【作者】 林美娟；

【导师】 陈如凯；

【作者基本信息】 福建农林大学 ， 作物栽培学与耕作学， 2007， 硕士

【摘要】 低温是限制植物分布与生长的重要因素，低温伤害是一种严重的自然灾害，全球每年因此造成农作物的损失高达数千亿美元。甘蔗是热带和亚热带作物，低温霜冻会对蔗糖生产造成极其严重的损失，既影响蔗糖的产量和蔗糖分，又给制糖生产造成困难：同时，影响种苗的数量和质量，造成种苗严重不足；影响次年的宿根蔗。甘蔗是一种具有高度杂合性的异源多倍体或非整倍体的无性繁殖作物，其遗传背景十分复杂，同时植物抗冻性属于多基因控制的性状，其抗冻性的遗传机理尚未明确，利用常规手段往往难以达到定向改良目的，利用现代生物学技术进行植物抗冻性分子改良具有高效性和针对性，可弥补常规育种技术的不足，加速高抗冻性植物新品种的选育进程。抗冻蛋白具有热滞活性，能抑制冰晶的重结品和改变冰品的生长方向等特性，在一定程度上能有效地防止低温对植物的伤害。本研究成功地从胡萝卜中克隆到抗冻蛋白基因，并通过原核表达研究该基因所编码的特异蛋白表达情况；针对目前基因枪转化法中存在的再生率低的问题，对轰击后的甘蔗愈伤组织的分化培养基进行优化；通过基因枪转化法，尝试将构建好的胡萝卜抗冻蛋白基因植物表达载体转化到优良甘蔗品种中，以期改良甘蔗的抗寒性，减轻低温对甘蔗生产造成的损失。主要研究结果如下：1．用同源克隆的方法，获得了胡萝卜抗冻蛋白基因开放编码框，全长999 bp，编码332个氨基酸，理论分子量为36 kDa。2．构建了胡萝卜抗冻蛋白基因的原核表达载体，转化大肠杆菌BL21。采用IPTG诱导，使宿主菌重组子表达特异的胡萝卜抗冻蛋白基因。3．采用正交实验设计方法得到了粤糖96／86愈伤组织在基因枪轰击后的最佳分化培养基配方：MS+1.0 mg／L 6-BA+1.0 mg／L KT+20 g／L蔗糖+6 g／L琼脂粉。平均分化率和健康苗数比原有再生体系分别提高了183％和1080％，差异均达极显著。4．构建了胡萝卜抗冻蛋白基因的真核表达载体，并进行遗传转化甘蔗研究，后续工作还在进行之中。更多还原

【Abstract】 Chilling injury of plant is one of the most serious natural disasters to agriculture and forestry production in the world, which can cause heavy yield loss to the global crops about hundreds of billions of dollars every year.Sugarcane is a tropical and subtropical crop, while low temperature and frost can cause devastating losses to sugarcane production, which not only affects the yield of sugarcane and sugar content but also causes difficulties to sugar production. Meanwhile, it also impacts the population and growth of seedlings, which results in serious shortage of seedlings and will affect the production of sugarcane ratoon in next year. However, sugarcane is a vegetative propagation crop, which has highly heterozygous allopolyploid or aneuploid chromosomes. Its genetic background is very complex. Moreover, the antifreezing capability of plant is controlled by polygenes, and there is no well-defined genetic mechanism for its freezing resistance. So it is very difficult to improve the target trait by conventional breeding methods. The application of modern biotechnology using genetic transformation can overcome the limitation of conventional breeding methods, and proved to be an efficient and targeted measure for genetic improvement of plant freezing resistance, and accelerates the selective process for high freezing resistance of new plant varieties.Antifreeze proteins (AFPs) are proteins that have the ability to change the growth directions and inhibit the recrystallization of ice, that is to say thermal hysteresis activity (THA), it can effectively prevent low temperature from damaging plants at a certain extent. In this study, one of the antifreeze protein genes was cloned from carrot successfully, and the prokaryotic expression of AFP (antifreeze protein of carrot) was also studied.In view of the current problem of calli low regeneration rate via microprojectile bombardment, the calli of sugarcane variety YT 96/86 after bombarded by microprojectile were studied for their good regeneration, and the optimum culture media was selected. A carrot gene encoding antifreeze protein was cloned, and its plant expression vector was attempted to transform into an excellent sugarcane variety by using microprojectile bombardment, in order to improve its antifreezing ability and reduce the loss of sugarcane production caused by low temperature.The results were as follows: 1. A carrot gene encoding antifreeze protein was obtained by cloning. The sequence analysis showed that the complete carrot antifreeze protein gene consists of 999 nucleotides and encodes a polypeptide consisting of 322 amino acids, and its molecular weight is 36 kDa in theory.2. The antifreeze protein gene (AFP gene) was cloned in the expression vector (pGEX-4T-3) of E.coli BL21, after IPTG induction, the expression of AFP gene in the recombinant strain was detected.3. The effects of different concentrations and combinations of 6-BA, KT, NAA and sugar on regeneration for the sugarcane variety YT 96/86 calli after bombarded by microprojectile had been studied by experiment of orthogonal design in this paper, and the optimum culture media was selected. The results showed that the optimum compounds and concentrations was MS + 1.0 mg/L 6-BA + 1.0 mg/L KT + 20 g/L sugar + 6g/L agar powder. The average rate of regeneration for calli and the quantity of health seedlings were increased 183 % and 1080 % respectively than original regeneration system, and their differences were both significant.4. An eukaryotic expression vector of carrot antifreeze protein gene had been established, and it was transformed into sugarcane via microprojectile bombardment. The follow-up study is on going.更多还原