Abstract: The low regeneration rate of alfalfa callus is one of the main factors limiting the genetic transformation efficiency of alfalfa. It has been found that GRF transcription factor family genes play an important role in promoting callus regeneration in a variety of plants. In this study,we used the AtGRF protein sequences as template to search in alfalfa genome database and obtained 9 MsGRFs genes containing miR396 target sites. Excepting MsGRF3a and MsGRF4a,the expression levels of the other 7 MsGRFs in embryonic callus of alfalfa were significantly higher than those in non-embryonic callus. To further investigate the effects of MsGRFs on callus regeneration efficiency of alfalfa,we transferred MIM396 gene into alfalfa and reduced the expression of miR396 in transgenic plants. The results showed that the regeneration time was significantly shortened,while the regeneration rate of callus of MIM396 transgenic plants was significantly increased. Compared to wild-type callus,the leaf inducing callus of MIM396 plants showed significantly higher expression of MsGRFs except MsGRF3a. The results indicated that miR396-MsGRF pathway played an important regulatory role in alfalfa callus regeneration,which provided candidate genes for improving the regeneration efficiency of alfalfa and breaking the restriction of genotype on regeneration of different alfalfa varieties.

Key words: miR396, MsGRFs, Medicago sativa, Callus, Regeneration

摘要： 紫花苜蓿(Medicago sativa L.)愈伤组织再生率受基因型限制，是影响苜蓿遗传转化效率的主要因素之一。研究发现，GRF转录因子家族基因在提高多种植物愈伤再生中起着重要促进作用。本试验以拟南芥(Arabidopsis thaliana)AtGRF序列为模板，在紫花苜蓿基因组序列中查找，获得了含有miR396靶点的9个MsGRFs基因。除MsGRF3a和MsGRF4a外，其余7个MsGRFs在紫花苜蓿胚性愈伤组织中的表达量均显著高于非胚性愈伤。为了进一步研究MsGRFs对紫花苜蓿愈伤再生效率的影响，我们在紫花苜蓿中转入MIM396基因，降低miR396的表达。结果显示：MIM396植株叶片愈伤组织再生时间显著缩短，再生率显著提高；与野生型愈伤组织相比，在MIM396植株叶片诱导的愈伤组织中，除MsGRF3a外，其余MsGRFs显著高表达。以上研究表明，miR396-MsGRF通路对苜蓿愈伤组织再生起着重要调控作用，这为提高苜蓿再生效率，并打破基因型对不同苜蓿品种再生的限制提供候选基因。

关键词: miR396, MsGRFs, 紫花苜蓿, 愈伤组织, 再生