改变天冬氨酸激酶和二氢吡啶二羧酸合成酶调控活性提高玉米中游离赖氨酸含量  

Improving Free Lysine Content of Maize by Changing Regulatory Activities of Aspartokinase and Dihydrodipicolinate Synthase

作　　者：赵倩[1] 于静娟[1] 朱登云[1] 敖光明[1] 

机构地区：[1]中国农业大学生物学院农业生物技术国家重点实验室,北京100094

出　　处：《农业生物技术学报》2004年第3期247-252,共6页Journal of Agricultural Biotechnology

基　　金：国家高技术研究发展计划(863)项目(No.2001AA212131)资助

摘　　要：以烟草(Nicotiana tabacum var．samsum)叶片为材料，提取RNA，分离mRNA，进行RT-PCR扩增编码二氢吡啶二羧酸合成酶(DHDPS)的cDNA片段。序列分析表明，得到971个核苷酸的片段。与发表的序列相比，成熟蛋白N-端编码区缺失了21个核苷酸，且第627位核苷酸发生了C到T的改变，但没有引起氨基酸的变化。将ahaps cDNA编码区的第410和411位核苷酸由AC突变为TT，这一突变使DHDPS成熟蛋白的第104位氨基酸由天冬酰胺变为异亮氨酸。以玉米(Zea mays L．)总DNA为模板，PCR扩增DHDPS叶绿体转运肽的编码序列，序列分析结果显示，扩增片段中含有DHDPS转运肽完整编码区162个核苷酸及成熟蛋白N-端编码区21个核苷酸。将突变后的础(天冬氨酸激酶)基因和dhdps cDNA与CaMV35S启动子、转运肽序列及潮霉素抗性基因连接，构建表达载体pRHAK和pRHDH，用基因枪轰击法将pRHAK和pRTDH转入玉米胚性愈伤组织，得到再生植株。经分子检测证明，目的片段已整合到玉米基因组中。T1代植株的游离氨基酸含量测定显示，部分转入础基因的植株中，叶片和种子中游离苏氨酸的含量提高3～5倍。部分转入dhdps cDNA的植株中，叶片和种子中游离赖氨酸含量分别提高了4倍和1倍。Total RNA was isolated from the leaves of tobacco (Nicotiana tabacum var. samsum ). The dihydrodipicolinate synthase (DHDPS) cDNA fragment was amplified by RT-PCR. Sequence analysis showed that it has 971 nt with a 21 nt deletion at the beginning of the mature protein coding region. The mutations were induced at 410 and 411 sites of coding region, which corresponds to a substitution of asparagine to isoleucine at position 104 from the first amino acid of DHDPS mature protein.Total DNA of maize (Zea mays L) was used as template to amplify the coding region of chloroplast transit peptides by PCR. Nucleotide sequence analysis indicated that the fragment has 162 nt coding for transit peptides (TP) and 21 nt coding for DHDPS amino end of mature protein.The expression vectors for altered ak (aspartokinase) and dhdps were constructed by linking with CaMV35S promoter, TP sequence and Hyg gene. The plasmid pRHAK and pRHDH were introduced into maize embryogenic callus by microprojectile bombardment and the transgenic plants were regenerated. Genomic PCR and Southern blotting analysis indicated that ak and dhdps genes have been integrated into genome of maize. As a result, leaves and mature seeds of ak transformants exhibited a 3~5 fold increase in the accumulation of free threonine. Expression of altered dhdps cDNA resulted in a 4-fold and 1-fold increase in the accumulation of free lysine in the leaves and mature seeds of transgenic plants, respectively.

关 键 词：天冬氨酸激酶 二氢吡啶二羧酸合成酶 定点突变 玉米 转化 游离赖氨酸 

分 类 号：S513.01[农业科学—作物学]