【作者】 殷东林；

【导师】 向本春； 祝建波；

【作者基本信息】 石河子大学 ， 生物化学与分子生物学， 2011， 硕士

【摘要】 VvSUC11和VvSUC12是葡萄蔗糖转运蛋白,已有的研究表明二者的功能是负责蔗糖从质外体向薄壁细胞装载,在葡萄成熟时果实糖分积累中起重要作用。在农业生产实践中,为了获得高产量,人们不仅要设法提高农作物光合作用形成的生物产量,而且要通过一定的措施来提高其经济器官的产量；因此,利用蔗糖转运蛋白来定向的提高农作物经济器官的产量,是一种调控库源关系的有效手段。为了研究蔗糖转运蛋白能否提高甜菜含糖量,本研究将葡萄蔗糖转运蛋白基因VvSUC11和VvSUC12与甘薯的甘薯贮藏蛋白(sporamin)基因的根部特异性启动子命名为SP1和SP2重组。以实验室保存的pCAMBIA2301为起始载体构建了用于转化甜菜的双价植物表达载体PCAMBIA2301-SP1-VvSUC11-SP2-VvSUC12。甜菜是两年生异交草本植物,因为其栽培驯化的时间较短,遗传背景狭窄,种质资源贫乏；特别是甜菜自交高度不亲和性,使得优良甜菜单株的基因型因不能遗传而很容易丢失,给优良选育材料品种的繁殖和保存带来了很大的困难。现代生物技术的发展,特别是基因工程的发展给甜菜育种方法开辟了一条新途径。可以通过植物组织培养和基因工程相结合的方法进行甜菜的分子育种。目前关于甜菜组织培养的众多研究表明,甜菜可以采取直接器官发生再生植株方式(不经历愈伤组织阶段)进行组织培养,而且用这种方式获得的再生苗更适合作为转化外源基因的受体材料。研究表明,甜菜的叶柄作为外植体,其再生频率较高,遗传转化后,其转化率也较高。本研究建立了甜菜叶柄高效直接再生体系。试验通过种植无菌甜菜苗、无菌苗预培养、叶柄的诱导分化培养,不定芽的产生和生根,获得了完整的再生植株,建立了甜菜再生体系。试验结果表明：甜菜种子消毒后在1/2MS培养基中生长10 d左右,取出幼苗除根后在预培养培养基(MS+6-BA0.5mg/L+NAA0.05mg/L)的中预培养50-60 d,然后取其叶柄作为外植体在诱导分化培养基(MS+6-BA0.5mg/L+NAA0.05mg/L)中进行不定芽诱导,不定芽的诱导率在50%以上；将不定芽移到生根培养基(MS+IBA2.0mg/L)中诱导生根,诱导生根率在90%以上。用农杆菌介导法将载体PCAMBIA2301-SP1-VvSUC11-SP2-VvSUC12转化甜菜KWS-9103品种,发现预培养4d,侵染时农杆菌的浓度OD600值为0.5附加0.005%表面活性剂：Silwet L-77,延迟筛选4d,转化效率最高,可达42%。对在卡那霉素中分化并生根的甜菜植株进行PCR和RT-PCR检测,证明了目的基因已整合到甜菜中并表达,为研究其能否提高甜菜的含糖量奠定了坚实的基础。更多还原

【Abstract】 VvSUC11 and VvSUC12 are sucrose transporters from grape berries. Studies showed that their function was the loading of sucrose from the apoplast into the parenchyma cells. They play a key role in the accumulation of sugar during this process of ripening of grapes.In agricultural production, in order to obtain high yield, people not only try to improve the biological yield of photosynthesis, but also to adopt certain measures to improve economic production. The use of sucrose transporters orientation to improve the economics of production is an effective mean to regulate the relationship between the sources to the library.To study whether sucrose transporters can improve the yields of sugar of sugar beet, we have recombined genes VvSUC11、VvSUC12 from the grapes, and root-specific promoters of sweet potato storage protein gene from sweet potato, named as SP1 and SP2. We have constructed a bivalent plant expression vector PCAMBIA2301-SP1-VvSUC11-SP2-VvSUC12 using PBI121 and pCAMBIA2301 as original vectors.Sugar beet is a 2-year-old, out crossing herbaceous plant. It is a poor seed resource because of shorter time of domestication cultivation and narrow genetic background. In particular, its high incompatibility in self-fertilization makes it easily to lose good single plant genotype causing lots of difficulties in breeding and preservation of the fine varieties. However, the development of modern biotechnology, particularly, the development of genetic engineering provides a new way in the breeding of sugar beet.. We can precede molecule breeding by the way of combining plant tissue culture and genetic engineering. At present, the large numbers of sugar beet tissue culture studies have shown that regeneration plants from the direct organogenesis (not through callus phase) and plant regeneration was a more suitable receptor plant for receiving foreign gene. Studies have shown that the rates regeneration of petioles of sugar bee as explants is higher, and after transformation, their rate of transformation is also higher.We have stetted up a high efficient direct regeneration system of its petioles. Through planting free vaccine seedlings, their pre-cultivating, induction of differentiation cultivating of petioles, adventitious buds obtained and rooting, regenerated plants obtained, a rapid propagation system of sugar beet was established. The results showed that sugar beet seeds were sterilized and germinated in the1/2MS for 10 d, then their seedlings were transferred to pre-culture medium (MS +6-BA 0.5mg/L+NAA0.05mg/L) for a 50～60 d pre-culture, and then their petioles were cut as explants and were transferred to the shoot-inducing medium(MS+6-BA0.5mg/L+NAA0.05mg /L) for induction of adventitious buds, induction rate of adventitious buds was more than 50%. Adventitious buds were placed onto the rooting medium (MS+IBA2.0mg/L) for rooting. Induction rate of rooting was more than 90%.We transformed the vector PCAMBIA2301-SP1-FvSUC11-SP2-VvSUC12 into sugar beet of KWS-9103 breeding line with the methodologies of Agrobacterium-mediated transformation. We have established the optimal genetic transformation protocol of sugar beet as followed:the explants pre-cultured for 4 days were immersed in Agrobacterium suspension of OD600= 0.5, supplemented with 0.005% Silwet L-77, and followed by 4-day culture on medium containing cefotaxime, then the buds were selected on medium containing kanamycin and cefotaxime. The percentage of kanamycin -resistant buds was as high as 42%. Results of PCR and RT-PCR proved that the aim genes had integrated into sugar beet genome and expressed. It will lay a solid foundation for studying whether sucrose transporters can improve the yields of sugar of sugar beet.更多还原