为了验证查尔酮还原酶基因CHR1在大豆苷元合成中的作用, 文章克隆了CHR1基因并构建了RNA干扰表达载体pCPB-CHR1-RNAi, 将载体转化受体大豆品种“吉农28”中, 以期抑制CHR1基因的转录。通过农杆菌介导的遗传转化和PCR检测得到4株T0 代阳性植株, 13株T1代阳性植株。Southern blotting结果表明, 功能元件以单拷贝的形式整合到大豆的基因组中。利用实时荧光定量PCR法(Quantitative real-time PCR, qRT-PCR)测定CHR1基因在mRNA水平上的表达量, 结果表明, 转基因大豆植株中CHR1的表达量与未转化的受体大豆相比降低了60%~99%; 高效液相色谱法(High performance liquid chromatography, HPLC)检测到合成大豆苷元过程中的前体物质异甘草素的含量降低了38.7%。该RNA干扰机制在转录水平上抑制了CHR1基因的表达。
吴楠, 王丕武, 李丹, 代力强, 郑成忠, 卢实, 才源, 张卓, 曲静, 夏海丰
. 大豆查尔酮还原酶基因CHR1的功能研究[J]. 遗传, 2014
, 36(7)
: 707
-712
.
DOI: 10.3724/SP.J.1005.2014.0707
To verify the function of chalcone reductase gene (CHR1) in soybean Daidzein synthesis, CHR1 gene in soybean was cloned, and an RNAi expression vector pCPB-CHR1-RNAi was constructed. Four transformed plants in T0 generation and thirteen transformed plants in T1 generation of soybean “Jinong28” were obtained by agrobacterium-mediated genetic transformation, in which transcriptions of CHR1 gene were depressed. Southern blotting showed the functional fragment of pCPB-CHR1-RNAi was integrated into the genome of recipient soybean in the form of a single copy. Detection of the transcription of CHR1 gene using quantitative real-time PCR (qRT-PCR) showed that the expression of CHR1 gene in transformed plants decreased 60%-99% compared to the recipient soybean, while the content of isoliquiritigenin, the precursors of daidzein, decreased 38.7%. These results indicate that RNA interference can suppress the transcription of CHR1 gene expression successfully.
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