野生茄子托鲁巴姆StLTPa7的克隆与分析

Cloning and Analysis of StLTPa7 from Solanum torvum

【目的】从野生茄子托鲁巴姆(Solanum torvum Swartz)中克隆非特异性脂质转移蛋白基因StLTPa7 cDNA,并解析其功能。【方法】采用同源基因设计引物,并根据RT-PCR方法克隆StLTPa7 cDNA序列;通过农杆菌浸染法转化烟草,构建StLTPa7过表达转基因烟草植株;采用菌丝生长速率法检测转基因烟草植株蛋白提取物对大丽轮枝菌的体外抑菌活性。【结果】托鲁巴姆中克隆获得1个StLTPa7 cDNA序列,该序列含有1个345 bp的开放阅读框,推测编码长114个氨基酸的蛋白,相对分子量为11.42 kD,理论等电点为9.01。StLTPa7受水杨酸(SA)和大丽轮枝菌诱导表达,在处理后24和48 h的表达量较高。为了分析StLTPa7对大丽轮枝菌的抗性,构建了过表达转基因烟草植株,共获得了4个PCR阳性转StLTPa7烟草株系。荧光定量PCR分析显示,该基因在转基因烟草株系L5和L7中过量表达。抑菌试验显示,转基因烟草株系L5蛋白提取物对大丽轮枝菌的抑菌率约为对照的2.5倍。【结论】从野生茄子托鲁巴姆中克隆到1个StLTPa7 cDNA序列,该基因与大丽轮枝菌的生长和增殖的抑制作用相关,可能参与植物对大丽轮枝菌的防卫过程。

【Objective】The objective of this study is to clone non-specific lipid transfer protein gene StLTPa7 from Solanum torvum and to analyze the function of the gene.【Method】 Primers were designed according to homologous cloning method.In order to clone StLTPa7,the gene cDNA was amplified by RT-PCR.Over-expression transgenic tobacco plants were generated via Agrobacterium-mediated transformation.Analysis on the inhibitory activity of proteins extracted from transgenic tobacco plants was done using mycelia growth rate method.【Result】StLTPa7 cDNA contained an ORF of 345 bp long and encoded a putative protein of 114 amino acids with a molecular weight of 11.42 kD and a theoretical pI of 9.01.StLTPa7 was induced to express by both salicylic acid（SA） and Verticillium dahliae and the level of transcript was the highest 24 to 48 h after treatment.To analyze the resistance of StLTPa7 to V.dahliae,the plants over-expressing StLTPa7 were generated by Agrobacterium-mediated transformation and 4 transgenic lines were identified by PCR.Quantitative RT-PCR indicated that the gene over-expressed in transgenic lines L5 and L7.Anti-fungal assay revealed that the inhibitory rate of the proteins extracted from the transgenic line L5 to V.dahliae was 2.5 times compared to the control【.Conclusion】StLTPa7 exhibits associated to inhibitory effect of V.dahliae growth,suggesting that it may be involved in plant defense against V.dahliae.