gene bar.The four plasmids were transformed to Agrobacterium tumefaciens LBA4404.
2.Regeneration and genetic transformation system for Nicotiana tabacum W38 and transgenic tobacco GW(transformed over expression gene PttGA20ox) were established and optimized. Subculture medium was MS + 6-BA 1.0 mg/L + NAA 0.1 mg/L + agar 5.5 g/L;Rooting medium was 1/2 MS + NAA 0.1 mg/L + agar 5.5 g/L;Differentiation medium was MS + 6-BA 2.0 mg/L + NAA 0.1 mg/L + agar 4.8 g/L.
3.The most effective genetic transformation procedure was established.The results of different level reagents tests showed that the best reagents concentrations for adventitious bud regeneration were Kan 75 mg/L,Glu 0.5 mg/L,for segment propagation and rooting were Kan 150 mg/L,Glu 1.0 mg/L,and the best concentration of Cef was 250 mg/L for all.The genetic transformation test showed that pre-culture time,late selection and Acetosyringone were unconspicuous for the transformation; and leaves immersed in the bacterium solution(OD_(600)=0.6) for 10-15 min,co-cultured for 2 d can get the highest frequency of transformation.
4.It showed that target gene had been integrated in genomic DNA of transgenic plants from the result of adventitious bud regeneration and molecular detection for transformed tobacco AW (transformed antisense NtGA20ox gene),RW(transformed inverted repeat NtGA20ox gene),AG (transformed antisense PttGA20ox gene),and RG(transformed inverted repeat PttGA20ox gene).
5.Growth analysis showed that the transgenic plants were dwarfism as the controls.The stems of tobacco AW and RW were thicker than the untransformed control plants,their internodes were lower, and leaves were bigger,their rooting time was deferred and roots were less than control.The transgenic plants AG and RG showed a similar physiological index to the tobacco AW and RW, except the change for stems were unobvious compared with their control GW.
6.Cytology comparison showed that the leaves cells of the transgenic plants were more compact than controls,and the length of stems cells was smaller than controls.
7.The endogenous hormone contents of ABA and ZR of the transgenic plants were similar to the controls,but the contents of IAA,GA_3 and GA_4 were lower than controls.In the experiment of sprinkling extrinsic GA,all the transgenic plants came back to length growth as controls;it showed that the decreased contents of GA by gene transform could be reversed.
引文
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