杜鹃红山茶CaCPI基因的克隆及过量表达提高烟草植株的抗虫性

Cloning CPI from Camellia azalea and Overexpressing to Tobacco Enhances Insect Tolerance

绝大多数观赏山茶花属于设施栽培植物,因环境湿热易造成病虫害出现,导致观赏价值降低,影响种植效益。为了提高抗虫性,根据山茶同源序列设计特异性引物,利用同源克隆和3＇,5＇-RACE技术,从杜鹃红山茶嫩叶组织中克隆出半胱氨酸蛋白酶抑制剂（Cysteine proteinase inhibitor,CPI）基因,命名为CaCPI,基因全长579 bp,开放阅读框306 bp,编码101个氨基酸,分子量为11.078 kDa,等电点p I=6.72。利用实时荧光定量PCR方法对杜鹃红山茶根、茎、叶中该基因的表达特性进行分析,结果表明,CaCPI基因在叶中的表达量较高,其次为茎,在根中表达量较低,比较显示叶的表达量分别约为茎的1.53倍,根的1.61倍。转基因烟草分析表明,过量表达CaCPI基因后,转基因烟草中目的基因表达量提高了55.84-174.83倍,并且转基因植株抗蚜虫能力获得提高,接虫5 d后植株上的蚜虫绝大多数死亡,累积死亡率约为90.75%,高达野生型的5.14倍。

The majority of ornamental camellias are cultivated plants in greenhouse,but pests and diseases are likely to occur in hot-damp environment,which leads to detrimental effects on ornamental and economic value. On the basic of homologous sequences of Camellia japonica,a Cysteine proteinase inhibitor（ CPI） gene was isolated from the tender leaf in C. azalea by the 3＇,5＇-RACE technology named CaCPI to improve the resistance of plants to the insect pest. The full-length c DNA of CaCPI was 579 bp,containing a 306 bp ORF which encodes 101 amino acids.The relative molecular weight of CaCPI protein was 11. 078 kDa,and its isoelectric point（ p I） was 6. 72. Expressions of CaCPI in root,stem and leaf of camellias were analyzed by fluorescent quantitative Real-time PCR. The results showed that the expression level of CaCPI in leaf was the highest,medium in stem and the lowest in root. It was also found that compared with stem and root,the expression level in leaf was about 1. 53-fold and 1. 61-fold,respectively. Furthermore,the expression levels of CaCPI in transgenic plants increased up to 55. 84-174. 83 times as high as wild type tobacco plants. In addition,overexpression of CaCPI enhanced insect tolerance in transgenic plants. Two genotypes plants were inoculated with aphids after 5 days indicated that overexpression of CaCPI gene significantly increased insect resistant to Homoptera Myzus persicae,cumulative mortality rate of aphids in transgenic tobacco plants was up to 90. 75% and about 5. 14 fold compared with wild type plants.