Abstract：Abstract Lily (Lilium brownii var. viridulum) is one of the main fresh cut flowers in the world which possesses both high ornamental and economic value. However, the yield will be greatly reduced because the low temperature inhibits the growth of the lily. To improve the antifreeze function of Lily and breed the new characters, Antifreeze protein gene (AFPⅢ) originates from Anarhichas minor which was codon optimized according to the codon usage bias in plants and the antifreeze gene (AFⅠ) was synthesized by the company. Then AFⅠ was inserted into Pcambia1390 vector by enzyme digestion and ligation with the Peanut chlorotic streak caulimovirus (PCISV) promoter. Thus the plants expression vector carrying AFⅠ gene was constructed. And then the P1390-PCISV-AFⅠ plasmid was transformed into Agrobacterium EHA105 by electric shock. Subsequently, AFⅠ gene was introduced into lily callus mediated by Agroinfection, and the results of PCR confirmed that the AFⅠ gene had been integrated into Oriental Lily cultivar "Conca D or"．Restoring growth test was carried out to examine the antifreezing function of the positive transgenic plants, and the results of the test revealed that experimental group had a great antifreezing function in -2 ℃ compared with the control group, and the Semi-quantitative reverse transcription PCR demonstrated that the expression of AFⅠ gene leads to the antifreezing function of transgenic plants. This study provides an important reference for the cultivation of new varieties of antifreeze plants.
[1] 蔡宣梅，郭文杰，张 洁，等. 2016. 木门百合试管鳞茎培养体系的建立[J]. 武夷科学, (32):110-114.（CAI ，GUO ，Z Jie et.al Establishment of tissue culture system of bulblets in tube of Lily Conca D'or [J] WUYI SCIENCE JOUＲNAL (32):110-114.） [2] 江勇, 贾士荣，费云标， 等. 1999. 抗冻蛋白及其在植物抗冻生理中的作用[J]. 植物学报, 41(7):677-685. （JIANG Yong ,JIA Shi_Rong ,FEI Yun,et.al .Antifreeze Proteins and Their Role in Plant Antifreeze [J] Acta Botanica Sinica Physiology，41(7):677-685.） [3] 刘友良，朱根海，刘祖祺. 1985. 植物抗冻性测定技术的原理与比较[J]. 植物生理学通报, (1):40-43.(Liu YL ,Zhu GH,Liu ZQ 1985. The principle and comparison of the determination technology of plant antifreeze plant physiology Comunications[J] (1)40-43) [4] 邱立明, 马纪. 2009. 昆虫抗冻蛋白基因的克隆与表达研究进展[J]. 昆虫知识, 46(6):837-845.( QIU Li-Ming, MA Ji, 2009. Recent advances in cloning and expression of the insect antifreeze protein genes. Chinese Bulletin of Entomology 46(6):837-845.) [5] 魏宪辉，陈玉珍，代焕琴, 等. 2007. 低温贮藏百合种球耐冻性检测[J]. 林业科技开发, 21(4).( WE IX ian- hui, CHEN Yu- zhen, DAIH uan- qin et.al. 2007.A Freez ing- tolerance Test of Four Lily Cu ltivars, Forest Science and technology development.21(4)) [6]文燕 2014抗冷冻蛋白AFPⅢ在玻璃化冷冻过程中对小鼠卵母细胞和胚胎保护作用的研究[D].硕士学位论文,山东大学,导师：邓晓惠pp.31-44(Wen Y 2014 The protective role of antifreeze protein Ⅲ on mature mouse oocyte and embryo in vitrification[D].Thesis for M.S ,Shangdong University, Suppervisor:Deng XH pp.31-44) [7]曾琴 2016 抗冷冻蛋白Ⅲ对家兔卵巢组织冷冻效果的影响 [D]硕士学位论文,成都中医药大学导师：刘伟信,pp.44-52（Zen Q 2016）The protective role of antifreeze protein Ⅲ on rabbit ovarian tissue [D]Thesis for M.S , Chengdu University of TCM ,Suppervisor:Liu WX pp.44-52) [8] Andorfer C.A, Duman J.G. 2000. Isolation and characterization of cDNA clones encoding antifreeze proteins of the Pyrochroid beetle Dendroides canadensis. Insect Physiol, 46(3):365-372 [9] Dexter，S.T. 1930. [J].PLANT Physical, (5):215-223. [10] Georges F, Saleem M, Cutler A J. 1990. Design and cloning of a synthetic gene for the flounder antifreeze protein and its expression in plant cells. Gene, 91 :159-165. [11] Hightower R, Baden C, Penzes E, et al. 1991. Expression of antifreeze proteins in transgenic plants. Plant Mol Biol, 17:1013-1021 [12] Rancourt D E, Walker V K, Davies P L. 1987. Flounder antifreeze protein synthesis under heat shock control in transgenic Drosophila melanogaster. Mol Cell Biol, 7:2188-2195
