【作者】 孔凡明；

【导师】 王文兵； 谭小力；

【作者基本信息】 江苏大学 ， 生物化学与分子生物学， 2008， 硕士

【摘要】 甘蓝型油菜(Brassica napus)是重要的经济作物之一。传统常规育种技术在油菜重要经济性状的遗传改良中起到重要作用,但随着生产需求对品种要求的日益提高,常规育种技术的局限性也日显突出。因此采用新技术将新的基因资源引入油菜十分必要。利用农杆菌介导的遗传转化引入外源基因为油菜产量、品质、抗性和其它性状的遗传改良提供了新途径,该技术也是现代生物学研究中了解特定基因功能与表达模式不可缺少的手段。但现有农杆菌介导的油菜转化方法还存在着转化效率不高、重复性差、转化周期长等问题,因而限制了该技术的广泛应用。因此,建立一个转化效率高、重复性好、转化周期短的油菜转化体系是势在必行的。血红蛋白(Hemoglobin)普遍存在于动物、植物、细菌、真菌、酵母、藻类、原生动物等生物体中。血红蛋白在动物体中的生理功能主要是结合氧并协助其运输,从而促进机体中氧的供应、呼吸链电子流量、ATP酶活力等;血红蛋白在微生物体中可以促进菌体的生长、蛋白质的合成、代谢产物的产生等;最近,也有文章报道血红蛋白能促进烟草的生长、提高烟草叶中叶绿素与尼古丁的含量等。本研究旨在用农杆菌(Agrobacterium tumefactiens)介导的转化方法,以甘蓝型油菜品种宁油12号的子叶节为外植体,优化一个转化周期短、重复性好的遗传转化体系。从而用优化了的遗传转化体系,将血红蛋白基因(SLR2097)导入油菜中,研究血红蛋白对油菜的影响。主要研究内容和结论如下:1.用含有携带GFP基因的植物双元表达载体pCAMBIA1303的农杆菌来侵染甘蓝型油菜品种宁油12号的子叶节外植体,从而优化油菜的遗传转化体系。实验结果显示,在含有浓度为4 mg/L的6-BA与0.04 mg/L的NAA的筛选培养基中获得了最高的小芽发育率为94.38%;而当用含有浓度为200μM的乙酰丁香酮与0.02%的silwet L77,OD值为0.8的农杆菌悬浮液来侵染油菜的子叶节10 min并在含有浓度为200μM的乙酰丁香酮与0.02%的silwet L77的培养基中共培养时,获得了最高的转化率为6.96%;转基因植株是用PCR技术、Southern blotting分析来鉴定的;在荧光显微镜下也观察到了转基因植株中的GFP基因的表达。2.文献表明血红蛋白能促进烟草的生长、提高烟草叶中叶绿素与尼古丁的含量。本研究以蓝细菌(Synechocystis sp.PCC 6803)为材料,采用PCR技术从蓝细菌的基因组DNA中克隆了SLR2097基因的ORF,此ORF全长375 bp,编码一个由123个氨基酸残基组成的多肽链,推测其编码的多肽链的相对分子质量为30.39kD,理论等电点为5.26。比较蓝细菌的SLR2097与其它13种生物的血红蛋白基因编码的氨基酸序列发现,这些生物的SLR2097基因编码的氨基酸序列的保守性非常低,除了有一个共同的基序F-[LF]-x(4)-[G]-G-[PAT]-x(2)-[YW]-x-[GSE]-x(1,5)-[LW]-x(3)-H之外,几乎再没有共同的序列。3.构建携带35S启动子的植物双元表达载体pCAMBIA1300-CaMV35S。并将SLR2097基因连入此表达载体上,转化农杆菌菌株LBA4404。然后,利用优化了的遗传转化体系,将SLR2097基因导入甘蓝型油菜中。用10 mg/L与20 mg/L的潮霉素来筛选转基因植株,并用PCR技术鉴定转基因植株。从观察结果来看,转基因油菜比野生型油菜要早熟。更多还原

【Abstract】 Brassica napus is one of the most important oil crops.Conventional breeding has made tremendous contribution to the improvement of economic trait.The increasing demand on production of diferent oils such as edible oil,industrial oil needs new technology in rapeseed breeding to further enhance both the productivity and adaptability of new cultivars.So it was necessary to adopt new technology to transfer the new genes into rapeseed.Genetic engineering offered new possibilities to improve the crop in various special characters that were difficult to obtain by traditional breeding procedures.Important traits,such as rapeseed yield,quality,disease and insect tolerance, male sterility,and the other special characaters,have been introduced into the crops by genetic transformation,and genetic transformation was also used to detect the function of different genes.However,it was very difficult to use the present Agrobacterium-mediated transformation in many genotypes because there existed serious low transformation frequency,low reproducibility and long-term transformation. Therefore,it is essential to found an efficient Agrobacterium-mediated transformation system that has short period.Hemoglobin existed in most organisms including animals,plants,protozoa,algae, bacteria and fungi.Hemoglobin in animal can bound and transport oxygen in order to improve provision of oxygen,fluence of electrons in respiratory chain,vigor of ATPase; and hemoglobin in microbacteria can promote growth of microbacteria,synthesis of protein and production of metabolite.Recently,a paper reports that Vitreoscilla hemoglobin can enhance growth of tobacco,the chlorophyll content and the nicotine content of it.Using cotyledonary nodes as explants,an efficient system for Agrobacterium-mediated transformation of Brassica napus cv."Ningyou12" was developed through optimizing the infection conditions and the concentrations of phytohormone.And then hemoglobin gene SLR2097 of Synechocystis was transformed into Brassica napus with the optimized transformation system in order to research the influence of SLR2097 gene on Brassica napus cv."Ningyou12".The major study contents and conclusions are as follows:1.Cotyledonary nodes of Brassica napus cv."Ningyou12" were infected with Agrobacterium tumefactiens containing expression vector pCAMBIA1303-GFP in order to optimize genetic transformation system.Different combinations of optical densities and infection durations and different concentrations of acetosyingone and silwet L77 were evaluated for the efficient infection of Agrobacterium.Maximum transformation frequency was 6.96%when cotyledonary nodes were infected for 10 min with Agrobacterium suspension of which the OD was 0.8 and then co-cultivated on the medium with 200μM acetosyingone and 0.02%silwet L77.In addition,concentrations of 6-benzyladenine and naphthalene acetic acid were also optimized.Maximum green plantlet frequency was 94.38%when cotyledonary nodes were cultivated on the medium containing 4 mg/L 6-BA and 0.04 mg/L NAA after co-cultivation.Evidences for transformation were confirmed by PCR analysis,Southern blotting analysis.The expression of GFP gene in transgenic plants was also observed with fluorescence microscope.2.The literatures indicated that hemoglobin can improve growth of transgenic tobacco and increase the chlorophyll content and the nicotine content of transgenic tobacco.The open reading frame of hemoglobin gene SLR2097 was cloned from genomic DNA of Synechocystis sp.PCC 6803.Sequence analysis indicated that the open reading frame of hemoglobin gene SLR2097 was only 375 bp,which encoded a 123 amino acid polypeptide chain belonging to the truncated hemoglobin family. Molecular weight of its deduced protein was 13.9kDa and theoretical pI of its deduced protein was 5.27.Multiple sequence alignment demonstrated that hemoglobin gene of Synechocystis shared low sequence identity with another twelve organisms,and there was only a common motif ’F-[L]-x(4)-[G]-G-[T]-x(2)-[Y]-x-[G]-[R]-x-[M]-x(3)-H’ among thirteen species.3.Expression vector pCAMBIA1300-CaMV35S was constructed successfully. And then SLR2097 was ligated into the vector pCAMBIA1300-CaMV35S.Finally,the pCAMBIA1300-CaMV35S containing SLR2097 was transformed into A.tumefactiens strain LBA4404.The hemoglobin gene SLR2097 of Synechocystis was transformed into Brassica napus with the optimized transformation system.And transgenic plants were identified with PCR analysis.Phenotype analysis of transgenic Brassica napus indicated that exogenous expression of hemoglobin gene SLR2097 led to the earlier maturity.更多还原