【作者】 陈桂信；

【导师】 吕柳新； 潘东明；

【作者基本信息】 福建农林大学 ， 果树学， 2005， 博士

【摘要】 (木奈)(Prunus salicina Lindl. var. cordata J.Y.Zhang et al)生产上普遍存在果肉的褐变现象,造成果实的食用品质和耐贮性下降,甚至丧失其商品价值。(木奈)果肉褐变是多酚类物质、多酚氧化酶(polyphenol oxidase,PPO)与氧结合起酶促反应的结果。本研究采用RT-PCR、RACE和染色体步移等分子生物学技术,从(木奈)嫩芽和果肉中分离PPO基因及5′端调控序列,为进一步采用反义RNA技术,进行遗传转化,解决(木奈)果肉褐变问题开辟一条新的遗传改良途径。主要试验结果如下: 1.建立了(木奈)基因组DNA的提取和纯化方法。为解决(木奈)组织器官内多酚类物质、多糖、色素等物质的干扰,本试验以(木奈)嫩芽为材料,加入10%(W/W)PVPP与材料充分研磨,提取缓冲液中β-巯基乙醇的浓度为1%(V/V);用氯仿/异戊醇(24:1)抽提裂解液2次,在所获得的上相中,加入1/10体积经65℃预热的NaCl/CTAB溶液,混匀,再用氯仿/异戊醇(24:1)连续抽提2次;在DNA粗提液中加入2/3体积的5M NaCl和3倍体积冰冷的无水乙醇沉淀DNA。结果表明,该方法能有效地去除(木奈)嫩芽中多酚和多糖等杂质,所得DNA样品比较纯净、完整,可以直接用于后续的PCR扩增、酶切和染色体步移。 2.建立了(木奈)嫩芽总RNA的提取和纯化方法。通过加入10%(W/W)PVPP与材料共研磨,并在TriZoL提取液中加入1%(V/V)β-巯基乙醇;在匀浆液中加入终浓度为20%乙醇沉淀多糖;在裂解液抽提的上相中加入终浓度为3MLiCl沉淀RNA;在RNA的粗提液中加入1/30体积的3M NaAc(pH5.2)和0.1倍体积的无水乙醇沉淀多糖。试验结果表明,该方法有效地去除了(木奈)嫩芽中多酚、多糖等杂质,获得纯净、完整的RNA样品,直接用于后续的RT-PCR和RACE反应。 3.建立了(木奈)果肉总RNA提取和纯化方法。提取缓冲液中的Tris-HCl和EDTA的pH值为8.8,CTAB的浓度为2.5%(W/V),并加入0.5%SDS(W/V),中和果肉中的有机酸;在裂解后的果肉样品中加入终浓度为15%(VN)无水乙醇,用氯仿抽提2次;在RNA的粗提液中加入1/30体积3M NaAc(pH5.2)和0.1倍体积无水乙醇沉淀多糖2次。试验结果表明,该更多还原

【Abstract】 There exists commonly browning of pulps in Nai (Primus salicina Lindl.var. cordata J.Y.Zhang et al.\ which decreases the edible quality and storage ability of fruit and even loses fruit commercial value. Browning of pulps results from enzymatic reaction by integration of polyphenolics, polyphenol oxidase (PPO) and oxygen. To resolve the problem of browning of pulps by anti-sense RNA technology, the PPO gene and its regulatory sequence of 5’ terminal were isolated from buds and pulps of Nai by RT-PCR, RACE and chromosome walking etc in the study for genetic transformation, which would create a new way of genetic modification of browning of pulps in Nai. The main results were as follows.1. The method for extraction and purification of genomic DNA from buds of Nai was developed.To remove secondary substances such as polyphenolics, polysaccharides and pigments etc in the tissues of Nai, the young buds were grinded with 10% (WAV) Polyvinylpolypyrrilodone (PVPP) together, and the concentration of β-mercaptoethanol in extraction buffer was 1% (V/V); the lysis solution was extracted twice with 24:1 chloroform : isoamyl alcohol; the aqueous phase obtained was mixed even with 1/10 volume NaCl/CTAB solution preheated at 65 ℃, then extracted twice again with 24:1 chloroform : isoamyl alcohol; in roughsolution of DNA, 2/3 volume of 5M sodium chloride and three volume of absolute ethanol were added to precipitate DNA. The results showed that the impurities such as polyphenolics and polysaccharides etc were effectively removed from the samples by the method, and the pure and intact DNA sample obtained was directly used in successive PCR amplification, digestion of endonucleases and chromosome walking etc.2. The method for extraction and purification of total RNA from buds of Nai was developed.The buds were grinded with 10% (WAV) PVPP together, and 1% (V/V) p-mercaptoethanol was added in Trizol extracting buffer; absolute ethanol was added to 20% (V/V) final concentration in homogenization solution to precipitate polysaccharides; Lithium Chloride was added to final concentration (3M) to precipitate RNA in the aqueous phase obtained after extracting with chloroform; in the rough solution of RNA, 1/30 volume of 3M sodium acetate (pH5.2) and 1/10 volume of absolute ethanol were added to precipitate polysaccharides. The results showed that the pure and intact RNA sample obtained was directly used in the successive RT-PCR and RACE reactions.3. The method for extraction and purification of total RNA from pulps of Nai was developed.To neutralize organic acid in pulps of Nai, in the extraction buffer, pH value of Tis-HCl and EDTA was 8.8, the concentration of CTAB was 2.5% (W/V), 0.5% SDS (W/V) was added; absolute ethanol was added to 20% (V/V) final concentration in the pulps lysis solution, and then extracted twice with 24:1 chloroform risoamyl alcohol; 1/30 volume of 3M sodium acetate (pH5.2) and 1/10 volume of absolute ethanol in rough solution of RNA to precipitate polysaccharides twice. The resultsshowed that the impurities such as polyphenolics, polysaccharides, and organic acids etc were effectively removed from pulps of Nai by the method, that the pure and intact RNA sample obtained was directly used in the successive RT-PCR and RACE reactions.4. The coding regions of PPO genes were cloned from buds and pulps of Nai by the techniques of RT-PCR, RACE and chromosome walking etcThe coding region of PPO gene cloned from Nai’s buds contained 1770 nucleotides coding for 589 amino acid residues; the coding region of PPO gene cloned from pulps of Nai contained 1794 nucleotides coding for 597 amino acid residues; each sequence of the amino acids contained one thylakoid-transit region and two copper-binding sites, i.e., CuA and CuB. Alignments for nucleotide sequences and deduced amino acid sequences of PPO from buds and pulps of Nai to those of other plants with DNAMAN software showed that the two PPO gene were higher homologous to some Rosaceae fruit trees, the phylogenetic tree was drawn according to the results of alignments for the sequences of the amino acids of PPOs in plants, the result showed that the PPOs of pulps from Nai, apricot and apple were clustered into one group, and that the PPOs from buds of Nai, peels and leaves of apple, and pulps of pear were clustered into another group, therefore, the PPO genes from buds and pulps of Nai were two members among the gene family, the sequences of nucleotides and amino acids of PPO from buds of Nai were repectively 62.7% and 50.4% homologous compared with those from the pulps. The intron was found in PPO gene of pulps, but PPO gene of buds had no intron.5.The regulatory sequence of 57 terminal in the PPO gene from buds of Nai was cloned by chromosome walking.更多还原