植物非生物逆境研究及蝴蝶兰新品种培育
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摘要
第一章低温诱导型的水稻OsFAD8基因的启动子OsFAD8P的克隆与响应分析
细胞膜膜脂蛋白的不饱和脂肪酸与饱和脂肪酸的相对含量影响着细胞膜的流动性,从而调节植物对低温的耐受性。本文克隆了OsFAD8的5’端编码序列,命名为OsFAD8P,为首次报道的水稻中冷诱导型启动子。同时研究了其在各种特定的条件下启动下游基因的表达及其表达量随温度的变化而改变的趋势。包含此启动子重组表达载体的构建,以及通过农杆菌介导的转基因的方法,转入草本及木本植物中可启动下游GUS蛋白表达,同时发现转基因水稻愈伤组织和拟南芥幼苗中GUS的表达量随处理温度降低和时间延长而增多。另外在拟南芥的不同发育时期,其在果荚中几乎观察不到表达量,但是在萌发5d的种子,7d的幼苗中可看到表达。然而在幼苗的根基部、靠近花序部位的茎中和叶子的经脉中有大量表达；叶肉细胞中几乎观察不到表达量。此启动子对ABA,6-BA等一些激素的调控也有响应。同时过表达GUS的拟南芥植株与对照相比生长发育正常。
为了研究从月季中分离的小分子热激蛋白Rchsp17.8的生理功能,把Rchsp17.8的编码框插入到表达载体PHB的35S组成型启动子后面,通过农杆菌GV3101转化烟草,获得转基因植株。在高温、干旱、渗透、高盐等胁迫条件下分别检测转基因烟草形态、失水率、电导率、脯氨酸含量及其内外源基因表达模式。结果显示：经胁迫处理后,与对照相比转基因烟草抗性表型明显,电导率较低,脯氨酸含量大幅度提高,失水率降低；P5CS基因的表达量上调,说明转Rchsp17.8基因烟草苗在高温以及高渗透胁迫下表现出明显的耐受性。
面对目前花卉业这个已经成为具有较大经济效益的产业,通过花卉品质的改变来提高花卉的观赏价值及经济价值越来越受到重视。花卉品质包括花形、花色、花香、花大小、花期长短等,其中尤为重要的被人们关注的品质是花色与花期的长短。花卉品质的优劣直接关系到其观赏价值与经济价值,然而蝴蝶兰属于高档商品花卉,因此蝴蝶兰花卉品质的改良也因此显得尤为重要,本实验通过基因枪转化以及农杆菌转化法转化PhCHS5以及PhF3’5’H基因以期获得花色优质蝴蝶兰新品种,从而提高蝴蝶兰的商业价值,另外首次共转多个基因,达到了蝴蝶兰花色改良的新尝试,进而为深入研究蝴蝶兰花色素相关基因表达的调节机制提供依据。
The relative quantity of two kinds of fatty acids including unsaturated and saturated fatty acids of membrane lipids are very important for plants to cope with the cold-stress, for the reason that it can regulate the fluidity of membrane. We cloned the 5'-region of gene OsFAD8, called OsFAD8P. It is the first time for the work of cold-induced promoter in Oryza sativa L.We investigated the expression of GUS which was promoted by this promoter on different situation and the expression trend of GUS quantity go with the change of the temperature. We cloned the cold-inducible genetic constructs:OsFAD8P-GUS, and conducted the genetic transformation on the herbage and woody plants by Agrobacterium-mediated transformation method. In transgenic callus tissue of rice and seedings of Arabidopsis thaliana, the promoter OsFAD8P promoted the expression of the down-stream gene when exposured to cold, and the quantity of GUS was more expressed as the temperature decreased and time prolonged. GUS was hardly detected in the pod, but it was observed after the seed of Arabidopsis thaliana germinated 5 days later and also in 7 days old seeding of the Arabidopsis thaliana. While it was abundantly expressed in the nervation of the leaves, stem near the inflorescence and base of the root. Little expression was detected in the mesophyll. And the promoter also sensitive to other abiotic-stress as well as hormone like ABA,6-BA, and the growth and development of the transgenic plants are normal as the WT.
Rchspl7.8 cloned from Chinese rose is belongs to a cytosolic class I sHSP, and it is induced under different stress. In order to study the physiological function of Rchspl7.8, we subcloned the ORF of Rchsp17.8 recombined after 35S:promoter into DH5α, then transformed into plant gene expression vector Agrobaterium rumefaciens, Leaf disc of tobacco was infected by Agrobaterium and three transformants were attained. We assessed the phenotype, relative electrical leakage, proline content, water lose rate under high temperature, osmotic stress, and salt stress respectively, and the transcript levels of P5CS gene was also investigated by semi-quantitative RT-PCR Results showed that transgenic line showed lower relative electrical leakage, more proline content, lower water lose rate, and the expressed of P5CS was elevated. Compared with the non-transgenic lines, transformed ones showed obvious resistance to high temperature and osmotic stress. The possible mechanism to different abiotic stress was also discussed.
The economic value of flowers are much more larger than before. So how to change the quality of them, it seems quite important. Quality of flowers is including: shape of flowers, the size of flowers, the color of flowers, the flowers fragrance, and the florescence time. And especially the color and the florescence time are appreciated by people. And Phalaenopsis are much more popular in the society as ornamental than before. In order to increase the commercial value of the Phalaenopsis, people pay much more attention to improve the quality of them from the aspects of color and time of flowering. We try to change the color of Phalaenopsis by transfer the gene PhCHS5 and PhFS'5'H, so as to obtain the new species of Phalaenopsis and improve the commercial value of the Phalaenopsis, at the same time, the co-transformation is a n ew try for the breeding in this study and it can be a further study for the gene regulation of Phalaenopsis.
细胞膜膜脂蛋白的不饱和脂肪酸与饱和脂肪酸的相对含量影响着细胞膜的流动性,从而调节植物对低温的耐受性。本文克隆了OsFAD8的5’端编码序列,命名为OsFAD8P,为首次报道的水稻中冷诱导型启动子。同时研究了其在各种特定的条件下启动下游基因的表达及其表达量随温度的变化而改变的趋势。包含此启动子重组表达载体的构建,以及通过农杆菌介导的转基因的方法,转入草本及木本植物中可启动下游GUS蛋白表达,同时发现转基因水稻愈伤组织和拟南芥幼苗中GUS的表达量随处理温度降低和时间延长而增多。另外在拟南芥的不同发育时期,其在果荚中几乎观察不到表达量,但是在萌发5d的种子,7d的幼苗中可看到表达。然而在幼苗的根基部、靠近花序部位的茎中和叶子的经脉中有大量表达；叶肉细胞中几乎观察不到表达量。此启动子对ABA,6-BA等一些激素的调控也有响应。同时过表达GUS的拟南芥植株与对照相比生长发育正常。
为了研究从月季中分离的小分子热激蛋白Rchsp17.8的生理功能,把Rchsp17.8的编码框插入到表达载体PHB的35S组成型启动子后面,通过农杆菌GV3101转化烟草,获得转基因植株。在高温、干旱、渗透、高盐等胁迫条件下分别检测转基因烟草形态、失水率、电导率、脯氨酸含量及其内外源基因表达模式。结果显示：经胁迫处理后,与对照相比转基因烟草抗性表型明显,电导率较低,脯氨酸含量大幅度提高,失水率降低；P5CS基因的表达量上调,说明转Rchsp17.8基因烟草苗在高温以及高渗透胁迫下表现出明显的耐受性。
面对目前花卉业这个已经成为具有较大经济效益的产业,通过花卉品质的改变来提高花卉的观赏价值及经济价值越来越受到重视。花卉品质包括花形、花色、花香、花大小、花期长短等,其中尤为重要的被人们关注的品质是花色与花期的长短。花卉品质的优劣直接关系到其观赏价值与经济价值,然而蝴蝶兰属于高档商品花卉,因此蝴蝶兰花卉品质的改良也因此显得尤为重要,本实验通过基因枪转化以及农杆菌转化法转化PhCHS5以及PhF3’5’H基因以期获得花色优质蝴蝶兰新品种,从而提高蝴蝶兰的商业价值,另外首次共转多个基因,达到了蝴蝶兰花色改良的新尝试,进而为深入研究蝴蝶兰花色素相关基因表达的调节机制提供依据。
The relative quantity of two kinds of fatty acids including unsaturated and saturated fatty acids of membrane lipids are very important for plants to cope with the cold-stress, for the reason that it can regulate the fluidity of membrane. We cloned the 5'-region of gene OsFAD8, called OsFAD8P. It is the first time for the work of cold-induced promoter in Oryza sativa L.We investigated the expression of GUS which was promoted by this promoter on different situation and the expression trend of GUS quantity go with the change of the temperature. We cloned the cold-inducible genetic constructs:OsFAD8P-GUS, and conducted the genetic transformation on the herbage and woody plants by Agrobacterium-mediated transformation method. In transgenic callus tissue of rice and seedings of Arabidopsis thaliana, the promoter OsFAD8P promoted the expression of the down-stream gene when exposured to cold, and the quantity of GUS was more expressed as the temperature decreased and time prolonged. GUS was hardly detected in the pod, but it was observed after the seed of Arabidopsis thaliana germinated 5 days later and also in 7 days old seeding of the Arabidopsis thaliana. While it was abundantly expressed in the nervation of the leaves, stem near the inflorescence and base of the root. Little expression was detected in the mesophyll. And the promoter also sensitive to other abiotic-stress as well as hormone like ABA,6-BA, and the growth and development of the transgenic plants are normal as the WT.
Rchspl7.8 cloned from Chinese rose is belongs to a cytosolic class I sHSP, and it is induced under different stress. In order to study the physiological function of Rchspl7.8, we subcloned the ORF of Rchsp17.8 recombined after 35S:promoter into DH5α, then transformed into plant gene expression vector Agrobaterium rumefaciens, Leaf disc of tobacco was infected by Agrobaterium and three transformants were attained. We assessed the phenotype, relative electrical leakage, proline content, water lose rate under high temperature, osmotic stress, and salt stress respectively, and the transcript levels of P5CS gene was also investigated by semi-quantitative RT-PCR Results showed that transgenic line showed lower relative electrical leakage, more proline content, lower water lose rate, and the expressed of P5CS was elevated. Compared with the non-transgenic lines, transformed ones showed obvious resistance to high temperature and osmotic stress. The possible mechanism to different abiotic stress was also discussed.
The economic value of flowers are much more larger than before. So how to change the quality of them, it seems quite important. Quality of flowers is including: shape of flowers, the size of flowers, the color of flowers, the flowers fragrance, and the florescence time. And especially the color and the florescence time are appreciated by people. And Phalaenopsis are much more popular in the society as ornamental than before. In order to increase the commercial value of the Phalaenopsis, people pay much more attention to improve the quality of them from the aspects of color and time of flowering. We try to change the color of Phalaenopsis by transfer the gene PhCHS5 and PhFS'5'H, so as to obtain the new species of Phalaenopsis and improve the commercial value of the Phalaenopsis, at the same time, the co-transformation is a n ew try for the breeding in this study and it can be a further study for the gene regulation of Phalaenopsis.
引文
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