过表达或沉默棉花GhPCBER基因株系的获得及其茎叶木质素和木脂素含量研究

编码苯基香豆满苄基醚还原酶(phenylcoumaran benzylic ether reductase,PCBER)的基因PCBER属于PIP亚家族,是苯丙烷代谢途径中参与木脂素合成的关键基因。该研究构建了棉花GhPCBER基因的植物过表达载体并转化拟南芥,同时构建了VIGS(virus induced gene silencing,病毒诱导的基因沉默)载体转化棉花,采用实时荧光定量PCR技术对GhPCBER基因在不同组织中的表达进行分析;对野生型和转基因植株茎叶组织中的木质素和木脂素含量进行测定分析。结果表明:(1)成功构建了GhPCBER植物过表达载体pGWB17-GhPCBRE以及基因沉默重组载体pTRV2-GhPCBER;经遗传转化获得6株转棉花GhPCBER基因抗性拟南芥植株,同时获得15株GhPCBER基因沉默棉花植株(5株为一组)。(2)PCR检测表明,6株转基因拟南芥均为过表达株系,其中株系1、2、3相对表达量更高,且在茎、叶组织中的表达量分别较野生型提高了7～14倍和6～16倍,表明GhPCBER基因成功在拟南芥中过表达;GhPCBER基因沉默棉花植株的茎、叶组织中的表达量分别比野生型棉株约下降12%和26%,表明烟草脆裂病毒(TRV)体系(pTRV2-GhPCBER)成功抑制了GhPCBER基因的表达。(3)转GhPCBER基因拟南芥茎、叶中木质素和木脂素含量较野生型均显著降低;GhPCBER基因沉默棉花植株茎、叶中木质素和木脂素含量较野生型均极显著降低;组织化学染色观察发现GhPCBER基因沉默棉花植株茎秆颜色明显比野生型染色浅,也证明沉默基因棉花植株茎秆中的木质素含量减少。(4)苯丙烷代谢通路中8个相关基因的实时荧光定量PCR分析发现,过表达或抑制GhPCBRE基因均会导致苯丙烷代谢途径发生重新定向。

Overexpression or Silenced Cotton GhPCBER Gene Lines and Study on Lignin and Lignan Contents in Leaves and Stems

The PCBER gene belongs to the PIP subfamily and is an important gene involved in the synthesis of lignans in the phenylpropane metabolic pathway. In this study, The GhPCBER plant over expression vector pGWB17 GhPCBRE was constructed and transformed into Arabidopsis thaliana, and gene silencing recombinant vector pTRV2 GhPCBER was also constructed and transformed into cotton. Real time quantitative PCR was used to analyze the expression of GhPCBER gene under different tissues.The lignin and lignan contents in stem and leaf tissues from transgenic A. thaliana and cotton plants were extracted and determined. The results showed that: (1) GhPCBER plant overexpression vector pGWB17 GhPCBRE and gene silencing recombinant vectors pTRV2 GhPCBER were successfully constructed. Six transgenic A. thaliana lines with overexpression GhPCBER were obtained by genetic transformation, and GhPCBER gene silenced cotton plants were obtained(5 strains as a group). (2) PCR analysis showed that six transgenic Arabidopsis thaliana lines were overexpressing lines, among which the relative expression of lines 1, 2 and 3 was higher, and the expression in stem and leaf tissues was 7-14 and 6-16 times higher than that of wild type, indicating that the GhPCBER gene was successfully overexpressed in A. thaliana; The expression levels of GhPCBER gene silencing cotton plants in stems and leaves were 12% and 26% lower than those in wild type cotton plants, respectively, indicating that the tobacco fragile virus (TRV) system (pTRV2 GhPCBER) successfully inhibited the expression of GhPCBER gene. (3) The contents of lignin and lignan in stems and leaves of GhPCBER transgenic A. thaliana were significantly lower than that of wild type; the contents of lignin and lignan in stems and leaves of GhPCBER gene silencing cotton plants were significantly lower than that of wild type; Histochemical staining observation showed that the color of the stem of GhPCBER gene silencing cotton plants was significantly lighter than that of wild type, which also proved that the lignin content in the stems of silenced cotton plants decreased; (4) Real time quantitative PCR analysis of eight related genes in the phenylpropanoid metabolic pathway revealed that overexpression or inhibition of the GhPCBRE gene resulted the reorientation of the phenylpropanoid metabolic pathway.