【作者】 施佳慧；

【导师】 李铎； 朱加进；

【作者基本信息】 浙江大学 ， 食品科学， 2009， 博士

【摘要】 番茄营养丰富,富含维生素C、有机酸、矿物质等营养成分,尤其是独特的番茄红素,吸引了越来越多的消费者,是难得的“水果蔬菜”,现已成为世界上第二大蔬菜作物。航天诱变育种技术是农作物诱变育种的新兴领域和重要手段,已广泛应用于各种植物的遗传操作改良过程,实现高效益的遗传改进。本课题的研究目的是通过对神舟五号搭载的航天育种番茄突变株变1(M1)、变2(M2)进行生理生化和营养功能的全面研究,并与原亲本CK进行比较,旨在为航天育种番茄乃至航天育种植物的大面积栽培、加工及开发利用提供依据,并推动航天育种产品的研究向纵深化方向发展。第一部分：航天育种番茄与亲本种间差异性比较通过对航天育种番茄和原亲本幼嫩叶片进行不同的DNA提取方法和PCR扩增体系进行比较,得到了最佳的组合参数,据此条件进行PCR扩增,并进行测序,然后通过软件进行碱基序列的分析。结果发现：航天育种番茄与原亲本DNA分子序列有八个碱基发生了变化,证实了航天诱变确使实验材料发生DNA序列的突变,说明了本实验中航天育种番茄和原亲本番茄已经发生了DNA水平上的变异。通过对每个航天育种突变株M1、M2及其亲本共105个番茄叶片样本建立训练模型。采用主成分分析法对光谱数据进行聚类分析,并将小波变换用于对大量光谱数据的压缩,同时结合神经网络建立番茄品种鉴别模型。用每个品种15个样本,共45个番茄叶片的样本进行预测。结果表明,波长范围在400～1000nm,不同番茄品种叶片的光谱图有明显区别。在可见光波段550nm处,亲本番茄品种和两突变株M1,M2有较显著的差别,在700nm以后的近红外部分,两突变株M1、M2的反射率值要大于亲本番茄。用该方法对航天育种番茄不同品种的鉴别正确率达到97.8%。从光谱学角度证明了航天育种番茄和亲本之间确实存在着差异。通过对航天育种突变株M1、M2及其亲本共105个番茄果实样本建立训练模型,采用偏最小二乘法对光谱特征信息进行提取,与神经网络结合建立番茄品种的鉴别模型,该模型将提取后的主成分作为神经网络的输入。并用每个品种15个样本,共45个番茄果实的样本进行预测。结果发现：三个品种果实的反射率在400-1000nm的变化趋势比较相近,不同番茄果实品种的光谱图有明显区别,亲本番茄的反射率值明显低于两个突变株M1、M2,突变株M1的反射率比M2更高。两个模型的鉴别正确率分别达到95.6%和97.8%,也同样从光谱学角度证明了航天育种番茄和亲本之间确实存在着差异。这也为航天育种番茄不同品种的快速鉴别提供了一种新方法。第二部分：航天育种番茄根系分泌物异同研究采用水培方式培育各品种番茄,并定期收集不同时期的根系分泌物,经过XAD-4大孔吸附树脂进行分离纯化,用气相色谱-液质联用(GC-MS)对主要低分子根系分泌物组成分进行分析。结果发现：不同番茄品种在不同时期根系分泌物组成差异显著,同一品种在不同时期根系分泌物组成也存在显著性差异。研究还发现了在航天育种番茄根系分泌物中存在一些如乙苯、二甲苯等有污染性的化合物。为研究航天诱变育种作物生态安全性提供了一定的理论依据。第三部分：航天育种番茄自身抗菌能力评估通过自然腐烂率和打孔腐烂率进行各品种番茄自身抗菌实验比较,结果发现航天育种番茄M2的自然腐烂率低于原亲本CK,抗自然外伤和抗链格孢菌和灰葡萄菌的能力均优于原亲本；而航天育种番茄品种M1的自然腐烂率高于原亲本,抗自然外伤、抗链格孢菌和灰葡萄菌的能力均劣于原亲本。通过不同生长阶段果实体内一些抗氧化保护酶类,如超氧化物歧化酶(SOD)、过氧化氢酶(CAT)酶活的测定,以及膜脂氧化终产物丙二醛(MDA)含量的测定,初步探讨了航天育种番茄自身抗菌能力机理。结果发现,航天育种番茄M2的SOD和CAT酶活一般都高于亲本CK, MDA值相对最低；而航天育种番茄M1的SOD和CAT的酶活相对来说变化幅度较大,但最终值也最低,MDA值则相对最高。这与航天育种番茄M2体内抑菌能力最强,而M1能力最弱的结果是吻合的。第四部分：航天育种番茄营养和功能评估通过高效液相色谱法(HPLC)、原子吸收光谱法、全自动氨基酸分析仪等方法,全面地分析了航天育种番茄营养成分。结果发现：与原亲本相比,航天育种番茄维生素C、番茄红素、总类黄酮、氨基酸、果胶、Ca、K等均发生了显著性的变化。尤其是航天育种番茄维生素C含量高达71.72mg/100g,几乎是原亲本番茄的1.5倍。番茄红素含量也达到8.99mg/100g,为原亲本的1.4倍多。说明航天育种番茄具有较高的营养价值。通过人体肿瘤细胞实验,用MTT法测定细胞增殖抑制率,流式细胞仪测定细胞凋亡和周期变化进行抑癌功能研究,考察航天育种番茄对结肠癌细胞系SW480、HT-29、SW620,血液病相关细胞株Raji、U937和K562作用情况。结果表明,航天育种番茄品种有抑制SW480和HT-29细胞增殖的作用,最高抑制率能达到30%,并能够诱导其凋亡；而对SW620的作用效果不明显,抑制率只有5%左右。航天育种番茄品种有抑制Raji、U937和K562细胞增殖的作用,最高抑制率为16%,并能够诱导Raji细胞的凋亡。综合研究发现,航天育种番茄品种M2对结肠癌细胞的作用效果最强,航天育种番茄品种M1对血液病细胞的作用效果最强,而原亲本则最弱。更多还原

【Abstract】 Tomato contains high amount of vitamin C, lycopene, folic acid and minerals etc., especially the lycopene which attracts more and more consumers for its specific medical value. It is an important "fruit and vegetable" crop of special economic. Demands for tomato going uptrend year by year make it become the second largest vegetable. Space mutation breeding used only in China supplies a favorable chance for breeding new species. It makes use of different environments between upper air and ground to place a premium on changes in biologic germ plasm, which can be the raw material of breeding new species. The objective of this study was to evaluate the physiological and biochemical properties, nutrition and function of tomatoes M1, M2 using space mutation breeding by Chinese SZ-5 spacecraft, and comparisons with their parent CK were done. It can provide basis for large area cultivation, processing, development and utilization of tomatoes even other plants using space mutation breeding, and then studies were promoted further.Part One:Study of differences between tomatoes M1, M2 and CKWe studied methods of genomic DNA of young leaves of tomatoes M1, M2 and CK extraction and different Polymerase Chain Reaction (PCR) amplification systems, and the optimum and improved system of reaction was obtained. DNA sequences were obtained according to the designed primers. The results of sequencing showed that eight base sequences change had taken place between CK and M1, M2. The study of molecular genetics in various materials could directly reflect the effects of space mutation breeding on plant hereditary. Our results demonstrated that there were some differences among CK, M1 and M2 at DNA level.In order to quickly analyze varieties of tomato via space mutation breeding with near infrared spectra. Firstly, principal component analysis was used to analyze the clustering of tomato leaf samples. Then abundant spectral data were compressed by wavelet transform and the model was built with radial basis function neural network. The model regarded the compressed data as the input of neural network input vectors and the training process was speed up. One hundred and five leaf samples of CK, M1 and M2 were selected randomly to build the training model, and forty five samples formed the prediction set. The results showed that, at the wavelength range of 400-1000nm, there were obvious differences in spectrogram among CK, M1 and M2. When in Visible light at 550nm, there were significant differences between M1, M2 and CK; While in the near-infrared part behind 700nm, the reflectance value of M1 and M2 were higher than CK. The discrimination rate of 97.8% was achieved by this method. It demonstrated that there were some differences among CK, M1 and M2 from pointview of spectroscopy.One hundred and five fruit samples of CK, M1 and M2 were built the training model. Characteristics of the pattern were analyzed by partial least square. The model was built with radial basis function neural network and regarded the compressed data as the input of neural network input vectors. Forty five samples formed the prediction set. The results showed that, the reflectance rate in this three kinds of tomatoes has similar trend, while there were obvious differences in spectrogram among CK, M1 and M2 at the wavelength range of 400-1000nm. The reflectance value of CK was lower than M1 and M2. The discrimination rate of these two models was achieved 95.6% and 97.8%. It offered a new approach to the fast discrimination of varieties of tomato via space mutation breeding.Part Two:Evaluation of root exudates assessment on tomatoes using space mutation breedingVarious tomatoes were cultivated using hydroponics method. When in seedling and florescence periods, roots were washed and root exudates were collected, respectively. The active residues were finally purified with XAD-4 resin exchange columns. Low-molecular-weight components in root exudates were analyzed using GC-MS. The results showed there were obvious variations on composition and quantity of root exudates of the same kind of plant but different genotypes, and the same plant at different growing stages. Besides, we also found some pollutants and harmful chemicals among these root exudates. Therefore, more attention should be paid on the safety of tomatoes by space mutation breeding for environment and human.Part Three:Evaluation of antibacterial capacity in vivo of tomatoes using space mutation breedingAntibacterial capacity in vivo of tomatoes were compared according to natural and perforated decay rate. The results showed that, natural decay rate of M2 was lower than CK, and natural anti-trauma and anti-alternata and Botrytis cinerea ability of M2 were better than the CK. While natural decay rate of M1 was higher than CK, and natural anti-trauma and anti-Alternata and Botrytis cinerea ability of M2 were worse than the CK.Activities of some antioxidant enzymes in vivo were determined through various stages of fruit growth, such as SOD, CAT, and MDA. Preliminary study was done to evaluate the mechanism of antibacterial capacity in vivo of tomatoes using space mutation breeding. The results showed that, the SOD and CAT value of M2 was usually higher than that of CK, with minimum value of MDA. The SOD and CAT value of M1 changed larger, but achieved minimum eventually, with maximum value of MDA. These were consistant with the results that M2 had the strongest antibacterial capacity in vivo, while M1 had the weakest ability.Part Four:Evaluation of nutrition and function of tomatoes using space mutation breedingTo determinate the main nutritional components of M1, M2, and CK, HPLC, Atomic absorption spectrophotometer, Automatic amino acid analyzer, UV spectrophotometry and other methods were used to determine the contents of nutriments. The results showed that, compared to CK, there were obvious changes in vitamin C, lycopene, flavonoids, amino acids, pectin, Ca, K and etc.. Especially the content of Vc in tomatoes using space mutation breeding reached 71.72mg/100g, lycopene to 8.99mg/100g, they were nearly the 1.5 times than that of the CK. The tomatoes using space mutation breeding had high nutrition value. Thus, it provided the scientific basis for a great culturing and the further comprehensives utilization.To evaluate the anti-cancer effect of the tomatoes using space mutation breeding, viability of cells were determined by MTT assay, and the cell-cycle profile and apoptosis induction were analyzed by Flow cytometry. The effects of tomatoes on uman colon cancer cell lines, SW480, HT-29, SW620 and human blood disease cell lines, Raji, U937 and K562 were evaluated. The results showed that, Ml and M2 can inhibit cell proliferation of SW480 and HT-29 cells. The maximum inhibitory rate reached 30%, and they can induced these cells cycle arrest. But they were ineffective on SW620 cell, and the inhibitory rate was only about 5%. Besides, M1 and M2 can inhibit cell proliferation of Raji, U937 and K562 cells. The maximum inhibitory rate was up to 16%, and they can induced Raji cell cycle arrest. These data suggested that M2 had the strongest effect on colon cancer cells, while M1 had the strongest effect on blood disease cells, and CK had the weakest effect on both these two kinds of cells.更多还原