Abstract Senescence, regarded as the last stage of tobacco leaf development, seriously affects the accumulation of the photosynthetic products along with degradation of chlorophyll and lipid, resulting in the decrease of the yield and the quality of the crop. Transgenic technology is an important technical means to cultivate new varieties with high yield and resistance, which is helpful to increase the yield of the unit area of the tobacco (Nicotiana tabacum). During the growth of tobacco, the delay of senescence can increase the amount of photosynthetic products, which could increase the yield of crop. After harvesting, senescence delay of the leaves can keep the freshness of tobacco, so as to solve the problem of storage and transportation. Therefore, research on leaf senescence regulation has a very important significance in agricultural production. In this work, the Agrobacterium-mediated transformation method were adopted to introduce BAS1 (phyB activation-tagged suppressor 1) gene driven by the SAG12 promoter into tobacco and 45 SAG12-BAS1 trangenic plants were obtained according to the result of resistance screening and PCR identification. Among these transgenic plants, eight transgenic tobacco plants showed that their leaves senility were delayed. Leaf chlorophyll content, protective enzyme activity and plant growth phase were observed and measured on the wild type and transgenic SAG12-BAS1 tobacco leaves during leaf senescence. The results showed that the chlorophyll content of transgenic SAG12-BAS1 tobacco plants was higher than that of the wild type from the top to the base. The results of content analysis of super-oxide dismutase (SOD) and malondialdehyde (MDA) investigated between the wild type and transgenic SAG12-BAS1 tobacco demonstrated that SOD activity in transgenic SAG12-BAS1 tobacco plants was 31.98 % higher than that of wild type and the content of MDA was 48.28% lower than that of the wild type. The conclusion that the senescence of transgenic tobacco delayed for 10~15 d than that of the wild type was drawn through the observation of tobacco growth development process. Besides, the result of cytokinin content detected between the wild type and transgenic tobacco at the beginning of leaf senescence showed that the cytokinin content of the wild type was 40.2% lower than that of transgenic plants. The above results indicated that transgenic SAG12-BAS1 tobacco have delayed the senescence process and that may relate with the increase of cytokinin content and protective enzyme activity and the initiation of senescence promoter. This study provides a theoretical basis for the further study of the function of SAG12-BAS1 gene, and it also provides the basis for the development of genetically modified anti-aging materials.
参 考 文 献 曹孟良, 周智, 张启发. 2000. PSAG12-IPT转基因植物的抗早衰研究[J]. 云南大学学报自然科学版, 23(1):73- 78.(Cao M L, Zhou Z, Zhang Q F. 2000. Studies on senescence-inhibition of Psagl2-ipt transgenic plants[J]. Journal of Yunnan University, 23(1):73- 78.) 曹显祖, 朱庆森, 顾自奋.1981. 关于杂交水稻结实率的研究[J]. 江苏农业科学, 1:1-7. (Cao X Z, Zhu Q S, Gu Z F. 1981. About hybrid rice seed setting rate study[J]. Jiangsu Agricultural Sciences, 1:1-7.)DOI:10.15889/j.issn.1002-1302. 付永彩, 丁月云, 刘新仿, 等. 1998. 抑制衰老的嵌合基因在水稻中的转化[J]. 科学通报, 43(18):1963-1967.(Fu Y C, Ding Y Y, Liu X F, et al. 1998. Chimeric gene conversion inhibiting senescence in rice[J]. Chinese Science Bulletin, 43(18):1963-1967.) 郭林霞, 董璇, 赵德刚. 2016. 转杜仲几丁质酶基因EuCHIT1番茄提高对灰霉病的抗性[J]. 植物生理学报, 52 (5): 703–714.(Guo L X, Dong X, Zhao D G. 2016. Transgenic tomato plants expressing a Eucommia ulmoides chitinase gene Eu-CHIT1 and their resistance to Botrytis cinerea[J]. Plant Physiol J, 52 (5): 703–714 )DIO: 10.13592/j.cnki.ppj.2016.0066 高俊风. 2006. 植物生理学实验指导书[M].北京:高等教育出版社:74-75.(Gao J F.2006. The Plant-Physiology Experiment Instructs[M]. Beijing. Higher Education Press. ) 李秋庆, 陈国菊, 曹必好, 等. 2015. 农杆菌介导PSAG12-BAS1基因转化辣椒的研究[J]. 辣椒杂志, 02:23-29.( Li Q Q, Chen G J, Cao B H, et al. 2015. Agrobacterium tumefaciens -mediated genetic transformation of hot pepper with psag12-ipt[J]. Pepper magazine, 02:23-29 ) 奚亚军, 张启发, 林拥军, 等. 2004. 利用农杆菌浸种法将叶片衰老抑制基因PSAG12-IPT导入普通小麦的研究[J]. 中国农业科学, 37(8):1235-1238.(Xi Y J, Zhang Q F, Lin Y J, et al. 2004. Introduction of leaf senescence-inhibiting gene PSAG12-IPT into common wheat via agrobacterium tumefaciens soaked seeds[J]. Scientia Agricultura Sinica, 37(8):1235-1238. ) 姚新转, 吕立堂, 赵德刚. 2016. 转拟南芥BAS1基因提高烟草光合特性[J]. 山地农业生物学报.(YaoX Z,Lv L T,Zhao D G. 2016. Improving photosynthetic characteristics of tobacco plants with BAS1 gene in transgenic Arabidopsis thaliana[J]. Journal of Mountain agriculture and biology. 已发表 ) 袁政, 张大兵. 2002. 植物叶片衰老的分子机制[J]. 植物生理学通讯, 38(5):417-422.(Yuan Z, Zhang D B. 2002. The Molecular Mechanism of Plant Leaf Senescence[J]. Plant Physiology Communications, 38(5):417-422.) DOI:10.13592/J.cnki.ppj. 章家长, 孙振远, 李召虎, 等. 2005. 转P(SAG12)-ipt基因结缕草的获得及其衰老特性分析[J]. 自然科学进展, 07：818-823.( Zhang J Z, Sun Z Y, Li S H, et al. 2005. Gene transfer P(SAG12)-ipt Zoysia sinica obtained and Its senescence characteristics analysis[J]. Progress in Natural Science, 07：818-823.) 邹冰杰, 吕立堂, 赵德刚. 2016. 超量表达拟南芥油菜素内酯基因BAS1对烟草维管组织发育的影响[J]. 基因组学与应用生物学, 35.(Zou B J, Lv L T, Zhao D G. 2016. Overexpression of Arabidopsis BAS1 gene effects of Brassinolide on tobacco tissue development dimension[J]. Genomics and Applied Biology, 35.) 郑 丽, 李名扬, 晁岳恩, 等. 2005. 根癌农杆菌介导 ipt 基因对切花菊的遗传转化[J]. 农业生物技术学报, 13 (1): 26~31(Zheng L, Li M Y, Chao Y E, et al. 2005. Genetic transformation of ipt gene to cut chrysanthemum mediated by agrobacterium tumefaciens. Journal of Agricultural Biotechnology, 13 (1): 26~31.) Buchanan-Wollaston, V., Ainsworth, C. 1997. Leaf senescence in Brassica napus: Cloning of senescence-related genes by subtractive hybridization. Plant Mol. Biol. 33, 821–834. DOI: 10.1023/A:1005774212410 Chang HS, Jones ML, Banowetz GM, et al. 2003. Overproduction of cytokinins in petunia flowers transformed with P-SAG12-IPT delays corolla senescence and decreases sensitivity to ethylene[J].Plant Physiology, 132(4):2174 Doi:10.1104/pp.900086 Gan, S., Amasino, R.M. 1995. Inhibition of leaf senescence by autoregulated production of cytokinin. Science 270, 1986–1988. Horsch RB, Fry JE, Hoffmann NL, et al. 1985. A simple and general method for transferring genes into plants. Science, 227: 1229~1231．DOI: 10.1126/science.227.4691.1229 Hareven, D., Gutfinger, T., Parnis, A., et al. 1996. The making of a compound leaf: Genetic manipulation of leaf architecture in tomato. Cell 84, 735–744.DOI:10.1016/S0092-8674(00)81051-X Jefferson RA, Kavanagh TA, Bevan MW .1987. GUS fusions: β-glu-curonidase as a sensitive and versatile gene fusion marker inhigher plants. EMBO J, 6 (13): 3901~3907 Marnett LJ 2002. Oxy radicals, lipid peroxidation and DNA damage.Toxicology, 181: 219–222 DOI:10.1016/S0300-483X(02)00448-1 Nam, H.G. 1997. The molecular genetic analysis of leaf senescence. Curr. Opin. Biotechnol. 8, 200–207.DOI:10.1016/S0958-1669(97)80103-6 Nooden, L.D., Guiamet, J.J., and John, I. 1997. Senescence mechanisms. Physiol. Plant. 101, 746–753. DOI: 10.1111/j.1399-3054.1997.tb01059.x
