小叶杨胆碱单氧化物酶(CMO)基因的克隆与转化杂种落叶松及表达  

Transformation of Hybrid Larch(Larix leptolepis× L. olgensis) with CMO Gene Cloned from Populus simonii cDNA Library

作　　者：张守攻[1] 韩素英[1,2] 汪泉[1] 刘国华[3] 周春娥[4] 齐力旺[1] 

机构地区：[1]中国林科院林业研究所细胞生物学实验室 [2]中国林科院森环森保所重点实验室,北京100091 [3]重庆文理学院生命科学系 [4]河南师范大学生命科学院

出　　处：《分子植物育种》2006年第4期483-488,共6页Molecular Plant Breeding

基　　金：国家973项目“树木育种的分子基础研究”(G1999016003);国家林业局948项目(98-4-04-02);国家转基因与产业化专项(J2002-B-005)与(JY03-B-28-02)资助.

摘　　要：甜菜碱是在生物界广泛存在的一类细胞相容性物质,它不仅能使细胞在干旱等胁迫条件下与环境保持渗透平衡,而且还具有稳定复杂蛋白质高级结构的能力。胆碱单氧化酶(CMO)在植物体中是催化胆碱转变为甜菜碱的关键酶,单独转化CMO基因的转基因植物可以完成甜菜碱的合成,起到抗盐、耐旱的作用。小叶杨是我国特有乡土树种,具有抗旱、耐瘠薄、适应性广、生长优良、寿命长等特性,在荒沙及黄土高原区均能生长。本研究以小叶杨为材料构建了干旱胁迫和正常生长条件下的cDNA文库,以特异性引物从中扩增出一条1269bp大小的DNA片段,经序列分析证实该片段编码胆碱单氧化物酶(CMO);将该片段构建入植物表达载体pBI121中,在落叶松杂交育种中利用花粉管通道法将带有该CMO基因的植物表达质粒转入杂种落叶松,收获球果取出种子,提取转化种子发芽长出幼芽的DNA,特异性PCR扩增和Southern、WesternBlotting检测证实落叶松中CMO基因的插入。因此,我们不仅克隆得到了小叶杨编码胆碱单氧化物酶(CMO)的基因,还将其导入杂种落叶松,得到CMO超重表达的转基因杂种落叶松。Betaine can be found widely in plants, it can protect the cell from the drought, and it can also keep the complex proteins＇ structure. Choline monooxygenase （CMO） is an important enzyme which catalysis the choline to the betaine. The plant only transformed the CMO gene can synthesize the betaine so that it can prevent the high salt and the drought. Populus simonii has the ability to live in the desert and the poor land, in our research, cDNA library was constructed from the drought-treated and normal Populus simonii materials. By using PCR with specific primers, a segment of 1 269bp was gained and identified as the gene encoding choline monooxygenase（CMO）. This segment was constructed into the plant expression vector pBI121. Then the constructed plasmid was transformed into hybrid larch （Larix leptolepis xL. olgensis） by using the Pollen Tube Pathway. The PCR amplification, Southern and Western Blotting hybridization methods and sequence analysis were used to identified the CMO gene in the transgenic hybrid larch （Larix leptolepisxL, olgensis）. Through the experiment, we not only got the CMO gene from the Populus simonii and transformed it to the Larix leptolepisxL, olgensis, but also got the CMO gene over-expressed transformed Larix leptolepisxL, olgensis.

关 键 词：小叶杨 CMO基因 转录因子 杂种落叶松 

分 类 号：S792.116[农业科学—林木遗传育种] S791.22[农业科学—林学]