半夏凝集素基因(pta)导入水稻及其表达的初步研究

A Primary Study of Transferring the Pinellia Tenata Agglutinin (pta) Gene into Rice and Expression

半夏凝集素基因是一种有重要价值的抗虫基因。利用RACE PCR技术克隆出半夏凝集素 (pta)基因 ,并将它构建到载体pCAMBIA1 30 5中形成双元载体 (含pta基因和hpt基因 )。利用农杆菌介导法将pta基因转入粳稻品种鄂宜 1 0 5、中花 1 2和籼稻品种E优 5 32成熟胚诱导的愈伤组织中。通过PCR检测 ,从 1 1 7株T0 代再生植株中筛选出 36株 (其中鄂宜 1 0 5、中花 1 2、E优 5 32分别为 1 9株、7株、1 0株 )转基因植株。对这些转基因后代植株进行Southernblot分析和RT PCR分析表明 :pta基因已经整合到受体细胞基因组中 ,并在转录水平上得到了有效表达。通过对转基因后代的遗传分析 ,成功地从T1 代表现为 1∶2∶1孟德尔分离的分离群体中筛选出 7个独立转基因水稻纯系 (其中包括 4个鄂宜 1 0 5、1个中花 1 2和 2个E优 5 32转基因纯系 )。对这 7个独立转基因水稻T2 代纯系进行褐飞虱生物抗性鉴定和田间隔离喂养实验 ,结果显示 ,这些转基因纯系对褐飞虱的存活率和发育进度均有显著的抑制作用。同时 ,实验结果还表明 2～ 5mg L的 2 ,4 D是愈伤组织诱导和生长的必需条件 ,而且受体的基因型对愈伤组织诱导率和转化频率均有显著影响 ,在同等条件下 。

Pinellia tenata agglutinin ( pta ) gene,which has significant insecticidal activities,was cloned from the inflorescence of Pinellia tenata by using RACE PCR.After constructed the binary vector that carried pta gene and hpt gene by linking to pCAMBIA1305 vector,the pta gene was introduced into mature seed derived calli of japonica rice Eyi105,Zhonghua12 and indica rice E you 532 through Agrobacterium tumefaciens mediated transformation.Thirty six positive transgenic plants (19 Eyi105,seven Zhonghua 12 and 10 E you 532,respectively) were identified by PCR from 117 T 0 regeneration plants.Southern blot assay and RT PCR analysis for the transgenic progenies indicated that the pta gene has been successfully introduced and integrated into the genome of the receptors and the pta gene has been expressing at the transcription level.Through genetic analysis,7 transgenic homozygous lines (4 Eyi105,1 Zhonghua 12 and 2 E you 532,respectively) were selected from the T 1 generation in which the pta gene showed 1∶2∶1 Mendel segregation.The pest resistance biological test and field rice brown planthoper separate feed test on the T 2 transgenic homozygous lines had indicated that the transgenic homozygous lines have significant insecticidal activities.It evidently inhibits the livability and viability for rice brown planthopper.In addition,this study showed that 2～5 mg/L 2,4 D was necessary for calli inducing and calli growing.And the genotype of the receptor was markedly affect on the calli induction frequency and transformation frequency.In the same condition,the calli induce frequency and transformation frequency of japonica is higher than indica.