摘要
基因枪等直接转化法可将去除质粒载体主干序列的外源基因表达框导入植物基因组 ,消除质粒主干序列对植物基因组的影响 ,同时由于转基因分子较小 ,比较容易实现多个基因的共转化 ,在植物基因工程育种上有重要的应用价值。研究了基因枪介导外源基因表达框 (包括启动子、基因开放阅读框和终止子 )转化水稻的影响因素和转基因的整合模式 ,结果表明 :(1)基因枪介导外源基因表达框转化水稻的频率约在 0 1%～ 0 5 %之间 ,非选择标记基因与选择标记基因的共转化频率约为 5 0 %～ 6 0 % ,增加基因表达框DNA浓度可提高转化率。相同基因构建物对不同品种水稻的转化率不同 ,基因构建物的侧边序列对基因枪转化的品种差异可能具有重要影响。 (2 )非选择标记基因cecropinB表达框在水稻基因组内整合模式简单 ,仅有 1～ 3个拷贝 ;筛选标记基因bar表达框却比完整质粒转化后的整合模式复杂得多 ,插入拷贝数在 4～ 14个之间。线形基因表达框的游离DNA末端和CaMV
Whole plasmids are used in both Agrobacterium mediated transformation and direct DNA transfer,generally leading to the integration of vector backbone sequences into the host genome along with the transgene(s).The undesirable vector backbone sequences may not only promote transgene rearrangements and affect transgene or endogenous gene expression negatively, but have disadvantage on the safe assessment of the transformants as 'desert DNA'. The direct DNA transforming systems can transfer minimal gene expression cassettes (promoter,open reading frame,terminator) into plant genome and generate 'safer' transformants,also it can delivery multiple genes of agronomic relevance to economically important crop plants.But there is seldom researching reports on the topic till now.The present paper studied some factors that affecting the transforming efficiency of liner gene expression cassettes to rice varieties by particle bombardment,and the integration patterns of the gene expression cassettes in rice genome were compared with that of the whole plasmids.The results showed:(1) The transforming frequency of gene expression cassettes to rice via particle bombardment is 0 1%～0 5%,the cotransforming frequency of non selectable gene is about 50%～60% when two separate gene expression cassettes were used for transformation.Increasing the DNA mole content can increase the transforming frequency and the beside sequences of gene constructs may play an important role on the variation of transforming efficiency between different rice varieties.(2) It's reported that the selectable and non selectable transgene expression cassettes generated low copy number transgenic plants with simple integration patterns.While our results showed that the non selectable cecropin B gene cassette generated simple integration patterns with 1～3 copies in the rice genome,but the selectable bar gene cassette which got 4～14 copies had much more complex integration patterns than that of the whole plasmids which got 1～3 copies only.As the bar gene is promoted by the CaMV35 promoter,in which there is a 19 bp palindromic sequence could act as recombination hot spot and lead to DNA rearrangement,we presumed that the transgene recombination events happened during the integration course have generated the complex Southern patterns of bar gene expression cassette.The recombination character,the heredity behavior and the expression law of gene expression cassettes in the rice genomes will be reported in our future papers.
基金
国家自然科学基金 (批准号 :3 9970 4713 9970 40 9)
浙江省自然科学基金(批准号 :3 0 0 2 2 0 )
农业部水稻生物学重点实验室开放项目~~
