黄瓜LDC克隆、表达载体的构建及烟草转化研究

Cloning and expression vector construction of cucumber LDC and transformation to tabacco

关键词:  黄瓜  赖氨酸脱羧酶  烟草  遗传转化

Key words:  cucumber  lysine decarboxylase  tabacco  genetic transformation

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.