我们的网站为什么显示成这样?

可能因为您的浏览器不支持样式,您可以更新您的浏览器到最新版本,以获取对此功能的支持,访问下面的网站,获取关于浏览器的信息:

|本期目录/Table of Contents|

黄瓜LDC克隆、表达载体的构建及烟草转化研究(PDF)

《广西植物》[ISSN:1000-3142/CN:45-1134/Q]

期数:
2015年02期
页码:
255-260
栏目:
遗传与育种
出版日期:
2015-04-20

文章信息/Info

Title:
Cloning and expression vector construction of cucumber LDC and transformation to tabacco
文章编号:
1000-3142(2015)02-0255-06
作者:
简 兴1 苗永美2* 隋益虎2 边卓吾2 陈存款2 黄春景2 李振兴2
1. 安徽科技学院 城建与环境学院, 凤阳 233100; 2. 安徽科技学院 生命科学学院, 凤阳 233100
Author(s):
JIAN Xing1 MIAO Yong-Mei2* SUI Yi-Hu2 BIAN Zhuo-Wu2 CHEN Cun-Kuan2 HUANG Chun-Jing2 LI Zhen-Xing2
1. College of Urban Construction and Environment, Anhui Science and Technology University, Fengyang 233100, China; 2. College of Life Sciences, Anhui Science and Technology University, Fengyang 233100, China
关键词:
黄瓜 赖氨酸脱羧酶 烟草 遗传转化
Keywords:
cucumber lysine decarboxylase tabacco genetic transformation
分类号:
Q943.2
DOI:
10.11931/guihaia.gxzw201312015
文献标识码:
A
摘要:
赖氨酸脱羧酶(LDC)催化赖氨酸脱羧形成尸胺。该研究采用RT-PCR技术从耐冷黄瓜‘Chipper’中分离到一段cDNA序列,利用DNAMAN6.0软件进行氨基酸序列分析,用BLASTp对蛋白的保守序列进行比对,用双酶切法和T4DNA连接酶构建了植物超表达载体pCAMBIA1304-LDC,通过在烟草不定芽诱导和生长培养基中添加不同浓度的潮霉素(Hyg)以确定不定芽分化和生长的筛选压力浓度,同时优化了菌液浓度、侵染时间和预、共培养时间4个农杆菌侵染条件。结果表明:该序列的CDS全长具有648 bp,编码216个氨基酸,BLASTp结果显示该蛋白具有赖氨酸脱羧酶的保守序列,认为分离到的序列为黄瓜LDC基因,在GenBank中的登录号是KC202438; 当在叶块不定芽诱导培养基中添加10 mg·L-1的Hyg时,芽诱导率明显降低,为5.41%; 当芽生长培养基中的Hyg浓度为20 mg·L-1时,芽苗成活率为33.33%,明显低于对照,因此认为10 mg·L-1和20 mg·L-1的Hyg浓度是抗性芽诱导和生长的筛选浓度; 农杆菌侵染时,烟草叶盘不需要预培养,农杆菌OD600值为0.6,侵染5 min、共培养4 d时,侵染效果最好,能获得较高的抗性芽分化率。获得的抗性植株移栽成活后,叶片DNA经过PCR鉴定,获得了29株转化植株,转化率达93.55%。转化植株的获得为下一步基因功能分析提供了材料。
Abstract:
Lysine decarboxylase(LDC)catalyzed the decarboxylation of lysine to form cadaverine. To research the molecular biology function of LDC gene,the cDNA of LDC was cloned by RT-PCR technique from chilling tolerant ‘Chipper’ cucumber treated under low temperature. The sequence of amino acids and conserved sequence of protein were analysed with software DNAMAN6.0 and by BLASTp. A plant expression vector pCAMBIA1304-LDC was constructed by the methods of double restriction enzyme digestion and T4DNA ligase. The selective concentrations of Hyg for adventitous buds induction and shoots growth were confirmed by adding differents concentration of Hyg to the media,and the infection condition of agrobacterium was optimized. The results showed that the coding sequence of the LDC gene had 648 bp in length,encoding 216 amino acids which contained lysine decarboxylase conserved sequence,so the sequence was firmly believed as LDC gene. The accession number was KC202438. The induction rate of adventitious buds reduced to 5.41% when the concentration of Hyg was 10 mg·L-1,and the survival rate was significantly lower than control group when adding 20 mg·L-1 Hyg into medium. The optimized transformation condition was no preculture,the suitable concentration OD=0.6,infection for 5 min,and coculture duration 4 d. and higher resistant buds differentiation rate were gained under the optimized condition. 29 T0 plants were identified by PCR technique on DNA level,the transformation rate reached 93.55%. The gene modified plants would be used for the research of LDC gene function in future.

参考文献/References

Aziz A,Martin-Tanguy J,Larher F. 1997. Plasticity of polyamine metabolism associated with high osmotic stress in rape leaf discs and with ethylene treatment[J]. Plant Growth Regul,21: 153-163
Gamarnik A,Frydman R. 1991. Cadaverine,an essential diamine for the normal root development of germinating soybean(Glycine max)seeds[J]. Plant Physiol,(97): 778-785
Kuznetsov V,Shorina M,Aronova E,et al. 2007. NaCl-and ethylene-dependent cadaverine accumulation and its possible protective role in the adaptation of the common ice plant to salt stress[J]. Plant Sci,172: 363-370
Li JA(李建安),Hu FM(胡芳名),Tan XF(谭晓风). 2006. Impact of hygromycin on Arabidopsis suspension cells and calli(拟南芥悬浮细胞及其愈伤组织对潮霉素的反应)[J]. J Centr S For Univ(中南林学院学报),26(3): 42-46
Liu J(刘俊). 2004. Regulation of polyamine metabolism and its relation to salt tolerance and signal transduction in plants(植物多胺代谢的调节与抗盐性的关系及多胺在其信号转导中的地位)[D]. Nanjing(南京): Naning Agriculture University(南京农业大学) Lu MH(逯明辉),Li XM(李晓明),Chen JF(陈劲枫),et al. 2005. Study on chilling tolerance of cucumber during germination and expression of lysine decarboxylase gene(黄瓜发芽期耐冷性与赖氨酸脱羧酶基因表达)[J]. Sci Agric Sin(中国农业科学),38(12): 2 492-2 495
Ma TC(马廷臣),Yu RR(余蓉蓉),Chen RJ(陈荣军),et al. 2013. Global genome experession change of transcription factors in conventional indica rice roots under different drought stress(全基因组表达分析不同强度干旱胁迫下常规籼稻根系转录因子表达变化)[J]. J Nucl Agric Sci(核农学报),27(9):1 258-1 269
Miao YM(苗永美). 2013. Estalishment of chilling tolerance evaluation system for cucumber(Cucumis sativus L.)and cloning and transformation of CsLDC(黄瓜耐冷评价体系的建立及CsLDC的克隆及转化黄瓜研究)[D]. Nanjing(南京): Naning Agriculture University(南京农业大学) Michael W,Thomas H,Hans-Martin S. 1979. A model mechanism for the enzymatic synthesis of lupin alkaloids[J]. Zeitschrift f�/i>252;r Naturfors,34: 704-708
Niemi K,Haggman H,Sarjala T. 2002. Effects of exogenous diamines on the interaction between ectomycorrhizal fungi and adventitious root formation in Scots pine in vitro[J]. Tree Physiol,22(6): 373-381
Somnuk B,Kae K,Emi I,et al. 2012. Lysine decarboxylase catalyzes the first step of quinolizidine alkaloid biosynthesis and coevolved with alkaloid production in Leguminosae[J]. The Plant Cell,24(3): 1 202-1 216
Tian MM(田苗苗),Liu YJ(刘艳菊),Li M(李敏),et al. 2011. Study on the construction of antisense expression vector of glycine max oleic acid desaturase gene and its transfer to Nicotiana tabacum(大豆fad基因反义表达载体构建及转化烟草研究)[J]. Guihaia(广西植物),31(1):111-116
Wang D(王丹),Zou L(邹莉),Wang Y(王义),et al. 2010. Study on the sensitivity of poplar to hygromycin(杨树对潮霉素的敏 感性研究)[J]. J Jilin Agric Univ(吉林农业大学学报),32(1): 47-50
Wang GL(王关林),Fang HJ(方宏筠). 1998. Thory and Technology of Plant Genetic Engineering(植物基因工程原理与技术)[M]. Beijing(北京): Science Press(科学出版社): 224
Yang M(杨猛),Zhuang WF(庄文锋),Wei S(魏湜),et al. 2013. Cloning of zmpepc gene from maize and transformation spring wheat(玉米苗期受低温胁迫蛋白表达差异研究)[J]. J Nucl Agric Sci(核农学报),27(11):1 742-1 748
Zhang WP(张万萍). 2009. Molecular cloning of a cold-tolerant gene and investigation of cold-tolerance related physiological mechinisms in Cucumis sativus( 黄瓜耐冷相关基因(CsLDC)克隆分析及外源生长物质调节耐冷性生理机制研究)[D]. Nanjing(南京): Naning Agriculture University(南京农业大学)
Zhang ZM(张哲敏),Sun P(孙萍),Wang WT(王旺田),et al. 2013. Bioinformatics analysis of CBF2 in three different chilling resistance grapes and construction of plant expression vector(三种不同抗冻性葡萄中CBF2基因的生物信息学分析及植物表达载体构建)[J]. Guihaia(广西植物),33(1):82-88
Zhang ZZ(张志忠),Wu J(吴菁),Hua LL(华吕柳). 2005. Studies on agrobacterium tumefaciens mediated gentic transformation of watermelon(新根癌农杆菌介导的西瓜遗传转化研究)[J]. J Fruit Sci(果树学报),22(2): 134-137

备注/Memo

备注/Memo:
收稿日期: 2014-02-09修回日期: 2014-05-17
基金项目: 安徽省科技厅青年基金(1208085QC55); 安徽省教育厅重点科研计划项目(KJ2013A074); 安徽省大学生创新创业训练计划项目(AH201310879097); 安徽科技学院第十批大学生创新课题(13XSZ52)。
作者简介: 简兴(1975-),男,广西桂林人,博士研究生,讲师,主要从事植物生物技术研究,(E-mail)jianx@ahstu.edu.cn。 *通讯作者: 苗永美,博士,讲师,主要从事蔬菜遗传育种及生物技术研究,(E-mail)mym416@163.com。
更新日期/Last Update: 2015-04-20