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

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866 bp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.

Clon ing of PAL Gene from Sophora japon ica, Construction of Anti2Sense Geneof SjPAL and Its Genetic Tran sforma tion in A rabidopsis

A PAL gene fragment with 866 bp length ( named as SjPAL ) was cloned from Sophora japonica by RTP-CR and using a pair of degenerated p rimers, which were designed basing on the sequence of other p lant PAL genesconserved region. The deduced SjPAL polypep tide showed high identities ( > 87% ) to other p lant PAL amino acidsvia sequence analysis, and contained the similar active sites in PAL p roteins of O ryza sativa and A rabidopsis.Phylogenetic tree analysis indicated that the SjPAL had a closer relationship with PAL p roteins from leguminous p lantspecies. RTP-CR analysis revealed that the relative abundance of SjPAL mRNA in root and stem was about threetimes as in leaf. Utilizing anti-sense RNA technology, SjPAL gene was inserted directionally into pB I121, a p lantexp ression vector, to construct an anti-sense fusion gene and a p lant exp ression vector pB I121-PAL. Genetictransformation to A rabidopsis was mediated by EHA105. Transgenic A rabidopsis lines were performed using PCR,Northern blot, PAL activity of per unit material, and total polyphenolic and flavonoid concentration analysis. Theresults showed that the exp ression level of PAL gene, PAL activities of per unitmaterial, and total polyphenolic andflavonoid concentration of transformed lines were all significantly lower than the wild control. The p resent studiesp rovided an experimental basis for further genetic transformation in S. japonica of SjPAL gene anti-sense exp ressionvector to imp rove its rooting ability in regeneration system via regulating its phenolic compound content.