国槐苯丙氨酸解氨酶基因的克隆、反义表达载体构建及遗传转化  

Cloning of PAL Gene from Sophora japonica,Construction of Anti-Sense Gene of SjPAL and Its Genetic Transformation in Arabidopsis

作　　者：许锋[1,2] 朱俊[1] 张风霞[1] 王燕[1] 程水源[3] 程述汉[2] 

机构地区：[1]长江大学园艺园林学院,湖北荆州434025 [2]山东农业大学园艺科学与工程学院,山东泰安271018 [3]黄冈师范学院生命科学与工程学院,湖北黄冈438000

出　　处：《林业科学研究》2008年第5期611-618,共8页Forest Research

基　　金：教育部新世纪优秀人才支持计划(NCET-04-0746);湖北省教育厅重大项目(Z200627002);湖北省自然科学基金(2002AB094);湖北省青年杰出人才基金(2003AB014)

摘　　要：根据其它植物PAL基因的保守区域,设计一对兼并引物,采用RT-PCR方法从国槐中克隆了一个长866bp的PAL基因片段,命名为SjPAL。序列分析发现SjPAL多肽与其它植物的PAL氨基酸序列高度同源(87%以上),且包含与水稻和拟南芥的PAL类似的活性位点。系统进化树分析表明SjPAL与豆科植物的PAL亲缘关系较近。RT-PCR结果显示,SjPAL在根和茎的表达量约为叶中的3倍。利用反义RNA技术将SjPAL基因克隆至植物表达载体pBI121,构建了SjPAL反义RNA植物表达载体pBI121-PAL,通过根癌农杆菌EHA105将其导入拟南芥基因组,对获得的抗性植株进行PCR鉴定、Northern杂交分析、PAL活性分析以及总多酚含量和类黄酮含量分析。结果表明,该反义RNA已整合到拟南芥基因组中,转基因拟南芥的PAL基因表达量、单位材料PAL活性、总多酚含量和类黄酮含量均显著低于野生型对照。本研究为下一步利用该基因反义表达载体转化国槐,通过调节酚类物质含量提高其在再生体系中的生根能力提供了试验依据。A PAL gene fragment with 866 bp length PCR and using a pair of degenerated primers, which （named as SjPAL） was cloned from Sophora japonica by RT- were designed basing on the sequence of other plant PAL genes conserved region. The deduced SjPAL polypeptide showed high identities （ 〉 87% ） to other plant PAL amino acids via sequence analysis, and contained the similar active sites in PAL proteins of Oryza sativa and Arabidopsis. Phylogenetic tree analysis indicated that the SjPAL had a closer relationship with PAL proteins from leguminous plant species. RT-PCR analysis revealed that the relative abundance of SjPAL mRNA in root and stem was about three times as in leaf. Utilizing anti-sense RNA technology, SjPAL gene was inserted directionally into pBI121, a plant expression vector, to construct an anti-sense fusion gene and a plant expression vector pBI121-PAL. Genetic transformation to Arabidopsis was mediated by EHA105. Transgenic Arabidopsis lines were performed using PCR,Northern blot, PAL activity of per unit material, and total polyphenolic and flavonoid concentration analysis. The results showed that the expression level of PAL gene, PAL activities of per unit material, and total polyphenolic and flavonoid concentration of transformed lines were all significantly lower than the wild control. The present studies provided an experimental basis for further genetic transformation in S. japonica of SjPAL gene anti-sense expression vector to improve its rooting ability in regeneration system via regulating its phenolic compound content.

关 键 词：国槐 生根能力 PAL基因 反义表达载体 遗传转化 总多酚 类黄酮 

分 类 号：S718.46[农业科学—林学]