利用VIGS技术初步验证WSR1基因功能的研究

Preliminary Validation of WSR1 Gene Function by VIGS Technology

为进一步了解小麦淀粉合成酶转录因子对淀粉合成的影响,以水稻 RSR1基因为探针,首先利用同源克隆技术和RACE技术获得了小麦 WSR1基因的全长cDNA;生物信息学分析表明,该基因含有AP2保守结构域。随后,以大麦条纹花叶病毒(BSMV)为载体,八氢番茄红素脱氢酶(PDS)基因为指示基因,利用VIGS技术和qRT-PCR技术对 WSR1基因的功能进行研究。结果表明,在接种5 d后, WSR1基因的相对表达量下降到50%, 25 d后下降到12%; WSR1基因沉默后的冀麦325籽粒内支链淀粉、总淀粉和抗性淀粉含量均较对照显著增加;直链淀粉含量、千粒重较对照极显著增加。以上结果表明 WSR1基因负向调控小麦的淀粉合成。

To further understand the effect of wheat starch synthase transcription factors on starch synthesis,the rice RSR1 gene was used as the probe to obtain the homologous RSR1 fragment from wheat genome. The full-length cDNA of the wheat WSR1 gene was cloned using RACE technology. The bioinformatics analysis showed that the gene contains an AP2 conserved domain,namely WSR1. The barley stripe mosaic virus(BSMV) was used as the vector,and the phytoene desaturase(PDS) gene was used as the indicator gene. The function of WSR1 gene was studied by virus induced gene silencing(VIGS) and qRT-PCR. The result showed that after five days of inoculation,the relative expression level of WSR1 gene was reduced to 50%,and remained at a relatively low level(about 12%) after 25 days of inoculation. The contents of amylopectin,total starch and resistant starch were much higher in the WSR1-silenced Jimai 325 than that in the control of Jimai 325 without BSMV:WSR1 transformation. The proportion of amylose was increased from 14.47% to 15.60% and the 1 000-grain weight was increased from 43.1 g to 45.8 g. It was demonstrated that the WSR1 gene negatively regulates starch synthesis in wheat.